Phylogeny of yeasts and related filamentous fungi within Pucciniomycotina determined from multigene sequence analyses.

In addition to rusts, the subphylum Pucciniomycotina (Basidiomycota) includes a large number of unicellular or dimorphic fungi which are usually studied as yeasts. Ribosomal DNA sequence analyses have shown that the current taxonomic system of the pucciniomycetous yeasts which is based on phenotypic criteria is not concordant with the molecular phylogeny and many genera are polyphyletic. Here we inferred the molecular phylogeny of 184 pucciniomycetous yeast species and related filamentous fungi using maximum likelihood, maximum parsimony and Bayesian inference analyses based on the sequences of seven genes, including the small subunit ribosomal DNA (rDNA), the large subunit rDNA D1/D2 domains, the internal transcribed spacer regions (ITS 1 and 2) of rDNA including the 5.8S rDNA gene; the nuclear protein-coding genes of the two subunits of DNA polymerase II (RPB1 and RPB2) and the translation elongation factor 1-α (TEF1); and the mitochondrial gene cytochrome b (CYTB). A total of 33 monophyletic clades and 18 single species lineages were recognised among the pucciniomycetous yeasts employed, which belonged to four major lineages corresponding to Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes and Mixiomycetes. These lineages remained independent from the classes Atractiellomycetes, Classiculomycetes, Pucciniomycetes and Tritirachiomycetes formed by filamentous taxa in Pucciniomycotina. An updated taxonomic system of pucciniomycetous yeasts implementing the 'One fungus = One name' principle will be proposed based on the phylogenetic framework presented here.

Introduction

Basidiomycetous yeasts are unicellular or dimorphic fungi that belong to the three lineages of the Basidiomycota, namely Pucciniomycotina, Ustilaginomycotina and Agaricomycotina (also previously known as Urediniomycetes, Ustilaginomycetes and Hymenomycetes, respectively) (Boekhout, 1991, Bauer et al., 2006, Hibbett et al., 2007, Boekhout et al., 2011). At present, yeasts in the Pucciniomycotina comprise 28 genera, including 19 teleomorphic and 9 anamorphic ones (Bauer et al., 2009, Boekhout et al., 2011, Turchetti et al., 2011, Toome et al., 2013, de García et al., 2015). Our understanding of the phylogenetic relationships of these basidiomycetous yeasts and their systematics largely improved due to sequence analysis of parts of the ribosomal DNA (rDNA) (Fell et al., 2000a, Scorzetti et al., 2002), but the full taxonomic consequences of these studies have not yet been made. For instance, teleomorphic and anamorphic genera are still treated separately, and many anamorphic genera, such as Rhodotorula and Sporobolomyces, are polyphyletic (Fell et al., 2000a, Scorzetti et al., 2002, Boekhout et al., 2011, Hamamoto et al., 2011, Sampaio, 2011a). Species of these two genera occur in three classes in the Pucciniomycotina, and some Rhodotorula species occur even in another subphylum Ustilaginomycotina (Boekhout et al., 2011, Sampaio, 2011a).

Earlier results using sequence analysis of the small subunit (SSU or 18S) rDNA indicated that the yeast members within Pucciniomycotina could be divided into four groups, designated as the , , and subbrunneus clusters (Hamamoto and Nakase, 2000, Nakase, 2000). Sequence analyses of the large submit (LSU or 26S) rDNA D1/D2 domains and the internal transcribed spacer (ITS) region showed similar results and four lineages named , , and (Fell et al., 2000b, Scorzetti et al., 2002). In the 5th edition of The Yeasts, a Taxonomic Study, all known Pucciniomycotina yeast species were classified into four classes, namely Agaricostilbomycetes (the Agaricostilbum lineage), Cystobasidiomycetes (the lineage), Microbotryomycetes (the and lineages) and Mixiomycetes (Boekhout ). The above listed studies provided a detailed grouping of species in many clades of these four classes, but the molecularly defined clades frequently lacked concordance or statistic support and many species remained unassigned (Boekhout ). With the advent of the ‘One Fungus = One Name’ concept (Hawksworth, 2011, Taylor, 2011, McNeill et al., 2012) the anamorphic taxa have to be combined with the teleomorphic ones into a single taxonomy. Thus the boundaries of the clades and genera have to be reassessed by analyzing a robust molecular data set. For many of the yeast members of Pucciniomycotina SSU rDNA sequences were not yet available and also some LSU rDNA D1/D2 and ITS data were missing. In addition, protein coding gene sequences have rarely been used in molecular phylogeny studies of basidiomycetous yeasts. The multigene analysis of the fungal kingdom as presented by the Assembling the Fungal Tree of Life (AFTOL) consortium (James ) and its derived taxonomy (Hibbett ) showed the potential of this kind of analysis to improve our understanding of fungal evolutionary relationships and taxonomy.

In the present work, we employed the six genes that were used in the AFTOL project (James ) and an additional mitochondrial gene, cytochrome b (CYTB) that was used in phylogenetic analyses of some basidiomycetous yeast genera (Biswas et al., 2001, Biswas et al., 2005, Yokoyama, 2005, Wang and Bai, 2008) to resolve the tree of life of the pucciniomycetous yeasts. The aim of this work is to recognise monophyletic clades and to improve the phylogeny and taxonomy of this group of eukaryotic microorganisms. In addition, by using available data, mainly generated from the AFTOL project (http://www.aftol.org/data.php), we also inferred the evolutionary relationships between the unicellular yeast taxa and the main groups of filamentous fungi in the Pucciniomycotina.

Materials and methods

Yeast and filamentous taxa employed

One hundred and ninety nine strains belonging to 184 yeast species within Pucciniomycotina were studied (Table 1). They were mostly type and authentic strains from CBS Fungal Biodiversity Centre (CBS-KNAW), Utrecht, The Netherlands, the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, and the Japan Collection of Microorganisms (JCM), RIKEN BioResource Center, Saitama, Japan. The type strains of all pucciniomycetous yeast species included in the latest edition of The Yeasts, a Taxonomic Study (Kurtzman ) were employed. In addition, fifteen pucciniomycetous yeast species that were published after the publication of that treatment were used in this study. Fifteen representative filamentous taxa from the Pucciniomycotina were employed as references and two taxa from Ustilaginomycotina were used as an outgroup (Table 1). The alignments and trees were deposited in TreeBASE (No. 18076).

Table 1

List of pucciniomycetous yeasts and selected reference filamentous taxa employed. The sequences with GenBank numbers in bold are determined in this study.

Lineage/Clade	Species	Strain number	ITS	D1D2	SSU	RPB1	RPB2	TEF1	CYTB
Agaricostilbomycetes
Agaricostilbales
Kondoaceae
Kondoa	Bensingtonia changbaiensis	AS 2.2310T	AY233339	AY233339	AY233339	KJ708024	KJ708147	KJ707751	KJ707585
	B. miscanthi	JCM 5733T	AF444516	AF189891	D38236	KJ708023	KJ708149	KJ707753	KJ707719
	B. phyllada	JCM 7476T	AF444514	AF189894	D38237	KJ708022	KJ708152	KJ707756	KJ707727
	B. sorbi	AS 2.2303T	AY233343	AY233343	AY233343	KJ708029	KJ708156	KJ707897	KJ707584
	B. subrosea	JCM 5735T	AF444565	AF189895	D38238	KJ708027	KJ708157	KJ707895	KJ707640
	B. thailandica	JCM 10651T	AB040114	EF384207	AB040114	KJ708026	KJ708159	KJ707898	KJ707661
	B. yuccicola	JCM 6251T	AF444518	AF189897	D38367	KJ708025	KJ708161	/	/
	Kondoa aeria	CBS 8352T	AF444562	AF189901	KJ708417	KJ708020	KJ708172	KJ707905	/
	K. malvinella	AS 2.1946T	AF444498	AF189903	D13776	KJ708021	KJ708173	KJ707896	KJ707568
Bensingtonia	Bensingtonia ciliata	AS 2.1945T	AF444563	AF189887	D38233	KF706509	KF706536	KF706486	KJ707567
	B. naganoensis	JCM 5978T	AF444558	AF189893	D38366	KJ707960	KJ708151	KJ707755	KJ707722
	B. pseudonaganoensis	AS 2.2601T	DQ224375	DQ224374	KJ708416	KJ707959	KJ708153	KJ707956	KJ707590
Agaricostilbaceae
ingoldii	B. ingoldii	JCM 7445T	AF444519	AF189888	D38234	KJ707961	KJ708148	KJ707752	KJ707726
	B. musae	JCM 8801T	AF444569	AF189892	D43946	KJ707963	KJ708150	KJ707754	KJ707743
Agaricostilbum	Agaricostilbum hyphaenes	CBS 7811	AF444553	AF177406	AY665775	KJ707965	KJ708145	KJ707749	KJ707645
	A. pulcherrimum	FO 29365 (ATCC MYA-4629)	AJ406402	GU291289	FJ641896	/	FJ623647	/	/
	Sterigmatomyces elviae	JCM 1822T	AF444551	AF177415	KJ708432	KJ707964	KJ708345	/	KJ707699
	S. elviae	JCM 1602	AB038053	KP216512	KP216516	KJ708077	KJ708208	KJ707852	AB040614
	S. halophilus	AS 2.1935T	AF444556	AF177416	D64119	KJ707962	/	KJ707890	KJ707566
Chionosphaeraceae
Chionosphaera	Chionosphaera apobasidialis	CBS 7430	AF444599	AF177407	U77662	/	KJ708163	KJ707883	KJ707641
	C. cuniculicola	CBS 10063	KJ778640	KJ708465	KJ708368	KJ707985	KJ708164	KJ707886	KJ707593
	C. cuniculicola	CBS 10065	KJ778641	KJ708466	KJ708369	KJ707984	KJ708165	KJ707887	KJ707594
Kurtzmanomyces	Kurtzmanomyces insolitus	JCM 10409T	AF444594	AF177408	KJ708424	KJ707986	KJ708175	KJ707893	KJ707685
	K. nectairei	AS 2.1950T	AF444494	AF177409	D64122	KJ707980	KJ708176	KJ707884	KJ707571
	K. tardus	JCM 10490T	AF444566	AF177410	KJ708425	KJ707992	KJ708177	KJ707885	KJ707686
sasicola	Sporobolomyces sasicola	AS 2.1933T	AF444548	AF177412	AB021688	KJ707990	KJ708335	KJ707900	KJ707565
	S. taupoensis	JCM 8770T	AF444592	AF177413	D66886	/	KJ708339	KJ707901	KJ707741
	S. xanthus	AS 2.1957T	AF444547	AF177414	D64118	KJ707993	KJ708343	KJ707902	KJ707573
lactophilus	S. lactophilus	JCM 7595T	AF444545	AF177411	AB021675	/	KJ708312	KJ707889	KJ707642
	S. lophatheri	CBS 11272T	AB126046	AB124561	AB126046	KJ707988	KJ708315	KJ707880	KJ707608
	Cystobasidiopsis nirenbergiae	BBA 65452T	GQ180106	FJ536254	/	/	/	/	/
Single-species lineage	Mycogloea nipponica	CBS 11308	KJ778629	KJ708456	KJ708370	KJ707982	KJ708194	KJ707882	KJ707609
Incertae sedis in Agaricostilbales
ruber	Sporobolomyces clavatus	AS 2.2318T	AY364839	AY364839	KJ708406	KJ707979	KJ708295	KJ707894	KJ707586
	S. diospyri	JCM 12157T	AB126047	AB124560	AB126047	KJ707989	KJ708298	KJ707904	KJ707696
	S. dracophylli	AS 2.1959T	AF444583	AF189982	D66882	KJ707987	KJ708299	KJ707879	KJ707575
	S. pyrrosiae	JCM 12159T	AB126045	AB124562	AB126045	KJ707981	KJ708330	KJ707903	KJ707697
	S. ruber	AS 2.1958T	AF444550	AF189992	AB021686	KJ707983	KJ708333	KJ707899	KJ707574
Single-species lineage	Bensingtonia sakaguchii	JCM 10047T	AF444626	AF363646	AB001746	KJ707958	KJ708155	KJ707891	KJ707671
Spiculogloeales
subbrunneus	Sporobolomyces coprosmicola	JCM 8767T	AF444576	AF189981	D66879	/	KJ708171	KJ707908	KJ707740
	S. dimmenae	JCM 8762T	AB038046	AB644404	D66881	KJ707991	KJ708297	KJ707907	KJ707739
	S. linderae	JCM 8856T	AF444582	AF189989	D66885	/	/	KJ707906	KJ707744
	S. novozealandicus	JCM 8756T	AB038048	KJ708467	KJ708443	KJ708073	KJ708319	KJ707851	KJ707738
	S. subbrunneus	JCM 5278T	AF444549	AF189997	AB021691	/	/	KJ707909	KJ707710
Mycogloea	Mycogloea sp.	TUBFO40962	/	AY512868	DQ198791	/	/	/	/
Spiculogloea	Spiculogloea sp.	TUB RB1040	/	AY512885	/	/	/	/	/
Cystobasidiomycetes
Cystobasidiales
minuta	Cystobasidium fimetarium	DB1489	/	AY512843	AY124479	/	/	LM644071	/
	Rhodotorula benthica	JCM 10901T	AB026001	AB026001	AB126647	KJ708081	KJ708214	KJ707842	KJ707691
	R. calyptogenae	JCM 10899T	AB025996	AB025996	AB126648	KJ708075	KJ708218	KJ707840	KJ707690
	R. laryngis	JCM 10953T	AB078500	AB078500	AB126649	KJ708055	KJ708240	KJ707824	KJ707619
	R. lysiniphila	JCM 5951T	AB078501	AB078501	AB126650	KJ708074	KJ708243	KJ707845	KJ707721
	R. minuta	AS 2.1516T	AF190011	AF189945	D45367	KJ708059	KJ708246	KJ707825	KJ707562
	R. pallida	JCM 3780T	AB078492	AF189962	AB126651	KJ708056	KJ708253	KJ707826	KJ707621
	R. pinicola	AS 2.2193T	AF444292	AF444293	AB126652	KJ708057	KJ708257	KJ707827	KJ707579
	R. slooffiae	JCM 10954T	AF444627	AF444722	AB126653	KJ708058	KJ708266	KJ707828	KJ707629
Single-species lineage	Occultifur externus	JCM 10725T	AF444567	AF189910	AB055193	KJ708060	KJ708199	KJ707829	KJ707689
Erythrobasidiales
Erythrobasidium	Erythrobasidium hasegawianum	AS 2.1923T	AF444522	AF189899	D12803	KF706506	KF706534	KJ707776	KJ707563
	Sporobolomyces elongatus	AS 2.1949T	AF444561	AF189983	AB021669	KJ708012	KJ708300	KJ707782	KJ707570
	S. yunnanensis	AS 2.2090T	AB030353	AB127358	AF229176	KJ708015	KJ708344	KJ707779	KJ707576
Bannoa	Bannoa sp.	MP 3490	DQ631900	DQ631898	DQ631899	/	DQ631901	DQ631902	/
	B. hahajimensis	JCM 10336T	AB035897	AB082571	AB035897	KJ708014	KJ708146	KJ707750	KJ707682
	Sporobolomyces bischofiae	JCM 10338T	AB035721	AB082572	AB035721	KJ708018	KJ708292	KJ707777	KJ707684
	S. ogasawarensis	JCM 10326T	AB035713	AB082570	AB035713	KJ708017	KJ708323	KJ707781	KJ707681
	S. syzygii	JCM 10337T	AB035720	AB082573	AB035720	KJ708011	KJ708338	KJ707778	KJ707683
Single-species clade	Cyrenella elegans	CBS 274.82	KJ778626	KJ708454	KJ708360	KJ708080	KJ708168	KJ707830	KJ707620
	Rhodotorula lactosa	CBS 5826T	AF444540	AF189936	D45366	KJ708016	KJ708239	/	AB040633
Naohideales
Naohidea	Naohidea sebacea	CBS 8477T	DQ911616	DQ831020	KP216515	KF706508	KF706535	KF706487	KJ707654
	N. sebacea	CBS122592	/	/	/	KJ708019	KJ708198	KJ707783	KJ707612
Incertae sedis in Cystobasidiomycetes
aurantiaca	Rhodotorula armeniaca	JCM 8977T	AF444523	AF189920	AB126644	KP216521	KJ708211	KJ707762	AB040615
	R. aurantiaca	JCM 3771T	AF444538	AF189921	KJ708436	KJ707970	KJ708212	KJ707757	AB040616
	Sporobolomyces kluyveri-nielii	JCM 6356T	AF444544	AF189988	AB021674	KJ707977	KJ708310	KJ707760	/
	S. phyllomatis	JCM 7549T	AF444515	AF189991	AB021685	KJ707976	KJ708328	KJ707761	KJ707728
	S. salicinus	JCM 2959T	AF444511	AF189995	AB021687	/	/	KJ707758	KJ707703
marina	Rhodotorula marina	JCM 3776T	AF444504	AF189944	AB126645	KJ707973	KJ708244	KJ707795	AB040635
	Sporobolomyces coprosmae	JCM 8772T	AF444577	AF189980	D66880	KJ707966	KJ708296	KJ707798	KJ707742
	S. foliicola	AS 2.2527T	AF444521	AF189984	AB021671	KJ707969	KJ708302	KJ707797	KJ707589
	S. gracilis	JCM 2963T	AF444578	AF189985	KJ708433	KJ707968	KJ708304	KJ707799	KJ707705
	S. oryzicola	JCM 5299T	AF444546	AF189990	AB021677	KJ707974	KJ708324	KJ707955	KJ707712
	S. symmetricus	AS 2.2299T	AY364836	AY364836	KJ708350	KJ707975	KJ708337	KJ707800	KJ707582
	S. vermiculatus	JCM 10224T	AB030335	AF460176	AB030322	KJ707967	KJ708342	KJ707801	KJ707675
Sakaguchia	Rhodotorula cladiensis	CBS 10878T	FJ008055	FJ008049	KJ708354	/	KJ708219	KJ707847	KJ707603
	R. lamellibrachii	CBS 9598T	AB025999	AB025999	AB126646	KJ708098	KJ708314	KJ707876	KJ707667
	R. meli	CBS 10797T	FJ807683	KJ708452	KJ708355	KJ708085	KJ708245	KJ707855	KJ707602
	R. oryzae	AS 2.2363T	AY335160	AY335161	KJ708352	KJ708100	KJ708250	KJ707853	KJ707587
	R. oryzae	AS 2.3289	KP216523	KJ708451	KJ708353	KJ708103	KJ708251	KJ707848	KJ707592
	Rhodotorula sp.	JCM 8162	KJ778625	KJ708453	KJ708356	KJ708079	KJ708268	KJ707858	KJ707732
	Sakaguchia dacryoidea	JCM 3795T	AF444597	AF189972	D13459	KJ708102	KJ708348	KP216514	KJ707709
	S. dacryoidea	CBS 7999	AF444571	AF444723	KJ708351	KJ708099	KJ708346	KJ707878	KJ707647
magnisporus	Rhodotorula bloemfonteinensis	CBS 8598T	EU075189	EU075187	KJ708359	KJ708082	KJ708215	/	KJ707657
	R. orientis	CBS 8594T	HM559719	HM559718	KJ708358	KJ708078	KJ708249	KJ707843	KJ707656
	R. pini	CBS 10735T	EU075190	EU075188	KJ708357	KJ708084	KJ708258	KJ707832	KJ707601
	Sporobolomyces magnisporus	JCM 11898T	AB112078	AB111954	KJ708428	KJ708013	KJ708317	KJ707780	KJ707695
Microbotryomycetes
Sporidiobolales
Rhodosporidium	Rhodosporidium babjevae	JCM 9279T	AF444542	AF070420	AB073270	/	/	KJ707874	KJ707746
	R. diobovatum	JCM 3787T	AF444502	AF070421	AB073271	KJ708091	KJ708277	KJ707865	KJ707708
	R. kratochvilovae	JCM 8171T	AF444520	AF071436	AB073273	KJ708095	KJ708205	KJ707863	KJ707733
	R. paludigenum	JCM 10292T	AF444492	AF070424	KJ708422	KJ708094	KJ708206	KJ707870	KJ707676
	R. sphaerocarpum	JCM 8202T	AF444499	AF070425	AB073275	KJ708086	KJ708207	KJ707867	KJ707734
	R. toruloides	CBS 349	AF444489	AF070426	X60180	KJ708090	KJ708278	/	KJ707623
	R. toruloides	AS 2.1389	KJ778637	KP216510	KJ708403	KJ708072	KJ708265	KJ707846	KJ707561
	Rhodotorula araucariae	JCM 3770T	AF444510	AF070427	KJ708435	KJ708096	KJ708209	KJ707862	AB041048
	R. dairenensis	CBS 4406T	AF444501	AY033552	KJ708411	/	KJ708276	KJ707866	KJ707625
	R. evergladiensis	CBS 10880T	FJ008054	FJ008048	KJ708398	/	KJ708228	KJ707834	/
	R. glutinis	JCM 8208T	AF444539	AF070429	X69853	/	/	KJ707869	AB040626
	R. graminis	JCM 3775T	AF444505	AF070431	X83827	KJ708093	KJ708234	KJ707868	AB040628
	R. mucilaginosa	JCM 8115T	AF444541	AF070432	AB021668	/	KJ708247	KJ707861	KJ707731
	R. pacifica	CBS 10070T	AB026006	AB026006	KJ708397	KJ708087	KJ708252	KJ707860	KJ707595
	R. taiwanensis	CBS 11729T	GU646862	GU646863	KJ708409	KJ708066	KJ708271	KJ707838	KJ707611
	Sporobolomyces alborubescens	JCM 5352T	AB030342	AF207886	KJ708440	KJ708089	KJ708289	KJ707864	KJ707714
Mixed Rhodosporidium/Sporidiobolus	Rhodosporidium azoricum	JCM 11251T	AB073229	AF321977	AB073269	KJ708053	KJ708202	KJ707813	KJ707693
	R. fluviale	JCM 10311T	AY015432	AF189915	AB073272	KJ708046	KJ708204	KJ707816	KJ707679
	R. lusitaniae	JCM 8547T	AY015430	AF070423	AB073274	KJ708047	/	KJ707812	KJ707737
	Rhodotorula colostri	CBS 348T	JN246563	AY372177	KJ708399	KJ708051	KJ708220	KJ707818	KJ707622
	Sporidiobolus microsporus	JCM 6882T	AF444535	AF070436	KJ708441	KJ708054	KJ708284	KJ707817	KJ707724
	S. ruineniae	JCM 1839T	AF444491	AF070434	AB021693	KJ708052	KJ708286	KJ707820	KJ707700
	Sporobolomyces nylandii	JCM 10213T	AB030323	AF387123	AB030319	KJ708050	KJ708321	KJ707822	KJ707674
	S. odoratus	JCM 11641T	KJ778638	AF387125	KJ708427	KJ708045	KJ708322	KJ707819	KJ707694
	S. poonsookiae	JCM 10207T	AB030327	AF387124	AB030320	KJ708048	KJ708329	KJ707821	KJ707672
Sporidiobolus	Sporidiobolus johnsonii	AS 2.1927T	AY015431	AF070435	L22261	KJ708105	/	KJ707914	KJ707564
	S. longiusculus	CBS 9655T	JN246566	KJ708464	KJ708400	KJ708109	KJ708282	KJ707929	KJ707668
	S. metaroseus	CBS 7683T	EU003482	EU003461	KJ708415	KJ708068	KJ708283	KJ707841	KJ707644
	S. pararoseus	JCM 5350T	AF417115	AF070437	AB021694	KJ708115	KJ708279	KJ707924	KJ707713
	S. salmonicolor	JCM 1841T	AY015434	AF070439	AB021697	KJ708114	KJ708287	KJ707923	KJ707701
	Sporobolomyces bannaensis	AS 2.2285T	AY274824	AY274823	KJ708405	KJ708120	KJ708290	KJ707934	KJ707581
	S. beijingensis	AS 2.2365T	AY364837	AY364837	KJ708407	KJ708116	KJ708291	KJ707919	KJ707588
	S. blumeae	JCM 10212T	AB030331	AY213010	AB030321	/	KJ708293	KJ707926	KJ707673
	S. carnicolor	JCM 3766T	AY069991	AY070008	KJ708434	KJ708117	KJ708294	KJ707912	KJ707707
	S. holsaticus	CBS 1522	AF444509	AF189975	AB021672	KJ708106	/	KJ707916	KJ707614
	S. japonicus	AS 2.2192T	AY069992	AY158640	/	KJ708123	KJ708307	KJ707932	KJ707578
	S. jilinensis	AS 2.2301T	AY364838	AY364838	KJ708450	KJ708111	KJ708308	KJ707913	KJ707583
	S. koalae	CBS 10914T	EU276008	EU276011	KP216519	KJ708063	KJ708311	KJ707850	KJ707604
	S. marcillae	JCM 6883T	AY015437	AF070440	KJ708442	KJ708112	KJ708318	KJ707933	KJ707725
	S. patagonicus	CBS 9658	AY552329	AY158656	KP216518	KJ708108	KJ708326	KJ707930	KJ707669
	S. patagonicus	CBS 9657T	AY552328	AY158655	KJ708421	KJ708110	KJ708325	KJ707928	KP216520
	S. phaffii	AS 2.2137T	AY069995	AY070011	KJ708404	KJ708113	KJ708327	KJ707918	KJ707577
	S. roseus	AS 2.1948T	AY015438	AF070441	X60181	KJ708119	KJ708331	KJ707917	KJ707569
	S. ruberrimus	CBS 7550T	AY015439	AF070442	KJ708402	KJ708121	KJ708332	KJ707915	KJ707643
	S. salmoneus	AS 2.2195T	AY070005	AY070017	KJ708401	KJ708107	KJ708334	KJ707920	KJ707580
Kriegeriales
Kriegeriaceae
Kriegeria	Kriegeria eriophori	CBS 8387T	AF444602	NR_119455	DQ419918	KJ708144	KJ708174	KJ707936	KJ707649
glacialis	Rhodotorula glacialis	CBS 10436T	EF151249	EF151258	KJ708381	KJ708067	KJ708233	KJ707831	KJ707597
	R. psychrophenolica	CBS 10438T	EF151246	EF151255	KJ708382	KJ708071	KJ708259	KJ707859	KJ707598
	R. psychrophila	CBS 10440T	EF151243	EF151252	KJ708383	/	KJ708260	KJ707833	KJ707599
Single-species lineage	Meredithblackwellia eburnea	CBS12589	JX508799	JX508798	JX508797	/	/	/	/
	Rhodotorula rosulata	CBS 10977T	EU872492	EU872490	KJ708384	KJ708083	KJ708263	KJ707854	KJ707607
Camptobasidiaceae
Glaciozyma	Glaciozyma antarctica	JCM 9057T	AF444529	AF189906	DQ785788	KJ708131	KJ708182	/	KJ707745
Leucosporidiales
Leucosporidium	Leucosporidium creatinivorum	JCM 10699	KJ778627	KJ708455	KJ708385	KJ708064	KJ708221	KJ707857	KJ707687
	L. creatinivorum	CBS 8620T	AF444629	AF189925	KJ708418	KJ708036	KJ708178	KJ707789	KJ707658
	L. fellii	JCM 9887T	AF444508	AF189907	KJ708449	KJ708030	KJ708184	KJ707784	KJ707748
	L. fragarium	JCM 3930	AF444530	AF070428	KJ708437	KJ708034	KJ708231	KJ707790	AB040623
	L. fragarium	CBS 6254T	AF444530	AF070428	KJ708413	KJ708031	KJ708179	KJ707791	AB040623
	L. golubevii	CBS 9651T	AY212987	AY212999	KJ708386	KJ708037	KJ708185	KJ707787	/
	L. intermedium	JCM 5291T	AF444630	AF189889	D38235	KJ708132	KJ708188	KJ707785	KJ707711
	L. muscorum	CBS 6921T	AF444527	AF070433	KJ708414	KJ708038	KJ708180	KJ707793	AB040638
	L. scottii	JCM 9052T	AF444495	AF070419	X53499	KJ708033	KJ708186	KJ707788	AB040658
	L. yakuticum	JCM 10701	AY212989	AF189971	KJ708426	KJ708032	KJ708274	KJ707794	KJ707688
	L. yakuticum	CBS 8621T	AY212989	AY213001	KJ708419	/	KJ708181	/	KJ707659
Microbotryales
Microbotryum	Microbotryum reticulatum	CBS 101451	KJ778630	KJ708457	KJ708389	KJ708040	KJ708189	KJ707806	KJ707596
	M. scabiosae	CBS 677.93	KJ708459	KJ708459	KJ708390	/	KJ708195	KJ707808	KJ707633
	M. scabiosae	CBS 176.24	KJ708458	KJ708458	KJ708391	KJ708039	KJ708190	KJ707810	KJ707615
	M. scorzonerae	CBS 685.93	KJ708461	KJ708461	KJ708392	/	KJ708191	KJ707804	KJ707635
	M. scorzonerae	CBS 364.33	KJ708460	KJ708460	KJ708393	KJ708043	KJ708196	KJ707805	KJ707624
	M. violaceum	CBS 143.21	KJ708462	KJ708462	KJ708388	KJ708042	KJ708192	KJ707811	KJ707613
	Sphacelotheca hydropiperis	CBS 179.24	KJ708463	KJ708463	KJ708394	KJ708041	KJ708281	KJ707807	KJ707616
	S. koordersiana	JAG 55	DQ832221	DQ832219	DQ832220	DQ832223	DQ832222	DQ832224	/
Single-species lineage	Rhodotorula hordea	JCM 3932T	AF444524	AF189933	AY657013	/	KJ708235	KJ707802	/
Heterogastridiales
Heterogastridium	Heterogastridium pycnidioideum	CBS 591.93	GU291276	GU291290	KJ708412	KJ708009	KJ708170	KJ707770	KJ707630
Incertae sedis in Microbotryomycetes
buffonii	Rhodotorula bogoriensis	JCM 1692T	AF444536	AF189923	KJ708363	KJ708130	KJ708216	KJ707949	AB040619
	R. buffonii	JCM 3929T	AF444526	AF189924	KJ708362	KJ708127	KJ708217	KJ707946	AB040620
	R. pustula	JCM 3934T	AF444531	AF189964	KJ708361	KJ708128	KJ708261	KJ707937	AB040642
tsugae	R. cresolica	JCM 10955T	AF444570	AF189926	KJ708365	KJ708135	KJ708222	KJ707942	/
	R. pilati	JCM 9036T	AF444598	AF189963	KJ708364	KJ708137	KJ708256	KJ707947	AB040641
	Sporobolomyces tsugae	JCM 2960T	AF444580	AF189998	AB021692	/	KJ708340	KJ707945	KJ707628
yarrowii	Rhodotorula silvestris	CBS 11420T	GQ121045	GQ121044	KJ708366	KJ708069	KJ708264	KJ707849	KJ707610
	R. straminea	CBS 10976T	EU872491	EU872489	KJ708367	KJ708065	KJ708269	KJ707844	KJ707606
	R. yarrowii	JCM 8232T	AF444628	AF189971	AB032658	/	KJ708275	KJ707938	KJ707735
griseoflavus	Sporobolomyces fushanensis	JCM 12422T	KP216522	AB176591	AB176530	KJ708142	KJ708303	KJ707944	KJ707698
	S. griseoflavus	JCM 5653T	AF444557	AF189986	D66884	KJ708143	KJ708305	KJ707950	KJ707717
yamatoana	Bensingtonia yamatoana	AS 2.1956T	AF444634	AF189896	D38239	KJ708141	KJ708160	KJ707948	KJ707572
	Rhodotorula arctica	CBS 9278	AB478857	AB478858	KJ708371	KJ708070	KJ708210	KJ707856	KJ707666
singularis	R. lignophila	CBS 7109T	AF444513	AF189943	KJ708372	KJ708139	KJ708241	KJ707953	KJ707637
	Sporobolomyces singularis	JCM 5356T	AF444600	AF189996	AB021690	KJ708140	KJ708336	KJ707957	KJ707716
Colacogloea	Colacogloea peniophorae	CBS 684.93	DQ202270	AY629313	DQ234565	DQ234569	DQ234550	DQ234566	/
	Rhodotorula cycloclastica	CBS 8448T	AF444732	AF444631	KJ708376	KJ707997	KJ708224	KJ707775	KJ707652
	R. diffluens	JCM 1695T	AF444533	AF075485	KJ708380	KJ708125	KJ708226	KJ707939	AB040621
	R. eucalyptica	CBS 8499T	EU075185	EU075183	KJ708377	KJ708061	KJ708227	KJ707839	KJ707655
	R. foliorum	JCM 1696T	AF444633	AF317804	KJ708378	KJ708126	KJ708230	KJ707941	AB040622
	R. philyla	JCM 3933T	AF444506	AF075471	KJ708438	KJ707995	KJ708254	KJ707772	KJ707631
	R. retinophila	CBS 8446T	AF444624	AF444730	KJ708373	KJ707994	KJ708262	KJ707771	KJ707651
	R. terpenoidalis	CBS 8445T	AF444623	AF444729	KJ708374	KJ707999	KJ708272	KJ707774	KJ707650
	Sporobolomyces falcatus	JCM 6838T	AF444543	AF075490	AB021670	KJ708124	KJ708301	KJ707943	KJ707723
vanillica	Rhodotorula ingeniosa	JCM 9031T	AF444534	AF189934	KJ708445	KJ708004	KJ708237	KJ707803	AB040631
	R. vanillica	JCM 9741T	AF444575	AF189970	KJ708448	KJ708005	KJ708273	KJ707809	KJ707747
sonckii	R. auriculariae	JCM 1597T	AF444507	AF189922	KJ708429	KJ708134	KJ708213	KJ707935	AB040617
	R. sonckii	JCM 3935T	AF444601	AF189969	KJ708439	KJ708118	KJ708267	KJ707911	AB040643
Curvibasidium	Curvibasidium cygneicollum	JCM 10310T	AF444490	AF189928	KJ708423	KJ708001	KJ708169	KJ707768	KJ707678
	C. cygneicollum	JCM 9029T	AB038090	KP216511	KJ708444	KJ708062	KJ708232	KJ707836	AB040625
	C. pallidicorallinum	CBS 9091T	AF444641	AF444736	KJ708420	KJ708000	KJ708167	KJ707767	KJ707665
	Rhodotorula nothofagi	JCM 9034	AF444537	AF189950	KJ708447	KJ708002	KJ708248	KJ707765	AB040639
Reniforma	Reniforma strues	CBS 8263T	AF444573	AF189912	KP216517	KJ708122	KJ708200	KJ707927	KJ707648
Single-species lineage	Pseudoleucosporidium fasciculatum	CBS 8786T	KJ778628	AY212993	KJ708387	KJ707998	KJ708183	KJ707769	/
	Rhodotorula crocea	CBS 2029T	FM957565	AY372179	KJ708410	KJ708007	KJ708223	KP216513	KJ707618
	R. ferulica	JCM 8231T	AF444528	AF363653	KJ708379	KJ708129	KJ708229	KJ707940	/
	R. hylophila	JCM 1805T	AF444622	AF363645	KJ708431	KJ708008	KJ708236	KJ707764	AB040630
	R. javanica	JCM 9032T	AF444532	AF189935	KJ708446	KJ708006	KJ708238	KJ707766	AB040632
	Sporobolomyces inositophilus	JCM 5654T	AF444559	AF189987	AB021673	KJ708136	KJ708306	KJ707951	KJ707718
Mixiomycetes
Mixia	Mixia osmundae	CBS 9802	DQ831010	DQ831009	D14163	KJ708076	KJ708193	KJ707837	KJ707670
Tritirachiomycetes
	Tritirachium oryzae	CBS 164.67	GQ329853	KF258732	JF779647	/	JF779648	JF779645	/
	Tritirachium sp.	CBS 473.93	JF779664	JF779649	JF779650	/	JF779646	JF779651	/
	Tritirachium sp.	CBS 265.96	JF779668	JF779652	JF779653	/	JF779654	/	/
Pucciniomycetes
	Chrysomyxa arctostaphyli	CFB22246	DQ200930	AY700192	AY657009	/	DQ408138	DQ435789	/
	Endocronartium harknessii	CFB22250	DQ206982	AY700193	AY665785	/	DQ234551	DQ234567	/
	Helicobasidium mompa	CBS 278.51	AY292429	AY254179	U77064	/	/	EF100614	/
	Insolibasidium deformans	TDB183-1	/	AF522169	AY123292	/	/	/	/
	Platygloea disciformis	IFO32431	DQ234556	AY629314	DQ234563	/	DQ234554	DQ056288	/
	Puccinia graminis tritici	CRL75-36-700-3/ECS	AF468044	AF522177	AY125409	XM_003334476	XM_003321826	XM_003333024	/
	Septobasidium canescens	DUKE:DAH(323)	DQ241446	DQ241479	DQ241410	/	/	/	/
Atractiellomycetes
	Helicogloea lagerheimii	FO 36341	AY512849	AY124476	/	/	/	/	/
	H. variabilis	KW 1540	L20282	U78043	/	/	/	/	/
	Platygloea vestita	DB 1280	AY512872	AY124480	/	/	/	/	/
Classiculomycetes
	Classicula fluitans	ATCC 64713	AY512838	AY124478	/	/	/	/	/
	Jaculispora submersa	CCM 8127	AY512853	AY124477	/	/	/	/	/
Ustilaginomycotina
	Rhodotorula phylloplana	JCM 9035T	AB038131	AF190004	AJ496258	KP322906	KP323063	KP323116	AB041051
	Ustilago maydis	CBS 504.76/IFM 49220	AF453938	AY854090	X62396	XM401478	AY485636	AY885160	AB040663

Sequencing and molecular phylogenetic analyses

A set of seven genes or loci were included in this study, including three rDNA regions, namely SSU, LSU D1/D2 domains and ITS (including 5.8S rDNA); three nuclear protein coding genes, namely the largest subunit of RNA polymerase II (RPB1), the second largest subunit of RNA polymerase II (RPB2), and translation elongation factor 1-α (TEF1); and the mitochondrial gene cytochrome b (CYTB). Sequencing of the ITS region and LSU D1/D2 domains were performed using methods described previously (Fell et al., 2000b, Wang and Bai, 2004). SSU rDNA sequences were determined according to Wang . Sequences of CYTB were obtained as described by Wang & Bai (2008). PCR and sequencing primers for RPB1, RPB2 and TEF1 are listed in Table 2. PCR amplification and sequencing of the three nuclear protein-coding genes were performed using methods described previously (Wang ). GenBank accession numbers for all the sequences determined in this study are listed in Table 1.

Table 2

PCR and sequence primers used.

Locus	Primers (5′–3′)
RPB1	RPB1-Af: GAR TGY CCD GGD CAY TTY GG
RPB1-Cr: CCN GCD ATN TCR TTR TCC ATR TA
RPB2	fRPB2-5F: GAY GAY MGW GAT CAY TTY GG
fRPB2-7cR: CCC ATR GCT TGY TTR CCC AT
bRPB2-6F: TGG GGY ATG GTN TGY CCY GC
gRPB2-6R: GCA GGR CAR ACC AWM CCC CA
TEF1	EF1-983F: GCY CCY GGH CAY CGT GAY TTY AT
EF1-2218R: AT GAC ACC RAC RGC RAC RGT YTG
EF1-1577F: CAR GAY GN TAC AAG ATY GGT GG
EF1-1567R: AC HGT RCC RAT ACC ACC RAT CTT
CYTB	E1M4: TGR GGW GCW ACW GTT ATT ACT A
E2mr3: GGW ATA GCA CGT ARA AYW GCR TA
18S rDNA	P1 F: ATC TGG TTG ATC CTG CCA GT
570 F: CGC GGT AAT TCC AGC TCC A
934 F: CTG CGA AAG CAT TTG CCA AGG
1272 F: ATG GCC GTT CTT AGT TGG T
U1 R: TGG AAT TAC CGC GGC TGC TGG CAC C
U2 R: CCG TCA ATT CCT TTA AGT TTC AGC C
U3 R: GAC GGG CGG TGT GTA CAA AGG GCA G
ITS + 5.8S rDNA	ITS1: GTC GTA ACA AGG TTT CCG TAG GTG
ITS4: TCC TCC GCT TAT TGA TAT GC
D1D2 of 26S rDNA	NL1: GCA TATC AAT AAG CGG AGG AAA AG
NL4: GG TCC GTG TTT CAA GAC GG

Sequences were aligned with the MAFFT program (Standley 2013) using the L-INS-I algorithm. The alignment datasets were analysed with Modeltest version 3.04 (Posada & Crandall 1998) using the Akaike information criterion (AIC) to find the most appropriate model of DNA substitution. A general time-reversible model of DNA substitution additionally assuming a percentage of invariable sites and Γ-distributed substitution rates at the remaining sites (GTR + I + G) was selected for Maximum likelihood (ML) and Bayesian inference (BI) analyses. ML analysis was conducted using RAxML-HPC 7.2.8 (Stamatakis 2006) with a rapid bootstrap analysis using a random starting tree and 1 000 bootstrap replicates searching for the best maximum-likelihood tree, and with GTRGAMMAI as the model of evolution. BI analysis was conducted using MrBayes 3.1.2 (Ronquist ) with the GTR + I + G model and 5 000 000 to 10 000 000 generations, two independent runs and four chains. The other parameters were set as default. The analysis was stopped when the standard deviation of split frequencies between the trees generated in the independent runs was below 0.01. Twenty five percent of these trees were discarded, the remaining were used to compute a 50 % majority rule consensus tree to obtain estimates for posterior probabilities. Maximum parsimony (MP) analysis was performed using PAUP* 4.0b10 (Swofford 2002) with a heuristic search with 1 000 random additions and TBR. Bootstrap analysis was performed from 1 000 replicates using 10 random additions and TBR for each replicate. The gaps in the alignment were treated as missing data. MulTrees and Steepest descent options were not in effect. A bootstrap percentage (BP) of ≥70 % or a Bayesian posterior probability (PP) of ≥0.9 was considered as significantly supported in all constructed trees in this study.

Results and discussion

Sequence data obtained

From the sequences of the yeast strains employed here, 98.4 % (188/191) TEF1, 98.9 % (174/176) RPB1, 97.9 % (186/190) RPB2, 87.1 % (162/186) CYTB, 51.8 % (102/197) SSU, 9.1 % (18/198) LSU D1/D2 and 8.1 % (16/198) ITS sequences were newly determined in this study and the remaining sequences were retrieved from GenBank (Table 1). PCR amplification and sequencing of rDNA regions were successful for all the species studied. The success ratios of PCR amplification and sequencing of the RPB1, RPB2, TEF1 and CYTB genes were 88 %, 91 %, 95 % and 93 %, respectively. The single gene sequences of the SSU rDNA, LSU rDNA D1/D2 domains, ITS + 5.8S rDNA, TEF1, RPB1, RPB2 and CYTB were aligned using the MAFFT algorithm (Standley 2013), resulting in alignments of 1 773, 646, 1 252, 1 023, 796, 1 270 and 387 nucleotide lengths, respectively. Different data sets consisting of the three rDNA regions, the four protein coding genes, and the combined seven genes, respectively, were constructed. When available, the corresponding sequences from representative filamentous taxa in Pucciniomycotina were also incorporated in the data sets. In addition, a data set of SSU and LSU rDNA D1/D2 sequences from the yeast strains employed in this study and those from the representative filamentous taxa compared in Bauer et al., 2006, Schell et al., 2011 and Toome was constructed, because of the scarcity of available ITS and protein gene sequences of filamentous taxa in the Pucciniomycotina. Each of the data sets was subjected to ML, MP and BI analyses. The trees obtained were visually compared to inspect the phylogenetic concordance among the taxa analysed, based on which monophyletic lineages and clades were recognised and defined (Table 3). As expected, among the trees drawn from different data sets analysed, the seven genes-based trees exhibited the highest resolution with strongest support values (Table 3). The backbones of the trees shown here were obtained from ML analysis. The seven genes-based ML tree was used as the primary basis for lineage and clade recognition and definition, and as the starting point for the subsequent comparison and discussion.

Table 3

Statistical supports to the monophyletic clades with multiple strains resolved in pucciniomycetous yeasts based on different data sets using different algorithms.

Lineage/Clade	Seven genes	rDNA	Protein genes	SSU + D1D2
BP1/BP2/PP	BP1/BP2/PP	BP1/BP2/PP	BP1/BP2/PP
Agaricostilbomycetes	nm/nm/nm	86/59/1.0	nm/nm/nm	88/89/1.0
Agaricostilbales	88/100/1.0	100/100/100	100/100/1.0	99/100/1.0
Kondoa	100/100/1.0	100/100/1.0	100/100/1.0	99/100/1.0
Bensingtonia	100/100/1.0	100/100/1.0	98/88/1.0	99/99/1.0
ingoldii	100/100/1.0	100/99/1.0	99/86/1.0	98/90/1.0
Agaricostilbum	100/100/1.0	99/100/1.0	100/100/1.0	71/66/ns
Chionosphaera	100/100/1.0	100/100/1.0	100/96/1.0	53/98/1.0
Kurtzmanomyces	100/98/1.0	59/ns/1.0	99/95/1.0	63/97/1.0
sasicola	100/100/1.0	100/100/1.0	100/100/1.0	99/100/1.0
lactophilus	88/100/1.0	100/100/1.0	100/100/1.0	99/100/1.0
ruber	100/100/1.0	100/100/1.0	100/100/1.0	99/99/1.0
Spiculogloeales	100/100/1.0	100/100/1.0	100/100/1.0	99/100/1.0
subbrunneus	100/100/1.0	100/100/1.0	100/100/1.0	100/100/1.0
Cystobasidiomycetes	100/98/1.0	100/100/1.0	99/100/1.0	99/100/1.0
Cystobasidiales	100/100/1.0	100/100/1.0	100/100/1.0	99/100/1.0
minuta (Cystobasidium)	98/98/1.0	71/83/1.0	95/91/1.0	75/79/1.0
Erythrobasidiales	100/nm/1.0	nm/nm/1.0	95/nm/1.0	nm/nm/nm
Erythrobasidium	100/nm/1.0	97/86/1.0	nm/nm/nm	93/91/1.0
Bannoa	100/100/1.0	100/100/1.0	100/100/1.0	99/100/1.0
Incertae sedis
aurantiaca	100/100/1.0	100/100/1.0	100/100/1.0	99/100/1.0
marina	100/99/1.0	100/100/1.0	100/78/1.0	99/93/1.0
Sakaguchia	100/100/1.0	81/56/1.0	99/100/1.0	84/85/1.0
magnisporus	100/97/1.0	97/78/1.0	91/76/1.0	98/96/1.0
Microbotryomycetes	100/100/1.0	100/100/1.0	99/59/1.0	99/100/1.0
Sporidiobolales	88/100/1.0	99/100/1.0	82/100/1.0	91/93/1.0
Rhodosporidium	100/100/1.0	100/100/1.0	88/nm/ns	94/97/1.0
MixedRhodosporidium/Sporidiobolus	88/89/1.0	100/100/1.0	82/nm/1.0	98/98/1.0
Sporidiobolus	100/100/1.0	100/100/1.0	74/nm/1.0	98/100/1.0
Kriegeriales	ns/nm/nm	nm/nm/nm	nm/nm/nm	nm/nm/nm
glacialis	99/100/1.0	86/92/1.0	100/93/ns	52/ns/1.0
Leucosporidiales	95/99/1.0	98/97/1.0	91/96/1.0	74/70/1.0
Leucosporidium	95/99/1.0	98/97/1.0	91/96/1.0	74/70/1.0
Microbotryales	81/nm/1.0	100/100/1.0	nm/nm/nm	66/74/ns
Microbotryum	100/100/1.0	100/100/1.0	100/100/1.0	ns/75/1.0
Incertae sedis
buffonii	100/100/1.0	100/100/1.0	100/100/1.0	99/100/1.0
tsugae	82/93/nm	nm/nm/nm	93/94/1.0	nm/nm/nm
yarrowii	100/100/1.0	100/100/1.0	100/100/1.0	99/99/1.0
griseoflavus	100/100/1.0	100/100/1.0	100/100/1.0	99/99/1.0
yamatoana	100/100/1.0	100/100/1.0	100/100/1.0	93/98/1.0
singularis	100/100/1.0	100/100/1.0	100/100/1.0	99/100/1.0
Colacogloea	99/89/1.0	67/86/ns	72/65/1.0	nm/nm/nm
vanillica	100/100/1.0	100/100/1.0	100/100/1.0	99/100/1.0
sonckii	100/100/1.0	100/100/1.0	100/100/1.0	99/100/1.0
Curvibasidium	100/100/1.0	100/100/1.0	100/100/1.0	99/100/1.0

Note. BP1 and BP2, bootstrap values from the maximum likelihood and maximum parsimony analyses, respectively; PP, Bayesian posterior probability; nm: not monophyletic; ns, not supported.

Major lineages

The higher-level phylogenetic classification of the Pucciniomycotina proposed in Aime and Bauer mainly based on SSU and LSU rDNA sequence analyses was adopted in Hibbett and Boekhout . They distinguished eight classes, namely Agaricostilbomycetes, Atractiellomycetes, Classiculomycetes, Cystobasidiomycetes, Cryptomycocolacomycetes, Microbotryomycetes, Mixiomycetes and Pucciniomycetes. Schell proposed a new class Tritirachiomycetes in this subphylum based on multiple gene analyses and septal pore ultrastructure to accommodate the anamorphic genus Tritirachium that was once classified in the Pezizomycotina (Ascomycota). This affiliation was recently confirmed by Manohar and Aime .

In agreement with Boekhout our phylogenetic analyses based on the seven-gene dataset showed that the majority of the yeast species employed belonged to four major lineages corresponding to Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes and Mixiomycetes (Fig. 1). The phylogenetic analyses of the three rDNA genes and four protein coding genes (Fig. 2, Fig. 3) showed a similar result to that obtained from the analysis of the seven-gene dataset. However, the position of the Spiculogloeales varied. In the seven genes-based tree this order showed a close relationship to the Mixiomycetes with 94–99 % BP and 1.0 PP support values (Fig. 4). The Mixiomycetes contains only one species Mixia osmundae, which is a fern parasite occurring on Osmunda ferns (Nishida et al., 1995, Nishida et al., 2011). The close affinity of the Spiculogloeales with Mixia osmundae was also revealed and strongly supported in the trees drawn from the four protein-coding genes (Fig. 3). However, in the trees constructed from the three rDNA regions, the Spiculogloeales formed a lineage basal to Agaricostilbomycetes with 59–91 % BP and 1.0 PP support values, while Mixia osmundae was located as a branch basal to the Microbotryomycetes lineage with 1.0 Bayesian PP support (Fig. 2).

Fig. 1

Phylogeny of yeast species in the Pucciniomycotina inferred from the combined sequences of the SSU rDNA, LSU rDNA D1/D2 domains, ITS regions (including 5.8S rDNA), RPB1, RPB2, TEF1 and CYTB. The tree backbone was constructed using maximum likelihood analysis. Bootstrap percentages of maximum likelihood and maximum parsimony analyses over 50 % from 1 000 bootstrap replicates and posterior probabilities of Bayesian inference above 0.9 are shown respectively from left to right on the deep and major branches resolved. Bar = 0.2 substitutions per nucleotide position.

Fig. 2

Phylogeny of yeast species in the Pucciniomycotina inferred from the combined sequences of the SSU rDNA, LSU rDNA D1/D2 domains, and ITS regions (including 5.8S rDNA). The tree backbone was constructed using maximum likelihood analysis. Bootstrap percentages (BP) of maximum likelihood and maximum parsimony analyses over 50 % from 1 000 bootstrap replicates and posterior probabilities (PP) of Bayesian inference above 0.9 are shown respectively from left to right on the deep and major branches and in the brackets following the clades resolved. The branches ending with filled diamonds represent single-species clades. Bar = 0.1 substitutions per nucleotide position. Note: ns, not supported (BP < 50 % or PP < 0.9); nm, not monophyletic.

Fig. 3

Phylogeny of yeast species in the Pucciniomycotina inferred from the combined sequences of RPB1, RPB2, TEF1 and CYTB. The tree backbone was constructed using maximum likelihood analysis. Bootstrap percentages (BP) of maximum likelihood and maximum parsimony analyses over 50 % from 1 000 bootstrap replicates and posterior probabilities (PP) of Bayesian inference above 0.9 are shown respectively from left to right on the deep and major branches and in the brackets following the clades resolved. The branches ending with filled diamonds represent single-species clades. Bar = 0.1 substitutions per nucleotide position. Note: ns, not supported (BP < 50 % or PP < 0.9); nm, not monophyletic.

Fig. 4

Phylogeny of yeast species in the Agaricostilbomycetes and Mixiomycetes inferred from the combined sequences of the SSU rDNA, LSU rDNA D1/D2 domains, ITS regions (including 5.8S rDNA), RPB1, RPB2, TEF1 and CYTB. The tree backbone was constructed using maximum likelihood analysis. Bootstrap percentages (BP) of maximum likelihood and maximum parsimony analyses over 50 % from 1 000 bootstrap replicates and posterior probabilities (PP) of Bayesian inference above 0.9 are shown respectively from left to right on the deep and major branches and clades resolved. The branches ending with filled diamonds represent single-species clades. Bar = 0.2 substitutions per nucleotide position. Note: ns, not supported (BP < 50 % or PP < 0.9); nm, not monophyletic.

The phylogenetic relationships between the yeast species and the filamentous fungal lineages recognised within Pucciniomycotina so far (Aime et al., 2006, Aime et al., 2014, Bauer et al., 2006, Boekhout et al., 2011, Schell et al., 2011) are shown in the tree constructed from the SSU and LSU rDNA D1/D2 domains sequences (Fig. 5). The yeast lineages mentioned above and the filamentous lineages, Atractiellomycetes, Classiculomycetes, Pucciniomycetes and Tritirachiomycetes, were separated as independent lineages. Microbotryomycetes exhibited a close relationship to the filamentous fungal lineage Classiculomycetes with moderate BP (56–79 %) and strong PP (1.0) support, being in agreement with Aime et al., 2006, Aime et al., 2014 and Bauer . However, the phylogenetic relationships among the remaining lineages were not confidently resolved. The Spiculogloeales was located as a deep lineage basal to the Agaricostilbomycetes with 88–89 % BP and 1.0 PP support (Fig. 5), being similar to the result shown in the tree based on the three rDNA regions (Fig. 2). This result suggests that the Spiculogloeales may represent a distinct class, supporting Bauer and Aime who indicated that the Agaricostilbomycetes might not be monophyletic and need to be separated into two classes because of the weakly supported monophyly of the class obtained from SSU rDNA sequence analysis.

Fig. 5

Phylogeny of yeast taxa and filamentous fungi in the Pucciniomycotina inferred from the combined sequences of SSU rDNA and LSU rDNA D1/D2 domains. The tree backbone was constructed using maximum likelihood analysis. Bootstrap percentages (BP) of maximum likelihood and maximum parsimony analyses over 50 % from 1 000 bootstrap replicates and posterior probabilities (PP) of Bayesian inference above 0.9 are shown respectively from left to right on the deep and major branches and clades resolved. The branches ending with filled diamonds represent single-species clades. Bar = 0.02 substitutions per nucleotide position. Note: ns, not supported (BP < 50 % or PP < 0.9).

Agaricostilbomycetes

The two orders Agaricostilbales and Spiculogloeales in this class (Aime et al., 2006, Aime et al., 2014, Bauer et al., 2006, Hibbett et al., 2007, Boekhout et al., 2011) were resolved with strong statistical support values in all the trees drawn from different data sets using different algorithms (Table 3, Fig. 2, Fig. 3, Fig. 4, Fig. 5). However, as shown above, the Spiculogloeales formed a sister lineage to Mixiomycetes, rather than to the Agaricostilbales in the trees drawn from the seven genes and the four protein-coding genes (Fig. 3, Fig. 4). The order Spiculogloeales was proposed by Bauer for a well-supported clade formed by two unidentified teleomorphic species, Spiculogloea sp. RB 1040 and Mycogloea sp. FO 40962, resulted from phylogenetic analyses of the joint SSU/LSU data set. Sporobolomyces (pro parte) was included in this order due to the fact that Sporobolomyces coprosmicola showed a close relationship with Spiculogloea sp. RB 1040 in the tree from the LSU rDNA sequences (Bauer ). In the Spiculogloeales lineage recognised from the seven-gene dataset obtained in this study, five anamorphic species of the genus Sporobolomyces, namely S. linderae, S. coprosmicola, S. subbrunneus, S. dimmenae and S. novazealandicus, formed the subbrunneus clade which was resolved and strongly supported in all the trees constructed in this study (Fig. 2, Fig. 3, Fig. 4, Fig. 5). The SSU and LSU rDNA D1/D2 tree showed that this clade was closely related with Spiculogloea sp. RB 1040and Mycogloea sp. FO 40962 formed a branch basal to Spiculogloea sp. RB 1040 and the subbrunneus clade with strong BP and PP support (Fig. 5). The species of Mycogloea shared some phenotypic characters with those of Spiculogloea, including the presence of dimorphism, mycoparasitism and presence of tremelloid haustorial cells subtended by clamp connections (Bandoni 1998). However, previous molecular analyses (Aime et al., 2006, Aime et al., 2014, Bauer et al., 2006) and this study (Fig. 5) indicated that Mycogloea does not appear monophyletic. The genus Spiculogloea contains four described species with S. occulta as the type (Roberts, 1996, Roberts, 1997, Hauerslev, 1999, Trichiès, 2006). However, molecular data are not available from any of them at present. Additional molecular analyses on a better taxonomic sampling including the type species are needed to resolve the phylogenetic placements of Mycogloea and Spiculogloea species.

In the Agaricostilbales lineage, nine well-supported clades with yeasts species occurred, namely , , , , , ingoldii, lactophilus, ruber and sasicola. In addition, Bensingtonia sakaguchii and a filamentous species, Mycogloea nipponica that has a yeast stage, were each recognised to represent a clade (Table 3, Fig. 2, Fig. 3, Fig. 4).

The clade contained two teleomorphic Agaricostilbum species and two anamorphic Sterigmatomyces species. The type species of both genera were included in this clade. Agaricostilbum species form synnemata-like basidiomata and have a stable yeast state with buds usually produced on short denticles (Wright, 1970, Wright et al., 1981, Bandoni and Boekhout, 2011). The Sterigmatomyces species produce conidia on stalks and appear to lack a filamentous stage (Fell, 1966, Fell, 2011a). Species of Agaricostilbum and Sterigmatomyces occurred together in trees drawn from the LSU rDNA D1/D2 domains (Fell ), ITS (Scorzetti ) and from all data sets generated in this study (Fig. 2, Fig. 3, Fig. 4, Fig. 5), suggesting that they represent a robust single clade.

The two Bensingtonia species, B. musae and B. ingoldii, which were assigned to the clade in Scorzetti and to the Agaricostilbaceae in Bauer and Boekhout , formed the ingoldii clade distinct from, but closely related to the clade with strong support values in all the trees obtained in this study (Fig. 2, Fig. 3, Fig. 4, Fig. 5). These two Bensingtonia species form ballistoconidia but do not form conidiogenous stalks (Nakase et al., 1989, Nakase et al., 2011, Takashima et al., 1995), thus being different from the Agaricostilbum and Sterigmatomyces species. Therefore, the two Bensingtonia species are assigned in a separate clade in this study.

The clade accommodated two Kondoa species including the type species of this genus, K. malvinella, and seven anamorphic species of the genus Bensingtonia (Table 1, Fig. 4). The clade contained B. ciliata, the type species of the genus, and two other species B. naganoensis and B. pseudonaganoensis. Each of the and the clades received strong support values in all the trees obtained from different data sets (Table 3, Fig. 2, Fig. 3, Fig. 4, Fig. 5). The clade was assigned to the Agaricostilbaceae in Bauer and Boekhout based on LSU rDNA sequence analyses. However, Wang indicated that this clade was closely related to the clade. The close relationship of this clade with the clade was strongly supported in the trees constructed from different data sets in this study (Fig. 3, Fig. 4, Fig. 5), suggesting that the clade should be assigned to the Kondoaceae.

From the species included in the Chionosphaeraceae in Bauer et al., 2006, Bauer et al., 2009 and Boekhout , five distinct clades and two single species lineages were distinguished (Table 3, Fig. 2, Fig. 3, Fig. 4, Fig. 5). The three anamorphic Kurtzmanomyces species including the type species of this genus formed a distinct clade closely related to the teleomorphic species Mycogloea nipponica that forms auricularioid basidia (Bandoni 1998). Though the latter has a Kurtzmanomyces-like state, the connection between Kurtzmanomyces and M. nipponica needs to be addressed further as discussed in Sampaio (2011b). The original description of M. nipponica based on a Japanese collection did not include a living culture (Bandoni 1998). The culture from which molecular data were obtained was isolated from a collection made in Taiwan (Kirschner ). It is not clear whether the Kurtzmanomyces species have a sexual Mycogloea-like stage and if the remaining five Mycogloea species (Bandoni 1998) have a Kurtzmanomyces-like yeast stage. The present and previous (Aime et al., 2006, Aime et al., 2014, Bauer et al., 2009) studies indicate that the genus Mycogloea is polyphyletic and species of this genus occur in the Agaricostibales and Spiculogloeales. Thus, at present, we consider it better to treat M. nipponica as representing a clade separated from the clade. The two teleomorphic Chionosphaera species including the generic type Ch. apobasidialis formed an independent clade with a close affinity to the clade and M. nipponica (Fig. 4). The genus Chionosphaera is characterised by holobasidia that are different from the gasteroid basidia of Mycogloea nipponica (Bandoni, 1998, Kwon-Chung, 2011).

The ten Sporobolomyces species in the family Chionosphaeraceae employed in this study were separated into three different clades, namely the sasicola clade with three species, the lactophilus clade with two species, and the ruber clade with five species (Table 1, Fig. 4). The lactophilus and sasicola clades showed a close relationship in all the trees obtained (Fig. 2, Fig. 3, Fig. 4, Fig. 5). The sasicola clade recognised in Scorzetti based on LSU rDNA D1/D2 sequence analysis included Sporobolomyces lactophilus, however, the inclusion of this species in the sasicola clade was not supported in the ITS tree (Scorzetti ). The close relationship of the three species in the sacicola clade and the two species in the lactophilus clade was not supported in the LSU rDNA D1/D2 tree constructed in Boekhout either. Thus, we prefer to maintain the lactophilus and the sasicola clades as distinct clades. Bauer described the teleomorphic genus Cystobasidiopsis with only one species, C. nirenbergiae, and showed that it clustered together with S. lactophilus based on neighbour-joining analysis of the LSU rDNA D1/D2 sequences. Our ML, MP and BI analyses of the LSU rDNA D1/D2 sequences also clustered C. nirenbergiae together with S. lactophilus and S. lophatheri with 71–98 % BP and 1.0 PP supports (data not shown). More sequence data are needed to confirm the relationship of C. nirenbergiae with the clade. The close relationship of the lactophilus and the sasicola clades with the and clades occurred in all trees obtained in this study, supporting that they belong to the Chionosphaeraceae.

The ruber clade was assigned to the Chionosphaeraceae in Boekhout , but its affinity to the other clades of this family mentioned above was not supported in this study. In trees drawn from the rDNA regions and the four protein-coding genes, the ruber clade was located as a sister lineage to the Agaricostibaceae and the Kondoaceae, respectively (Fig. 2, Fig. 3). In the seven genes-based tree, this clade was resolved as a sister lineage to the other families within Agaricostilbomycetes (Fig. 4), which suggests that the ruber clade represents a separate family in this class.

Bensingtonia sakaguchii was consistently located as a separate lineage basal to the family Chionosphaeraceae in different trees with strong BP and PP support values (Fig. 2, Fig. 3, Fig. 4, Fig. 5). Phenotypically, this species has Q9 as the major ubiquinone that differs from the other species in the Chionosphaeraceae that have Q10 (Boekhout ).

Cystobasidiomycetes

This class mainly consists of taxa known from yeast stages only. Three orders, Cystobasidiales, Erythrobasidiales and Naohideales, were distinguished by Aime et al., 2006, Aime et al., 2014, Bauer et al., 2006 and Boekhout based on LSU rDNA sequence analyses. However, the circumscription of the Erythrobasidiales in Aime is different from that in the latter two studies. In addition to the three orders, we observed four more sister clades in the Cystobasidiomycetes in the tree from the seven genes (Fig. 6), which were also largely resolved and supported in the trees from the rDNA and the four protein gene datasets (Fig. 2, Fig. 3).

Fig. 6

Phylogeny of yeast species in the Cystobasidiomycetes inferred from the combined sequences of SSU rDNA, LSU rDNA D1/D2 domains, ITS regions (including 5.8S rDNA), RPB1, RPB2, TEF1 and CYTB. The tree backbone was constructed using maximum likelihood analysis. Bootstrap percentages (BP) of maximum likelihood and maximum parsimony analyses over 50 % from 1 000 bootstrap replicates and posterior probabilities (PP) of Bayesian inference above 0.9 are shown respectively from left to right on the deep and major branches and clades resolved. The branches ending with filled diamonds represent single-species clades. Bar = 0.05 substitutions per nucleotide position. Note: ns, not supported (BP < 50 % or PP < 0.9); nm, not monophyletic.

The teleomorphic species Naohidea sebacea in the Naohideales formed a basal branch in the Cystobasidiomycetes in all the trees constructed in this study (Fig. 2, Fig. 3, Fig. 5, Fig. 6), being in agreement with Boekhout and Sampaio & Chen (2011). This species is mycoparasitic, forms cream-colored colonies, has ‘simple’ septal pores and reproduces by long and slender basidia without probasidia (Oberwinkler, 1990, Sampaio and Chen, 2011).

The Cystobasidiales proposed in Bauer contains two teleomorphic genera, Cystobasidium and Occultifur, and some anamorphic Rhodotorula species based on SSU and LSU rDNA sequence analyses. Recently, Yurkov confirmed the close relationship of nine described Rhodotorula species in the clade with Cystobasidium fimetarium, the type species of the genus, based on ML analysis of SSU, ITS, LSU rDNA D1/D2 and TEF1 sequences. They transferred the Rhodotorula species to the genus Cystobasidium. The monophyly of the clade was shown in all the trees generated in this study with strong support values (Fig. 2, Fig. 3, Fig. 5, Fig. 6). Though the separation of Occultifur externus from the other taxa in the Cystobasidiales was not resolved in Sampaio & Oberwinkler (2011) based on LSU rDNA D1/D2 sequence analysis, it was located as a distinct branch basal to the clade in all the trees obtained in this study (Fig. 2, Fig. 3, Fig. 5, Fig. 6), being in agreement with Nagahama et al., 2006, Boekhout et al., 2011 and Yurkov . C. fimetarium and O. externus share some morphological characters, including the presence of clamp connections and haustoria, a similar basidial morphology and mode of basidiospore germination. The former species differs, however, from the latter by the presence of probasidia (Sampaio et al., 1999, Scorzetti et al., 2002, Sampaio and Oberwinkler, 2011). The phylogenetic and phenotypic comparisons suggest that O. externus represents a separate clade.

The yeast species with hydrogenated coenzyme Q10 system (Q-10H2) formed two clades in the Erythrobasidiales, namely the and clades, which was proposed by Bauer . The clade included a teleomorphic species Bannoa hahajimensis, an undescribed Bannoa species MP 3490 (Scorzetti ) and three Sporobolomyces species (Table 1, Fig. 6). The clade contained the monotypic teleomorphic genus Erythrobasidium and two Sporobolomyces species (Table 1, Fig. 6). The close phylogenetic relationship of the two clades was resolved in almost all the trees obtained, but their sexual life cycles are distinguishable. Erythrobasidium hasegawianum produces unicellular basidia without mating (Hamamoto 2011, Hamamoto ), while Bannoa hahajimensis produces unicellular basidia on a clamp connection formed after mating (Hamamoto ).

Two anamorphic species Rhodotorula lactosa and Cyrenella elegans were located as basal branches to the two clades in the Erythrobasidiales in the trees drawn from the seven genes and the four protein coding genes (Fig. 3, Fig. 6). The affinity of R. lactosa with the Erythrobasidiales was also supported in the rDNA trees, which located R. lactosa as a sister branch to the clade (Fig. 2). This result is consistent with Boekhout and Sampaio (2011a), though the major CoQ of R. lactosa is Q-9 (Yamada & Kondo 1973). The phylogenetic position of Cy. elegans remains uncertain. In contrast to the results obtained from the seven-gene and four protein coding gene sequence analyses, this species was located in a branch basal to the Cystobasidiales and Erythrobasidiales in the tree obtained from the three rDNA genes with strong support (Fig. 2), being in agreement with the result shown in Sampaio (2011c) based on LSU rDNA D1/D2 sequence analysis. Cy. elegans is an unusual species as it forms conidia with radiate appendages resembling those of aquatic hyphomycetes. It also forms clamp connections in the hyphae and teliospores, although germination of teliospores with basidia has not been observed (Gochenaur, 1981, Sampaio, 2011c). The phylogenetic and phenotypic comparisons suggest that Cy. elegans represents an independent lineage in Cystobasidiomycetes.

The marina clade included Rhodotorula marina and five Sporobolomyces species (Table 1, Fig. 6). Interestingly, all Sporobolomyces species in this clade form nearly symmetrical ballistoconidia, differing from the other Sporobolomyces species that typically form asymmetrical ballistoconidia (Shivas and Rodrigues de Miranda, 1983, Wang and Bai, 2004). The aurantiaca clade contained two Rhodotorula and three Sporobolomyces species (Table 1, Fig. 6). The marina and aurantiaca clades were also recognised in Scorzetti et al., 2002, Nagahama et al., 2006 and Boekhout . A close relationship of these two clades was shown in the tree from the three rDNA genes (Fig. 2), but was not supported in the trees from the four protein-coding genes and the seven genes (Fig. 1, Fig. 3). Species from these two clades were included in the Erythrobasidiales in Aime . This conclusion, however, was not supported in the present study. In the rDNA and the four protein-coding genes-based trees, the position of these two clades varied (Fig. 2, Fig. 3). In the seven genes-based tree, the marina and aurantiaca clades were resolved as sister lineages to the Erythrobasidiales (Fig. 6).

The magnisporus clade consisted of Sporobolomyces magnisporus and three Rhodotorula species described recently by Pohl . Sporobolomyces magnisporus was assigned to the Erythrobasidiales in Boekhout . The close relationship of the magnisporus clade with the Erythrobasidiales was shown in Pohl and in the rDNA genes-based tree in this study (Fig. 2). However, in the trees from the four protein coding genes and the seven genes, the relationships of the magnisporus clade with the other clades in Cystobasidiomycetes were not resolved (Fig. 3, Fig. 6).

The clade included the monotypic teleomorphic genus Sakaguchia and five anamorphic Rhodotorula species (Table 1, Fig. 6). This clade was consistently resolved and strongly supported in all the trees constructed in this (Table 3, Fig. 6) and previous studies (Nagahama et al., 2006, Boekhout et al., 2011). The genus Sakaguchia was treated as ‘incertae sedis’ in Aime et al., 2006, Bauer et al., 2006 and Boekhout , but was assigned to the Erythrobasidiales in Fell (2011b). The close phylogenetic relationship of the clade with the clades in Cystobasidiomycetes was not resolved in any of the trees generated in this study (Fig. 2, Fig. 3, Fig. 5, Fig. 6). Furthermore, Sakaguchia dacryoidea produces teliospores (Yamada et al., 1994, Fell and Statzell-Tallman, 1998), that are different from the sexual structures of Bannoa and Erythrobasidium species in the Erythrobasidiales. Our results suggest that the clade together with the marina, aurantiaca and magnisporus clades represent lineages distinct from the currently recognised orders in the Cystobasidiomycetes.

Microbotryomycetes

More than half of the yeast species compared in this study belong to the class Microbotryomycetes. Within this class, six and nine clades were distinguished by Scorzetti and Boekhout , respectively. Five orders, namely Heterogastridiales, Kriegeriales, Leucosporidiales, Microbotryales and Sporidiobolales, have been proposed in this class mainly based on SSU, LSU and ITS-5.8S rDNA sequence analyses (Sampaio et al., 2003, Aime et al., 2006, Aime et al., 2014, Bauer et al., 2006, Hamamoto et al., 2011, Toome et al., 2013). These orders were also recognised in this study. In addition to the clades that could be assigned to the five orders, we observe a considerable number of clades that did not belong to any of the orders.

The Sporidiobolales was resolved as a monophyletic group with strong BP and PP support values (Table 3, Fig. 7). Three clades, namely , and mixed / clades (Fig. 7), are in agreement with Boekhout . The clade was composed of nine Rhodotorula and six Rhodosporidium species and Sporobolomyces alborubescens, including the type species of the former two genera (Rhodotorula glutinis and Rhodosporidium toruloides). The clade contained 15 Sporobolomyces and five Sporidiobolus species, including the type species of these two genera (Sporobolomyces roseus and Sporidiobolus johnsonii). The mixed / clade consisted of nine species from the four genera mentioned above (Table 1, Fig. 7). The three clades were well-supported in the trees drawn from the seven-gene and the rDNA datasets with 100 % BP and 1.0 PP supports (Fig. 2, Fig. 7). In the tree derived from the four protein coding gene dataset, each of the three clades was also resolved as monophyletic group by ML and BI analyses with strong support values (Table 3), but was not resolved as a monophyletic group by MP analysis (Fig. 3).

Fig. 7

Phylogeny of yeast species in the Microbotryomycetes inferred from the combined sequences of SSU rDNA, LSU rDNA D1/D2 domains, ITS regions (including 5.8S rDNA), RPB1, RPB2, TEF1 and CYTB. The tree backbone was constructed using maximum likelihood analysis. Bootstrap percentages (BP) of maximum likelihood and maximum parsimony analyses over 50 % from 1 000 bootstrap replicates and posterior probabilities (PP) of Bayesian inference above 0.9 are shown respectively from left to right on the deep and major branches and clades resolved. The branches ending with filled diamonds represent single-species clades. Bar = 0.05 substitutions per nucleotide position. Note: ns, not supported (BP < 50 % or PP < 0.9); nm, not monophyletic.

The Leucosporidiales included two teliospore-forming yeast genera, namely Leucosporidium and Mastigobasidium, and the anamorphic genus Leucosporidiella (Table 2, Fig. 7). The latter was proposed by Sampaio as the anamorphic counterpart of Leucosporidium to accommodate the Rhodotorula species that belong to the Leucosporidiales. In this study, the described Mastigobasidium, Leucosporidium and Leucosporidiella species except Leucosporidium fasciculatum were located in the monophyletic clade, which was resolved in all the trees constructed from different data sets (Fig. 2, Fig. 3, Fig. 5, Fig. 7). The assignment of Leucosporidium fellii and Mastigobasidium intermedium to the Leucosporidiales is uncertain in Sampaio because of their clustering with the Microbotryales in the Bayesian Markov chain Monte Carlo (MCMC) analysis of LSU rDNA D1/D2 sequences. The affinity of L. fellii and Ma. intermedium with the clade was also not supported in Boekhout . In the present study, the close relationship of these two species within the clade was resolved and strongly supported in all the trees obtained (Fig. 2, Fig. 3, Fig. 5, Fig. 7), being in agreement with Yurkov and de García . Yurkov described Leucosporidium drummii, that produces hyphae without clamp connections and intercalary teliospores. The teliospores germinate with either typical basidia for species of the genus Leucosporidium or produce, depending on the conditions, hyphae that originated from curved metabasidia similar to those of Mastigobasidium intermedium (Golubev 1999, Sampaio et al., 2003, Yurkov et al., 2012). Recently, Laich described an anamorphic species as Leucosporidium escuderoi f.a. based on the new code for fungal nomenclature (McNeill ). de García transferred the species of the genera Mastigobasidium and Leucosporidiella into the genus Leucosporidium and proposed a new genus Pseudoleucosporidium to accommodate the species Leucosporidium fasciculatum. Another Leucosporidium species, L. antarcticum, was transferred to the genus Glaciozyma which was proposed for a group of psychrophilic yeasts from various cold environments, such as soil, seawater and sediment, in Antarctica and European glaciers (Turchetti ). Recently, a new species Glaciozyma litorale was isolated from silt, alga and coastal sand in the White Sea intertidal zone, supporting the psychrophilic nature of this genus (Kachalkin, 2014). The genus Glaciozyma was assigned to the family Camptobasidiaceae in the Kriegeriales by Toome based on LSU rDNA D1/D2 sequence analysis.

Six species from the order Kriegeriales proposed by Toome were employed in this study, including Glaciozyma antarctica representing the family Camptobasidiaceae, and Kriegeria eriophori and four Rhodotorula species representing the family Kriegeriaceae (Table 1). These species were located together in a cluster in the seven-gene tree (Fig. 7). The affinity of G. antarctica with the species in the Kriegeriaceae was not supported by ML and MP analyses. In the rDNA and the four protein-coding genes-based trees, G. antarctica was not located in the same cluster with the Kriegeriaceae species (Fig. 2, Fig. 3), suggesting that the order Kriegeriales defined by Toome may not be monophyletic. Among the four Rhodotorula species in this order, R. glacialis, R. psychrophenolica and R. psychrophila (Margesin ) formed a strongly supported clade labeled as glacialis in all the trees obtained (Table 3, Fig. 2, Fig. 3, Fig. 5, Fig. 7). The close relationship between the monotypic teleomorphic genus Kriegeria and the glacialis clade was shown in different trees, but the statistic support values were low or lacking (Table 3), suggesting they represent separate clades. The species Rhodotorula rosulata formed a branch basal to the and the glacialis clades in the trees from the seven genes and the three rDNA genes with 100 % BP and 1.0 PP supports values (Fig. 2, Fig. 7), suggesting that R. rosulata represents another clade in the Kriegeriales. Toome showed that R. rosulata was closely related to Meredithblackwellia eburnea in their ML analysis of LSU, SSU and ITS sequences. These authors, however, did not transfer R. rosulata to Meredithblackwellia because of the lack of statistic support. The relationship between R. rosulata and Me. eburnea needs to be addressed further.

Within the Microbotryales as defined by Bauer only one known anamorphic yeast species Rhodotorula hordea was included based on LSU rDNA D1/D2 sequence analysis (Boekhout et al., 2011, Sampaio, 2011a). In agreement with Boekhout this species was located as a basal branch of the order with strong support value in the trees from the seven genes (Fig. 7) and the rDNA genes (Fig. 2) in this study. However, in the tree from the four protein-coding genes, the affinity of the species with the Microbotryales was not resolved (Fig. 3). The closest relative of R. hordea is Ustilentyloma fluitans, a parasite of Glyceria (Graminiae) plants (Vánky 2002). In the LSU rDNA D1/D2 domains, R. hordea differs from Ustilentyloma fluitans by only one mismatch (Sampaio 2011a), suggesting that the former represents a yeast stage of U. fluitans or a closely related Ustilentyloma species. No yeast species is included in the Heterogastridiales which includes the genus Heterogastridium.

The species that could not be assigned to any recognised orders in Microbotryomycetes formed 10 clades and 7 monotypic lineages. In addition to the four Rhodotorula species which were included in the clade in Boekhout and Sampaio (2011a), two Rhodotorula species and Sporobolomyces falcatus (Table 2, Fig. 7) were included in this clade together with the dimorphic mycoparasite Colacogloea peniophorae, which forms minute basidiocarps in nature (Sampaio ). In the phylogenetic trees obtained from the seven genes, Rhodotorula foliorum, Rhodotorula diffluens and Sporobolomyces falcatus clustered in the clade (Fig. 7). Though the affinity of these three species with this clade was weak or not supported in the trees from the rDNA genes (Fig. 2, Fig. 5), this was supported in the tree from the four protein-coding genes (Fig. 3).

The clade contained two teleomorphic Curvibasidium species (Table 1). Leucosporidium fasciculatum was located basal to this clade with 100 % BP and 1.0 PP support values in the trees from the seven genes, the rDNA and the four protein-coding genes (Fig. 2, Fig. 3, Fig. 7). The close relationship of L. fasciculatum with the clade was also shown in previous studies (Sampaio et al., 2004, Boekhout et al., 2011, Sampaio, 2011e), however, in contrast to Curvibasidium, L. fasciculatum lacks clamp connections and forms septate basidia (phragmobasidia) (Sampaio 2011d). Therefore, L. fasciculatum has been placed in a new genus Pseudoleucosporidium by de García . The vanillica clade contained two Rhodotorula species as recognised by Sampaio and Boekhout . The and vanillica clades and L. fasciculatum were located basal to the Leucosporidiales in the trees from the seven-genes with moderate PP support values (Fig. 7) and in the tree from the rDNA genes with strong supports by all algorithms employed (Fig. 2). However, in the tree from the four protein-coding genes, the close relationships of these two clades with the Leucosporidiales were not resolved (Fig. 3). Phenotypically, the Curvibasidium species form non-septate basidia, which is a unique feature in the Pucciniomycotina (Sampaio ).

Among the species tentatively assigned to the yamatoana/Leucosporidium antarcticum group in Boekhout , three (Kriegeria eriophori, Camptobasidium hydrophilum and Leucosporidium antarcticum) were assigned to the Kriegeriales by Toome . From the remaining species of this group, four clades and two single-species lineages were distinguished in this study (Fig. 7). The buffonii clade contained three Rhodotorula species, the tsugae clade included Sporobolomyces tsugae and two Rhodotorula species, and the yarrowii clade comprised three Rhodotorula species. These three clades clustered together in the ML and MP trees based on the seven genes with weak ML BP support (Fig. 7). The BI tree from the seven genes, and the trees from the rDNA and the four protein coding genes did not support the close relationship of these three clades (Fig. 2, Fig. 3, Fig. 7). Rhodotorula cresolica was located in the tsugae clade in the tree from the four protein-coding genes with 93–94 % BP and 1.0 pp support values (Fig. 3). This phylogeny was also supported by the ML and MP analyses of the seven genes, though not supported in the BI tree from the seven genes and the trees from the rDNA dataset (Fig. 2, Fig. 5, Fig. 7).

The griseoflavus clade containing two Sporobolomyces species, the yamatoana clade with Bensingtonia yamatoana and Rhodotorula arctica, and the singularis clade with Sporobolomyces singularis and Rhodotorula lignophila, clustered together with high BP and PP values in all the phylogenetic trees constructed (Fig. 2, Fig. 3, Fig. 5, Fig. 7). Each of these clades received strong support values in the trees. Sporobolomyces inositophilus was located in the same cluster with these three clades with strong support values (Fig. 7), however, its relationship to each of the clades was not resolved by ML and BI, suggesting that this species may represent a separate clade. In addition, species of the griseoflavus and yamatoana clades were characterised by the presence of Q10 and Q9, respectively, supporting their separation as two clades.

Rhodotorula auriculariae located in the yamatoana/Leucosporidium antarcticum group in Boekhout was shown to be closely related with Rhodotorula sonckii, which was located as a basal branch of the Microbotryomycetes in Boekhout . The sonckii clade formed by these two species clustered with the Microbotryales and Heterogastridiales in the tree from the seven genes (Fig. 7). The close relationship of this clade with the Microbotryales was also supported in the tree from the rDNA genes (Fig. 2), but not supported in the tree from the four protein-coding genes (Fig. 3). The relationship of the sonckii clade with the Heterogastridiales was not resolved in the analyses of the rDNA and the four protein-coding genes (Fig. 2, Fig. 3). Rhodotorula ferulica was also placed in the yamatoana/Leucosporidium antarcticum group by Boekhout . This species was located basal to the clade in the ML tree from the seven genes but the BP support was lack. This relationship was, however, not resolved by the other algorithms used in this study (Fig. 7, Table 3).

The following Rhodotorula species, R. crocea, R. hylophila, and R. javanica, occupied isolated positions in the Microbotryomycetes with their closest relatives not being resolved. Their phylogenetic positions changed in different trees constructed from different data sets using different algorithms (Fig. 2, Fig. 3, Fig. 5, Fig. 7). The species Reniforma strues, which was located at the deepest branch in the Microbotryomycetes in Boekhout , exhibited a relationship with Heterogastridium pycnidioideum (Heterogastridiales) in the trees from the seven and the rDNA genes with strong BP and PP support values (Fig. 2, Fig. 7). However, the position of the former was uncertain in the tree from the four protein-coding genes (Fig. 3). Reniforma strues is a morphologically unique anamorphic yeast species, forming reniform cells and buds (Pore and Sorenson, 1990, Pore and Fell, 2011).

Conclusion

The molecular phylogeny of yeasts and related dimorphic and filamentous basidiomycetes in the Pucciniomycotina was inferred based on analyses of sequences of seven genes using different phylogenetic algorithms. The major phylogenetic groupings of pucciniomycetous yeasts observed in previous studies based on the LSU rDNA D1/D2 domains or ITS-5.8S sequences (Fell et al., 2000b, Scorzetti et al., 2002, Boekhout et al., 2011) were confirmed in the present study. In each of the major groups, more robust topologies with higher resolution were achieved in this study than obtained before. The yeast taxa employed were assigned into four major lineages, namely Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes and Mixiomycetes. These lineages are independent from Atractiellomycetes, Classiculomycetes, Cryptomycocolacomycetes, Pucciniomycetes and Tritirachiomycetes that are formed by filamentous taxa in the Pucciniomycotina.

The orders distinguished in previous studies except the Kriegeriales were all resolved as monophyletic groups in this study. The order Spiculogloeales was resolved as a sister lineage of Mixiomycetes, rather than of the order Agaricostilbales in the Agaricostilbomycetes. This suggests that the Spiculogloeales may represent a new class in Pucciniomycotina. In the Cystobasidiomycetes, four independent groups with sisterhood to the orders Cystobasidiales, Erythrobasidiales, and Naohideales were resolved, suggesting that additional orders remain to be discerned in this class. In addition to the five existing orders Heterogastridiales, Kriegeriales, Leucosporidiales, Microbotryales, and Sporidiobolales in the class Microbotryomycetes, several groups that seem to represent new orders were recognised. The boundaries of some of these new groups remain to be defined.

A total of 33 monophyletic clades and 18 single species lineages were recognised among the pucciniomycetous yeasts employed in this study (Table 1, Table 3). As shown previously, the majority of the currently anamorphic genera are polyphyletic. For example, Rhodotorula and Sporobolomyces species occurred in 17 and 23 clades, respectively. These genera and related teleomorphic ones need to be redefined. A considerable number of new genera need to be proposed to accommodate the monophyletic clades that do not include any generic type species. The next step will be to propose an updated taxonomic system for yeasts and related taxa within Pucciniomycotina based on the phylogenetic framework presented here and to implement the ‘One fungus = One Name’ principle.