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Phylogenetic classification of yeasts and related taxa within Pucciniomycotina

Most small genera containing yeast species in the Pucciniomycotina (Basidiomycota, Fungi) are monophyletic, whereas larger genera including Bensingtonia, Rhodosporidium, Rhodotorula, Sporidiobolus and Sporobolomyces are polyphyletic. With the implementation of the “One Fungus = One Name” nomenclatural principle these polyphyletic genera were revised. Nine genera, namely Bannoa, Cystobasidiopsis, Colacogloea, Kondoa, Erythrobasidium, Rhodotorula, Sporobolomyces, Sakaguchia and Sterigmatomyces, were emended to include anamorphic and teleomorphic species based on the results obtained by a multi-gene phylogenetic analysis, phylogenetic network analyses, branch length-based methods, as well as morphological, physiological and biochemical comparisons. A new class Spiculogloeomycetes is proposed to accommodate the order Spiculogloeales. The new families Buckleyzymaceae with Buckleyzyma gen. nov., Chrysozymaceae with Chrysozyma gen. nov., Microsporomycetaceae with Microsporomyces gen. nov., Ruineniaceae with Ruinenia gen. nov., Symmetrosporaceae with Symmetrospora gen. nov., Colacogloeaceae and Sakaguchiaceae are proposed. The new genera Bannozyma, Buckleyzyma, Fellozyma, Hamamotoa, Hasegawazyma, Jianyunia, Rhodosporidiobolus, Oberwinklerozyma, Phenoliferia, Pseudobensingtonia, Pseudohyphozyma, Sampaiozyma, Slooffia, Spencerozyma, Trigonosporomyces, Udeniozyma, Vonarxula, Yamadamyces and Yunzhangia are proposed to accommodate species segregated from the genera Bensingtonia, Rhodosporidium, Rhodotorula, Sporidiobolus and Sporobolomyces. Ballistosporomyces is emended and reintroduced to include three Sporobolomyces species of the sasicola clade. A total of 111 new combinations are proposed in this study.


INTRODUCTION
The subphylum Pucciniomycotina (Phylum Basidiomycota, Kingdom Fungi) presently includes eight classes, and four of these, namely the Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes and Mixiomycetes, contain taxa with a dominant yeast stage (Aime et al. 2006, Hibbett et al. 2007. So far 28 genera with yeast states have been proposed within Pucciniomycotina , Turchetti et al. 2011, Toome et al. 2013. Most of these genera are monophyletic, whereas five genera, namely Bensingtonia, Rhodosporidium, Rhodotorula, Sporidiobolus and Sporobolomyces are polyphyletic . Bensingtonia, Rhodotorula and Sporobolomyces species are placed in various classes of Pucciniomycotina , Hamamoto et al. 2011, Sampaio 2011, Yurkov et al. 2015. Species of the teleomorphic genera Rhodosporidium and Sporidiobolus together with some asexual Rhodotorula and Sporobolomyces species are nested within Sporidiobolales , Sampaio 2011. With the implementation of the "One Fungus = One Name" nomenclatural principle (Hawksworth 2011, Taylor 2011, McNeill et al. 2012) these five polyphyletic genera need to be revised.
Several studies using molecular analyses of ribosomal DNA sequences have provided a detailed grouping of species in clades among the four classes that contain yeast and yeast-like species (Hamamoto & Nakase 2000, Nakase 2000), but many species remained unassigned . Thus the boundaries of the clades and genera have to be reassessed by analysing a robust molecular dataset. In another study we analysed seven gene fragments, namely SSU (18S) rRNA, LSU (26S/28S) rRNA D1/D2 domains, the ITS region (including the 5.8S rRNA), RPB1 (the largest subunit of DNA polymerase II), RPB2 (the second largest subunit of DNA polymerase II), TEF1 (translation elongation factor 1-α) and CYTB (cytochrome b) that placed most pucciniomycetous yeast species into 51 wellsupported clades . These data are used here to address the taxonomic affiliations of those fungi. We propose 26 of the 51 recognised clades as new taxa at the genus, family and class levels based on a phylogenetic and taxonomic analysis of the combined seven genes-based and the enlarged LSU rRNA gene datasets. The assessment of taxonomic ranks followed the branch length-based methods as described in Liu et al. (2015).

Strains and molecular phylogenetic analyses
Multi-gene data of the yeast strains used were taken from a previous study . As described previously  multi-gene phylogenetic trees constructed from Maximum likelihood (ML), Maximum parsimony (MP) and Bayesian inference (BI) analyses of a dataset comprising nucleotide sequences of the ITS region (including the 5.8S rRNA), the D1/D2 domains of the LSU rRNA, the SSU rRNA, and the RPB1, RPB2, TEF1 and CYTB genes is used here to address the taxonomy of the pucciniomycetous yeasts. Fifty-one clades that may be equal to the generic rank, including 16 singlespecies lineages, were recognised among the pucciniomycetous yeasts used in the previous study with strong statistical support values in all trees drawn using different phylogenetic algorithms . In order to detect the reliability of those 51 clades, a phylogenetic network approach was employed to infer the relationships between those pucciniomycetous yeasts. The seven genes-based phylogenetic network was constructed in SplitsTree4 (Huson & Bryant 2006) using the ConsensusNetwork algorithm with default parameter settings. The seven single-gene ML trees used in the phylogenetic network analysis were constructed using RAxML-HPC 7.2.8 (Stamatakis 2006) using the parameter settings described previously .
The supplementary LSU rRNA gene (D1/D2 domains) sequence dataset containing data from newly published pucciniomycetous yeast species and a few additional filamentous teleomorphic taxa was constructed and subjected to constrained maximum likelihood (ML) and maximum parsimony (MP) analyses based on the topology of a seven genes-based dataset taken from . The LSU sequences were aligned with MAFFT version 7 and the G-INS-i option (Standley K 2013). Constrained phylogenetic analyses were only inforced for species previously analysed using seven DNA loci. Only bipartitions that received at least 85% bootstrap support during fast bootstrapping of the seven genes-based dataset  conducted with Pthreads-parallelised RAxML version 8.1.24 (Stamatakis 2014) were used as a backbone constraint for LSU phylogenetic inference. Fast bootstrapping in conjunction with the autoMRE bootstopping criterion (Pattengale et al. 2009) and subsequent search for the best tree (Stamatakis et al. 2008) were conducted using the GTRCAT model approximation. MP bootstrapping with 1 000 replicates was conducted with TNT version 1.1/June 2015 (Goloboff et al. 2008). The alignments and trees were deposited in TreeBASE (No. 18537).

Quantitative assessment of taxonomic ranks
The modified Generalized Mixed Yule Coalescent (GMYC) method (Humphreys & Barraclough 2014) was applied iteratively to identify higher evolutionary significant units (higher ESUs) above the species levels in the pucciniomycetous yeasts as done before for tremellomycetous fungi (Liu et al. 2015). Firstly, the overall GMYC analysis was carried out for the simulation at class level, and secondly nested analyses were run for the higher ESUs identification at family level for each clade. Outgroup samples were excluded from the dataset using the drop.tip command in ape (Paradis 2006). A chronogram was calculated from the ML-based tree using the penalised likelihood method (Sanderson 2002) as implemented in the chronopl command in ape (Paradis 2006). The chronogram was then analysed using a modified GMYC package in SPLITS in R (version 2.10, www.cran.r-project.org) using the single threshold method. In the case of clades with a small number of samples the modified GMYC approach would not result in significant differences simply because of sampling size: these were marked as 'NA' (not analysed).
Phylogenetic rank boundary optimisation (PRBO), a phylogenetic variant of clustering optimisation (Göker et al. 2009, Stielow et al. 2011, was conducted based on taxonomy-based reference information as described in Liu et al. (2015). A reduced classification including twelve putatively reliable genera of pucciniomycetous yeasts was chosen as reference taxonomy. The resulting optimal upper boundaries for the divergence of each taxonomic rank were then applied back to the entire dataset. These boundaries for each taxonomic rank were compared with boundaries estimated from the entire classification (Table 1). For each newly proposed or already established taxon, maximum subtree height (MaSH) of its corresponding clade, absolute deviation and significant deviation (Sigdev) from the threshold optimal for the reliable taxa were calculated. One hundred bootstrap replicates were applied to obtain the 95% confidence intervals for the boundaries to detect the significances of the divergences from the optimal range for each taxonomic rank.
The seven genes-based ML tree used in  was employed as the basis for the PRBO and iterative modified GMYC analyses. The taxa within Ustilaginomycotina were used as outgroup and taxa within Pucciniomycetes were used as ingroup in the above two analyses. Note that none of the two methods was followed strictly in the current study. Where possible, wider circumscriptions of taxa were chosen to lower the number of taxonomic changes suggested; moreover, where possible, clades with distinct phenotypic or ecological features were proposed as new taxa (Liu et al. 2015). Additionally, already established taxa were kept unless they appeared evidently non-monophyletic.

Taxonomic units addressed by the iterative modified GMYC and PRBO analyses
The pucciniomycetous yeast species belong to four recognised classes, namely Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes and Mixiomycetes , Hibbett et al. 2007. The overall modified GMYC analysis supported the Agaricostilbomycetes without Spiculogloeales as a class in agreement with the indication that Spiculogloeales may represent a new class within Pucciniomycotina based on the seven genes-based phylogenetic analyses . Thus Spiculogloeomycetes is proposed as a new class to accommodate the order Spiculogloeales.
The nested analyses of the GMYC approach identified five families in the class Agaricostilbomycetes (Table 2), including the PHYLOGENETIC CLASSIFICATION OF YEASTS AND RELATED TAXA www.studiesinmycology.org recognised families Agaricostilbaceae, Chionosphaeraceae and Kondoaceae, a new Agaricostilbales family 1 (ruber clade) and a new Agaricostilbales family 2 (Bensingtonia sakaguchii lineage). Because only B. sakaguchii occurs in the new Agaricostilbales family 2, this family is not proposed in this study.
Consequently, B. sakaguchii is placed into a new genus (see Taxonomy), which is presently treated as 'incertae sedis' in the Agaricostilbales.
Nine clades were indentified at the family level within Cystobasidiomycetes by the nested analyses of GMYC ( Table 2). The presently accepted families Cystobasidiaceae, Erythrobasidiaceae and Naohideaceae belong to Cystobasidiales, Erythrobasidiales and Naohideales, respectively. The genus Occultifur is separated from Cystobasidiaceae as a family in the GMYC analyses, but we presently prefer to keep this genus in the Cystobasidiaceae due to the low number of taxa in this genus. The Cyrenella and Rhodotorula lactosa lineages were suggested as two new families in the Erythrobasidiales by the GMYC nested analyses, however, these two lineages represent single species each, and, therefore, we temporarily placed them as 'incertae sedis' in the Erythrobasidiales. The aurantiaca and marina clades were grouped into one family in the GMYC nested analyses, which is not supported by the phylogenetic analysis of seven genes that showed the two clades as a paraphyletic group . The magnisporus and Sakaguchia clades were identified as families in agreement with the phylogenetic analysis of seven genes .
Within Microbotryomycetes two families, namely Leucosporidiaceae and Microbotryaceae, were supported by the nested analyses of GMYC approach ( Table 2). The family Sporidiobolaceae in the Sporidiobolales was divided into three families represented by the Sporidiobolus clade, the Rhodosporidium clade and the mixed Rhodosporidium/ Sporidiobolus clade, respectively. We preserve the current taxonomic status of Sporidiobolaceae because the phenotype of these three clades is similar and it forms a strongly supported lineage in the phylogenetic analysis of seven genes . The Kriegeriaceae and Camptobasidiaceae in the Kriegeriales were grouped into a single family in the nested GMYC analyses. However, the Camptobasidiaceae, including Glaciozyma antarctica, clustered together with the Kriegeriaceae lacking support value in the ML analysis and they did not occur in the same cluster in the MP and BI analyses (Wang et al. 2015a, Fig. 1 of this study). Consequently the two families are maintained in this study.
Ten clades and seven single-species lineages in Microbotryomycetes could not be assigned to presently recognised families and orders . These clades are strongly divergent from each other and seem to have a sister relationship to the known families and orders within Microbotryomycetes (Fig. 1). The species Rhodotorula hylophila, R. javanica, R. crocea and Reniforma strues were not included in the nested GMYC analyses because they occurred outside the Microbotryomycetes in the overall GMYC analysis. In the nested GMYC analyses, the griseoflavus, yamatoana, singularis clades and Sporobolomyces inositophilus were identified as one family that was supported by the phylogenetic analysis of seven genes with strong support values ; the buffonii, tsugae and yarrowii clades were assigned to one family, but this was weakly supported by the seven genes ML analysis (57 % BP), lacking support in the BI analysis and were not supported by the MP analysis ( Fig. 1), and consequently, they are not treated as a single family in this study; the other clades and the single-species lineages were identified as separate families in the nested GMYC analyses (Table 2).
Among 51 pucciniomycetous yeast clades suggested as genera in the previous multi-gene phylogenetic study (Wang Table 1. PRBO results showing the divergences, if any, of the proposed genera from the optimal range of divergences for their rank as inferred from selected reference data.

Taxa
Rank MaSH Deviation Sigdev   Mixiomycetes not supported

NA Mixia
Note: NA means "not analysed". In the overall GMYC analysis, Naohideales is a separate class from Cystobasidiomycetes; Spiculoglomycetes and Mixiomycetes were identified as one class; Heterogastridium, Rhodotorula hylophila, and Reniforma form a separate class from Microbotryomycetes; Rhodotorula javanica and Rhodotorula crocea form another separate class from Microbotryomycetes. Fig. 1. Phylogenetic tree inferred using the combined sequences of SSU rRNA, LSU rRNA D1/D2 domains, ITS regions (including 5.8S rRNA), RPB1, RPB 2, TEF1 and CYTB, depicting the phylogenetic positions of existing yeast taxa and new genera (in bold) within Pucciniomycotina. 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 0.95 are shown respectively from left to right on the deep and major branches. Bar = 0.2 substitutions per nucleotide position. Note: ns, not supported (BP < 50 % or PP < 0.9); nm, not monophyletic. The new taxa are in bold. PHYLOGENETIC CLASSIFICATION OF YEASTS AND RELATED TAXA www.studiesinmycology.org et al. 2015a), only few were found to deviate from the optimal range of divergences as determined in the PRBO analysis, and were found to significantly deviate (Table 1), which supports the preliminary taxonomic conclusions from the multi-gene phylogenetic analysis. Twenty-six of them represent currently described genera. The others are proposed as new genera ( Fig. 1) based on the phylogenetic analyses, PRBO analysis and phenotypic comparisons ( Table 1, Table 3 and Figs 2, 3) presented in this study.

Phylogenetic analyses
Only 156 species from 184 ones used in the seven genes-based tree  were selected to construct the phylogenetic network, because some protein genes were not available for all species. As a result all clades recognised in the seven genes-based tree could be recognised in the network approach. The network result showed that the five classes containing yeast species, viz. Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes, Mixiomycetes and Spiculogloeomycetes, remain separated ( Fig. 2A). The 51 clades in the seven genesbased tree are also separated without any reticulation . This result confirmed the reliability of the combined phylogenetic analysis of the seven genes.
For a better understanding of the phylogenetic relationships between Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes, Mixiomycetes and Spiculogloeomycetes, and to include recently described species, an enlarged LSU rRNA gene dataset was analysed. Thereby, the LSU dataset analysed by  was enlarged from 184 to 242 sequences containing both sexual (e.g. Camptobasidium, Cystobasidium, Glaciozyma, Kondoa and Ustilentyloma) and asexual (e.g. Rhodotorula, Sporobolomyces and Occultifur) genera and species . The constrained ML analysis of the enlarged LSU dataset was used to place species known from LSU rRNA gene sequences in the phylogenetic clades previously recognised in the analysis based on the seven DNA-loci. Results from the LSU analysis were not used to challenge the results of the multi-gene study , but to investigate the stability of the taxonomy in undersampled clades.
The enlarged analysis of the LSU rRNA gene dataset suggests that the number of single-species lineages in Microbotryomycetes is likely to increase in the future, since many sequences representing potentially new species could not be assigned to any of the clades recognised . In agreement with previous studies , Weiß et al. 2004, our results showed that LSU alone is not sufficient to resolve many clades in Microbotryomycetes (Fig. 6).

Type order: Agaricostilbales Oberw. & R. Bauer
This class contains the order Agaricostilbales. Our previous multi-gene sequence analyses indicated that nine well support clades, namely Agaricostilbum, Bensingtonia, Chionosphaera, Kondoa, Kurtzmanomyces, ingoldii, lactophilus, ruber, sasicola, and two species Bensingtonia sakaguchii and Mycogloea nipponica, occurred in the Agaricostilbales . These clades which are delimited at the generic rank are supported by the PRBO analysis (Table 1), the phylogenetic network analysis (Fig. 2B), and the analysis of the enlarged LSU rRNA gene dataset (Fig. 4). The genera Chionosphaera and Kurtzmanomyces are well-established genera. The genera Sterigmatomyces, Cystobasidiopsis and Kondoa are emended to include both teleomorphic and anamorphic species in the Agaricostilbum, lactophilus and Kondoa clades, respectively. Ballistosporomyces is emended and reintroduced to include species in the sasicola clade. Jianyunia gen. nov., Pseudobensingtonia gen. nov. and Ruinenia gen. nov. are proposed to accommodate the species in the Bensingtonia sakaguchii, ingoldii and ruber clades, respectively. Ruineniaceae fam. nov. is proposed to accommodate the genus Ruinenia based on results from the phylogenetic analysis of seven genes ( Fig. 1) and GMYC analyses ( This order was proposed to accommodate the family Agaricostilbaceae (Oberwinkler & Bauer 1989). The Agaricostilbaceae, Chionosphaeraceae and Kondoaceae were accepted in this order by Bauer et al. (2006). Here we propose Ruineniaceae and Jianyunia as 'incertae sedis' in the Agaricostilbales.
Sexual reproduction observed in some species. Basidia occur predominantly in synnemata-like basidiomata. Hyphae, basidia and basidiospores relatively thick-walled. Basidiospores often attached to a budding locus (Bandoni & Boekhout 2011). Colonies cream and butyrous. Budding cells present or not, some of them produce one or more stalk-like conidiophores with blastoconidia separating at a septum in the mid-region of the stalk on the parent cell. Ballistoconidia not produced. Major CoQ system Q-9. This genus is proposed to accommodate the ingoldii clade containing two species that previously belonged to the genus Bensingtonia . Member of the Agaricostilbaceae. The genus is mainly circumscribed by the Table 3.

Rhodotorula phylloplana AF190004
Classiculomycetes (    phylogenetic analysis of seven genes, in which it formed a sister lineage to the genus Sterigmatomyces within Agaricostilbaceae (Figs 1, 4). This genus is phylogenetically distinct from the Bensingtonia clade that contains the type species of Bensingtonia, B. ciliata, that belongs to the family Kondoaceae   Fig. 4

of this study).
Sexual reproduction not known. Colonies greyish-yellow or dark yellow and butyrous. Budding cells present. Pseudohyphae present or not. Ballistoconidia present, ellipsoidal or kidneyshaped. Major CoQ system Q-9. Note: The species of Pseudobensingtonia do not form conidiogenous stalks, which are present in the anamorphic species of Sterigmatomyces (Nakase et al. 1989, Takashima et al. 1995. This family is characterised by teleomorphic members with gasteroid basidia with simultaneous basidiospore production per basidium (Oberwinkler & Bandoni 1982). Notes: Mycogloea nipponica was placed in this family based on a multi-gene analyses  and an analysis of the enlarged LSU rRNA gene dataset (Fig. 4) Type species: Ballistosporomyces xanthus Nakase et al.
This genus is emended and reintroduced to include species of the sasicola clade , which occurred as a well-supported clade related to the genus Cystobasidiopsis within Chionosphaeraceae (Figs 1, 4). The genus Ballistosporomyces was erected by Nakase et al. (1989) and included Ba. xanthus (= Sporobolomyces xanthus), the type of Ballistosporomyces, and Ba. ruber (= Sporobolomyces ruber). This genus was treated as a synonym of Sporobolomyces (Boekhout 1991). Our analyses showed that Ba. xanthus (S. xanthus) is located in the sasicola clade, whereas Ba. ruber (S. ruber) occurs in the ruber clade that is phylogenetically distinct from the family Chionosphaeraceae (Fig. 1). Thus, here we emend and reintroduce Ballistosporomyces as a genus to include the species of the sasicola clade.
Sexual reproduction unknown. Colonies orange to pale yellowish-brown and butyrous. Budding cells present. Hyphae and pseudohyphae not formed. Ballistoconidia present, allantoid.
Note: Sporobolomyces ruber (Ba. ruber), which is located in the ruber clade ( Fig. 1), is proposed as a new combination in Ruinenia (Fig. 4). This genus is emended to include species of the genera Kondoa and Bensingtonia hitherto classified in the Kondoa clade , which occurred as a well supported clade that is phylogenetically distinct from the genus Bensingtonia within Kondoaceae (Figs 1, 4). Thus all Bensingtonia species included in the Kondoa clade will be transferred into the genus Kondoa based on the "One Fungal = One Name" principle (Hawksworth 2011, Taylor 2011, McNeill et al. 2012 This genus agrees with the ruber clade . Member of Ruineniaceae (Agaricostilbales, Agaricostilbomycetes). The genus is mainly circumscribed by the phylogenetic analysis of seven genes, in which it occurred as a well supported clade distinct from the other genera within Agaricostilbales (Figs 1, 4).
Sexual reproduction not known. Colonies orange-red or salmonpink, and butyrous. Budding cells present. Hyphae and pseudohyphae present or not. Ballistoconidia present, ellipsoidal, reniform to falcate. Major CoQ system Q-10. Note: The species of Ruinenia (i.e. ruber clade) form salmonorange to red colonies, which are a unique feature in the Agaricostilbomycetes (Fig. 3A). Additionally, five sequences representing potential new species of this genus were obtained from public databases (Fig. 4). This genus agrees with the Bensingtonia sakaguchii lineage . Member of Agaricostilbales (Agaricostilbomycetes). The genus is mainly circumscribed by the phylogenetic analysis of seven genes, in which it occurred as a single-species lineage distinct from the other genera within Agaricostilbales (Figs 1, 4).

Member of
Pucciniomycotina. The class is mainly circumscribed by the phylogenetic analysis of seven genes, in which it formed a deep well supported lineage with affinity to Mixiomycetes within Pucciniomycotina (Fig. 1). The diagnosis of the class Spiculogloeomycetes is based on the description of the order Spiculogloeales . The nomenclature of this class is based on the Spiculogloeales. This class contains species of Spiculogloea and some species of Mycogloea and Sporobolomyces (Aime et al. 2006. Phyllozyma gen. nov. is proposed to accommodate the Sporobolomyces species in the subbrunneus clade based on the phylogenetic analysis of seven genes (Fig. 1), PRBO (Table 1), phylogenetic network analysis (Fig. 2B) and the analysis of the enlarged LSU rRNA gene dataset (Fig. 4).
The name Spiculogloeaceae was validated by Denchev (2009) to include the taxa of Spiculogloeales . Note: The species of the genus Spiculogloea and Mycogloea are not listed here because cultures of Spiculogloea and Mycogloea, except for Mycogloea nipponica that is located in the Chionosphaeraceae, are presently not available. Moreover, nucleotide sequence data for type species of these genera are not available from public databases. Etymology: The genus is named based on the habitat as all species from this clade were isolated from the leaves of plants.
This genus agrees with the subbrunneus clade . Member of the Spiculogloeaceae (Spiculogloeales, Spiculogloeomycetes). The genus is mainly circumscribed by the phylogenetic analysis of seven genes and the analysis of the enlarged LSU rRNA gene dataset, in which it occurred as a well supported clade (Figs 1, 4). Species in the subbrunneus clade are associated with plant leaves (Hamamoto et al. 2011) and are ecologically different from the teleomorphic species Spiculogloea spp. and Mycogloea spp., which are mycoparasites with tremelloid haustorial cells (Roberts 1996, Bauer 2004, Weiß et al. 2004. Sexual reproduction not known. Colonies pale yellowishbrown, reddish-orange and butyrous. Budding cells present. Hypha and pseudohyphae present or not. Ballistoconidia present, ellipsoidal, fusiform or sickle-shaped. Major CoQ system Q-10. Note: S. productus and S. corallinus were not included in our previous phylogenetic study . These two species were placed in the subbrunneus lineage closely related to S. subbrunneus and S. dimnenae based on the sequence analysis of the D1/D2 domains of LSU rRNA (Furuya et al. 2012, Fig. 4 of the present study), and hence, they are recombined in the genus Phyllozyma.
Type order: Cystobasidiales R. Bauer et al.
Three orders, namely Cystobasidiales, Erythrobasidiales and Naohideales, as well as the aurantiaca, magnisporus, marina and Sakaguchia clades occur within Cystobasidiomycetes . As the relative positioning of these five groups could not be resolved with certainty ( Fig. 1) we suggest to treat the four clades as families but not to assign them to any order. Instead, we place them as 'incertae sedis' within Cystobasidiomycetes. Buckleyzymaceae fam. nov. with Buckleyzyma gen. nov, Microsporomycetaceae fam. nov. with Microsporomyces gen. nov., Symmetrosporaceae fam. nov. with Symmetrospora gen. nov., and Sakaguchiaceae fam. nov. are proposed to accommodate the taxa in the aurantiaca, magnisporus, marina and Sakaguchia clades, respectively (Figs 1, 5).
This order was proposed to accommodate the family Cystobasidiaceae that is characterised by nanometer-fusion mycoparasitism with tremelloid haustorial cells and septal pores with cystosomes . Note: R. portillonensis that is phylogenetically located in the Cystobasidium clade was recently proposed by Laich et al. (2013). This species was placed in this genus in the analysis of the enlarged LSU rRNA gene dataset (Fig. 5)

5)
Occultifur kilbournensis (2011), but only O. externus was included in our previous phylogenetic study  as neither living cultures nor molecular data are available for the other two species. Three new asexual members of the genus Occultifur (Fig. 5), namely O. brasiliensis, O. kilbournensis and O. tropicalis, were recently described by Gomes et al. (2015), Kurtzman & Robnett (2015) and Khunnamwong et al. (2015), respectively.
This order accommodates the genera Erythrobasidium and Bannoa that have non-tremelloid haustorial cells, septal pores without cystosomes, and coenzyme CoQ10 (H2), as well as some Rhodotorula and Sporobolomyces species ). The genus Cyrenella was not placed in this order by Aime et al. (2006), Bauer et al. (2006) and Hibbett et al. (2007). This genus, however, formed a well supported clade with the genera Erythrobasidium and Bannoa, and Rhodotorula lactosa, which is placed in a new genus Hasegawazyma (Fig. 1). Thus, this order includes the family Erythrobasidiaceae and the genera Cyrenella and Hasegawazyma, which are treated as 'incertae sedis' in the Erythrobasidiales.
Family Erythrobasidiaceae Denchev, Mycol. This genus is emended to include species of Bannoa and related Sporobolomyces species that hitherto were classified in the Bannoa clade , which occurred as a well supported clade related to Erythrobasidium within Erythrobasidiales (Figs 1, 5).
Sexual reproduction observed in some species. Clamp connections present. Teliospores not formed. Unicellular basidia arise laterally on a clamp connection, or terminally at the hyphae. Cells of the basidia germinate with hyphae, from which yeast cells originate. Colonies orange to salmon-red. Budding cells present. Pseudohyphae absent. Ballistoconidia present or not, ovoid and ellipsoidal. Major CoQ system Q-10(H2). This genus is emended to include species of Erythrobasidium and related Sporobolomyces species that hitherto were classified in the Erythrobasidium clade , which occurred as a well supported clade closely related to Bannoa within Erythrobasidiales (Figs 1, 5).
This genus corresponds to the Rhodotorula lactosa lineage (Wang et al. 2015a, Fig. 5 of this study). Member of Erythrobasidiales (Cystobasidiomycetes). The genus is mainly circumscribed by the phylogenetic analysis of seven genes and the analysis of the enlarged LSU rRNA gene dataset, in which it occurred as a single-species lineage distinct from the other genera within Cystobasidiomycetes (Figs 1, 5). This genus differs from Bannoa, Cyrenella and Erythrobasidium by having ubiquinone Q-9.

Type family: Naohideaceae Denchev
This order was proposed to accommodate the genus Naohidea, which is characterised by nanometer-fusion mycoparasitism with intracellular haustoria (tremelloid) and septal pores without cystosomes .
The name Naohideaceae was validated by Denchev (2009) to include the taxa of Naohideales ).
The genus Naohidea, representing the order Naohideales, produces cream-coloured cultures that are different from all other taxa in the Cystobasidiomycetes, which form pink to orange-red pigmented colonies (Sampaio & Chen 2011, Fig. 3B of this study). Etymology: The genus name relates to the type species, Sporobolomyces magnisporus that produces small (micro-) ballistoconidia.
This genus agrees with the magnisporus clade . Member of the Microsporomycetaceae (Cystobasidiomycetes). The genus is mainly circumscribed by the phylogenetic analysis of seven genes and the analysis of the enlarged LSU rRNA gene dataset, in which it occurred as a sister clade to Cystobasidiales and Sakaguchia within the Cystobasidiomycetes (Figs 1, 5).
Sexual reproduction not known. Colonies orange or salmoncoloured and butyrous. Budding cells present. Pseudohyphae present or not. Ballistoconidia present or not, ellipsoidal, allantoid to amygdaliform. Major CoQ system Q-10. This genus is emended to include Sakaguchia dacryoidea and related Rhodotorula species that hitherto were classified in the Sakaguchia clade , which occurred as a well supported clade within the Cystobasidiomycetes (Figs 1, 5).
Sexual reproduction in some species. Clamp connections present. Teliospores laterally or terminally on the hyphae. Teliospores germinate with two-to four-celled metabasidium with lateral and terminal basidiospores. Colonies red or orange-red and butyrous. Budding cells present. Pseudohyphae or true hyphae present or not. Ballistoconidia not produced. Major CoQ system Q-10. Note: Our analyses suggest that Rhodotorula sp. JCM 8162 represents a potential new species of this genus, as revealed by the multi-gene analyses  and the analysis of the enlarged LSU rRNA gene dataset (Fig. 5).
This class presently contains four orders that contain yeast species, namely Kriegeriales, Leucosporidiales, Microbotryales and Sporidiobolales (Aime et al. 2006, Hibbett et al. 2007, Toome et al. 2013). Ten clades and seven single-species lineages do not belong to the above listed orders , and they may be assigned into eleven families based on modified GMYC analysis ( Table 2) in conjunction with the phylogenetic analysis of seven genes (Fig. 1). However, most of these suggested families are monotypic or poorly sampled (Table 2). Thus, we propose Chrysozymaceae fam. nov. to accommodate the griseoflavus, yamatoana, singularis and Sporobolomyces inositophilus clades, and Colacogloeaceae fam. nov. to cover the Colacogloea clade because these two proposed families have more taxa than the other clades and have strong support values (Fig. 1). The other six clades and six single-species lineages are proposed to be included in twelve genera that at the higher rank are considered as 'incertae sedis' within the Microbotryomycetes at present. The species Rhodotorula svalbardensis (Singh et al. 2014) was not included in the seven genes-based phylogenetic analysis  and has no proper placement in the constrained LSU analysis (Fig. 6). The phylogenetic placement of this species needs to be addressed by a robust molecular analysis. Thus, we leave it as Rhodotorula svalbardensis pro tem. in this study. The term "pro tem." (pro tempore) was proposed in Wang et al. (2015b) and indicates a temporary taxonomic placement. This family was proposed to accommodate the sexual plant parasite Kriegeria, the asexual genus Meredithblackwellia, and six related Rhodotorula species (Toome et al. 2013). The species R. pilati was placed in the Kriegeriaceae by Toome et al. (2013), but this affiliation was not supported in the muligene study by . This species was located in the tsugae clade . The other five Rhodotorula species in the Kriegeriaceae are reclassified into two new genera Phenoliferia and Yamadamyces. Notes: Species of Phenoliferia (i.e. the glacialis clade) have similar assimilation patterns of carbon and nitrogen compounds (Margesin et al. 2007). They share the ability to grow on raffinose as the sole carbon source but not on maltose. In contrast, closely related taxa, such as Kriegeria eriophori and Rhodotorula rosulata, have different assimilation properties and are able to utilise maltose, but not raffinose (Table 3). R. himalayensis was not included in our previous phylogenetic study . This species was found to be closely related to R. psychrophila and R. glacialis based on the sequence analyses of LSU rRNA D1/D2 domains and ITS (Shivaji et al. 2008, Turchetti et al. 2011, Toome et al. 2013, Singh et al. 2014, Fig. 6  Notes: The species R. rosulata was found to differ from K. eriophori in the utilisation of three carbon sources, namely methyl-α-D-glucoside, L-arabinose and myo-inositol (Table 3). The micromorphology of the species resembles that of Me. eburnea and K. eriophori with budding cells remaining connected to each other at the base and forming clusters (or rosettes) of cells.
The family Camptobasidiaceae was described to accommodate a teleomorphic aquatic fungus Camptobasidium (Marvanov a & Suberkropp 1990) that was placed in the Atractiellales (Atractiellomycetes) by Moore (1996). Aime et al. (2006) indicated that Camptobasidium belongs to Microbotryomycetes rather than Atractiellomycetes based on rRNA sequence analysis. Note: Only one species of the genus Glaciozyma, namely G. antarctica, was employed in our previous study  because G. litoralis, G. martini and G. watsonii were only recently published (Turchetti et al. 2011, Kachalkin 2014. These species were included in the analysis of the enlarged LSU rRNA gene dataset (Fig. 6), and the genus Glaciozyma received high support in ML and MP analyses.
This order was proposed to accommodate phytoparasitic taxa lacking colacosomes (lenticular bodies) and teliospores (Bauer et al. 1997. The order includes the families Microbotryaceae and Ustilentylomataceae. No yeast species are included in the family Microbotryaceae, but yeast stages of taxa in this family occur (Fig. 3D).
This family was proposed to include taxa with 'simple' septal pores in the Microbotryales (Bauer et al. 1997). Note: The species of the genera Aurantiosporium and Fulvisporium are not listed here because no yeast phase has been observed in these two genera. Representatives of these genera were included in the analysis of the enlarged LSU rRNA gene dataset (Fig. 7). Note: Mi. collariae is a yeast species within Ustilentylomataceae, which was recently described to accommodate two strains isolated from the intestine of a plant bug (Collaria oleosa, Miridae, Heteroptera, Insecta) by Suh et al. (2012). This anamorphic species is distantly related to any teleomorphic parasitic taxa in this family (Fig. 7). Type species: Ustilentyloma pleuropogonis Savile.
This genus was originally described for teleomorphic fungi occurring on Poaceae (Savile & Parmelee 1964, V anky 2002. Here it is emended to include free-living yeast species with unknown sexual states. Notes: The close relatives of R. hordea are plant parasite species of Ustilentyloma based on the sequence analysis of the D1/D2 domains of the LSU rRNA (Sampaio 2011, Fig. 7 of this study) and the ITS (including 5.8S rRNA) region. Nucleotide sequences of R. hordea differ from Ustilentyloma fluitans (GenBank number: AF009882 and KC994459) and Ustilentyloma brefeldii (GenBank number: EF622016) by 1 and 15 positions in the D1/D2 domains of the LSU rRNA, respectively. However, R. hordea differs from U. fluitans by 78 mismatches and gaps (12 %) in the ITS region (GenBank number: AY212990 and KC994460), and by the assimilation of D-galactose, L-sorbose, D-ribose, lactose, erythritol, ribitol and protocatechuic acid (Sampaio 2011). The genus Ustilentyloma established by Savile & Parmelee (1964) contains U. brefeldii, U. fluitans, U. oreochloae and U. pleuropogonis. All species of this genus are parasites infecting leaves of Poaceae. R. hordea isolated from the leaves of six-row barley (Hordeum vulgare subsp. hexastichium, Poaceae) may have a similar ecology as a phyllosphere inhabitant as the species of Ustilentyloma, although it is not known to be a parasite on barley. One possibility may be that R. hordea represents a yeast stage of a dimorphic phytoparasite that is distinct from U. fluitans. Sequences of U. pleuropogonis, the type species of Ustilentyloma, are not available at present, but the above mentioned sequence analysis of U. brefeldii and U. fluitans indicated that this genus may be monophyletic (Fig. 7). The morphological characters of this genus also support its monophyletic nature (V anky 2002). Based on the above discussion we prefer to propose R. hordea as a new combination of Ustilentyloma. The name Rhodotorula hordea was chosen to replace Candida graminis when it was transferred to Rhodotorula by Weijman et al. (1998) because the specific epithet 'graminis' was already used for the species Rhodotorula graminis.
Here we reuse this epithet for Ustilentyloma graminis.
This order was proposed to include the family Sporidiobolaceae (Sampaio et al. 2003).
This genus is emended to include Rhodotorula species and their sexual counterpart Rhodosporidium in the Rhodosporidium clade , which is a well supported clade within Sporidiobolaceae (Sporidiobolales). The Rhodosporidium clade is composed of Rhodotorula glutinis, the type species of Rhodotorula, and Rhodosporidium toruloides, the type species of Rhodosporidium (Figs 1, 8). The name Rhodotorula is older than Rhodosporidium, and has taxonomic priority over the latter.
Sexual reproduction observed in some species. Clamp connections present. Teliospores may be formed and produce transversely septate basidia. The basidiospores ovoid, passively released and germinate by budding. Colonies red and butyrous. Budding cells present. Pseudohyphae or true hyphae present or not. Ballistoconidia formed or not, ellipsoidal. Major CoQ systems Q-9 or Q-10. This genus is emended to include species of Colacogloea and related Rhodotorula species in the Colacogloea clade , which occurred as a well supported clade within Microbotryomycetes (Figs 1, 6).
Sexual reproduction observed in some species. Teleomorphic taxa mycoparasitic, and the sexual state develops only in the host. Basidiocarps minute, pulvinate to effuse, and mucoidgelatinous. Basidia auricularioid (i.e., transversely septate). Hyphae thin-walled, hyaline, with clamp connections, and grow intrahymenially in host fructifications. Septal pores 'simple'. Colacosomes (or lenticular body) occur at the interface between the parasite and the host . Colonies cream, mucoid or butyrous. Budding cells present. Ballistoconidia not formed. Major CoQ system Q-10.

Suggestion for new species descriptions
In the future descriptions of new species in the genera Rhodotorula, Sporobolomyces and Bensingtonia should be restricted to the clades containing the respective type species (Figs 1,4,8).
In the case of unclassified Microbotryomycetes or 'incertae sedis', none of the aforementioned generic names should be used to describe new species, and new genera have to be introduced following a robust phylogenetic analysis utilising several independent DNA loci or whole-genome comparisons (e.g. . Our results show that using the LSU rRNA gene alone is not sufficient to resolve the high-level phylogenetic relationships in Microbotryomycetes.