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Persoonia : Molecular Phylogeny and Evolution of Fungi logoLink to Persoonia : Molecular Phylogeny and Evolution of Fungi
. 2014 May 23;33:41–47. doi: 10.3767/003158514X682313

Moniliellomycetes and Malasseziomycetes, two new classes in Ustilaginomycotina

Q-M Wang 1,✉, B Theelen 2, M Groenewald 2, F-Y Bai 1,2, T Boekhout 1,2,3,4
PMCID: PMC4312936  PMID: 25737592

Abstract

Ustilaginomycotina (Basidiomycota, Fungi) has been reclassified recently based on multiple gene sequence analyses. However, the phylogenetic placement of two yeast-like genera Malassezia and Moniliella in the subphylum remains unclear. Phylogenetic analyses using different algorithms based on the sequences of six genes, including the small subunit (18S) ribosomal DNA (rDNA), the large subunit (26S) rDNA D1/D2 domains, the internal transcribed spacer regions (ITS 1 and 2) including 5.8S rDNA, the two subunits of RNA polymerase II (RPB1 and RPB2) and the translation elongation factor 1-α (EF1-α), were performed to address their phylogenetic positions. Our analyses indicated that Malassezia and Moniliella represented two deeply rooted lineages within Ustilaginomycotina and have a sister relationship to both Ustilaginomycetes and Exobasidiomycetes. Those clades are described here as new classes, namely Moniliellomycetes with order Moniliellales, family Moniliellaceae, and genus Moniliella; and Malasseziomycetes with order Malasseziales, family Malasseziaceae, and genus Malassezia. Phenotypic differences support this classification suggesting widely different life styles among the mainly plant pathogenic Ustilaginomycotina.

Keywords: fungi, molecular phylogeny, smuts, taxonomy, yeasts

INTRODUCTION

Basidiomycota (Dikarya, Fungi) contains three main phylogenetic domains, namely the subphyla Agaricomycotina, Pucciniomycotina and Ustilaginomycotina(Hibbett et al. 2007). Within Ustilaginomycotina three classes have been proposed, namely Ustilaginomycetes, Exobasidiomycetes and Entorrhizomycetes (Begerow et al. 1997, 2006, Bauer et al. 2001, 2006). Other researchers, however, questioned the status of Entorrhizomycetes and considered them incertae sedis among Basidiomycota (Matheny et al. 2006, Hibbett et al. 2007).

Ustilaginomycotina comprises plant pathogenic fungi (smuts), which are mostly dimorphic and present a yeast stage during the live cycle, and asexual fungi which are known only as yeasts or yeast-like species, e.g. Acaromyces spp., Pseudozyma spp., Meira spp., Tilletiopsis spp. and Malassezia spp. (Boekhout 1991, 1995, Boekhout et al. 1995, 2003, 2011, Begerow et al. 2000, 2006, Fell et al. 2000). Pseudozyma spp. belong to Ustilaginomycetes and the genera Acaromyces, Meira and Tilletiopsis to Exobasidiomycetes (Begerow et al. 2006, Hibbett et al. 2007, Boekhout et al. 2011). The taxonomic position of Malassezia spp., that is an important inhabitant of the human and animal skin microbiota (Guého-Kellermann et al. 2010, Sugita et al. 2010, Gaitanis et al. 2012, Findley et al. 2013), is not settled. It has been proposed that the genus belongs to Ustilaginomycotina (Begerow et al. 2000, Xu et al. 2007), but its final affiliation within this group remained problematic. The genus was treated to represent a distinct order Malasseziales in the Exobasidiomycetes based on molecular phylogenetic analyses of the nuclear ribosomal RNA genes alone or in combination with protein genes (Begerow et al. 2000, 2006, Bauer et al. 2001, Weiß et al. 2004). However, Matheny et al. (2006) suggested that the Malasseziales might be affiliated with Ustilaginomycetes. Therefore, Hibbett et al. (2007) excluded Malasseziales from Exobasidiomycetes and treated it as incertae sedis in Ustilaginomycotina.

The yeast-like genus Moniliella was described by Stolk & Dakin (1966). In the following year the genus Trichosporonoides was introduced by Haskins & Spencer (1967). Both have a basidiomycetous affinity because they can hydrolyse urea, show a positive Diazonium Blue B (DBB) staining and have multi-lamellar cell walls. The septal pores of the species studied so far (M. oedocephala, M. spathulata, M. suaveolens) show a diversity of septa. Moniliella suaveolens has typical dolipores with an arch of endoplasmic reticulum, M. spathulata has a micropore-like structure and M. oedocephala has both (Haskins 1975, Martínez 1979). Boekhout (1998) and de Hoog & Smith (1998a, b) indicated that Trichosporonoides was probably synonymous with Moniliella. Later Rosa et al. (2008) confirmed that both genera were indeed congeneric based on 26S rRNA D1/D2 domain sequence analysis and, consequently, transferred all species into the genus Moniliella. These authors were, however, not sure about the phylogenetic relationships with either Ustilaginomycotina or Agaricomycotina.

In order to clarify the phylogenetic affiliations of Malassezia and Moniliella, we performed phylogenetic analyses based on the six genes that were used to address the higher-level phylogeny of the Fungi (James et al. 2006, Hibbett et al. 2007). Our results demonstrated that Malassezia and Moniliella belong to Ustilaginomycotina where they form deep and well-supported lineages with a sister relationship to both Ustilaginomycetes and Exobasidiomycetes. We therefore propose two new classes, Malasseziomycetes and Moniliellomycetes for these lineages, with additional support from phenotypic characters.

MATERIALS AND METHODS

Taxon sampling

Almost all the recognised Malassezia and Moniliella species were employed (Table 1). Reference species representing Pucciniomycotina, Agaricomycotina and currently recognised classes of Ustilaginomycotina were selected based on sequence availability for the six genes used in the AFTOL1 project (http://aftol.org/data.php) (Table 1). Sequence data generated in this study or data retrieved from GenBank were mostly from type strains of the taxa compared.

Table 1.

Taxa sampled and sequence accession numbers employed (those in bold are determined in this study).

Species Strain D1D2 ITS SSU RPB1 RPB2 EF1-α
Ustilaginomycetes
     Cintractia limitata HAJB 10488 DQ645506 DQ645508 DQ645507 DQ645510 DQ645509 DQ645511
     Melanotaenium endogenum AFTOL ID1918 DQ789979 DQ789981 DQ789980 – – –
     Melanotaenium euphorbiae voucher HUV17733 JN367314 JN367289 JN367342 – – JN367365
     Rhodotorula acheniorum AS 2.3198T AF190001 AB038128 AJ496256 KF706499 KF706522 KF706474
     Schizonella melanogramma CBS 174.42 DQ832210 DQ832212 DQ832211 DQ832214 DQ832213 DQ832215
     Sporisorium reilianum CBS 131460 KF706430 KF706438 KF706441 – KF706511 KF706472
     Urocystis colchici AFTOL-ID1647 DQ838576 DQ839596 DQ839595 – DQ839597 DQ839598
     Urocystis eranthidis voucher hmk292 JN367324 JN367299 JN367352 JN367428 – JN367375
     Ustanciosporium gigantosporum HRK 023 JN367325 JN367300 JN367353 JN367429 – JN367376
     Ustilago hordei CBS 131470 KF706429 KF706437 KF706442 KF706498 KF706521 KF706473
     Ustilago maydis CBS 504.76/MS 115 AF453938 AY854090 X62396 XM401478 AY485636 AY885160
     Ustilago tritici CBS 669.70 DQ094784 DQ846894 DQ846895 DQ846897 DQ846896 DQ846898
Exobasidiomycetes
     Exobasidium gracile DSM 4460 DQ663699 DQ663700 DQ785786 DQ663702 DQ663701 DQ663703
     Exobasidium rhododendri CBS 101457 DQ667151 DQ667153 DQ667152 DQ667155 DQ667154 DQ667156
     Jaminaea angkoriensis C5b EU587489 EU604147 EU604148 – – –
     Microstroma juglandis CBS 287.63 AF009867 DQ789988 DQ789987 DQ789990 DQ789989 DQ789991
     Quambalaria cyanescens CBS 876.73 DQ317616 DQ317623 KF706440 – KF706531 KF706485
     Rhamphospora nymphaeae CBS 72.38 DQ831032 DQ831034 DQ831033 – DQ831035 DQ831036
     Tilletiaria anomala CBS 436.72 AJ235284 DQ234558 AY803752 DQ234571 AY803750 DQ835991
CBS 607.83T AJ235282 AB025704 KF706451 – KF706530 KF706483
     Tilletiopsis washingtonensis CBS 544.50T AJ235278 DQ835994 AJ271382 – DQ835995 DQ835996
Malasseziomycetes
     Malassezia caprae CBS10434T AY743616 AY743656 KF706456 KF706495 KF706513 KF706467
     Malassezia dermatis CBS 9169T AB070365 AY390284 KF706452 KF706490 KF706532 KF706461
     Malassezia equina CBS 9969T AY743621 KF706439 KF706454 KF706492 KF706515 KF706463
     Malassezia furfur CBS 1878T AF063214 AY743634 KF706457 KF706497 KF706516 KF706469
     Malassezia globosa CBS 7966T AF064025 AY387132 – KF706493 KF706518 KF706465
     Malassezia japonica CBS 9431T EF140672 EF140669 KF706458 – KF706514 KF706464
     Malassezia nana CBS 9558T EF140673 EF140667 KF706453 KF706491 KF706510 KF706462
     Malassezia obtusa CBS 7876T AB105197 AY387137 KF706455 – KF706519 KF706470
     Malassezia pachydermatis CBS 1879T AY743605 AB118941 DQ457640 DQ785792 DQ408140 DQ028594
     Malassezia restricta CBS 7877T AF064026 AY743636 EU192367 KF706496 KF706520 KF706471
     Malassezia slooffiae CBS 7956T AJ249956 AY743633 KF706459 – – –
     Malassezia sympodialis CBS 7222T AF064024 AY743632 KF706460 – – –
     Malassezia yamatoensis CBS 9725T AB125263 AB125261 – KF706494 KF706512 KF706466
Moniliellomycetes
     Moniliella acetoabutens CBS 169.66T AF335523 EU252153 KF706443 KF706500 KF706523 KF706476
     Moniliella madida CBS 240.79T AF335522 – KF706447 KF706502 KF706525 KF706478
     Moniliella megachiliensis CBS 190.92T EF137916 KF706433 KF706448 KF706501 KF706524 KF706477
     Moniliella mellis CBS 350.33T EU545185 – KF706446 – KF706528 KF706481
     Moniliella nigrescens CBS 269.81T AF335527 KF706436 – KF706504 KF706527 KF706480
     Moniliella oedocephalis CBS 649.66T AF335521 KF706435 KF706449 – – KF706484
     Moniliella pollinis CBS 461.67T AF335525 KF706434 KF706450 KF706505 KF706529 KF706482
     Moniliella spathulata CBS 241.79T AF335526 KF706432 KF706444 KF706503 KF706526 KF706479
     Moniliella suaveolens CBS 126.42T AF335520 KF706431 KF706445 – – KF706475
Pucciniomycotina
     Bensingtonia ciliata AS 2.1945T AF189887 AF444563 D38233 KF706509 KF706536 KF706486
     Chrysomyxa arctostaphyli CFB22246 AY700192 DQ200930 AY657009 – DQ408138 DQ435789
     Endocronartium harknessii CFB22250 AY700193 DQ206982 AY665785 – DQ234551 DQ234567
     Erythrobasidium hasegawianum AS 2.1923T AF189899 AF444522 D12803 KF706506 KF706534 KF706488
     Naohidea sebacea CBS 8477 DQ831020 DQ911616 – KF706508 KF706535 KF706487
     Platygloea disciformis IFO32431 AY629314 DQ234556 DQ234563 – DQ234554 DQ056288
     Puccinia graminis CRL75-36-700-3/ECS AF522177 AF468044 AY125409 XM_003334476 XM_003321826 XM_003333024
     Sporidiobolus salmonicolor CBS 490T AF070439 AY015434 AB021697 KF706507 KF706533 KF706489
Agaricomycotina
     Auriculibuller fuscus CBS 9648T AF444763 AF444669 KF036604 KF036314 KF036727 KF036999
     Bulleromyces albus CBS 501T AF075500 AF444368 X60179 KF036334 KF036745 KF037016
     Boletellus projectellus MB03-118 AY684158 AY789082 AY662660 AY788850 AY787218 AY879116
     Dacryopinax spathularia GEL 5052 AY701525 AY854070 AY771603 AY857981 AY786054 AY881020
     Filobasidiella depauperata CBS 7841T AF487884 EF211248 AJ568017 KF036417 KF036885 KF037150
Wallemia clade
     Wallemia ichthyophaga EXF994/EXF1059 DQ847516 AY302523 AY741382 DQ847522 DQ847519 DQ847525
     Wallemia muriae MZKI-B952/EXF1054 DQ847517 AY302534 AY741381 DQ847523 DQ847520 DQ847526
     Wallemia sebi EXF483 DQ847518 AY328917 AY741379 DQ847524 DQ847521 DQ847527
Ascomycota
     Aleuria aurantia OSC 100018 AY544654 DQ491495 NG013139 DQ471120 DQ247785 DQ466085
     Aspergillus nidulans NRRL 2395/FGSC4 EF652445 AY373888 U77377 XM_653321 XM_677297 XM_656730
     Neurospora crassa NRRL 13141/ICMP 6360 AY681158 AY681193 AY046271 XM_959004 AF107789 AF402094
     Schizosaccharomyces pombe 972H- CU329672 CU329672 CU329672 D13337 X56564 NC_003421
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PCR and DNA sequencing

Genomic DNA was extracted from the living cultures grown on the Yeast Extract Peptone Dextrose (YPD) plate using the method as described by Bolano et al. (2001). A set of six genes were selected, including three protein-coding genes, namely RPB1 (the largest subunit of RNA polymerase II), RPB2 (the second largest subunit of RNA polymerase II) and EF1-α (translation elongation factor 1-α), and three rRNA genes namely small subunit (SSU or 18S) ribosomal DNA (rDNA) D1/D2 domains of the large subunit (LSU or 26S) rDNA, and the ITS 1+2 regions (including 5.8S rDNA) of the rDNA. PCR and sequencing of the rRNA genes were performed using the methods described previously (Fell et al. 2000, Scorzetti et al. 2002, Wang et al. 2003). PCR and sequencing primers for RPB1, RPB2 and EF1-α are listed in Table 2. PCR parameters for amplifying RPB1 and RPB2 were as follows: an initial denaturation step at 94 °C for 4 min; 36 cycles of denaturation at 94 °C for 1 min, annealing at 50–52 °C for 1 min and extension at 72 °C for 1 min; and a final extension step of 8 min at 72 °C. Amplification of the EF1-α gene used a touchdown PCR: an initial denaturation at 94 °C for 4 min; an annealing temperature of 62 °C in the first cycle, successively reducing the Tm by 1 °C per cycle over the next nine cycles to reach a final Tm of 52 °C, which is used in the remaining 30 cycles and extension at 72 °C for 1 min; and a final extension step of 8 min at 72 °C. Cycle sequencing was performed using the ABI BigDye cycle sequencing kit (QIAGEN, Valencia, California). Electrophoresis was done on an ABI PRISM 3710 or 3730 DNA sequencer.

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
EF1-α 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
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Molecular phylogenetic analyses

Sequences were aligned with the Clustal X program (Thompson et al. 1997). The alignment datasets were firstly analysed with Modeltest v. 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 further analyses. Maximum likelihood (ML) analyses were conducted in MEGA v. 5 (Tamura et al. 2011). Maximum parsimony (MP) analysis was conducted in PAUP v. 4.0b10 (Swofford 2002) where the support of the branching topologies was derived from 1 000 replicates with 10 random additions. Three to four ascomycetous species were used as outgroups. Bayesian posterior probability analyses were conducted in MrBayes v. 3.2 (Ronquist et al. 2012) with parameters set to 1 000 000 generations, four runs and four chains. The chains were heated to 0.25 and a stop value of 0.01 was used. The alignment matrix was deposited in TreeBASE (www.treebase.org) with submission ID 14907.

RESULTS

A total of 108 new sequences were generated from 31 species in this study (Table 1). New sequences and those retrieved from GenBank generated from the same species were concatenated in different combinations to form three separate datasets as follows: 1) a rRNA gene dataset formed by SSU, LSU D1/D2 and ITS (including 5.8S rDNA); 2) a protein gene dataset consisted of RPB1, RPB2 and EF1-α; and 3) a six-gene dataset formed by the combination of the former two datasets as used in James et al. (2006). All the datasets were subjected to ML, MP and Bayesian analyses respectively and the topologies of the trees obtained were visually examined for phylogenetic concordance.

In the majority of the trees, four distinct monophyletic clades, namely Exobasidiomycetes, Malassezia, Moniliella and Ustilaginomycetes, were consistently resolved within Ustilaginomycotina (Table 3, Fig. 1, 2). The Malassezia, Moniliella and Ustilaginomycetes clades each received 1.0 post probability and 99–100 % bootstrap support in the trees constructed using different algorithms based on the six-gene dataset. These three clades were also clearly resolved and strongly supported (1.0 post probability and 92–100 % bootstrap values) in the trees drawn from the rRNA gene and the protein gene datasets, except for the Ustilaginomycetes clade which received weak (51–65 %) bootstrap support in the ML and MP trees drawn from the rRNA gene dataset (Table 3, Fig. 2). The Exobasidiomycetes was resolved as a monophyletic clade in the trees constructed from the rRNA gene and the six-gene datasets, but only received strong support in the Bayesian and ML trees drawn from the rRNA gene dataset. This clade was shown to be non-monophyletic in all the trees drawn from the protein gene dataset (Table 3, Fig. 1, 2). In the Bayesian and ML trees (Fig. 2d, e), Tilletiopsis fulvescens and Tilletiaria anomala formed a separate branch, while the remaining taxa of the Exobasidiomycetes formed an unsupported (0.6 post probability) group in the Bayesian tree (Fig. 2d) and two separate groups in the ML tree (Fig. 2e). In the MP tree (Fig. 2f), two Exobasidium species formed a separate branch from the other Exobasidiomycetes taxa, which in turn formed an unsupported group.

Table 3.

Statistical support values for the major clades resolved in Ustilaginomycotina.

Clade Three rRNA genes
Three protein genes
Combined six genes
PP MLBP MPBP PP MLBP MPBP PP MLBP MPBP
Exobasidiomycetes 1.0 76 < 50 nm nm nm 1.0 51 < 50
Malassezia 1.0 100 100 1.0 100 100 1.0 100 100
Moniliella 1.0 100 100 1.0 100 100 1.0 100 100
Ustilaginomycetes 1.0 51 65 1.0 92 98 1.0 99 99
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MLBP = bootstrap percentage from maximum likelihood analysis.

MPBP = bootstrap percentage from maximum parsimony analysis.

PP = posterior probability from Bayesian analysis.

Nm = not monophyletic.

Fig. 1.

Fig. 1

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Phylogenetic tree constructed using Bayesian analysis of the combined sequences of 18S rDNA, 26S rDNA D1/D2 domains, ITS regions (including 5.8S rDNA), RPB1, RPB2 and EF1-α depicting the phylogenetic placements of genera Moniliella and Malassezia within the Ustilaginomycotina. Branch lengths are scaled in terms of expected numbers of nucleotide substitutions per site. Bayesian posterior probabilities and bootstrap percentages from 1 000 replicates of maximum likelihood and maximum parsimony analyses are shown respectively from left to right on the deep and major branches resolved.

Fig. 2.

Fig. 2

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Phylogenetic trees based on datasets of rRNA genes including 18S, D1/D2 domains of 26S and ITS-5.8S (a, b and c); protein genes including RPB1, RPB2 and EF1-α (d, e and f) and the combined six genes (g, h and i) using Bayesian (a, d and g), maximum likelihood (b, e and h) and maximum parsimony (c, f and i) analyses, showing the major clades resolved within the Ustilaginomycotina. Bayesian posterior probabilities above 0.7 or bootstrap percentages over 50 % from 1 000 replicates are shown.

The phylogenetic relationships among the Exobasidiomycetes, Malassezia, Moniliella and Ustilaginomycetes clades were clearly resolved by Bayesian analysis of the six-gene dataset, showing that the Exobasidiomycetes clade was located basal to the other three clades; Malassezia and Moniliella were sister clades and Ustilaginomycetes was basal to them. Their relationships were all strongly supported by 1.0 post probability (Fig. 1, 2g). However, the relationships among the four clades within Ustilaginomycotina were largely unresolved in the other trees (Fig. 2).

DISCUSSION

Basidiomycetous species in the subphylum Ustilaginomycotina are usually dimorphic, producing a saprobic haploid yeast phase and a parasitic dikaryotic hyphal phase. A considerable number of cultivable ustilaginomycetous fungi are only known by asexual yeasts and yeast-like microbes and are classified mainly based on physiological, biochemical and molecular criteria commonly used for yeasts, forming a taxonomic system hitherto independent from that of filamentous fungi (Boekhout 1991, Boekhout et al. 2011). Molecular methods recently showed the affiliation of these yeasts with various filamentous smuts and merged the two groups into a unified taxonomic system (Bauer et al. 2001, Begerow et al. 2000, 2006, Weiß et al. 2004, Matheny et al. 2006, Boekhout et al. 2011). However, the fine phylogenetic positions of the yeast and yeast-like groups have been a matter of debate. Here we show that the Malassezia and Moniliella clades present two deeply rooted lineages within the smut fungi (Ustilaginomycotina, Basidiomycota, Fungi). They also possess unique phenotypic (morphological, ultrastructural, physiological and biochemical) characters distinct from those of Ustilaginomycetes and Exobasidiomycetes.

The genus Moniliella was originally classified in the order Moniliales of fungi imperfecti (Stolk & Dakin 1966). A sexual morph has not been observed (de Hoog 1979a) and all members of the genus have greyish to olivaceous black colonies, yeast-like growth, hyphae that disarticulate in arthroconidia (Martinez et al. 1979, de Hoog 1979b), and CoQ-9 as the major ubiquinone (de Hoog et al. 2011). All species, except M. fonsecae, can ferment glucose and some species also galactose, sucrose and/or raffinose (Martínez et al. 1979, de Hoog et al. 2011), which among Basidiomycota is a rare trait. Some species are considered xerophilic (Hocking & Pitt 1981, de Hoog et al. 2011) and most are known from industrial settings, food stuffs, fats, oils and acids or substrates with low water activity or were isolated from flowers in tropical forest ecosystems (de Hoog et al. 2011). Thus their origin and ecology also differs from the vast majority of Ustilaginomycotina that typically are plant pathogens (Begerow et al. 2006). Glucose, galactose and mannose (low) were found to be present in the cell walls of Moniliella species, whereas xylose and fucose were absent (Weijman 1979), which led this author to conclude that the genus may not be related to the Agaricomycotina. Ustilaginomycotina, as analysed thus far, have glucose as the dominant cell wall sugar, with some galactose and mannose, but without xylose (Dörfler 1990, Boekhout et al. 1992, Bauer et al. 1997, 2001, Prillinger et al. 2011, van der Klei et al. 2011), thus agreeing with the cell wall composition as known from Moniliella species.

The septal ultrastructure of the Moniliella species as investigated so far revealed a rather complex pattern with dolipores in M. suaveolens, micro-pore-like structures in M. spathulata and both types occurring in M. oedocephala. The dolipores resemble those of Agaricomycetes but with an arch of endoplasmic reticulum instead of parenthesomes (Haskins 1975, Martinez 1979, Bauer et al. 2006, van Driel et al. 2009). These differences in pore structures may be growth stage dependent, and differ between yeast-like, hyphal and arthroconidial stages, but they may also be caused by the fixation protocols used. The dolipore structure is quite different from the simple pore with membrane caps observed in the Ustilaginomycotina and the simple pore without membrane apparatus typical for the Pucciniomycotina (Boekhout et al. 1992, Bauer et al. 1997, 2001, 2006, van der Klei et al. 2011). The biochemical and ultrastructural characters made the taxonomic and phylogenetic positions of Moniliella elusive for a long time. However, our results clearly show that the Moniliella species belong to the Ustilaginomycotina with strong support (Fig. 1, 2).

Our analyses showed that the species of Malassezia formed a highly supported deep lineage in the Ustilaginomycotina that we rank at the class level. This is further sustained by the unique monopolar budding, the thick helicoidal cell wall, the lipid dependency of most Malassezia species and the lipophily of M. pachydermatis (Guého-Kellermann et al. 2011). Malassezia species occur commonly on human and animal skin, but metagenomic data also revealed the presence of the species in some unexpected habits, such as forest soil, nematodes or corals (Guého-Kellermann et al. 2010, Gaitanis et al. 2012). The genome comparison between Ustilago maydis and M. globosa revealed some major differences in the enzyme armamentarium used by human inhabiting Malassezia yeasts that differs from that of the plant pathogen U. maydis (Xu et al. 2007, Sun et al. 2013).

Our analyses of the three rRNA genes and the combined six genes indicated that Exobasidiomycetes seem to be monophyletic when the Malassezia clade is excluded, but it remains polyphyletic in the analysis of the three protein-coding genes. Begerow et al. (1997, 2000) and Bauer et al. (2001) showed that the Exobasidiomycetes were weakly supported or with 56–85 % bootstrap support values based on the LSU rDNA analysis. Begerow et al. (2006) indicated that Exobasidiomycetes were paraphyletic using a combined analysis of SSU, D1/D2, ITS, atp6 and β-tubulin sequences, but this lineage was found to be monophyletic when the SSU data were excluded, but with weak bootstrap support. Matheny et al. (2006) indicated that the taxa within Exobasidiomycetes except the Malassezia clustered together with or without statistical support based on the different gene combination datasets. Thus, for a better understanding of the phylogeny of Exobasidiomycetes further analyses using more molecular data or genomic data are needed.

In addition to the Clustal X, we also used the MAFFT program (Katoh & Standley 2013) to align the sequences and the Gblocks program (Talavera & Castresana 2007) to remove ambiguously aligned blocks from the alignments. The alignments produced by the MAFFT and those treated by the Gblocks were all subjected to Bayesian, ML and MP analyses. The consensus was that the Malassezia, Moniliella and Ustilaginomycetes taxa were respectively resolved as strongly supported monophyletic clades in all the trees obtained, while the Exobasidiomycetes was monophyletic without statistic support in the trees drawn from the six-gene dataset but polyphyletic in the trees drawn from the rRNA gene and the protein gene datasets (data not shown). The polyphyletic nature of the Exobasidiomycetes was magnified by using the new programs, implying that this group may not represent a single class.

In conclusion, multiple gene phylogenetic analyses and phenotypic comparisons suggest that the species of Malassezia and Moniliella form two independent deep lineages representing sister groups to the recognised classes Ustilaginomycetes and Exobasidiomycetes within Ustilaginomycotina. Therefore, we propose two new classes to accommodate these fungi.

TAXONOMY

Monilielliomycetes Q.M. Wang, F.Y. Bai & Boekhout, class. nov. — MycoBank MB805229

Type order. Moniliellales.

Etymology. The nomenclature of the class is derived from the generic name of Moniliella Stolk & Dakin, Antonie van Leeuwenhoek 32: 399. 1966.

Member of Ustilaginomycotina. Sexual morph unknown. Colonies are smooth or velvety, greyish to olivaceous black. Budding cells are ellipsoidal and form terminally on true hyphae that disarticulate with artroconidia. Pseudohyphae and chlamydospores may be present. Cell walls are multi-lamellar. Hyphal septa typically possess dolipores with an arch of endoplasmic reticulum, but ‘micropore’-like structures may also be present. Sugars are fermented by most species. Nitrate is assimilated. Urease and diazonium blue B (DBB) reactions are positive. Coenzyme Q-9 is present. Xylose and fucose are absent from whole-cell hydrolysates.

Moniliellales Q.M. Wang, F.Y. Bai & Boekhout, ord. nov. — MycoBank MB805230

Type family. Moniliellaceae.

The diagnosis of the order Moniliellales is based on the description of the class Monilielliomycetes. The nomenclature of the order is based on the genus Moniliella Stolk & Dakin, Antonie van Leeuwenhoek 32: 399. 1966.

Moniliellaceae Q.M. Wang, F.Y. Bai & Boekhout, fam. nov. — MycoBank MB805231

Type genus. Moniliella Stolk & Dakin, Antonie van Leeuwenhoek 32: 399. 1966.

The diagnosis of the family Moniliellaceae is based on the description of the order Moniliellales. The nomenclature of the family is based on the genus Moniliella Stolk & Dakin, Antonie van Leeuwenhoek 32: 399. 1966.

Malasseziomycetes Boekhout, Q.M. Wang & F.Y. Bai, class. nov. — MycoBank MB805514

Type order. Malasseziales R.T. Moore with family Malasseziaceae Denchev & R.T. Moore, Mycotaxon 110: 379. 2009, and genus Malassezia Baillon (1889).

Etymology. The nomenclature of the class is derived from the order name of Malasseziales R.T. Moore, Bot. Mar. 23: 371. 1980, emend. Begerow et al., Mycol. Res. 104: 59. 2000.

Member of Ustilaginomycotina. Sexual morph unknown. Cells are globose, ovoid or cylindrical. Budding is typically monopolar on a more or less broad base, enteroblastic and percurrent. The cell wall is multi-lamellate, and the inner layer of the cell wall is corrugated with a groove spiralling from the bud site. The species are lipid dependent or lipophilic. Sugars are not fermented. Urease and diazonium blue B (DBB) reactions are positive. Coenzyme Q-9 is formed. Xylose is absent in whole-cell hydrolysates.

Acknowledgments

This study was supported by grants No. 31010103902 and No. 30970013 from the National Natural Science Foundation of China (NSFC), KSCX2-YW-Z-0936 from the Knowledge Innovation Program of the Chinese Academy of Sciences and grant No. 10CDP019 from the Royal Netherlands Academy of Arts and Sciences (KNAW). TB is supported by grant NPRP 5-298-3-086 of Qatar Foundation. The authors are solely responsible for the content of this manuscript.

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