Antonie van Leeuwenhoek (2014) 105:933–942 DOI 10.1007/s10482-014-0149-9

ORIGINAL PAPER

Three new anascosporic genera of the Saccharomycotina: Danielozyma gen. nov., Deakozyma gen. nov. and Middelhovenomyces gen. nov Cletus P. Kurtzman • Christie J. Robnett

Received: 9 January 2014 / Accepted: 2 March 2014 / Published online: 16 March 2014  Springer International Publishing Switzerland (outside the USA) 2014

Abstract Three new non-ascosporic, ascomycetous yeast genera are proposed based on their isolation from currently described species and genera. Phylogenetic placement of the genera was determined from analysis of nuclear gene sequences for D1/D2 large subunit rRNA, small subunit rRNA, translation elongation factor-1a and RNA polymerase II, subunits B1 and B2. The new taxa are: Deakozyma gen. nov., type species Deakozyma indianensis sp. nov. (type strain NRRL YB-1937, CBS 12903); Danielozyma gen. nov., type species Danielozyma ontarioensis comb. nov. (type strain NRRL YB-1246, CBS 8502); D. litseae comb. nov. (type strain NRRL YB-3246, CBS 8799); Middelhovenomyces gen. nov., type species Middelhovenomyces tepae comb. nov. (type strain NRRL Y-17670, CBS 5115) and M. petrohuensis comb. nov. (type strain NRRL Y-17663, CBS 8173). Keywords Danielozyma
Deakozyma
Middelhovenomyces
Candida
New yeast genera

C. P. Kurtzman (&)
C. J. Robnett Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604, USA e-mail: [email protected]

Introduction Circumscription of yeast genera has often been based on morphological and physiological characters that were perceived as unique. With the introduction of molecular comparisons, many genera were found to be polyphyletic and required recircumscription based on these new data, such as seen for Kluyveromyces (Kurtzman & Robnett 2003) and Pichia (Kurtzman et al. 2008). Despite uncertainties in description of ascosporic genera from phenotype, genus assignment of anascosporgenous (non-ascosporogenous) species of the Saccharomycotina has proved even more elusive. The solution has been placement of these species in the polyphyletic anamorphic genus Candida or in one of several other phenotypically circumscribed non-ascosporic genera. In the last major treatment of Candida (Lachance et al. 2011), there were nearly 400 described species, and since then, approximately 100 additional species of Candida have been proposed. Consequently, species of Candida represent nearly half of all known ascomycetous yeasts and phylogenetic analyses show these species to be distributed throughout the Saccharomycotina (e.g., Lachance et al. 2011). The newly revised Botanical Code, which was renamed the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code) (McNeill et al. 2012), now requires that fungi have only one valid name, and the expectation is that assignment of species to genera will be based on phylogeny. When

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both the sexual and the asexual states of a species are known, if they have individual names, only one of the names is now valid. Further, teleomorphic genus names do not have priority over anamorphic names. For example, the type species of the genus Candida is C. vulgaris (:C. tropicalis) and the type species for the ascosporic state of this clade is Lodderomyces elongisporus. Because clade members C. tropicalis and C. albicans are far better known than L. elongisporus, it seems likely that the genus name retained for this clade will be Candida, and Lodderomyces will become a synonym. However, most species of Candida are not members of the C. tropicalis clade and must be reassigned to other genera. For many species, such as C. thaimueangensis, which is closely related to Pichia membranifaciens (Kurtzman et al. 2008), genus placement is already recognized and only a formal transfer as a new combination is needed. However, based on current phylogenetic studies, many Candida species are not members of described clades and must be assigned to new genera. Because species of Candida span the phylogenetic breadth of the Saccharomycotina, and some represent quite divergent lineages, numerous new genera will be required for classification of these species. Presently, most yeast species have been recognized as distinct based on analysis of D1/D2 LSU rRNA gene sequences and from ITS sequences. Each of these two sequences seldom includes more than 600 nucleotides and the phylogenetic resolution from the sequences is often inadequate for assignment of species to genera. Consequently, multigene analyses are usually required to recognize clades for circumscription of new genera. Where species placement is based on multigene datasets and the species represent well isolated monophyletic lineages, it seems appropriate to describe new genera. An opposing argument might be that because there are often no defining phenotypic characters, the only means for recognizing species assigned to these new genera is through multigene sequencing. While correct, this requirement also now applies to many well recognized ascosporic genera, most of which have been recircumscribed from gene sequence comparisons (Kurtzman 2011). In the present study, two non-ascosporic strains representing a new species were recognized from multigene sequence analysis, but this species is not a member of presently recognized genera or the C. tropicalis clade of Candida. In view of the genetic

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isolation of this species, we are proposing that it be assigned to a new genus. In the course of our analyses, two additional clades of species, which are now assigned to Candida, were recognized as isolated lineages unrelated to C. tropicalis or to any described genera, and these two clades are also being proposed as new genera.

Materials and methods Phenotypic characterization of strains Species examined in this study are listed in Table 1 with GenBank accession numbers for the genes sequenced. Microscopic examination and determination of physiological reactions were by standard methods (Kurtzman et al. 2011) with growth tests conducted in liquid media and incubated for 4 weeks at 25 C. The composition of all culture media used is given in Kurtzman et al. (2011). DNA isolation and phylogenetic analysis Methods for DNA isolation and sequencing of the internal transcribed spacer (ITS) and nuclear genes for large subunit (LSU) rRNA, small subunit (SSU) rRNA, translation elongation factor-1a (EF-1a), RNA polymerase II B subunits B1 (RPB1) and B2 (RPB2) were reported earlier (Kurtzman and Robnett 1998a, 2003, 2013). Briefly, freeze-dried cells from 48-h YM agar grown cultures were broken in a Bullet Blender (Next Advance) using 0.1 mm glass beads, and the cells were then suspended in 29 CTAB buffer (1 M Tris–Cl, pH 8.4, 10 ml; 5 M NaCl, 28 ml; 0.5 M EDTA, pH 8.0, 5 ml; CTAB, 2 g; H2O to 100 ml). Beads and cell debris were removed by centrifugation and the DNA was precipitated from the supernatant. Following DNA isolation, regions for each of the genes to be sequenced were amplified by PCR and the resulting amplicons were sequenced using the ABI BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and an ABI 3730 automated DNA gene analyzer according to the manufacturer’s instructions. Gene lengths of approximately 600–1,000 nucleotides were amplified with overlap between sections and then edited and assembled as a complete sequence. Gene sequences were aligned using the program Muscle, which is included in MEGA, version 5.2

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Table 1 Strains of new species compared, reference taxa and gene sequence accession numbers Culture collection no.a

GenBank DNA sequence accession numbers

NRRL

CBS

D1/D2

SSU

EF-1a

RPB1

RPB2

Ambrosiozyma monosporaTS

Y-1484

2554

EU011590

JQ698881

JQ699031

JQ713013

JQ698945

Barnettozyma populiTS

Y-12728

8094

EF550277

JQ698877

EF552501

JQ713006

JQ698938

Candida abiesophila

Y-11514

5366

EF550212

JQ698894

JQ699051

JQ713033

JQ698965

Candida bromeliacearum

Y-27811

10002

KF964135

KF964117

KF964128

KF977586

KF977593

Candida incommunis

Y-17085

5604

KF964136

KF964118

KF964129

KF977587

KF977594

Candida sorboxylosa

Y-17669

6378

KF964137

KF964119

KF964130

KF977588

KF977595

Cephaloascus fragransTS

Y-6742

121.29

JQ689052

JQ698916

JQ699079

JQ713063

JQ698995

Clavispora lusitaniaeTS

Y-11827

6936

JQ689030

JQ698900

JQ699057

JQ713040

JQ698972

Y-2156

5644

EF550328

JQ698878

EF552552

JQ713008

JQ698940

YB-3246

8799

AF271086

KF964120

KF964131

KF977589

KF977596

Danielozyma ontarioensisTS

YB-1246

8502

AF017244

KF964121

KF964132

KF977590

KF977597

Deakozyma indianensisTS,

YB-1937

12903

KF964138

KF964122

KF964133

KF977591

KF977598

YB-3980

12904

KF964139

KF964123

KF964134

KF977592

KF977599

Species

Cyberlindnera americana

TS

Danielozyma litseae

Debaryomyces hansenii

b

TS

Y-7426

767

JQ689041

JQ698910

JQ699068

JQ713052

JQ698984

Dekkera bruxellensisTS Diddensiella santjacobensis

Y-12961 Y-17667

74 8183

JQ689028 JQ689062

JQ698898 AB018150

JQ699055 GU597343

JQ713037 JQ713074

JQ698969 JQ699006

Dipodascus albidusTS

Y-12859

766.85

GU597326

GU597336

GU597348

JQ713086

JQ699018

Hyphopichia burtoniiTS

Y-1933

2352

GU597324

JQ698903

GU597339

JQ713043

JQ698975 JQ698970

Kodamaea ohmeri

TS

Y-1932

5367

GU597323

GU597327

GU597338

JQ713038

Komagatella pastorisTS

Y-1603

704

JQ689069

JQ698928

JQ699092

JQ713084

JQ699016

Komagataella phaffii

Y-7556

2612

AF017407

EF550394

EF552480

GQ327957

KF977600

Komagataella populi

YB-455

12362

JN234404

KF964124

JN234408

JN234410

KF977601

Komagataella pseudopastoris

Y-27603

9187

AF403149

EF550393

EF552479

GQ327956

KF977602

Komagataella ulmi

YB-407

12361

KF964140

KF964125

JN234407

JN234409

KF977603

Kregervanrija fluxuum

TS

YB-4273

2287

EF550268

JQ698897

JQ699054

JQ713036

JQ698968

Kuraishia capsulataTS

Y-1842

1993

EF550270

JQ698883

JQ699033

JQ713015

JQ698947

Kurtzmaniella cleridarumTS

Y-48386

8793

JQ689038

JQ698907

JQ699065

JQ713049

JQ698981

Lodderomyces elongisporusTS

YB-4239

2605

JQ689035

JQ698906

JQ699062

JQ713046

JQ698978

Metschnikowia bicuspidataTS

YB-4993

5575

JQ689032

JQ698902

JQ699059

JQ713042

JQ698974

TS

Meyerozyma guilliermondii Middelhovenomyces petrohuensis

Y-2075 Y-17663

2030 8173

JQ689047 JQ689061

JQ698913 KF964126

JQ699074 GU597342

JQ713058 JQ713073

JQ698990 JQ699005

Middelhovenomyces tepaeTS

Y-17670

5115

JQ689063

KF964127

JQ699087

JQ713075

JQ699007

Millerozyma farinosaTS

Y-7553

185

JQ689046

AB054281

JQ699073

JQ713057

JQ698989

Nakazawaea holstiiTS

Y-2155

4140

JQ689055

JQ698919

JQ699082

JQ713066

JQ698998

Ogataea minuta

TS

Y-411

1708

EU011618

JQ698880

JQ699030

JQ713012

JQ698944

Pachysolen tannophilusTS

Y-2460

4044

JQ689056

JQ698920

JQ699083

JQ713067

JQ698999

Peterozyma toletanaTS

YB-4247

2504

JQ689057

JQ698921

JQ699084

JQ713068

JQ699000

Phaffomyces opuntiaeTS

Y-11707

7010

JQ689068

JQ698927

JQ699091

JQ713083

JQ699015

Pichia membranifaciensTS

Y-2026

107

EF550227

JQ698896

JQ699053

JQ713035

JQ698967

Priceomyces haplophilusTS

Y-7860

2028

JQ689039

JQ698908

JQ699066

JQ713050

JQ698982

Saccharomycopsis capsularis

TS

Y-17639

2519

JQ689010

JQ698884

JQ699034

JQ713016

JQ698948

Saturnispora disporaTS

Y-1447

794

JQ689027

JQ698895

JQ699052

JQ713034

JQ698966

Scheffersomyces stipitisTS

Y-7124

5773

JQ689044

JQ698912

JQ699071

JQ713055

JQ698987

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Table 1 continued Culture collection no.a

GenBank DNA sequence accession numbers

NRRL

CBS

D1/D2

SSU

Schizosaccharomyces pombeTS

Y-12796

356

JQ689077

JQ698936

EF552572

JQ713095

D13337

Spathaspora passalidarumTS

Y-27907

10155

JQ689036

DQ232894

JQ699063

JQ713047

JQ698979

Spencermartinsiella europaeaTS

Y-48265

11730

GU597325

GU597333

GU597345

JQ713076

JQ699008

TS

JQ699001

Species

Sporopachydermia lactativora

EF-1a

RPB1

RPB2

Y-11591

6192

JQ689058

JQ698922

JQ699085

JQ713069

Starmerella bombicolaTS

Y-17069

6009

JQ689065

JQ698924

JQ699088

JQ713080

JQ699012

Sugiyamaella smithiaeTS

Y17850

7522.2

DQ438218

GU597334

GU597346

JQ713078

JQ699010

Trichomonascus petasosporus

YB-2092

9602

JQ689064

GU597332

GU597344

JQ713077

JQ699009

Trigonopsis variabilisTS

Y-1579

1040

JQ689074

JQ698933

JQ699097

JQ713091

JQ699023

Wickerhamiella domercqiae

TS

Y-6692

4351

DQ438240

GU597335

GU597347

JQ713079

JQ699011

Wickerhamomyces canadensisTS

Y-1888

1992

JQ689007

EF550438

EF552524

JQ713007

JQ698939

Yamadazyma philogaeaTS

Y-7813

6696

JQ689048

JQ698914

JQ699075

JQ713059

JQ698991

Yarrowia lipolyticaTS

YB-423

6124

JQ689067

JQ698926

JQ699090

JQ713082

JQ699014

Zygoascus hellenicusTS

Y-7136

5839

JQ689060

GU597328

GU597340

JQ713071

JQ699003

TS type species of the genus a

NRRL, ARS Culture Collection, National Center for Agricultural Utilization Research, Peoria, IL, USA; CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands

b GenBank accession numbers of ITS sequences for Danielozyma litseae: NRRL YB-1937 = KJ476205, NRRL YB3980 = KJ476206

(Tamura et al. 2011), and the alignments were visually adjusted. Regions of uncertain alignment were removed for some analyses and included much of the D2 domain of LSU and small sections of SSU and RPB2. Each of the five gene sequences was analyzed individually and in various combinations using the maximum likelihood, maximum parsimony and neighbor-joining programs included in MEGA ver. 5.2. Bootstrap support was determined from 1,000 replicates.

Wickerhamiella (Fig. 1), which were not included in the previous analysis. The conflict in placement may also be influenced by large deletions in the LSU and SSU rRNA genes that are common to NRRL YB-1937 and species of Komagataella. Candida incommunis, C. bromeliacearum and C. sorboxylosa have similar deletions and also appear related to Komagataella from D1/D2 sequence analysis (Fig. 90.3, Lachance et al. 2011). For this reason, these three species were included in the present study. Analysis of individual genes (D1/D2, SSU, EF-1a, RPB1, RPB2), as well as datasets that included only the rRNA genes and the

Results and discussion Three isolated clades of non-ascosporic, ascomycetous yeasts are proposed for placement in three new genera: 1. NRRL YB-1937 and NRRL YB-3980, 2. C. litseae NRRL YB-3246 and C. ontarioensis NRRL YB-1246, 3. C. petrohuensis NRRL Y-17663 and C. tepae NRRL Y-17670. Strain NRRL YB-1937 was recognized as a new species from multigene sequence analysis (Kurtzman and Robnett 2010) in which it was placed near the genus Komagataella. In the present study, the species is near the genera Yarrowia, Starmerella and

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Fig. 1 Phylogenetic placement of the genera Danielozyma, c Deakozyma and Middelhovenomyces among representative genera of the Saccharomycotina as determined from maximum likelihood analysis of concatenated gene sequences from D1/D1 LSU rRNA, SSU rRNA, EF-1a, RPB1 and RPB2. Schizosaccharomyces pombe was the designated outgroup species in the analysis. Bootstrap values (1,000 replicates) [50 % are given at branch nodes, and strain accession numbers are NRRL. The ML analysis is based on the general time reversible model (GTR ? G ? I) (Nei and Kumar 2000) and the tree with the highest log likelihood (-139,462.9083) is shown. All positions with less than 95 % site coverage were eliminated resulting in a total of 6,259 positions in the final dataset. Scale = substitutions per site

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937 100

Candida abiesophila Y-11514 Saturnispora dispora Y-1447 Kregervanrija fluxuum YB-4273 Pichia membranifaciens Y-2026 95 Candida sorboxylosa Y-17669 90 88 Ambrosiozyma monospora Y-1484 Ogataea minuta Y-411 98 Dekkera bruxellensis Y-12961 66 Kuraishia capsulata Y-1842 99 100 Komagataella pastoris Y-1603 100 Komagataella ulmi YB-407 Komagataella phaffii Y-7556 Komagataella populi YB-455 100 100 Komagataella pseudopastoris Y-27603 98 Cyberlindnera americana Y-2156 Barnettozyma populi Y-12728 100 100 100 Phaffomyces opuntiae Y-11707 Starmera amethionina Y-10978 100 Wickerhamomyces canadensis Y-1888 99 Nakazawaea holstii Y-2155 100 Pachysolen tannophilus Y-2460 Peterozyma toletana YB-4247 Saccharomycopsis capsularis Y-17639 99 Metschnikowia bicuspidata YB-4993 61 71 Candida bromeliacearum Y-27811 Clavispora lusitaniae Y-11827 99 Kodamaea ohmeri Y-1932 Danielozyma ontarioensis YB-1246 74 Danielozyma litseae YB-3246 98 Scheffersomyces stipitis Y-7124 69 77 Priceomyces haplophila Y-7860 Millerozyma farinosa Y-7553 Meyerozyma guilliermondii Y-2075 57 Yamadazyma philogaea Y-7813 Lodderomyces elongisporus YB-4239 100 93 Hyphopichia burtonii Y-1933 98 Kurtzmaniella cleridarum Y-48386 Debaryomyces hansenii Y-7426 80 78 Spathaspora passalidarum Y-27907 Cephaloascus fragrans Y-6742 Sporopachydermia lactativora Y-11591 Dipodascus albidus Y-12859 91 Sugiyamaella smithiae Y-17850 76 Spencermartinsiella europaea Y-48265 76 Diddensiella santjacobensis Y-17667 Middelhovenomyces tepae Y-17670 65 Middelhovenomyces petrohuensis Y-17663 100 Zygoascus hellenicus Y-7136 100 Wickerhamiella domercqiae Y-6692 99 Starmerella bombicola Y-17069 78 Yarrowia lipolytica YB-423 Candida incommunis Y-17085 Deakozyma indianensis YB-3980 100 50 100 Deakozyma indianensis YB-1937 Trichomonascus petasosporus YB-2092 Trigonopsis variabilis Y-1579 Schizosaccharomyces pombe Y-12796 100 100

0.1

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protein coding genes, showed that NRRL YB-1937, NRRL YB-3980, C. incommunis, C. bromeliacearum and C. sorboxylosa are not associated with Komagataella. Consequently, earlier analyses were apparently influenced by deletions in gene sequences and the species composition of the dataset. Candida incommunis, C. abiesophila and C. sorboxylosa appear to represent isolated lineages in the present five-gene analysis (Fig. 1), and as later discussed, these three taxa will be considered for future placement in three new genera. The placement of C. bromeliacearum will require analysis with all described species of Clavispora and Metschnikowia. The remaining two clades considered in this study, C. litseae/C. ontarioensis and C. petrohuensis/C. tepae, are shown in Fig. 1 to be well separated from other genera (clades), and the genetic isolation of these two lineages was also demonstrated in an earlier multigene study that included all known species of the genera neighboring these two clades (Kurtzman and Robnett 2007). Species placements were essentially identical whether the sequences were analyzed by maximum likelihood, maximum parsimony or neighbor-joining. In view of these results, we propose the following three new genera and one new species.

Deakozyma Kurtzman & Robnett gen. nov.

Fig. 2 Deakozyma indianensis NRRL YB-1937. a Budding cells and a pseudohypha, 3 days, YM agar, 25 C, b true hyphae with constricted septa, 7 days, yeast morphology agar, 25 C. Bar = 10 lm for both figures

Description of the genus Growth is by multilateral budding and from formation of pseudohyphae and true hyphae. An ascosporic state is unknown. Fermentation of sugars is absent. Growth occurs with many common pentoses, hexoses, disaccharides and sugar alcohols but is absent with methanol and hexadecane. Nitrate may be utilized as a sole source of nitrogen. The genus can be resolved from other members of the Saccharomycotina by gene sequence analysis. Phylogenetic placement: Saccharomycetales, Saccharomycotina, Ascomycota. From the analysis presented in Fig. 1, the genus Deakozyma is most closely related to Yarrowia, Wickerhamiella, Starmerella and Trichomonascus. Etymology: The genus is named in honor of the late Professor Tibor Dea´k, Department of Microbiology and Biotechnology, Corvinus University of Budapest, Hungary, for his outstanding studies of the yeast

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microbiota of foods and strategies for controlling food spoilage by yeasts (e.g., Dea´k 2007). Type species: Deakozyma indianensis Kurtzman & Robnett. MycoBank number: MB 807335. Deakozyma indianensis Kurtzman & Robnett sp. nov. Description of the species (based on NRRL YB-1937 and NRRL YB-3980) On YM agar after 3 days at 25 C, cells are ellipsoidal to elongate (2–4.5 9 3–12 lm), divide by multilateral budding and occur singly, in pairs and in short chains (Fig. 2a). Colony growth is dull, white, partially butyrous, not raised and with occasional outgrowths of hyphae. After 7 days at 25 C, pseudohyphae and

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true hyphae form under the coverglass of a Dalmau plate with yeast morphology agar. True hyphae are somewhat constricted at the septa (Fig. 2b). Aerobic growth on morphology agar is dull, white, and the surface is highly convoluted with ridges of growth. Margins are entire.

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Etymology: the species name indianensis denotes that the type strain was isolated from a substrate collected in the State of Indiana, USA. MycoBank number: MB 807336.

Danielozyma Kurtzman & Robnett gen. nov. Examination for ascosporulation NRRL YB-1937 and NRRL YB-3980 were grown alone and as a mixture on YM, 5 % malt extract and RG agar media at 17 and 25 C. The cultures were examined at approximately weekly intervals for 2 months, but neither conjugation nor ascospore formation was observed. Fermentation was absent for glucose, galactose, maltose, sucrose, lactose, raffinose and trehalose. Growth occurred on the following carbon sources: glucose, sucrose, galactose, trehalose, maltose, melezitose, methyl-a-D-glucoside, cellobiose, L-sorbose, D-xylose, D-arabinose, ethanol, glycerol, ribitol, Dmannitol, D-glucitol, myo-inositol, DL-lactate, succinate, citrate, D-gluconate, D-glucosamine, N-acetyl-Dglucosamine, 2-keto-D-gluconate and 5-keto-D-gluconate. Carbon sources not supporting growth were inulin, raffinose, melibiose, lactose, soluble starch, salicin, L-rhamnose, L-arabinose, D-ribose, methanol, erythritol, galactitol, hexadecane and saccharate. Nitrate and cadaverine supported growth, and growth was present in media with 0.1 % cycloheximide. Growth was absent in vitamin-free medium, in 10 % NaCl/5 % glucose and at 37 C. Gelatin was not hydrolyzed and extracellular starch-like compounds were not formed. Type strain: NRRL YB-1937 is preserved as a lyophilized preparation in the ARS Culture Collection, National Center for Agricultural Utilization Research, Peoria, IL, USA, and as CBS 12903, with the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands. NRRL YB-1937 was isolated by L. J. Wickerham from the eggs of an unidentified insect, McCormick’s Creek State Park, Spencer, Indiana, USA. A second strain, NRRL YB-3980 (CBS 12904), also isolated by L. J. Wickerham, was from the rotting log of an unidentified tree species, Gull Lake, Michigan, USA. Nucleotide differences between the two strains: D1/D2 LSU rRNA = 0, SSU rRNA = 0, EF1a = 1, RPB1 = 0, RPB2 = 6, ITS = 0. GenBank accession numbers for sequences from the two strains are given in Table 1.

Description of the genus Growth is by multilateral budding and from formation of pseudohyphae and true hyphae. Blastoconidia may develop on pseudohyphae as well as on true hyphae, often on small denticles. Hyphae may form endoconidia. An ascosporic state is unknown. Sugars are fermented. Growth occurs with many common pentoses, hexoses, disaccharides, sugar alcohols and hexadecane but not on methanol. Nitrate is not utilized as a sole source of nitrogen. The genus can be separated from other members of the Saccharomycotina by gene sequence analysis. Phylogenetic placement: Saccharomycetales, Saccharomycotina, Ascomycota. The genus Danielozyma appears most closely related to Kodamaea (Fig. 1). In an earlier study, D. ontarioensis and D. litseae were weakly associated with Sporopachydermia (Kurtzman & Robnett 2007). Etymology: The genus is named in honor of Dr. Heide-Marie Daniel, Earth and Life Institute, Mycology, Mycothe`que de l’Universite´ catholique de Louvain, Belgium, for innovative molecular taxonomic studies of yeasts (e.g., Daniel and Meyer 2003). Type species: Danielozyma ontarioensis (Kurtzman & Robnett) Kurtzman & Robnett. MycoBank number: MB 807332. Danielozyma ontarioensis (Kurtzman & Robnett) Kurtzman & Robnett comb. nov. Basionym: Candida ontarioensis Kurtzman & Robnett (1998b). Can. J. Microbiol. 44:966. Type strain: NRRL YB-1246 (CBS 8502). MycoBank No. MB 807333. Danielozyma litseae (Kurtzman) Kurtzman & Robnett comb. nov. Basionym: Candida litseae Kurtzman (2001). Antonie van Leeuwenhoek 79:358. The original spelling of the

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species epithet litsaeae was found to be an orthographic variant. Type strain: NRRL YB-3246 (CBS 8799). MycoBank No. MB 807334. The genus Danielozyma presently has two assigned species, D. ontarioensis and D. litseae. D. ontarioensis was circumscribed from four strains that had been isolated from the frass of insect larvae in spruce trees (Picea sp., Picea engelmannii) growing in Canada and the USA (Kurtzman and Robnett 1998b), and D. litseae is known from a single strain that had been isolated from the frass of an insect that had bored into the wood of Litsea polyantha, near New Delhi, India (Kurtzman 2001). As reported in the original descriptions, neither species formed ascospores. Middelhovenomyces Kurtzman & Robnett gen. nov. Description of the genus Growth is by multilateral budding and from formation of pseudohyphae. An ascosporic state is unknown. Fermentation of sugars is absent. Growth may occur with pentoses, hexoses, disaccharides and sugar alcohols, but reactions are often weak or slow. Growth is absent with methanol and hexadecane, and nitrate is not utilized as a sole source of nitrogen. The genus can be distinguished from other members of the Saccharomycotina by gene sequence analysis. Phylogenetic placement: Saccharomycetales, Saccharomycotina, Ascomycota. The genus Middelhovenomyces is most closely related to Diddensiella and Zygoascus (Fig. 1). Etymology: The genus Middelhovenomyces is named in honor of Dr. Wouter J. Middelhoven, Wageningen University, The Netherlands, for his studies of new yeasts and their novel metabolic properties (e.g., Middelhoven 2006; Middelhoven and Kurtzman 2003). Type species: Middelhovenomyces tepae (Grinbergs) Kurtzman & Robnett. MycoBank number: MB 807337. Middelhovenomyces tepae (Grinbergs) Kurtzman & Robnett comb. nov. Basionym: Candida tepae Grinbergs (1967). Arch. Mikrobiol. 56:202.

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Type strain: NRRL Y-17670 (CBS 5115). MycoBank No. MB 807338. Middelhovenomyces petrohuensis (C. Ramı´rez & A. Gonza´lez) Kurtzman & Robnett comb. nov. Basionym: Candida petrohuensis Ramı´rez & Gonza´lez (1984). Mycopathologia 88:99. Type strain: NRRL Y-17663 (CBS 8173). MycoBank No. MB 807339. The genus Middelhovenomyces presently has two assigned species, M. tepae and M. petrohuensis. M. tepae is known from three strains that had been isolated from rotted wood in the Valdivian forest of Chile. Two of the strains represent C. antillancae and C. bondarzewiae, which were shown to be synonyms of M. tepae from D1/D2 LSU rRNA gene sequence analysis (Kurtzman and Robnett 1998a). M. petrohuensis is also known from three strains that had been isolated from rotted wood in the Valdivian forest. Two of these strains represent C. ancudensis and C. drimydis, which were shown to be synonyms of M. petrohuensis from D1/D2 LSU rRNA gene sequence analysis (Kurtzman and Robnett 1998a). Ascosporulation was not reported in the original descriptions of M. tepae and M. petrohuensis or in descriptions of their synonyms, and examination in the present study did not detect ascospores in strains of these species. As noted earlier, Candida species that are not part of the C. tropicalis/C. albicans clade will require reclassification as specified under the new Code, and this will come about from transfers as new combinations to currently described genera and from placement in new genera when the species are not members of presently known genera. Although reassignment of species is required by the new Code, there is no specified completion date, thus allowing time for more extensive analyses of some species groups. During this interim period, new non-ascosporic species can still be placed in Candida, but with the understanding that the species will be reassigned to a phylogenetically defined genus as more robust datasets become available. A factor in the transfer of Candida species to other genera is that recognition of genera from phylogenetic analysis is not always clear and guidelines will need to be developed. Genus circumscription from molecular criteria is affected by many factors, which include the number of genes analyzed, possible differences in substitution rates for the genes under study and perhaps

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the number of species included in the analysis. Consequently, there is no simple distance formula for defining a genus, as was also found for definition of yeast (Kurtzman and Robnett 1998a) and mold species (Taylor et al. 2000). Most phylogenetically circumscribed yeast genera appear as strongly supported clades in phylogenetic trees (e.g., Kurtzman 2011). Because some genera are monotypic, such as Pachysolen and Babjeviella, a concern is whether the single species representing the genus is clearly separate from other genera. In the case of the preceding two genera, multigene sequence analysis provided strong support that these two taxa are isolated from neighboring genera (Kurtzman and Robnett 2013). The placement of C. incommunis and C. abiesophila is perhaps less clear. As seen in Fig. 1, C. incommunis may represent a monotypic sister genus of Deakozyma, and C. abiesophila probably represents a sister genus of Saturnispora, rather than being divergent members of Deakozyma and Saturnispora, respectively. Until further evidence is available to confirm isolation from their apparent sister genera, these two species can remain in the genus Candida. Another concern is whether or not closely related species have been inadvertently excluded from analysis, which may affect interpretation of genus boundaries. Because sequences from multiple genes are not available for many yeasts, an initial approach to investigate possible exclusion of species is to compare all known species from the readily available diagnostic D1/ D2 and ITS sequences. Although these sequences are usually insufficient for circumscribing genera, the analysis will allow detection of species that may be congeneric. The tentative genus assignment can then be tested from multigene analysis as was done in this study for C. incommunis, C. bromeliacearum and C. sorboxylosa, which initially appeared from D1/D2 analysis to be near the genus Komagataella. Various estimates suggest that less than 1 % of extant fungi are known (e.g., Blackwell 2011, and references therein) and absence of these species may affect genus circumscription. Using placement of the large number of new species described in the last decade as a guide, it seems just as likely that undiscovered species will either represent new genera or will be members of extant genera rather than having an intermediate placement between sister genera. From the foregoing, it is apparent that genus circumscription is an evolving concept that awaits refinement from inclusion of additional species and expanded gene sequence analyses. Nonetheless, this uncertainty

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does not preclude placement of well separated monophyletic species and groups of species in new genera. Acknowledgments We thank Nathane Orwig for sequence determinations and Todd Ward for helpful comments. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. The USDA is an equal opportunity provider and employer.

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