Meredithblackwellia eburnea gen. et sp. nov., Kriegeriaceae fam. nov ...

Mycologia, 105(2), 2013, pp. 486–495. DOI: 10.3852/12-251 # 2013 by The Mycological Society of America, Lawrence, KS 66044-8897

Meredithblackwellia eburnea gen. et sp. nov., Kriegeriaceae fam. nov. and Kriegeriales ord. nov.—toward resolving higher-level classification in Microbotryomycetes Merje Toome1,2

INTRODUCTION

Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803

The external surface of aerial plant parts (phyllosphere or phylloplane) accommodates a highly diverse microbial community. A high number of various prokaryotic and eukaryotic organisms have been found to colonize plant surfaces, showing adaption to this habitat to be a successful evolutionary trend that has taken place frequently and independently several times (Andrews and Harris 2000). Among fungi, yeasts are the most commonly isolated active phylloplane organisms; filamentous fungal species, on the other hand, are recovered more often from plant surfaces in the dormant spore stage (Nakase 2000, Whipps et al. 2008). It has been shown that many phylloplane yeasts could protect plants from pathogens and therefore promote plant growth (e.g. McCormack et al. 1994, Buck 2002); nevertheless, some of them are thought to be parasitic. Because only a few species have been studied in detail, the ecology of the majority of yeasts on plants is poorly understood and their composition may significantly vary depending on climate conditions or the surrounding microbial communities (Andrews and Harris 2000). Several studies examining the diversity of yeasts on plants have found that the most frequently recovered species are yeasts from Pucciniomycotina, mostly belonging to the genera Sporobolomyces, Rhodotorula and Bensingtonia (e.g. Nakase 2000). Most of these have ballistosporic spore discharge, a feature characteristic of many basidiomycetes (Kirk et al. 2008), including yeasts and species with yeast states in Pucciniomycotina. Historically these yeasts have been placed into form-genera based on their carbon assimilation abilities and colony pigmentation (Kurtzman et al. 2011). Recent advances in reconstructing the phylogenetic relationships of Pucciniomycotina (e.g. Aime et al. 2006) now allow the application of molecular phylogenetics to facilitate the integration of anamorphic yeasts within the teleomorph-based classification. For example, species placed in Sporobolomyces occur across most of the yeast-forming Pucciniomycotina classes and species placed in Rhodotorula can be found in Ustilaginomycotina as well as Pucciniomycotina (Sampaio 2004, Scorzetti et al. 2002). The type species for both Rhodotorula (R. glutinis (Fresen.) F.C. Harrison) and Sporobolomyces

Robert W. Roberson School of Life Sciences, Arizona State University, Tempe, Arizona 85287

M. Catherine Aime1 Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803

Abstract: A field survey of ballistosporic yeasts in a Neotropical forest yielded a new species isolated from a fern leaf. The isolate is a cream-colored butyrous yeast that reproduces by budding. Budding occurs at both the apical and basal cell poles; occasionally multiple budding events co-occur, giving rise to rosette-like clusters of cells at both poles of the yeast mother cell. DNA sequences of large and small subunit and the internal transcribed spacer regions of the nuclear ribosomal DNA cistron indicated an affinity to Microbotryomycetes, Pucciniomycotina. A new genus, Meredithblackwellia, is proposed to accommodate the new species, M. eburnea (type strain MCA4105). Based on phylogenetic analyses, Meredithblackwellia is related to Kriegeria eriophori, a sedge parasite, to an aquatic fungus Camptobasidium hydrophilum and to several recently described anamorphic yeasts that have been isolated from plant material or psychrophilic environments. Morphological and ultrastructural studies confirm the relatedness of M. eburnea to these taxa and prompted the re-evaluation of higher-level classification within Microbotryomycetes. We propose here a new order, Kriegeriales, and place two families, Kriegeriaceae fam. nov. and Camptobasidiaceae R.T. Moore, within it. Our study re-emphasizes the need for systematic revision of species described in Rhodotorula. Key words: basidiomycete yeasts, fungal taxonomy, phylloplane, simple septate basidiomycetes

Submitted 11 Jul 2012; accepted for publication 21 Sep 2012. 1 Current address: Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907. 2 Corresponding author. E-mail: [email protected]

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TOOME ET AL.: HIGHER-LEVEL CLASSIFICATION IN MICROBOTRYOMYCETES (S. salmonicolor (B. Fisch. & Brebeck) Kluyver & C.B. Niel) are now known to belong to Sporidiobolales in Microbotryomycetes (Scorzetti et al. 2002), highlighting the necessity for additional taxonomic studies that will integrate the ex-Sporobolomyces s.s. and exRhodotorula s.s. species into a natural classification. Microbotryomycetes is the second largest class in Pucciniomycotina with more than 200 described species. Microbotryomycetes primarily contains the ‘‘anther smuts’’, smut-like species formerly classified in Ustilaginomycotina, and numerous anamorphic yeasts (Bauer et al. 2006). At present, the class contains four orders: Microbotryales—predominantly teliospore-forming plant parasites; Sporidiobolales and Leucosporidiales—anamorphic or teliosporeforming yeasts isolated from various habitats and surfaces; and Heterogastridiales—hyphal fungi isolated from decaying plant material and mushrooms (Aime et al. 2006, 2012). Although there have been great improvements in circumscribing a natural Microbotryomycetes over the past decade, the placement of almost a quarter of the species within it remains unresolved. For instance, Kriegeria, Camptobasidium and Colacogloea, all monotypic genera, are shown to be members of Microbotryomycetes (Aime et al. 2006) but are still classified incertae sedis within the class as are many anamorphic species, especially those currently placed in either Rhodotorula or Leucosporidium. In this paper we describe a new yeast genus and species that was isolated from a fern phylloplane in western Guyana and provide a threelocus phylogenetic analysis of Microbotryomycetes. As a result, one new order and one new family, Kriegeriales and Kriegeriaceae, are described, and the higher-level placement of many species previously placed incertae sedis is resolved. MATERIALS AND

METHODS

Sample collection and isolation.—The yeast strain described in this study (collection No. MCA4105) was isolated from the leaf surface of an unidentified fern at a permanent base camp in the Pakaraima Mountains in western Guyana (5u18904.80N, 59u54940.40W; 710 m) on 28 May 2010. The leaf was cut into small pieces that were attached with a thin layer of petroleum jelly to the inner lid of a Petri dish containing potato dextrose agar media (PDA) with added chloramphenicol (1 mL L21), to avoid bacterial growth. Plates were monitored daily by eye for presence of colonies, which were transferred with sterile toothpicks into 2 mL microtubes containing the same media. These isolates were kept in the microtubes until transferred to laboratory conditions, where pure cultures were streaked out and stored on PDA at 4 C and as glycerol stocks at 280 C for long-term storage. The culture is deposited in Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity

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Centre, the American Type Culture Collection (ATCC) and the Agricultural Research Service Culture Collection (NRRL) under deposit Nos. CBS12589, ATCC MYA-4884 and NRRL Y-48821 respectively. Physiological and morphological characterization.—Physiological, biochemical and morphological properties of the isolate were determined following Kurtzman et al. (2011). The fermentation of glucose and the assimilation of carbon and nitrogen compounds were determined at 25 C over 4 wk. The effect of temperature was determined during 2 wk at 4, 20, 25 and 30 C on agar plates containing yeast malt agar (YMA) and cornmeal Agar (CMA) media. Morphological characters of the isolate were observed on YMA and CMA plates at 1, 2 and 3 wk after inoculations. To describe microscopic characters, the culture was incubated in YM broth and PDA media for 5 d and actively budding yeast cells were viewed with a Zeiss (Carl Zeiss, Inc., Thornwood, New York) dissecting light microscope with a 403 objective. For standard differential interference contrast (DIC) observations, cells from a 5 d old culture on PDA were directly observed. For nuclear division observations, actively growing cells from the same culture were fixed with freshly prepared 4% formaldehyde in phosphate buffered saline (PBS, 0.05 M, pH 6.8) for 30 min at room temperature, washed in PBS, and stained in 49,6-diamidino-2-phenylindole (DAPI; Sigma, St Louis, Missouri) at 0.1 mg/mL in H20 for 5 min. Cells were rinsed in H20 and mounted on glass slides in 90% glycerol—10% 0.1 M PBS (pH 8.6)—2% N-propyl gallate (Sigma) and viewed with DIC and epifluorescence optics. All images were viewed on an Axioscope microscope (Carl Zeiss, Inc.) and observed with a Plan-Neofluor 1003/1.3 (oil immersion) objective and captured with a Roper Cool SNAP ES digital camera (Roper Scientific Inc., Tucson, Arizona) with MetaMorph 6.0/6.1 software (Universal Imaging Corp., Downingtown, Pennsylvania). To describe the ultrastructure of MCA4105, cells were grown as monolayers on a thin, sterile, deionized dialysis membrane overlying PDA at 23 C. After approximately 24 h growth, cells and supporting membranes were trimmed with a razorblade to approximately 5 3 5 mm, removed from the agar surface and immediately cryofixed by plunging rapidly into liquid propane cooled to 2186 C with liquid nitrogen (Hoch 1986, Roberson and Fuller 1988). Cryofixed cells were freeze substituted in acetone containing 2% OsO4 at 285 C for 48–72 hr. While in the substitution solution, the cells were slowly warmed to room temperature, rinsed with 100% acetone and infiltrated with epoxy resin. Infiltrated cells were flat-embedded between a Teflon-coated glass slide and Teflon strips and polymerized at 60 C for 24 h. After resin polymerization, selected hyphae were sectioned on a Leica Ultracut microtome (Leica Microsystems Inc., Bannockburn, Illinois) and post stained for 5 min in 2% uranyl acetate in 50% ethanol and for 3 min in lead citrate. Sections were examined with a FEI CM12S transmission electron microscope (TEM) (FEI Electronics Instruments Co., Mahwah, New Jersey) at 80 kV coupled to a Gatan 689 CCD digital camera (1024 3 1024 pixel area; Gatan Inc., Pleasanton, California). For all imaging methods, final

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figures were constructed with Adobe Photoshop 7.0 (Adobe Systems Inc., San Jose, California). DNA extraction, sequencing and phylogenetic analysis.—DNA was extracted from colonies actively growing on PDA with Promega Wizard Genomic DNA Purification Kit (Promega, Madison, Wisconsin). PCR reactions were carried out in 25 mL reactions that contained 12.5 mL GoTaq Master Mix (Promega), 1.25 mL each primer (10 mM), 9 mL molecular grade water and 1 mL DNA template. Amplifications of the internal transcribed spacer (ITS) region, large (LSU) and small (SSU) subunit of the nuclear ribosomal DNA (rDNA) were conducted with primer pairs ITS1F (Gardes and Bruns 1993)/ITS4 (White et al. 1990), 5.8SR/LR6 (Vilgalys and Hester 1990) and NS1/NS4 and NS3/NS8 (White et al. 1990) respectively. Amplification conditions for the ITS region were 5 min at 95 C followed by 35 cycles of 30 s at 94 C, 45 s at 45 C and 45 s at 72 C, ended with a 7 min extension at 72 C. The same PCR conditions were used for LSU and SSU, except that for both the elongation step was extended to 1 min and for SSU annealing was conducted at 55 C for 30 s. Sequencing of amplified fragments was performed by Beckman Coulter, Inc. (Danvers, Massachusetts) with the same primers that were used for amplification. The SSU, LSU and ITS sequences of MCA4105 are deposited in GenBank under accession numbers JX508797, JX508798 and JX508799 respectively. Sequences were edited with Sequencher 4.10.1 (Gene Codes Corp.) and blasted in GenBank (www.ncbi.nlm.nih. gov) with BLASTn for initial identification, which indicated that the isolate belonged to Pucciniomycotina. Thereafter the LSU sequence was analyzed within the dataset of Aime et al. (2006), which supported placement within Microbotryomycetes. New datasets were constructed for each sequenced locus by aligning the closest available blast matches with at least 94% identity and a selection of other members of Microbotryomycetes. To determine the phylogenetic position within the Microbotryomycetes, the first dataset combined all three loci for 16 Microbotryomycetes species, including exemplars from each described order and Mixia osmundae as an outgroup, because this is one potential sister group to Microbotryomycetes in prior studies (Aime et al. 2006). A second dataset was created for family positioning containing LSU and ITS sequences of 18 taxa found to be the most closely related to MCA4105 by BLASTn and prior phylogenetic analyses. SSU was not included in this dataset due to lack of available sequences for selected taxa. Leucosporidium scottii was included to test monophyly of Kriegeriales, and Microbotryum violaceum was chosen as an outgroup based on the results from analysis of dataset 1. Sequences were aligned with the MUSCLE algorithm in MEGA 5.0 (Kumar et al. 2008). In dataset 1, the sequences of LSU, ITS and SSU were aligned along 587, 533 and 1349 base pairs respectively. For dataset 2, ambiguously aligned regions were excluded from analyses and therefore LSU and ITS sequences were aligned along 453 and 404 base pairs respectively. The alignments are available in TreeBASE (treebase.org) under submission number 13340. Details of sequences used for these analyses are presented (SUPPLEMENTARY TABLE I).

The maximum likelihood (ML) analyses were conducted in RAxML-HPC2 7.2.8 via the CIPRES Science Gateway (Miller et al. 2010) using the 2k option for bootstrap analysis. Bayesian posterior probability analyses were conducted in MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) with parameters set to 5 000 000 generations, four runs and four chains. The chains were heated to 0.25 and a stop value of 0.01 was used. Maximum parsimony (MP) analyses were conducted in PAUP* 4.0b10 (Swofford 2002) where the support of the branching topologies derived from 1000 replicates with 10 random additions.

RESULTS The isolated yeast is fast-growing, forming white butyrous colonies on agar media (FIG. 1a). Microscopic studies showed that MCA4105 reproduces by budding, which occurs on a narrow base from each pole, although occasionally lateral budding was observed (FIG. 1b, h). Cells produce up to three buds at the same time either on the same or opposite apex and multiple budding events give rise to rosette-like clusters (FIG. 1c–e). Ultrastructural and nuclear division studies showed that during budding the nucleus moves into the forming bud (FIG. 1f–i), and during mitosis one spindle pole remains in the bud while the other one migrates back into the mother cell (FIG. 1j, k). Microscopy studies also showed that MCA4105 cells contain numerous vacuoles and lipid bodies (FIGS. 1b, c, 2a). Additionally, ultrastructure studies revealed subgloboid spindle pole bodies with an internal flattened layer and numerous mitochondria (FIG. 2a–c). In actively budding cells, increased Golgi complexes and numerous vesicles were found in the immediate proximity of the tip of the bud, and newly forming buds were surrounded with a thick layer of extracellular matrix (FIG. 2e, f). Budding is enteroblastic, and after the bud matures a new membrane and cell wall form to close the budding site (FIG. 2g–i). The LSU sequence of MCA4105 shares 97% identity (1035 of 1066 bp) with Rhodotorula sp. FK.2.1. (GenBank No. FN400943; unpubl), isolated from the nectar of Viola sp. in Germany, and Rhodotorula sp. KBP 3844 (No. FN400942; unpubl), isolated from a lichen on Picea abies in Russia. The SSU region shared highest identity (99%; 1558 of 1571 bp) with the plant parasite Kriegeria eriophori (DQ419918; Kumar et al. 2007) and 98% identity (1542 of 1571 bp) with Glaciozyma antarctica (DQ785788; Matheny et al. 2006) and Leucosporidium sp. AY30 (GQ336996; unpubl), the latter two isolated from ice and water. The closest identities in the ITS region were with uncultured Rhodotorula isolates (GU931760, GU931719; Amend et al. 2010) from house dust in Canada that shared 84% identity (545 and 541 of 652 bp respectively), and Rhodotorula sp.

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FIG. 1. Morphological characteristics of Meredithblackwellia eburnea (HOLOTYPE, MCA4105). a. 7 d old culture on YMA media, showing the smooth butyrous culture with entire margins and single colonies with elevated center. Bar 5 1 cm. b. Apically budding yeast cells and non-budding cells with vacuoles (asterisk) and lipid bodies (white arrow). Bar 5 10 mm. c. Numerous cells forming a rosette. Vacuoles are noted (asterisks). Bar 5 10 mm. d. Apical budding (arrowheads) occurring simultaneously from both poles. Bar 5 5 mm. e. Three buds forming from the same site (arrowhead). Bar 5 5 mm. f–k. Nuclear division observed in the cells using epifluorescence microscopy and associated DIC images. Note the nucleus moving into the bud (h, i) and then back to the mother cell (j, k). Bar 5 10 mm.

USM-PSY62 (HM545717; unpubl) from Antarctic sea with 83% identity (540 of 652 bp). Phylogenetic analyses place MCA4105 with K. eriophori in a lineage that is distinct from the other described orders of Microbotryomycetes (FIG. 3A) and sister to Camptobasidiaceae (FIG. 3B). MCA4105 forms a distinct lineage, sister to Rhodotorula rosulata (FIG. 3B) and a new genus and species are erected to accommodate this isolate. TAXONOMY Kriegeriales Toome & Aime, ord. nov. MycoBank MB801220 Etymology: The name of the order is derived from the genus Kriegeria Bres. (Bresadola 1891), which contains the earliest described known member.

Diagnosis: Members of Pucciniomycotina, Microbotryomycetes, with simple pore septa and subgloboid spindle pole bodies. Hyphae, when present, may have clamp connections. Asexual reproduction occurs mostly by polar budding or, in one species, by production of aquatically adapted tetraradiate spores. Teleomorph variable where known either aquatic or plant parasitic and sorus-forming. Basidiospores are sessile or singly formed on sterigmata, on transversely septate basidia. Known members of the Kriegeriales are plant parasites, aquatic fungi or saprobes from cold, temperate and tropical environments. The majority

of members are known as anamorphic non-pigmented yeasts; a teleomorphic state is known only for K. eriophori and C. hydrophilum. The order accommodates two families: Kriegeriaceae Toome & Aime and Camptobasidiaceae R.T. Moore. Kriegeriaceae Toome & Aime, fam. nov. MycoBank MB801221 Type: Kriegeria Bres. (Bresadola 1891). Etymology: The name of the family is derived from the genus Kriegeria.

Diagnosis: Members of Pucciniomycotina, Microbotryomycetes, Kriegeriales, with simple pore septa and subgloboid spindle pole bodies. Anamorphic states grow as colorless to cream-colored yeasts that reproduce most commonly by enteroblastic polar budding and form chains or rosettes. The nuclear division takes place in the daughter cell. Some anamorphs form hyphae and pseudohyphae. Teleomorphic state, where known, hyphal, plant parasitic and sorus-forming. Basidiospores form singly on transversely septate metabasidium, which also may produce yeast cells. To date three genera are recognized: Kriegeria Bres. and its anamorph, Zymoxenogloea D.J. McLaughlin and Double´s, and Meredithblackwellia Toome & Aime. Known species are K. eriophori (5 Z. eriophori), Rhodotorula glacialis, R. himalayensis, R. pilati, R. psychrophenolica, R. psychrophila, R. rosulata, Rhodo-

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FIG. 2. Ultrastructural characters of Meredithblackwellia eburnea (HOLOTYPE, MCA4105). a. Mature yeast cell with several vacuoles (V) and lipid bodies (arrows). Bar 5 1 mm. b. Nucleus (N) and nucleolus (NU) and a mitochondrion (M). Bar 5 0.2 mm. c. A subgloboid spindle pole body with a flat internalized layer (white arrow) and radiating microtubules (black arrows). The nucleus is indicated (N). Bar 5 0.2 mm. d. A mitochondrion (M) and a layered structure (white arrow head), resembling a microscala. Bar 5 0.1 mm. e. Cell forming two buds at the same time. Note the thick layer of extracellular matrix (asterisk). Bar 5 0.2 mm. f. Golgi complex (G) with numerous vesicles (white arrows) in the tip of a forming bud. Bar 5 0.2 mm. g–h. Cconnection between the mother and daughter cell after the bud has fully matured. Bars 5 0.5 and 0.2 mm respectively. i. Bud scar, showing the cell wall layers of the mother cell (black arrowheads) and a new cell wall formed after the daughter cell detached. Bar 5 0.2 mm.

torula sp. KRP3844, Rhodotorula sp. FK.2.1, Rhodotorula sp. CBS11784 and Meredithblackwellia eburnea. Meredithblackwellia Toome & Aime, gen. nov. MycoBank MB801222 Type: Meredithblackwellia eburnea Toome & Aime, this paper. Etymology: Meredithblackwellia, in honor of Meredith Blackwell, Boyd Professor, Louisiana State University, for her vast contributions to yeast research.

Diagnosis: Members of Pucciniomycotina, Microbotryomycetes, Kriegeriales, Kriegeriaceae with subgloboid spindle pole bodies with a flat internalized layer. Anamorphic yeasts that grow as cream-colored butyrous colonies on PDA and YMA. Cells are elongated, bud mostly on a narrow base from each pole, and form rosettes of semi-attached cells. Cells contain numerous vacuoles and lipid bodies and nuclear division occurs inside the bud. Teleomorph not known.

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FIG. 3. Phylogenetic trees illustrating the position of Meredithblackwellia eburnea within Microbotryomycetes. a. Maximum likelihood tree based on the sequences of LSU, SSU and ITS (first dataset), showing the position of M. eburnea in Microbotryomycetes and the five orders within it. The topology was rooted with Mixia osmundae. b. Maximum likelihood tree based on LSU and ITS regions (second dataset) of Kriegeriales, showing the position of M. eburnea and composition of the families Camptobasidiaceae and Kriegeriaceae. The topology was rooted with Microbotryum violaceum. On both trees the numbers above the branch provide bootstrap (left) and maximum parsimony (right) values and the Bayesian posterior probability value is given under the branch. GenBank accession numbers for used sequences are presented in SUPPLEMENTARY TABLE I.

Meredithblackwellia eburnea Toome & Aime, sp. nov. FIGS. 1, 2 MycoBank MB801223 Etymology: eburnea, from Latin, eburneus, referring to the cream (ivory, yellowish white) color of the colonies in culture.

Diagnosis: Meredithblackwellia eburnea is similar to Rhodotorula rosulata Golubev & Scorzetti, differing in that M. eburnea does not form a pseudomycelium and is able to assimilate D-ribose and a-methyl-D-glucoside, and unable to assimilate L-rhamnose, myo-inositol, succinate and ethanol. While M. eburnea can grow in the absence of vitamins, R. rosulata cannot. Type: GUYANA: Region 8, Potaro-Siparuni: Pakaraima Mountains, Upper Potaro River Basin, , 15 km east of Mount Ayanganna, 5u18904.80N, 59u54940.40W; 710 m, from surface of undetermined fern leaf, 28

May 2010, leg. M.C. Aime, MCA4105 (NRRL Y-48821 – HOLOTYPE as lyophilized tissue; CBS 12589 and ATCC MYA-4884 – ISOTYPES). On YMA and PDA media, the colonies are smooth, butyrous and creamy white with entire margins. Single colonies are round with elevated center. After 3 d in YM broth, cells are elongate, slightly fusiform, 3.9–5.2 3 12.6–17.6 mm (av. 4.7 3 14.8 mm), with width/length ratio of 0.25/0.4 (av. 0.32). The cells contain several vacuoles and lipid bodies (FIG. 2a), and are found either singly or in rosettes (FIG. 1b, c). Budding is enteroblastic and occurs on a narrow base from each pole; up to three buds have been observed developing from the same cell at the same time. Spindle pole bodies are subgloboid with a flat internalized layer (FIG. 2c, d). Optimal growth is at 20–25 C.

492 TABLE I.

MYCOLOGIA Comparison of physiological characteristics of Meredithblackwellia eburnea and other members of Kriegeriales 1a

2b

3c

4d

5e

6f

7g

8h

9i

10j

Carbon source D-Galactose D-Ribose D-Xylose L-Arabinose D-Arabinose L-Rhamnose Melezitose Glycerol myo-Inositol DL-Lactate Citrate

2k + w 2 + w + + 2 + 2

2 2 w 2 2 + + w + d d

+ + + + 2 + + + 2 d +

+ 2 w + 2 2 + 2 2 2 +

2 2 n/a n/a 2 n/a n/a 2 2 2 2

2 2 2 2 2 2 + 2 2 2 2

2 2 n/a 2 2 + 2 2 2 2 2

+ 2 w/w/2 2 2 v + n/a v

+ v v v 2 2 w 2 2 n/a v

v 2 v 2 2 2 2 + 2 n/a 2

Nitrogen source Nitrate (K) Nitrite (Na) Ethylamine

2 2 +

+ + +

+ + +

+ 2 +

+ 2 +

+ 2 +

+ 2 +

+ w w

+ w w

+ + n/a

Others 50% D-Glucose w/o vitamins Max growth T C

w + 30

n/a 2 25

2 + 25

2 n/a 22

2 n/a 20

2 n/a 15

2 n/a 20

2 + 20

2 + 20

2 + 17

a

1. M. eburnea. 2. Rhodotorula rosulata (Golubev and Scorzetti 2010). c 3. Kriegeria eriophori (Kurtzman et al. 2011). d 4. R. himalayensis (Shivaji et al. 2008). e 5. R. glacialis (Margesin et al. 2007). f 6. R. psychrophila (Margesin et al. 2007). g 7. R. psychrophenolica (Margesin et al. 2007). h 8. Glaciozyma martinii (Turchetti et al. 2011). i 9. G. watsonii (Turchetti et al. 2011). j 10. G. antarctica (Kurtzman et al. 2011). k + 5 positive; 2 5 negative; w 5 weak positive; d 5 delayed growth; v 5 variable; n/a 5 data not available. b

Fermentation ability is negative. The following carbon compounds are assimilated: D-glucose, Dribose, D-arabinose, sucrose, maltose, trehalose, amethyl-D-glucoside, cellobiose, salicin, arbutin, melezitose, glycerol, D-mannitol, D-glucono-1.5-lactone, DLlactate and quinic acid. Weak growth was detected on D-galactose, L-sorbose, D-xylose, L-arabinose, L-rhamnose, ribitol, xylitol and ethanol. No growth occurs on D-glucosamine, melibiose, lactose, raffinose, inulin, soluble starch, D-glucitol, galactitol, myo-inositol, succinate, citrate, methanol or propane-1.2-diol. Among nitrogen compounds, ethylamine is assimilated; weak growth was detected on L-lysine and Dglucosamine. No growth occurs in the presence of 10% or 16% NaCl or 60% glucose; however, weak growth was detected on 50% glucose. The culture grows well in vitamin free media (some assimilation results are in TABLE I). Material examined: MCA4105 (NRRL Y-48821 5 CBS 12589 5 ATCC MYA-4884).

Camptobasidiaceae R.T. Moore The family was established by R.T. Moore to accommodate Camptobasidium hydrophilum (Moore 1996) and originally was placed in Atractiellales. Our phylogenetic analysis shows that this family belongs to Kriegeriales (FIG. 1B). Camptobasidiaceae contains two known genera: Camptobasidium Marvanova´ & Suberkropp (Mycologia 82:209, 1990) and Glaciozyma Turchetti, Connell, Thomas-Hall & Boekhout (Extremophiles 15:579, 2011). Known members of the Camptobasidiaceae are C. hydrophilum, G. antarctica, G. watsonii, G. martini, Leucosporidium sp. AY30 and Antarctic yeast CBS8941. DISCUSSION The phylogenetic analyses of the first dataset of LSU, SSU and ITS rDNA show that MCA4105 belongs to Pucciniomycotina, Microbotryomycetes, forming a supported ordinal clade, Kriegeriales ord. nov. with

TOOME ET AL.: HIGHER-LEVEL CLASSIFICATION IN MICROBOTRYOMYCETES the sedge parasite Kriegeria eriophori (FIG. 3A). Expanded sampling shows Kriegeriales comprise two family clades, the previously described Camptobasidiaceae and Kriegeriaceae fam. nov. The family Kriegeriaceae accommodates K. eriophori, a hyphal fungus with a white yeast state (Double´ s and McLaughlin 1992), several anamorphic white yeasts that have been isolated from plant surfaces from temperate and tropical climates and glacier-associated white yeasts. All these species form elongated yeast cells with polar budding, which sometimes occurs in small chains or rosettes. Camptobasidiaceae, originally a monotypic family placed in Atractiellales (Moore 1996) but shown to belong to Microbotryomycetes incertae sedis in Aime et al. (2006), is a supported member of Kriegeriales (FIG. 3B). Camptobasidiaceae contains a teleomorphic aquatic hyphal fungus Camptobasidium hydrophilum (Marvanova´ and Suberkropp 1990) and the recently described genus Glaciozyma, which contains psychrophilic white yeasts isolated from Antarctic sea water or glacier sediments (Turchetti et al. 2011). Although Glaciozyma species grow mostly as yeast, they often also form hyphae and teliospores. In addition to the taxa mentioned above, several anamorphic white Rhodotorula spp. are referable to Kriegeriales (FIG. 3B). Morphologically M. eburnea resembles R. rosulata, especially in the formation of rosettes in culture (Golubev and Scorzetti 2010; FIG. 1c). Unlike R. rosulata, however, M. eburnea has not been observed to form pseudomycelium in culture. The two species share 95% identity (527/556 bp) at the LSU and 88% (410/464 bp) at the ITS, and phylogenetic analyses show these species are clearly related and may even be congeneric (FIG. 3B). However, we defer transferring R. rosulata to Meredithblackwellia in the absence of strong molecular support (FIG. 3B) given that the aim of this study was to describe M. eburnea and resolve higher-level placement for several species previously placed incertae sedis. Nonetheless, it is clear that in the future the description of new genera in Kriegeriales will be necessary for several species currently placed in Rhodotorula s.l. There exists little ultrastructural data for members of Kriegeriales. However, the only species to have been studied in detail, K. eriophori, has been shown to produce subgloboid spindle pole bodies with a flat internalized layer (Swann et al. 1999), which closely resemble those observed in M. eburnea (FIG. 2c). This spindle pole body type is also described as characteristic for other Microbotryomycetes (Celio et al. 2006). Although mitosis has been studied in a relatively small number of basidiomycete yeasts (e.g. McCully and Robinow 1972, McLaughlin et al. 1996), our study confirms the present view that nuclear division in the

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yeasts of Microbotryomycetes occurs in the bud (FIG. 1f–k). One additional feature of note was the presence of a layered structure in M. eburnea (FIG. 2d) that somewhat resembles the microscala described in other select basidiomycete yeasts (McLaughlin 1990). However, microscala have not been found in any other species in Microbotryomycetes and the microscala-like structures in M. eburnea seem to lack the cross connections between layers that are characteristic for these structures. The great variety of habitats from which the isolates with the closest BLASTn matches to M. eburnea were recovered suggests that Kriegeriales is an ecologically diverse group of fungi. Based on the species known to date, representatives of Camptobasidiaceae have been isolated from water and glacier sediments. Kriegeriaceae species, on the other hand, are mostly found in association with plants in temperate climates but also in association with glaciers. Therefore, M. eburnea, as a fungus isolated in the Neotropics, represents a new ecozone distribution for this group. Because habitats such as ice, Antarctic sea, or leaf surfaces in tropical areas are poorly studied for fungal diversity, it is expected that further studies of these and other undersampled habitats will increase species discovery in Microbotryomycetes. ACKNOWLEDGMENTS This study was supported by Assembling the Fungal Tree of Life (AFTOL) project NSF DEB-0732968. We thank Terry Henkel for facilitating long term research in Guyana, Claire Whittaker for general laboratory assistance and Tomas Rush, Sebastian Albu, Gregory Heller and Jorge Diaz for assistance with the assimilation experiments. We are grateful for the help of David Lowry and Ricardo Reyes from the School of Life Sciences Electron Microscope Facility at Arizona State University with TEM and DAPI staining sample preparation and David McLaughlin for his expertise in TEM image interpretation. Anonymous reviewers made useful suggestions on earlier versions of this manuscript, and Else Vellinga provided advice on Latin. This is number 196 in the Smithsonian’s Biological Diversity of the Guiana Shield Program publication series.

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