Mycol. Res. 99 (ID): 1239-1246 (1995) Printed in Great Britain 1239

A new pleomorphic ascomycete, Calyptrozyma arxii gen. et sp. nov., isolated from the human lower oesophagus

TEUN BOEKHOUT!, HENRI ROElJMANS' AND FRANS SPAAy3

1 Centraalbureau voor Schimmelcultures, Division, ]ulianalaan 67, 2628 BC Delft, The Netherlands 2 Centraalbureau voor Schimmelcultures, P.o. Box 273, 3740 AG Baarn, The Netherlands 3 Lehrstuhl Spezielle Botanik und Mykologie, Universitiit Tabingen, 72076 Tabingen, Germany

A new pleomorphic ascomycete, Calyptrozyma arxii, isolated from the human lower oesophagus, is described. Initial growth results in yeast cells, followed by formation of hyphae. occurs by blasto-, aleurio- and arthroconidia. In addition, unicellular to multicellular conidia-like cells occur, which initially are somewhat thick-walled. Finally, the outer wall layers of these cells become loose. Sexual reproduction is by eight-spored asci formed on aggregations of generative hyphae. Although no ascomata are formed, the belongs to the Euascomycetes, as was deduced from septal ultrastructure and partial ribosomal RNA sequences.

A fungus isolated from a patient with oesophagitis and white agar, and oatmeal agar (Gams et al., 1987). Preparations and exudate, turned out to represent a new pleomorphic yeast-like measurements were made in water. ascomycete. It was isolated together with species of Nuclei were stained using 4'-6' -diamidino-2-phenylindole and Aureobasidium and probably grows saprotrophically. (DAP!) according to Coleman et al. (1981) and examined using Initially, only vegetative growth was observed, revealing a Zeiss AXioskop epifluorescence microscope with filter 01. the highly pleomorphic nature of the fungus. Its asexual stage walls were stained using 5 % aqueous ca!cofluor white and comprised yeast cells, pseudohyphae, true hyphae, blasto-, examined using the same microscope with filter 05. aleurio- and arthroconidia, and unicellular to multicellular, Nutritional physiological tests were performed as described branched and somewhat thick-walled -like structures by Van der Walt & Yarrow (1984). Assimilation of carbon with a loosening outer . The sexual stage of the life sources was tested in liquid medium, whereas assimilation of cycle, observed on diluted yeast extract-malt extract agar, nitrogen compounds was investigated using auxanograms. consisted of naked asci, formed on aggregations of ascogenous For electron microscopy, cells were fixed in 0'5 % w / v hyphae, occurring along conjugating hyphae. potassium permanganate, stained with 1'5 % uranyl acetate in As the fungus could not be identified with any described 50% ethanol, dehydrated through a graded series of ethanoL genus of endomycetalean , nor with little-developed and embedded in Spurr's resin. taxa belonging to the Euascomycetes, it is described here as For analysis of cell wall carbohydrates, whole-cell hydro• a new genus and species. lyzates were analysed using gas-liquid chromatography/mass spectroscopy as described by Weijman et al. (1982) and Weijman & Golubev (1987). MATERIALS AND METHODS Coenzyme Q was extracted and purified as described by Yamada & Kondo (1971,1973). The number of isoprenologues Strain studied was determined with reverse-phase thin-layer chromatography The strain, CBS 354.92, was isolated from the lower using Merck HPTLC RP-18 F254s plates and a mixture of oesophagus of a 67-year-old woman from Lake Placid, New acetone and acetonitrile (80:20, v/v) as the developing York u.s.A., suffering from oesophagitis and white exudate. It solvent (Nakase & Suzuki, 1985). The separated components was maintained on 1% w/v yeast extract-D'5% w/v were detected under ultraviolet light at 254 nm. peptone-4% w/v glucose agar (YPGA) slants at 10°C. DNA was isolated from three to five day-old, rotary-shaken Morphology was studied on a variety of media at room cultures (150 rpm), grown at 25° in YPG-broth. Cells were temperature: yeast morphology agar (YMoA, Difco), com washed in tap water and saline EDTA (0'1 M NaCl, meal agar (CMA, Difco), yeast extract-malt extract agar 0'15 M EDTA) and broken with a French Press. DNA was (YMA, Difco), 10 times diluted YMA, YPGA, potato-dextrose isolated and purified using the method of Britten et a!. (1970). agar (PDA, Oxoid), 5 % malt extract agar (MEA, Difco), V8• Molar percentage G + C was analysed according to Owen & juice agar (Gams et a!', 1987), ten times diluted V8-extract Lapage (1976), using a Perkin and Elmer UV/Vis spectro- New pleomorphic ascomycete, Calyptrozyma arxii 1240

3

4

7

10 IJ-rn

Figs 1-7. Morphology of Calypfrozymaarxii. Fig. 1. Yeast cells on YMoA. Fig. 2. Hyphae with aleurioconidia on YPGA. Fig. 3. Yeast cell growing into on YMoA. Fig. 4. Blasto- and arthroconidia-like cells on 2% MEA. Fig. 5. Thick-walled conidia-like cells with a loosening outer cell wall on YMA. Fig. 6. Asci on globules of gametangial coils and conjugating hyphae on 10 times diluted YMA. Fig. 7. Conjugating yeast and hyphal cells on 10 times diluted YMA (bar indicates 10 ~m). 1. Boekhout, H. Roeijmans and F. Spaay 1241

Figs 8-18. Fig. 8. Budding yeast cells (YPGA. 24 h, bar = 10 ~m). Fig. 9. CalcoAuor white-stained yeast cells (YPGA. 24 h, bar = 10 ~m). Fig. 10. Blastoconidia and arthroconidia-like cells (YPGA, 8 wk bar = 10 j.lm). Fig. 11. DAPI stained nucleus in mother cell (YPGA, 24 h, bar = 10 ~). Fig 12. DAr! stained nucleus in bud (YPGA, 24 h, bar = 10 ~m). Fig. 13. DAr! stained nuclei in bud and mother cell (YrGA, 24 h, bar = 10 ~m). Fig. 14. Conidia-like cells with a loosening outer cell wall (10 times diluted YMA, 25 d, bar = 10 ~m). Fig. 15. Young vacuolated asci on globule of ascogenous hyphae. (10 times diluted YMA, 25 d, bar = 10 ~m.) Fig. 16. Asci with clusters of (10 times diluted YMA, 25 d, bar = 10 >-1m). Fig. 17. Cluster of germinating ascospores (10 times diluted YMA, 25 d, bar = 10 ~m). Fig. 18. Septal ultrastructure (KMnO4' bar = 1 j.lm). New pleomorphic ascomycete, Caiyptrozyma arxii 1242

photometer. Temperature increase was 0'5° min-1 and the Tm Table 1. Nutritional physiology of Calyptrozyma arrii value was calculated automatically using the first derivative of Fermentation (25°): absent the melting curve. Candida parapsilosis (CBS 604) was used as a reference. Assimilation of Assimilation of Isolation of ribosomal RNA (rRNA) was performed as carbon compounds (25 0) nitrogen compounds follows. Cells grown in 2 % YPG-broth on a rotary shaker for o.glucose + nitrate + 2 d at 25° were broken in 4 M guanidinium thiocyanate using o-galactose + nitrite + a French Press. The rRNA was purified by centrifugation in a o-sorbose d ethylamine + caesium chloride gradient as described by Maniatis et ai. o.glucosamine -,w L.lysine (1982). The rRNA subunits were separated by non-denatur• o-ribose - .w cadaverine + o-xylose + creatine ating 3'2% acrylamide gel electrophoresis. Sequencing of L-arabinose + creatinine the region of the small subunit rRNA was accomplished with o-arabinose - ,w imidazole a specific primer 5'-ACGGGCGGTGTGTAC (position 1627) L-rhamnose d (Kurtzman & Liu, 1990) and the dideoxynucleotide chain sucrose + termination method (Sanger et al., 1977; Lane et al., 1985). maltose + a,a trehalose + Nucleotide fragments were separated on 8 %-8 M urea gels methyl a-glucoside + and visualized by autoradiography. cellobiose + Sequences (position 1320-1619) were aligned using Align salicin + Plus 2'0 (Scientific & Educational Software) and corrected with arbutin + a word processor. Data from additional species were obtained melibiose + lactose from a database (De Rijk et ai., 1992). raffinose + Reconstruction of the phylograms was done using melezitose + neighbour-joining (Saitou & Nei, 1987) and maximum inulin d,w parsimony using MEGA 1'0 (Kumar et al., 1993). Confidence starch + values for individual branches were determined by bootstrap glycerol d meso-erythritol analysis using 1000 replications (Felsenstein, 1985). ribitol xylitol L-arabinitol RESULTS o-glucitol + o-mannitol + Morphology galactitol +,d myo-inositol +,d After 10 d on YMoA, colonies were ca 6 mm diam. (14 mm glucono a-lactone +,d after 2 I d), flat, with the surface slightly pustulate, wrinkled or 2-keto-o-gluconate ridged, somewhat shiny, pale yellowish, yellowish brown to o-gluconate cream, moist to tough, and with a narrow flattened, sharply o-glucuronate + o-galacturonate + delimited, crenulate margin. The reverse was concolorous. On oL-lactate + YPGA the surface was dull, irregular, with transverse ridges succinate + and locally with white hirsute pustules. On MEA, CMA and citrate YMA submerged mycelial sectors occurred at the margin of methanol the colony. On PDA, V8-juice, and diluted V8-juice agars ethanol + propane 1,2 dial + colonies were yeast-like and formed submerged at butane 2,3 dial the margin. (+ = growth, - = no growth, w = weak growth, d = delayed Three-day-old colonies at YMoA consisted mainly of yeast growth after 2 or 3 wk) cells, which were ellipsoidal, cylindrical, allantoid, somewhat Growth without vitamins +; resistance to 0'01 % cycloheximide dumb-bell-shaped or ogival, 6-9'5 x 2-4 11m (3-5-8 x 2-5 11m growth with 50% glucose -; starch production -; acetic acid production on YPGA) Figs 1, 8). Budding occurred enteroblastically at the -; urease activity + ; reaction with Diazonium Blue B-; growth between poles of the cells, usually on short denticles, but also on a 4 and 30° +; growth at 35° slow, no growth at 37°. broad base. Proliferation was sympodial, leaving visible scars. The buds were frequently elongate forming branched hyphae (Fig. 3). Subglobular, uninucleate cells, 6-10 11m diam. were After ca 10 days, clavate aleurioconidia, 6-10 x 3-4 IJm, present, which germinated to form uninucleate ellipsoidal had formed laterally along the hyphae (Fig. 2). The aleurio• yeast cells. During initial growth of the bud, the nucleus conidia were observed to germinate while still attached to remained in the mother cell. At approximately metaphase all the hypha forming either yeast cells or hyphae attaining a the DNA appeared to be present in the bud, whereas during size of 9-20 x 3-4 \-1m. In shaken YM or YPG broth telophase the daughter nucleus appeared to migrate back into (ISO rpm, 25°) yeast cells formed branched or unbranched the mother cell (Figs II- 13). Lateral and transverse septa of pseudohyphae or hyphae, with cells up to ca 170 x 2'5• the yeast cells stained with calcofluor white. However, the cell 3 IJm. Hyphae were slender, composed of cells 6-60 x 1'5• walls of the daughter cells were found to stain less intensely 3'5 IJrn, and on diluted YMA, YMoA and CMA were (Fig. 9). observed to disarticulate forming arthroconidia-like cells (Fig. T. Boekhout, H. Roeijmans and F. Spaay 1243

Fig. 19. Ultrastructure of conidia-like cells with a loosening outer cell wall, and endogenously formed conidia (KMn04, bar = 1 lffil).

4). Ellipsoidal to fusiform sessile blastoconidia were formed on surrounded by a fibrous capsule. The cell wall had a thin hyphae or on lateral branches or denticles (YMoA, diluted electron-dense outer layer, whereas the transverse septa had a YMA). One to five-celled, branched or unbranched, cylindrical thin electron-transparent central layer. The septa attenuate cells with a loosening outer cell wall (initially giving these towards a central pore, which was occluded by an electron• cells a thick-walled impression) occurred solitarily or in dense body, and surrounded by Woronin bodies (Fig. IS). The clusters, laterally and terminally on the hyphae on YMA, cylindrical cells with a loosening outer cell wall showed diluted YMA, CMA, PDA, and MEA (Figs 5, 14). The endogenously formed septate endospores, which were released cell lumina of these cells measured 10-30 x 2-4 ~m; enteroblastically (Fig. 19). occurring enteroblastically with slender hyphae In the DNA melting curve two peaks were observed. or yeast cells. Anastomoses frequently occurred between Mol% G + C of the main peak (nDNA) was found to be hyphae, yeast cells and between these various types of cells 53'7±0'5, and that of the minor peak (mtDNA) 2S'9± 1'0. (Figs 6, 7). Whole-cell hydrolyzates contained the following monomers: After growth for ca 3 wk on diluted YMA at room fucose, galactose, glucose, rhamnose and mannose; xylose temperature, many short, curved and finger-like gametangial was not detected. The coenzyme Q comprised ten iso• coils were formed along the hyphae, which finally formed up prenologues. to ca 40 !-lm wide aggregations of narrow generative hyphae. Partial ISS rRNA sequences, using parsimony and One to several, clavate to cylindrical, eight-spored asci, neighbour-joining (Kimura 2-parameter distances) for phylo• 30-47 x 10-15 !-lm, developed from these hyphal aggre• genetic reconstruction (Kumar et al., 1993), suggested that the gations (Figs 6, IS, 16). Young asci were highly vacuolated fungus belonged to the Euascomycetes. It did not form a (Fig. IS). Dehiscence of the mature asci appeared to occur by terminal branch with any of the investigated representatives apical dissolution of the cell wall (Fig. 16). The ascospores of the main groups of Ascomycetes, namely Ascosphaeriales were ellipsoidal, smooth-walled, 5-7'S x 3'4-5 !-lm, and were (Ascosphaera apis (Maasen ex Claussen) Olive & Spiltoir), released in clusters (Figs 16-17). Ascospores germinated at Onygenales ( rubrum (Castell.) Sabour.), Eurotiales their apex forming yeast cells (Fig. 17). Ascomata were not ( f/avus (KI6cker) Stolk & Samson), Leotiales observed. (Sclerotinia sclerotiorum (Lib.) de Bary), Sordariales (Sordaria fimicola (Roberge ex Desm.) Ces. & De Not.), Hypocreales (Hypomyces chrysospermus Tul.), nor with representatives of Physiology, ultrastructure, mol% G + C, carbohydrate Endomycetales (Dipodascus albidus Lagerh. and Saccharomyces composition, coenzyme Q and partial ribosomal RNA cerevisiae Meyen ex E. C. Hansen) (Fig. 20). These orders were sequences found to be strongly supported when several representatives Data on the nutritional physiology are presented in Table of genera supposedly belonging to these orders were included I. The cell walls were of medium electron density, possibly for comparison (data not shown). New pleomorphic ascomycete, Calyptrozyma arxii 1244

Eremascus albus Species of Eidam are similar to C. arxii due to the Talaromyces flavus presence of naked asci (Arx, 1972) and a similar septal ultrastructure (Kreger-van Rij et al., 1974). However, they Ascosphaera apis L-----Calyptrozyma arxii differ from the present fungus by their spherical asci formed L------Sclerotinia sclerotiorum solitarily on conjugating hyphae or short gametangial coils, L------,--::-::4 Hypom)'ces chrysospermus the presence of basipetally formed conidia originating on 100 Sordaria fimicola elongated conidiogenous cells (Arx, 1972), and dissimilar L- Taphrina deformans I- ____=_~ Dipodascus albidus partial rRNA sequences. 100 Saeeharomyees cerevisiae Some ascomycetous yeasts, currently classified in the L------Mueor racemosus Endomycetales (Arx & Van der Walt, 1987; Kurtzman, 1993), show morphological similarities with the present Fig. 20. Evolutionary of Calyptrozyma and representatives of main groups of ascomycetes based on partial 18S ribosomal RNA fungus. 5tephanoascus M. T. Sm., Van der Walt & Johannson sequences (position 132G-1619) using the maximum parsimony and Zygoascus M. T. Sm. are similar in their initial yeast-like method with the heuristic search option from MEGA. growth, followed by hyphal development, and formation upon conjugation (Smith et al., 1976; de Hoog et aI., 1985; Smith, 1986). Both 5tephanoascus and Zygoascus, as well as other genera of the Endomycetaceae Schroeter, have galeate Calyptrozyma arxii Boekhout & Spaay, gen. et sp. nov. ascospores (Redhead & Malloch, 1977; Arx & Van der Walt, Diagnosis generico-specifica 1987). Calyptrozyma arxii differs also from species in these Ascomyces pleomorphicus. Thallus vegetativus primum zymoideus, genera in septal ultrastructure, due to the presence of e cellulis gemmantibus, pseudohyphis et praecipue hyphis constans, diaphragm-like septa with Woronin bodies. In contrast, septa aleurioconidia, cellulae arthroconidiorum similes et blastoconidia of 5tephanoascus and Zygoascus have micropores (Smith, 1986). formata. Secundum hyphas protuberantiae formantur conidiorum Other yeast-like ascomycetous genera showing some similes, septatae, ramosae vel simplices, fere crassitunicatae, pariete morphological similarities with C. arxii are Dipodascus Lagerh. exteriore deinde relaxato, e quibus cellulae endogenae propagantur. and Galactomyces Redhead & Malloch, due to the formation of Anastomoses inter hyphas frequentes. Glomera gametangialia in globulos hypharum generativarum transformata, e quibus asci nudL arthroconidia. However, formation of asci in these genera cylindrici, odospori oriuntur. Ascosporae ellipsoideae glomeratae, occurs by fusion of gametangia, their septa have micropores cellulas zymosas germinantes. Ascomata omnino absentia. Septa (Kreger-van Rij & Veenhuis, 1974; de Hoog et al., 1986), and porc typi ascomycetum perforata. Coenzymum Q-I0 adest. Parietes the coenzyme Q has 9 isoprenoids (Kuraishi et aI., 1985; hydrolysatae fucosum, galadosum, glucosum, rhamnosum, man• Yamada et al., 1987). nosum continentes. Acidum nucleicum 53'7% G + C continens. Like Calyptrozyma arxii, Yarrowia lipolytica (Wick. Kurtzman Fermentatio, excretum amyloideum et readio colorantis DBB & Herman) Van der Walt & Arx (1980) is one of the few absentes. Ureasium formatur. Temperatura inter 4 et 30° crescere yeast-like ascomycetes showing urease activity using standard potest. test methods (Barnett et al., 1990; Kreger-van Rij, 1984). This Species typica Calyptrozyma arxii Boekhout & Spaay. Holotypus in species does not seem to be closely related, due to the herb. CBS 5453; vivus CBS 354.92 praeservatur. formation of 1-2(-4) variably shaped ascospores, a number of physiological differences (Barnett et aI., 1990), the presence of septal micropores (Kreger-van Rij & Veenhuis, 1973) and DISCUSSION coenzyme Q-9 (Yamada & Kondo, 1972). Moreover, according Calyptrozyma arxii is a highly pleomorphic ascomycete. to Booth & Vishniac (1987), Yarrowia lipolytica is, in fad, Vegetative growth is by yeast cells, pseudohyphae, aleurio• urease-negative, and this may be a further physiological conidia, arthroconidia and blastoconidia, as well as septate difference. hyphae on which septate, branched or unbranched, somewhat Our molecular data do not suggest a close phylogenetic thick-walled conidia-like cells occur with a loosening outer cell relationship between C. arxii and the Endomycetales as it wall. Naked, clavate, eight-spored asci originate from aggre• cannot be identified with any of the known endomycetalean gations of generative hyphae occurring along conjugating taxa. hyphae. Ascospores germinate with the formation of yeast Species of Botryoascus Arx are similar to C. arxii due to the cells. presence of naked, one-celled asci which are not formed on The strain could not be identified with any known ascophores (Redhead & Malloch, 1977). However, this genus endomycetalean yeast species, nor with yeast-like Euasco• differs by the presence of galeate ascospores (Arx & Van der mycetes. Using morphological and physiological data all Walt, 1987). The asci and ascospores of C. arxii are similar, known yeast species differed in ten or more tests when to some extent, with those found in species of Lasiobolidium entered in the yeast identification program of Barnett et al. Malloch & Cain, which in contrast to C. arxii are formed in (1990). Surprisingly, species keyed out first were hetero• c1eistothecia (Malloch & Cain, 1971). basidiomycetous yeasts belonging to genera like The anamorphic cycle of C. arxii shows morphological Klitz., Leucosporidium Fell, Statzell, 1. L. Hunter & Phaff, Bullera similarities with several described anamorphic fungi. Conidia Derx and Behrend. formed by Hormographiella verticillata Guarro, Gene & E. None of the ascomycetous yeast genera described fits the Gueho (Guarro et al., 1992) show some resemblance with the morphological and physiological characteristics of this fungus. unicellular to multicellular, branched or unbranched conidium- T. Boekhout, H. Roeijmans and F. Spaay 1245 like cells found in C. arxii. According to its description, Hoog, G. S. de, Rantio-Lehtimiiki, A H. & Smith, M. T. (1985). Blastobotrys; H. verticillata is characterized by the presence of distinct Sporo/hrix and Trichosporiella: generic delimitation, new species, and a S/ephanoascus teleomorph. An/onie van Leeuwenhoek 51, 79-109. conidiophores (Guarro et al., 1992). The aleurioconidia and Hoog, G. S. de, Smith, M. T. & Gueho, E. (1986). A revision of the genus biastoconidia occurring in C. arxii show some resemblance and its teleomorphs. Studies in 29, 1-131. with those of the anamorphs of Stephanoascus ciferrii (Smith et Kreger-van Rij, N. J. W. (1984). The Yeasts, a Taxonomic Study, 3rd ed. aT., 1976) and of species of Trichosporiella Kamyschko (de Elsevier: Amsterdam, The Netherlands. Hoog et al., 1985). The sessile or short-stalked aleurioconidia Kreger-van Rij. N. J. W. & Veenhuis, M. (1973). Electron microscopy of septa of C. arxii resemble those found in species of in ascomycetous yeasts. 39, 481-490. Kreger-van Rij, N.]. W. & Veenhuis, M. (1974). and septa in the genus Corda and allied genera (van Oorschot, 1980). Dipodascus. Canadian journal of Botany 52, 1335-1338. Partial 18S ribosomal RNA sequences suggest that C. arxii Kreger-van Rij, N.]. W., Veenhuis, M. & Leemburg-van der Graa£. C. A belongs to the Euascomycetes. Both parsimony and neighbour• (1974). Ultrastructure of hyphae and ascospores in the genus Eremascus joining methods of phylogeny reconstruction resulted in the Eidam. Antonie van Leeuwenhoek 40, 533-542. same topology of the tree. However, none of the compared Kumar, S.. Tamura, K. & Nei, M. (1993). MEGA: Molecular Evolutionary Genetics Analysis, version J.D. The Pennsylvania State University: University major groups of Ascomycetes formed a terminal branch with Park, U.SA C. arxii. Our data suggest that C. arxii may belong to a Kuraishi, H., Katayama-Fujimura, Y., Sugiyama, J. & Yokoyama, T. (1985). separate order. However, any definite decision needs molecular Ubiquinone systems in fungi. I. 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(Accepted 16 February 1995)