A Novel Heterothallic Ascomycetous Yeast Species: Stephanoascus smithiae, Teleomorph of Candida edax

System. Appl. Microbiol. 17,237-246 (1994) © Gustav Fischer Verlag, Stuttgart· Jena . New York

A Novel Heterothallic Ascomycetous Yeast Species: Stephanoascus smithiae, T eleomorph of Candida edax G. GIMENEZ-]URADO h , A.

J.

CIDADAo2 and A. BEI]N-VAN DER WAAIJ3

The Portuguese Yeast Culture Collection, Laboratory of Microbiology Laboratory of Cell Biology: Gulbenkian Insititute of Science, 2781 Oeiras Codex, Portugal 3 Yeast Division of the Centraalbureau voor Schimmelcultures, Julianalaan 67 A, 2628 BC Delft, The Netherlands 1

2

Received January 23, 1994

Summary The new species Stephanoascus smithiae was found to be heterothallic and to constitute the perfect state of Candida edax, a yeast of ascomycetous affinity. The presence of asci crowned with an apical cell, the formation of galeate or hemispherical ascospores, and copious development of true mycelium with denticulate conidiogenous cells, assigns the teleomorphic state to the genus Stephanoascus Smith, van der Walt et Johannsen. Morphological and physiological characters of Steph. smithiae, including the ability to utilize non-conventional sources of carbon, were compared with those of other hyphal yeast species in the genera Stephanoascus, Zygoascus, Arxula, and with other related anamorphic hypha I ascomycetous yeasts which showed striking phenotypical resemblances with the perfect forms examined. Further differentiation was based on the determination of mol% G+C content and extent of DNA relatedness. Different physiological characteristics, maximum temperatures for growth, and significantly low relative DNA reassociation values with the type strains of Steph. ciferrii (34-38%) and Steph. farinosus (1-3%) indicated that it represents a novel taxon. Similar phenotypic characteristics, a positive mating reaction and a high degree of DNA relatedness demonstrated conspecificity with C. edax. The description of the new teleomorph is given and possible relationships with other taxa are discussed.

Key words: Yeast - Taxonomy - Hyphal ascomycetous yeasts - Stephanoascus smithiae - Uncommon physiological characters

Introduction Two haploid strains were isolated from soil which mate to produce a perfect species resembling Stephanoascus. The genus Stephanoascus (Smith et aI., 1976) was established to accommodate Stephanoascus ciferrii, the teleomorph of Candida ciferrii, a yeast species with a conidiogenesis compatible with the expanded concept for the genus Sporothrix Hektoens et Perkins as delimited by De Hoog et a1. (1985). Smith et a1. (1976) mentioned that one strain identified as Sporothrix catenata mated with a mating type of Steph. ciferrii. Associations between Stephanoascus species and Sporothrix anamorphs were reinforced with the description of a second teleomorphic state, Stephanoascus farinosus (De Hoog et aI., 1985), which was based on a strain originally identified as Sporothrix fungorum, but considered to be different from its type " Corresponding author

strain. These discoveries stimulated further interest in the relationship between yeast-like representatives of the genera Sporothrix and the phenotypically related Blastobotrys and strains originally described as yeasts. The carbohydrate composition of whole cells and the fine structure of the septa (Smith and Batenburg-van der Vegte, 1985) led Weijman and de Hoog (1985) to recognize three distinct groups within the hyphomycete genus Sporothrix: Sporothrix sect. Sporothrix, Sporothrix sect. Farinosa Weijman and de Hoog and Sporothrix sect. Luteoalba Weijman and de Hoog, comprising fungi of ascomycetous, hemiascomycetous and basidiomycetous affinity, respectively. The analysis of the major ubiquinone (coenzyme Q) system by Yamada and Smith (1985) and further studies of the fine structure (Smith and Batenburg-van der Vegte, 1986) revealed two groups within Sporothrix sect. Farinosa, one comprising species with a simple pore plus Woro-

238

G. Gimenez-Jurado, A.J. Cidadao and A. Beijn-van der Waaij

nin bodies and a Q-10(H 2 ), thus bearing a phylogenetic affinity with hyphal Ascomycetes, in particular to the genus Ophiostoma, and the second comprising species with single or scattered micropores, CoQ-9 and related to the genus Stephanoascus or other Hemiascomycetes. The latter included anamorphs of Stephanoascus as well as Sp. (oliorum and Sp. guttuli(ormis, for which no teleomorphs are known. Based on these findings, Smith and Batenburgvan der Vegte (1986) proposed that all species of Sporothrix sect. Farinosa associated with hyphal Ascomycetes should be t;ansferred to Sporothrix sect. Sporothrix. Smith (1986) described a second filamentous genus, Zygoascus, with the single species Z. hellenicus representing the perfect state of Candida hellenica, C. inositophila and C. steatolytica. Like Steph. ci(errii, this species is heterothallic, forms persistent asci with hemispherical to hatshaped ascospores, produces CoQ-9 and hyphal septa with micropores. The number of asci per conjugation bridge and the absence of an apical cell allows differentia-

tion of this genus from Stephanoascus. Accordingly, Von Arx and van der Walt (1987) proposed the exclusion of the genera Stephanoascus, Zygoascus and Yarrowia, another genus with septal micropores and CoQ-9 system, from the Endomycetaceae, restricting this family of the Endomycetales to genera with smooth, hat-shaped ascospores, and evanescent asci. Yarrowia can be distinguished from the other two genera by the presence of deliquescent asci, ascospores lacking an electron-dense base as determined by transmission electron microscopy, and a positive urease reaction which is very rare in ascomycetous yeasts. Middelhoven et al., (1985) reported that Steph. ciferrii and the arthroconidial species Arxula adeninivorans, two species commonly found in soil, were among the few ascomycetous yeasts capable of utilizing uric acid, adenine, n-alkylamines or diamines as sole sources of carbon and energy. The taxonomic relevance of the capacity to utilize uncommon substrates, not included in the conventional set for yeast classification, as both carbon or nitrogen

Table 1. Strains examined and their origin Species

Stephanoascus Steph. ciferrii Steph. farinosus Steph. smithiae

Zygoascus Z. hellenicus

IGC 1 Number

CBS 2 Number

Origin

3818 4164!T 4165 IT 4592 T 4646!T 4647!T 5052

4856 5295 6699 140.71 7522.1 7522.2

Neck of cow, Netherlands Pig, Netherlands Soil, South Africa Carpophore of Hirneola auricula-judae, Netherlands Soil, Brazil Soil, Brazil Rotten wood, U.S.A.

3483 4838 IT 4839 IT 3665 5024

4099 6736 5839

Fermenting grape must, Greece Washings of ion-exchange resin, Japan Udder of mastitic bovine, South Africa unknown Olive brine, Portugal

8244 6697

Soil, Netherlands Soil, South Africa

8184 8169 8168 1898 8172 6064 5657 5604 8005 8183 5721

Alpechfn, Spain Rotten trunk of Nothofagus dombeyii, Chile Rotten trunk of Eucryphia cordifolia, Chile Blood of mink, Canada Brown rot of trunk of Nothofagus dombeyii, Chile Liver of bat Mormoops megalophyl/a, Colombia Insect tunnel in Sclerocarya caffra, South Africa Grape must, Japan High-moor peat, USSR Rotten trunk of Nothofagus dombeyii, Chile Sputum, Chile Dried wine lees, Portugal

Arxula A. adeninivorans A. terrestris Candida C. auringiensis C. bertae var. bertae C. bertae var. chiloensis C. blankii C. castrensis C. chiropterorum C. edax C. incommunis C. paludigena C. santjacobensis C. valdiviana Candida sp. nov. 3 1

2 3

4831 T 4636 T 4637 T 3410T 4832 T 4857 4183 T 4837 4834 T 4835 T 4858 T 4854

IGC, Instituto Gulbenkian de Ciencia, Portuguese Yeast Collection; T, Type strain; IT, Isotypes. CBS, Centraalbureau voor Schimmelcultures, Netherlands. Fonseca, 1992.

Stephanoascus smithiae sp. nov., the telemorph of Candida edax

sources has been suggested in certain cases (Bos and de Bruyn, 1973; Hofmann and Schauer, 1988; Middelhoven et a1., 1985, 1989; Fonseca, 1992). In the present study, phenotypical and genomic comparisons of inositol-positive, filamentous, anamorphic and teleomorphic ascomycetous yeast species with similar conidiogenesis were undertaken in order to elucidate their taxonomical relationships. The sexual state of Candida edax is described.

Fig. 1. Stephanoascus smithiae IGC 4646 X IGC 4647 on 5% (Difco) malt extract agar. 1000X. (a) Asci at different stages of development. Arrow indicates the apical cell. (b) One-spored ascus with apical cell arising laterally on hyaline hyphae. Insert bars represent 10 !tm.

239

Materials and Methods Strains. The strains examined and their. origin are listed in Table 1. The holotype of Stephanoascus smithiae was isolated from soil, collected in Brazil, by direct inoculation into liquid mineral media (Van Uden, 1967) with 0.5% (v/v) methanol and adjusted to pH 3.5 to discourage bacrerial growth. Isolated colonies were obtained and purified on agar plates containing glucose (1 %), yeast extract (0.3%), malt extract (0.3%), and peptone (0.5% ). However, purified colonies were not able to grow

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G. Gimenez-Jurado, A.J. Cidadao and A. Beijn-van der Waaij

on medium with methanol as sole carbon and energy source. An additional strain was isolated from a sample of rotten wood collected in Atlanta, U.S.A., and kindly offered by J. P. Sampaio. All strains were maintained on slopes of YM agar. Isolation of mating types. Some colonies of soil isolates were ascosporogenous. Mating types were obtained from a mixed culture utilizing a heat-shock treatment at 60°C followed by subsequent plating at 1 min intervals on YM agar. Surviving single colonies, from samples taken after 15-20 min, were randomly selected and transferred to YM agar slopes. Mating experiments were performed on 5% malt extract agar. Based on results of fertile mating reactions, IGC 4646, IGC 4183 and IGC 5052 were arbitrarily designated as mating type a, and IGC 4647 represented the opposite mating type alpha. Characteristics of the strains. Morphological and physiological characters were determined using the methods described by Van der Walt and Yarrow (1984). Growth on N-hexadecane, butylamine, hexylamine, adenine, xanthine, allan to in, acetoin, uric acid and butane-2,3-diol was evaluated according to the methods described by Middelhoven et al. (1989). The methods used for DNA isolation are those described earlier (Gimenez-Jurado et aI., 1990). Determinations of the guanine plus cytosine of the nuclear DNA were done following the method of Marmur and Doty (1962) with a Gilford Response UV-VIS spectrophotometer, and its Thermal Programming software, using nDNA from Candida parapsilosis CBS 604 (mol% G+C = 40.5) as reference. The extent of DNA-DNA reassociation was determined spectrophotometrically using the same instrument, following the procedures described by Seidler and Mandel (1971), as modified by Kurtzman et al. (1980). Ubiquinone isoprenologues were extracted from cells of Steph. smithiae (IGC 4646) following the methods referenced earlier (Gimenez-Jurado et aI., 1990). Transmission electron microscopy. Steph. smithiae mating types were mixed and grown on 5% Difco malt extract agar for 1 week at 25°C, Hyphae and asci containing mature ascospores were fixed in 3 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 hour at 4°C, postfixed in 1% Os04 in the same buffer, also for 1 hour at 4°C, and processed for Epon embedding. Ultrathin sections were double stained with uranyl acetate and lead citrate, and observed and micrographed with a Jeol100CX transmission electron microscope operating at 80kV.

Description Stephanoascus smithiae Gimenez-Jurado sp.nov.

Species heterothallica, status ascigerus Candidae edax Van der Walt et Nel. In medio liquido cum dextroso et peptono et extracto levedinis post 3 dies 25°C cellulae sunt cylindratae, ovoideae, aut longiovoidae pro maxima parte irregulares (2.0-12.0) x (1.0-2.0) ~m, singulae vel binae. Reproductio vegetativa per gemmas enteroblasticas. Post 7 dies 25°C sedimentum sparsim et velum repente formantur. Cultura in striis in medio agaro cum dextroso et peptono et extracto levedinis post 7 dies ad 25°C, color albus ad cremeum vergens, pagina laevis vel leviter striata, saepe fimbriata cum mycelium et in centro elevata. Coloniae nitidae, butyrosae, margo laevis ad crematum. Post 1 mensem coloniae rugosae et sexus oppositus color fulvus in centro. Mycelia vere tenua formantur cum septis ad intervalla regularia et conidia ovata vel globosa exorientia hyphae dentiformis, saepe plus quam unum, alteruter intercalaris aut apicalis praesens. Asci observati in agaro cum extracto malti post con-

jugationem sexorum oppositorum, IGC 4646 x IGC 4647. Asci formantur post coniunctionem hypharum etsi non observata, praesens prae-ascus globosus aut sphaericus in evagitione coniunctionis hypharum. Asci maturi constant ascosporis hemisphaericis vel petasiformis (5.0-8.0) X (3.0-5.0) ~m, alteruter intercalaris vel terminalis oriens, cellula apicali coronata unde gemmae secundariae formantur. Fermentatio nulla. Assimilat glucosum, galactosum, L-sorbosum, Dglucosaminum, D-ribosum, D-xylosum, L-arabinosum, Darabinosum, L-rhamnosum, sucrosum, maltosum, trehalosum, a-methyl-D-glucosidum, cellobiosum, salicinum, arbutin urn, melibiosum, lactosum, raffinosum, melezitosum, inulinum, amylum solubile (lente), glycerolum, ierythritolum, ribitolum, xylitolum, D-glucitolum, D-mannitolum, galactitolum, i-inositolum, D-glucon-a-lactonum, 2-keto-D-gluconatum, D-gluconatum, D-glucoronatum, acidum succinicum, acidum citricum, acidum mtartaricum, acidum malicum, acidum glutamicum, ethanolum, butano-2,3-diolum, N-hexadecanum, N-butylaminum, N-hexylaminum, N-acetyl-glucosaminum, stachyosum et palatinosum. Assimilat kalii nitratis, sodii nitrosi, ethyl amini, cadaverini, D-glucosamini et acidum glutamicum. Vitamina externa ad crescentiam necessaria sunt. Temperatura maxima crescentiae 35-36°C. Materiae amyloidae non formantur. Ureum non finditur. Proportio molar is guanini plus cytosini in acido deoxyribonucleico 47.0 ± 0.2 per centum (IGC 4646) et 47.1 ± 0.1 (IGC 4647). Ubiquinonum majus Q-9 (IGC 4646). Typus. Isotypi IGC 4646 et IGC 4647, ex solo in botanic horto Rio de Janeiro brasiliensi isolati. Holotypus cultura conjugata IGC 4646 X IGC 4647, exsiccata in collectione zymotica lusitana, et vivus in collectione zymotica lusitana, Oeiras, et in collectione zymotica Delphis Batavorum (CBS 7522.1 et CBS 7522.2) praeservatus. Growth in glucose (2%) yeast extract (0.5%) peptone (1 %) broth. After 3 days at 25°C cells vary in shape from cylindrical to ovoidal, elliptical or obovate, although mainly irregular, measuring (2-12) X (1-2)~m, occurring singly or in pairs and presenting holoblastic-multilateral budding. After 1 week at 25°C a scanty sediment and a thin dry creeping pellicle are formed. Growth on glucose (2%) yeast extract (0.5%) peptone (1 %) agar. After 1 week at room temperature strains form white to cream compact colonies with smooth to slightly striated or wrinkled surfaces, often fringed with mycelium and elevated at the centre. Colonies from all strains were either glossy or shiny, butyrous and had relatively smooth or crenate margins. After 1 month, they became more ornamented with plicate to rugose or wrinkled surfaces and one of the mating types developed a tan or fawn colour at the centre of the colonies. Slender true mycelium developed with septa at regular intervals, and oval blastospores arising from denticle-like structures occurred along the hyphae, either intercalary or terminal. Dalmau plate cultures on corn-meal agar. After 7 days at 25°C growth under aerobic conditions showed copious development of true, branched, septate mycelium with a

242

G. Gimenez-Jurado, A.J. Cidadao and A. Beijn-van der Waaij

Fig. 2. Stephanoascus smithiae IGC 4646 x IGC 4647. TEM micrographs showing: (a) Longitudinal section through a mature ascus with septum delimiting mother ascus cell from elongated apical cell. (b) Transverse section through mature ascus with one hat-shaped ascospore. (c) Longitudinal section of cell wall showing septum and typical ascomycetous cell wall ultrastructure. The presence of a single, central, micropore in the cross wall is also suggested (see arrow). Insert bars represent 1.0 ~m.

ical or hat-shaped, with a thick basal brim. Asci were persistent and remained intact even in cultures that were three-months-old, though the ascospores were larger. Physiological characteristics. Fermentation capability, growth on carbon and nitrogen sources, as well as other properties are summarized in Table 2. Fine structure. Examination of ultrathin sections through mature asci revealed one hat-shaped ascospore with a thick electron-dense base on the ventral and dorsal sides of the spore (Fig.2a and b). Longitudinal sections

through mature asci confirmed the presence of an elone gated daughter or apical cell delimited from the mother ascus cell via the formation of a septum (Fig.2b). The ultrastructure of the cell wall is ascomycetous. The cell was is typically two-layered with a thick, electron-translucent inner layer and a thin, electron-dense outer layer (Fig.2c). Features of the fine structure of mycelial septa suggest the presence of a single, centrally located micropore, although no micropores could clearly be observed. Etymology. The specific epithet smithiae, latin gen. of

Stephanoascus smithiae sp. nov., the telemorph of Candida edax Table 2. Physiological characteristics of Stephanoascus smithiae sp'.nov.

241

Fermentation not observed Assimilation of carbon sources D. Glucose D-Galactose L-Sorbose D-Glucosamine D-Ribose D-Xylose L-Arabinose D-Arabinose L-Rhamnose Sucrose Maltose a, a-Trehalose Me-a-D-glucoside Cellobiose Salicin Arbutin Melibiose Lactose Raffinose Melezitose Inulin

+ + + + + + + + + + + + + + + + + + + +

Starch Glycerol m-Erythritol Ribitol Xylitol D-Glucitol D-Mannitol Galactitol m-Inositol D-Glucono-1,5 -lactone 2-keto-D-gfuconic acid D-Gluconic acid D-Glucuronic acid DL-Lactic acid Succinic acid Citric acid L-Tartaric acid D-Tartaric acid m- Tartaric acid Saccharic acid Malic acid

+W + + + + + + + + + + + + + + +D +D

Mucic acid L-Glutamic acid Methanol' Ethanol Butane-2,3-diol N-Hexadecane N-Butylamine N-Hexylamine Adenine Xanthine Uric acid Allantoin Acetoin Betaine N -Me-Glucamine N-Acetyl-Glucosamine Lysine Stachyose Palatinose 3-0-Methylglucose 2-Deoxy-D-Glucose

+ + + + + +

+ + +

Assimilation of nitrogen sources Nitrate Nitrite Ethylamine Cadaverine Creatine

+ + + +

Creatinine D-Glucosamine Betaine L-Glutamic acid N-Me-Glucamine

+ +

Other Characteristics Growth in vitamin-free medium Growth on 50% glucose yeast-extract agar Formation of starch-like compounds Hydrolysis of urea Colour reaction with Diazonium Blue B Growth with 0.01 % cycloheximide Growth with 0.1 % cycloheximide Growth in 10% NaCl/5% glucose Growth at 30°C Growth at 35 °C Growth at 3rC Lipolytic activity: Tween 80 Olive oil (5% v/v) Major ubiquinone Mol% G+C of nDNA

Positive Absent Negative Negative Negative Positive Positive Positive Positive Positive Negative Absent Absent 9 47.0 ± 0.2 (IGC 4646) and 47.2 ± 0.1 (IGC 4647)

(-) negative; (+) positive; (+ W) weak positive response; (+ D) delayed positive response.

width of (1.5-4.0) !lm and outgrowth or projections occurring at regular intervals along the hyphae, often with ovoid to globose blastoconidia (3.0-4.5) x (2.0-3.0) !lm. Sections of the septate hyphae may be coiled. Arthroconidia were not formed. Budding cells were scarce. Formation of asci and ascospores. Asci with ascospores were observed after 1 week incubation at 25°C on maltextract agar upon mating of strains IGC 4646 and IGC 4647 (Fig. 1) Ascus formation presumably occurs following fusion of hyphae, although fusion of hyphal cell walls

was not observed, giving rise to a globose or spherical preascus. Mature asci consisting of one hemispherical or helmet-shaped ascospore, measuring (5.0-8.0) x (3.0-5.0) !lm, emerged either terminally or intercalary on the hyphae, crowned with a persistant apical cell (Fig. 1) measuring (2.0-6.0) X (2.0-3.0) !lm, from which buds often arise. Ascospore shape varied with the angle of observation. From a rear view ascospores appeared to be spherical with spiny outgrowths and a thick sheath. However, from a lateral view spores appeared to be hemispher-

Stephanoascus smithiae sp. nov., the telemorph of Candida edax

243

Table 3. Differential characteristics of Stephanoascus and related taxa Part A. Utilization of selected compounds as carbon and energy sources! Species

Hex

Bty

Hyl

Ade

Xan

Ur

All

+W +W +

+

+

+

+

+ +

+

+

+

+

+

+ +

+ +

Ace

2,3-BD L-Tar3

m-Tar 3

Teleomorphs Stephanoascus Steph. ciferrii Steph. farinosus Steph. smithiae Zygoascus Z. hellenicus

n.d.

n.d. +D

+WD +WD

+ +

+ +W

+

+ +D

+ +

+ +

+

+W + + + +W +

+W +W +W +W +

+W

+W

V

Anamorphs Arxula A. adeninivorans A. terrestris Candida C. auringiensis C. bertae var. bertae C. bertae var. chiloensis C. blankii C. castrensis C. chiropterorum C. edax C. in communis C. paludigena C. santjacobensis C. valdiviana Candida sp. nov.

+ +

+ + + +W + +W +

+ + + + + + + + +

+W + + + + + +

+ + + +W

+W +W

+ +

+D

+ +D

+

+

+

Part B. Morphological and other characteristics Species

Septal ultrastructure

Steph. ciferrii Steph. farinosus Steph. smithiae Z. hellenicus A. adeninivorans A. terrestris C. auringiensis C. bertae var. bertae C. bertae var. chiloensis C. blankii C. castrensis C. chiropterorum C. edax C. incommunis C. paludigena C. santjacobensis C. valdiviana Candida sp. nov.

scattered micropores 4 one central micropore 4 one central micropore one central micropore s scattered micropores 6 scattered micropores 7 n.d. n.d. n.d. n.d. n.d. n.d. one central micropore n.d. n.d. n.d. n.d. n.d.

Arthroconidia

+

Two-celled aSCI

CoQ

Mol% G+C

Tmax (0C)

+ + +

94 94 9 95 97 97 n.d. n.d. n.d. n.d. n.d. n.d 9 89 n.d. n.d. n.d. n.d.

46.2-47.0 49.2±0.7 47.0-47.4 43.5 -44.9 5 48.5 ±0.5 45.0 8 46.5 ±0.9 45.6±0.3 45.8±0.1 54.1-55 9 44.3±0.1 48.0 9 47.4±0.1 48.9 9 37.8 ±0.2 48.5 ±0.6 55.3 9 40.0± 0.310

40-42 25-26 35-36 37-38 42-44 37-38 38-39 35-36 30-34 42-44 35-36 38-39 35-36 36-37 33-34 30-31 30-31 42-44

n.a. n.a. n.a. n.a. n.a. n.a n.a. n.a n.a n.a. n.a. n.a. n.a. n.a.

! Abbreviations: Hex = N-hexadecane, Bty = N-butylamine, Hyl = N-hexylamine, Ade = Adenine, Xan = Xanthine, Ur = Uric acid, All = Allantoin, Ace = Acetoin, 2,3,-BD = Butane-2,3-diol, L-Tar = L-tartaric acid, m-Tar = meso-tartaric acid, +, present; -, absent; ?, presence of arthroconidia not confirmed; n.a., not applicable; n.d., not determined. 2 At least two strains studied unless only one isolate is known for that species. 3 Data from Fonseca (1992). 7 Van der Walt et al. (1990). 4 Smith and Batenburg-Van de Vegte (1989). 8 Gueho et al. (1985). 5 Smith (1986). 9 Barnett et al. (1983). 6 Middelhoven et al. (1984). 10 A. Fonseca, personal communication.

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G. Gimenez-Jurado, A.J.Cidadao and A. Beijn-van der Waaij

Smith was chosen in honour of M. Th. Smith for her enormous contribution to the uncovering and understanding of filamentous ascomycetous genera, in particular Stephanoaseus, Zygoaseus and Arxula. Origin of strains studied. The holotype of the species is a dried specimen of a sporulating culture of the two mating types IGC 4646 and IGC 4647. The isotypes IGC 4646 and IGC 4647 were isolated in January 1990, from soil collected in the Horto botanical garden, Rio de Janeiro, Brazil. They have been deposited (living and dried) in the Portuguese Yeast Culture Collection in Oeiras (Portugal), and under number CBS 7522.1 and CBS 7522.2 in the Yeast Division of the Centraalbureau voor Schimmelcultures in Delft (Netherlands). Discussion The formation of gale ate ascospores within a persistent, globose, two-celled ascus arising from the fusion of two or more hyphal cells characterize the new species as a member of the genus Stephanoaseus Smith et al. (1976). Stephanoaseus and Zygoaseus are genera which closely resemble one another, but which can be differentiated by the number of asci per conjugation bridge and the presence or absence of mature asci with apical cells (Smith, 1986). Since the new species has asci with apical cells and no more than one ascus per conjugation bridge, it was considered adequate to classify our isolates as Stephanoaseus. Within the genus (Table 3), Steph. farinosus can be readily distinguished from Steph. smithiae by its psychrophilic character (Tmax = 25°C), slow fermentation of Dglucose and homothallism (De Hoog et aI., 1985), a character that is unique to this species, while the new species and Steph. eiferrii differ in numerous assimilation tests, maximum temperature for growth (T max = 40-42 0C), number of ascospores per asci and number and distribution of septal micropores (Smith et aI., 1976). Determination of the molar percentage G + C content (Table 3) of the three species in this genus showed no significant

differences between Steph. smithiae (47.0-47.4 mol%) and Steph. eiferrii (46.8-47.0 mol%), but a substanially higher G + C value was found for Steph. farinosus (49.2 mol%). Relative nDNA-nDNA reassociation (Table 4) between the isotype strains of Steph. smithiae (IGC 4646, IGC 4647) and the isotype strain of Steph. eiferrii (IGC 4165) gave values that ranged between 34 and 38%. However, the significant DNA complementarity did not correlate with the phenotypic and fertility data, which jus" tifies the separation of these taxa. In the present study, a group of anamorphic ascomycetous yeasts showing striking phenotypical similarities with the new Stephanoaseus teleomorph and a second teleomorphic genus Zygoaseus were selected for comparison. The imperfect taxa included in this study, belonging to the genera Candia or Arxula (Table 1), have no known sexual stages, produce copious amounts of septate hyphae with condidia borne on denticles and all have the ability to grow on myo-inositol as a sole source of carbon. This preliminary survey yielded two Candida species, Candida edax van der Walt and Nel (1968) and Candida bertae Ramirez and Gonzalez (1984), virtually identical in phenotype with the new Stephanoaseus teleomorph. Subsequent determination of the molar % (G+C) content revealed that C. edax shares a similar DNA base composition (47.4% ± 0.1), while those of C. bertae var. bertae (45.6 ± 0.2 mol%) and var. ehiloensis (45.8 ± 0.1 mol%) are significantly lower. Determinations of DNA-DNA reassociation between the isotypes of Steph. smithiae and the corresponding type strains gave values of 95-99 % with C. edax and 11-15% extent of relatedness with the two varieties of C. bertae, thus demonstrating conspecificity with the former species. Mating studies performed between the type strain of C. edax (IGC 4183) and the isotype strains of Steph. smithiae resulted in a reaction with the mating type of Steph. smithiae designated as alpha (IGC 4647), producing mature asci with viable ascospores. In view of the results we may conclude that C. edax represents the anamorph of Steph. smithiae. For comparative purposes, 18 species belonging to the teleomorphic genera Stephanoaseus and Zygoaseus, to the

% DNA-DNA reassociation

with Steph. smithiae 1

Species

Strain

IGC 4646

IGC 4647

IGC IGC IGC IGC

4646 4165 4592 4839

38±4.1 1±2.0 9±0.9

99±0.5 34 ± 1.4 3±0.5 4±0.5

IGC 4183 IGC 4636 IGC 4637

95±0.2 14±1.7 15 ± 1.5

99 ± 1.0 13 ± 1.2 11 ±0.3

Teleomorphs

Stephanoascus smithiae Stephanoascus eiferrii Stephanoascus farinosus Zygoascus hellenicus Anamorphs

Candida edax Candida bertae var. bertae Candida bertae var. chiloensis 1

Mean ± s.d. based on three determinations.

Table 4. Extent of DNA relatedness between Stephanoascus smithiae and closely related anamorphic and teleomorphic yeast species

Stephanoascus smithiae sp. nov., the telemorph of Candida edax

anamorphic genus Arxula, and including all inositol-assimilating Candida species, were tested for utilization of nhexadecane, n-butylamine, n-hexylamine, adenine, xanthine, uric acid, allantoin, acetoin, and butane-2,3-diol as sole sources of carbon and energy. The results are summarized on Table 3 (part A). Twelve out of the 18 species can grow on n-hexadecane, not an uncommon property among yeasts. According to Bas and de Bruyn (1973) hydrocarbon-assimilating species have three characteristics in common: petite-negative, ability to grow on lysine medium and predominance of the coenzyme Q-9 system. More recently Hofmann and Schauer (1988) extended this correlation to include a few ascomycetous yeasts with CoQ8, such as Hyphopichia burtonii and Candida incommunis. In a study of yeasts growing on purines and amines Middelhoven et al. (1985) concluded that among ascomycetous yeast species utilization of uric acid or amines as sole carbon source is restricted to hydrocarbon-positive species. While this pattern was observed in all species of the genera Stephanoascus and Arxula, which contain ubiquinone Q-9 system, as well as in Yarrowia (Hofmann and Schauer 1988), another ascomycetous hyphal yeast genus with CoQ-9, this correlation did not hold for C. auringiensis, C. castrensis, C. paludigena, Candida sp. nov., and Z. hellenicus (Table 3, part A). Steph. farinosus assimilates nhexadecane, but it does not grow on any of the alkylmonamines or diamines tested, viz. ethylamine, n-butylamine and putrescine (Middelhoven et aI, 1989). In addition, this species has the uncommon feature among the group of yeasts examined of not growing on myo-inositol (Barnett et al., 1983). The position of this taxon in the genus Stephanoascus seems somewhat tenuous considering its distinctive characteristics. Nevertheless, ascospore morphology, ubiquinone Q-9 system and fine structure are consistent with its placement in that genus. Hydrocarbon-positive Candida species include C. bertae, C. blankii, and C. valdiviana, and weak growth was obtained for C. chiropterorum and C. incommunis. C. santjacobensis holds an unusual position among this group of inositolpositive species (with the exception of Steph. farinasus) in that it does not assimilate any of the substrates tested. C. bertae, phenotypically similar to Steph. smithiae, can be differentiated from the new species by its ability to utilize xanthine, uric acid and L-tartaric acid. Steph. smithiae can be further distinguished from C. bertae var. bertae by the capacity to grow on butane-2,3-diol and from varietY chiloensis by being capable of using hexylamine as carbon source. Utilization of adenine, like N-hexadecane, appears to be an unusual characteristic among yeasts. Middelhoven et al. (1984) isolated a new species, Arxula adeninivorans, in selective media with adenine as the sole source of carbon, nitrogen and energy. Since then, adenine assimilation has been demonstrated for a number of ascomycetous yeasts and yeast-like fungi, namely Steph. ciferrii, Arx. terrestris, one strain of Geotrichum fermentans and Blastobotrys proliferans (Middelhoven et a!., 1989). In the present investigation another yeast with this characteristics was recognized, C. chiropterorum. In addition to adenine, this species has the unusual capacity to assimilate allantoin as

245

sole source of carbon, a characteristic only known to occur, among the yeasts, in the species Steph. ciferrii (Middelhoven et aI., 1985). Even among yeast-like fungi, this property has only been found in B. proliferans and in one strain of G. fermentans. All strains capable of utilizing adenine as a carbon and energy source can also grow on their degradation products, xanthine and uric acid. Acetoin assimilation was found in three yeast species. C. incommunis, C. paludigena and Z. hellenicus, although in the latter it was a variable property. Assimilation of this compound in the Dipodascaceae was reported in B. proliferans, Galactomyces reessii, G. candidum, G. klebahnii, several species in Dipodascus and Lipomyces starkeyi as well as in several species included in the Euascomycetes (Middelhoven et aI., 1989). There appears to be a correlation between adenine, allantoin, and acetoin assimilation in yeasts or yeast-like fungi and the presence of CoQ-9 system. In a recent survey of yeasts capable of utilizing tartaric acid as carbon source, Fonseca (1992) demonstrated that this caracteristic is primarily observed in yeasts of basidiomycetous affinity. The few ascomycetous yeasts that were able to utilize at least one of the tartrate isomers belonged to the species Steph. smithiae, C. bertae, C. paludigena and Candida sp. nov., data corroborated by our results. As pointed out by others, assimilation of nonconventional compounds may prove to be a valuable taxonomic tool for distinguishing certain species or groups of orgalllsms. Recently, Steph. ciferrii, Arx. adeninivorans and Arx. terrestris have been shown to be closely related by molecular phylogeny studies based on the analysis of partial sequences of 18S and 26S ribosomal RNAs (Yamada et al., 1990), in addition to other common taxonomically important characteristics: CoQ-9 system, relatively high optimum temperature, xerotolerance, multiperforate septa and blastoconidia borne on denticles. As shown above, several anamorphic species classified in Candida show striking morphological and physiological resemblances with the genera Stephanoascus, Zygoascus and possibly Yarrowia. These perfect genera were recently excluded from the Endomycetaceae (Von Arx and van der Walt, 1987) and so far no attempt has been made to classify these hyphal yeasts in a separate family. Questions concerning the relationships within this group of yeasts, which includes three perfect and two imperfect genera, and the links to species classified in Sporothrix sect. Farinosa as delimited by Smith and Batenburg-van der Vegte (1986) need to be addressed in the future by applying useful morphological and physiological criteria as well as molecular biology methods and knowledge of their ecology. Acknowledgments. The authors are indebted to Prof. Isabel Spencer-Martins for highly valuable advise and critical reading of the manuscript, to Maria foao Nolasco for the soil sample that permitted the isolation of the new teleomorph, to Ema Fonseca,

Gulbenkian Institute of Science, for HPLC analysis of the extracted ubiquinones of Steph. smithiae, and to David Yarrow, Yeast Division, CBS, Delft, for providing the type strains used in this study and for critical reading of the manuscript.

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G. Gimenez-Jurado, A.J. Cidadao and A. Beijn-van der Waaij

References Barnett, J. A., Payne, R. W., Yarrow, D.: In: Yeasts: Characteristics and identification. Cambridge, Cambridge Univ. Press., 1983 Bos, P., de Bruyn, j. C: The significance of hydrocarbon assimilation in yeast identification. Antonie van Leeuwenhoek 39, 99-107 (1973) De Hoog, G. S., Rantio-Lehtimiiki, A. H., Smith, M. Th.: Blastobotrys, Sporothrix and Trichosporiella: generic delimitation, new species, and a Stephanoascus teleomorph. Antonie van Leeuwenhoek 51, 79-109 (1985) Fonseca, A.: Utilization of tartaric acid and related compounds by yeasts: taxonomic implications. Can. J. Microbiol. 38, 1242-1251 (1992) Gimenez-Jurado, G., Placido, T., Cidadao, A.j., Cabe~a-Silva, C, Fonseca, E., Roeijmans, H.j., Eijk, G. W., van Uden, N.: Kurtzmanomyces tardus sp. nov., a new anamorphic yeast species of basidiomycetous affinity. Antonie van Leeuwenhoek 58, 129-135 (1990) Gueho, E., Tredick, j., Phaff, H.j.: DNA relatedness among species of Geotrichum and Dipodascus. Can. J. Bot. 63, 961-966 (1985) Hofmann, K. H., Schauer, F.: Utilization of phenol by hydrocarbon assimilating yeasts. Antonie van Leeuwenhoek 54, 179-188 (1988) Kurtzman, C P., Smiley, M.j., Johnson, Cj., Wickerham, L.j., Fuson, G. B.: Two closely related heterothallic species, Pichia amylophila and Pichia mississipiensis: Characterization by hybridization and deoxyribonucleic acid reassociation. Int. J. System. Bacteriol. 30, 208-216 (1980) Marmur, j., Doty, P.: Determination of the base composition of DNA from its thermal denaturation temperature. J. Mol. BioI. 5, 109-118 (1962) Middelhoven, W.j., Hoogkamer-Te, N., Kreger-van Rij, N. j. W.: Trichosporon adeninovorans sp. nov., a yeast species utilizing adenine, xanthine, uric acid, putrescine and primary n-alkylamines as sole source of carbon, nitrogen and energy. Antonie van Leeuwenhoek 50, 369-378 (1984) Middelhoven, W.j., de Kievit, H., Biesbroek, A. L.: Yeasts species utilizing uric acid, adenine, n-alkylamines or diamines as sole source of carbon and energy. Antonie van Leeuwenhoek 51, 289-301 (1985) Middelhoven, W.j., de Hoog, G. S., Notermans, D.: Carbon assimilation and extracellular antigens of some yeast-like fungi. Antonie van Leeuwenhoek 55, 165-175 (1989) Ramirez, C, Gonzalez, A.: Two new species and one variety of nitrate-utilizing mycelial Candida isolated from decayed wood

in the evergreen rainy Valdivian forest of southern Chile. Mycopathologia 88, 55-60 (1984) Seidler, R.J., Mandel, M.: Quantitative aspects of deoxyribonucleic acid renaturation: Base composition, site of chromosome replication and polynucleotide homologies. J. Bacteriol. 106, 608-614 (1971) Smith, M. Th., van der Walt, j. P., Johannsen, E.: The genus Stephanoascus gen. nov. (Ascoideaceae). Antonie van Leeuwenhoek 42, 119-127 (1976) Smith, M. Th., Batenburg-van der Vegte, W. H.: Ultrastructure of septa in Blastobotrys and Sporothrix. Antonie van Leeuwenhoek 51, 121-128 (1985) Smith, M. Th., Batenburg-van der Vegte, W. H.: Additional information on the ultrastructure in the genus Sporothrix. J. Gen. Appl. Microbiol. 32, 549-552 (1986) Smith, M. Th.: Zygoascus hellenicus gen. nov., sp. nov., the teleomorph od Candida hellenica (= C inositophila = C steatolytica). Antonie van Leeuwenhoek 52, 25-37 (1986) Van der Walt, j. P., Nel, E. E.: Candida edax sp.n. Antonie van Leeuwenhoek 34, 106-108 (1968) Van der Walt, J. P., von Arx, j. A., Liebenberg, N.v.d. W.: Multiperforate septa in Geotrichum and Dipodascus. S. Afr. J. Bot. 2, 184-186 (1983) . Van ~er Walt, j.P., Yarrow, D.: Methods for the isolation, mamtenance, classification, and identification of yeasts, pp. 45-104. In: The Yeasts: A Taxonomic Study. Amsterdam, Elsevier Science Publ., 1984 Van der Walt, J. P., Smith, M. Th., Yamada, Y.: Arxula gen. nov. (Candidaceae), a new anamorphic, arthroconidial yeast genus. Antonie van Leeuwenhoek 57,59-61 (1990) Van Uden, N.: Transport-limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition. Archiv fur Mikrobiologie 58, 155-168 (1967) Von Arx, j. A., van der Walt, j. P.: Ophiostomatales and Endomycetales, pp. 167. In: The Expanding Realm of Yeast-like Fungi. Amsterdam, Elsevier Science Pub!., 1987 Weijman, A. C M., de Hoog, G. S.: Carbohydrate patterns and taxonomy of Sporothrix and Blastobotrys. Antonie van Leeuwenhoek 51,111-120 (1985) Yamada, Y., Smith, M. Th.: The Coenzyme Q system in strains of species in the genera Stephanoascus and Sporopachydermia (Saccharomycetaceae). Trans. mycol. Soc. Japan 26, 247-251 (1985) Yamada, Y., Nogawa, C: The molecular phylogeny of the ascomycetous yeast genus Arxula Van der Walt, Smith et Yamada based on the partial sequences of 185 and 26S ribosomal ribonucleic acids. 14th International Specialized Symposium on Yeasts, Smolenice, Czechoslovakia, Abstract p.27 (1990)

G. Gimenez-Jurado, Laboratory of Microbiology, Gulbenkian Institute of Science, Apartado 14, P-2781 OEIRAS Codex, Portugal