Antonie van Leeuwenhoek 59: 81-93, 1991. �9 1991 Kluwer Academic Publishers. Printed in the Netherlands.

BuUeromyces genus novum (Tremellales), a teleomorph for BuUera alba, and the occurrence of mating in Bullera variabilis

Teun Boekhout x, Alvaro Fonseca 2 & Wilma H. Batenburg-van der Vegte 3 1 Centraalbureau voor Schimmelcultures, Yeast Division, Julianalaan 67, 2628 BC Delft, The Netherlands; : Laboratory of Microbiology, Gulbenkian Institute of Science, 2781 Oeiras Codex, Portugal; 3 Kluyver Laboratory for Biotechnology, Department of Microbiology and Enzymology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands

Received 9 May 1990; Accepted 19 November 1990

Key words: Bullera, Bulleromyces, taxonomy, morphology, teleomorph, ultrastructure

Abstract

Mating is observed in Bullera alba and B. variabilis, resulting in the formation of dikaryotic mycelium with clamps, haustorial branches, and lateral and terminal dikaryotic, clavate, lageniform or subglobose cells. These cells develop in B. alba into tremellaceous phragmobasidia. Karyogamy has been observed in young non-divided basidia. Germination of the phragmobasidia occurred by acropetal chains of yeast cells, ballistospores or hyphae. Septal pores are dolipores with parenthesomes made up of U-shaped vesicles (Tremellales type). For the teleomorph of B. alba a new genus, Bulleromyces, is proposed, with only one species, viz. Bulleromyces albus.

Introduct ion

Ballistospore-forming, heterobasidiomycetous im- perfect yeasts, belonging to the genera Sporobolo- myces and Bullera, were thus far classified in the Sporobolomycetaceae, because of the presence of ballistospores (Derx 1948; Von Arx et al. 1977). However, in recent years cell wall composition and

the coenzyme Q system proved to be different among these two genera (Weijman & Rodrigues de Miranda 1983; Nakase & Suzuki 1986; Suzuki & Nakase 1988). Further it turned out that perfect states of several species of Sporobolomyces, viz. S. salmonicolor and S. shibatanus, belong to the ge- nus Sporidiobolus, which is characterized by the

presence of a clamped dikaryotic mycelium, on which teliospores are formed (Fell & Statzell Tall- man 1980, 198t). Van der Walt (1973) recognized Aessosporon dendrophilum as the perfect state of

Bullera dendrophila. This author assumed that di- ploidization was effected by somatogamous auto- gamy, leading to the formation of teliospores. No dikaryotic mycelium was present. Bandoni (1987) reported mating, and the formation of a dikaryotic mycelium among strains of B. alba. However, he did not observe teliospores and/or basidia.

In the present investigation the life cycle of Bul- lera was studied by means of mating experiments among strains of species of Bullera. This resulted in mating, and formation of dikaryotic mycelia in both B. alba and B. variabilis. Moreover, in B. alba tremellaceous phragmobasidia were observed, in which karyogamy occurred. This state is described as the teleomorph of the genus Bullera, for which the name Bulleromyces is proposed.

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C

Fig. 1. Morphology of Bulleromyces albus and the dikaryophase of Bullera variabilis on cornmeal agar. A-C, Bulleromyces albus. (A) Hyphae with lateral clavate to lageniform cells, and phragmobasidia (CBS 6032 • CBS 7441). (B) Germinating basidia (CBS 6032 x CBS 7441). (C) Conjugation between yeast cells, and haustorial branch (arrow) (CBS 7440 x CBS 7441). (D) Bullera variabilis, dikaryotic hyphae with haustorial branches (arrows), clavate, fusiform to lageniform cells, and subglobose cells (CBS 7347 x CBS 7367). Bar indicates 10 t~m.

Materials and methods

The following strains were studied in the present investigation: Bullera alba CBS 500, type strain, ex rust-infected straw of Hordeum ]ubatum, USA; CBS 501, ex air in dairy, USA; CBS 502, ex air in dairy, USA; CBS 6302, ex grass in Everglades, Florida, USA; CBS 7440 (ICG 4539), ex dead leaf of Platanus sp., Portugal; CBS 7441 (ICG 4538), ex

leaf of Ligustrum sp., Portugal; CBS 7503 (ICG 4560), ex leaf of Juglans regia, Portugal; UBC 70- 8014, Canada; UBC 70-8080, Canada; UBC 70- 8082, Canada. Of Bullera variabilis the following strains were used: CBS 7354 (JCM 3914), ex dead base of bamboo culm, Canada; CBS 7347 (JCM 5275), type strain, ex dead leaf of Oryza sativa, Japan; CBS 7364 (JCM 5984), ex dead leaf of Sasa species, Japan; CBS 7365 (JCM 7265), ex dead leaf

of Miscanthus sinensis, Japan; CBS 7366 (JCM 7266), ex dead leaf of Miscanthus sinensis, Japan; CBS 7367 (JCM 5661), ex dead leaf of Miscanthus sinensis, Japan.

Strains were maintained on 1% yeast extract-0.5 % peptone-4% glucose agar (YPGA) or 5% malt ex- tract agar (MEA). Matings were performed at cornmeal agar (CMA, Difco) or potatodextrose agar (PDA, Oxoid) at 17~ or room temperature as follows: two mating partners were thoroughly mixed near the margin of the plates, after which some streaks were made on the plates, or two mat- ing partners were mixed in a suspension, which was poured on the plates. The plates were weekly in- vestigated during two months.

Comparative nutritional tests were performed according to Van der Walt & Yarrow (1984) at 17 ~ C.

For ultrastructural studies hyphae were fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.0) to which 4% tannic acid was added, fol- lowed by fixation with 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.0) for 60 rain. Mate- rial was also fixed with 1.5% v/v aequeous potassi- um permanganate for 20 min. All these treatments were followed by staining with 1.5 % uranyl acetate in 50% v/v ethanol for 30rain, followed by de- hydration through a graded ethanol series and em- bedding in Spurr's resin.

For SEM unfixed hyphae were examined in fro- zen hydrated state with low temperature SEM (Staugaard et al. 1989).

Karyology was studied by the mithramycine/eth- idium bromide method as described by Barlogie et al. (1976), or on HC1-Giemsa stained preparations (Streiblov~i 1988). Preparations were investigated by fluorescence microscopy using a Zeiss Axioskop with a Ploemopak fluorescence device with filter combination 14 (excitation 590 rim). Photographs were made with a Zeiss M35 photocamera using Ilford PanF 50 ASA films.

Results

Strains of Bullera alba mated only on CMA. Varia- ble hyphal development was observed in the differ-

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ent matings. Transfer of hyphae and/or conjugated cells to PDA resulted in more extensive hyphal development. Two groups of strains were found compatible. The first mating group comprised CBS 500, CBS 7441 and CBS 7503, the second group consisted of CBS 501, CBS 6302, CBS 7440 and UBC 70-8014. Compatible strains formed lateral submerged mycelium after mating. Mating in- volved formation of conjugation tubes, followed by the subsequent formation of dikaryotic mycelium (Figs 1A-C, 2D-E).

The dikaryophase was made up of slender, regu- larly branched hyphae, measuring 40-130 x 1.5- 3.0/zm, and which were regularly septate. Some- times hyphae formed lateral, wart-like outgrows. True clamps were present at the septa. At part of the clamps small, c. 3.5-5.0 • 2.5-3.5/zm, clavate cells occurred, on which slender, c. 1/zm wide, branched or unbranched, haustorial branches were formed (Fig. 1C). The haustorial branches and the cells from which they originated were monokaryot- ic. Besides these haustorial branches, larger cla- vate, lageniform, ovoidal to subglobose cells oc- curred laterally or terminally at the hyphae. These cells measured 5.0-10.0 • 4.0-7.0 ~m, were dika- ryotic, clamped at their base, and sometimes grew terminally with clamped hyphae of normal width (Figs 1A, 2D, 3B, 4C). Frequently, these cells de- veloped into subglobose or clavate basidia, mea- suring 9.0-18.0 x 7.0-12.5/zm (Figs 1A-B, 2F-E, 3C, 4B). The number of basidia formed in each mating was highly variable. The basidia were strongly vacuolated and refractile. They finally di- vided, more or less longitudinally, into two to four cells. However, the mode of septation of the basi- dia was rather variable. Apart from strictly longitu- dinally septate phragmobasidia, also obliquely or transversely septate phragmobasidia frequently oc- curred.

Small, young basidia were still dikaryotic, but during enlargement they became monokaryotic (Figs 3C-D). The nuclei of these monokaryotic basidia were distinctly larger, if compared with the nuclei of the isolated yeast cells or the nuclei of the dikaryon. Prior to division of the basidium, meiosis seems to occur. As a consequence, the individual cells of the phragmobasidium were monokaryotic.

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Germination of the phragmobasidia occurred in a variable manner. In most cases short acropetal chains of monokaryotic ellipsoidal, fusiform or cy- lindrical yeast cells, measuring 5.0-17.0 x 2.5- 5.0/xm, were formed on short denticles or on atten- uated or somewhat fusiform sterigmata of up to c. 18/~m long (Figs 1A-B). A second mode of germi- nation was the formation of rotationally or slightly bilaterally symmetrical ballistospores, which mea- sure 4.5-8.0 • 3.7-5.5 ~m (Fig. 1B), on sterigmata of variable length (up to c. 12/xm). The third ob- served type of germination was the formation of hyphae. These terminated with an attenuate apex on which subglobose ballistospores were formed, or with a blunt apex on which either laterally or terminally yeast ceils originated. The meiospores were monokaryotic, and the size of the nuclei was found comparable to that of the nuclei in the dika- ryophase or the solitary yeast phase. The meios- pores frequently occurred aggregated in clusters.

Conjugation occurred also between hyphae (Fig. 4A). Strain CBS 7441 deviated from the other strains in being partially self sporulating. In this case conjugation tubes, clamped dikaryotic myceli- um, haustorial branches, globose to elongate cells and few basidia were formed.

Septal pores were investigated in the following three mating combinations: CBS 7440x CBS 7441, CBS 500 x CBS 7440, and CBS 6302 x CBS 7441. The septa of the resulting hyphae contained dolipores with one central transverse electron dense band in the pore canal, and a transverse strand of an endoplasmic reticulum-like structure just outside the pore canal. The parenthesome con- sisted of U-shaped vesicles in which the inner mem- brane was thickened (Fig. 5B)

Fig. 2. Morphology of Bulleromyces albus and the dikaryophase of Bullera variabilis on cornmeal agar. (A-C) Bullera variabilis (CBS 7347 x CBS 7367). (A-B) Lateral and terminal subglo- bose cells. (C) Haustorial branch. (D-G) Bulleromyces albus. (D) Mycelium with damps and lateral, clavate, subglobose to lageniform cells, which may grow out terminally with hyphae (CBS 500 x CBS 6302). (E) Young basidia and ballistospore (arrow). (F-G) Germinating phragmobasidia (E-G all CBS 6302 • CBS 7441). Bars indicate 10tzm.

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Table 1. Nutritional physiological characteristics of mating part- ners of Bulleromyces albus. Growth after 3 weeks at 17~ (+ growth positive, d growth delayed, w growth weak, - no growth, brackets indicate occasional deviations from general pattern).

D-Glucose + Glucono-8-1acton + , d D-Galactose + (d) 2-Keto-D-gluconate + L-Sorbose + , d ( - ) D-Gluconate + (d) D-Glucosamine + ( - ) D-Glucuronate + D-Ribose + DL-Lactate + , w D-Xylose + Succinate + L-Arabinose + Citrate + D-Arabinose + Methanol - L-Rhamnose + Ethanol v Sucrose + Glycol - Maltose + Propane-l , 2-diol - ct, ct Trehalose + Butane-2, 3-diol - Methyl ct-glucoside + Nitrate - Cellobiose + Nitrite - Salicin + (d) Ethylamine + ( - ) Arbutin + L-Lysine + Melibiose + ( - ) Cadaverine v Lactose + (d) Creatine - Raffinose + Creatinine - Melezitose + Imidazole - Inulin + , d w/o Vitamins - Soluble Starch + ( - ) w/o Inositol + Glycerol + , d w/o Pantothenate + Meso-erythritol v w/o Biotin + Ribitol + w/o Thiamin - Xylitol + w/o Biotin + Thiam. - L-Arabinitol + , d w/o Pyridoxine + D-Glucitol + (d) w/o Pyrid. + Thiam. - D-Mannitol + (d) w/o Niacin + Galactitol + , d ( - ) w/o PABA + Myo-inositol +

Nutritional physiological characteristics of Bul- lera alba are presented in Table 1.

In Bullera variabilis mating was only observed on CMA between CBS 7347 and CBS 7367. Conjuga- tion resulted in a regularly branched, dikaryotic mycelium with clamps at the septa (Fig. 1D). Cells measured 40-120 • 1.8--3.0/zm. At clamps c. 1/~m wide haustorial branches were formed on a clavate base, measuring 3.5-5.0 x 2.5-3.5/zm (Fig. 1D, 2C). The haustorial branches were monokaryotic. Dikaryotic globose, clavate, cylindrical to lageni- form cells, measuring 7-27 x 4.0-6.5/zm, were formed laterally and terminally on the hyphae (Figs 1D, 2A-B). No basidial development was ob- served.

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Fig. 3. Karyology of Bulleromyces albus on cornmeal agar. (A) Dikaryotic hyphae (CBS 6302 • CBS 7441). (B) Lateral dikaryotic lageniform cells (CBS 7440 x CBS 7441). (C) Dikaryotic hyphae with young, dikaryotic basidium (arrow) (CBS 6302 x CBS 7441). (D) Monokaryotic basidium, note intensity of fluorescence (CBS 500 x CBS 7440). Bars indicate in 10~m.

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Fig. 4. Morphology of Bulleromyces albus (SEM). (A) Anastomosis between hyphae (• 4.500). (B) Young phragmobasidium with clamp (• 2.800). (C) Lateral clavate to lageniform cells, which grow out as hyphae (x 3.600) (all CBS 500 • CBS 7440).

Septal pores were dolipores with up to six, trans- verse, electron dense bands inside the pore canal, and an electron dense band just outside the pore canal. Parenthesomes made up of U-shaped ves- icles (Tremellales type) were observed after fixa- tion with Glutaraldehyde and OsO4 (Fig. 5A). However, after fixation with KMnO4, no parenthe- some structure was seen. In this case strands and vesicles of endoplasmic reticulum occurred in the cytoplasm near the electron dense band just out- side the pore canal.

Discussion

Mating in Bullera alba, resulting in dikaryotic my- celium with haustorial branches, has already been observed by Bandoni (1987). However, that author did not observe basidia and septal pore structures. The longitudinally, obliquely or occasionally trans- versely septate basidia, formed by compatible mat- ing strains of B. alba, clearly point to a Tremellace- ous relationship. This is supported by the presence of haustorial branches (Bandoni 1987). Tremel-

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Fig. 5. Septal ultrastructure of Bulleromyces and the dikaryophase of Bullera variabilis. (A) B. variabilis (CBS 7347 x CBS 7367, x 100.000, Glutaraldehyde + Tannic Acid). (B) BuUeromyces albus (CBS 500 x CBS 7440, • 100.000, Glutaraldehyde + Tannic Acid).

lales, according to Bandoni (1984, 1987), are char- acterized by haploid yeast phases, dolipore septa, Tremella type of parenthesomes consisting of U- shaped vesicles, basidia containing 2 or 4 meios- pores, and, as additional criteria, they frequently grow as mycocoparasites, and form haustorial branches.

Tremellales affinity for the anamorph-genus Bullera has been suggested earlier. Flegel (1976) reported the formation of subglobose, apiculate ballistospores, similar to those formed in Bullera, in the yeast phase of Sirobasidium magnum. Ban- doni (1987) assigned a dikaryotic form of a Bullera species to the Tremellales. The nucleotide se-

quence of the 5S rRNA of Bullera alba was also found to be similar to that of other Tremellales, Exidiales and Agaricales (Gottschalk & Blanz 1985; Blanz & Unseld 1987). This latter characteristic dearly separates Bullera from Sporobolomyces.

The dolipore and parenthesome complex we ob- served in the dikaryotic hyphae of B. alba and B. variabilis is typical for Tremellales (Bandoni 1987; Moore 1987), and it is similar to the septal pores observed in e.g. Tremella fuciformis (Khan 1976), T. brasiliensis (Moore 1978), T. mesenterica (Moore 1978; Patrignani et al. 1984), Sirobasidium magnum (Moore 1979), and Filobasidium capsuli- genum (Kreger-van Rij & Veenhuis 1971; Moore & Kreger-van Rij 1972). This pore structure also dis- tinguishes Bullera from Bensingtonia, Sporobolo- myces and Sporidiobolus, which have diaphragma- like septa attenuating towards a central pore (Kreger-van Rij & Veenhuis 1971; Boekhout, un- publ.).

Haustorial branches formed in the dikaryophase of Bullera originate on clamps, and are monoka- ryotic, as in Sterigmatosporidium (Kraepelin & Schulze 1982) and Christiansenia pallida (Oberwin- kler et al. 1984). Probably this monokaryotic char- acter controls the outgrowth of narrow haustorial branches. It strongly reminds one of the formation of narrow germ tubes in monokaryotic yeast phases of Itersonilia, whereas in the dikaryophase of that genus such narrow outgrowths are not formed (Boekhout, unpubl.). The occurrence of mono- karyotic haustorial branches also indicates the presence of some monokaryotization mechanism of the dikaryophase of Bullera, which also seems to appear in Sterigmatosporidium and Christiansenia pallida.

Strains of B. alba can be divided into three groups using compatability as a criterium. Group one comprises strains CBS 500, CBS 7441 and CBS 7503, a second group comprises CBS 501, CBS 6302, CBS 7440 and UBC 70-8014, and a third group consists of CBS 502, UBC 70-8080 and UBC 70-8082. Strains belonging to the first two groups are compatible and mating occurs between, but not among these groups. Strains belonging to the third group do not show any mating responses. This indicates the presence of at least two mating types.

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However, strains of the third group also belong to B. alba as can be concluded from morphological, physiological and biochemical data (Boekhout, un- publ.). Because strains belonging to the third group do not show any mating response we infer the presence of more then two mating types. Prob- ably a tetrapolar mating system is operative as this also occurs in other Tremellales (Bandoni 1963, 1965, 1987; Flegel 1976; Wong 1987). For further elucidation of the mating system, isolation of meiospores, subculturing and further mating ex- periments are necessary. CBS 7441 is partially self- sporulating, and forms a clamped mycelium with haustorial branches. The presence of both hetero- thallic and self-sporulating strains is also known in other heterobasidiomycetous yeasts, e.g. Filobasi- diella neoformans (Erke 1976; Kwon-Chung 1978; Schmeding et al. 1981), and Leucosporidium (Fell et al. 1969).

Of the six strains of B. variabilis only the combi- nation CBS 7347 • CBS 7367 formed conjugation tubes, followed by formation of dikaryotic myceli- um with haustorial branches, bearing lateral and terminal, clavate, lageniform or subglobose cells. No basidial development has been seen. The rea- son for this incompatibility is not known, but it may be due to genetic and/or environmental factors. Absence of karyogamy has also been observed in dikaryons of Itersonilia perplexans (Olive 1952; So- well & Korf 1960; Boekhout 1991). However, from a morphological point of view, the dikaryotic phase of B. variabilis is very similar to that of B. alba.

Septal pores of the dikaryophase of B. variabilis are dolipores with electron-dense banding inside the pore canal, and a band of electron dense mate- rial at each end of the pore canal. Parenthesomes are made up of U-shaped vesicles. The pore struc- ture of the dikaryon of B. variabilis is similar to that observed in B. alba. The presence of a parenthe- some made up of U-shaped vesicles clearly points towards a relationship within the Tremellales.

Aessosporon dendrophilum van der Walt was considered to represent the teleomorph of Bullera dendrophila. Thick-walled, lipid-rich resting spores of the latter species were interpreted as teliospores, in which diploidization was supposed to occur by somatogamous autogamy (van der Walt

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1973). No dikaryophase was found, and no experi- mental evidence for the occurrence of haploid and diploid generations of cells was presented. Conse- quently, the distinction of Aessosporon has to be rejected.

Bulleromyces clearly differs from other hetero- basidiomycetous teleomorph yeast taxa by the for- mation of ballistospores, the presence of longitudi- nally or obliquely septate phragmobasidia, and the absence of teliospores and chlamydospores. Other non-teliospore forming genera, Filobasidium and Filobasidiella, share the presence of xylose in the cell walls and a similar coenzyme Q-10 system. But these genera can be readily distinguished by one- celled, clavate basidia (Kwon-Chung & Fell 1984a, 1984b; Kwon-Chung 1987). The life cycle of Ster- igmatosporidium Kraepelin & Schulze (1982) agrees in many respects with that of Bulleromyces. Both genera have a coenzyme Q-10 system (Yama- da & Banno 1984; Nakase & Suzuki 1986), xylose in the cell walls (Yamada et al. 1988), and form a dikaryotic clamped mycelium after conjugation, on which dikaryotic, clavate cells are formed. Ster- igmatosporidium differs from Bulleromyces by the formation of dikaryotic chlamydospores, the site of karyogamy, formation of conidia on sterigma-like outgrowths, and the absence of ballistospores (Kraepelin & Schulze 1982).

We prefer to place Bulleromyces in the Tremel- lales, rather than in the Filobasidiales, because of 1. formation of typically Tremellales basidia, and 2. presence of dolipores with parenthesomes of the Tremellales type. However, these two orders share many similarities, e.g. the presence of xylose in cell walls (von Arx & Weijman 1979; D6rfler 1990), coenzyme Q-9 or Q-10 composition (Yamada & Kondo 1973; Yamada et al. 1987), dolipore mor- phology (Khan & Kimbrough 1982; Moore 1987), and the presence of haustorial branches. Parenthe- some morphology on its own cannot be used to separate these two orders, as the parenthesome of Filobasidium capsuligenum is made up of U-shaped vesicles, whereas pores of Filobasidium floriforme lack parenthesomes (Moore & Kreger-van Rij 1972). In conclusion only basidium morphology seems a useful criterion to separate these two or- ders. From that point of view, the Tremellales have

longitudinally, obliquely or, more rarely, trans- versely septated basidia (Jiilich 1981, Bandoni 1987), and the Filobasidiales have one-celled cy- lindrical or capitate basidia (Jiilich 1981; Oberwin- kler 1987; Kwon-Chung 1987).

Taxonomic part

Bulleromyces Boekhout & A. Fonseca genus novum

Hyphae dikaryotic, regularly branched, and sep- tare; clamps present; haustorial branches mono- karyotic, branched or unbranched, formed on cla- vate bodies on clamps; young basidia dikaryotic, subglobose, clavate or ovoidal; after karyogamy basidia enlarging, becoming longitudinally, obliquely or rarely transversely septate, 2-4-celled; cells of basidia germinating either with hyphae or sterig- mata at the end of which monokaryotic ballist0s- pores can be formed, or with acropetal chains of monokaryotic yeast cells, formed on short denti- cles or somewhat longer fusiform sterigmata; septa having dolipores with parenthesomes; parenthe- somes made up of U-shaped vesicles (Tremellales type); xylose present in cell walls, which are elec- tron-dense and lamellate; coenzyme Q-10; hetero- thallic, but self-sporulating strains occurring; ana- morph Bullera Derx.

Type: Bulleromyces albus Boekhout & A. Fonseca spec. nov. (Figs 1-5)

Bulleromyces albus Boekhout & A. Fonseca spec. nov. (Figs 1-5)

Dikaryon formation following mating of appropri- ate mating partners of Bullera alba. Hyphae dika- ryotic, regularly branched, made up of cells mea- suring 40-130 • 1.5-3.0/~m, sometimes short wart-like outgrows occurring laterally or terminal- ly; clamps present; haustorial branches monoka- ryotic, formed on c. 3.5-5.0 • 2.5-3.5/xm basal clavate bodies, which originate at clamps; dika- ryotic clavate, ellipsoidal or cylindrical cells, mea- suring 5.0-10.0 x 4.0-7.0/~m, arising laterally or terminally on hyphaea or hyphal branches, some-

times forming dikaryotic hyphae of normal width, or developing into basidia; basidia sessile or short- stalked, occurring singly or in small clusters, basi- dia becoming vacuolated after karyogamy, enlarg- ing up to 9.0-18.0 • 7.0-12.5/xm, and becoming four-celled due to longitudinal, oblique or, more rarely, transverse septation; germination of basidia occurring by formation of ellipsoidal, fusiform or cylindrical yeast ceils, measuring 5.0-17.0 • 2.5- 5.0tzm, formed either solitarily or in acropetal chains, which may proliferate sympodially, or by formation of rotationally, or somewhat bilaterally symmetrical ballistospores, measuring 4.5-8.0 • 3.7-5.5/zm, on sterigmata or terminally and lat- erally on hyphae; meiospores monokaryotic, oc- curring frequently in clusters; mol.% G + C 53.5- 55.5. Nutritional physiological characteristics are presented in Table 1. Type: Due to the presence of different mating types the combination CBS 500 • CBS 6302 is deposited as holotypus in herb. CBS.

At present the life cycle of only one species, Bulleromyces albus, is known. Mating of two strains (CBS 7347 x CBS 7367) of Bullera varia- bilis resulted also in conjugation and dikaryon for- mation. Monokaryotic haustorial branches and lat- eral dikaryotic subglobose, clavate or lageniform cells were also formed. But in this case neither karyogamy nor basidium formation was observed. Consequently, a full description of the life cycle of Bullera variabilis cannot be presented at this time. Mating experiments performed in all other known Bullera species did not yield any mating response (Boekhout, unpubl.). Therefore, we consider Bul- leromyces albus at present as the only known spe- cies of Bulleromyces. But it may be expected, that after isolation and mating of more strains belong- ing to Bullera variabilis and other Bullera species, the number of species of Bullerornyces will soon increase.

Latin diagnoses

Bulleromyces Boekhout & A. Fonseca

Hyphae dikaryoticae; fibulae adsunt; rami hausto- riales monokaryotici, ad protrusiones claviformes

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e fibulis oriundas. Basidia longitudine vel oblique vel raro transversaliter septata, 2-4 cellularia; seu hyphis, seu sterigmatibus gemmantia; e sterigmati- bus seu ballistosporae monokaryoticae, rotunda- tae vel modice bilaterales seu catenae acropetales cellularum zymaticarum, monokaryoticarum, sub- globosarum vel ellipsoidalium vel fusiformium vel cylindricarum oriuntur. Septa doliporis et paren- thesomatibus preadita; parenthesomata vesiculas U-formes continentia. Parietes cellularum xylosum continentes. Coenzyma Q-10. Fungi heterothallici, sed etiam culturae monosporales fertiles inveniun- tur. Anamorphosis Bullera Derx.

Species typica Bulleromyces albus Boekhout & A. Fonseca.

Bulleromyces albus Boekhout & A. Fonseca

Hyphae dikaryoticae e copulatione Bullerae albae oriuntur. Rami haustoriales monokaryotici; rami lat- erales vel partes terminales dikaryotici clavati vel ellipsoidei vel cylindrici, 5.0-10.0• 4.0-7.0~m, nonnumquam hyphas dikaryoticas, latitudine nor- males, vel basidia formantes. Basidia sessilia vel breviter stipitata, singula vel pauca acervata, longi- tudine vel oblique vel raro transversaliter septata, gemmantia seu catenis acropetalibus cellularum zymaticarum, monokaryoticarum, subglobosa- rum, vel ellipsoidalium vel fusiformium vel cylin- dricarum, 5.0-17.0 x 2.5-5.0/xm, seu ballistospo- ris rotundatis vel modice bilateralibus, 4.5-8.0 • 3.7-5.5/xm, e summis sterigmatibus vel lateraliter. Mol. % G + C 53.5-55.5. Typus: CBS 500 • CBS 6306, depositus in Herb. CBS.

Acknowledgements

Drs. W. Gams, G.S. de Hoog, M.Th. Smith (CBS, Baarn and Delft respectively, the Netherlands), and N. van Uden (Instituto Gulbenkian de Cien- cia, Oeiras, Portugal) are greatly acknowledged for critical reading of the manuscript and stimulating discussions, as is G. Gim6nez (Instituto Gulben- kian de Ciencia) for putting strain ICG 4560 (CBS 7503) at our disposal. Dr. W. Gams kindly provid-

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ed the latin diagnosis, which is greatly appreciated. Marjolein van der Horst contributed in the prep- aration for cryoscanning electronmicroscopy.

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