J. Gen. Appl. Microbiol., 53, 167–175 (2007)

Full Paper

Asterotremella gen. nov. albida, an anamorphic tremelloid yeast isolated from the agarics lycoperdoides and Asterophora parasitica

Hansjörg Prillinger,1,* Ksenija Lopandic,1 Takashi Sugita,2 and Michael Wuczkowski1

1 University of Natural Resources and Applied Life Sciences, Institute of Applied Microbiology (IAM), Austrian Center of Biological Resources and Applied Mycology (ACBR), Muthgasse 18, 1190 Wien, Austria 2 Meiji Pharmaceutical University, Department of Microbiology, Noshio, Kiyose 204–8588, Japan

(Received October 23, 2006; Accepted March 12, 2007)

Using a genotypic approach (PCR-fingerprinting, DNA/DNA reassociation, partial sequences of the 26S rDNA gene, complete sequences of the 18S rDNA gene, and sequences of the internal transcribed spacers) five tremelloid yeast isolates from the agarics Asterophora lycoperdoides and A. parasitica were shown to be conspecific with ramirezgomezianus. It was not possible to distinguish the yeast strains from A. lycoperdoides and A. parasitica using se- quences from the intergenic spacer (IGS1). Phylogeny based on the 26S (D1/D2-domain), ITS1- 5.8S-ITS2 and complete 18S rDNA demonstrated that C. ramirezgomezianus is closely related to several additional Cryptococcus (C. humicola, C. longus, C. musci, C. pseudolongus) within the Trichosporonales. A new genus, Asterotremella, and a new family, Asterotremellaceae were introduced for Cryptococcus species clustering within the Trichosporonales having a ubiquinone Q-9. Cryptococcus ramirezgomezianus is a synonym of Asterotremella albida.

Key Words—Asterotremella gen. nov. albida; Asterotremellaceae fam. nov.; Cryptococcus humicola; Hymenomycetes; ; Trichosporonales; 18S-26S-ITS-IGS r-DNA

Introduction al., 1988, 1989; Prillinger, 1987a). The term tremelloid was suggested by Oberwinkler (Laaser et al., 1988; When culturing the pileitrama of young fruit bodies Oberwinkler pers. comm.) for budding yeasts having a of the two mycoparasitic agarics, Asterophora lycoper- cup-shaped parenthesom. The yeasts appeared infre- doides (Bull.: Fr.) Ditmar and A. parasitica (Bull.: Fr.) quently after 8–16 days in axenic cultures on different Singer, two tremelloid yeasts were detected (Laaser et media. With A. lycoperdoides the isolation procedure was successfully repeated in the following years, lead- * Address reprint requests to: Dr. Hansjörg Prillinger, Univer- ing to yeast strains from three different localities in sity of Natural Resources and Applied Life Sciences, Institute of Bavaria, Germany. In subcultures of the original yeast Applied Microbiology (IAM), Austrian Center of Biological Re- isolates from both species, Asterophora fruit bodies sources and Applied Mycology (ACBR), Muthgasse 18, A11190 developed after 4–6 weeks (Prillinger, 1986). Attempts Wien, Austria. to isolate yeasts from Asterophora chlamydospores of Tel: 43–1–36006 6207 Fax: 43–1–3697615 E-mail: [email protected] artificially grown fruit bodies were successful in our Abbreviations: ITS, internal transcribed spacer; IGS, inter- first A. lycoperdoides mycelial strain (Prillinger, 1986). genic spacer. The yeast isolates of the two agarics A. lycoperdoides 168 PRILLINGER et al. Vol. 53 and A. parasitica could not be distinguished using each primer, 5–20 ng of DNA preparation and 1.25 U physiological tests (Laaser et al., 1989). These test BioTherm-DNA-Polymerase (GeneCraft). A total of 36 showed also that Cryptococcus humicola and Trich- amplification cycles were performed in a Trio-Ther- osporon cutaneum were the closest relatives. Restric- moblock TB1 thermocycler (Biometra): 98°C, 15 s; tion fragment analysis of the mitochondrial DNA 59°C, 1 min; 72°C, 2 min. The reaction was completed (Prillinger, 1987a) and of the ribosomal DNA (Laaser by a last elongation step at 72°C for 10 min. To remove et al., 1989) showed insignificant differences between the remaining primers and nucleotides, the PCR pro- the two yeast strains. These differences, however, ducts were purified by QIAquick PCR Purification Kit could not be corroborated by PCR-fingerprinting (Qiagen). A 611 bp long fragment was sequenced with (Prillinger, unpublished data). primers NL1 (GCATATCAATAAGCGGAGGAAAAG) To clarify the taxonomic and phylogenetic relation- and NL4 (GGTCCGTGTTTCAAGACGG) (White et al., ship of the A. lycoperdoides and A. parasitica yeast 1990) on an ABI 3100 Avant Sequencer (Applied isolates we have performed DNA/DNA reassociation Biosystems, Foster City, USA). Sequences of the experiments and DNA sequencing of the following ri- strains HB81 (AM265553) and HB82 (AM265554) bosomal DNA regions: 18S rDNA; D1/D2-domain of were deposited at the EMBL data library. 26S rDNA; ITS1-5.8-ITS2 and IGS1. We propose a Sequencing of 18S rRNA encoding gene. A frag- new genus for Cryptococcus species with a ment of approximately 2 kb was amplified with the ubiquinone Q-9 clustering within the Trichosporonales primers NS0 (TATCTGGTTGATCCTGCC) and ITS2p (Takashima et al., 2001). (GCTGCGTTCTTCATCGATGC) under the conditions described in the former paragraph. Primers used for Materials and Methods the sequencing of a 1,795 bp long fragment of the 18S rRNA encoding gene were those as described by Strains.Yeast isolates from the agaric Lopandic et al. (2004). Sequences of the strains HB81 Asterophora lycoperdoides (MB 215CBS 170.86): (AJ496260) and HB82 (AJ496261) were deposited at HB 81CBS 10411 leg. H. Prillinger, 10.10. 1984, the EMBL data library. Pirkensee near Regensburg, Bavaria, Germany; HB Sequencing of ITS regions. A fragment of approx. 466CBS 10413 leg. U. Passauer, 20. 9. 1994, Of- 2 kb was amplified by the primers 18/1184 (GACT- fensee near Ebensee, O.Ö. Austria. Yeast isolates CAACACGGGGAAACTC) and NL4 (GGTCCGTGT- from the agaric fungus A. parasitica (MB 217CBS TTCAAGACGG) under the above described condi- 683.82): HB 82CBS 7989 leg. H. Prillinger, 19. 9. tions. A 496 bp long fragment of the ITS1-5.8S-ITS2 1985, Paintner Forst near Regensburg, Bavaria, Ger- region was sequenced with primers ITS1p (TCCG- many; HB 467CBS 10414 and HB 468CBS 10415 TAGGTGAACCTGCGG) and ITS4p (TCCTCCGCT- leg. H. Schmid, 20. 9. 1994, Offensee near Ebensee, TATTGATATGC) (White et al., 1990). Sequences of O.Ö. Austria. Cryptococcus ramirezgomezianus CBS the strains HB81 (AM265555) and HB82 (AM265556) 2839TJCM 1460THB 1256T; leg C. Ramirez were deposited at the EMBL data library. Gómez, rotten toadstool, France. Additional yeast Sequencing of IGS1 region. The IGS1 regions strains and accession numbers are listed in Table 1. were determined according to Sugita et al. (2002) by DNA preparation. Genomic DNA was extracted using the primers 26SF (ATCCTTTGCAGACGACT- and purified as described by Lopandic et al. (2004). TGA) and 5SR (AGCTTGACTTCGCAGATCGG). Am- Partial sequencing of 26S rRNA encoding gene. plification was conducted under the following condi- Sequencing of the D1/D2 domain of 26S rDNA was tions: initial denaturation at 94°C, 3 min, followed by carried out as described by Lopandic et al. (2004) with 30 cycles of 94°C, 30 s, 57°C, 30 s and 72°C, 1 min minor modification. A fragment of about 2 kb was am- and a final extension at 72°C, 10 min. The 687 bp plified by polymerase chain reaction with the primers long IGS1 sequences of strains HB81 (AM265557), ITS3p (GCATCGATGAAGAACGCAGC) and YAM2 HB82 (AM265558), HB466 (AM265559) and HB467 (CGACTTCCCTTATCTACATT). The PCR was per- (AM265560) were deposited at the EMBL data library. formed in 50 ml reaction mixture containing Tris-based Phylogenetic analysis. Sequences of the 26S buffer pH 8.8, 4.5 mM MgSO4, 0.2 mM of each de- (D1/D2 region), 5.8S-ITS and 18S rDNA were com- oxynucleotide triphosphate (Peqlab), 0.1 pmol ml1 of bined and aligned using the ClustalX program (Thomp- 2007 Asterotremella gen. nov. 169

Table 1. Yeast and fungal isolates used in this study with their respective Genbank accession numbers.

Genbank accession numberc Other Strain ACBR No.a designationb 18S rDNA 26S rDNA 5.8S-ITS

Agaricus campestris DQ113914 AY207134 Asterophora lycoperdoides MB 215 CBS 170.86 AJ496254 AF223190 A. parasitica MB 217 CBS 683.82 AJ496255 AF223191 AF357038 Asterotremella albida HB 81 CBS 10411 AJ496260 AM265553 AM265555 A. albida HB 82 CBS 7989 AJ496261 AM265554 AM265556 A. albida HB 466 AM265559 A. albida HB 467 AM265560 A. albida HB 468 A. albida (Cryptococcus ramirezgomezianus) HB 1256T CBS 2839T AB039285 AB126584 AB035578 A. (Cryptococcus) humicola CBS 571T AB032637 AF189836 AB035572 A. (Cryptococcus) longa CBS 5920T AB035586 AB126585 AB035577 A. (Cryptococcus) pseudolonga CBS 8297T AB051047 AB126587 AB051048 Bullera dendrophila CBS 6074T D31649 AF189870 AF444443 Cryptococcus aerius CBS 155T AB032614 AF075486 AF145324 C. albidus CBS 142T AB032616 AF075474 AF14532 C. amylolentus CBS 6039T AB032619 AF105391 AF444306 C. curvatus CBS 570T AB032626 AF189834 AB035574 C. daszewskae CBS 5123T AB035582 AB126588 AB035580 C. fragicola CBS 8898T AB035588 AB126585 AB035588 C. musci CBS 8899T AB039378 AB126586 AB035579 C. neoformans ATCC 24067 L05428 Filobasidium uniguttulatum CBS 1730T AB032664 AF075468 AB032692 Guehomyces pullulans CBS 2532T AB001766 AF105394 AF444417 Kurtzmanomyces nectairei CBS 6405T D64122 AF177409 AF444494 Sporobolomyces roseus CBS 486T X60181 AF070441 AB030351 Tremella globospora Un. Of Calif. U00976 Trichosporon brassicae CBS 6382T AB001731 AF075521 AF444436 T. caseorum HB 819T CBS 9052T AJ319754 AJ319757 AJ319758 T. cutaneum CBS 2466T AB001753 AF075483 AF444325 T. domesticum CBS 8280T AB001754 AF075512 AF444414 T. faecale CBS 4828T AB001728 AF105395 AF444419 T. gracile CBS 8189T AB001756 AF105399 AF444440 T. inkin CBS 5585T AB001757 AF105396 AF444420 T. lactis HB 865T CBS 9051T AJ319755 AJ319756 AJ319759 Trichosporon laibachii CBS 5790T AB001760 AF075514 AF444421 Trichosporon loubieri CBS 7065T AB001759 AF075522 AF444438 Trichosporon moniliiforme CBS 2467T AB001761 AF105392 AF444415 Trichosporon mucoides CBS 7625T AB001763 AF075515 AF444423 Trichosporon ovoides CBS 7556T AB001765 AF075523 AF444439 Trichosporon porosum CBS 2040T AB051045 AF189833 AB51046 Tsuchiyaea wingfieldii CBS 7118T D64121 AF177404 AF444327

a ACBR: Oesterreichisches Zentrum fuer Biologische Resourcen, Institut für Angewandte Mikrobiologie, Universität für Bodenkul- tur, Wien, Austria. b CBS: Centraalbureau voor Schimmelcultures, Uppsalalaan, The Netherlands; ATCC: American Type Culture Collection, 10801 University Boulevard, Manassas (VA), USA. c Accession numbers of the sequences determined in the present work are indicated in bold. 170 PRILLINGER et al. Vol. 53 son et al., 1997). Resulting alignment was visually cor- rected. Phylogenetic relationships were estimated by parsimony analysis from the software PAUP 4.0b10 (Swofford, 2002) treating all alignment gaps as miss- ing data. Maximum parsimony trees were inferred using heuristic search with branch-swapping option and tree bisection-reconnection. Confidence values for individual branches were determined by a bootstrap analysis with 100 repeated samplings of the data. DNA-DNA reassociation. DNA-DNA reassociation experiments were carried out using the microplate method with chemiluminometric detection (Sugita et al., 1994). PCR-fingerprinting. Amplification reactions were performed in 50 ml volumes containing Tris-based buffer pH 8.8, 0.015% Bovine serum albumin (Roche; heat treated before use at 75°C for 15 min), 0.15 mM of each deoxynucleotide triphosphate (Peqlab), 0.2 pmol ml1 primer (a bacteriophage M13 core sequence (GAGGGTGGCGGTTCT) (Genxpress), 5–20 ng of Fig. 1. PCR-fingerprinting using primer M13. T template DNA and 1.25 U BioTherm-DNA-Polymerase CBS 2839 type strain of Asterotremella albida (Cryptococcus ramirezgomezianus); CBS 571T type strain of Asterotremella (GeneCraft). Polymerase chain reaction (PCR) was humicola. HB 81, HB 466 Yeast isolates from Asterophora ly- performed in a MJ Research thermal cycler (PTC200) coperdoides; HB 82, HB 467, HB 468 Yeast isolates from A. programmed for the initial denaturation at 94°C for parasitica. 2min, and 37 cycles of: 94°C/40 s, 39°C/70 s, 72°C/90 s, with a final extension of 72°C/10 min. A fragicola, C. daszewskae) were proposed. The name 20 ml aliquot of the reaction mixture was mixed with C. ramirezgomezianus nom. nov. was introduced for PCR-stop solution (30% sucrose, 0.2% Bromophenol Sporobolomyces albidus Ramirez Gomez JCM 1460T, T blue, 0.1 M EDTA) and run on a 1.5% agarose gel. The CBS 2839 (Takashima et al., 2001). gel image was photographed under UV and analyzed by the Bio-Rad gel analysis system (Bio-Rad Labora- PCR-fingerprinting tories, Inc., Vienna, Austria). For PCR-fingerprinting we used the primer M13. Fig- ure 1 demonstrates convincingly that the yeast isolates of A. lycoperdoides (HB 81, HB 466) and A. parasitica Results and Discussion (HB 82, HB 467, HB 468) are conspecific with C. Genotypic identification using partial sequences of the ramirezgomezianus (CBS 2839T). As already shown 26S rDNA (D1/D2-region) by Takashima et al. (2001) C. ramirezgomezianus and In basidiomycetous yeasts Fell et al. (2000) sug- C. humicola (CBS 571T) are two distinct species. gested that strains that differ by two or more nu- cleotides in the D1/D2 region of the 26S rDNA repre- Genotypic identification using the ITS1-5.8S-ITS2 re- sent different species. Our yeast isolates from the gion agarics A. lycoperdoides (HB 81) and A. parasitica The ITS1 and ITS2 region of the two tremelloid (HB 82) show 100% identity with Cryptococcus yeast isolates from A. lycoperdoides (HB 81) and ramirezgomezianus. Our data corroborate the close A. parasitica (HB 82) were identical. C. ramirez- phylogenetic relationship between C. ramirez- gomezianus differed in one base pair within the ITS1 gomezianus and C. humicola. Sugita et al. (2000) and region in the position 120 (one transversion, T instead Takashima et al. (2001) have recently shown that C. of A). The ITS2 region was identical in the three yeast humicola is a heterogeneous species. Five novel strains. species (C. longus, C. pseudolongus, C. musci, C. 2007 Asterotremella gen. nov. 171

Table 2. DNA-DNA reassociation values determined Phylogenetic characterization using complete se- between Asterotremella albida JCM1460T and yeast quences from the 18S rDNA isolates from Asterophora lycoperdoides Figure 2 shows that the species C. longus, C. (HB 81, HB 466) and A. parasitica pseudolongus, C. humicola, C. musci, and C. ramirez- (HB 82, HB 467). gomezianus form a homogeneous clade with 86%

Strains HB 81 HB 82 HB 466 HB 467 JCM 1460T bootstrap support within the Trichosporonales. In ad- dition all these species are characterized by a HB 81 100 101 97 94 88 ubiquinone Q-9 (Takashima et al., 2001). The HB 82 95 100 92 93 94 ubiquinone Q-10 Cryptococcus species C. curvatus, C. HB 466 94 94 100 94 93 daszewskae, C. fragicola cluster outside this clade in- HB 467 102 97 98 100 90 termingled with Trichosporon species. The mycelial T JCM 1460 90 89 92 94 100 strains of Asterophora lycoperdoides and A. parasitica cluster with Agaricus campestris ().

Genotypic identification using the IGS1 region Recently Sugita et al. (2002) showed that the analy- From Fig. 2 it can be seen that the genus Crypto- sis of the widely diverged IGS sequences may be a coccus is heterogeneous. This was already shown by useful tool for differentiating between phylogenetically Guého et al. (1993), Takashima and Nakase (1999) very closely related species. The IGS1 region com- and Fell et al. (2000). Whereas the neotype species of prises 687 bp. It was not possible to separate the two Cryptococcus, C. neoformans, clusters within the yeast isolates from A. lycoperdoides (HB 81, HB 466) Tremellales, species phylogenetically related to C. hu- or A. parasitica (HB 82, HB 467). We were not able to micola (Takashima et al., 2001) cluster within the corroborate the data of Laaser et al. (1989) which Trichosporonales (Fig. 2). On the basis of our phyloge- showed a restriction fragment length polymorphism of netic data (Fig. 2 and Fig. 3) and ubiquinone Q-9 a the ribosomal DNA. new genus Asterotremella and a new family Astero- were proposed. Figure 3 comprises a DNA-DNA reassociation phylogenetic tree based on three different genes (com- DNA-DNA reassociation experiments were per- plete 18S, 5.8S/alignable ITS, and 26S rDNAs [D1/D2 formed with the following yeast strains: HB 81, HB domain]). The Asterotremellaceae form a homoge- 466; HB 82, HB 467 and C. ramirezgomezianus (Table neous clade with 100% bootstrap support near the dif- 2). Price et al. (1978) examined yeast species in four ferent Trichosporon species. The ubiquinone Q-10 genera and proposed that strains having 80% or species Cryptococcus curvatus, C. daszewskae, C. greater DNA relatedness were conspecific, an opinion hagleriorum (Middelhoven et al., 2003) and C. fragi- expressed earlier by Martini and Phaff (1973) from cola appear heterogeneous (Fig. 2 and Fig. 3). Addi- their studies of Kluyveromyces spp. According to tional genes have to be sequenced (Kurtzman, 2003) Kurtzman (1998) strains showing DNA relatedness to clarify their taxonomic position. 70% are usually conspecific. DNA-DNA reassocia- tion values were in the range of 88–102% (Table 2). Latin diagnosis of Asterotremella Prillinger, Lopandic & From these data we conclude that the yeast isolates Sugita gen. nov. from A. lycoperdoides and A. parasitica are conspe- Carposomata absentia: Fungi Trichosporonalium cific and belong to the species C. ramirezgomezianus. ubiquinono majore Q-9. Haustoria tremelloidea absen- tia. Cellulae sphaeroideae, ovoideae vel elongatae, Physiological characteristics gemmis polaribus prolificantes; pseudomycelium et The yeast isolates from A. lycoperdoides and A. par- mycelium verum formantur. Coloniae in medio solido asitica are physiologically indistinguishable (Laaser et albae vel cremeae. D-Glucuronicum et inositolum as- al., 1989). Different from the Asterophora yeasts, C. similantur, at non kalii nitratum. Fermentatio nulla. ramirezgomezianus is able to utilize soluble starch Parietes cellularum xylosum continent. Diazonium (Takashima et al., 2001). caeruleum B positivum, ureum finditur. Materia amy- loidea iodophila formatur. Thiaminum ad crescendum 172 PRILLINGER et al. Vol. 53

Fig. 2. Maximum parsimonious tree indicating phylogenetic position of the genus Asterotremella within the order Trich- osporonales. This is one of six most parsimonious trees based on complete 18S rDNA sequences (number of characters NC1,746, constant characters CC1,304, parsimony uninformative characters PUC188, parsimony informative characters PIC254, CI0.615, RI0.735, RC0.452). The bootstrap factors 50% are not shown. S. roseus and K. nectairei are the outgroup species. necessarium. Diazonium blue B and urease reaction are positive. Typus generis: Asterotremella albida (Ramirez Extracellular amyloid substances are produced. Thi- Gómez) Prillinger, Lopandic & Sugita comb. nov. amine is necessary for growth.

Genus description Etymology Anamorphic yeasts clustering within the Trich- Astero refers to asterisk; a Tremella with an asterisk osporonales having a ubiquinone Q-9. Tremelloid showing no mycoparasitic interactions; haustoria are haustoria are lacking. Cells are spheroidal, ovoidal or absent. Fertile fruiting-bodies appear in a mixed cul- elongate. Reproduction by polar budding; pseudo- and ture of Asterophora chlamydospores and correspond- true hyphae are present. Colony color on solid media ing yeast cells (Fig. 4). may be white or cream; all species utilize D-glu- curonate and inositol. Potassium nitrate is not utilized. The Asterophora/Asterotremella system may be an Fermentation is lacking. Xylose is present in cell walls. ideal model to study the evolution of mating types. On 2007 Asterotremella gen. nov. 173

Fig. 3. Maximum parsimonious tree describing phylogenetic position of the genus Asterotremella within the order Trich- osporonales. This is one of three most parsimonious trees (number of characters NC2,736, constant characters CC2,161, parsimony unin- formative characters PUC251, parsimony informative characters PIC324, CI0.611, RI0.728, RC0.445) generated by analy- sis of a dataset containing sequences from 18S rDNA, ITS1-5.8S-ITS2 and 26S rDNA (D1/D2-region). The bootstrap factors 50% are not shown. C. amylolentus and G. pullulans are the outgroup species. the basis that the Asterotremella yeasts have lost their et al. (1993, 2002). mycoparasitic action we speculate that transcription factors moved from Asterotremella to Asterophora dur- Species accepted ing mating-type evolution. In Neurospora crassa the 1. Asterotremella albida (Ramirez Gómez) Prillinger, gene product of the mating-type locus A displays a re- Lopandic et Sugita comb. nov. Basionym: gion of similarity to the MAT a1 polypeptide of Saccha- Sporobolomyces albidus Ramirez Gómez. Microbiol. romyces cerevisiae, whereas the mt a-1 polypeptide is Esp. 10, 215–247, 1957. Synonym: Cryptococcus homologues to the shorter Schizosaccharomyces ramirezgomezianus Takashima, Sugita, Shinoda et pombe Mat-Mc polypeptide (Glass et al., 1990; Metz- Nakase. enberg, 1990; Staben and Yanofsky, 1990). An evolu- 2. Asterotremella humicola (Daszewska) Prillinger, tion from parasitism to homothallism and heterothal- Lopandic et Sugita comb. nov. Basionym: Torula humi- lism was proposed by Prillinger (1987b) and Prillinger cola Daszewska. Bull. Soc. Bot. Genève Sér. 2, 4, 174 PRILLINGER et al. Vol. 53

Family description Tremelloid yeasts clustering within the Trich- osporonales having a ubiquinone Q-9. Tremelloid haustoria are lacking. Cells are spheroidal, ovoidal or elongate; pseudo- and true hyphae are present. Colony color on solid media may be white or cream; all species utilize D-glucuronate and inositol. Potassium nitrate is not utilized. Fermentation is lacking. Xylose is present in cell walls. Diazonium blue B and urease re- action are positive. Extracellular amyloid substances are produced. Thiamine is necessary for growth. The following Cryptococcus species having a ubiquinone Q-10 and clustering within the Trich- osporonales are excluded from the Asterotremel- laceae: C. curvatus, C. haglerorum, (Middelhoven et

Fig. 4. Well developed fruit-bodies of Asterophora lycoper- al., 2003) C. daszewskae and C. fragicola. doides appear from the original yeast isolation plate. Acknowledgments

255–316, 1912. This work was funded by the Austrian Science Fund (FWF, project: P13876-B12). For the IGS-sequences of HB 81 and HB 3. Asterotremella longa (Takashima, Sugita, Shinoda 82 we thank Dr. O. Molnár. Dr. M. Weiß has prepared the Latin et Nakase) Prillinger, Lopandic et Sugita comb. nov. diagnosis. Dr. K. Vánky helped with nomenclatural problems. Basionym: Cryptococcus longus Takashima, Sugita, Mrs. U. Aulestia Vargas helped to translate a Spanish study. Shinoda et Nakase. Int. J. Syst. Evol. Microbiol. 51, 2199–2210, 2001. References 4. Asterotremella musci (Takashima, Sugita, Shinoda et Nakase) Prillinger, Lopandic et Sugita comb. nov. Fell, J. W., Boekhout, T., Fonseca, A., Scorzetti, G., and Statzell-Tallman, A. (2000) Biodiversity and systematics of Basionym: Cryptococcus musci Takashima, Sugita, basidiomycetous yeasts as determined by large-subunit Shinoda et Nakase. Int. J. Syst. Evol. Microbiol. 51, rDNA D1/D2 domain sequence analysis. Int. J. Syst. Evol. 2199–2210, 2001. Microbiol., 50, 1351–1371. 5. Asterotremella pseudolonga (Takashima, Sugita, Glass, N. L., Grotelueschen, J., and Metzenberg, R. L. (1990) Shinoda et Nakase) Prillinger, Lopandic et Sugita Neurospora crassa mating type region. Proc. Natl. Acad. comb. nov. Basionym: Cryptococcus pseudolongus Sci. USA, 87, 4912–4916. Takashima, Sugita, Shinoda et Nakase. Int. J. Syst. Guého, E., Improvisi, L., Christen, R., and de Hoog, G. S. Evol. Microbiol. 51, 2199–2210, 2001. (1993) Phylogenetic relationships of Cryptococcus neofor- mans and some related basidiomycetous yeasts deter- mined from partial large subunit rRNA sequences. Antonie Asterotremellaceae Prillinger, Lopandic, Sugita fam. van Leeuwenhoek, 63, 175–189. nov. Kurtzman, C. P. (1998) Nuclear DNA hybridization: Quantitation Cellulae sphaeroideae, ovoideae vel elongatae; of close genetic relationships. In The Yeasts, A Taxonomic pseudomycelium et mycelium verum formantur. Colo- Study, 4th ed., ed. by Kurtzman, C. P. and Fell, J. W., Else- niae in medio solido albae vel cremeae. D-Glucuron- vier, Amsterdam, pp. 63–68. icum et inositolum assimilantur, at non kalii nitratum. Kurtzman, C. (2003) Phylogenetic circumscription of Saccha- Fermentatio nulla. Parietes cellularum xylosum conti- romyces, Kluyveromyces and other members of the Sac- nent. Familia Trichosporonalium ubiquinono majore charomycetaceae, and the proposal of the new genera Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma Q-9. Haustoria tremelloidea absentia. Diazonium and Zygotorulaspora. FEMS Yeast Res., 1607, 1–13. caeruleum B positivum, ureum finditur. Materia amy- Laaser, G., Jahnke, K.-D., Prillinger, H., Bauer, R., Hoffmann, loidea iodophila formatur. Thiaminum ad crescendum P., Deml, G., and Oberwinkler, F. (1988) A new tremelloid necessarium. Typus familiae: Asterotremella Prillinger, yeast isolated from Asterophora lycoperdoides (Bull.: Fr.) Lopandic, Sugita. Ditm. Antonie van Leeuwenhoek, 54, 57–74. 2007 Asterotremella gen. nov. 175

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