International Journal of Systematic and Evolutionary Microbiology (2004), 54, 1401–1407 DOI 10.1099/ijs.0.03037-0 Curvibasidium cygneicollum gen. nov., sp. nov. and Curvibasidium pallidicorallinum sp. nov., novel taxa in the Microbotryomycetidae (Urediniomycetes), and their relationship with Rhodotorula fujisanensis and Rhodotorula nothofagi José Paulo Sampaio,1 Wladyslav I. Golubev,2 Jack W. Fell,3 Mário Gadanho1 and Nikita W. Golubev4 Correspondence José Paulo Sampaio jss@fct.unl.pt 1 Centro de Recursos Microbiológicos, Secção Autónoma de Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal 2 Russia Collection of Microorganisms, Institute for Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino 142290, Russia 3 Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Key Biscayne, FL 33419, USA 4 Mendeleev Chemical–Technological University, Moscow 125820, Russia Strains of Rhodotorula fujisanensis (Basidiomycota, Urediniomycetes, Microbotryomycetidae), including the type strain, are sexually compatible and produce clamped mycelium with teliospores. However, as teliospore germination had not been documented, the complete sexual cycle was not known. During the course of this work, the basidial stage of R. fujisanensis was characterized. In addition, mating studies employing isolates that were identified preliminarily as Rhodotorula nothofagi, a species that is related closely to R. fujisanensis, yielded mycelium with teliospores, which formed basidia and basidiospores. The new data were evaluated by using several criteria, including the available molecular phylogenetic framework for the Microbotryomycetidae. Curvibasidium gen. nov. is described here, to accommodate two teleomorphs: Curvibasidium cygneicollum sp. nov. (CBS 4551T), which is described as the sexual stage of R. fujisanensis, and Curvibasidium pallidicorallinum sp. nov. (CBS 9091T), which is related closely to R. nothofagi, but does not represent its sexual stage. The dimorphic basidiomycetes are distributed among several lineages of the Basidiomycota (Swann & Taylor, 1995; Sampaio & Fonseca, 2002; Scorzetti et al., 2002; Sampaio & Bauer, 2003). They constitute a remarkably diverse group of fungi, based on DNA sequence divergence studies, ultrastructural markers, ecological adaptations and metabolic properties. The yeast stage allows the colonization of a wide range of ecological niches and is also important for dispersion. The filamentous stage tends to be more resilient, particularly when specialized resting structures, such as teliospores, are formed. Hyphae are also important in the development of the sexual stage and in the establishment of mycoparasitism or phytoparasitism. Published online ahead of print on 5 March 2004 as DOI 10.1099/ ijs.0.03037-0. Many species of basidiomycetous yeasts are known only in their asexual phase and are classified in genera such as Rhodotorula Harrison or Cryptococcus Vuillemin. However, mating studies have allowed the disclosure of the sexual stage of species that were previously classified in these genera. Typically, the teleomorphic stage is initiated with conjugation between two compatible yeast cells, followed by production of mycelium. When environmental conditions are adequate, basidia and basidiospores are produced. The investigation of new sexual states allows a better Abbreviation: ITS, internal transcribed spacer. The GenBank/EMBL/DDBJ accession numbers for the sequences described in this work are as follows: AY383746, AY383747 and AY383748 (complete ITS region sequences of Curvibasidium pallidicorallinum CBS 6231, VKM Y-2861 and VKM Y-1135, respectively); AY383749 (complete ITS region sequence of Rhodotorula nothofagi A45); and AY383750 and AY383751 (partial 26S rRNA gene sequences of Rhodotorula ingeniosa CBS 6728 and Rhodotorula sp. CBS 4858, respectively). 03037 G 2004 IUMS Printed in Great Britain 1401 J. P. Sampaio and others understanding of the evolutionary processes that underlie the present diversity of dimorphic basidiomycetes, including the phylogenetic relationships between and within anamorphic genera. Sexual compatibility is known for certain strains of Rhodotorula fujisanensis (Soneda) Johnson & Phaff, and clamped mycelium with teliospores has been documented (Fell et al., 1984; Barnett et al., 1990; Boekhout & Fell, 1995). However, until now, teliospore germination had not been reported and, therefore, the complete sexual cycle was not known. In the present report, the basidial stage of R. fujisanensis is characterized. In addition, the study of another sexual stage, obtained by using isolates that were identified preliminarily as R. nothofagi Ramı́rez & González, a species related closely to R. fujisanensis, is also presented. The data were evaluated by using several criteria, including the molecular phylogenetic framework that is available for the Microbotryomycetidae. A new genus, Curvibasidium gen. nov., is described, to accommodate the two teleomorphs. The relationship of Curvibasidium with R. fujisanensis and R. nothofagi is discussed. For microscopy, cultures were grown on MYP agar (malt extract, 0?7 % w/v; yeast extract, 0?05 % w/v; soytone peptone, 0?25 % w/v; agar, 1?5 % w/v) at room temperature (20–23 uC) and studied with an Olympus BX50 microscope, using phase-contrast optics. For determination of sexual compatibility, pairs of 2–4-day-old cultures were crossed on MYP agar, potato dextrose agar (PDA) and corn-meal agar (CMA), incubated at room temperature and examined regularly for the production of mycelium and teliospores. Teliospore germination required a prolonged resting stage (9–12 months). After this period, agar blocks containing the teliospores were transferred to 2 % water agar and observed regularly under the microscope. Physiological and biochemical characterization was carried out according to techniques described by Yarrow (1998). Additional assimilation tests were performed by using aldaric acids and aromatic compounds as described by Fonseca (1992) and Sampaio (1999), respectively. For determination of the extent of DNA homology, total genomic DNA was extracted and purified by using procedures described previously (Sampaio et al., 2001). For DNA–DNA reassociation experiments, a Gilford Response UV-VIS spectrophotometer and its thermal programming software were used and the methods of Kurtzman et al. (1980) were followed. For rRNA gene sequence analysis, total DNA was extracted by using the protocol of Sampaio et al. (2001) and amplified by using primers ITS5 (59-GGAAGTAAAAGTCGTAACAAGG-39) and LR6 (59-CGCCAGTTCTGCTTACC-39). Cycle sequencing of the 600–650 bp region at the 59 end of the 26S rRNA gene D1/D2 domains employed forward primer NL1 (59-GCATATCAATAAGCGGAGGAAAAG-39) and reverse primer NL4 (59-GGTCCGTGTTTCAAGACGG-39). 1402 The internal transcribed spacer (ITS) region was sequenced by using the forward primer ITS1 (59-TCCGTAGGTGAACCTGCGG-39) and the reverse primer ITS4 (59-TCCTCCGCTTATTGATATGC-39). Sequences were obtained with an Amersham Pharmacia ALF Express II automated sequencer by using standard protocols. Alignments were made with MegAlign (DNAStar) and corrected visually. Heuristic maximum-parsimony analysis was employed (100 rounds of heuristic search with tree bisection–reconnection branch-swapping, starting from trees that were obtained by random addition of sequences, multrees option on, deepest descent option off) and was validated by using 1000 rounds of bootstrap analysis (Felsenstein, 1985). Maximumparsimony and bootstrap calculations used PAUP* software (Swofford, 2001). Latin diagnosis of Curvibasidium Sampaio et W. Golubev gen. nov. Fungi dimorphi Microbotryomycetidarum. Mycelium poris simplicibus fibulatis. Colacosomata nulla. Teliosporae globosae, holobasidiis germinant. Basidiosporae ovoideae ad bacilliformes, sessiles, gemmis germinant. Ballistoconidia nulla. Fermentatio nulla. D-Glucuronatum nitratumque non assimilantur. Productio compositorum amylo similium nulla. Systema CoQ 9 dominans. Polysaccharida extracellularia fucosum rhamnosumque continent. Description of Curvibasidium Sampaio & W. Golubev gen. nov. Curvibasidium (Cur.vi.ba.si9di.um. N.L. neut. n. Curvibasidium referring to the curved shape of the basidium). Dimorphic. Belongs to the subclass Microbotryomycetidae. Mycelium with clamp connections. Simple septal pore. Colacosomes are not produced. Spherical teliospores germinate, producing a holobasidium. Sessile, ovoid-tobacilliform basidiospores are released passively and germinate by budding. Ballistoconidia are not formed. Fermentative ability is absent. No assimilation of D-glucuronate or nitrate occurs. Starch-like compounds are not formed. Dominant CoQ system is 9. Extracellular polysaccharides contain fucose and rhamnose. Phylogenetic placement, as deduced by analysis of the D1/D2 domains of 26S rRNA gene, is shown in Fig. 1. The type species is Curvibasidium cygneicollum Sampaio. Latin diagnosis of Curvibasidium cygneicollum Sampaio sp. nov. Fungus dimorphus. Hyphae 1?5–2?5 mm diametro, conjugatione culturarum compatibilium procreantur. Septa fibulata, colacosomata nulla. Teliosporae plerumque globosae, 12–18 (22) mm diametro, terminales vel intercalares. Basidia stipitata, curvata, matura retroflexa, 6–8 (10)615–22 (26) mm, aseptata, basidiosporas sessiles, ovoideas ad bacilliformes in appendicibus basidialibus brevibus procreant. Basidiosporae gemmis germinant. Cellulae zymosae longe ovoideae ad cylindraceae, (1?5) 2–36(5) 7–12 (15) mm. International Journal of Systematic and Evolutionary Microbiology 54 Curvibasidium gen. nov. Fig. 1. Phylogenetic tree of Curvibasidium and related taxa of the Microbotryomycetidae. Maximum-parsimony analysis of an alignment of the D1/D2 region of the 26S rRNA gene. The topology was rooted with Naohidea sebacea, Occultifur externus, Rhodotorula minuta and Sakaguchia dacryoidea. Numbers on branches are bootstrap values (1000 replicates; values below 50 % are not shown). GenBank accession numbers of the sequences are indicated after strain numbers. Cultura in striis cremea ad aurantiaca. Characteres biochemici physiologicique Curvibasidii cygneicolli in http://www.crem. fct.unl.pt/dimorphic_basidiomycetes/Databases/databases.htm describuntur. Description of Curvibasidium cygneicollum Sampaio sp. nov. Curvibasidium cygneicollum (cyg.nei.col9lum. L. n. cygnus swan; L. n. collum neck; N.L. n. cygneicollum swan-neck, http://ijs.sgmjournals.org referring to the typical shape of the stalked basidium, which resembles the neck of a swan). Dimorphic. Hyphae (1?5–2?5 mm in diameter) are formed after mating of sexually compatible strains. Two mating types are known (see Table 1). Clamp connections are present, but colacosomes are lacking. Teliospores are usually spherical [12–18 (22) mm in diameter], terminal or intercalary (Fig. 2a). Basidia are produced after a prolonged resting stage of the teliospores. Basidia are stalked, curved and bent over the stalk when matured (Fig. 2b). Stalks 1403 J. P. Sampaio and others Table 1. Strain numbers, isolation source and sexuality of the two novel species of Curvibasidium described in this study and of R. nothofagi Species/strain* C. cygneicollum CBS 4551Td CBS 7950 CBS 8056 PYCC 4187 CBS 6371 PYCC 4694 CBS 8163§ CBS 7949 CBS 7951 CBS 8264 PYCC 4705 PYCC 4445 PYCC 4968 PYCC 4947 VKM Y-2859 JI 06 JI 21 C. pallidicorallinum VKM Y-2284T (=CBS 9091T) CBS 6231 VKM Y-2861 VKM Y-1135 VKM Y-2860 PTZ 185 R. nothofagi CBS 8166T A45 Isolation source Hare faeces, Mount Fuji, Japan Wood of Quercus suber, Arrábida, Portugal Berry of Vitis coignetiae (wild grape), Japan Flower of Acacia sp., Portugal Leaf, France Rotten wood, Arrábida, Portugal Rotten trunk of Laurelia sempervirens, Futrono, Chile Dry leaf, Arrábida, Portugal Tree root, Gerês, Portugal Leaf of Acacia sp., New Zealand Caterpillar nest, Sintra, Portugal Woodland soil, Lisbon, Portugal Tree trunk, Arrábida, Portugal Rotten tree trunk, Lisbon, Portugal Herbaceous plants, Prioksko-terrasny Biosphere Reserve, Moscow region, Russia Fruiting body of Exidiopsis sp., Caramulo, Portugal Fruiting body of Exidiopsis sp., Caramulo, Portugal SexualityD MT A1 MT A1 MT A1 MT A1 MT A2 MT A2 ANA SF|| ANA ANA ANA ND ANA ANA MT A2 ND ND Gramineous plant, Moscow region, Russia Frass of Ruguloscolytus rugulosus Herbaceous plants, Prioksko-terrasny Biosphere Reserve, Moscow region, Russia Air, Kishinev, Moldavia Herbaceous plants, Prioksko-terrasny Biosphere Reserve, Moscow region, Russia Herbaceous plants, Prioksko-terrasny Biosphere Reserve, Moscow region, Russia MT MT MT MT MT MT A1 A1 A2 A2 A1 A1 Rotten trunk of Nothofagus obliqua, Futrono, Chile Sea water off Faro, south of Portugal ANA ANA *CBS, Centraalbureau voor Schimmelcultures, Yeast Division, Utrecht, The Netherlands; PYCC, Portuguese Yeast Culture Collection, FCT-UNL, Portugal; VKM, All-Russian Collection of Microorganisms, Pushchino, Russia; JI, PTZ personal collections of J. Inácio and W. I. Golubev, respectively; A, collection of yeasts isolated from aquatic environments by M. Gadanho and J. P. Sampaio. DANA, Anamorphic; MT, mating type; ND, not determined. dType strain of R. fujisanensis (Soneda) Johnson & Phaff. §Type strain of Rhodotorula futronensis (Ramı́rez & González) Roeijmans et al. ||Originally this strain was self-fertile, but currently it lacks the ability to produce mycelium with teliospores. measure 1?5–2?5615–35 (50) mm. Basidia [6–8 (10)615– 22 (26) mm] are devoid of septa and produce sessile, ovoidto-bacilliform basidiospores [1?5–266–12 (20) mm] on short basidial appendages (Fig. 2b). Each basidium has two to four sites of basidiospore formation and multiple basidiospores are formed at each site. Basidiospores germinate by budding. Yeast cells are long and ovoid to cylindrical [(1?5) 2–36(5) 7–12 (15) mm] (Fig. 2a). Streak cultures are cream-coloured or pale orange, butyrous and semi-dull. Colony margins are entire, and fringed with pseudomycelium in some cases. Physiological and biochemical features of C. cygneicollum are available at http://www.crem.fct.unl.pt/dimorphic_basidiomycetes/ Databases/databases.htm and tests that allow its differentiation from Curvibasidium pallidicorallinum are depicted in Table 2. The phylogenetic placement of C. cygneicollum is 1404 shown in Fig. 1. C. cygneicollum is sensitive to the mycocins produced by Rhodotorula glutinis and Rhodotorula mucilaginosa, but insensitive to mycocins secreted by Rhodotorula pallida and by species of the genera Cryptococcus, Cystofilobasidium, Filobasidium and Sporidiobolus. Microscopic slides from the crossing of CBS 4551T and CBS 6371, showing mycelium, teliospores, basidia and basidiospores, were deposited in the Portuguese Yeast Culture Collection under number ZP-01-03 (holotype). As the physiological and molecular characterization of a mixed culture presents obvious difficulties, we propose that strain CBS 4551T, isolated from hare droppings collected at Mount Fuji, Japan, should be designated as the type strain of C. cygneicollum. This strain is deposited in the Centraalbureau voor Schimmelcultures, Yeast Division, International Journal of Systematic and Evolutionary Microbiology 54 Curvibasidium gen. nov. Fig. 2. Line drawings of different developmental stages of Curvibasidium cygneicollum (a, b), Curvibasidium pallidicorallinum (c, d) and Leucosporidium fasciculatum (e, f). Yeast cells (after 4–6 days on MYP agar), mycelium and teliospores (after 1–2 weeks on PDA) (a, c, e) are shown; for Curvibasidium pallidicorallinum, the detail of the clamp connections is presented. Germinated teliospores, basidia and sessile basidiospores (b, d, f) are shown. Note the holobasidia of Curvibasidium cygneicollum and Curvibasidium pallidicorallinum and the phragmobasidia of L. fasciculatum. Bar, 10 mm. Utrecht, The Netherlands, and in the Portuguese Yeast Culture Collection, FCT-UNL, Portugal, as PYCC 3116T. Table 2. Physiological characteristics that allow differentiation between C. cygneicollum, C. pallidicorallinum, R. nothofagi and L. fasciculatum Species: 1, C. cygneicollum; 2, C. pallidicorallinum; 3, R. nothofagi; 4, L. fasciculatum. D, Delayed results. The complete dataset of physiological and biochemical features is available at http://www.crem. fct.unl.pt/dimorphic_basidiomycetes/Databases/databases.htm. Characteristic 1 2 3 4 Sucrose Maltose a,a-Trehalose m-Hydroxybenzoic acid Gallic acid Catechol DL-Lactic acid Cadaverine Growth at 30 uC 2 2 2 2 + 2 2 + 2 2 2 2 2 + 2 2 2 2 2 + 2 + 2 + + + + + 2 + 2 + 2 http://ijs.sgmjournals.org D 2 2 Latin diagnosis of Curvibasidium pallidicorallinum W. Golubev, Fell et N. Golubev sp. nov. Fungus dimorphum. Hyphae 1?5–2?5 mm diametro, conjugatione culturarum compatibilium procreantur, fibulatae. Colacosomata nulla. Teliosporae plerumque globosae (10– 19 mm diametro) interdum ovoideae, pyriformes (9–16610– 21 mm), terminales vel intercalares. Basidia unicellulata, 3?5–5 (6?5)630–50 (70) mm, curvata. Basidiosporae sessiles, bacilliformes, 2?5–3?566–15 (19) mm, in appendicibus brevibus lateris convexi basidii procreantur, gemmis germinantes. Cellulae zymosae longe ovoideae ad cylindraceae, 2–565–11 mm. Cultura in striis pallide corallina ad roseocremea. Characteres biochemici physiologicique Curvibasidii palleocorallini in http://www.crem.fct.unl.pt/dimorphic_ basidiomycetes/Databases/databases.htm describuntur. Description of Curvibasidium pallidicorallinum W. Golubev, Fell & N. Golubev sp. nov. Curvibasidium pallidicorallinum (pal.lid.i.cor.al9li.num. L. adj. pallidus pale; L. n. corallium coral; L. adj. pallidicorallinum pale coral, referring to the pinkish-cream colour of cultures of this species). 1405 J. P. Sampaio and others Dimorphic. Hyphae (1?5–2?5 mm in diameter) are formed after mating of sexually compatible strains. Two mating types are known (see Table 1). Clamp connections are present and medallion-shaped (Fig. 2c). Colacosomes are absent. Teliospores are usually spherical (10–19 mm in diameter), sometimes ovoid or pear-shaped (9–166 10–21 mm), terminal or intercalary (Fig. 2d). Basidia are one-celled [3?5–5 (6?5)630–50 (70) mm], curved and produced after a prolonged resting stage of the teliospores (Fig. 2d). Sessile, bacilliform basidiospores [2?5–3?566–15 (19) mm] are produced on short basidial appendages that originate on the convex side of the basidium (Fig. 2d). Each basidium has one to four sites of basidiospore formation and multiple basidiospores are formed at each site. Basidiospores germinate by budding. Yeast cells are long and ovoid to cylindrical (2–565–11 mm) (Fig. 2c). Streak cultures are pale orange to pinkish-cream, flat, smooth, butyrous, semi-dull or glistening. Colony margins are entire. Physiological and biochemical features of C. pallidicorallinum are available at http://www.crem.fct.unl.pt/dimorphic_ basidiomycetes/Databases/databases.htm and tests that allow its differentiation from C. cygneicollum and R. nothofagi are depicted in Table 2. The phylogenetic placement of C. pallidicorallinum is shown in Fig. 1. C. pallidicorallinum is sensitive to the mycocins produced by R. glutinis and R. mucilaginosa, but insensitive to mycocins secreted by R. pallida and by species of the genera Cryptococcus, Cystofilobasidium, Filobasidium and Sporidiobolus. Microscopic slides from the crossing of VKM Y-2284T and VKM Y-2861, including mycelium, teliospores, basidia and basidiospores, were deposited in the Portuguese Yeast Culture Collection under number ZP-02-03 (holotype). As the physiological and molecular characterization of a mixed culture presents obvious difficulties, we propose that strain VKM Y-2284T, isolated from a gramineous plant collected in the Moscow region, Russia, should be designated the type strain of C. pallidicorallinum. This strain is deposited in the Russia Collection of Microorganisms (VKM), Institute for Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russia. Two strains have also been deposited in the Centraalbureau voor Schimmelcultures (CBS), Yeast Division, Utrecht, The Netherlands: CBS 9091T (=VKM Y-2284T) and CBS 9642 (=VKM Y-2861). The life cycles of C. cygneicollum and C. pallidicorallinum were investigated on MYP agar, CMA and PDA. Conjugation of opposite mating types resulted in the formation of true mycelium with clamp connections. In C. pallidicorallinum, clamp connections have a peculiar medallion shape (Fig. 2c). For both species, teliospores are abundant 1 week after inoculation, but their germination requires a prolonged resting stage. For C. cygneicollum, crossings made on PDA plates and incubated for 2 weeks at room temperature were sealed and maintained at 15 uC for 9 months. After that period, small agar blocks containing teliospores were transferred to 2 % water agar and 1406 incubated at room temperature. Teliospores germinated approximately 1 week after transfer to water agar. Although the same procedures were also successful for C. pallidicorallinum, the original observation of the complete life cycle of this species employed CMA. Agar pieces containing 1month-old teliospores were soaked in distilled water and maintained at 5 uC for 1 year. Teliospore germination occurred on transfer of the agar blocks to 2 % water agar and incubation for 1 week at room temperature. The type strain of C. pallidicorallinum has mycocinogenic activity (Golubev, 1992). Its killing patterns are unique, as they include not only urediniomycetous yeasts (Rhodosporidium, Rhodotorula and Sporidiobolus), but also hymenomycetous yeasts (Cryptococcus, Cystofilobasidium, Filobasidium and Itersonilia). This is the first report of mycocins that are active against yeasts of two different classes. Phylogenetic placement The phylogenetic position of Curvibasidium was inferred by comparing the D1/D2 domains of the 26S rRNA gene sequences of the two species with representative members of the Microbotryomycetidae (Fig. 1). C. cygneicollum differed from C. pallidicorallinum by three substitutions. The sequences of the two species showed no intraspecific variability. The closest relative of Curvibasidium was Leucosporidium fasciculatum Bab’eva & Lisichkina, with 19 mismatches with respect to C. cygneicollum and 22 mismatches with respect to C. pallidicorallinum. The association of L. fasciculatum with Curvibasidium received strong statistical support (Fig. 1). As L. fasciculatum is not related to Leucosporidium scottii Fell, Statzell, Hunter & Phaff, this species was excluded from the circumscription of the order Leucosporidiales (Sampaio et al., 2003). We reexamined the micromorphological features of L. fasciculatum (Fig. 2e, f) and confirmed the observations made by Bab’eva & Lisichkina (2000). The mycelium of L. fasciculatum, in contrast to that of Curvibasidium, lacks clamp connections (Fig. 2e) and the basidia are septate (phragmobasidia) (Fig. 2f). As the holobasidium of Curvibasidium is a unique trait in the Microbotryomycetidae, this feature is an important diagnostic property for the new genus and is probably a derived characteristic. The absence of this characteristic in L. fasciculatum prevented us from transferring this species to Curvibasidium. Moreover, Curvibasidium has CoQ 9 (Goto & Oguri, 1983), which is an uncommon feature in the Microbotryomycetidae. However, this chemotaxonomic marker has not yet been determined for L. fasciculatum. From nutritional and physiological perspectives, L. fasciculatum is also wellseparated from Curvibasidium (Table 2). We consider that a study of additional teleomorphic species related to Curvibasidium and L. fasciculatum is needed before a change in the scope of Curvibasidium, or the creation of a new genus for L. fasciculatum, is made. Another important feature of Curvibasidium is the absence of colacosomes (also referred to as lenticular bodies). Among members of the Microbotryomycetidae that are International Journal of Systematic and Evolutionary Microbiology 54 Curvibasidium gen. nov. depicted in Fig. 1, the plant parasites of the Microbotryales and Kriegeria eriophori Bresadola lack colacosomes, as do the non-plant parasites Camptobasidium hydrophilum Marvanová & Suberkropp, Leucosporidium antarcticum Fell, Statzell, Hunter & Phaff and L. fasciculatum. The presence of colacosomes has been related to a mycoparasitic life strategy (Bauer & Oberwinkler, 1991). These subcellular structures have only been reported for certain dimorphic basidiomycetes classified in the Microbotryomycetidae, such as Colacogloea Oberwinkler & Bandoni, the Sporidiobolales (Rhodosporidium Banno and Sporidiobolus Nyland), the Leucosporidiales (L. scottii, Leucosporidium fellii GiménezJurado & van Uden and Mastigobasidium Golubev) and Heterogastridium Oberwinkler & Bauer, which lacks a yeast stage (Kreger-van Rij & Veenhuis, 1971; Sampaio et al., 2003). Anamorph–teleomorph connections As the type strain of R. fujisanensis is a mating strain of C. cygneicollum, it is evident that C. cygneicollum is the sexual stage of R. fujisanensis. A similar connection was suggested for C. pallidicorallinum and R. nothofagi on the basis of phylogenetic analysis of the D1/D2 domains (Fig. 1). However, several attempts to mate the type strain of R. nothofagi with the available sexually compatible strains of C. pallidicorallinum invariably gave negative results. The relationships of R. nothofagi, C. pallidicorallinum and C. cygneicollum were investigated further by analysing the complete ITS region (ITS1+5?8S rRNA gene+ITS2). No variability was detected for C. cygneicollum in the ITS region, whereas for C. pallidicorallinum–R. nothofagi, four sites were variable (a phylogenetic tree is available at http:// www.crem.fct.unl.pt/dimorphic_basidiomycetes/Databases/ databases.htm). In the analysis of the ITS data, the four sexually compatible strains of C. pallidicorallinum were separated from the two asexual strains (the type strain of R. nothofagi and strain A45). Nuclear DNA–DNA reassociation experiments also supported this separation, as homology between the type strains of C. pallidicorallinum and R. nothofagi was not high and ranged from 40 to 46 % in three independent essays. Therefore, on the the basis of the lack of sexual compatibility and the ITS and DNA reassociation data, we consider that R. nothofagi is not the asexual state of C. pallidicorallinum. R. nothofagi can be distinguished from C. pallidicorallinum on the basis of the ITS sequences, the absence of mating and the ability to grow at 30 uC. In both C. cygneicollum and C. pallidicorallinum, sexual compatibility seems to be biallelic, as crossing experiments suggested the presence of only two mating types. Six strains of C. cygneicollum (Table 1) are regarded as anamorphic, as they did not react sexually with the two mating types of this species. Acknowledgements We thank M. Weiß (University of Tübingen, Germany) for preparing the Latin diagnoses. M. 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