Abstract
Abbreviations: ITS, internal transcribed spacer
The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are AB085795AB085812.
In addition to these strains, five other strains in the C. laurentii complex were included in this study. Four were strains of opposite mating types from wheat and corn (JCM 9909, JCM 9910, JCM 9911, JCM 9912) isolated by Kurtzman (1973). These strains were identified as C. laurentii, but did not mate with the type strains of C. laurentii, C. laurentii var. flavescens or C. laurentii var. magnus (Kurtzman, 1973). Guého et al. (1993) confirmed this heterogeneity by sequence analysis of the D1/D2 region of the 26S rDNA. A final strain, JCM 5945, isolated by Tokuoka et al. (1985) from a flower, was also included. This paper clarifies the taxonomic assignment of these strains.
Yeast strains.The strains used in this study are listed in Table 1. They were stock cultures identified as C. laurentii in the Centraalbureau voor Schimmelcultures (CBS) and the Japan Collection of Microorganisms (JCM).
Table 1. Strains used in this study
Morphological, physiological and biochemical characteristics.
Most of the morphological, physiological and biochemical characteristics were examined according to the methods of Yarrow (1998). The assimilation of nitrogen compounds was investigated on solid media using starved inoculum. Vitamin requirements were determined by the method of Komagata & Nakase (1967). The maximum growth temperature was determined in YM broth (Difco) using thermoregulated metal block heaters.
Major ubiquinones.
Cells were grown in 500 ml Erlenmeyer flasks containing 250 ml YM broth on a rotary shaker at 150 r.p.m. at 25 °C and were harvested in the early stationary growth phase and then washed with distilled water. Extraction, purification and identification of ubiquinones were carried out according to the method of Nakase & Suzuki (1986).
DNA base composition.
Cells were grown as described above and were harvested in the exponential growth phase and then washed with distilled water and freeze-dried. Isolation and purification of nuclear DNA were done according to Takashima & Nakase (2000). The DNA base composition was determined by HPLC after enzymic digestion of DNA to deoxyribonucleosides as described by Tamaoka & Komagata (1984). The DNA-GC kit (Yamasa Shoyu) was used as the quantitative standard.
Sequencing and phylogenetic analysis.
Nuclear DNA was extracted by the method of Makimura et al. (1994). The 18S rDNA and ITS regions, including 5·8S rDNA, were amplified by PCR according to Sugita & Nakase (1999). The D1/D2 region of the 26S rDNA was amplified according to Kurtzman & Robnett (1997). PCR products were sequenced directly using an ABI Prism BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems) and analysed with an Applied Biosystems sequencer model 310 according to the manufacturer's instructions. Reference sequences used for the phylogenetic study were obtained from the DDBJ/GenBank/EMBL database. Sequences were aligned with those of related species using CLUSTAL W version 1.8 (Thompson et al., 1994) and checked visually. Phylogenetic trees were constructed using the neighbour-joining method (Saitou & Nei, 1987). Evolutionary distances were calculated according to Kimura (1980). Sites where gaps existed in any sequences were excluded. Bootstrap analyses (Felsenstein, 1985) for the neighbour-joining method were performed from 100 random resamplings.
The phylogenetic relationships among C. laurentii strains used in this study are shown in Fig. 1. Strains JCM 9066T, CBS 318T, CBS 942T and JCM 9909 belong to phylogenetic group I. Bulleromyces albus Boekhout & A. Fonseca, Bullera hannae Hamamoto & Nakase, Bullera pseudoalba Nakase & Suzuki, Bullera penniseticola Takashima & Nakase and Bullera unica Hamamoto & Nakase were included in this cluster. The type strain of C. laurentii, JCM 9066T, clustered with CBS 2174 and CBS 8648, as shown previously (Sugita et al., 2000). Sequences of the ITS region and D1/D2 region of the 26S rDNA in CBS 2174 and CBS 8648 were identical, although three and one base differences, respectively, were detected between each of these two strains and JCM 9066T. Physiological and biochemical characteristics were almost the same among them. Based on these results, CBS 2174 and CBS 8648 were classified as C. laurentii.
|
The type strain of Torula flavescens, CBS 942T, belongs to phylogenetic group I and is thought to be distinct from C. laurentii. Torula flavescens was described by Saito in 1922 and was transferred to the genus Torulopsis as Torulopsis flavescens (Saito) Lodder in 1934, to Cryptococcus flavescens (Saito) Skinner in 1950 and then to C. laurentii var. flavescens (Saito) Lodder & Kreger-van Rij in 1952. Since the treatment by Rodrigues de Miranda (1984), this species has been viewed as a synonym of C. laurentii. As described previously, CBS 942T is distinct from C. laurentii based on sequence analyses. Strain CBS 8645, a clinical isolate, and Cryptococcus nodaensis G60 showed high sequence similarity to CBS 942T (99·1 % for the D1/D2 region of 26S rDNA and 100 % for overall ITS region) (Sugita et al., 2000). It has been stated previously that CBS 942 and CBS 8645 are the same species as C. nodaensis; however, C. nodaensis is a nomen invalidum because no Latin description has been given (Sato et al., 1999). CBS 8645 was identified as C. flavescens.
The ITS sequences of JCM 9909, 9910, 9911 and 9912, and the D1/D2 region of JCM 9909 were identical to those of C. flavescens CBS 942T. The four former strains were isolated from wheat or corn and their mating was observed by Kurtzman (1973). Although these strains were reported to represent an anamorphic species because the complete life cycle was not determined, it is assumed that some morphological characteristics are similar to those of Bulleromyces albus. When the complete life cycle of these strains is observed, it will be described as a teleomorph of C. flavescens. In this paper, these strains have been identified as C. flavescens based on the results of sequencing analyses and physiological and biochemical properties.
The type strain of Torula aurea, CBS 318T, belongs to phylogenetic group I and is thought to be distinct from C. laurentii and C. flavescens. Torula aurea was described by Saito in 1922, was transferred to Chromotorula aurea by F. C. Harrison in 1928 and then to Rhodotorula aurea by Lodder in 1934. Lodder & Kreger-van Rij (1952) considered this species to be a member of the C. laurentii complex. Phaff & Fell (1970) treated this species as a member of C. laurentii var. flavescens. Since no strains have been reported to be phylogenetically close to this one, it has been described as Cryptococcus aureus comb. nov. in this paper. JCM 5945, which was isolated from a flower and identified as C. laurentii (Tokuoka et al., 1985), was reclassified as C. aureus based on identical ITS1 and ITS2 sequences and on physiological and biochemical properties.
Phylogenetic group II
Phylogenetic group II consisted of CBS 973T, CBS 2409T, CBS 2993 and CBS 6578. Cryptococcus victoriae (Montes et al., 1999) also belongs to phylogenetic group II. This group constituted a cluster with Bullera globispora and C. dimennae in phylogenetic trees based on sequences of 18S rDNA and the D1/D2 region of the 26S rDNA. The type strain of Rhodotorula peneaus, CBS 2409T, which was described by Phaff et al. (1952) and placed in C. laurentii var. flavescens by Phaff & Fell (1970), was shown to be a distinct species. The name Cryptococcus peneaus comb. nov. is proposed for this strain.
Torulopsis carnescens was described by Verona & Luchetti (1936) and treated as a synonym of C. laurentii by Lodder & Kreger-van Rij (1952). Our previous paper (Sugita et al., 2000) stated that the ITS region and D1/D2 region of the 26S rDNA of the type strain of this species, CBS 973T, had sequences identical to those of Trimorphomyces papilionaceus Bandoni & Oberwinkler (AF075491, source strain CBS 445.92). When the 18S rDNA sequence of CBS 973T was determined and analysed using a phylogenetic tree, the position of this strain was far from Trimorphomyces papilionaceus (AF053716, source strain RJB 75-9458-B). To resolve this conflict, attempts to determine the sequence and to confirm the morphology of CBS 445.92 were made but, unfortunately, a living culture could not be obtained. Strain CBS 200.94, labelled Trimorphomyces papilionaceus, had a D1/D2 region that was identical to sequence AF075491 (source strain CBS 445.92), but the distinct morphological characteristic papilionaceus' was not observed in this strain (G. Okada, personal communication). Recently, the sequence data of AF075491 have been corrected using another strain (CBS 443.92). Whereas Trimorphomyces papilionaceus JCM 9899, JCM 11071 and JCM 11072 were almost identical and differed by 1213 bp from CBS 443.92 in the D1/D2 region of their 26S rDNA, these strains had more than 67 base differences when compared with CBS 973T. CBS 973T apparently does not belong to Trimorphomyces papilionaceus.
It is very interesting that the 18S rDNA of CBS 973T was identical to that of Taphrina maculans in the database (AB000953, source strain CBS 427.69). Sjamsuridzal et al. (1997) assumed that this strain might have been misidentified as Taphrina based on morphological, chemotaxonomic or physiological and biochemical characteristics, in addition to the sequence analysis. Since Taphrina maculans CBS 427.69 is not available for distribution from the CBS, this strain has not been compared with CBS 973T in our laboratory, but it is assumed that CBS 973T, Taphrina maculans CBS 427.69 and Taphrina californica CBS 374.39 (Sjamsuridzal et al., 1997) are closely related strains.
The morphological, physiological and biochemical characteristics of CBS 973T closely matched those of Torulopsis carnescens described by Verona & Luchetti (1936), although lactose was not assimilated according to their description. In this paper, CBS 973T is described as C. carnescens as enough data were obtained to confirm the identity of this strain. The taxonomic position of Taphrina maculans CBS 427.69 and Taphrina californica CBS 374.39 should be studied further.
Recently, Vishniac (2002) proposed two species, Cryptococcus heimaeyensis and Cryptococcus tephrensis, for isolates from Iceland. C. heimaeyensis was phylogenetically closely related to CBS 2993 and C. tephrensis was closely related to CBS 6578. CBS 2993 was isolated from bronchus of a lung patient in France by G. Cochet (strain no. 237) and CBS 6578 was isolated from sea water by J. W. Fell (ML 24-237); these two strains were respectively deposited in the CBS in November 1957 and March 1972. Both strains were tentatively identified as C. laurentii and kept in the CBS. Since only two and three base differences, respectively, were detected in the D2 regions of 26S rDNA in the former and latter pair, DNADNA reassociation experiments will be necessary to confirm the taxonomic positions of CBS 2993 and CBS 6578.
As shown in Table 2, the physiological and biochemical characteristics of the various species in the C. laurentii complex are very similar, but members of phylogenetic group I are distinguishable by the combination of assimilation patterns of D-glucosamine, N-acetyl-D-glucosamine, DL-lactic acid, 1,2-propanediol and sodium nitrite and vitamin requirements. C. carnescens, C. peneaus and C. victoriae could be differentiated by assimilation patterns of L-sorbose, inulin, D-glucosamine, N-acetyl-D-glucosamine, glucono-δ-lactone, DL-lactic acid, potassium nitrate, sodium nitrite, ethylamine hydrochloride and cadaverine dihydrochloride. Cryptococcus sp. CBS 2993 could hydrolyse fat, indicating that this is a unique strain within the C. laurentii complex.
Table 2. Physiological and chemotaxonomic characteristics among the C. laurentii complex Strains/species: 1, C. laurentii JCM 9066T, CBS 2174 and CBS 8648; 2, C. aureus CBS 318T and JCM 5945; 3, C. flavescens CBS 942T, CBS 8645, JCM 9909, JCM 9910, JCM 9911 and JCM 9912; 4, C. carnescens CBS 973T; 5, C. peneaus CBS 2409T; 6, C. victoriae (data from Montes et al., 1999); 7, Cryptococcus sp. CBS 2993; 8, Cryptococcus sp. CBS 6578. +, Positive; -, negative; L, latent; W, weak; LW, latent and weak; ND, no data; st, stimulative. All strains examined in this study can assimilate ammonium sulfate as a sole nitrogen source and are positive for production of starch-like substances, urease, Diazonium blue B complex and assimilation of glucose, galactose, sucrose, maltose, cellobiose, melibiose, raffinose, melezitose, soluble starch, D-xylose, L-arabinose, 2- and 5-ketogluconic acids and saccharate. All strains are negative for fermentation of glucose, acid production from glucose, liquefaction of gelatin and assimilation of methanol, hexadecane and 2,3-butanediol.
In this study, several species were identified as members of the genus Cryptococcus, although they should be reclassified into other appropriate genera in the future because they are considerably phylogenetically distant from Cryptococcus neoformans (type species of the genus) (Fell et al., 2000; Takashima & Nakase, 1999).
Taxonomic treatment
Cryptococcus laurentii (Kufferath) C. E. Skinner, in Am Midl Nat 43, 249 (1950).
≡Torula laurentii Kufferath, in Ann Soc R Sci Méd Nat Brux 74, 45 (1920).
≡Torulopsis laurentii (Kufferath) Lodder, in Verh K Ned Akad Wet Afd Natuurkd Sect II 32, 160 (1934).
≡Rhodotorula laurentii (Kufferath) Hasegawa, Banno & Yamauchi, in J Gen Appl Microbiol 6, 212 (1960).
Cryptococcus aureus (Saito) Takashima, Sugita, Shinoda & Nakase comb. nov.
≡Torula aurea Saito, in Mitt Jpn J Bot 1, 44 (1922).
≡Chromotorula aurea (Saito) F. C. Harrison, in Trans R Soc Can Sect V 22, 202 (1928).
≡Rhodotorula aurea (Saito) Lodder, in Verh K Ned Akad Wet Afd Natuurkd Sect II 32, 125 (1934).
Type strain: CBS 318T (=ATCC 32063T =IFO 0372T =NRRL Y-1582T).
Cryptococcus flavescens (Saito) C. E. Skinner (1950).
≡Torula flavescens Saito, in Mitt Jpn J Bot 1, 43 (1922).
≡Torulopsis flavescens (Saito) Lodder, in Verh K Ned Akad Wet Afd Natuurkd Sect II 32, 166 (1934).
≡Cryptococcus laurentii (Kufferath) C. E. Skinner var. flavescens (Saito) Lodder & Kreger-van Rij in The Yeasts, a Taxonomic Study, p. 381 (1952).
Cryptococcus carnescens (Verona & Luchetti) Takashima, Sugita, Shinoda & Nakase comb. nov.
≡Torulopsis carnescens Verona & Luchetti, in Boll Reg Inst Super Agrar Pisa 280 (1936).
Type strain: CBS 973T (=ATCC 32064T =MUCL 30641T =NRRL Y-1503T).
Cryptococcus peneaus (Phaff, Mrak & Williams) Takashima, Sugita, Shinoda & Nakase comb. nov.
≡Rhodotorula peneaus Phaff, Mrak & Williams, in Mycologia 44, 438 (1952).
Type strain: CBS 2409T (=ATCC 13546T =MUCL 30643T =NRRL Y-2005T).
The authors sincerely thank Dr G. Okada, Japan Collection of Microorganisms, for his contribution on the morphology of T. papilionaceus and Professor Emeritus Junta Sugiyama for making a taxonomic treatment in this paper.Footnotes
†Present address: Central Research Unit, National Centre for Genetic Engineering and Biotechnology (BIOTEC), NSTDA, 113 Phahonyothin Road, Pathumthani 12120, Thailand.References
Fell, J. W., Boekhout, T., Fonseca, A., Scorzetti, G. & Statzell-Tallman, A. (2000). Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. Int J Syst Evol Microbiol 50, 13511371.[Abstract]
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783791.[CrossRef]
Guého, E., Improvisi, L., Christen, R. & de Hoog, G. S. (1993). Phylogenetic relationships of Cryptococcus neoformans and some related basidiomycetous yeasts determined from partial large subunit rRNA sequences. Antonie van Leeuwenhoek 63, 175189.[CrossRef][Medline]
Harrison, F. C. (1928). A systematic study of some torulae. Trans R Soc Can Sect V 22, 187225.
Hasegawa, T., Banno, I. & Yamauchi, S. (1960). A taxonomic study on the genus Rhodotorula. J Gen Appl Microbiol 6, 196215.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111120.[CrossRef][Medline]
Komagata, K. & Nakase, T. (1967). Microbiological studies on frozen foods. V. General properties of yeasts isolated from frozen foods. J Food Hyg Soc Jpn 8, 5357 (in Japanese).
Kufferath, H. (1920). Peut-on obtenir du moût de bière alcalin? Ann Soc R Sci Méd Nat Brux 74, 1646.
Kurtzman, C. P. (1973). Formation of hyphae and chlamydospores by Cryptococcus laurentii. Mycologia 65, 388395.[Medline]
Kurtzman, C. P. & Robnett, C. J. (1997). Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol 35, 12161223.[Abstract]
Lodder, J. (1934). Die anaskosporegenen Hefen, I. Hälfte. Verh K Ned Akad Wet Afd Natuurkd Sect II 32, 1256.
Lodder, J. & Kreger-van Rij, N. J. W. (1952). The Yeasts, a Taxonomic Study. Amsterdam: North Holland.
Makimura, K., Murayama, Y. S. & Yamaguchi, H. (1994). Detection of a wide range of medically important fungi by polymerase chain reaction. J Med Microbiol 40, 358364.[Abstract]
Montes, M. J., Belloch, C., Galiana, M., Garcia, M. D., Andrés, C., Ferrer, S., Torres-Rodriguez, J. M. & Guinea, J. (1999). Polyphasic taxonomy of a novel yeast isolated from Antarctic environment; description of Cryptococcus victoriae sp. nov. Syst Appl Microbiol 22, 97105.[Medline]
Nakase, T. & Komagata, K. (1971). Significance of DNA base composition in the classification of yeast genus Candida. J Gen Appl Microbiol 17, 121130.
Nakase, T. & Suzuki, M. (1986). Bullera megalospora, a new species of yeast forming large ballistospores isolated from dead leaves of Oryza sativa, Miscanthus sinensis, and Sasa sp. in Japan. J Gen Appl Microbiol 32, 225240.
Phaff, H. J. & Fell, J. W. (1970). Genus 3. Cryptococcus Kützing emend. Phaff et Spencer. In The Yeasts, a Taxonomic Study, 2nd edn, pp. 10881145. Edited by J. Lodder. Amsterdam: North Holland.
Phaff, H. J., Mrak, E. M. & Williams, O. B. (1952). Yeasts isolated from shrimp. Mycologia 44, 431451.
Rodrigues de Miranda, L. (1984). Genus 5. Cryptococcus Kützing emend. Phaff et Spencer. In The Yeasts, a Taxonomic Study, 3rd edn, pp. 845872. Edited by N. J. W. Kreger-van Rij. Amsterdam: Elsevier.
Saito, K. (1922). Untersuchungen über die atmosphärischen Pilzkeime. Mitt Jpn J Bot 1, 154.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406425.[Abstract]
Sato, I., Kobayashi, H., Hanya, Y., Murakami, S., Scorzetti, G. & Fell, J. W. (1999). Cryptococcus nodaensis sp. nov., a yeast isolated from soil in Japan that produces a salt-tolerant and thermostable glutaminase. J Ind Microbiol Biotechnol 22, 127132.[CrossRef]
Sjamsuridzal, W., Tajiri, Y., Nishida, H., Thuan, T. B., Kawasaki, H., Hirata, A., Yokota, A. & Sugiyama, J. (1997). Evolutionary relationships of members of the genera Taphrina, Protomyces, Schizosaccharomyces, and related taxa within the archiascomycetes: integrated analysis of genotypic and phenotypic characters. Mycoscience 38, 267280.[CrossRef]
Skinner, C. E. (1950). Generic name for imperfect yeasts, Cryptococcus or Torulopsis? Am Midl Nat 43, 242250.[CrossRef]
Sugita, T. & Nakase, T. (1999). Non-universal usage of the leucine CUG codon and the molecular phylogeny of the genus Candida. Syst Appl Microbiol 22, 7986.[Medline]
Sugita, T., Nishikawa, A., Ikeda, R. & Shinoda, T. (1999). Identification of medically relevant Trichosporon species based on sequences of internal transcribed spacer regions and construction of a database for Trichosporon identification. J Clin Microbiol 37, 19851993.
Sugita, T., Takashima, M., Ikeda, R., Nakase, T. & Shinoda, T. (2000). Intraspecific diversity of Cryptococcus laurentii as revealed by sequences of internal transcribed spacer regions and 28S rRNA gene and taxonomic position of C. laurentii clinical isolates. J Clin Microbiol 38, 14681471.
Takashima, M. & Nakase, T. (1999). Molecular phylogeny of the genus Cryptococcus and related species based on the sequences of 18S rDNA and internal transcribed spacer regions. Microbiol Cult Coll 15, 3547.
Takashima, M. & Nakase, T. (2000). Four new species of the genus Sporobolomyces isolated from leaves in Thailand. Mycoscience 41, 6577.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125128.[CrossRef]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.
Tokuoka, K., Ishitani, T., Goto, S. & Komagata, K. (1985). Identification of yeasts isolated from high-sugar foods. J Gen Appl Microbiol 31, 411427.
Vancanneyt, M., Coopman, R., Tytgat, R., Hennebert, G. L. & Kersters, K. (1994). Whole-cell protein patterns, DNA base compositions and coenzyme Q types in the yeast genus Cryptococcus Kützing and related taxa. Syst Appl Microbiol 17, 6575.
Verona, O. & Luchetti, G. (1936). Ricerche microbiologiche su di alcuni vini ed alcune uve delle marche. Boll Reg Inst Super Agrar Pisa 12, 256311.
Vishniac, H. S. (2002). Cryptococcus tephrensis, sp. nov., and Cryptococcus heimaeyensis, sp. nov.; new anamorphic basidiomycetous yeast species from Iceland. Can J Microbiol 48, 463467.[CrossRef][Medline]
Yamada, Y. & Kondo, K. (1973). Coenzyme Q system in the classification of the yeast genera Rhodotorula and Cryptococcus, and the yeast-like genera Sporobolomyces and Rhodosporidium. J Gen Appl Microbiol 19, 5977.
Yarrow, D. (1998). Methods for the isolation, maintenance and identification of yeasts. In The Yeasts, a Taxonomic Study, 4th edn, pp. 77100. Edited by C. P. Kurtzman & J. W. Fell. Amsterdam: Elsevier.