- Email: [email protected]
Systematic study of basidiomycetous yeasts – evaluation of the ITS regions of rDNA to delimit species of the genus Rhodosporidium1
FEMS Yeast Research 2 (2002) 409^413
www.fems-microbiology.org
Systematic study of basidiomycetous yeasts ^ evaluation of the ITS regions of rDNA to delimit species of the genus Rhodosporidium1 Makiko Hamamoto b
a;
, Takahiko Nagahama b , Miki Tamura
a
a Japan Collection of Microorganisms, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan Deep-sea Microorganism Research Group, Japan Marine Science and Technology Center (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
Received 16 October 2001 ; received in revised form 10 April 2002; accepted 11 April 2002 First published online 28 May 2002
Abstract Nucleotide sequences of internal transcribed spacer (ITS) regions were determined to establish the guidelines for species identification in the genus Rhodosporidium. Forty-two strains of nine species of the genus Rhodosporidium were used for ITS (ITS1 and ITS2) analysis. Intraspecific length polymorphisms and sequence variations were observed within R. azoricum, R. diobovatum, R. paludigenum, R. sphaerocarpum and R. toruloides, while no variation was observed within R. babjevae and R. kratochvilovae. Based on comparison of the levels of intraspecific and interspecific sequence similarity, strains with identical sequences were considered to represent a single species and strains with 92% or lower similarity of ITS sequences were considered to be distinct species in the genus Rhodosporidium. 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Yeast; Internal transcribed spacer region ; Molecular systematics ; Rhodosporidium
1. Introduction The analysis of 18S rDNA sequences revealed that the basidiomycetes can be divided into three classes: the Hymenomycetes, Urediniomycetes and Ustilaginomycetes [1]. This class level taxonomy is correlated with morphological (septal pore ultrastructure) and chemotaxonomic (cellular carbohydrate composition) characteristics. After the establishment of the class level taxonomy for basidiomycetes, many yeast researchers have made their e¡orts to discuss the systematics at the order or generic level mainly based on the analysis of 18S rDNA or 26S rDNA sequences. For example, 18S rDNA sequences of some anamorphic genera of basidiomycetous yeasts were analyzed in order to obtain a phylogenetic perspective of the generic level classi¢cation. These studies indicated that ¢ve genera, Bensingtonia, Bullera, Cryptococcus, Sporobolomyces and Tilletiopsis, are polyphyletic in the 18S rDNA-based tree [2^6]. For basidiomycetous yeasts, analyses of ribosomal
* Corresponding author. Tel. : +81 (48) 467-9560; Fax : +81 (48) 462-4617. E-mail address : [email protected] (M. Hamamoto). 1
This paper is related to 21st ISSY.
DNA internal transcribed spacer (ITS) and intergenic spacer (IGC) regions, and the D1/D2 region of 26S rDNA, are carried out by many yeast researchers aiming at species level taxonomy. In current yeast systematics, for species in which a sexual life cycle is not found, the DNA reassociation value has been considered one of the most e¡ective criteria for di¡erentiating species. However, DNA complementarity values are relative values in estimating species di¡erentiation. Therefore, such values cannot be utilized as the database of yeast identi¢cation. Furthermore, there are sometimes ‘intermediate values’ between closely related strains in the DNA^DNA reassociation experiments. In such cases, the results cannot be clearly interpreted as conspeci¢c or not. The use of distinctive sequence data for species would provide other useful information for species level taxonomy. For example, the combined analysis of ITS and IGS regions indicated the usefulness of molecular markers to establish di¡erences between genealogically closely related yeast species [7,8]. However, these studies have not clearly indicated speci¢c guidelines as to the number or percentage of changes in these regions. Fell et al. [9] suggested that strains with identical D1/D2 sequences are considered a single species, and that strains that di¡ered by two or more nucleotides of the D1/D2 region of 26S rDNA represented di¡erent taxa. However, they also reported that some species whose
1567-1356 / 02 / $22.00 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 1 5 - 0
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M. Hamamoto et al. / FEMS Yeast Research 2 (2002) 409^413
D1/D2 sequences were identical could be clearly separated at the species level based on the analysis of their ITS sequences [9]. It suggests that the ITS sequences may be more useful to discriminate yeast species than the D1/D2 sequences of 26S rDNA. In this paper, nucleotide sequences of the ITS (ITS1 and ITS2) region of nine currently known species of the genus Rhodosporidium which are de¢ned by their sexual life cycles were determined and analyzed to establish guidelines for species classi¢cation in basidiomycetous yeasts.
2. Materials and methods Strains used in this study are listed in Table 1. Cells for DNA extraction were grown on YM agar (Difco, USA) for 3^7 days at 17 or 25‡C. Methods for genomic DNA extraction, determination and analysis of the nucleotide sequence of the ITS regions were performed as described previously [5]. Sequences of the ITS regions were analyzed for both ITS1 and ITS2, not including 5.8S rDNA.
Table 1 Rhodosporidium strains used in this study Species R. azoricum R. babjevae
R. diobovatum
R. £uviale R. kratochvilovae
R. lusitaniae R. paludigenum R. sphaerocarpum
R. toruloides
Strain no.a
Originb T
JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM
11251 11252 9279T 9280 9281 9282 9283 3786 3787T 10290
JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM
10311T 8171T 8172 10449 10450 11253 11254 11255 11256 11257 11258 11259 11260 11261 11262 8547T 10292T 10293 3791 8202T 9055 10294
JCM JCM JCM JCM JCM JCM JCM
10020 10021 10022 10049 10050 10295 10296
T
IGC 5062 IGC 5084 VKM Y-2275T VKM Y-2276 VKM Y-1310 VKM Y-1629 VKM Y-1631 S. Goto YK 223 S. Goto YK 225T IFO 0688 IGC 4914 CBS 6568T IAM 13072T IAM 13073 IGC 4818 IGC 4819 J. P. Sampaio ZP J. P. Sampaio ZP J. P. Sampaio ZP J. P. Sampaio ZP J. P. Sampaio ZP IGC 4793 IGC 5085 IGC 5244 IGC 5591 IGC 5606 CBS 7604T IFO 10547T IFO 10548 S. Goto YK 221 IAM 12261T IFO 1939 IFO 1438 IGC 5414 J. P. Sampaio ZP IFO 0559 IFO 0880 IFO 1236 IFO 0388 IFO 1235 IFO 10032 IFO 10034
17 26 182 210 246
417
Sexualityc Isolation source
DDBJ accession no. of ITS1^5.8S rDNA^ITS2
A1 A2 A1 A2 A1 A2 A2 A2 A1 A1 A1 ss ss ss A1 A2 A2 A2 A2 A2 anamorph ss A2 A1 anamorph anamorph ss A2 A1 A1 A2 ss A2 ss A1 A1 A2 A2 A1 A1 A1 A2
AB073229 AB073230 AB073231 AB073232 AB073233 AB073234 AB073235 AB073236 AB049025 AB073238 AB073239 AB073240 AB073241 AB073242 AB073243 AB073244 AB073245 AB073246 AB073247 AB073248 AB073249 AB073250 AB073251 AB073252 AB073253 AB073254 AB073255 AB073256 AB073257 AB073258 AB049026 AB073259 AB073260 AB073261 AB073262 AB049028 AB073263 AB073264 AB073265 AB073266 AB073267 AB073268
Soil, Portugal Soil, Portugal Herbaceous plant, Russia Herbaceous plant, Russia Silage, Ukraine Soil, Russia Soil, Russia Seawater, USA Seawater, USA Portugal Wood, Portugal Water, USA Unknown Unknown Woodchips, Portugal Moss, Portugal Leaf, Portugal Pine cone, Portugal Polipore fungus, Portugal Plant litter, Portugal Seawater, Portugal Grasshopper, Portugal Soil, Portugal Culture contaminant, Portugal Sputum Air, Japan Soil, Portugal Juncus roemerianus, USA Rhizophora mangle, USA Seawater, Antarctica Seawater, Antarctica Seawater, Antarctica Salt farm, Japan Salt farm, Slovenia Mud from salt marsh, Portugal Wood pulp, Sweden Soil, Japan Unknown Air, Japan Unknown Unknown Unknown
a
JCM, Japan Collection of Microorganisms, Saitama, Japan. CBS, Centraalbureau voor Schimmelcultures, Yeast Division, Utrecht, The Netherlands; IAM, Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan; IFO, Institute for Fermentation, Osaka, Japan; IGC, Portuguese Yeast Culture Collection, FCT-UNL, Portugal; VKM, All-Russian Collection of Microorganisms, Moscow, Russia. c ss, self-sporulating. b
FEMSYR 1490 7-8-02
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411
Fig. 1. Intra- and interspeci¢c relationships in the genus Rhodosporidium based on ITS1 and ITS2 sequences. The unrooted dendrogram was constructed using the neighbor-joining method as described previously [5]. Numbers by nodes are bootstrap percentages (values 6 50% not shown) derived from 1000 replicates.
Within R. azoricum, R. diobovatum, R. paludigenum and R. sphaerocarpum, two distinct ITS sequence types were identi¢ed, and four within R. toruloides. Intraspeci¢c sequence variations are represented alphabetically for convenience. Within R. azoricum, type A included strain JCM 11251T and type B included strain JCM 11252. Within R. diobovatum, type A included strain JCM 3787T and type B included strains JCM 3786, JCM 10290 and IGC 4914. Within R. paludigenum, type A included strain JCM 10292T and type B included strain JCM 10293. Within R. sphaerocarpum, type A included strains JCM 3791, JCM 8202T and JCM 10294 and type B included strains JCM 9055, IGC 5414 and ZP 417. Within R. toruloides, type A included strains JCM 10020, JCM 10049 and JCM 10050, type B included strains JCM 10021 and JCM 10022, type C included strain JCM 10295, and type D included strain JCM 10296. Levels of sequence similarity between the distinct ITS types were 99.7% within R. azo-
3. Results and discussion The nucleotide sequences of ITS regions (ITS1 and ITS2) of the 42 strains shown in Table 1 were determined. The sizes of ITS were 361 bp in R. azoricum, 361 bp in R. babjevae, 372 bp in R. diobovatum, 361 bp in R. £uviale, 360 bp in R. kratochvilovae, 359 bp in R. lusitaniae, 361^ 362 bp in R. paludigenum, 366 bp in R. sphaerocarpum and 363^365 bp in R. toruloides. The unrooted dendrogram in Fig. 1 was constructed from datasets aligned by CLUSTAL W version 1.81 on the basis of 386 sites. 3.1. Intraspeci¢c analysis of ITS Intraspeci¢c sequence variations were observed in R. azoricum, R. diobovatum, R. paludigenum, R. sphaerocarpum and R. toruloides, while no intraspeci¢c sequence variation was observed in R. babjevae and R. kratochvilovae. Table 2 Interspeci¢c ITS sequence similarity of Rhodosporidium species Species
1. 2. 3. 4. 5. 6. 7. 8. 9.
R. R. R. R. R. R. R. R. R.
azoricum babjevae diobovatum £uviale kratochvilovae lusitaniae paludigenum sphaerocarpum toruloides
% ITS sequence similarity 2
3
4
5
6
7
8
9
76.0
74.9^75.7 91.2
98.3 77.9 76.2^77.5
76.6 92.1 86.2 79.1
92.0 81.0 78.7^79.4 88.8 83.6
79.8 85.6^86.3 81.9^82.5 83.7 91.4 84.1
78.7^79.2 87.5^88.2 80.5^81.1 84.2^84.9 86.6^87.2 84.0^84.7 88.7
76.4^77.4 78.8^79.9 77.8^80.4 77.0^79.9 81.7^86.1 79.2^80.9 85.0^87.3 80.3^83.2 -
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M. Hamamoto et al. / FEMS Yeast Research 2 (2002) 409^413
ricum, 99.5% within R. diobovatum, 99.7% within R. paludigenum, 99.2% within R. sphaerocarpum and 94.8^97.8% within R. toruloides. In spite of the considerable intraspeci¢c variation in the ITS sequence of R. toruloides, strains in types A, B, C and D formed a distinct group in the unrooted dendrogram based on ITS sequences (Fig. 1), that could discriminate R. toruloides from the other eight species in the genus Rhodosporidium. Mating experiments were performed between the mating strains of R. toruloides used in this study, and mycelia with clamp connections and distinctive teliospores were observed (data not shown). Therefore, the levels of sequence similarity (94.8^97.8%) in R. toruloides are considered to represent a single species. 3.2. Interspeci¢c analysis of ITS A pairwise analysis of nine Rhodosporidium species revealed that interspeci¢c similarity values ranged from 74.9 to 98.3% (Table 2). All species in the genus Rhodosporidium were distinguished from one another at levels of 92% or lower similarity of ITS sequence, except the high level of sequence similarity (98. 3%) between R. azoricum and R. £uviale. 3.3. Evaluation of sequences of ITS region for species delimitation of Rhodosporidium species Members of the teleomorphic genus Rhodosporidium are characterized by asexual reproduction by budding without ballistoconidia, the sexual reproduction by the formation of teliospores, the absence of xylose in whole-cell hydrolysates, the presence of Q-9 or Q-10 as the major ubiquinone isoprenologue, the inability to ferment sugars, and positive diazonium blue B and urease reactions. The currently known nine species belonging to this genus are wellde¢ned based on their fertility [10,11]. Sampaio et al. [12] studied R. kratochvilovae and related anamorphic species by the polyphasic taxonomic approach and revealed that all strains examined were members of R. kratochvilovae. They investigated ITS sequences for three R. kratochvilovae strains and found no base pair di¡erences among them. In our study, 14 R. kratochvilovae strains examined for ITS sequences had no intraspeci¢c variation. Therefore, strains with identical ITS sequences are considered to represent a single species. On the ITS-based dendrogram (Fig. 1), nine species in the genus Rhodosporidium formed a tight phylogenetic group. They were distinguished from one another at levels of 92% or lower similarity of ITS sequence, except the high level of sequence similarity (98.3%) between R. azoricum and R. £uviale (Table 2). The type strains of R. azoricum and R. £uviale had similar G+C contents, produced similar basidia, and only two nucleotide di¡erences were found in the D1/D2 region of the 26S rDNA between them [11]. However, the low levels of DNA complemen-
tarity and the di¡erent banding patterns based on DNA ¢ngerprinting using the microsatellite-primed PCR approach were obtained between R. azoricum and R. £uviale [11]. R. azoricum in which only two strains are known is heterothallic, while R. £uviale in which only one strain is known is homothallic. Mating experiments were performed between strains of R. azoricum and R. £uviale, but we could not establish whether the three strains represented a single species. Additional data are required to con¢rm the relationship between R. azoricum and R. £uviale. Between the genealogically closely related yeast species of the genus Tilletiopsis, the ETS sequences in the IGS region were more useful for species separation than the ITS sequences (unpublished data). Fell and Blatt [7] and Diaz and Fell [8] also have reported that the IGS region is useful and may be required for strain separation in the genera Xanthophyllomyces, Pha⁄a and Mrakia in addition to the ITS region. Consequently, in general, strains with identical ITS (ITS1 and ITS2) sequences were considered to represent a single species and strains with levels of 92% or lower similarity of ITS sequences were considered to be di¡erent species in the genus Rhodosporidium. Finally, recent developments of molecular biological techniques are revolutionizing our knowledge about the natural relationships among basidiomycetous yeasts. We expect that the construction of yeast systematics from molecular phylogenetic perspectives will contribute to the establishment of the lower-level taxonomy.
Acknowledgements We acknowledge Dr. J.P. Sampaio, Universidade Nova de Lisboa, Portugal, for kindly providing strains from IGC and his valuable isolates.
References [1] Swann, E.C. and Taylor, J. (1995) Phylogenetic perspectives on basidiomycete systematics: evidence from the 18S rRNA gene. Can. J. Bot. 73 (Suppl. 1), S862^S868. [2] Takashima, M., Suh, S.-O. and Nakase, T. (1995) Phylogenetic relationships among species of the genus Bensingtonia and related taxa based on the small subunit ribosomal DNA sequences. J. Gen. Appl. Microbiol. 41, 131^141. [3] Suh, S.-O., Takashima, M., Hamamoto, M. and Nakase, T. (1996) Molecular phylogeny of the ballistoconidium-forming anamorphic yeast genus Bullera and related taxa based on small subunit ribosomal DNA sequences. J. Gen. Appl. Microbiol. 42, 501^509. [4] Takashima, M. and 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. Collect. 15, 35^47. [5] Hamamoto, M. and Nakase, T. (2000) Phylogenetic analysis of the ballistoconidium-forming yeast genus Sporobolomyces based on 18S rDNA sequences. Int. J. Syst. Evol. Microbiol. 50, 1373^ 1380.
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M. Hamamoto et al. / FEMS Yeast Research 2 (2002) 409^413 [6] Takashima, M. and Nakase, T. (1996) A phylogenetic study of the genus Tilletiopsis, Tilletiaria anomala and related taxa based on the small subunit ribosomal DNA sequences. J. Gen. Appl. Microbiol. 42, 421^429. [7] Fell, J.W. and Blatt, G.M. (1999) Separation of strains of the yeasts Xanthophyllomyces dendrorhous and Pha⁄a rhodozyma based on rDNA IGS and ITS sequence analysis. J. Ind. Microbiol. Biotech. 23, 677^681. [8] Diaz, M.R. and Fell, J.W. (2000) Molecular analyses of the IGS and ITS regions of rDNA of the psychrophilic yeasts in the genus Mrakia. Antonie van Leeuwenhoek 77, 7^12. [9] Fell, J.W., Boekhout, T., Fonseca, A., Scorzetti, G. and Statzell-Tallman, A. (2000) Biodiversity and systematics of basidiomycetous
413
yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. Int. J. Syst. Evol. Microbiol. 50, 1351^1371. [10] Fell, J.W. and Statzell-Tallman, A. (1998) Rhodosporidium Banno. In: The Yeasts, A Taxonomic Study, 4th edn. (Kurtzman, C.P. and Fell, J.W., Eds.), pp. 678^692. Elsevier, Amsterdam. [11] Gadanho, M., Sampaio, J.P. and Spencer-Martins, I. (2001) Polyphasic taxonomy of the basidiomycetous yeast genus Rhodosporidium : R. azoricum sp. nov.. Can. J. Microbiol. 47, 213^221. [12] Sampaio, J.P., Gadanho, M., Santos, S., Duarte, F.L., Pais, C., Fonseca, A. and Fell, J.W. (2001) Polyphasic taxonomy of the basidiomycetous yeast genus Rhodosporidium : Rhodosporidium kratochvilovae and related anamorphic species. Int. J. Syst. Evol. Microbiol. 51, 687^697.
FEMSYR 1490 7-8-02
www.fems-microbiology.org
Systematic study of basidiomycetous yeasts ^ evaluation of the ITS regions of rDNA to delimit species of the genus Rhodosporidium1 Makiko Hamamoto b
a;
, Takahiko Nagahama b , Miki Tamura
a
a Japan Collection of Microorganisms, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan Deep-sea Microorganism Research Group, Japan Marine Science and Technology Center (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
Received 16 October 2001 ; received in revised form 10 April 2002; accepted 11 April 2002 First published online 28 May 2002
Abstract Nucleotide sequences of internal transcribed spacer (ITS) regions were determined to establish the guidelines for species identification in the genus Rhodosporidium. Forty-two strains of nine species of the genus Rhodosporidium were used for ITS (ITS1 and ITS2) analysis. Intraspecific length polymorphisms and sequence variations were observed within R. azoricum, R. diobovatum, R. paludigenum, R. sphaerocarpum and R. toruloides, while no variation was observed within R. babjevae and R. kratochvilovae. Based on comparison of the levels of intraspecific and interspecific sequence similarity, strains with identical sequences were considered to represent a single species and strains with 92% or lower similarity of ITS sequences were considered to be distinct species in the genus Rhodosporidium. 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Yeast; Internal transcribed spacer region ; Molecular systematics ; Rhodosporidium
1. Introduction The analysis of 18S rDNA sequences revealed that the basidiomycetes can be divided into three classes: the Hymenomycetes, Urediniomycetes and Ustilaginomycetes [1]. This class level taxonomy is correlated with morphological (septal pore ultrastructure) and chemotaxonomic (cellular carbohydrate composition) characteristics. After the establishment of the class level taxonomy for basidiomycetes, many yeast researchers have made their e¡orts to discuss the systematics at the order or generic level mainly based on the analysis of 18S rDNA or 26S rDNA sequences. For example, 18S rDNA sequences of some anamorphic genera of basidiomycetous yeasts were analyzed in order to obtain a phylogenetic perspective of the generic level classi¢cation. These studies indicated that ¢ve genera, Bensingtonia, Bullera, Cryptococcus, Sporobolomyces and Tilletiopsis, are polyphyletic in the 18S rDNA-based tree [2^6]. For basidiomycetous yeasts, analyses of ribosomal
* Corresponding author. Tel. : +81 (48) 467-9560; Fax : +81 (48) 462-4617. E-mail address : [email protected] (M. Hamamoto). 1
This paper is related to 21st ISSY.
DNA internal transcribed spacer (ITS) and intergenic spacer (IGC) regions, and the D1/D2 region of 26S rDNA, are carried out by many yeast researchers aiming at species level taxonomy. In current yeast systematics, for species in which a sexual life cycle is not found, the DNA reassociation value has been considered one of the most e¡ective criteria for di¡erentiating species. However, DNA complementarity values are relative values in estimating species di¡erentiation. Therefore, such values cannot be utilized as the database of yeast identi¢cation. Furthermore, there are sometimes ‘intermediate values’ between closely related strains in the DNA^DNA reassociation experiments. In such cases, the results cannot be clearly interpreted as conspeci¢c or not. The use of distinctive sequence data for species would provide other useful information for species level taxonomy. For example, the combined analysis of ITS and IGS regions indicated the usefulness of molecular markers to establish di¡erences between genealogically closely related yeast species [7,8]. However, these studies have not clearly indicated speci¢c guidelines as to the number or percentage of changes in these regions. Fell et al. [9] suggested that strains with identical D1/D2 sequences are considered a single species, and that strains that di¡ered by two or more nucleotides of the D1/D2 region of 26S rDNA represented di¡erent taxa. However, they also reported that some species whose
1567-1356 / 02 / $22.00 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 1 5 - 0
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410
M. Hamamoto et al. / FEMS Yeast Research 2 (2002) 409^413
D1/D2 sequences were identical could be clearly separated at the species level based on the analysis of their ITS sequences [9]. It suggests that the ITS sequences may be more useful to discriminate yeast species than the D1/D2 sequences of 26S rDNA. In this paper, nucleotide sequences of the ITS (ITS1 and ITS2) region of nine currently known species of the genus Rhodosporidium which are de¢ned by their sexual life cycles were determined and analyzed to establish guidelines for species classi¢cation in basidiomycetous yeasts.
2. Materials and methods Strains used in this study are listed in Table 1. Cells for DNA extraction were grown on YM agar (Difco, USA) for 3^7 days at 17 or 25‡C. Methods for genomic DNA extraction, determination and analysis of the nucleotide sequence of the ITS regions were performed as described previously [5]. Sequences of the ITS regions were analyzed for both ITS1 and ITS2, not including 5.8S rDNA.
Table 1 Rhodosporidium strains used in this study Species R. azoricum R. babjevae
R. diobovatum
R. £uviale R. kratochvilovae
R. lusitaniae R. paludigenum R. sphaerocarpum
R. toruloides
Strain no.a
Originb T
JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM
11251 11252 9279T 9280 9281 9282 9283 3786 3787T 10290
JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM
10311T 8171T 8172 10449 10450 11253 11254 11255 11256 11257 11258 11259 11260 11261 11262 8547T 10292T 10293 3791 8202T 9055 10294
JCM JCM JCM JCM JCM JCM JCM
10020 10021 10022 10049 10050 10295 10296
T
IGC 5062 IGC 5084 VKM Y-2275T VKM Y-2276 VKM Y-1310 VKM Y-1629 VKM Y-1631 S. Goto YK 223 S. Goto YK 225T IFO 0688 IGC 4914 CBS 6568T IAM 13072T IAM 13073 IGC 4818 IGC 4819 J. P. Sampaio ZP J. P. Sampaio ZP J. P. Sampaio ZP J. P. Sampaio ZP J. P. Sampaio ZP IGC 4793 IGC 5085 IGC 5244 IGC 5591 IGC 5606 CBS 7604T IFO 10547T IFO 10548 S. Goto YK 221 IAM 12261T IFO 1939 IFO 1438 IGC 5414 J. P. Sampaio ZP IFO 0559 IFO 0880 IFO 1236 IFO 0388 IFO 1235 IFO 10032 IFO 10034
17 26 182 210 246
417
Sexualityc Isolation source
DDBJ accession no. of ITS1^5.8S rDNA^ITS2
A1 A2 A1 A2 A1 A2 A2 A2 A1 A1 A1 ss ss ss A1 A2 A2 A2 A2 A2 anamorph ss A2 A1 anamorph anamorph ss A2 A1 A1 A2 ss A2 ss A1 A1 A2 A2 A1 A1 A1 A2
AB073229 AB073230 AB073231 AB073232 AB073233 AB073234 AB073235 AB073236 AB049025 AB073238 AB073239 AB073240 AB073241 AB073242 AB073243 AB073244 AB073245 AB073246 AB073247 AB073248 AB073249 AB073250 AB073251 AB073252 AB073253 AB073254 AB073255 AB073256 AB073257 AB073258 AB049026 AB073259 AB073260 AB073261 AB073262 AB049028 AB073263 AB073264 AB073265 AB073266 AB073267 AB073268
Soil, Portugal Soil, Portugal Herbaceous plant, Russia Herbaceous plant, Russia Silage, Ukraine Soil, Russia Soil, Russia Seawater, USA Seawater, USA Portugal Wood, Portugal Water, USA Unknown Unknown Woodchips, Portugal Moss, Portugal Leaf, Portugal Pine cone, Portugal Polipore fungus, Portugal Plant litter, Portugal Seawater, Portugal Grasshopper, Portugal Soil, Portugal Culture contaminant, Portugal Sputum Air, Japan Soil, Portugal Juncus roemerianus, USA Rhizophora mangle, USA Seawater, Antarctica Seawater, Antarctica Seawater, Antarctica Salt farm, Japan Salt farm, Slovenia Mud from salt marsh, Portugal Wood pulp, Sweden Soil, Japan Unknown Air, Japan Unknown Unknown Unknown
a
JCM, Japan Collection of Microorganisms, Saitama, Japan. CBS, Centraalbureau voor Schimmelcultures, Yeast Division, Utrecht, The Netherlands; IAM, Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan; IFO, Institute for Fermentation, Osaka, Japan; IGC, Portuguese Yeast Culture Collection, FCT-UNL, Portugal; VKM, All-Russian Collection of Microorganisms, Moscow, Russia. c ss, self-sporulating. b
FEMSYR 1490 7-8-02
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Fig. 1. Intra- and interspeci¢c relationships in the genus Rhodosporidium based on ITS1 and ITS2 sequences. The unrooted dendrogram was constructed using the neighbor-joining method as described previously [5]. Numbers by nodes are bootstrap percentages (values 6 50% not shown) derived from 1000 replicates.
Within R. azoricum, R. diobovatum, R. paludigenum and R. sphaerocarpum, two distinct ITS sequence types were identi¢ed, and four within R. toruloides. Intraspeci¢c sequence variations are represented alphabetically for convenience. Within R. azoricum, type A included strain JCM 11251T and type B included strain JCM 11252. Within R. diobovatum, type A included strain JCM 3787T and type B included strains JCM 3786, JCM 10290 and IGC 4914. Within R. paludigenum, type A included strain JCM 10292T and type B included strain JCM 10293. Within R. sphaerocarpum, type A included strains JCM 3791, JCM 8202T and JCM 10294 and type B included strains JCM 9055, IGC 5414 and ZP 417. Within R. toruloides, type A included strains JCM 10020, JCM 10049 and JCM 10050, type B included strains JCM 10021 and JCM 10022, type C included strain JCM 10295, and type D included strain JCM 10296. Levels of sequence similarity between the distinct ITS types were 99.7% within R. azo-
3. Results and discussion The nucleotide sequences of ITS regions (ITS1 and ITS2) of the 42 strains shown in Table 1 were determined. The sizes of ITS were 361 bp in R. azoricum, 361 bp in R. babjevae, 372 bp in R. diobovatum, 361 bp in R. £uviale, 360 bp in R. kratochvilovae, 359 bp in R. lusitaniae, 361^ 362 bp in R. paludigenum, 366 bp in R. sphaerocarpum and 363^365 bp in R. toruloides. The unrooted dendrogram in Fig. 1 was constructed from datasets aligned by CLUSTAL W version 1.81 on the basis of 386 sites. 3.1. Intraspeci¢c analysis of ITS Intraspeci¢c sequence variations were observed in R. azoricum, R. diobovatum, R. paludigenum, R. sphaerocarpum and R. toruloides, while no intraspeci¢c sequence variation was observed in R. babjevae and R. kratochvilovae. Table 2 Interspeci¢c ITS sequence similarity of Rhodosporidium species Species
1. 2. 3. 4. 5. 6. 7. 8. 9.
R. R. R. R. R. R. R. R. R.
azoricum babjevae diobovatum £uviale kratochvilovae lusitaniae paludigenum sphaerocarpum toruloides
% ITS sequence similarity 2
3
4
5
6
7
8
9
76.0
74.9^75.7 91.2
98.3 77.9 76.2^77.5
76.6 92.1 86.2 79.1
92.0 81.0 78.7^79.4 88.8 83.6
79.8 85.6^86.3 81.9^82.5 83.7 91.4 84.1
78.7^79.2 87.5^88.2 80.5^81.1 84.2^84.9 86.6^87.2 84.0^84.7 88.7
76.4^77.4 78.8^79.9 77.8^80.4 77.0^79.9 81.7^86.1 79.2^80.9 85.0^87.3 80.3^83.2 -
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ricum, 99.5% within R. diobovatum, 99.7% within R. paludigenum, 99.2% within R. sphaerocarpum and 94.8^97.8% within R. toruloides. In spite of the considerable intraspeci¢c variation in the ITS sequence of R. toruloides, strains in types A, B, C and D formed a distinct group in the unrooted dendrogram based on ITS sequences (Fig. 1), that could discriminate R. toruloides from the other eight species in the genus Rhodosporidium. Mating experiments were performed between the mating strains of R. toruloides used in this study, and mycelia with clamp connections and distinctive teliospores were observed (data not shown). Therefore, the levels of sequence similarity (94.8^97.8%) in R. toruloides are considered to represent a single species. 3.2. Interspeci¢c analysis of ITS A pairwise analysis of nine Rhodosporidium species revealed that interspeci¢c similarity values ranged from 74.9 to 98.3% (Table 2). All species in the genus Rhodosporidium were distinguished from one another at levels of 92% or lower similarity of ITS sequence, except the high level of sequence similarity (98. 3%) between R. azoricum and R. £uviale. 3.3. Evaluation of sequences of ITS region for species delimitation of Rhodosporidium species Members of the teleomorphic genus Rhodosporidium are characterized by asexual reproduction by budding without ballistoconidia, the sexual reproduction by the formation of teliospores, the absence of xylose in whole-cell hydrolysates, the presence of Q-9 or Q-10 as the major ubiquinone isoprenologue, the inability to ferment sugars, and positive diazonium blue B and urease reactions. The currently known nine species belonging to this genus are wellde¢ned based on their fertility [10,11]. Sampaio et al. [12] studied R. kratochvilovae and related anamorphic species by the polyphasic taxonomic approach and revealed that all strains examined were members of R. kratochvilovae. They investigated ITS sequences for three R. kratochvilovae strains and found no base pair di¡erences among them. In our study, 14 R. kratochvilovae strains examined for ITS sequences had no intraspeci¢c variation. Therefore, strains with identical ITS sequences are considered to represent a single species. On the ITS-based dendrogram (Fig. 1), nine species in the genus Rhodosporidium formed a tight phylogenetic group. They were distinguished from one another at levels of 92% or lower similarity of ITS sequence, except the high level of sequence similarity (98.3%) between R. azoricum and R. £uviale (Table 2). The type strains of R. azoricum and R. £uviale had similar G+C contents, produced similar basidia, and only two nucleotide di¡erences were found in the D1/D2 region of the 26S rDNA between them [11]. However, the low levels of DNA complemen-
tarity and the di¡erent banding patterns based on DNA ¢ngerprinting using the microsatellite-primed PCR approach were obtained between R. azoricum and R. £uviale [11]. R. azoricum in which only two strains are known is heterothallic, while R. £uviale in which only one strain is known is homothallic. Mating experiments were performed between strains of R. azoricum and R. £uviale, but we could not establish whether the three strains represented a single species. Additional data are required to con¢rm the relationship between R. azoricum and R. £uviale. Between the genealogically closely related yeast species of the genus Tilletiopsis, the ETS sequences in the IGS region were more useful for species separation than the ITS sequences (unpublished data). Fell and Blatt [7] and Diaz and Fell [8] also have reported that the IGS region is useful and may be required for strain separation in the genera Xanthophyllomyces, Pha⁄a and Mrakia in addition to the ITS region. Consequently, in general, strains with identical ITS (ITS1 and ITS2) sequences were considered to represent a single species and strains with levels of 92% or lower similarity of ITS sequences were considered to be di¡erent species in the genus Rhodosporidium. Finally, recent developments of molecular biological techniques are revolutionizing our knowledge about the natural relationships among basidiomycetous yeasts. We expect that the construction of yeast systematics from molecular phylogenetic perspectives will contribute to the establishment of the lower-level taxonomy.
Acknowledgements We acknowledge Dr. J.P. Sampaio, Universidade Nova de Lisboa, Portugal, for kindly providing strains from IGC and his valuable isolates.
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M. Hamamoto et al. / FEMS Yeast Research 2 (2002) 409^413 [6] Takashima, M. and Nakase, T. (1996) A phylogenetic study of the genus Tilletiopsis, Tilletiaria anomala and related taxa based on the small subunit ribosomal DNA sequences. J. Gen. Appl. Microbiol. 42, 421^429. [7] Fell, J.W. and Blatt, G.M. (1999) Separation of strains of the yeasts Xanthophyllomyces dendrorhous and Pha⁄a rhodozyma based on rDNA IGS and ITS sequence analysis. J. Ind. Microbiol. Biotech. 23, 677^681. [8] Diaz, M.R. and Fell, J.W. (2000) Molecular analyses of the IGS and ITS regions of rDNA of the psychrophilic yeasts in the genus Mrakia. Antonie van Leeuwenhoek 77, 7^12. [9] Fell, J.W., Boekhout, T., Fonseca, A., Scorzetti, G. and Statzell-Tallman, A. (2000) Biodiversity and systematics of basidiomycetous
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