Impact of Polyphasic dpproach of Phenotypic and Genotypic Characters in Yeast Identilication TA B. THUANI*, HIROKO KAWASAKI, DERLENE S. ATTILI2, VANDERIEI CANHOS2, AKIRA YOKOTA1 ANd JUNIA SUGIYAMA1 tlns:titute of Molecular and Cellular Biosciences, The Univer;ity of Tolqo, Yayoi 1-1-1, Bunlqto-ku, Tbkyo 113, Japan, zFundagdo Tropical de Pesquisas e Tbcnologia " AndrdTbsello ", Campinas, S4 Brazil Annual Report of lCBiotech Vol. 19 (1996) lnternational Center for Biotechnology, Osaka University ':'i
Transcript
Impact of Polyphasic dpproach of Phenotypic and GenotypicCharacters in Yeast Identilication
TA B. THUANI*, HIROKO KAWASAKI, DERLENE S. ATTILI2, VANDERIEICANHOS2, AKIRA YOKOTA1 ANd JUNIA SUGIYAMA1
tlns:titute of Molecular and Cellular Biosciences, The Univer;ity of Tolqo, Yayoi 1-1-1,Bunlqto-ku, Tbkyo 113, Japan, zFundagdo Tropical de Pesquisas e Tbcnologia" AndrdTbsello ", Campinas, S4 Brazil
Annual Report of lCBiotech Vol. 19 (1996)
lnternational Center for Biotechnology, Osaka University
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Impact of Polyphasic Approach of Phenotypic and GenotypicCharacters in Yeast Identification
TA B. THUANI*, HIROKO KAWASAKI, DERLENE S. AITILI2, VANDERLEI P.
CANHOS2, AKIRA YOKOTA1 ANd JUNTA SUGIYAMA1
llnstitute of Molecular and Cellular Biosciences, The [Jniversity of Toteyo, Yayoi 1-1-1,Bunlqto-ku, Tolqo 113, Japan, 2Fundagdo Tropical de pesquisas e kcnorogia"Andr 6Tos ello ", Campinas, SP, Brazil
Polyphasic taxonomy including molphological, physiological, chemotaxono-mical, and molecular characterization are effective to the elucidation of theidentity of yeasts. In these studies, we applied such an approach to theidentification of some yeast isolates. Three yeast strains were isolated fromfern leaves (isolate 21) and from soil (isolates s-3-17 and S-3-19), which werecollected in Brazil in November 1993 by J. Sugiyama. A data set based onphenotypic characters such as DBB, urease activity, and extracellular DNaseactivity tests, ubiquinone system, sugar composition in the cell wall indicateda basidiomycete affinity of the isolatos: e.g., positive for the DBB, ureaseactivity, and extracellular DNase activity tests in s-3-17, s-3-19 and 21; majorubiquinone Q-10, no xylose in the cell wall,60.8 mowo G+C in S-3-17;58.7molvo G+c in s-3-19, and 48.7 mol%o in isolate 21. Further studies onmorphological characters showed all isolates having the multilayered cell walland enteroblastic budding. The data indicate a basidiomycete affinity of theisolates. Phylogenetic analysis based on 18S rDNA sequencing as a genotypiccharacter suggests isolates s-3-17 and S-3-19 are closely related toRhodotorulamucilaginosa. Isolate 21, producing ballistospores, also belongs to abasidiomycete and is closeiy related to sporobolomyces roseus. we arso madechemotaxonomic characterization of 25 species of the archiascomycete generaTaphrina and Protomyces having a yeast state. Among them ,,Taphrina
californica" and "L maculans" had Q-10 as the major ubiquinone, xylose inthe cell wall, and 49.3 and 46.7 molvo G+c, respectively. Morphorogicallythey were characterized by the multilayered cell wall and enteroblastic budding.The data suggest a basidiomycete affinity. Moreover, evidences from phenotypiccharacters and molecular phylogeny as a genotypic character indicate that"Taphrina californica" and"T. maculans" belong to the basidiomycetes, andthey are closely related to species of the genera Tremeila, Buileromyces,F i lobas idie I la, T richos p o ro n, and F ilobasidium.
Research work in the "International Post-Graduate University Course in Microt*rlogy"lupported by the Japanese Government, UNESCO and ICRO (International Cell ResearthOrganization).*Present address: The Centre for Mycological Research, National Hanoi University, HanoiCity, Vietnam.
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Introduction
Impact of a polyphasic approach of phenotypic and genotypic data is extremely great infungal systematics (1). The former placement of Mixia osmundae as an ascomycete memberis one of such examples. Integration of molecular and morphological evidence clearly indicatedthatM. osmundae is a member of the urediniomycete lineage within the Basidiomycota (2).
The strains focused in this study were isolated from fern leaves (isolate 21) and from soil(isolates 5-3-17 and 5-3-19), which were collected in Brazil in November L93,by J. Sugiyamain cooperation with G. Okada (Japan Collection of Microorganisms, RIKEN) and Brazilianmycologists and botanists. The materials and strain data have been described in the previousreports by Chatanon et al. (3) and Battsetseget al. (4).
The strains were identified by the polyphasic analysis of morphological, ultrastructural,physiological, biochemical and chemotaxonomical characters as the phenotypic criteria, andDNA base composition and 18S rDNA sequence divergence as tlre genotypic criteria- Pnesented
in this account are the impact of such a polyphasic approach of phenotypic and genotypiccharacters in yeast identification. We also investigated chemotaxonomic features of 25species of the archiascomycete genera Taphrina and Protomyces which have a yeast state.These data have been evaluated in this report from various aspects in yeast systematics.
Material and Methods
Strains examined
Strains S-3-17 and S-3-19 were isolated from soil and strain 2l was isolated from fernleaves, both collected in the Atlantic Rain Forest ('sooretama') of Brazil [For details, see theprevious report by Chatanon et al. (3)1. Chemotaxonomic characteristics of twenty-fivespecies of archiascomycete were examined as shown in Table 2. In addition to these, strainsof basidiomycetes: Rllodosporidium toruloidesHM13469r,Rhodotorula glutinis IAM 14386r,
Leucosporidium toruloides IAM 13043r, and Sporidiobolus johnsonii JCM 1840r were usedfor comparative study, and Saccharomyces cerevisiae IAM 4178, Cystofilobasidiwninfirmominiatum IAM 13500, and Cryptococcus bhutanensis IAM 14321 were used forphysiological tests as reference smins. Strains with a superscript "T" and "NT'indicate thesrains derived from the holotype and neotype, respectively.
M o rp ho lo gi c al c haracte r s
Strains were cultivated using the following four media: yeast-malt extract (YM) agar,potato-dextrose agar (PDA), corn meal agar (CMA), and YM broth, which have been usedfor the moqphological characteristics in "The yeasts, a taxonomic study" (5) and "Yeasts:characteristics and identification" (6).
U ltras tructur al c har ac t e r s
The cell wall ultrastructure and the mode of conidiogenesis were observed with atransmission electron microscope. The young cells cultivated in YM broth medium, wereharvested by centrifugation, fixed in 3%o-glutataldehyde for 2 hr, washed with distilled water
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5 times and postfixed with 27o potassium permanganate for 2 hr, rinsed with distilled waterseveral times at room temperature, and then embedded in agar and the agar blocks were cutinto small pieces. These cells in the agar blocks were dehy&ated in increasing concentrationof ethanol and acetone, and finally embedded in Spurr's low viscosity medium. Ultra-thinsection was cut by a diamond knife and poststained with uranyl acetate for 2hr and then withReynolds lead citrate solution for 10 min. The sections were examined with a JEOL 1210electron microscope at an accelation voltage of 80 kV (2).
P hysiolo gical c haracter s
Physiological characteristics of the strains were examined according to the methodsdescribed by Kreger-van Rij (5), and Barnett et al. (6). The urease test, assimilation ofinositol, and formation of starch-like compounds were examined by using solid medium, andthe results were recorded after 8-l0-day-incubation at25oC. The test for the extracellulardeoxyribonuclease @Nase) activity was ca:ried out following to Sen and Komagata (7). Thetest for the ability of fermentation by using broth medium was also done.
Diazoniurn Blue B (DBB ) test
strains were grown on YM plates for 7 to 10 days at 25"c. The DBB reagent wasprepared immediately before use by dissolving the DBB salt in chilled 0.25 M Tris-HClbuffer (pH 7.0 ). The reagent DBB (2 to 4 drops ) was applied directly to surface of colonies,and the color reaction was recorded (8).
C he mot axo no mi c c har ac t e r s :Determination of cell wall sugar composition
The cells grown in YM broth medium under continuous shaking at27oC for 3 to 7 days,were harvested by centifugation, and washed twice with distilled water. Cell walls wereprepared according to the modified method of Prillingeret al. (9) as follows: Cells weresuspended in 0.1 M phosphate buffer (p}J7.2), together with glass beads, disrupted by aBrown Cell Homogenizer (2 - 5 min) until no intact cells are present under the light microscope.
The cell debris were centrifuged at 1,000 rpm for 10 min to exclude intact cells and glassbeads, then centrifuged at 15,000 rpm for 20 min, washed with phosphate buffer, and finallycentrifuged at 15,000 rpm for 20 min. The pellets obtained were supended in water, appliedonto the top of 607o sucrose solution, and centrifuged at 15,000 rpm, 60 min. Cell wallfractions were washed three times with cold water, centrifuged at 15,000 rpm for 20 min, andfreeze-dried. The dried cell walls were hydrolyzed in 2N trifluoroacetic acid (IFA) at 100oCfor 16 hr. Afterreduction with NaBflo, the sugars were acefylated with acetic anhydride/pyridine(1:1). The alditol acetate derivatives were analyzed with gas-liquid chromatography by usingShimadzu GC-8A, with nitrogen gas as a carrier and a column of Rtx 2330 (0.32 mm x 30m). The column temperature was increased from 77soc to 240oC. Sugars were estimated onthe basis of sample coincidence with the relative retention times for the alditol acetatederivatives of the neutral monosaccharide standards. We also checked sugar component bythinJayer chromotography ( 10).
Determination of the major ubiquinone system
The strains were cultivated in YM broth medium. After 2 days, the cells were harvested
by centrifugation, and washed twice with distilled water. After treatment the cells with
alkali-pyrogalloll, the ubiquinones were extracted two times with hexane and partially purified
by thin layer chromatography according to the procedure of Kuraishiet al. (11)' The purified
ubiquinones were determined from the retention times of reversed phase high-performance
liquid chromatogram (1 1).
M ol e c ul ar c harac t e r is ti c s :
Determination of DNA base composition
The cells were grown in 5 liter flasks containing 1.5 liter of YM broth medium under
shaking at 27oC for 30 to 48 hr . The cells were harvested by centrifugation and washed
twice with distilled water. The cells were disrupted by freezing in liquid nitrogen and then by
homogenizer. After disruptting the cells, we used benzyl chloride to destroy the cells, and
then used BECKMAN TL-100 ultracentrifuge for 16 to18 hr. The DNA was isolated and
purified according to the isolation method of genomic DNAs using benzyl chloride (12).
DNA base composition was calculated from the relative amount of nucleosides of peak areas
on a high-perfomance liquid chromatogmm (HPLC) (13).
Determination of rDNA sequences and phylogenetic analysis
Method for preparation of DNA was described previously (DNA base composition
analysis). Ribosomal RNA gene was amplyfied by PCR using primers P-l (5'-
ATCTGGTTGATCCTG CCAGT-3' ) and P-2 (5' -GATCCTICCG CAGGTTCACC-3' ). The
condition of PCR was 94"C for 3 min, 1 cycle, 94oC for 30 sec, 55oC for 30 sec, 72"C for 2
min, 30 cycles, andT2oC for 5 min, 1 cycle. The PCR products generated wi& Takara EX
Taq polymerase were cloned by using Original TA Cloningl Kit (Introgen Corporation, CA,
USA). Preparation of plasmid DNA from Escherichia coli was used QIAprep Spin Plasimd
Kit (QIAGEN GmbH, Germany and QIAGEN Inc, CA, USA). The sequencing reactions
were done with SequiThermrM Long-Readru Cycle Sequencing Kits-LC (Epicetre
Technologies, WI, USA). Sequencing was carried out on a LI-COR model 4000 DNA
sequencer and image analyzer with 6Vo acrylamide gels fl-ong Ranger Gel Concentrate;
ATBiochem, PA, USA). The phylogenetic tree was reconstructed by applying the algorithm
of the neighbour-joining method (14) to Knuc values by using the software "CLUSTAL
1ry,'(15). A total of 1,162 positions was considered excluding positions with gaps and deletions.
The bootstrap procedure by Felsenstein (16) was used to resampling the data 1,000 times.
Results
Morphological characteristics are summarized in Table 1. TEM micrographs of cell wall
structure and mode of conidiogenesis are shown in Fig. 1. Results of cell wall sugar composition
are shown in Fig. 2-5. The results of some physiological and chemotaxonomic characters
were neumerated in the descriptions given below. Chemotaxonomic charaeteristics of
archiascomycetes are shown in Table 2,and a molecular phylogeny based on 18S rDNA
sequences data analysis shown in Fig. 6 comparing the reference data of basidiomycetes
yeast (fabte 3). The descriptions of the three isolates S-3-17, S-3-19, 27,'"Taphrina californica"
and "Taphrina maculans" are given below.
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Table 1. Morphological characteristics of srrains studies
Growth on YM broth medium:After two or three days at 25oC, the cells are round to oval, occur single, the budding isprincipally enteroblastic, the cell wall is composed of multilayered, and the colony color isred.Growth on YMagar medium:After two days at 25oC, the cells are similar in size to those in YM broth. Colonies are pink,the surface is smooth; after 5 to 7 days colonies become extremely mucoid and reddish incolor, developing a yellow pigmentation.Crowth on PDA medium:After one day at 25oC, the cells grow very fast as same as in other media. The vegetativerepncduction occurs also by budding.Growth on corn meal agar (CMA) medium:Neither pseudomycelium nor true mycelium is formed. No ballistospores are formed after 20days at 25oC. No teliospores are formed.Growth at 37"C: positiveFormation of carotenoid pigments: positiveFermentation ability : negativeFormation of starch-like compounds: negativeAssimilation of inositol: negativeUrease activity: positiveDNase activity: positiveDBB color reaction: positiveXylose in the cell wall: absentDNA base composition: 59.7 molVo Gy HPLC)Major ubiquinone system: Q-10Habitat: soil
Description of strain 2l
Growth on YM broth medium:After two days at 25oC, the cells are a ellipsoidal and occur singly or in pairs. Budding isprincipally enteroblastic, the cell wall is composed of multilayers, and the colony color is red.
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Fig. 1. Transmission elecrron micrographs showing the cell wall structure and
conidiogenesis. A, isolate S-3-17; B, isolate 21 C,"Taphrinafarlowit'CBS 376.39; and D, "Taphrina maculanl' CBS 427.69- N, nucleus.Arrows indicate the multilayered cell waIIs.
5i Ip 33 5 q e 5 = ebg I5 ;5 3 0 d E 3 33& Ida 1,,
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h q Q -1 oq r r H + o g n q g c) v-1 oq r:..1 <t \q q \ c.f og l*Q €o.o s riviJ oi di E, ri o + 'j 6i ri oi oi oid d vj.i -.: ltterrh€h€ahhrrrm€€nrhrE€n€o ld
, oqd)9-.,16)qno?q.-: , qn99q\qc9v1v1 , tq lu*O{o{nhrP6 6oa--9r€6 oa 16
$ E s il r i E *; ix r i $: x i= x: H H s k; lg.dNNNr+ l{:: - 13'11IB
Fig. 6. Phylogenetic tree based on the nuclear 18S rDNA sequence data. ]ree.rya9- constructed from the evolutionary distance(18) by using the neighbor-joiningmethod (14) The total number of nucleotides compared was i,162 afteralignment. The bootstrap values were derived from 1,000 resamplings.I, the ustilaginomycete lineage; II, the urediniomycete lineage;and trI, the hymenomycete lineage.
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l,
Table 3. List of SSU IRNA accession numbers offungal srainsused in this study
Species Stain No. Accession No.
Athelia bomdacinaAuricularia auriculaAuricularia p o ly tr i c haBensingtonia intermediaB e nsi ng to nia y amato anaBensingnnia phylladaBulleromyces albusCalocera corneaCronartiwn ribicolaCystofi lobasidium cap itatumDacrymyces chrysospermwDacrymyces stillatusE rythr o b as idi utn has e gaw ia numF i lob as idium fl o r ifo r meFilob asidie lla ne oformansHeterotextus alpinusLe uc o s po r idium lar i-mar i niLe uc os po r idiurn s c o t t i iMixia osmundaeMrakia frigidaPeridermium harknes s eiiRhodospo ridium dacryoidesR ho dos p o r i di um t or ul oi de sRhodotorula glutinisRhodotorula graminisR ho do tor ula muc il a g i no s aS c hizophyll um c o mrnuneSpongipellis unicolorSporidiobo I us j o hn s o ni iSporobolomyces roseus'Taphrina califo r nica "Tremella foliaceaTremella globosporaTremella morifurmisTrichosporon cutaneumUstilago hordeiUstilago maydis
Abbreviations: RUR, Russel Tree Farm; JCM, Japan Collection of Microorganisms,SIKEI-I, Wl!o, Saitama, Japan; MUCL, Myc6rheque de l'Universire Citholique'lpuvain-la-Neuve, Belgium; NCYC, Natiorial Colli:ction of yeast Cultures, 'Norwich, ]]$,- UC, Departrnent of Plant Biology, University of Califomia,Berkeley, USA.
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Growth on YM agar medium:After two days at 25oC, the cells morphology is the same as in liquid medium. Colonies are
pink, the surface is soft and after 4 to 7 days colonies become reddish in color.Growth on PDA medium:After one day at 25oC, the cells grow yery fast, the sueak culture at three days is bright red
to orange red and develops a yellow pigmentation, and the cells are the same other mediums.
The vegetative reproduction is also by budding.Growth on com meal agar (CMA) medium:Neither pseudomycelium nor true mycelium is formed. Ballistospores are formed after 3 to 5
days at 25"C (by the ballistospore-fall method).Growth at 37oC: negativeFormation of carotenoid pigments: positiveFermentation ability: ne gativeFormation of starch-like compounds: negativeAssimilation of inositol: weakUrease activity: positiveDNase activity: weakDBB color reaction: positiveXylose in the cell wall: absentDNA base composition: 48.7 molTo Oy HPLC)Major ubiquinone system: Q-10.Habitat: fern leaves.
Description of "Taphrina californica" CBS 374.39 and "Taphrina maculars" CBS 427.69
Growth on YM broth medium:After eight to ten days cultured at 22oC, the cells are spheroidal to ovoidal and mainiy single,the budding is principally enteroblastic, and the cell wal1 is multilayered.Growth on YM agar medium:After three to five days at 22oC, the cells of young cultures are same as YM broth. the
surface is smooth and soft, after 8 to 10 days colonies become light cream color.Growth on PDA medium:After three days at 22oC, the cell morphology is the samo as the other media. The vegetativereproduction is also by budding.Growth on corn meal agar medium:No ballistospores are formed after 20 days at 22"C (by the baliistospores - fall technique).Neither pseudomycelium nor true mycelium is formed.Growth at 37o C: negativeFermentation ability: negativeFormation of starch-like compounds: negativeAssimilation of inositol: negativeUrease activity : positiveDNase activity: negativeDBB color reaction: positiveXylose in the cell wall: present
All isolates showed a basidiomycete affinity based on the dau such as: the presences ofmultilayered cell walls, enteroblastic budding scars, major ubiquinone Q-10, and positive
reaction for DBB color, urease activity, and extracellular DNase activity tests. But the strain
21 showed weak extracellular DNase activity and had a lower G+C content. Based on the
chemotaxonomic characteristics and 18S rDNA sequences data analysis, three yeast isolates
were divided into two groups. Group 1 comprises strains 5-3-17 and S-3-19 which are
closely related to the genus Rhodotorula. Group 2 comprises only strain 21 which is closely
related to the genus Sporobolomyces.
On the studies of "Taphrina califurnica" and"Taphrina maculars", we have found that
they are defined by the following characteristics: multilayered cell wall, enteroblastic budding,
DBB positive, urease activity positive, and presence of xylose in the cell wall, which indicate
a basidiomycete affinity. However, they had a lower C+C content of DNA and showed
negative extracellular DNase activity. Based on chemotaxonomic characteristics and 18S
rDNA sequence data analysis we can put them into group 3, which is closely related tospecies of the genera Tremella, Bulleromyces (anamorph: Bullera), Filobasidiwn, andFilobasidiella (anamorph.' Cryptococcus), Trichosporon belonging to the basidiomycetes @ig.o.
Group 1: (strains S-3-17 and S-3-19)
Morphological characteristics of strains S-3-17 and S-3-19 were shown in Table 1 whichindicated that these two isolates had the multilayered cell wall and enteroblastic budding(Fig. 1). Neither pseudomycelium nor true mycelium is formed. Beside that, these
indentification is supported by chemotaxonomic data, particularly based on the presence ofQ-10 as the major ubiquinone system and DNA base composition with 60.8 molVo forS-3-17, and 58.7 molVo for S-3-19. Furthermore, they showed positive DBB test, urease and
extracellular DNase activity, characteristics of basidiomycetes. Together with the other
charactericstics such as: no formation of starch-like compound, no assimilation of inositol,
they showed some characters similar to the genus Rhodotorula . Studies on cell wall sugar
component of isolateb indicated that: the mannose is dominant, presence of glucose, galactose
and fucose, and absence of xylose in the cell wall (Fig. 2). The presence or absence offucose, galactose, rhamnose and xylose as well as the ratio of glucose to mamnose afterhydrolysis of purified yeast cell wall are valuable characteristics to assign yeasts or yeast
states of ascomycetes and basidiomycetes phylogenetically (9). However, based on 18S
rDNA sequence analysis and morphological and chemotaxonomic characters, strains S-3-17
and S-3-19 were suggested to be closely related to Rhodotorula mucilaginosa (Fig. 6).
Croup 2:
The strain 21 also showed a basidiomycetes affinity in having characteristics such as:
Q-10 major ubiquinone system, DBB color test positive, urease activiry positive, other cfiaracters
such as no fermentation, no formation of starch-like compound, weak assimilation of inositol.But the isolate 21 showed weak extracellular DNase activity and DNA base composition 48.5
molVo. Studies of cell wall sugar of strain 21 indicated that mannose is dominant, in additionto the presence of fucose, glucose, galactose, and the absence of xylose (Fig. 3). Futher
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investigation indicated that the strain 21 had some morphological characteristics such as:
multilayered cell wall and enteroblastic budding scars as shown by (Fig. 1), and no
pseudomycelium and true mycelium formation. On the other hand, we observed ballistospores
in this strain (Table 1). The isolate 21 indicated typical characteristics of the genus
Sporobolomyces. Morever, from the data of 18S rDNA sequenceing together with some
characters as mentioned above, the strain 21 was found to be closely related a Sporobolomyces
roseus (Frg. 6).
Group 3: "Taphrina californica" CBS 374.39 ar.d"Taphrina maculans" CBS 427.69
When we investigated chemotaxonomic characteristics of 25 species of archiascomycetes,
we found that species such as: "Taphrina farlowit' , "7. californica" , and "T. maculans" ,
showed interesting characteristics. According to Sugiyama et al. (8), in the cellular carbohydrate
profiles, especially the presence of xylose, even in small amounts, is significant taxonomically.
As shown in Table 2, almost all srrains of archiascomycetesTaphrina andProtonyces species
have glucose in dominant in addition to rhamnose, mannose, and galactose. But in Taphrina
farlowii CBS 376.39, mannose and glucose are dominant, and a small amount of galactose is
present, but the rhamnose is absent. In addition to these characteristics, Sugiyamaet al. (17)
suggested that the phylogenetic position of "7. farlowii" is closely related to some species ofthe genus Candida. '"Taphrina califurnica" and "7. macula,ns" showed some characters as
follows: positive reaction for the DBB color and urease activity tests, Q-10 as the major
ubiquinone, 49.3 mol%o C+i in '7. californica" and 46.7 mol?o in"T. maculans". Nofermentation ability, no formation of starch-like compouds formation, no assimilation ofinositol, and no extracellular DNase activity were also found. Futhermore, as the cell wall
sugar component, "Taphrina californica" and "7. macularu" contained xylose in the cellwall (Figs. 4 and 5). By comparing this character and other taxonomic criteria, these twospecies were indicated to have a basidiomycete affinity. Morphological characteristics as
shown in Table 1 and TEM micrographs (Fig. 1) indicated that &ese species had &e multilayered
cell wall and enteroblastic budding. Neither pseudomycelium nor true mycelium is formed-
From the phylogenetic analysis based on 18S rDNA sequence data (Frg. O and some evidences
of taxonomic criteria suggest that these two species '"T. califurnica" and "7. maculans" are
not member of archiascomycetes, but to be closely related to the tremellalean fungi. Thereforc,
both species should be transferred from the Ascomycota to the Basidiomycota.
In conclusion, the polyphasic analysis ofphenotypic and genotypic characters is indispensable
to clarify the identity of strains of yeasts and yeast-like fungi.
Acknowledgements
We thank Dr. A. Hirata, Institute of Molecualr and Cellular Biosciences, The Universityof Tokyo, by whom the TEM micrographs were taken. We also thank the curators of theculture collections, ATCC, CBS, IFO and IMI for supplying the authentic cultures. Thiswork was supponed in part by the Japan International Cooperation Agency (V.P.C and J.S.).
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References
1) Sugiyama, J., and H. Nishida:In Biodiversity and Evolution (Ami, R. et al.,eds.),pp.177-195, The National Science Museum Foundation, Tokyo (1995).
2) Nishida, H., K. Ando, Y. Ando, A. Hirata, and J. Sugiyama: Can. J. Bot.,73 (Suppl 1),
s660-s606 (1995).
3) Chatanon, L., R. Kikawa, H. Kawasaki, D. Attili, V.P. Canhos, H. Kuraishi, and J.
Sugiyama: Annual Reports of ICBiotech. Vol. 17 (manuscript submitted) (1994).
4) Bansetseg, C., Y. Tajiri, D. S. Attili, V. P. Canhos, H. Kuraishi, and J. Sugiyama:Annual Reports of ICBiotech, Vol. i8 (manuscript submitted) (1995).
5) Kreger-van Rij, N. J. W. (ed.) The Yeasts, A Taxonomic Study, Third edition (1984).
6) Barnett, J. A., R. W. Payne, and D. Yarrow: TheYeasts: Characteristics and Idetification.Second edition, Cambridge Univ. Press, Cambridge (1990).
7) Sen, K. and K. Komagata: J. Gen. Appl. Microbiol.,25, 127-137 (1979).
8) Sugiyama, J., M. Fukagawa, S. Chiu, and K. Komagata: J. Gen. Appl. Microbiol.,3l,s19-550 (1985).
9) Prillinger, H., F. Oberwinkler, C. Umile, K. Tlachac, R. Bauer, C. Dorfler, and E.Taufratzhofer: J. Gen. Appl. Microbiol.,39, 1-34 (1993).
10) Hasegawa, T., M. Takizawa, and S. Tanida: J. Gen. Appl. Microbiol.,29,319-322(1983).
l1) Kuraishi, H., Y. Katayama-Fujimura, J. Sugiyama, and T. Yokoyama: Trans. Mycol.Soc. Japan, 26, 383-395, (1985).
12) Vancanneyt, M., R. Coopman, R. Tytgat, J. F. Berny, G. L. Hennebert, and K. Kersters:J. Gen. Microbiol., 38, 363-371 (1992).
13) Mesbah, M., U. Premachandran, and W. B. Whitman: Int. J. Syst. Bacteriol., 39,159-16714) Saitou, N., and M. Nei: Mol. Biol. Evol.,4, 406-425 (1987).15) Thompson, J. D., D. C. Higgins, and T. J. Gibson: Nucleic Acids Res., 22,4673-4680
(1994).
16) Felsenstein, J.: Evolution, 39, 783-'191(1985).17) Sugiyama, J., Y. Tajiri, W. Sjamsuridzal, and H. Nishida:1n Symposium Program &
Abstract Book of the 9th International Symposium on Yeasts, 25-30 Aug. 1996, Sydney,p.9 (i996).
18) Kimura, M.: J. Mol. Evol., 16, 111-120.
19) Kuraishi, H., J. Sugiyama, and Y. Yamada: Bull. JFCC, 7, 117-133 (1991).
