two new ascomycetous anamorphic yeast species related to candida friedrichii—candida jaroonii sp....
TRANSCRIPT
ORIGINAL PAPER
Two new ascomycetous anamorphic yeast species relatedto Candida friedrichii—Candida jaroonii sp. nov.,and Candida songkhlaensis sp. nov.—isolated in Thailand
Yumi Imanishi Æ Sasitorn Jindamorakot Æ Kozaburo Mikata Æ Akira Nakagiri ÆSavitree Limtong Æ Wanchern Potacharoen Æ Morakot Tanticharoen ÆTakashi Nakase
Received: 24 July 2007 / Accepted: 2 April 2008 / Published online: 19 April 2008
� Springer Science+Business Media B.V. 2008
Abstract In a study of yeast diversity in Thailand,
eight strains of hitherto undescribed anamorphic yeasts
were isolated: four from insect frass, two from
Marasmius sp. fruiting bodies, one from a flower, and
one from jackfruit exudates. Phylogenetic analysis of
the D1/D2 domain of 26S ribosomal NA nucleotide
sequences indicated that the eight strains represented
two new species related to Candida friedrichii. Genetic
separation of the two new species was further supported
by DNA–DNA hybridization analysis, which resulted in
between-species similarity values of less than 48%, and
by electrophoretic karyotyping. The two new species are
C. jaroonii sp. nov. (type strain, ST-300T = NBRC
103209T = BCC 11783T = CBS 10790T) and C. song-
khlaensis sp. nov. (type strain, ST-328T = NBRC
103214T = BCC 11804T = CBS 10791T).
Keywords Candida jaroonii � Candida
songkhlaensis � New ascomycetous yeast species �Thailand
Introduction
Ascomycetous yeasts such as Saccharomyces cerevi-
siae are well known for their ability to ferment sugars
and therefore have been used to produce various
fermented foods around the world. However, many yet
undiscovered types of yeast may not only have the
ability to ferment but also to metabolize. Recently, to
better understand yeast diversity and to identify novel
functions of yeasts, microorganism diversity in the
tropical regions of Asia, mainly southeastern Asia, has
been studied, because various areas accommodate
specific plants associated with many yeast species, and
new yeast species are expected to be isolated from
these regions (Nakase 2001).
New ascomycetous yeasts have been isolated and
described in Thailand during the past fifteen years, such
as Candida stellimalicola (Suzuki et al. 1994), Citer-
omyces simensis (Nagatsuka et al. 2002), Candida
krobiensis, Candida sithepensis, Pichia siamensi
(Limtong et al. 2004), P. thermomethanolica (Limtong
et al. 2005), and Tetrapisispora namnaonensis (Sum-
pradit et al. 2005). We found 79 undescribed
ascomycetous species by analysis of 667 isolates
from several kinds of samples such as flowers, fruits,
Y. Imanishi (&) � K. Mikata � A. Nakagiri � T. Nakase
NITE Biological Resource Center (NBRC), National
Institute of Technology and Evaluation (NITE), 2-5-8,
Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan
e-mail: [email protected]
S. Jindamorakot � W. Potacharoen � M. Tanticharoen �T. Nakase
National Center for Genetic Engineering and
Biotechnology (BIOTEC) NSTDA, Thailand Science
Park, 113 Phaholyothin Rd., Klong 1, Klong Luang,
Pathumthani 12120, Thailand
S. Limtong
Department of Microbiology, Faculty of Science,
Kasetsart University, Bangkok 10900, Thailand
123
Antonie van Leeuwenhoek (2008) 94:267–276
DOI 10.1007/s10482-008-9242-2
leave, mushrooms, insect frass and etc. in various
places of Thailand (Nakase et al. 2006); some of
which have already been proposed as new species,
such as Candida easanensis, C. pattaniensis, and
C. nakhonratchasimensis (Jindamorakot et al. 2004)
and Pichia nongkratonensis (Nakase et al. 2005).
The relationships among yeast species and the
sources and locations from which yeasts are isolated,
are important and have been the subject of many studies
(Lachance et al. 2001, 2003; Boekhout 2005; Suh et al.
2005). In particular, many types of yeast, including
undescribed species, have been isolated from the guts of
various beetles and from insect frass (Morais et al. 1995;
Kurtzman and Robnett 1998; Lachance et al. 2001;
Brysch-Herzberg and Lachance 2004; Suh et al. 2005).
Direct relationships between yeasts and insect evolution
have not yet been shown, but yeasts are expected to be
associated with insects. In a study of yeast diversity in
Thailand, yeasts were isolated from insect frass, mosses,
flowers, mushrooms, and other sources (Nakase et al.
2006), and here we propose two new species.
Materials and methods
Yeast strains used in this study
The yeast isolates used in this study are listed in
Table 1. They were all isolated by direct streaking on
YM plates (1% glucose, 0.5% peptone, 0.3% yeast
extract, 0.3% malt extract, 1.5% agar) as described by
Jindamorakot et al. (2004). Candida jaroonii ST-620
was isolated from a sample collected in central
Thailand, C. jaroonii ST-163 was collected in the
northeast, and the other six strains were collected in
the south of Thailand. Annual precipitation is lower
and seasonal variations in temperature are greater in
northeast Thailand than in the other two areas.
Central and southern Thailand experience heavy
rainfall and have a typical tropical climate. The
central part of the country is flat, whereas the
southern part is sandwiched between the Andaman
Sea and the Gulf of Thailand.
Examination of morphological, physiological,
and biochemical characteristics
Morphological, physiological, and biochemical char-
acteristics were investigated according to the methods
described by Yarrow (1998). For the observation of
pseudohyphae, slide cultures were used, also as
described in Yarrow (1998). All strains were grown
on cornmeal agar (1.7% cornmeal agar from Nissui,
Tokyo) for 7 days at 25�C.
Mating tests
Strains to be mated were grown on YM slants at 25�C
for 3 days. Cells of the two strains were taken from
the YM slants using loops and were mixed and spread
on a cornmeal agar slant and then incubated at 25 or
10�C. The mating reaction was examined at 3- or
4-day intervals for up to 1 month and then again
2 months later. Mating tests were performed on all
possible pairs of strains.
Major ubiquinone analysis
Cells were grown in 1,000-ml flasks containing
400 ml of YPD broth (2% glucose, 2% peptone, 1%
yeast extract) on a rotary shaker at 120 rpm and 28�C
for 2 days and were harvested by centrifugation in the
early stationary growth phase. The cells were washed
three times with distilled water and freeze-dried. One
gram of freeze-dried cells was stirred into 60 ml of a
mixture of chloroform and methanol (2:1) for one
night, and then cells were removed by filtration. The
filtrate was evaporated to dryness, and the residue
was dissolved in 1 ml of acetone and concentrated
with N2 gas. The concentrate was dissolved in 0.3 ml
of 99.9% ethanol and analyzed by high-performance
liquid chromatography (HPLC). HPLC analysis was
carried out as described by Mikata and Yamada
(1999).
Sequencing of the D1/D2 domain of 26S
ribosomal DNA, the internal transcribed
spacer (ITS) region and phylogenetic analysis
Genomic DNA was prepared using Dr. GenTLE for
Yeast (Takara, Japan) according to the manufac-
turer’s protocol. Polymerase chain reaction (PCR)
was performed according to methods described for
the amplification of the D1/D2 domain of 26S
ribosomal DNA (rDNA) (Kurtzman and Robnett
1997) or ITS region (White et al. 1990). Purification
of PCR products and determination of the sequences
were performed as described by Imanishi et al.
268 Antonie van Leeuwenhoek (2008) 94:267–276
123
(2007). Phylogenetic trees were constructed by the
neighbor-joining method (Saitou and Nei 1987) using
a CLUSTAL W software package (Thompson et al.
1994). Bootstrap values (Felsenstein 1985) were
calculated from 1,000 trials.
The Genbank accession numbers of D1/D2 26S
sequences are shown in Fig. 1 and those of ITS
sequences are AB360437-360442, AB360444 and
AB360445.
DNA base composition (mol% G + C)
and DNA–DNA hybridization
A loopful of cells was inoculated into 40 ml YPD
broth and grown at 25�C with shaking at 100 rpm
overnight. Cells were collected in a 20-ml tube by
centrifugation and used for genomic DNA prepara-
tion. The genomic DNA was prepared according to
the method of Holm et al. (1986) as modified by
Kaneko and Banno (1991). The base composition was
determined by HPLC as described by Tamaoka and
Komagata (1984). DNA–DNA hybridization was
performed with the photobiotin microplate-hybrid-
ization method of Kaneko and Banno (1991).
Hybridizations were performed at 38�C with at least
three repetitions.
Electrophoretic karyotyping
Cells were collected from 5 ml of a culture grown
overnight in YPD broth. Preparation of agar plugs
containing chromosomal DNA, electrophoresis, and
detection of the chromosomal bands were performed
as described by Imanishi et al. (2007).
Results and discussion
Taxonomy
All isolates had a negative result in the diazonium
blue B (DBB) reaction and are therefore considered
to be ascomycetous yeasts. Ascospores were not
observed either in cultures or mating tests. These
results and macroscopic and microscopic character-
istics suggest that the isolates belong to the genus
Candida.
In the phylogenetic tree based on the nucleotide
sequences of the D1/D2 domain of 26S rDNA,
Table 1 Summary of yeasts examined in this study
Isolation
No.
Collection numbers Source Location Year of isolation
NBRC No. BCC No. CBS No.
C. jaroonii
ST-163 103212 8399 Flower Phu-Wao (Nong Khai), Thailand Feb. 2001
ST-300T 103209T 11783T 10790T Insect frass Kao-Yaow (Pattani), Thailand Mar. 2001
ST-365 103210 15077 Marasmius sp. Krung Ching Waterfall
(Nakhon Si Tammarat), Thailand
Sep. 2001
ST-366 103211 15078 Marasmius sp. Krung Ching Waterfall
(Nakhon Si Tammarat), Thailand
Sep. 2001
ST-620 103213 15303 Exudate of Jackfruit Thong Pha Phum Waterfall
(Kanchanaburi), Thailand
Nov. 2003
C. songkhlaensis
ST-328T 103214T 11804T 10791T Insect frass Nam Tok Tone-Nga-Chang Waterfall
(Songkhla), Thailand
Mar. 2001
ST-329 103215 11805 Insect frass Nam Tok Tone-Nga-Chang Waterfall
(Songkhla), Thailand
Mar. 2001
ST-333 103216 11809 Insect frass Nam Tok Tone-Nga-Chang Waterfall
(Songkhla), Thailand
Mar. 2001
NBRC: National Institute of Technology and Evaluation Biological Resource Center, Japan. BCC: Biotech Culture Collection, National
Center for Genetic Engineering and Biotechnology, Thailand. CBS: Centraalbureau voor Schimmelcultures, The Netherlands
Antonie van Leeuwenhoek (2008) 94:267–276 269
123
isolates were related to Candida friedrichii and allied
species and were separated into two species (Fig. 1):
one containing ST-163, ST-300, ST-365, ST-366, and
ST-620 (C. jaroonii); the other containing ST-328,
ST-329, and ST-333 (C. songkhlaensis). The best
match for the strains was C. friedrichii, according to
Fig. 1 Phylogenetic relationships inferred from nucleotide
sequences of the D1/D2 domain of 26S rDNA by the neighbor-
joining method. Bootstrap values were calculated from 1,000
replicates, and values below 60% were omitted. All sequences
were edited to the longest common region (501 bp). The
sequence accession numbers are shown in parentheses. The bar
indicates the sequence dissimilarity value of 0.01 substitutions/
site. Saccharomyces cerevisiae NRRL Y-12632T was used as
an out-group
270 Antonie van Leeuwenhoek (2008) 94:267–276
123
the results of a BLAST search using the D1/D2
domain sequences. In the phylogenetic tree based on
the sequences of the internal transcribed spacer (ITS)
of the rDNA (results not shown), the same eight
strains were separated into the same two species as
found by analyses of the D1/D2 domain of 26S
rDNA.
Candida jaroonii differed from C. friedrichii by
six substitutions (99.5% similarity); and from C. song-
khlaensis by two substitutions (99.8% similarity) in
their D1/D2 domain sequences. In their ITS
sequences, C. jaroonii differed from C. friedrichii
by 30 substitutions (96.9% similarity), and from
C. songkhlaensis by 6 substitutions (99.4% similar-
ity). These differences suggest that the two new taxa
are closely related to, but different species from
C. friedrichii. Within the same species, the D1/D2
domain sequences were identical, but one substitution
was found in the ITS region. These two species and
C. friedrichii showed similar mol% G + C values,
from 32.9 to 34.8% (Table 2). In the DNA–DNA
hybridization experiments, low homology values,
ranging from 22 to 48%, were found between the
two species, whereas high values (over 87%) were
observed among isolates of each species (Table 2).
Chromosomal DNA profiles examined by pulsed field
gel electrophoresis were clearly different between the
two species (Fig. 2). On the basis of these results, we
conclude that these two species represent two differ-
ent new species. In physiological characteristics, the
two species can assimilate D-Gluconic acid and
D-Glucosamine, and neither melibiose, Raffinose
nor Galactitol in contrast to C. friedrichii (Table 3).
Table 2 DNA relatedness
Strains G + C DNA relatedness (%)
1 2 3 4 5 6 7 8 9
C. jaroonii
1. ST-300T 34.1 100 100 97 85 99 47 48 47 36
2. ST-163 34.7 – 100 99 93 93 40 37 41 26
3. ST-365 34.1 – – 100 88 87 33 33 36 21
4. ST-366 34.1 – – – 100 91 22 23 24 13
5. ST-620 34.8 – – – – 100 27 35 31 31
C. songkhlaensis
6. ST-328T 34.3 45 42 23 45 36 100 91 94 27
7. ST-329 34.5 – – – – – – 100 95 31
8. ST-333 34.7 – – – – – – – 100 19
C. friedrichii
9. NBRC 10277T 32.9–33.7a 13 17 9 16 15 10 16 12 100
a Data from Meyer et al. (1998)
Fig. 2 Electrophoretic karyotyping of the isolates. Lanes 1
and 9, S. cerevisiae (size standard); lane 2, ST-43; lanes 3, 4
and 5, C. jaroonii ST-300, ST-365 and ST-366; lanes 6, 7 and
8, C. songkhlaensis ST-328, ST-329 and ST-333
Antonie van Leeuwenhoek (2008) 94:267–276 271
123
The utilization of propane-1, 2-diol is different
between the two species with a positive reaction in
C. songkhlaensis while C. jaroonii cannot assimilate
this compound (Table 3).
Ecology
Strains of C. jaroonii were isolated from various
substrates and locations. For instance, ST-163 was
isolated from a flower found in northeast Thailand
and ST-300 was isolated from insect frass in the south
(Table 1). The two strains SA5S11 (EF 460617.1)
and FN16S13 (EF 460530.1), isolated from soil in
Taiwan, are likely to belong to the same species as
Table 3 Phenotypic characteristics of the two new species and
C. friedrichii
Characteristic Species
C.songkhlaensis
C.jaroonii
C.friedrichiia
Fermentation
Glucose + + +
Galactose + + s/-
Sucrose - - -
Maltose - - -
Lactose - - -
Raffinose - - -
Cellobiose + + N
Assimilation
Glucose + + +
Galactose + + +
L-Sorbose -/L + +
Sucrose + + +
Maltose + + +
Cellobiose + + +
Trehalose + + +
Lactose - - -
Melibiose - - +
Raffinose - - +
Melezitose + + +
Inulin - - -
Soluble starch -/w -/w -
D-Xylose + + +
L-Arabinose +/L + +
D-Arabinose + + v
D-Ribose + + +
Table 3 continued
Characteristic Species
C.songkhlaensis
C.jaroonii
C.friedrichiia
L-Rhamnose - -/+ -
Ethanol L +/L +
Glycerol + + +
Erythritol +/L + +
Ribitol + + +
Galactitol - - +
D-Mannitol + + +
D-Glucitol + + +
a-Methyl-D-
glucoside
+ + +
Salicin + + +
Glucono-D-lactone + + +
2-Ketogluconic
acid
+ + +
5-Ketogluconic
acid
- - N
DL-Lactic acid L/Lw L/Lw V
Succinic acid + + +
Citric acid + + +
Inositol - - -
D-Glucuronic acid - - -
D-Galacturonic acid - - -
Xylitol + + +
D-Gluconic acid + + -
D-Glucosamine + +/L -
Propane-1,2-diol Lw - -
Butane-2,3-diol - - -
Methanol - - -
Hexadecane + + L
Potassium nitrate - - -
Ethylamine + + +
L-Lysine + + +
Cadaverine + + +
Vitamins free - - -
10% NaCl + 5%
glucose
+ + +
Growth temperature
(37�C)
+ + -
Growth temperature
(40�C)
- - -
DBB reaction - - -
a Data from Meyer et al. (1998); +: positive; w: weak growth;
-: negative; L: latent; N: no data; V: variable
272 Antonie van Leeuwenhoek (2008) 94:267–276
123
their D1/D2 26S sequences are identical to that of
C. jaroonii isolates from Thailand (Fig. 1). There-
fore, C. jaroonii seems to be associated with various
substrates and to be a common yeast species in the
natural environment in Southeast Asia.
Three strains of C. songkhlaensis were isolated
from insect frass in Nam Tok Tone-Nga-Chang
Waterfall. We attempted to isolate strains of C. song-
khlaensis from insect frass and other substrates from
other areas in Thailand but were unable to obtain any
strains. Therefore, the distribution of this species may
be localized. We were unable to determine the insect
species associated with the frass from which C. song-
khlaensis was isolated. By identifying this insect
species, we may be able to locate C. songkhlaensis
strains more efficiently, and the relationship among
this species, the substrate, and the location may
become more apparent.
Candida jaroonii, Imanishi, Jindamorakot, Nakagiri,
Limtong et Nakase sp. nov.
In liquido ‘‘YM’’ post dies 3 ad 25�C, cellulae
vegetativae ovoideae, ellipsoideae, cylindricae aut
Fig. 3 Photomicrographs
of vegetative cells and
pseudohyphae of
C. jaroonii ST-300 (a, b)
and C. songkhlaensisST-328 (c, d) grown in YM
broth for three days (a, c)
and on cornmeal agar for
7 days (b, d) at 25�C. Scale
bar = 10 lm
Antonie van Leeuwenhoek (2008) 94:267–276 273
123
elongatae, (2–4.5 9 2–9.5) lm (Fig. 3a), singulae
aut binae. In agro farina Zea mays confecto pseudo-
mycelium formatur (Fig. 3b). In medio agaro post
dies 7 ad 25�C, coloniae infimo-convexae, lenis cum
cremea.
Glucosum, galactosum et cellobiosum fermen-
tantur, et non, sucrosum, maltosum, lactosum nec
raffinosum.
Glucosum, galactosum, L-sorbosum, sucrosum,
maltosum, cellobiosum, trehalosum, melezitosum,
D-xylosum, L-arabinosum, D-arabinosum, D-ribosum,
L-rhamnosum (vel nullum), ethanolum (vel lente),
glycerolum, erythritolum, ribitolum, D-mannitolum,
D-glucitolum, a-methyl-D-glucosidum, salicinum,
glucono-d-lactonum, acidum 2-ketogluconicum,
DL-lacticum (lente vel nullum, vel lente et exiguum),
succinicum, acidium citricum, xylitolum, D-glucona-
tum, D-glucosaminum (vel lente) et hexadecanum
assimilantur, at non lactosum, melibiosum, raffinosum,
inulinum, amylum solubile (vel exiguum), galactitolum,
acidum 5-ketoglucosicum, inositolum, acidum D-glu-
curonicum, acidum D-galacturonicum, propanum 1,2
diolum, butanum 2,3 diolum nec methanolum. Ethyla-
minum, cadaverinum et L-lysinum assimilantur at non
nitricum. Ad crescentiam vitaminae externae necessaria
sunt. Non crescere potest in 10% natrium chloridum/5%
glucoso. Augmentum in 37�C. Diazonium caeruleum
B non respodens. Systema coenzymatis Q-9 adest.
Guaninum et cytosinum acidi deoxyribonucleati
34.1 mol%.
Typus stirps ST-300 (NBRC 103209T, BCC 11783 T,
CBS 10790T and Mycobank number 511065) isolatus
ex pulverize lingo insecto, in Kao-Yaow (Pattani),
Thailandia.
Description of Candida jaroonii, Imanishi,
Jindamorakot, Nakagiri, Limtong et Nakase sp. nov.
Growth in YM broth: after 3 days at 25�C, cells
are ovoidal to long ovoidal, ellipsoidal, cylindrical, or
elongate, 2-4.5 9 2–9.5 lm, and occur singly or in
pairs (Fig. 3a). Pseudomycelia are often observed.
Thin pellicle and sediment are produced. On YM agar
media, after 7 days at 25�C, the colonies are low
convex, smooth, and cream-colored.
Slide culture on cornmeal agar: Well-developed
pseudomycelia are abundantly produced (Fig. 3b).
Physiological and biochemical characteristics were
summarized in Table 3.
Ubiquinone type: Q-9.
The G + C content of the nuclear DNA:
34.1 mol% (by HPLC).
Nucleotide sequences of the D1/D2 domain of
26S rDNA (DQ404511, DQ404512, DQ404493,
AB292057.1, AB292056.1) at position 454: A; and
at position 462:G.
The type strain: ST-300 (NBRC 103209T, BCC
11783 T, CBS 10790T and Mycobank number 511065)
isolated from insect frass collected at Kao-Yaow
(Pattani), Thailand, in March 2001.
Etymology: The specific epithet is derived from
‘‘Dr. Jaroon Kumnuanta’’, for his many contributions
to yeast science in Thailand.
Candida songkhlaensis, Imanishi, Jindamorakot,
Nakagiri, Limtong et Nakase sp. nov.
In liquido ‘‘YM’’ post dies 3 ad 25�C, cellulae
vegetativae sphaericae, ovoideae, ellipsoideae, cylin-
dricae vel elongatae, (2–5 9 7) lm, singulae aut
binae (Fig. 3c). In agro farina Zea mays confecto
pseudomycelium formatur (Fig. 3d). In medio agaro
post dies 7 ad 25�C, coloniae infimo-convexae, lenis
cum cremea.
Glucosum, galactosum et cellobiosum fermentan-
tur, et non, sucrosum, maltosum, lactosum nec
raffinosum.
Glucosum, galactosum, sucrosum, maltosum, cel-
lobiosum, trehalosum, melezitosum, D-xylosum,
L-arabinosum (vel lente), D-arabinosum, D-ribosum,
ethanolum (vel lente), glycerolum, erythritolum,
ribitolum, D-mannitolum, D-glucitolum, a-methyl-
D-glucosidum, salicinum, glucono-d-lactonum, aci-
dum 2-ketogluconicum, DL-lacticum (lente vel lente
et exiguum), acidum succinicum, acidium citricum,
xylitolum, D-gluconatum, D-glucosaminum, propa-
num 1,2 diolum (lente et exiguum) et hexadecanum
assimilantur, at non L-sorbosum (vel lente), lactosum,
melibiosum, raffinosum, inulinum, amylum solubile
(vel exiguum), L-rhamnosum, galactitolum, acidum
5-ketogluconicum, inositolum, acidum D-glucuroni-
cum, acidum D-galacturonicum, butanum 2,3 diolum
nec methanolum. Ethylaminum, cadaverinum
et L-lysinum assimilantur at non nitricum. Ad cres-
centiam vitaminae externae necessaria sunt. Non
crescere potest in 10% natrium chloridum/5% gluc-
oso. Augmentum in 37�C. Diazonium caeruleum B
non respodens. Systema coenzymatis Q-9 adest.
Guaninum et cytosinum acidi deoxyribonucleati
34.3–34.7 mol%.
274 Antonie van Leeuwenhoek (2008) 94:267–276
123
Typus stirps ST-328 (NBRC 103214T, BCC 11804T,
CBS 10791T and Mycobank number 511066) isolatus
ex pulverize lingo insecto, in Nam Tok Tone-Nga-
Chang Waterfall (Songkhla), Thailandia.
Description of Candida songkhlaensis, Imanishi,
Jindamorakot, Nakagiri, Limtong et Nakase sp. nov.
Growth in YM broth: after 3 days at 25�C, cells
are spherical, ovoidal, ellipsoidal, cylindrical, or
elongate, 2–5 9 7 lm, and occur singly, in pairs, or
in chains (Fig. 3c). Pseudomycelia often observed.
Thin pellicle and sediment are produced. On YM agar
media after 7 days at 25�C, the colonies are low
convex, smooth, and cream-colored.
Slide culture on cornmeal agar: Well-developed
pseudomycelia are abundantly produced (Fig. 3d).
Physiological and biochemical characteristics were
summarized in Table 3.
Ubiquinone type: Q-9.
The G + C content of the nuclear DNA: 34.3–34.7
mol% by HPLC.
Nucleotide sequences of the D1/D2 domain of 26S
rDNA (DQ404499, DQ404500, DQ404503) at posi-
tions 454 and 462: T.
The type strain: ST-328 (NBRC 103214T, BCC
11804T, CBS 10791T and Mycobank number 511066)
isolated from insect frass collected at Nam Tok Tone-
Nga-Chang Waterfall (Songkhla), Thailand, in March
2001.
Etymology: The specific epithet is derived from
‘‘Songkhla’’, the place of isolation.
Acknowledgements We thank Mr. S. Ninomiya for technical
support and Dr. K. Shimizu of the University of Chiba for
valuable suggestions on the manuscript.
References
Boekhout T (2005) Gut feeling for yeasts. Nature 434:449–450
Brysch-Herzberg M, Lachance MA (2004) Candida bombi-phila sp. nov., a new asexual yeast species in the
Wickerhamiella clade. Int J Syst Evol Microbiol 54:
1857–1859
Felsenstein J (1985) Confidence limits on phylogenies: an
approach using the bootstrap. Evolution 39:783–791
Holm C, Meeks-Wagner DW, Frangman WL, Botstein D
(1986) A rapid, efficient method for isolating DNA from
yeast. Gene 42:169–173
Imanishi Y, Ueda-Nishimura K, Mikata K (2007) Two new
species of Kazachstania that form ascospores connected
by a belt-like intersporal body: Kazachstania zonata and
Kazachstania gamospora. FEMS Yeast Res 7:330–338
Jindamorakot S, Am-in S, Thuy TT, Duy ND, Kawasaki H,
Potacharoen W, Limtong S, Tanticharoen M, Nakase T
(2004) Candida easanensis sp. nov., Candida pattaniensissp. nov. and Candida nakhonratchasimensis sp. nov.,
three new species of yeasts isolated from insect frass in
Thailand. J Gen Appl Microbiol 50:261–269
Kaneko Y, Banno I (1991) Reexamination of Saccharomycesbayanus strains by DNA–DNA hybridization and elec-
trophoretic karyotyping. IFO Res Comm 15:30–41
Kurtzman CP, Robnett CJ (1997) Identification of clinically
important ascomycetous yeasts based on nucleotide
divergence in the 50 end of the large-subunit (26S) ribo-
somal DNA gene. J Clin Microbiol 35:1216–1223
Kurtzman CP, Robnett CJ (1998) Three new insect-associated
species of the yeast genus Candida. Can J Microbiol
44:965–973
Lachance MA, Starmer WT, Rosa CA, Bowles JM, Barker JS,
Janzen DH (2001) Biogeography of the yeasts of
ephemeral flowers and their insects. FEMS Yeast Res
1:1–8
Lachance MA, Bowles JM, Starmer WT (2003) Geography and
niche occupancy as determinants of yeast biodiversity: the
yeast–insect–morning glory ecosystem of Kıpuka Puaulu,
Hawaii. FEMS Yeast Res 4:105–111
Limtong S, Srisuk N, Yongmanitchai W, Kawasaki H,
Yurimoto H, Nakase T, Kato N (2004) Three new ther-
motolerant methylotrophic yeasts, Candida krabiensis sp.
nov., Candida sithepensis sp. nov., and Pichia siamensissp. nov., isolated in Thailand. J Gen Appl Microbiol
50:119–127
Limtong S, Srisuk N, Yongmanitchai W, Yurimoto H, Nakase
T, Kato N (2005) Pichia thermomethanolica sp. nov., a
novel thermotolerant, methylotrophic yeast isolated in
Thailand. Int J Syst Evol Microbiol 55:2225–2229
Meyer SA, Payne RW, Yarrow D (1998) Candida Berkhout.
In: Kurtzman CP, Fell JW (eds) The yeasts, a taxonomic
study, 4th edn. Elsevier, Amsterdam
Mikata K, Yamada Y (1999) The ubiquinone system in
Hasegawaea japonica (Yukawa et Maki) Yamada et Banno:
a new method for identifying ubiquinone homologs from
yeast cells. Inst Ferment Osaka Res Commun 19:41–46
Morais PB, Martins MB, Klaczko LB, Mendonca-Hagler LC,
Hagler AN (1995) Yeast succession in the Amazon fruit
Parahancornia amapa as resource partitioning among
Drosophila spp. Appl Environ Microbiol 61:4251–4257
Nagatsuka Y, Kawasaki H, Limtong S, Mikata K, Seki T
(2002) Citeromyces siamensis sp. nov., a novel halotol-
erant yeast isolated in Thailand. Int J Syst Evol Microbiol
52:2315–2319
Nakase T (2001) Ballistosporous yeasts found in the phyllo-
sphere of Asia. In: Nga BH, Tan HM, Suzuki K (eds)
Microbial diversity in Asia. World Scientific Publishing
Co., pp 3–40
Nakase T, Jindamorakot S, Am-in S, Kawasaki H, Potacharoen
W, Tanticharoen M (2005) Pichia nongkratonensis sp.
nov., a new species of ascomycetous yeast isolated from
insect frass collected in Thailand. Mycoscience 46:
192–195
Nakase T, Jindamorakot S, Am-in S, Potacharoen W, Tanti-
charoen M (2006) Yeast biodiversity in tropical forests of
Asia. In: Peter G, Rosa CA (eds) The yeast handbook
Antonie van Leeuwenhoek (2008) 94:267–276 275
123
biodiversity and ecophysiology of yeasts. Springer-
Verlag, pp 443–460
Saitou N, Nei M (1987) The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol
Evol 4:406–425
Suh SO, Nguyen NH, Blackwell M (2005) Nine new Candidaspecies near C. membranifaciens isolated from insects.
Mycol Res 109:1045–1056
Sumpradit T, Limtong S, Yongmanitchai W, Kawasaki H, Seki
T (2005) Tetrapisispora namnaonensis sp. nov., a novel
ascomycetous yeast species isolated from forest soil of
Nam Nao National Park, Thailand. Int J Syst Evol
Microbiol 55:1735–1738
Suzuki M, Nakase T, Komagata K (1994) Candida stellimali-cola, a new species of anamorphic yeast isolated from star
apple in Thailand. J Gen Appl Microbiol 40:115–121
Tamaoka J, Komagata K (1984) Determination of DNA base
composition by reversed-phase high-performance liquid
chromatography. FEMS Microbiol Lett 25:125–128
Thompson JD, Higgins DG, Gibson TJ (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 11:4673–4680
White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and
direct sequencing of fungal ribosomal RNA genes for
phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ,
White TJ (eds) PCR protocols: a guide to methods and
applications. Academic Press, Inc., New York, pp 315–322
Yarrow D (1998) Methods for the isolation, maintenance and
identification of yeasts. In: Kurtzman CP, Fell JW (eds) The
yeasts, a taxonomic study, 4th edn. Elsevier, Amsterdam
276 Antonie van Leeuwenhoek (2008) 94:267–276
123