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: imanishi-yumi@nite.go.jp

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.

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