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S
TUDIES IN
M
YCOLOGY
50:
397–400.
2004.
397
Tetrapisispora fleetii sp. nov., a new member of the Saccharomycetaceae
Cletus P. Kurtzman
1*
, Adele Statzell-Tallman
2
and Jack W. Fell
2
1
National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815
N. University Street, Peoria, Illinois;
2
Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600
Rickenbacker Causeway, Key Biscayne, Florida U.S.A.
*Correspondence: C.P. Kurtzman, kurtzman@ncaur.usda.gov
Abstract: A new yeast species, Tetrapisispora fleetii (ex-type strain NRRL Y-27350, CBS 8957, ML 4554), is proposed
based on an isolate from a food-processing plant in Georgia, U.S.A. Genus assignment and distinction from recognized
species is based on phylogenetic analysis of nucleotide sequences from ITS and domains D1/D2 of the large subunit (26S)
rDNA.
Taxonomic novelty: Tetrapisispora fleetii Kurtzman, Statzell-Tallman & Fell sp. nov.
Key words: molecular systematics, new yeast species, Tetrapisispora fleetii.
INTRODUCTION
The genus Tetrapisispora Ueda-Nishim. & Mikata
was proposed by Ueda-Nishimura & Mikata (1999) to
accommodate Kluyveromyces phaffii van der Walt and
three related new species: Tetrapisispora arboricola
Ued.-Nishim. & Mikata, T. iriomotensis Ued.-Nishim.
& Mikata, and T. nanseiensis Ued.-Nishim. & Mikata.
The four species form a distinct clade within the
Saccharomyces Meyen ex E.C. Hansen complex of
species when analyzed from nucleotide divergence in
the small subunit (18S) rDNA. The close relationship
of these four species was verified from a multigene
analysis of the Saccharomyces complex (Kurtzman &
Robnett 2003), which also showed that Kluyveromy-
ces blattae Henninger & Windisch is a basal member
of the Tetrapisispora clade. For this reason, K. blattae
was transferred to the genus Tetrapisispora (Kurtzman
2003).
In the present work, we describe a new species of
Tetrapisispora, which was recognized from sequence
analysis of ITS and the D1/D2 domains of large
subunit (26S) rDNA. This species was isolated from a
food-processing plant in northeastern Georgia and sent
to the University of Miami for identification. We
propose the name Tetrapisispora fleetii for this new
ascosporogenous species.
MATERIALS AND METHODS
Phenotypic characterizations followed the procedures
listed by Yarrow (1998). D1/D2 and ITS rDNA mo-
lecular sequencing employed methods presented by
Fell et al. (2000) and Kurtzman & Robnett (1998).
The ITS and D1/D2 sequences were analyzed phy-
logenetically by maximum parsimony and neighbour-
joining with the Kimura 2-parameter distance correc-
tion using the programmes of PAUP 4.0 (v. 63a)
(Swofford 1998). Sequence data for Tetrapisispora
fleetii (NRRL Y-27350, CBS 8957, ML 4554) were
deposited with GenBank: D1/D2 = AY645662; ITS =
AY645663. The D1/D2 sequences of the other species
included in the analysis were from the studies of
Kurtzman & Robnett (1998, 2003).
RESULTS
The proposed new species of Tetrapisispora was
determined to be novel from phylogenetic analysis of
nucleotide sequences from the domains D1/D2 of the
large subunit rDNA. The dataset used in the analysis
included all known ascomycetous yeast species
(Kurtzman & Robnett 1998, and subsequent GenBank
entries), and the analysis placed the species in the
genus Tetrapisispora near T. phaffii (Fig. 1). Both
maximum parsimony and neighbour-joining analyses
gave essentially the same tree. A further analysis
compared ITS sequences, but because of the large
number of indels in the dataset, about half of the
nucleotides in ITS1 and ITS2 had to be removed to
achieve a reliable alignment. Both maximum parsi-
mony and neighbour-joining analyses gave ITS trees
congruent with the D1/D2 trees.
Tetrapisispora fleetii Kurtzman, Statzell-Tallman
& Fell, sp. nov. MycoBank MB500099. Figs 2–6.
Etymology: The species is named in honor of Prof. dr
Graham Fleet, University of New South Wales, Aus-
K
URTZMAN ET AL
.
398
tralia, for his extensive and outstanding research with
yeasts, food microbiology and biotechnology.
In agaro malti post dies 3 ad 25 ºC, cellulae vegetativae
ellipsoideae (1.5–3.5 × 2.8–6 µm) ad elongatae (1.8–3 × 3–
7 µm), singulae aut binae. Gemmatio multilateralis. Raro
pseudomycelium tenuiter formatur. Asci per conjugationem
cellularum distinctarum vel e cellula cum gemma, 2
ascosporas continentes. Ascosporae sphaericae vel
ellipsoideae. Species homothallica.
Glucosum et galactosum fermentantur. Sucrosum,
maltosum, lactosum, raffinosum, et trehalosum non
fermentantur. Glucosum, galactosum, ribitolum (lente) et
D-gluconas assimilantur. Non assimilantur L-sorbosum,
sucrosum, maltosum, cellobiosum, trehalosum, lactosum,
melibiosum, raffinosum, melezitosum, inulinum, amylum
solubile, D-xylosum, L-arabinosum, D-arabinosum, D-
ribosum, L-rhamnosum, D-glucosaminum, N-acetyl-D-
glucosaminum, methanolum, ethanolum, glycerolum,
erythritolum, galactitolum, D-mannitolum, D-glucitolum, -
methyl-D-glucosidum, salicinum, 2-keto-D-gluconas, 5-
keto-D-gluconas, D-glucuronas, saccharatum, DL-acidum
lacticum, acidum succinicum, acidum citricum, inositolum,
hexadecanum et potassii nitratum. Non crescit in substrato
10 % sal / 5 % glucosi continente. Amylum non formatur.
Non crescit in 50 % glucoso addito. Vitamina externa
crecentiae necessaria. Temperatura 37 ºC cressit. Species
nova a speciebus aliis sequentiis nucleotidicis D1/D2 26S
rDNA et ITS rDNA distinguenda.
Typus: NRRL Y-27350 (CBS 8957, ML 4554) designat
stirpem typicam, isolatus in Georgia, U.S.A., lyophilus
depositus in Collectione Culturarum ARS (NRRL), Peoria,
Illinois U.S.A.
Growth on 5 % malt extract agar: After 3 d at 25 ºC,
the cells are ellipsoidal (1.5–3.5 × 2.8–6 µm) to short-
elongate (1.8–3 × 3–7 µm), and occur singly or in
pairs (Fig. 2). Budding is multilateral. Growth is
tannish-white, semiglistening and butyrous.
Dalmau plate culture on morphology agar: After 7 d
at 25 ºC, true hyphae were not formed under the
coverglass, but occasional poorly differentiated
strands of pseudohyphae were detected (Fig. 3).
Aerobic growth is tannish-white, semiglistening and
butyrous in texture. Colonies are low convex with a
depressed centre. Margins are smooth to finely lobed.
Ascospore formation. Ascospore formation occurred
on YM and yeast morphology agars after 7–10 d at 25
ºC. Ascosporulation was not abundant on these two
media but was absent on 5 % ME and McClary’s
acetate agars. Asci, which become deliquescent at
maturity, may be unconjugated or show conjugation
between independent cells or between a cell and its
bud (Fig. 4). Only two ascospores are formed in each
ascus. The ascospores are either spherical (Fig. 5) or
short-ellipsoidal (Fig. 6). The species may be
homothallic as indicated by the presence of conjuga-
tion between a cell and its bud. To further test this
Table 1. Fermentation, assimilation and other growth reactions of Tetrapisispora fleetii.*
Fermentation:
Glucose + Maltose – Trehalose –
Galactose + Lactose –
Sucrose – Raffinose –
Assimilation:
Glucose + L-Arabinose – D-Mannitol –
Galactose + D-Arabinose – D-Glucitol –
L-Sorbose – D-Ribose – -Methyl-D-glucoside –
Sucrose – L-Rhamnose – Salicin –
Maltose – D-Glucosamine
N-Acetyl-D-glucosamine – D-Gluconate +
Cellobiose – Methanol – DL-Lactate –
Trehalose – Ethanol – Succinate –
Lactose – Glycerol – Citrate –
Melibiose – Erythritol – Inositol –
Raffinose – Ribitol + Hexadecane –
Melezitose – Galactitol – Nitrate –
Inulin – Vitamin-free –
Soluble starch –
D-Xylose –
Additional assimilation tests and other growth characteristics:
2-Keto-D-gluconate – DBB –
5-Keto-D-gluconate – Gelatin liquefaction –
Saccharate – Growth at 37°C –
10 % NaCl + 5% glucose – D-Glucuronate –
Starch formation – Urease –
50 % (w/w) glucose-yeast extract agar –
* + = positive, – = negative, w = weak.
T
ETRAPISISPORA FLEETII SP
.
NOV
.
399
T. blattae
NRRL Y-10934
U69580
T. iriomotensis
NRRL Y-27309
AY046106
T. nanseiensis
NRRL Y-27310
AY046104
Lachancea kluyveri
NRRL Y-12651
U68552
Kluyveromyces marxianus
NRRL Y-8281
U94924
T. arboricola
NRRL Y-27308
AY046105
T. fleetii
NRRL Y-27350
AY645662
T. phaffii
NRRL Y-8282
U69578
16
100
54
99
43
23
25
12
6
16
16
12
11
8
24
29
Fig. 1. Phylogenetic tree showing placement of Tetrapisis-
pora fleetii among species of the genus Tetrapisispora with
reference species Lachancea kluyveri and Kluyveromyces
marxianus (outgroup species in the analysis) as represented
by the single most parsimonious tree derived from maxi-
mum parsimony analysis of nucleotide sequences from 26S
rDNA domains D1/D2. Branch lengths, proportional to
nucleotide substitutions, are given below the branches and
bootstrap values, based on 1000 replicates, are given above
the branches. Frequencies under 50 % are not presented.
Tree length = 241, consistency index = 0.768, retention
index = 0.643, parsimony informative characters = 87. All
taxa are represented by ex-type strains.
possibility, 24 single-ascospore isolates were obtained
by micromanipulation. Six of the spores germinated
and produced colonies that formed two-spored asci.
These results suggest that the species is homothallic,
but because the asci form only two ascospores, it is
not certain that ascosporulation was preceded by
meiosis.
Fermentation, assimilation and other growth charac-
teristics: Table 1.
Type strain: The ex-type strain was isolated in 1999 by an
anonymous collector as a random culture swipe from
equipment in a food-processing plant located in northeast-
ern Georgia, U.S.A. The strain was deposited at CBS,
NCAUR and the University of Miami as CBS 8957, NRRL
Y-27350, ML 4554, respectively.
DISCUSSION
The approximately 70 species placed in the Sac-
charomycetaceae have been assigned to 11 phyloge-
netically circumscribed genera on the basis of multi-
gene sequence analyses (Kurtzman 2003, Kurtzman &
Robnett 2003). Some of these genera, such as Zygo-
saccharomyces B.T.P. Barker and Torulaspora Lind-
ner, can be recognized from phenotype, but others,
such as Tetrapisispora and Kazachstania Zubkova,
cannot be differentiated from phenotype.
Species of the latter two genera differ from one
another in ascospore morphology, as well as in persis-
tence or deliquescence of the asci. Many of the species
ferment and assimilate few carbon compounds, further
limiting diagnostic characters. For Tetraspisispora,
individual species can be recognized through a com-
bination of growth reactions and morphology, and
these diagnostic characters are given in Table 2.
Figs 2–6. Tetraspisispora fleetii NRRL Y-27350. 2.
Budding cells, 5 % ME agar after 3 d. 3. Sparingly differ-
entiated pseudohyphae, aerobic growth, yeast morphology
agar after 7 d. 4. Conjugating cells, 5 % ME agar after 4 d.
5. Pair of spherical ascospores, YM agar after 10 d. 6. Pair
of ovoid ascospores, YM agar after 10 d. Incubation was at
25 ºC for all cultures. Scale bar = 5 µm for all figures.
K
URTZMAN ET AL
.
400
Table 2. Diagnostic characteristics for species of Tetrapisispora.*
Species
Growth/Morphology T. arboricola T. blattae T. fleetii T. iriomotensis T. nanseiensis T. phaffii
Trehalose + – – – – –
Glycerol + v – + + +
Ribitol – – + – – –
D-Gluconate v – + + – +
Vitamin-free – – – w – –
37 °C – – + – – –
Ascus per del del del per del
* + = positive, – = negative, v = strain variable (+/-), w = weakly positive, per = persistent, del = deliquescent.
Phylogenetically, T. fleetii is strongly supported
within the genus Tetrapisispora (Fig. 1) and shares a
branch with T. phaffii. However, internal branch
support is weak, and branch swapping within the
genus can be anticipated with the addition of more
species and the inclusion of additional genes in an
analysis.
ACKNOWLEDGEMENTS
Research at the University of Miami was supported by a
grant from the National Science Foundation (U.S.A.) DEB
0206521. The mention of firm names or trade products does
not imply that they are endorsed or recommended by the
U.S. Department of Agriculture over other firms or similar
products not mentioned.
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