International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2364–2369
DOI 10.1099/ijs.0.061499-0
Hanseniaspora nectarophila sp. nov.,
a yeast species isolated from ephemeral flowers
Neža Čadež,1 Fernando C. Pagnocca,2 Peter Raspor1 and Carlos A. Rosa3
Correspondence
Neža Čadež
[email protected]
1
Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana,
Jamnikarjeva 101, 1000 Ljubljana, Slovenia
2
Centre for the Study of Social Insects, UNESP – São Paulo State University, Rio Claro, SP, Brazil
3
Departamento de Microbiologia, ICB, C.P. 486, Universidade Federal de Minas Gerais,
Belo Horizonte, Minas Gerais, 31270-901, Brazil
Seven apiculate yeast strains that were isolated from the flowers of Syphocampylus corymbiferus
Pohl in Brazil are genetically, morphologically and phenotypically distinct from recognized species
of the genera Hanseniaspora and Kloeckera. Genetic discontinuities between the novel strains
and their closest relatives were found using a networking approach based on the concatenated
sequences of the rRNA gene (internal transcribed spacer and D1/D2 of the LSU), and the
protein-coding genes for actin and translation elongation factor-1a. Phylogenetic analysis based
on the rRNA and the actin gene placed the novel species represented by the strains in close
relationship to Hanseniaspora meyeri and Hanseniaspora clermontiae. PCR fingerprinting with
microsatellite primers confirmed the genetic heterogeneity of the novel species. The name
Hanseniaspora nectarophila sp. nov. is proposed, with UFMG POG a.1T (5ZIM 2311T5CBS
13383T) as the type strain; MycoBank no. MB807210. As the current description of the genus
does not allow the presence of multilateral budding, an emended diagnosis of the genus
Hanseniaspora Zikes is proposed.
Apiculate yeasts that belong to the genus Hanseniaspora
and its anamorph Kloeckera can cause fermentative spoilage
once they are introduced onto overripe and senescent fruit
(Phaff & Starmer, 1987). The vectors for their dispersal are
generally Drosophila species that are attracted to certain
fermented substrates, where they feed, oviposit and pick up
spoilage microbiota (Brito da Cunha et al., 1957; Miller &
Phaff, 1962; Morais et al., 1992; Chandler et al., 2012).
Apiculate yeasts belong to the early colonizers of decaying
fruit because they can grow rapidly, and this ecological
advantage enables them to predominate over the fruit
surface microbiota (Brito da Cunha et al., 1957). Later in
the process of fruit deterioration, these Hanseniaspora–
Kloeckera species are replaced by other yeast species, due to
their limited ability to assimilate various carbon sources
(Morais et al., 1995).
The genera Hanseniaspora and Kloeckera comprise, at
the time of writing, 17 recognized species that have been
delineated based on DNA–DNA hybridization of their
Abbreviations: EF-1a, translational elongation factor-1a; ITS, internal
transcribed spacer; MST, minimum spanning tree.
The GenBank/EMBL/DDBJ accession numbers for the sequences
determined in this study are listed in Table S1.
One supplementary table and three supplementary figures are available
with the online version of this paper.
2364
whole genomes (Meyer et al., 1978; Cadez et al., 2003;
Jindamorakot et al., 2009) or on sequencing of the D1/D2
domain of the LSU rRNA gene (Chang et al., 2012). This
classification has been confirmed by several phylogenetic
studies (Yamada et al., 1992; Boekhout et al., 1994; Kurtzman,
2003), although a single phylogenetic marker that can reliably
reconstruct the relationships between species of the genus
Hanseniaspora has not yet been described (Cadez et al., 2006).
As the rates for nucleotide substitutions in the LSU D1/D2
region between closely related species of Hanseniaspora are
lower than between the majority of yeast species (Kurtzman &
Robnett, 1998), a polyphasic approach for species delineation
must be applied.
In the present study, we report on the isolation of seven
apiculate yeast strains from flowers of Syphocampylus
corymbiferus (Campanulaceae) in Brazil that are genetically,
morphologically and phenotypically distinct from recognized species of the genera Hanseniaspora and Kloeckera.
All seven strains of apiculate yeasts were isolated from
flowers collected at the São Sebastião do Ribeirão Grande
farm in the Atlantic rainforest of the Pindamonhangaba
municipal area in the State of São Paulo, Brazil (22u 449 280
W 45u 289 190 S), in June 2006. Seven flowers of S.
corymbiferus were aseptically sampled using sterile plastic
bags. Yeasts were cultured within a few hours of sampling.
The nectar region of the flowers was scraped gently with a
Downloaded from www.microbiologyresearch.org by
061499 G 2014 IUMS
IP: 88.99.165.207
On: Sun, 18 Jun 2017 05:58:39
Printed in Great Britain
Hanseniaspora nectarophila sp. nov.
sterile loop and streak-inoculated on yeast extract-malt
extract agar (YMA; glucose 1 %, peptone 0.5 %, malt extract
0.3 %, yeast extract 0.3 %, agar 2 % and chloramphenicol
10 mg%). The strains were deposited in the Culture Collection of Microorganisms and Cells of the Universidade
Federal de Minas Gerais, Brazil (UFMG). Details of the
strains used in this study, their origins and GenBank
accession numbers are listed in Table S1 available in the
online Supplementary Material. The strains were phenotypically characterized by standard methods, according to
Kurtzman et al. (2011). Sporulation was investigated on
yeast potato-glucose (Sigma), 3 and 5 % malt extract (Difco;
Becton, Dickinson and Company) and YMA at 26 uC over
3 weeks. DNA was extracted from the cultures grown on
yeast potato-glucose agar plates for 2 days, using MasterPure
Yeast DNA Purification kits (Epicentre). Three microsatellite primers, (ATG)5, (GTG)5 and (GACA)4, were used in
PCR amplification reactions, as described previously (Cadez
et al., 2002). The thermal cycler was programmed for 35
cycles of 1 min at 94 uC, 1 min at 48 uC for primer (ATG)5,
1 min at 52 uC for (GTG)5 or 1 min at 43 uC for (GACA)4,
followed by 2 min at 72 uC. The PCR products were
separated on 2.5 % agarose gels by electrophoresis at 180 V
for 40 min. Ethidium-bromide-stained gels were documented by GelDoc 2000 (Bio-Rad) and processed using
BioNumerics 7.1 (Applied Maths). Similarities between the
combined fingerprints were calculated using the Pearson’s
product moment correlation coefficient (r), based on the
overall densitometric profiles of the banding patterns.
Cluster analysis of the pair-wise values was performed using
the UPGMA algorithm.
The internal transcribed spacer (ITS) and the LSU D1/D2
domain of the rRNA gene, the protein-coding genes for
actin and translation elongation factor 1 alpha (EF-1a) were
amplified and sequenced as detailed by Cadez et al. (2006),
except that the sequences were determined by a commercial
sequencing facility (Macrogen Inc., South Korea). The
relationships among the yeast strains were established by the
minimum spanning tree (MST) network creation method,
using BioNumerics 7.1, and by parsimony network analysis,
using the TCS 1.21 program (Clement et al., 2000). Gapped
positions were excluded from the analysis. For the phylogenetic analysis, the sequences were aligned using CLUSTAL X
(Thompson et al., 1997). The most-parsimonious trees were
generated by the PAUP* 4.0b10 software package (Swofford,
2002). Bootstrap support for the trees was determined from
1000 replications.
Species delineation, phylogenetic placement and
ecology
The seven strains formed ascospores on all of the media
tested, although sporulation was most abundant on 3 %
malt agar. The strains differed by two or fewer nucleotide
substitutions (¡0.35 % sequence divergence) in their
ITS region and D1/D2 LSU rRNA gene, and by up to five
substitutions (¡0.7 % divergence) in the partial sequences
of the protein-coding genes for actin and EF-1a. Further
http://ijs.sgmjournals.org
evidence for the conspecificity of the strains was confirmed
by PCR fingerprinting with three microsatellite primers
(Fig. S1). The seven strains shared similar PCR fingerprint
profiles, but were different from the type strains and other
representative strains of species of the genera Hanseniaspora
and Kloeckera.
Furthermore, a BLAST similarity search with the D1/D2 LSU
confirmed that the Brazilian strains belong to the apiculate
yeast family Saccharomycodaceae. Their closest relatives
were Hanseniaspora meyeri, Hanseniaspora clermontiae,
Hanseniaspora opuntiae and Hanseniaspora guilliermondii
from which they differed by four nucleotide substitutions
(0.7 % sequence divergence). Although four nucleotide
substitutions is not indicative of a separate species according
to the generalizations of Kurtzman & Robnett (1998), we
sought for further evidence of a novel species because lower
evolutionary rates in the D1/D2 region in comparison to the
overall genetic divergence for the Hanseniaspora uvarum–H.
meyeri–H. clermontiae and H. guilliermondii–Hanseniaspora
lachancei–H. opuntiae–Hanseniaspora pseudoguilliermondii
species complexes have been observed (Cadez et al., 2003,
2006). As the discriminating capacity of the protein-coding
genes for resolving relationships between closely related
species is better than that of the ribosomal gene (Daniel &
Meyer, 2003; Cadez et al., 2006) we used actin and EF-1a
genes as additional phylogenetic markers.
Speciation can be operationally observed by significant
genetic discontinuities between populations as a consequence of the interruption of gene flow between species
(Lachance et al., 2010). To observe these genetic discontinuities among the Brazilian strains and 21 strains of diverse
origin belonging to closely related species, we applied an
MST networking approach that combined all strains in a
single, most-parsimonious network (Bandelt et al., 1999;
Posada & Crandall, 2001). The distances between the strains
reflected the genetic relatedness based on the concatenated
sequences of the rRNA gene (ITS and D1/D2 LSU) and the
protein-coding genes for actin and EF-1a (Fig. 1). The
strains from Brazil segregated in a well-separated subnetwork that was connected by a long branch to its closest
relative (H. meyeri; 79 nt, 2.7 %). Divergences between
the other three species (H. clermontiae, H. opuntiae and
H. guilliermondii) differing by four nucleotide substitutions
from the novel strains along the D1/D2 sequences were even
higher, and ranged from 85 substitutions (2.9 %) for H.
clermontiae to 109 substitutions (3.7 %) for H. guilliermondii
(connections not shown). The boundary between the
Brazilian strains and their relatives was confirmed by using
a statistical parsimony network analysis (Posada & Crandall,
2001) of concatenated protein-coding and ribosomal datasets
at the 95 % connection limit. However, when the analysis was
conducted with the ITS and D1/D2 LSU sequences only,
shown by Lachance et al. (2010, 2011) to delimit species,
the Brazilian strains remained in a single network with
their closest relatives. Therefore, the genetic divergence
of the Brazilian strains was further confirmed by PCR
fingerprinting. The novel strains segregated from the type
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 05:58:39
2365
N. Čadež and others
NCAIM Y.00725
Hanseniaspora
meyeri
CBS 8734T
6
2
CBS 8773
8
31
Hanseniaspora
opuntiae
CBS 8823
59
6
FST, UNSW E90
CBS 8815
CBS 8821T
9
Hanseniaspora
clermontiae CBS 8822
5
ZIM 623
5
7
13
CBS 8820
CBS 8733T
MY970
NCAIM Y.00727
79
55
Hanseniaspora
guilliermondii
31
Hanseniaspora
nectarophila sp. nov.
UFMG POG 6.1b
Hanseniaspora
pseudoguilliermondii
UFMG POG 13.1
4
3
7
8
CBS 465T
10
CBS 95
7
UFMG POG a.1T
UFMG POG 13.2
5
UFMG POG J6.1
53
CBS 8772T
Hanseniaspora
lachancei
MY703
8
CBS 2567
CBS 466
CBS 8819
UFMG POG 9.1
5
CBS 8818T
UFMG POG 12
Fig. 1. MST of concatenated sequences of the ITS and D1/D2 LSU of the rRNA gene, and the protein-coding genes for
actin and EF-1a (length 2887 nt) of the strains of Hanseniaspora nectarophila sp. nov., isolated from flowers of
Siphocampylus corymbiferus in Brazil, and their closely related species H. meyeri, H. clermontiae, H. opuntiae, H.
lachancei, H. pseudoguilliermondii and H. guilliermondii. Numbers on the connecting lines show the number of
substitutions between strains. Distances between the strains are proportional to phylogenetic distances. The type strains are
shown in bold.
strains and representative strains of all species of the
genera Hanseniaspora and Kloeckera at a low similarity
value (60 %) (Figs S1– S3).
For the phylogenetic placement of the novel strains, a
concatenated dataset of the ITS and D1/D2 LSU of the
rRNA gene, and the actin gene was used to construct
the most-parsimonious tree with high statistical support
(Fig. 2). The novel strains clustered together with H. meyeri
and H. clermontiae within the Hanseniaspora valbyensis
clade.
The results presented here support the prediction that the
studied strains are genetically distinct from other species of
Hanseniaspora–Kloeckera, and therefore that they represent a
novel species, for which we propose the name Hanseniaspora
nectarophila sp. nov. (MycoBank no. MB807210).
The seven strains of H. nectarophila were isolated from
flowers of S. corymbiferus. The novel species was isolated
from all of the seven flowers sampled, which suggests that
this ephemeral substrate is its ecological niche. Pigmented
yeasts were also recovered from these flowers. These yeasts
can use the nectar produced in the flowers as a nutrient
source. This novel species probably uses insects that visit
these flowers as their vectors.
2366
Identification
As shown in Fig. 3, H. nectarophila is morphologically
unique, as the mature buds mostly remained adhered to the
mother cell. Also, even 48 h of cultivation either in yeast
extract-malt extract liquid medium (Fig. 3a) or on YMA
plates (Fig. 3b) gave rise to clusters of cells. Furthermore,
budding of the cells was not strictly bipolar, but arose
multilaterally as well. Based on this observation we propose
to emend the diagnosis of the genus Hanseniaspora Zikes
from ‘Budding is bipolar’ to ‘Budding is mostly bipolar’.
H. nectarophila produced one to two round ascospores per
ascus (Fig. 3c), a feature shared with H. uvarum, but not
with its closely related taxa.
Physiologically, the strains of H. nectarophila can be
differentiated from their closest relatives H. meyeri, H.
clermontiae, H. opuntiae, H. lachancei, H. pseudoguilliermondii, H. uvarum and H. guilliermondii by their ability to
ferment and assimilate trehalose (both slowly) and from
Hanseniaspora thailandica by their inability to assimilate
D-gluconate. Nevertheless, for the unambiguous discrimination of H. nectarophila from other species of
Hanseniaspora–Kloeckera, sequencing of the ITS and D1/
D2 LSU rRNA gene is suggested.
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 64
IP: 88.99.165.207
On: Sun, 18 Jun 2017 05:58:39
Hanseniaspora nectarophila sp. nov.
Wickerhamomyces anomalus CBS 5759NT
(AJ508511, AY046221, U74592)
Kloeckera taiwanica CBS 11434T
(KF958102, FJ873604, EF653942)
100
100
Hanseniaspora vineae CBS 2171T
(AM039459, AJ512443, U84224)
Hanseniaspora osmophila CBS 313T
83
(AM039455, AJ512431, U84228)
Hanseniaspora occidentalis var. occidentalis CBS 2592T
100
(AM039463, AJ512429, U84225)
Hanseniaspora occidentalis var. citrica CBS 6783T
(AM039464, AJ973092, AJ973101)
Hanseniaspora singularis ST-244T
(KF958094, KF980889, FJ391977)
76
Kloeckera lindneri CBS 285T
80
(AM039454, AJ512430, U84226)
Hanseniaspora valbyensis CBS 479T
100
(AM039458, AJ512434, U73596)
Kloeckera hatyaiensis ST-476T
(KF958085, KF958035, DQ404528,)
Hanseniaspora uvarum CBS 314T
100
(AM039456, AJ512432, U84229)
Hanseniaspora thailandica ST-464T
Hanseniaspora pseudoguilliermondii CBS 8772T
100
(AM039457, AJ512437, AJ512455)
Hanseniaspora opuntiae CBS 8733T
(AM039465, AJ512435, AJ512453)
80
Hanseniaspora guilliermondii CBS 465T
(AM039457, AJ512433, U84230)
55
Hanseniaspora lachancei CBS 8818T
(AM039469, AJ512439, AJ512457)
100
Hanseniaspora meyeri CBS 8734T
(AM039466, AJ512436, AJ512454)
Hanseniaspora clermontiae CBS 8821T
(AM039472, AJ512441, AJ512452)
Hanseniaspora valbyensis clade
(KF958103, AB501148, DQ404527)
100
Hanseniaspora nectarophila sp. nov. UFMG POG 12
(KF958099, KF958051, KF958076)
58
56 Hanseniaspora nectarophila sp. nov. UFMG POG 13.1
(KF958098, KF958050, KF958075)
Hanseniaspora nectarophila sp. nov. UFMG POG 13.2
10
(KF958100, KF958052, KF958077)
88
Hanseniaspora nectarophila sp. nov. UFMG POG 6.1b
100
Hanseniaspora nectarophila sp. nov. UFMG POG a.1T
(KF958096, KF958048, KF958073)
(KF958095, KF958047, KF958072)
85 Hanseniaspora nectarophila sp. nov. UFMG POG 9.1
(KF958101, KF958053, KF958078)
Hanseniaspora nectarophila sp. nov. UFMG POG J6.1
(KF958097, KF958049, KF958074)
Fig. 2. Phylogenetic tree showing the placement of Hanseniaspora nectarophila sp. nov. within the genera
Hanseniaspora–Kloeckera based on sequences of the ITS and the D1/D2 LSU of the rRNA gene, and the proteincoding gene for actin, with GenBank accession numbers listed in parentheses. One of the four most-parsimonious trees
is presented (tree length, 1647; consistency index, 0.6928; retention index, 0.7998). Bootstrap percentages from 1000
replicates are shown. Wickerhamomyces anomalus CBS 5759T was used as the outgroup. Bar, number of nucleotide
substitutions.
Description of Hanseniaspora nectarophila
Čadež, Pagnocca, Raspor and Rosa sp. nov.
Hanseniaspora nectarophila [nec.ta.ro9phi.la; Gr. n. nektar
the drink of the gods, honey; Gr. adj. philos loving; N.L. fem.
adj. nectarophila referring to the isolation source (nectar) of
the strains of the species].
http://ijs.sgmjournals.org
In yeast extract-malt extract liquid medium after 48 h at
25 uC, cells are apiculate, ovoid to elongate, 3.5–8.0 mm6
1.8–5.0 mm, and occur singly, in pairs or in short chains.
Budding is mostly bipolar. A sediment is present. After
1 month, a very thin ring is formed. After 1 month at 25 uC,
streak culture on malt agar is cream coloured, butyrous,
smooth, glossy, and flat to slightly raised at the centre, with
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 05:58:39
2367
N. Čadež and others
xylitol, L-arabinitol, D-glucitol, D-mannitol, galactitol, myoinositol, D-gluconate, D-glucuronate, D-galacturonate,
DL-lactate, succinate, citrate, methanol, ethanol, 1,2-propanediol, 2,3-butanediol and hexadecane. Assimilation of
nitrogen compounds is positive for ethylamine, lysine and
cadaverine; negative for potassium nitrate, sodium nitrite,
creatine, creatinine, glucosamine and imidazole. No growth
in vitamin-free medium. Growth occurs at 30 uC, but not at
35 uC. Growth with 10 % NaCl and with 0.1 % cycloheximide is positive, but growth is absent with 16 % NaCl, on
50 % (w/w) glucose-yeast extract agar and with 1 % acetic
acid. The diazonium blue B reaction is negative.
(a)
(b)
The type strain, UFMG POG a.1T (5ZIM 2311T5CBS
13383), was isolated from a flower of Siphocampylus
corymbiferus collected at the São Sebastião do Ribeirão
Grande farm in the Atlantic rainforest of the
Pindamonhangaba municipal area in the State of São
Paulo, Brazil, in June 2006. The MycoBank number is MB
807210.
Acknowledgements
(c)
Fig. 3. Micrographs of budding cells and ascospores of
Hanseniapora nectarophila sp. nov. UFMG POG a.1T. Cells were
grown in yeast extract-malt extract broth (a) and on YMA (b), with
one or two round ascospores (c) formed on 3 % malt extract after
21 days at 26 6C. Bars, 10 mm.
We kindly acknowledge M.Th. Smith (Centraalbureau voor
Schimmelcultures, the Netherlands), S. Jindamorakot (BIOTEC
Culture Collection, Thailand), D. Dlauchy and G. Péter (National
Collection of Agricultural and Industrial Micro-Organisms, Hungary),
A. L. Beh and G. H. Fleet (Food Science and Technology, University of
New South Wales, Australia) and W. Geißdörfer (Universitätsklinikum
Erlangen, Germany) for providing cultures. This study was supported
by funding from the Slovenian Research Agency (P4-0116 and MRICUL ZIM, IP-0510). This study was also funded by the Conselho
Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq
– Brazil, awarded to F. C. P., proc. 302615/2008 and C. A. R., proc.
560715/2010-2), Fundação de Amparo a Pesquisa do Estado de Minas
Gerais (FAPEMIG, proc. APQ-02163-11, awarded to C. A. R.), and the
Financiadora de Estudos e Projetos (FINEP, proc. 2084/07, awarded to
C. A. R.). We also thank Casa da Floresta Assessoria Ambiental, Brazil,
and the owners of the São Sebastião do Ribeirão Grande farm, Brazil,
for the use of their facilities during sample collection, and Dr Reinaldo
Monteiro for identification of the plant species.
References
Bandelt, H. J., Forster, P. & Röhl, A. (1999). Median-joining networks
an undulate margin. On slide culture with corn meal agar
after 7 days at 25 uC, a rudimentary pseudomycelium
is formed. Asci containing one to two warty, spherical
ascospores (1.4–3.4 mm) after 2 weeks or more are observed
on 3 and 5 % malt extract, and on potato-glucose agar and
YMA, at 26 uC. Ascospores are not released from the ascus.
Glucose and a,a-trehalose (slow) are fermented; D-galactose, maltose, sucrose and lactose are not fermented. The
carbon compounds that are assimilated are glucose, a,
a-trehalose (weakly or weakly and slowly), cellobiose,
salicin, arbutin, glucono-d-lactone and 2-ketogluconate;
no growth occurs on galactose, L-sorbose, glucosamine,
D-ribose, D-xylose, L-arabinose, D-arabinose, L-rhamnose,
sucrose, maltose, methyl a-D-glucoside, melibiose, lactose,
raffinose, melezitose, starch, glycerol, erythritol, ribitol,
2368
for inferring intraspecific phylogenies. Mol Biol Evol 16, 37–48.
Boekhout, T., Kurtzman, C. P., O’Donnell, K. & Smith, M. T. (1994).
Phylogeny of the yeast genera Hanseniaspora (anamorph Kloeckera),
Dekkera (anamorph Brettanomyces), and Eeniella as inferred from
partial 26S ribosomal DNA nucleotide sequences. Int J Syst Bacteriol
44, 781–786.
Brito da Cunha, A., El-Tabey Shehata, A. M. & de Oliveira, W. (1957).
A study of the diets and nutritional preferences of tropical species
Drosophila. Ecology 38, 98–106.
Cadez, N., Raspor, P., de Cock, A. W., Boekhout, T. & Smith, M. T.
(2002). Molecular identification and genetic diversity within species of
the genera Hanseniaspora and Kloeckera. FEMS Yeast Res 1, 279–289.
Cadez, N., Poot, G. A., Raspor, P. & Smith, M. T. (2003). Hanseniaspora
meyeri sp. nov., Hanseniaspora clermontiae sp. nov., Hanseniaspora
lachancei sp. nov. and Hanseniaspora opuntiae sp. nov., novel apiculate
yeast species. Int J Syst Evol Microbiol 53, 1671–1680.
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 64
IP: 88.99.165.207
On: Sun, 18 Jun 2017 05:58:39
Hanseniaspora nectarophila sp. nov.
Cadez, N., Raspor, P. & Smith, M. T. (2006). Phylogenetic placement
of Hanseniaspora–Kloeckera species using multigene sequence analysis
with taxonomic implications: descriptions of Hanseniaspora pseudoguilliermondii sp. nov. and Hanseniaspora occidentalis var. citrica var.
nov. Int J Syst Evol Microbiol 56, 1157–1165.
Chandler, J. A., Eisen, J. A. & Kopp, A. (2012). Yeast communities of
diverse Drosophila species: comparison of two symbiont groups in the
same hosts. Appl Environ Microbiol 78, 7327–7336.
Chang, C. F., Huang, L. Y., Chen, S. F. & Lee, C. F. (2012). Kloeckera
taiwanica sp. nov., an ascomycetous apiculate yeast species isolated
from mushroom fruiting bodies. Int J Syst Evol Microbiol 62, 1434–
1437.
Clement, M., Posada, D. & Crandall, K. A. (2000). TCS: a computer
program to estimate gene genealogies. Mol Ecol 9, 1657–1659.
Daniel, H. M. & Meyer, W. (2003). Evaluation of ribosomal RNA and
actin gene sequences for the identification of ascomycetous yeasts. Int
J Food Microbiol 86, 61–78.
Jindamorakot, S., Ninomiya, S., Limtong, S., Yongmanitchai, W.,
Tuntirungkij, M., Potacharoen, W., Tanaka, K., Kawasaki, H. &
Nakase, T. (2009). Three new species of bipolar budding yeasts of
the genus Hanseniaspora and its anamorph Kloeckera isolated in
Thailand. FEMS Yeast Res 9, 1327–1337.
Kurtzman, C. P. (2003). Phylogenetic circumscription of Saccharomyces,
Kluyveromyces and other members of the Saccharomycetaceae, and
the proposal of the new genera Lachancea, Nakaseomyces, Naumovia,
Vanderwaltozyma and Zygotorulaspora. FEMS Yeast Res 4, 233–245.
Kurtzman, C. P. & Robnett, C. J. (1998). Identification and phylogeny
of ascomycetous yeasts from analysis of nuclear large subunit (26S)
ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73, 331–
371.
Kurtzman, C. P., Fell, J. W. & Boekhout, T. (2011). Methods for
isolation, phenotypic characterization and maintenance of yeasts. In
The Yeasts, a Taxonomic Study, 5th edn, vol. 1, pp. 87–110. Edited by
C. P. Kurtzman, J. W. Fell & T. Boekhout. Amsterdam: Elsevier.
Lachance, M. A., Dobson, J., Wijayanayaka, D. N. & Smith, A. M. E.
(2010). The use of parsimony network analysis for the formal
http://ijs.sgmjournals.org
delineation of phylogenetic species of yeasts: Candida apicola, Candida
azyma, and Candida parazyma sp. nov., cosmopolitan yeasts associated
with floricolous insects. Antonie van Leeuwenhoek 97, 155–170.
Lachance, M. A., Wijayanayaka, T. M., Bundus, J. D. & Wijayanayaka,
D. N. (2011). Ribosomal DNA sequence polymorphism and the
delineation of two ascosporic yeast species: Metschnikowia agaves and
Starmerella bombicola. FEMS Yeast Res 11, 324–333.
Meyer, S. A., Smith, M. T. & Simione, F. P., Jr (1978). Systematics of
Hanseniaspora Zikes and Kloeckera Janke. Antonie van Leeuwenhoek
44, 79–96.
Miller, M. W. & Phaff, H. J. (1962). Successive microbial populations in
Calimyrna figs. Appl Microbiol 10, 394–400.
Morais, P. B., Hagler, A. N., Rosa, C. A., Mendonca-Hagler, L. C. &
Klaczko, L. B. (1992). Yeasts associated with Drosophila in tropical
forests of Rio de Janeiro, Brazil. Can J Microbiol 38, 1150–1155.
Morais, P. B., Martins, M. B., Klaczko, L. B., Mendonça-Hagler,
L. C. & Hagler, A. N. (1995). Yeast succession in the Amazon fruit
Parahancornia amapa as resource partitioning among Drosophila spp.
Appl Environ Microbiol 61, 4251–4257.
Phaff, H. J. & Starmer, W. T. (1987). Yeasts associated with plants,
insects and soils. In The Yeasts, 2nd edn, vol. 1. Edited by A. H. Rose
& J. S. Harrison. New York: Academic Press.
Posada, D. & Crandall, K. A. (2001). Intraspecific gene genealogies:
trees grafting into networks. Trends Ecol Evol 16, 37–45.
PAUP*: Phylogenetic analysis using parsimony
(and other methods), version 4. Sunderland, MA: Sinauer Associates.
Swofford, D. L. (2002).
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. &
Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
strategies for multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Res 25, 4876–4882.
Yamada, Y., Maeda, K. & Banno, I. (1992). The phylogenetic
relationships of the Q6-equipped species in the teleomorphic apiculate
yeast genera Hanseniaspora, Nadsonia, and Saccharomycodes based on
the partial sequences of 18S and 26S ribosomal ribonucleic acids. J Gen
Appl Microbiol 38, 585–596.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 05:58:39
2369