IJSEM Papers in Press. Published September 1, 2014 as doi:10.1099/ijs.0.068403-0
1
Wickerhamiella allomyrinae f.a., sp. nov., a yeast species
2
isolated from the gut of rhinoceros beetle Allomyrina dichotoma
3 4
Yong-Cheng Ren, Yun Wang, Liang Chen, Tao Ke and Feng-Li Hui
5 6
School of Life Science and Technology, Nanyang Normal University, Nanyang 473061,
7
PR China
8 9 10
Correspondence: Feng-Li Hui E-mail: [email protected]
11 12
Running title: Wickerhamiella allomyrinae f.a., sp. nov.
13
The journal's contents category : New Taxa -Eukaryotic Micro-organisms
14
Abbreviations: ITS, internal transcribed spacer; LSU, large subunit.
15 16
The GenBank/EMBL/DDBJ accession numbers for the sequences of the D1/D2 domains
17
of the large subunit rRNA gene and the ITS regions of Wickerhamiella allomyrinae sp.
18
nov. NYNU 13920T are KJ152751 and KJ152752, respectively.
19
1
1
Abstract Two strains of Wickerhamiella allomyrinae f.a., sp. nov. were isolated from the
2
gut of Allomyrina dichotoma (Coleoptera: Scarabeidae) collected from the Baotianman
3
National Nature Reserve, Nanyan, Henan Province, China. Sequence analyses of the
4
D1/D2 domains of the large subunit (LSU) rRNA gene revealed that this new species was
5
located in the Wickerhamiella clade (Saccharomycetes, Saccharomycetales), with the
6
three described species of the genus Candida, namely, Candida musiphila, Candida
7
spandovensis and Candida sergipensis, as its closest related species. The novel species
8
differed from these three species by 9.3–9.8% sequence divergence (35–45 nucleotide
9
substitutions) in the D1/D2 sequences. The species could also be distinguished from the
10
closely related species, C. musiphila, C. spandovensis and C. sergipensis, by growth on
11
vitamin-free medium and at 37 °C. The type strain is Wickerhamiella allomyrinae sp. nov.
12
NYNU 13920T (= CICC 33031T= CBS 13167 T).
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
2
1
The ascomycetous yeast genus Wickerhamiella is closely related to the genera
2
Spencermartinsiella, Starmerella,
3
Zygoascus based on concatenated gene sequences for the nearly entire large subunit (LUS)
4
rRNA, small subunit (SSU) rRNA, translation elongation factor-1a (EF-1a), and RNA
5
polymerase II, subunits 1 (RPB1) and 2 (RPB2) (Kurtzman & Robnett, 2013). The type
6
strain of the genus was initially described as Torulopsis domercqiae (as Torulopsis
7
domercqii) by van der Walt & van Kerken (1960). Van der Walt & Liebenberg
8
established the new genus Wickerhamiella in 1973, and transferred T. domercqiae to this
9
genus as Wickerhamiella domercqiae after examining two additional strains and
10
observing conjugation and ascospore formation in two of the three available strains (van
11
der Walt & Liebenberg, 1973). In the fifth edition of “The Yeasts, A Taxonomic Study,”
12
Wickerhamiella
13
Wickerhamiella cacticola, W. domercqiae, Wickerhamiella lipophila and Wickerhamiella
14
occidentalis (Lachance & Kurtzman, 2011). There are more than 17 Candida species that
15
are currently members of the genus Wickerhamiella on the basis of sequence-based
16
phylogenetic analyses (Lachance & Kurtzman, 2011; Badotti et al., 2013). Subsequently,
17
several additional members of the genera Wickerhamiella have been proposed, such as
18
Wickerhamiella pagnoccae (Barbosa et al., 2012), Wickerhamiella dulcicola and
19
Wickerhamiella cachassae (Badotti et al., 2013), Wickerhamiella slavikovae and
20
Wickerhamiella goesii (Hagler et al., 2013), Wickerhamiella kiyanii and Wickerhamiella
21
fructicola (Dayo-Owoyemi et al., 2014). Given that yeasts of the genus Wickerhamiella
22
are physiologically similar in the utilization of carbon and nitrogen compounds,
23
separating them is difficult on the basis of phenotypic characteristics. Therefore, the
24
species identification should be based on ribosomal DNA sequence comparisons.
accommodated
Sugiyamaella, Trichomonascus, Yarrowia
five
species:
Wickerhamiella
and
australiensis,
25 26
During a study on yeasts associated with insects, we isolated a large number of yeasts
27
from the digestive tract of insects as well as from related substrates, including rotting
28
wood, frass and galleries (Hui et al., 2013; Chen al., 2013). The majority of the yeasts
29
belonged to several major clades in the Saccharomycotina; some of these species have
30
been identified as novel species in earlier papers (Hui et al., 2012; Chen et al., 2013; Hui
31
et al., 2013; Hui et al., 2013a). Amongst the insect associates, we focused on two strains 3
1
of an asexual ascomycetous yeast species from the gut of rhinoceros beetle Allomyrina
2
dichotoma in China. Sequence analysis of the D1/D2 domains of the large subunit (LSU)
3
gene revealed that the aforementioned strains represent an undescribed anamorphic yeast
4
species belonging to the Wickerhamiella clade. In this paper, we describe this new
5
species as Wickerhamiella allomyrinae f.a., sp. nov.
6 7
The strains belonging to the proposed novel species, NYNU 13915 and NYNU13920T,
8
were isolated from the gut of two individuals of A. dichotoma in September 2013. Adult
9
insects were collected from the Baotianman National Nature Reserve near Nanyang
10
(approximate coordinates: 33°27′N and 111°48′E), which has a typical transitional
11
climate from the northern subtropical zone to the warm temperate zone in central China.
12 13
The methods for yeast isolation were detailed by Nguyen et al. (2006) and Urbina et al.
14
(2013). The insects were usually placed in Petri dishes for 1–3 days without food prior to
15
dissection. Withholding food helps to eliminate some contaminating organisms that
16
might be isolated from the gut. Each insect individual was surface disinfected by washing
17
in 70% ethanol (5 min), 5% bleach (5 min) and sterile water (10 min) prior to dissection.
18
The aseptically removed gut contents and saline wash solution were plated separately on
19
acidified yeast extract-malt extract (YM) agar (0.3% yeast extract, 0.3% malt extract,
20
0.5% peptone, 1% glucose, 2% plain agar, adjusted to pH 3.5 with HCl), and incubated at
21
25 °C for 3–4 days. Single yeast colonies were purified at least twice, and stored in 15%
22
glycerol at –80 °C and/or on YM agar at 4 °C.
23 24
The morphological observations and metabolic tests that constitute the standard yeast
25
description were performed according to established methods (Barnett et al., 2000;
26
Kurtzman et al., 2011). Assimilation tests for carbon and nitrogen sources were
27
performed in liquid media. Starved inocula were used in nitrogen and vitamin
28
assimilation tests. Strains were examined for ascosporulation on the following agar media
29
incubated at 15 °C and 25 °C for 1–4 weeks: YM agar, 5% malt extract agar, corn meal
30
agar and YCBAS agar (1.1% yeast carbon base, 0.01% ammonium sulphate and 1.8%
31
agar). 4
1 2
Genomic DNA was extracted using Ezup Column Yeast Genomic DNA Purification Kit
3
according to the manufacturer’s protocol (Sangon Biotech, Shanghai, China). The D1/D2
4
domains of the LSU rRNA gene and ITS regions were amplified by PCR, and sequenced
5
using primers NL1 and NL4 (Kurtzman & Robnett, 1998) and ITS1 and ITS4 (White et
6
al., 1990), respectively. Both DNA strands were sequenced, and the reactions were
7
carried out using a Dye Terminator cycle sequencing kit (Applied Biosystems,
8
Warrington).
9 10
The sequences were compared pairwise using BLAST search (Altschul et al., 1997), and
11
aligned with the sequences of related species retrieved from GenBank using the multiple
12
alignment program CLUSTAL_X version 1.81 (Thompson et al., 1997). A phylogenetic
13
tree based on LSU D1/D2 sequences was constructed using the neighbour-joining method
14
in MEGA 5.0 (Tamura et al., 2011). The evolutionary distance data were calculated from
15
Kimura’s two-parameter model (Kimura, 1980) in the neighbour-joining analyses. All
16
sites containing gaps in the alignment were excluded. Dipodascus magnusii NRRL Y-
17
17563T and Starmerella bombicola NRRL Y-17069T were used as outgroups. Confidence
18
levels of the clades were estimated from bootstrap analysis (1,000 replicates) (Felsenstein
19
1985), and only values above 50% were recorded on the resulting tree. Reference
20
sequences were retrieved from GenBank under the accession numbers indicated in the
21
tree.
22 23
Sequence Comparison and Species Delineation
24
The two strains of W. allomyrinae sp. nov. were found to share identical sequences in
25
both D1/D2 and ITS regions. Sequence analyses of the D1/D2 domains of the LSU rRNA
26
gene revealed that this new species was closely related to species in the Wickerhamiella
27
clade (Saccharomycetes, Saccharomycetales). In terms of pairwise sequence similarity,
28
the close matches of W. allomyrinae sp. nov. were Candida musiphila, Candida
29
spandovensis and Candida sergipensis. The D1/D2 sequences of the novel species
30
showed a sequence divergence of 9.3% (35 substitutions and 17 gaps over 548 bases)
31
from its closest relative C. musiphila. The novel species also differed from the other two 5
1
close relatives, C. spandovensis and C. sergipensis, by sequence divergences of 9.4% and
2
9.8%, respectively. For the ITS region, this new species differed by sequence divergence
3
of 13.3% and 21.4% from C. musiphila and C. spandovensis, respectively. However,
4
pairwise sequence analysis with C. sergipensis could not be performed because its ITS
5
sequences are not currently available from either the NCBI GenBank database or the CBS
6
database.
7
A phylogenetic analysis based on the D1/D2 domains of the LSU rRNA gene sequences
8
indicated that W. allomyrinae sp. nov. forms a subclade with C. spandovensis and
9
C. sergipensis (Fig. 1). The bootstrap support for this subclade was relatively low (86%,
10
Fig. 1). The high degree of sequence divergence among the described species of the
11
subclade could be the result of many unknown species yet to be discovered, and also
12
explain the low branch support of this subclade. More importantly, the new species
13
occupies a basal position with respect to C. spandovensis and C. sergipiensis, indicating
14
that the phylogenetic species concept applies in the present case.
15 16
Since the two isolates of W. allomyrinae sp. nov. were located in the Wickerhamiella
17
clade, which contains six sexual species assigned to the genus Wickerhamiella, special
18
efforts were made to induce their sexual state. However, the strains did not produce
19
ascospores or exhibit conjugation on the most common sporulation media (YM agar, 5%
20
malt extract agar, corn meal agar and YCBAS agar), alone or mixed in pairs, at 15 °C and
21
25 °C for 1–4 weeks. In spite of this result, the novel species was assigned to the genus
22
Wickerhamiella in conformance with the provisions of the Melbourne Code (Norvell,
23
2011).
24 25
W. allomyrinae sp. nov. exhibited a narrow range of carbon and nitrogen assimilation,
26
which is typical for species in the Wickerhamiella clade. However, the species could be
27
distinguished from the closely related species, C. musiphila, C. spandovensis and
28
C. sergipensis, by growth on vitamin-free medium and at 37 °C, which was positive for
29
the new species and negative for the three closely related species (Table 1). Furthermore,
30
W. allomyrinae sp. nov. could be distinguished from C. musiphila in terms of ability to
31
assimilate salicin, succinate and citrate, but not D-xylose. The new species also differed 6
1
from C. spandovensis by positive assimilation of salicin, inability to ferment glucose and
2
galactose and the lack of assimilation of L-arabinose, xylitol, galactitol or ethanol. The
3
new species was easily separated from C. sergipensis based on the ability to assimilate
4
salicin and succinate, and the inability to assimilate xylitol, galactitol or ethanol.
5 6
Most species in the Wickerhamiella clade are highly specialised nutritionally and
7
ecologically, and some of them have a strong association with flowers and floricolous
8
insects (Lachance et al., 2011; Lachance & Kurtzman, 2011; Barbosa et al., 2012). For
9
example, Lachance et al. (1998) described five species in the Wickerhamiella clade,
10
including two asexual taxa, Candida drosophilae and Candida lipophila, isolated from
11
flowers of Ipomoea acuminata and its associated insect Drosophila floricola. Recent
12
samplings of yeasts revealed that the sugar cane plant may be a new habitat for some
13
yeasts in this clade. W. slavikovae and W. goesii were isolated from sugar cane plants
14
(Hagler et al., 2013), whereas W. dulcicola and W. cachassae were isolated from sugar
15
cane juice and must, respectively (Badotti et al., 2013). Of the 53 yeast strains isolated in
16
this study, Candida maltose, Candida boleticola and Trichosporon moniliiforme were the
17
most frequently isolated species from the gut of rhinoceros beetles. In comparison, only
18
two isolates of W. allomyrinae sp. nov. were obtained from the environment we tested,
19
making it more difficult to speculate about the habitat of this novel species. However,
20
two other species, namely, Candida sp. BG02-7-18-027A-1-2 and Candida sp. BG01-7-
21
26-006A-1-1, in the same subclade have been isolated from the gut of beetles, suggesting
22
that this group of species may occur in beetle guts and similar substrates in the
23
Baotianman Reserve.
24 25
Description of Wickerhamiella allomyrinae Hui, Ren, Wang, Chen & Ke sp. nov.
26
Wickerhamiella allomyrinae (al.lo.my.ri'nae. N.L. gen. n. allomyrinae refers to the genus
27
of the host beetle, Allomyrina dichotoma).
28
In YM broth after 3 days at 25 °C, the cells are ovoid and variable in size (2–4 × 3–5 μm),
29
and occurred singly or in pairs. Budding is multilateral (Fig. 2). A sediment formed after
30
one month, but no pellicle was observed. On YM agar after 3 days at room temperature,
31
colonies are white, convex, smooth and opalescent, with an entire edge. In Dalmau plates 7
1
after two weeks on corn meal agar, pseudomycelia or true mycelia are not formed. No
2
asci or signs of conjugation were observed after growth on the most common sporulation
3
media. Fermentation of glucose is negative. Glucose, galactose, L-sorbose, sucrose,
4
maltose, salicin, arbutin, raffinose, glycerol, ribitol, glucitol, mannitol, D-galacturonate,
5
succinate and citrate are assimilated. No growth occurs in D-glucosamine, D-ribose, D-
6
xylose, L-arabinose, D-arabinose, L-rhamnose, α, α-trehalose, methyl α-D-glucoside,
7
cellobiose, melibiose, lactose, melizitose, inulin, soluble starch, erythritol, xylitol, L-
8
arabinitol, galactitol, myo-inositol, 2-keto-D-gluconate, 5-keto-D-gluconate, D-gluconate,
9
D-glucuronate, DL-lactate, methanol and ethanol as sole carbon sources. Assimilation of
10
nitrogen compounds: positive for ethylamine, L-lysine and D-tryptophan, and negative
11
for nitrate, nitrite, cadaverine, creatine, creatinine, glucosamine and imidazole. Growth in
12
vitamin-free medium is positive. Growth is observed at 37 °C, but not at 40 °C. Growth
13
in the presence of 10% NaCl plus 5% glucose, 0.01% cycloheximide and 1% acetic acid
14
are negative. Acid formation on chalk agar is positive. Starch-like compounds are not
15
produced. Urease activity and Diazonium Blue B reactions are negative.
16 17
The type strain, NYNU 13920T, was isolated from the gut of A. dichotoma collected from
18
the Baotianman National Nature Reserve, Nanyan, Henan Province, China. It has been
19
deposited in the collection of the Yeast Division of the Centraalbureau voor
20
Schimmelcultures, Utrecht, the Netherlands, as strain CBS 13167T and in the China
21
Centre of Industrial Culture Collection, Beijing, China, as strain CICC 33031T. The
22
Mycobank number is MB 809413.
23 24 25 26 27 28 29
8
1
Acknowledgments
2
This work was supported by the National Natural Science Foundation of China
3
(31370073) and the Research Planning Project of Basic and Advanced Technology of
4
Henan Province, China (122300410032).
5 6 7
Correspondence and requests for materials should be addressed to: Feng-Li Hui, College
8
of Life Science and Technology, Nanyang Normal University, 1638 Wolong Road,
9
Nanyang, China. Tel.: +86 377 63525086; Fax: +86 377 63525086; E-mail:
10
[email protected]
11
9
1
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Tables Table 1. Physiological characteristics differentiating Wickerhamiella allomyrinae sp. nov. from closely related species
Species: 1, W. allomyrinae sp. nov.; 2, C. musiphila (data from Wang et al., 2008); 3, C. spandovensis (Lachance et al., 2011); 4, C. sergipensis (Lachance et al., 2011). +, Positive; –, negative; d, delayed; s, slow; v, variable; n, not determined. Yeast species Characteristic 1
2
3
4
Glucose
–
–
+
v
Galactose
–
–
s
–
D-Xylose
–
d
v
–
L-Arabinose
–
–
+
v
Salicin
+
–
–
+
Xylitol
–
n
+
+
Galactitol
–
–
+
+
Succinate
+
–
+
–
Citrate
+
–
s
–
Ethanol
–
–
+
+
Vitamin-free medium
+
–
–
n
37 °C
+
–
–
–
Fermentation of
Assimilation of
Other tests
13
Figure Legends Fig. 1 Phylogenetic tree derived from neighbour-joining analysis based on the D1/D2 domains of large subunit rRNA gene sequence showing the placement of Wickerhamiella allomyrinae sp. nov. and other relevant species. Dipodascus magnusii NRRL Y-17563T and Starmerella bombicola NRRL Y-17069T were used as outgroups. Bootstrap values of above 50% are given at nodes based on 1,000 replications. The scale bar represents 2% sequence difference. Fig. 2 Photomicrographs of Wickerhamiella allomyrinae sp. nov. NYNU 13920T. Budding cells grown on YM agar for 3 days at 25 °C. Bar, 10 μm.
14
Candida azyma CBS 6826T (EF536346) Candida azyma UWOPS 95-693.4 (EF601042) Candida sp. UWOPS 95-805.2 (EF601043) Candida sp. UWOPS 95-863.2 (EF601041) 70 Candida azymoides UFMG R287T (DQ985171) 56 Candida parazyma UWOPS 99-724.2T (EF601046) Candida sp. EJ3M02 (EF653941) Candida alocasiicola AS2.3484T (EU284106) T 81 Wickerhamiella dulcicola TO-L15 (JQ180255) 52 Candida LCF-01 NN12L02 (HQ623501) 89 Candida sp. UWOPS 03-446.4 (EU443389) Wickerhamiella cachassae D5L7T (JQ180256) Candida sp. UL100 (HQ641271) Candida vanderwaltii CBS 5524T (EU443388) Candida sp. GE1L05 (FJ527060) Wickerhamiella goesii IMUFRJ 52102T (JN790617) 61 T 72 90 Wickerhamiella occidentalis UWOPS 91-698.4 (AF046037) T 72 Wickerhamiella lipophila UWOPS 91-681.3 (AF046040) 93 Wickerhamiella sp. 9E1 (AM946760) 99 Wickerhamiella australiensis UWOPS 05-260.2T (EF536348) Wickerhamiella cacticola NRRL Y-27362T (AF046035) 96 Candida jalapaonensis UFMG-T05-210T (EU580139) 70 Wickerhamiella kiyanii FB1-1DASPT (JX978398) 82 Wickerhamiella pagnoccae UFMG F18C1T (HQ593535) 54 Candida drosophilae UWOPS 91-716.3T (EU443387) 70 Candida sp. 9A2 (FM178292) 73 Candida sp. UWOPS 00-102.1 (AF313351) 100 Candida sp. EVN1238 CM122/05 (FR853155) 99 100 Candida sp. CBS 2275 (AY536215) Candida pararugosa NRRL Y-17089T (U62306) Candida sp. BG99-8-18-1-3-1 (AY242245) 90 61 Candida hasegawae NBRC 102566T (AB306510) 61 Wickerhamiella fructicola H10YT (JX978400) 61 Candida kazuoi NBRC 102565T (AB306509) Candida galacta NRRL Y-17645T (DQ438239) Candida sp. BG02-7-21-004C-1-1 (AY520293) 57 Candida bombiphila CBS 9712T (AJ620185) 85 97 Wickerhamiella domercqiae NRRL Y-6692T (DQ438240) Wickerhamiella cf. domercqiae UWO(PS)00-107.1 (AF313369) 99 Wickerhamiella cf. domercqiae UWO(PS)00-192.1 (AF313368) 84 Candida sp. 107 (EF141077) Candida sp. SJ-1 (EF653272) 91 Candida sorbophila NRRL Y-7921T (DQ438229) 98 Candida infanticola CBS11938 (HQ695009) 73 Candida sp. SN-102 (EF621560) 97 Candida sp. UWO(PS)00-136.3 (AF313356) Candida musiphila AS2.3479T (EU284104) Wickerhamiella allomyrinae NYNU 13920 (KJ152751) 86 85 Candida sergipensis UFMG-R188T (AF397405) Candida spandovensis NRRL Y-17761T (DQ438228) Candida sp. BG02-7-18-027A-1-2 (AY520352) 95 Candida sp. BG01-7-26-006A-1-1 (AY242275) Wickerhamiella slavikovae IMUFRJ 52096T (FJ463264) Candida versatilis NRRL Y-6652T (DQ438242) Starmerella bombicola NRRL Y-17069T (HQ111052) Dipodascus magnusii NRRL Y-17563T (JQ689070) 67 74 97 71
0.02
80
73
15
Fig. 1
Fig. 2
16
1
Wickerhamiella allomyrinae f.a., sp. nov., a yeast species
2
isolated from the gut of rhinoceros beetle Allomyrina dichotoma
3 4
Yong-Cheng Ren, Yun Wang, Liang Chen, Tao Ke and Feng-Li Hui
5 6
School of Life Science and Technology, Nanyang Normal University, Nanyang 473061,
7
PR China
8 9 10
Correspondence: Feng-Li Hui E-mail: [email protected]
11 12
Running title: Wickerhamiella allomyrinae f.a., sp. nov.
13
The journal's contents category : New Taxa -Eukaryotic Micro-organisms
14
Abbreviations: ITS, internal transcribed spacer; LSU, large subunit.
15 16
The GenBank/EMBL/DDBJ accession numbers for the sequences of the D1/D2 domains
17
of the large subunit rRNA gene and the ITS regions of Wickerhamiella allomyrinae sp.
18
nov. NYNU 13920T are KJ152751 and KJ152752, respectively.
19
1
1
Abstract Two strains of Wickerhamiella allomyrinae f.a., sp. nov. were isolated from the
2
gut of Allomyrina dichotoma (Coleoptera: Scarabeidae) collected from the Baotianman
3
National Nature Reserve, Nanyan, Henan Province, China. Sequence analyses of the
4
D1/D2 domains of the large subunit (LSU) rRNA gene revealed that this new species was
5
located in the Wickerhamiella clade (Saccharomycetes, Saccharomycetales), with the
6
three described species of the genus Candida, namely, Candida musiphila, Candida
7
spandovensis and Candida sergipensis, as its closest related species. The novel species
8
differed from these three species by 9.3–9.8% sequence divergence (35–45 nucleotide
9
substitutions) in the D1/D2 sequences. The species could also be distinguished from the
10
closely related species, C. musiphila, C. spandovensis and C. sergipensis, by growth on
11
vitamin-free medium and at 37 °C. The type strain is Wickerhamiella allomyrinae sp. nov.
12
NYNU 13920T (= CICC 33031T= CBS 13167 T).
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
2
1
The ascomycetous yeast genus Wickerhamiella is closely related to the genera
2
Spencermartinsiella, Starmerella,
3
Zygoascus based on concatenated gene sequences for the nearly entire large subunit (LUS)
4
rRNA, small subunit (SSU) rRNA, translation elongation factor-1a (EF-1a), and RNA
5
polymerase II, subunits 1 (RPB1) and 2 (RPB2) (Kurtzman & Robnett, 2013). The type
6
strain of the genus was initially described as Torulopsis domercqiae (as Torulopsis
7
domercqii) by van der Walt & van Kerken (1960). Van der Walt & Liebenberg
8
established the new genus Wickerhamiella in 1973, and transferred T. domercqiae to this
9
genus as Wickerhamiella domercqiae after examining two additional strains and
10
observing conjugation and ascospore formation in two of the three available strains (van
11
der Walt & Liebenberg, 1973). In the fifth edition of “The Yeasts, A Taxonomic Study,”
12
Wickerhamiella
13
Wickerhamiella cacticola, W. domercqiae, Wickerhamiella lipophila and Wickerhamiella
14
occidentalis (Lachance & Kurtzman, 2011). There are more than 17 Candida species that
15
are currently members of the genus Wickerhamiella on the basis of sequence-based
16
phylogenetic analyses (Lachance & Kurtzman, 2011; Badotti et al., 2013). Subsequently,
17
several additional members of the genera Wickerhamiella have been proposed, such as
18
Wickerhamiella pagnoccae (Barbosa et al., 2012), Wickerhamiella dulcicola and
19
Wickerhamiella cachassae (Badotti et al., 2013), Wickerhamiella slavikovae and
20
Wickerhamiella goesii (Hagler et al., 2013), Wickerhamiella kiyanii and Wickerhamiella
21
fructicola (Dayo-Owoyemi et al., 2014). Given that yeasts of the genus Wickerhamiella
22
are physiologically similar in the utilization of carbon and nitrogen compounds,
23
separating them is difficult on the basis of phenotypic characteristics. Therefore, the
24
species identification should be based on ribosomal DNA sequence comparisons.
accommodated
Sugiyamaella, Trichomonascus, Yarrowia
five
species:
Wickerhamiella
and
australiensis,
25 26
During a study on yeasts associated with insects, we isolated a large number of yeasts
27
from the digestive tract of insects as well as from related substrates, including rotting
28
wood, frass and galleries (Hui et al., 2013; Chen al., 2013). The majority of the yeasts
29
belonged to several major clades in the Saccharomycotina; some of these species have
30
been identified as novel species in earlier papers (Hui et al., 2012; Chen et al., 2013; Hui
31
et al., 2013; Hui et al., 2013a). Amongst the insect associates, we focused on two strains 3
1
of an asexual ascomycetous yeast species from the gut of rhinoceros beetle Allomyrina
2
dichotoma in China. Sequence analysis of the D1/D2 domains of the large subunit (LSU)
3
gene revealed that the aforementioned strains represent an undescribed anamorphic yeast
4
species belonging to the Wickerhamiella clade. In this paper, we describe this new
5
species as Wickerhamiella allomyrinae f.a., sp. nov.
6 7
The strains belonging to the proposed novel species, NYNU 13915 and NYNU13920T,
8
were isolated from the gut of two individuals of A. dichotoma in September 2013. Adult
9
insects were collected from the Baotianman National Nature Reserve near Nanyang
10
(approximate coordinates: 33°27′N and 111°48′E), which has a typical transitional
11
climate from the northern subtropical zone to the warm temperate zone in central China.
12 13
The methods for yeast isolation were detailed by Nguyen et al. (2006) and Urbina et al.
14
(2013). The insects were usually placed in Petri dishes for 1–3 days without food prior to
15
dissection. Withholding food helps to eliminate some contaminating organisms that
16
might be isolated from the gut. Each insect individual was surface disinfected by washing
17
in 70% ethanol (5 min), 5% bleach (5 min) and sterile water (10 min) prior to dissection.
18
The aseptically removed gut contents and saline wash solution were plated separately on
19
acidified yeast extract-malt extract (YM) agar (0.3% yeast extract, 0.3% malt extract,
20
0.5% peptone, 1% glucose, 2% plain agar, adjusted to pH 3.5 with HCl), and incubated at
21
25 °C for 3–4 days. Single yeast colonies were purified at least twice, and stored in 15%
22
glycerol at –80 °C and/or on YM agar at 4 °C.
23 24
The morphological observations and metabolic tests that constitute the standard yeast
25
description were performed according to established methods (Barnett et al., 2000;
26
Kurtzman et al., 2011). Assimilation tests for carbon and nitrogen sources were
27
performed in liquid media. Starved inocula were used in nitrogen and vitamin
28
assimilation tests. Strains were examined for ascosporulation on the following agar media
29
incubated at 15 °C and 25 °C for 1–4 weeks: YM agar, 5% malt extract agar, corn meal
30
agar and YCBAS agar (1.1% yeast carbon base, 0.01% ammonium sulphate and 1.8%
31
agar). 4
1 2
Genomic DNA was extracted using Ezup Column Yeast Genomic DNA Purification Kit
3
according to the manufacturer’s protocol (Sangon Biotech, Shanghai, China). The D1/D2
4
domains of the LSU rRNA gene and ITS regions were amplified by PCR, and sequenced
5
using primers NL1 and NL4 (Kurtzman & Robnett, 1998) and ITS1 and ITS4 (White et
6
al., 1990), respectively. Both DNA strands were sequenced, and the reactions were
7
carried out using a Dye Terminator cycle sequencing kit (Applied Biosystems,
8
Warrington).
9 10
The sequences were compared pairwise using BLAST search (Altschul et al., 1997), and
11
aligned with the sequences of related species retrieved from GenBank using the multiple
12
alignment program CLUSTAL_X version 1.81 (Thompson et al., 1997). A phylogenetic
13
tree based on LSU D1/D2 sequences was constructed using the neighbour-joining method
14
in MEGA 5.0 (Tamura et al., 2011). The evolutionary distance data were calculated from
15
Kimura’s two-parameter model (Kimura, 1980) in the neighbour-joining analyses. All
16
sites containing gaps in the alignment were excluded. Dipodascus magnusii NRRL Y-
17
17563T and Starmerella bombicola NRRL Y-17069T were used as outgroups. Confidence
18
levels of the clades were estimated from bootstrap analysis (1,000 replicates) (Felsenstein
19
1985), and only values above 50% were recorded on the resulting tree. Reference
20
sequences were retrieved from GenBank under the accession numbers indicated in the
21
tree.
22 23
Sequence Comparison and Species Delineation
24
The two strains of W. allomyrinae sp. nov. were found to share identical sequences in
25
both D1/D2 and ITS regions. Sequence analyses of the D1/D2 domains of the LSU rRNA
26
gene revealed that this new species was closely related to species in the Wickerhamiella
27
clade (Saccharomycetes, Saccharomycetales). In terms of pairwise sequence similarity,
28
the close matches of W. allomyrinae sp. nov. were Candida musiphila, Candida
29
spandovensis and Candida sergipensis. The D1/D2 sequences of the novel species
30
showed a sequence divergence of 9.3% (35 substitutions and 17 gaps over 548 bases)
31
from its closest relative C. musiphila. The novel species also differed from the other two 5
1
close relatives, C. spandovensis and C. sergipensis, by sequence divergences of 9.4% and
2
9.8%, respectively. For the ITS region, this new species differed by sequence divergence
3
of 13.3% and 21.4% from C. musiphila and C. spandovensis, respectively. However,
4
pairwise sequence analysis with C. sergipensis could not be performed because its ITS
5
sequences are not currently available from either the NCBI GenBank database or the CBS
6
database.
7
A phylogenetic analysis based on the D1/D2 domains of the LSU rRNA gene sequences
8
indicated that W. allomyrinae sp. nov. forms a subclade with C. spandovensis and
9
C. sergipensis (Fig. 1). The bootstrap support for this subclade was relatively low (86%,
10
Fig. 1). The high degree of sequence divergence among the described species of the
11
subclade could be the result of many unknown species yet to be discovered, and also
12
explain the low branch support of this subclade. More importantly, the new species
13
occupies a basal position with respect to C. spandovensis and C. sergipiensis, indicating
14
that the phylogenetic species concept applies in the present case.
15 16
Since the two isolates of W. allomyrinae sp. nov. were located in the Wickerhamiella
17
clade, which contains six sexual species assigned to the genus Wickerhamiella, special
18
efforts were made to induce their sexual state. However, the strains did not produce
19
ascospores or exhibit conjugation on the most common sporulation media (YM agar, 5%
20
malt extract agar, corn meal agar and YCBAS agar), alone or mixed in pairs, at 15 °C and
21
25 °C for 1–4 weeks. In spite of this result, the novel species was assigned to the genus
22
Wickerhamiella in conformance with the provisions of the Melbourne Code (Norvell,
23
2011).
24 25
W. allomyrinae sp. nov. exhibited a narrow range of carbon and nitrogen assimilation,
26
which is typical for species in the Wickerhamiella clade. However, the species could be
27
distinguished from the closely related species, C. musiphila, C. spandovensis and
28
C. sergipensis, by growth on vitamin-free medium and at 37 °C, which was positive for
29
the new species and negative for the three closely related species (Table 1). Furthermore,
30
W. allomyrinae sp. nov. could be distinguished from C. musiphila in terms of ability to
31
assimilate salicin, succinate and citrate, but not D-xylose. The new species also differed 6
1
from C. spandovensis by positive assimilation of salicin, inability to ferment glucose and
2
galactose and the lack of assimilation of L-arabinose, xylitol, galactitol or ethanol. The
3
new species was easily separated from C. sergipensis based on the ability to assimilate
4
salicin and succinate, and the inability to assimilate xylitol, galactitol or ethanol.
5 6
Most species in the Wickerhamiella clade are highly specialised nutritionally and
7
ecologically, and some of them have a strong association with flowers and floricolous
8
insects (Lachance et al., 2011; Lachance & Kurtzman, 2011; Barbosa et al., 2012). For
9
example, Lachance et al. (1998) described five species in the Wickerhamiella clade,
10
including two asexual taxa, Candida drosophilae and Candida lipophila, isolated from
11
flowers of Ipomoea acuminata and its associated insect Drosophila floricola. Recent
12
samplings of yeasts revealed that the sugar cane plant may be a new habitat for some
13
yeasts in this clade. W. slavikovae and W. goesii were isolated from sugar cane plants
14
(Hagler et al., 2013), whereas W. dulcicola and W. cachassae were isolated from sugar
15
cane juice and must, respectively (Badotti et al., 2013). Of the 53 yeast strains isolated in
16
this study, Candida maltose, Candida boleticola and Trichosporon moniliiforme were the
17
most frequently isolated species from the gut of rhinoceros beetles. In comparison, only
18
two isolates of W. allomyrinae sp. nov. were obtained from the environment we tested,
19
making it more difficult to speculate about the habitat of this novel species. However,
20
two other species, namely, Candida sp. BG02-7-18-027A-1-2 and Candida sp. BG01-7-
21
26-006A-1-1, in the same subclade have been isolated from the gut of beetles, suggesting
22
that this group of species may occur in beetle guts and similar substrates in the
23
Baotianman Reserve.
24 25
Description of Wickerhamiella allomyrinae Hui, Ren, Wang, Chen & Ke sp. nov.
26
Wickerhamiella allomyrinae (al.lo.my.ri'nae. N.L. gen. n. allomyrinae refers to the genus
27
of the host beetle, Allomyrina dichotoma).
28
In YM broth after 3 days at 25 °C, the cells are ovoid and variable in size (2–4 × 3–5 μm),
29
and occurred singly or in pairs. Budding is multilateral (Fig. 2). A sediment formed after
30
one month, but no pellicle was observed. On YM agar after 3 days at room temperature,
31
colonies are white, convex, smooth and opalescent, with an entire edge. In Dalmau plates 7
1
after two weeks on corn meal agar, pseudomycelia or true mycelia are not formed. No
2
asci or signs of conjugation were observed after growth on the most common sporulation
3
media. Fermentation of glucose is negative. Glucose, galactose, L-sorbose, sucrose,
4
maltose, salicin, arbutin, raffinose, glycerol, ribitol, glucitol, mannitol, D-galacturonate,
5
succinate and citrate are assimilated. No growth occurs in D-glucosamine, D-ribose, D-
6
xylose, L-arabinose, D-arabinose, L-rhamnose, α, α-trehalose, methyl α-D-glucoside,
7
cellobiose, melibiose, lactose, melizitose, inulin, soluble starch, erythritol, xylitol, L-
8
arabinitol, galactitol, myo-inositol, 2-keto-D-gluconate, 5-keto-D-gluconate, D-gluconate,
9
D-glucuronate, DL-lactate, methanol and ethanol as sole carbon sources. Assimilation of
10
nitrogen compounds: positive for ethylamine, L-lysine and D-tryptophan, and negative
11
for nitrate, nitrite, cadaverine, creatine, creatinine, glucosamine and imidazole. Growth in
12
vitamin-free medium is positive. Growth is observed at 37 °C, but not at 40 °C. Growth
13
in the presence of 10% NaCl plus 5% glucose, 0.01% cycloheximide and 1% acetic acid
14
are negative. Acid formation on chalk agar is positive. Starch-like compounds are not
15
produced. Urease activity and Diazonium Blue B reactions are negative.
16 17
The type strain, NYNU 13920T, was isolated from the gut of A. dichotoma collected from
18
the Baotianman National Nature Reserve, Nanyan, Henan Province, China. It has been
19
deposited in the collection of the Yeast Division of the Centraalbureau voor
20
Schimmelcultures, Utrecht, the Netherlands, as strain CBS 13167T and in the China
21
Centre of Industrial Culture Collection, Beijing, China, as strain CICC 33031T. The
22
Mycobank number is MB 809413.
23 24 25 26 27 28 29
8
1
Acknowledgments
2
This work was supported by the National Natural Science Foundation of China
3
(31370073) and the Research Planning Project of Basic and Advanced Technology of
4
Henan Province, China (122300410032).
5 6 7
Correspondence and requests for materials should be addressed to: Feng-Li Hui, College
8
of Life Science and Technology, Nanyang Normal University, 1638 Wolong Road,
9
Nanyang, China. Tel.: +86 377 63525086; Fax: +86 377 63525086; E-mail:
10
[email protected]
11
9
1
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sp.
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Metschnikowia
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and
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26
Elsevier. 77–100.
27
12
Tables Table 1. Physiological characteristics differentiating Wickerhamiella allomyrinae sp. nov. from closely related species
Species: 1, W. allomyrinae sp. nov.; 2, C. musiphila (data from Wang et al., 2008); 3, C. spandovensis (Lachance et al., 2011); 4, C. sergipensis (Lachance et al., 2011). +, Positive; –, negative; d, delayed; s, slow; v, variable; n, not determined. Yeast species Characteristic 1
2
3
4
Glucose
–
–
+
v
Galactose
–
–
s
–
D-Xylose
–
d
v
–
L-Arabinose
–
–
+
v
Salicin
+
–
–
+
Xylitol
–
n
+
+
Galactitol
–
–
+
+
Succinate
+
–
+
–
Citrate
+
–
s
–
Ethanol
–
–
+
+
Vitamin-free medium
+
–
–
n
37 °C
+
–
–
–
Fermentation of
Assimilation of
Other tests
13
Figure Legends Fig. 1 Phylogenetic tree derived from neighbour-joining analysis based on the D1/D2 domains of large subunit rRNA gene sequence showing the placement of Wickerhamiella allomyrinae sp. nov. and other relevant species. Dipodascus magnusii NRRL Y-17563T and Starmerella bombicola NRRL Y-17069T were used as outgroups. Bootstrap values of above 50% are given at nodes based on 1,000 replications. The scale bar represents 2% sequence difference. Fig. 2 Photomicrographs of Wickerhamiella allomyrinae sp. nov. NYNU 13920T. Budding cells grown on YM agar for 3 days at 25 °C. Bar, 10 μm.
14
Candida azyma CBS 6826T (EF536346) Candida azyma UWOPS 95-693.4 (EF601042) Candida sp. UWOPS 95-805.2 (EF601043) Candida sp. UWOPS 95-863.2 (EF601041) 70 Candida azymoides UFMG R287T (DQ985171) 56 Candida parazyma UWOPS 99-724.2T (EF601046) Candida sp. EJ3M02 (EF653941) Candida alocasiicola AS2.3484T (EU284106) T 81 Wickerhamiella dulcicola TO-L15 (JQ180255) 52 Candida LCF-01 NN12L02 (HQ623501) 89 Candida sp. UWOPS 03-446.4 (EU443389) Wickerhamiella cachassae D5L7T (JQ180256) Candida sp. UL100 (HQ641271) Candida vanderwaltii CBS 5524T (EU443388) Candida sp. GE1L05 (FJ527060) Wickerhamiella goesii IMUFRJ 52102T (JN790617) 61 T 72 90 Wickerhamiella occidentalis UWOPS 91-698.4 (AF046037) T 72 Wickerhamiella lipophila UWOPS 91-681.3 (AF046040) 93 Wickerhamiella sp. 9E1 (AM946760) 99 Wickerhamiella australiensis UWOPS 05-260.2T (EF536348) Wickerhamiella cacticola NRRL Y-27362T (AF046035) 96 Candida jalapaonensis UFMG-T05-210T (EU580139) 70 Wickerhamiella kiyanii FB1-1DASPT (JX978398) 82 Wickerhamiella pagnoccae UFMG F18C1T (HQ593535) 54 Candida drosophilae UWOPS 91-716.3T (EU443387) 70 Candida sp. 9A2 (FM178292) 73 Candida sp. UWOPS 00-102.1 (AF313351) 100 Candida sp. EVN1238 CM122/05 (FR853155) 99 100 Candida sp. CBS 2275 (AY536215) Candida pararugosa NRRL Y-17089T (U62306) Candida sp. BG99-8-18-1-3-1 (AY242245) 90 61 Candida hasegawae NBRC 102566T (AB306510) 61 Wickerhamiella fructicola H10YT (JX978400) 61 Candida kazuoi NBRC 102565T (AB306509) Candida galacta NRRL Y-17645T (DQ438239) Candida sp. BG02-7-21-004C-1-1 (AY520293) 57 Candida bombiphila CBS 9712T (AJ620185) 85 97 Wickerhamiella domercqiae NRRL Y-6692T (DQ438240) Wickerhamiella cf. domercqiae UWO(PS)00-107.1 (AF313369) 99 Wickerhamiella cf. domercqiae UWO(PS)00-192.1 (AF313368) 84 Candida sp. 107 (EF141077) Candida sp. SJ-1 (EF653272) 91 Candida sorbophila NRRL Y-7921T (DQ438229) 98 Candida infanticola CBS11938 (HQ695009) 73 Candida sp. SN-102 (EF621560) 97 Candida sp. UWO(PS)00-136.3 (AF313356) Candida musiphila AS2.3479T (EU284104) Wickerhamiella allomyrinae NYNU 13920 (KJ152751) 86 85 Candida sergipensis UFMG-R188T (AF397405) Candida spandovensis NRRL Y-17761T (DQ438228) Candida sp. BG02-7-18-027A-1-2 (AY520352) 95 Candida sp. BG01-7-26-006A-1-1 (AY242275) Wickerhamiella slavikovae IMUFRJ 52096T (FJ463264) Candida versatilis NRRL Y-6652T (DQ438242) Starmerella bombicola NRRL Y-17069T (HQ111052) Dipodascus magnusii NRRL Y-17563T (JQ689070) 67 74 97 71
0.02
80
73
15
Fig. 1
Fig. 2
16
