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Cystobasidium tubakii and Cystobasidium ongulense, new basidiomycetous yeast species isolated from East Ongul Island, East Antarctica
m y c o s c i e n c e 5 8 ( 2 0 1 7 ) 1 0 3 e1 1 0
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Cystobasidium tubakii and Cystobasidium ongulense, new basidiomycetous yeast species isolated from East Ongul Island, East Antarctica Masaharu Tsuji a,*, Megumu Tsujimoto a,b, Satoshi Imura a,b a
National Institute of Polar Research (NIPR), 10-3 Midori-cho, Tachikawa, Tokyo 190-8518, Japan Department of Polar Science, SOKENDAI (The Graduate University for Advanced Studies), 10-3 Midori-cho, Tachikawa, Tokyo 190-8518, Japan
b
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abstract
Article history:
Two new cold-adapted yeast species, Cystobasidium tubakii sp. nov. and Cystobasidium
Received 17 July 2016
ongulense sp. nov., were isolated from soil collected from East Ongul Island, East Antarctica,
Received in revised form
during the 49th Japanese Antarctic Research Expedition (JARE 49). Molecular analysis based
4 November 2016
on large subunit (LSU) D1/D2 domain and the combined sequences of small subunit (SSU)
Accepted 6 November 2016
rDNA, internal transcribed spacer (ITS) region, LSU D1/D2 domain and TEF1 sequences
Available online 10 December 2016
showed that these species are novel. Both species could grow at sub-zero temperatures and in vitamin-free media. These characteristics were likely obtained by the yeasts to survive
Keywords:
oligotrophic environments such as that in Antarctica. This is the first report of new fungal
Antarctic yeast
species isolated from near the Syowa station in the 60-y history of JARE.
Cystobasidiomycetes
© 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
LSU D1/D2 domain Syowa station
1.
Introduction
Antarctica is the southernmost landmass on Earth and has an area of approximately 14 million km2, making it the fifthlargest continent in the world. Approximately 98% of Antarctica is covered by ice and snow, and temperatures in coastal areas usually range from 5 C to 35 C (Ravindra and Chaturvedi 2011). Under such sub-zero conditions, the growth of cold-adapted fungi and concomitant decomposition of organic compounds play an important role in the nutrient cycle of Polar region ecosystems (Welander 2005).
East Ongul Island (69 10 S, 39 350 E) is located at the east side of the Lu¨tzow Holm Bay, East Antarctica. Syowa station has been established on this island since 1957, as the base of the Japanese Antarctic Research Expedition (JARE). To date, over 1000 fungal species from 421 genera have been isolated and recorded from Antarctica (Bridge and Spooner 2012); the list of known species includes 68% ascomycetes, 23% basidiomycetes, and 5% zygomycetes, with the final 4% comprising various other lineages. Previously, 12 ascomycetous and four basidiomycetous species have been reported from the vicinity of Syowa station (Soneda 1961; Tubaki 1961a, b; Tubaki and Asano 1965). More recently, 14
* Corresponding author. Fax: þ81 42 528 3492. E-mail address: [email protected] (M. Tsuji). http://dx.doi.org/10.1016/j.myc.2016.11.002 1340-3540/© 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
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genera of microfungi were isolated from willow wood located near the station (Hirose et al. 2013). However, these are the only reports to have described fungi isolated from near the station vicinity. Therefore, despite previous research on the mycoflora of East Ongul Island, knowledge of the fungal species inhabiting the island likely remains incomplete. Basidiomycetous yeasts have been widely reported to represent the dominant fungi in Polar regions (Singh et al. 2013; Tsuji et al. 2013b, 2016). Many of these yeasts have been found only in their asexual stage and are classified as anamorphic genera Cryptococcus Vuillemin or Rhodotorula Harrison (Boekhout et al. 2011). The genus Cystobasidium (Lagerheim) Neuhoff represents species previously classified as the Rhodotorula minuta clade (Sampaio 2011; Wang et al. 2015; Yurkov et al. 2015). In this study, four yeast colonies, light pinkish in color, were isolated from East Ongul Island, East Antarctica. Based on physiological testing and molecular analysis using the large subunit 26S rDNA (LSU D1/D2 domain) and internal transcribed spacer (ITS) region sequences, these strains were classified into two new basidiomycetous yeast species in the genus Cystobasidium, for which the names C. tubakii sp. nov. and C. ongulense sp. nov. are proposed.
2.
Materials and methods
2.1.
Sampling sites and sample collection
A total of 226 surface soil samples were collected from the entire of East Ongule Island near the Syowa station. The collected samples were immediately stored at 20 C until used. Total carbon and nitrogen concentrations in the 226 soil samples were measured using a CN analyzer (SUMIGRAPH NC-220F, Sumika Chemical Analysis Service, Tokyo, Japan); the average of the carbon and nitrogen concentrations were 0.063 ± 0.087% and 0.0025 ± 0.0023%, respectively.
2.2.
Isolation of strains
Each 0.1 g untreated soil sample was directly placed on potato dextrose agar (PDA, Difco, Becton Dickinson Japan, Tokyo) containing 50 mg/mL chloramphenicol and incubated at 10 C for a period of up to 3 wk. Yeast samples were chosen for isolation based on colony morphology. Each colony of a
different morphology was purified by repeated streaking on fresh PDA. The cultures of Cystobasidium tubakii and C. ongulense are deposited at the Japan Collection of Microorganisms (JCM), Riken, Japan, at the HUT Culture Collection (HUT), Hiroshima University, Japan, as well as at the Biological Resource Center, National Institute of Technology and Evaluation (NBRC), Japan. The detail of deposit numbers is shown in Table 1.
2.3.
Sequencing and phylogenetic analysis
DNA was extracted from yeast colonies, using an ISOPLANT II kit (Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer's protocols. The extracted DNA was amplified by polymerase chain reaction (PCR), using KOD-plus DNA polymerase (Toyobo, Osaka, Japan). Five fragment genes or loci, i.e., the small subunit (SSU) rDNA, LSU D1/D2 domains, ITS region, translation elongation factor 1-a (TEF1), and the mitochondrial gene cytochrome b (CYTB) were amplified in this study. The ITS region and LSU D1/D2 domain primers and conditions for PCR were described previously (Tsuji et al. 2013a). SSU rDNA primers were reported by Wang et al. (2003), primers for CYTB as previously described by Wang and Bai (2008), and TEF1 primers by Wang et al. (2015). The amplified DNA fragments were purified using Sephacryl S400HR (SigmaeAldrich Japan, Tokyo). Sequences were determined using an ABI prism 3130xl Sequencer (Applied Biosystems, Life Technologies Japan, Tokyo). GenBank accession numbers for all the sequences analyzed in this study are listed in Table 1. The sequences were aligned with the MAFFT program ver. 7.273 (Katoh and Standley 2013) using the LeINSeI algorithm. The alignments were deposited in TreeBASE (Submission ID: S19510). Maximum likelihood (ML) with an HYKþGþI model and maximum parsimony (MP) analysis with a TBR model were performed using MEGA7 (Kumar et al. 2016). Bayesian inference (BI) was constructed using MrBayes 3.2.5 (Ronquist et al. 2012) with a GTRþIþG model and 5,000,000 generations, two independent runs, and four chains. The other parameters were set as the default. We discarded 25% of these trees, with the remainder used to compute a 50% majority rule consensus tree to estimate posterior probabilities. A bootstrap analysis with 1000 replicates was performed to estimate the confidence of the tree nodes and a bootstrap percentage (BP) of 50% or Bayesian posterior probability (BPP) of 0.9 was considered supportive in all constructed trees in this study.
Table 1 e List of the Cystobasidium strains examined in this study. Strains
Isolation source
Cystobasidium tubakii JCM 31526T ¼ HUT7413T ¼ NBRC 112503T
Soil
JCM 31529 ¼ HUT7416 ¼ NBRC 112504
Soil
Cystobasidium ongulense JCM 31527T ¼ HUT7414T ¼ NBRC 112505T
Soil
JCM 31528 ¼ HUT7415 ¼ NBRC 112506
Soil
Locality
Accession numbers ITSþLSU rDNA
SSU rDNA
TEF1
CYTB
East Ongul Island, Antarctica East Ongul Island, Antarctica
LC155913
LC158350
LC158352
LC158354
East Ongul Island, Antarctica East Ongul Island, Antarctica
LC155915
LC158351
LC158353
LC158355
LC155914
LC155916
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We also determined the sequence similarity and nucleotide variation in the ITS region and LSU D1/D2 domain among the species most closely related with C. tubakii and C. ongulense, using the EMBOSS water alignment tool (http://www.ebi. ac.uk/Tools/psa/emboss_water/nucleotide.html).
2.4.
Physiological characteristics
The effect of temperature on the growth of fungi was determined on PDA plates in the range from 3 C to 37 C. The assessment of carbon assimilation was performed in glass vials with yeast nitrogen base liquid media for carbon assimilation tests according to following standard methods (Kurtzman et al. 2011), with incubation for 2 wk at 17 C. Assimilation of nitrogen and other physiological tests were also carried out in glass vials according to the protocols described by Kurtzman et al. (2011). Strains were examined for a sexual state after growth on the following media, which were incubated at 17e20 C: YM agar (3 g/L yeast extract, 3 g/L malt extract, 5 g/L peptone, 10 g/L glucose, and 20 g/L agar), 5% malt extract agar (50 g/L malt extract and 30 g agar/L), and corn meal agar (Difco).
3.
Results and discussion
A total of 293 fungal strains were isolated from the soil samples collected on East Ongul Island, East Antarctica. Among
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these strains, four were classified as genus Cystobasidium (Phylum Basidiomycota, subphylum Pucciniomycotina) according to sequence similarity of the ITS region and the LSU D1/D2 domain. Based on phylogenetic analysis of LSU D1/D2 domain sequences, strains JCM 31526T and JCM 31529 are branched from C. pallidum (Lodder) A. Yurkov, A. Kachalkin, H.M. Daniel, M. Groenewald, D. Libkind, V. de Garcia, P. Zalar, D. Gouliamova, T. Boekhout & D. Begerow; this branch is supported with 85% BP, 82% BP, and 0.96 BPP by ML, MP, and BI analyses, respectively (Fig. 1). Strains JCM 31527 and JCM 31528 are also € l) A. Yurkov, A. Kachalkin, divided from C. laryngis (Reierso H.M. Daniel, M. Groenewald, D. Libkind, V. de Garcia, P. Zalar, D. Gouliamova, T. Boekhout & D. Begerow. The clade of JCM 31527 and JCM 31528 exhibited 59% BP, 70% BP and 0.99 BPP scores by ML, MP, and BI analyses, respectively (Fig. 1). As they are considered to represent two new basidiomycetous yeast species in the genus Cystobasidium, strains JCM 31526T and JCM 31529 were named C. tubakii sp. nov., and strains JCM 31527 and JCM 31528 were named C. ongulense sp. nov. In addition, according to the LSU D1/D2 phylogenetic analysis, C. pallidum and C. benthicum (Nagah., Hamam., Nakase & Horikoshi) A. Yurkov, A. Kachalkin, H.M. Daniel, M. Groenewald, D. Libkind, V. de Garcia, P. Zalar, D. Gouliamova, T. Boekhout & D. Begerow were determined as the species most closely related to C. tubakii and C. laryngis, C. pinicola (F.Y. Bai, L.D. Guo & J.H. Zhao) A. Yurkov, A. Kachalkin, H.M. Daniel, M. Groenewald, D. Libkind, V. de Garcia, P. Zalar, D. Gouliamova, T. Boekhout & D. Begerow, and C. ritchiei A.M. Yurkov,
Fig. 1 e Phylogenetic tree based on the LSU D1/D2 domain. Maximum likelihood analysis of the LSU D1/D2 domain sequences of all Cystobasidium tubakii sp. nov., C. ongulense sp. nov., and closely related species. Cystobasidium tubakii strains and C. ongulense strains investigated in this study are highlighted in bold font. Sakaguchia dacryoidea CBS 6353 was designated as the outgroup. The tree backbone was constructed by maximum likelihood analysis with MEGA7. Bootstrap percentages of maximum likelihood and maximum parsimony analyses over 50% from 1000 bootstrap replicates and posterior probabilities of Bayesian inference above 0.9 are shown from left on the branches. The scale bar represents 0.02 substitutions per nucleotide position. ns, not supported (bootstrap percentages < 50% or Bayesian inference < 0.9).
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Kachalkin, H.M. Daniel, M. Groenew., T. Boekhout & D. Begerow were identified as the closest species to C. ongulense. Therefore, the LSU D1/D2 and ITS regions sequences of two new Cystobasidium species were compared with those of the closest related species. The interspecific nucleotide substitution in the LSU D1/D2 domain comprised 9 or 10 nucleotide substitutions between C. tubakii and C. benthicum or C. pallidum, respectively. Cystobasidium ongulense exhibited 5 and 9 nucleotide variations compared with C. laryngis and C. pinicola, respectively. Furthermore, C. pallidum was shown to differ by 6 nucleotides with C. benthicum and exhibited 5 nucleotides differences compared with C. ritchiei (Table 2). With respect to the nucleotide similarities and substitutions in the ITS region sequences, C. tubakii demonstrated 17 nucleotides changes and 96.9% sequence similarity with C. benthicum, as well as 118 nucleotide substitutions and 80.4% sequence similarity with C. pallidum. The ITS region sequence of C. ongulense identified 7 and 11 nucleotides substitutions in addition to 98.7% and 97.9% sequence similarity compared with those of C. laryngis and C. pinicola, respectively. In addition, C. laryngis showed 6 nucleotides differences and 98.9% similarity with C. pinicola in the ITS region sequence (Table 3). Previously, Yurkov et al. (2015) attempted phylogenetic analysis within the genus Cystobasidium using the combined sequences of SSU rDNA, ITS region, LSU D1/D2 domain, and TEF1 according to the ML method. This analytic method strongly supported classification of the genus Cystobasidium. Therefore, in the current study, we amplified and obtained SSU rDNA and TEF1 fragment sequences of the two new Cystobasidium species, as well for use in calculating their phylogenetic placement. From this, C. tubakii was supported with 76% BP, 68% BP and 0.96 BPP by ML, MP and BI analyses,
respectively, and C. ongulense was indicated by 97% BP, 97% BP, and 1.0 BI (Fig. 2). Therefore, considering the results of the phylogenetic analysis of LSU D1/D2 domain, the combined sequences of SSU rDNA, ITS region, LSU D1/D2 domain and TEF1, and nucleotide variations of LSU D1/D2 domain and ITS region sequences, C. tubakii and C. ongulense should be considered as representing novel species. The genus Cystobasidium is characterized by the following features: yeast cells are ovoid to elongated. The streak cultures are often pink to orange in color. D-Glucuronate is assimilated, whereas nitrate is not. Starch-like compounds are not produced. Fermentation abilities are absent (Sampaio and Oberwinkler 2011). As C. tubakii and C. ongulense, which were isolated from the soil of East Ongul Island, exhibited these characteristics, they were confirmed to belong to the genus Cystobasidium. Members of C. tubakii were able to utilize sucrose, melezitose, starch, and salicin, but were not able to assimilate sorbose. In contrast, C. pallidum could not assimilate sucrose, melezitose, and salicin. Comparison of the physiological characterization between C. tubakii and C. benthicum, indicated that they showed similar carbon assimilation patterns, but C. benthicum could not assimilate salicin. The carbon assimilation patterns of members of C. ongulense were highly similar to that of C. laryngis. However, starch was weakly assimilated and cellobiose and DL-lactate were not utilized by members of C. ongulense (Table 4). The optimum growth temperature of C. tubakii and C. ongulense was 15e17 C and 20 C, respectively. Cystobasidium tubakii could not grow at 30 C, whereas C. pallidum was able to grow at 30 C and C. benthicum could grow at 37 C (Table 5). The maximum growth temperature of C. ongulense was 30 C which is the same maximum growth temperature with C. laryngis and C. pinicola
Table 2 e Number of nucleotide substitutions in the LSU D1/D2 region sequence among the species most closely related to Cytobasidium tubakii and C. ongulense.
C. C. C. C. C. C. C.
tubakii ongulense benthicum laryngis pallidum pinicola ritchiei
C. tubakii
C. ongulense
C. benthicum
C. laryngis
C. pallidum
C. pinicola
C. ritchiei
e 21 10 17 9 15 19
21 e 15 5 17 8 16
10 15 e 10 6 9 13
17 5 10 e 12 7 11
9 17 6 12 e 11 5
15 8 9 7 11 e 12
19 16 13 11 5 12 e
Each value indicates the number of nucleotide substitutions (nt) between the indicated species pairs.
Table 3 e Nucleotide substitution and sequence similarity in the ITS region sequence among the species most closely related to Cytobasidium tubakii and C. ongulense.
C. C. C. C. C. C. C.
tubakii ongulense benthicum laryngis pallidum pinicola ritchiei
C. tubakii
C. ongulense
C. benthicum
C. laryngis
e 28 (94.8) 17 (96.9) 28 (94.8) 118 (80.4) 24 (95.4) 27 (95.0)
28 (94.8) e 23 (95.8) 7 (98.7) 129 (78.7) 11 (97.9) 11 (98.0)
17 (96.9) 23 (95.8) e 18 (96.9) 115 (80.5) 13 (97.4) 18 (96.5)
28 (94.8) 7 (98.7) 18 (96.9) e 124 (79.6) 6 (98.9) 12 (98.0)
C. pallidum 118 129 115 124 e 122 125
(80.4) (78.7) (80.5) (79.6) (79.9) (79.5)
C. pinicola
C. ritchiei
24 (95.4) 11 (97.9) 13 (97.4) 6 (98.9) 122 (79.9) e 10 (98.1)
27 (95.0) 11 (98.0) 18 (96.5) 12 (98.0) 125 (79.5) 10 (98.1) e
Each value indicates that the number of nucleotide substitutions (nt) between the indicated species pairs. Values in parentheses indicate the sequence similarity (%) between pairs of species.
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Fig. 2 e Phylogenetic reconstruction of Cystobasidium tubakii and C. ongulense using 18S rDNA, ITS region, LSU D1/D2 domain, and the TEF1 gene. Maximum likelihood analysis of the 18S rDNA, ITS, LSU D1/D2 domain and TEF1 sequences of all strains of C. tubakii, C. ongulense and their closely related species. Strains of C. tubakii and C. ongulense investigated in this study are highlighted in bold font. Sakaguchia dacryoidea CBS 6353 was designated as the outgroup. The tree backbone was constructed by maximum likelihood analysis using MEGA7. Bootstrap percentages of maximum likelihood and maximum parsimony analyses over 50% from 1000 bootstrap replicates and posterior probabilities of Bayesian inference above 0.9 are shown to left on the branches. The scale bar represents 0.02 substitutions per nucleotide position. ns, not supported (bootstrap percentages < 50% or Bayesian inference < 0.9).
Table 4 e Comparison of physiological characterizations among species of the genus Cytobasidium.
Cystobasidium benthicum C. calyptogenae C. fimetarium C. laryngis C. lysinophilum C. minutum C. oligophagum C. ongulense C. pallidum C. pinicola C. psychroaquaticum C. ritchiei C. slooffiae C. tubakii
Nitrate
D-glucuronate
Sucrose
Melezitose
Starch
Cellobiose
Salicin
L-Sorbose
DL-Lactate
¡ ¡
þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ v þ w þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ
þ w w þ w w
w þ þ þ w þ ¡ w v þ þ ¡
þ þ þ s þ w þ þ v þ
w v þ ¡ þ v w s ¡
þ þ v þ ¡ d v w s w
Main physiology test results for the characteristics of C. ongulense, C. tubakii, and related species are shown. Physiological data of related species were taken from Sampaio (2011), Sampaio and Oberwinkler (2011), Yurkov et al. (2015) and this study. þ, positive; w, weak; s, slow; , negative; v, variable; n, no data. Cystobasidium tubakii and C. ongulense investigated in this study are highlighted in bold font.
(Table 5). Moreover, we investigated the extracellular enzyme secretion by C. tubakii and C. ongulense using API zym (Biomerieux, Tokyo, Japan) and found that both species also have esterase, lipase, and b-glucosidase (Supplementary Table S1). After incubation for 10 d on YM agar at 17e20 C, yeast cells of C. tubakii were ovoid to elongated (3e4 mm 4e5 mm) and divided by polar budding (Fig. 3A). The cell morphology of C. ongulense also showed ovoid to elongated and the cell size was 3e3.5 mm 4e6 mm, which proliferation by polar budding after 10 d on YM agar at 17e20 C (Fig. 3B). Furthermore, neither species exhibited sexual activity, or produced ballistoconidia and pseudohyphae. Additionally, the results of a BLAST homology search, indicated that Cystobasidium sp. CBS 9086 and Basidiomycota sp. 3 HFP-2013 isolate A25M013 exhibited 100% matching over the LSU D1/D2 domain sequence with that of C. tubakii. CBS
9086 was isolated from Antarctic water and A25M013 was collected from King George Island, Antarctica (Scorzetti et al. 2002). These results suggest that C. tubakii may be distributed over a wide area in Antarctica. Conversely, C. ongulense exhibited the same LSU D1/D2 domain sequence as that of Cystobasidium sp. CCFEE 5633, Cystobasidium CCFEE 5628, Cystobasidium sp. CBS 8923, C. laryngis TP-Snow-Y131 and C. cf. laryngis DBVPG 10053. CCFEE 5628 and 5633 were collected from Mount Gran Sasso (Italy), CBS 8923 was isolated from Antarctica, TP-Snow-Y131 was obtained from snow in Tibet (China), and DBVPG 10053 was collected from the Italian alps (Scorzetti et al. 2002; Selbmann et al. 2014). Consequently, C. ongulense was considered to inhabit various cold region. All strains investigated in this study exhibited good growth in the vitamin-free medium, whereas other known type strains of Cystobasidium spp. could not grow in the medium.
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Table 5 e Comparison of additional growth tests and other characterizations among species of the genus Cytobasidium. Growth on vitaminfree
Optimal growth Growth at Growth at Growth at Growth at Fermentation 25 C 30 C 37 C ability temperature 3 C
Cell size (mm)
Cystobasidium benthicum C. calyptogenae C. fimetarium C. laryngis
n n n n
n n n n
þ þ þ þ
þ þ þ v
þ þ
2e3.5 3e5 2e3.5 3e6 2e3 5e7 2.5e4 3e8
C. lysinophilum C. minutum C. oligophagum C. ongulense C. pallidum C. pinicola C. psychroaquaticum
þ
n n 28e30 C 20 C n n n
n n n þ n n n
þ þ þ þ þ þ þ
þ þ þ w þ þ
þ ¡
¡
2e3 4e5 1.5e3 4e6 2e4 3e5 3e3.5 £ 4e6 2e4 4e6.5 2e3.5 2.5e7.5 4e5 6e9
C. ritchiei C. slooffiae C. tubakii
þ
n n 15e17 C
n n þ
þ þ þ
þ ¡
¡
¡
2.5e4 5e7 2e3.5 3e5 3e4 £ 4e5
Main characteristics of C. ongulense, C. tubakii, and related species are shown. Data in bold were collected in this study. Other physiological data were taken from Sampaio (2011), Sampaio and Oberwinkler (2011), Yurkov et al. (2015), and this study. þ, positive; w, weak; s, slow; , negative; v, variable; n, no data. Cystobasidium tubakii and C. ongulense investigated in this study are highlighted in bold font.
Fig. 3 e Morphology of Cystobasidium tubakii and C. ongulense. Cystobasidium tubakii (A) and C. ongulense (B) grown on YM agar for 10 d at 17e20 C. Bars 10 mm.
Cystobasidium tubakii and C. ongulense were found to grow under subzero temperature conditions. Based on the average total carbon and nitrogen concentrations in the soil samples, East Ongul Island constituted an oligotrophic environment. Thus, in comparison to related species, members of C. tubakii and C. ongulense were considered to have obtained these growth abilities to survive in an oligotrophic environments such as that of East Ongul Island. This is the first report of new fungal species isolated from near the Syowa station in the 60-y history of JARE.
3.1.
Taxonomy
Cystobasidium tubakii M. Tsuji, Tsujimoto & S. Imura, sp. nov. Fig. 3A. Mycobank no.: MB817702. Etymology: “tubakii” refers to the late mycologist Keisuke Tubaki, who was the first to report fungi from near the Syowa station, East Antarctica, to pay honor to his pioneering work.
Type: ANTARCTICA, East Ongul Island, 22 Dec 2007, isolated from soil, East Ongul Island, East Antarctica by M. Tsujimoto & S. Imura (holotype: strain JCM 31526T preserved in a metabolically inactive state at Japan Collection of Microorganisms, Riken, Japan; ex-type culture: HUT7413T and NBRC 112503T; paratype: JCM 31529, HUT7416, NBRC 112504); ITS and LSU D1/D2 domain: LC155913 (JCM 31526T), LC155914 (JCM 31529), SSU rDNA: LC158350 (JCM 31526T), TEF1: LC158352 (JCM 31526T), CYTB: LC158354 (JCM 31526T). Streak culture after 1 wk on 5% malt extract agar at 15e17 C: shiny, mucilaginous, smooth, entire margin. Yeast cells after 10 d on YM agar ovoid to elongated, 3e4 mm 4e5 mm, proliferating by polar budding. Sexual activity was not observed. Ballistoconidia and pseudohyphae not produced. Sugars not fermented. Assimilation of carbon compounds: D-glucose, sucrose, galactose (weak), trehalose (weak), melezitose, starch (weak), cellobiose (weak), salicin, D-xylose, ethanol, glycerol, ribitol, DL-lactate (weak), succinic acid, D-gluconate, Dglucuronate, N-acetyl-D-glucosamine, D-xylitol. Inulin, raffinose, melibiose, lactose, maltose, methyl-a-D-glucoside, L-
m y c o s c i e n c e 5 8 ( 2 0 1 7 ) 1 0 3 e1 1 0
sorbose, L-rhamnose, L-arabinose, D-arabinose, D-ribose, methanol, erythritol, galactitol, D-mannitol, D-glucitol, myoinositol, D-glucosamine, citric acid, potassium nitrate, and sodium nitrate are not assimilated. Growth on 5% glucose medium with 10% NaCl (w/v), 50% (w/v) glucose medium and in vitamin-free medium. Growth absent on 0.01% cyclohexamide. Maximum growth temperature at 25 C and optimal growth at 15e17 C. Growth on PDA at 3 C. Habitat: soil of East Ongul Island, East Antarctica in the vicinity of Syowa station, JARE. Cystobasidium ongulense M. Tsuji, Tsujimoto & S. Imura, sp. nov. Fig. 3B. Mycobank no.: MB817703. Etymology: “ongulense” refers to the geographic origin of this species. Type: ANTARCTICA, East Ongul Island, 22 Dec 2007, isolated from soil, East Ongul Island, East Antarctica by M. Tsujimoto & S. Imura (holotype: strain JCM 31527T preserved in a metabolically inactive state at Japan Collection of Microorganisms, Riken, Japan; ex-type culture: HUT7414T and NBRC 112505T; paratype: JCM 31528, HUT7415, NBRC 112506); ITS and LSU D1/D2: LC155915 (JCM 31527T), LC155916 (JCM 31528), SSU rDNA: LC158351 (JCM 31527T), TEF1: LC158353 (JCM 31527T), CYTB: LC158355 (JCM 31527T). Streak culture after 1 wk on YM agar at 17e20 C: shiny, mucilaginous, smooth, entire margin. Yeast cells after 10 d on YM agar ovoid to elongated, 3e3.5 mm 4e6 mm, proliferating by polar budding. Sexual activity was not observed. Ballistoconidia and pseudohyphae not produced. Sugars not fermented. Assimilation of carbon compounds: D-glucose, sucrose, trehalose, melezitose, starch (weak), salicin, D-xylose, ethanol, glycerol, ribitol, succinic acid (weak), D-gluconate, D-glucuronate, D-xylitol, L-arabinose, D-arabinose, Dmannitol, D-glucitol. No growth on inulin, raffinose, melibiose, galactose, lactose, maltose, methyl-a-D-glucoside, cellobiose, Lsorbose, L-rhamnose, D-ribose, methanol, erythritol, galactitol, DL-lactate, N-acetyl-D-glucosamine, myo-inositol, D-glucosamine, citric acid, potassium nitrate, or sodium nitrate. Growth on 5% glucose medium with 10% NaCl (w/v), 50% (w/v) glucose medium and in vitamin-free medium. Growth absent on 0.01% cyclohexamide. Maximum growth temperature at 30 C and optimal growth at 20 C. Growth on PDA at 3 C. Habitat: soil of East Ongul Island, East Antarctica in the vicinity of Syowa station, JARE.
Disclosure All necessary permits were obtained for the described field studies. Permission to undertake field studies and collect samples was granted by the Ministry of the Environment of Japan.
Acknowledgments This work was carried out as part of the Science Program of the 49th JARE. It was supported by NIPR under MEXT, Japan
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and SOKENDAI. This work was supported by a JSPS Grant-inAid for Challenging Exploratory Research (No. 16K12643), Young Scientists (A) (No. 16H06211) to M. Tsuji and Scientific Research (B) (No. 18310024) to S. Imura. The preparation of this paper was supported by an NIPR publication subsidy. We are grateful to Kenichi Watanabe for his technical support.
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.myc.2016.11.002.
references Sampaio JP, Bandoni RJ, Fell JW, KwonBoekhout T, Fonseca A, Chung KJ, 2011. Discussion of teleomorphic and anamorphic basidiomycetous yeasts. In: Kurtzman CP, Fell JW, Boeckhout T (eds), The yeast, a taxonomic study, 5th edn. Elsevier, Amsterdam, pp 1339e1372. Bridge PD, Spooner BM, 2012. Non-lichenized Antarctic fungi: transient visitors or members of a cryptic ecosystem? Fungal Ecology 5: 381e394; http://dx.doi.org/10.1016/ j.funeco.2012.01.007. Hirose D, Tanabe Y, Uchida M, Kudoh S, Osono T, 2013. Microfungi associated with withering willow wood in ground contact near Syowa Station, East Antarctica for 40 years. Polar Biology 36: 919e924; http://dx.doi.org/10.1007/s00300-0131320-x. Katoh K, Standley DM, 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772e780; http:// dx.doi.org/10.1093/molbev/mst010. Kumar S, Stecher G, Tamura K, 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870e1874; http://dx.doi.org/ 10.1093/molbev/msw054. Kurtzman CP, Fell JW, Boekhout T, Robert V, 2011. Methods for isolation, phenotypic characterization and maintenance of yeasts. In: Kurtzman CP, Fell JW, Boeckhout T (eds), The yeast, a taxonomic study, 5th edn. Elsevier, Amsterdam, pp 87e111. Ravindra R, Chaturvedi A, 2011. Antarctica. In: Singh VP, Singh P, Haritashya UK (eds), Encyclopedia of snow, ice and glaciers. Springer, Dordrecht, pp 45e53. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, € hna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP, 2012. Ho MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539e542; http://dx.doi.org/10.1093/sysbio/sys029. Sampaio JP, 2011. Rhodotorula Harrison (1928). In: Kurtzman CP, Fell JW, Boeckhout T (eds), The yeast, a taxonomic study, 5th edn. Elsevier, Amsterdam, pp 1873e1927. Sampaio JP, Oberwinkler F, 2011. Cystobasidium (Lagerheim) Neuhoff (1924). In: Kurtzman CP, Fell JW, Boeckhout T (eds), The yeast, a taxonomic study, 5th edn. Elsevier, Amsterdam, pp 1419e1422. Scorzetti G, Fell JW, Fonseca A, Stazell-Tallman A, 2002. Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions. FEMS Yeast Research 2: 495e517; http://dx.doi.org/10.1111/ j.1567-1364.2002.tb00117.x. Selbmann L, Zucconi L, Onofri S, Cecchini C, Isola D, Turchetti B, Buzzini P, 2014. Taxonomic and phenotypic characterization of yeasts isolated from worldwide cold rock-associated
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habitats. Fungal Biology 118: 61e71; http://dx.doi.org/10.1016/ j.funbio.2013.11.002. Singh P, Tsuji M, Singh SM, Roy U, Hoshino T, 2013. Taxonomic characterization, adaptation strategies and biotechnological potential of cryophilic yeasts from ice cores of Midre nbreen glacier, Svalbard, Arctic. Cryobiology 66: 167e175; Love http://dx.doi.org/10.1016/j.cryobiol.2013.01.002. Soneda M, 1961. On some yeasts from the Antarctic region. Special Publications from the Seto Marine Biological Laboratory 1: 1e10. Tsuji M, Fujiu S, Xiao N, Hanada Y, Kudoh S, Kondo H, Tsuda S, Hoshino T, 2013a. Cold adaptation of fungi obtained from soil and lake sediment in the Skarvsnes ice-free area, Antarctic. FEMS Microbiology Letters 346: 121e130; http://dx.doi.org/ 10.1111/1574-6968.12217. Tsuji M, Uetake J, Tanabe Y, 2016. Changes in the fungal community of Austre Brøggerbreen deglaciation area, Ny Alesund, Svalbard, High Arctic. Mycoscience 57: 448e451; http:// dx.doi.org/10.1016/j.myc.2016.07.006. Tsuji M, Yokota Y, Shimohara K, Kudoh S, Hoshino T, 2013b. An application of wastewater treatment in a cold environment and stable lipase production of Antarctic basidiomycetous yeast Mrakia blollopis. PLoS One 8: e59376; http://dx.doi.org/ 10.1371/journal.pone.0059376. Tubaki K, 1961a. On some fungi isolated from the Antarctic materials. Special Publications from the Seto Marine Biological Laboratory 14: 1e9. Tubaki K, 1961b. Note on some fungi and yeasts from Antarctica. Antarctic Record (Tokyo) 11: 161e162. Tubaki K, Asano I, 1965. Additional species of fungi isolated from the Antarctic materials. Japanese Antarctic Research Expedition Scientific Reports. Series E 27: 1e12.
Wang QM, Bai FY, 2008. Molecular phylogeny of basidiomycetous yeasts in the Cryptococcus luteolus lineage (Tremellales) based on nuclear rRNA and mitochondrial cytochrome b gene sequence analyses: proposal of Derxomyces gen. nov. and Hannaella gen. nov., and description of eight novel Derxomyces species. FEMS Yeast Research 8: 799e814; http://dx.doi.org/10.1111/ j.1567e1364.2008.00403.x. Wang QM, Bai FY, Zhao JH, Jia JH, 2003. Bensingtonia changbaiensis sp. nov. and Bensingtonia sorbi sp. nov., novel ballistoconidiumforming yeast species from plant leaves. International Journal of Systematic and Evolutionary Microbiology 53: 2085e2089; http:// dx.doi.org/10.1099/ijs.0.02736-0. Wang QM, Groenewald M, Takashima M, Theelen B, Han PJ, Liu XZ, Boekhout T, Bai FY, 2015. Phylogeny of yeasts and related filamentous fungi within Pucciniomycotina determined from multigene sequence analyses. Studies in Mycology 81: 27e53; http://dx.doi.org/10.1016/ j.simyco.2015.08.002. Welander U, 2005. Microbial degradation of organic pollutants in soil in a cold climate. Soil and Sediment Contamination 14: 281e291; http://dx.doi.org/10.1080/15320380590928339. Yurkov AM, Kachalkin AV, Daniel HM, Groenewald D, Libkind D, de Garcia V, Zalar P, Gouliamova DE, Boekhout T, Begerow D, 2015. Two yeast species Cystobasidium psychroaquaticum f.a. sp. nov. and Cystobasidium rietchieii f.a. sp. nov. isolated from natural environments, and the transfer of Rhodotorula minuta clade members to the genus Cystobasidium. Antonie van Leeuwenhoek 107: 173e185; http://dx.doi.org/10.1007/s10482014-0315-0.
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Cystobasidium tubakii and Cystobasidium ongulense, new basidiomycetous yeast species isolated from East Ongul Island, East Antarctica Masaharu Tsuji a,*, Megumu Tsujimoto a,b, Satoshi Imura a,b a
National Institute of Polar Research (NIPR), 10-3 Midori-cho, Tachikawa, Tokyo 190-8518, Japan Department of Polar Science, SOKENDAI (The Graduate University for Advanced Studies), 10-3 Midori-cho, Tachikawa, Tokyo 190-8518, Japan
b
article info
abstract
Article history:
Two new cold-adapted yeast species, Cystobasidium tubakii sp. nov. and Cystobasidium
Received 17 July 2016
ongulense sp. nov., were isolated from soil collected from East Ongul Island, East Antarctica,
Received in revised form
during the 49th Japanese Antarctic Research Expedition (JARE 49). Molecular analysis based
4 November 2016
on large subunit (LSU) D1/D2 domain and the combined sequences of small subunit (SSU)
Accepted 6 November 2016
rDNA, internal transcribed spacer (ITS) region, LSU D1/D2 domain and TEF1 sequences
Available online 10 December 2016
showed that these species are novel. Both species could grow at sub-zero temperatures and in vitamin-free media. These characteristics were likely obtained by the yeasts to survive
Keywords:
oligotrophic environments such as that in Antarctica. This is the first report of new fungal
Antarctic yeast
species isolated from near the Syowa station in the 60-y history of JARE.
Cystobasidiomycetes
© 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
LSU D1/D2 domain Syowa station
1.
Introduction
Antarctica is the southernmost landmass on Earth and has an area of approximately 14 million km2, making it the fifthlargest continent in the world. Approximately 98% of Antarctica is covered by ice and snow, and temperatures in coastal areas usually range from 5 C to 35 C (Ravindra and Chaturvedi 2011). Under such sub-zero conditions, the growth of cold-adapted fungi and concomitant decomposition of organic compounds play an important role in the nutrient cycle of Polar region ecosystems (Welander 2005).
East Ongul Island (69 10 S, 39 350 E) is located at the east side of the Lu¨tzow Holm Bay, East Antarctica. Syowa station has been established on this island since 1957, as the base of the Japanese Antarctic Research Expedition (JARE). To date, over 1000 fungal species from 421 genera have been isolated and recorded from Antarctica (Bridge and Spooner 2012); the list of known species includes 68% ascomycetes, 23% basidiomycetes, and 5% zygomycetes, with the final 4% comprising various other lineages. Previously, 12 ascomycetous and four basidiomycetous species have been reported from the vicinity of Syowa station (Soneda 1961; Tubaki 1961a, b; Tubaki and Asano 1965). More recently, 14
* Corresponding author. Fax: þ81 42 528 3492. E-mail address: [email protected] (M. Tsuji). http://dx.doi.org/10.1016/j.myc.2016.11.002 1340-3540/© 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
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genera of microfungi were isolated from willow wood located near the station (Hirose et al. 2013). However, these are the only reports to have described fungi isolated from near the station vicinity. Therefore, despite previous research on the mycoflora of East Ongul Island, knowledge of the fungal species inhabiting the island likely remains incomplete. Basidiomycetous yeasts have been widely reported to represent the dominant fungi in Polar regions (Singh et al. 2013; Tsuji et al. 2013b, 2016). Many of these yeasts have been found only in their asexual stage and are classified as anamorphic genera Cryptococcus Vuillemin or Rhodotorula Harrison (Boekhout et al. 2011). The genus Cystobasidium (Lagerheim) Neuhoff represents species previously classified as the Rhodotorula minuta clade (Sampaio 2011; Wang et al. 2015; Yurkov et al. 2015). In this study, four yeast colonies, light pinkish in color, were isolated from East Ongul Island, East Antarctica. Based on physiological testing and molecular analysis using the large subunit 26S rDNA (LSU D1/D2 domain) and internal transcribed spacer (ITS) region sequences, these strains were classified into two new basidiomycetous yeast species in the genus Cystobasidium, for which the names C. tubakii sp. nov. and C. ongulense sp. nov. are proposed.
2.
Materials and methods
2.1.
Sampling sites and sample collection
A total of 226 surface soil samples were collected from the entire of East Ongule Island near the Syowa station. The collected samples were immediately stored at 20 C until used. Total carbon and nitrogen concentrations in the 226 soil samples were measured using a CN analyzer (SUMIGRAPH NC-220F, Sumika Chemical Analysis Service, Tokyo, Japan); the average of the carbon and nitrogen concentrations were 0.063 ± 0.087% and 0.0025 ± 0.0023%, respectively.
2.2.
Isolation of strains
Each 0.1 g untreated soil sample was directly placed on potato dextrose agar (PDA, Difco, Becton Dickinson Japan, Tokyo) containing 50 mg/mL chloramphenicol and incubated at 10 C for a period of up to 3 wk. Yeast samples were chosen for isolation based on colony morphology. Each colony of a
different morphology was purified by repeated streaking on fresh PDA. The cultures of Cystobasidium tubakii and C. ongulense are deposited at the Japan Collection of Microorganisms (JCM), Riken, Japan, at the HUT Culture Collection (HUT), Hiroshima University, Japan, as well as at the Biological Resource Center, National Institute of Technology and Evaluation (NBRC), Japan. The detail of deposit numbers is shown in Table 1.
2.3.
Sequencing and phylogenetic analysis
DNA was extracted from yeast colonies, using an ISOPLANT II kit (Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer's protocols. The extracted DNA was amplified by polymerase chain reaction (PCR), using KOD-plus DNA polymerase (Toyobo, Osaka, Japan). Five fragment genes or loci, i.e., the small subunit (SSU) rDNA, LSU D1/D2 domains, ITS region, translation elongation factor 1-a (TEF1), and the mitochondrial gene cytochrome b (CYTB) were amplified in this study. The ITS region and LSU D1/D2 domain primers and conditions for PCR were described previously (Tsuji et al. 2013a). SSU rDNA primers were reported by Wang et al. (2003), primers for CYTB as previously described by Wang and Bai (2008), and TEF1 primers by Wang et al. (2015). The amplified DNA fragments were purified using Sephacryl S400HR (SigmaeAldrich Japan, Tokyo). Sequences were determined using an ABI prism 3130xl Sequencer (Applied Biosystems, Life Technologies Japan, Tokyo). GenBank accession numbers for all the sequences analyzed in this study are listed in Table 1. The sequences were aligned with the MAFFT program ver. 7.273 (Katoh and Standley 2013) using the LeINSeI algorithm. The alignments were deposited in TreeBASE (Submission ID: S19510). Maximum likelihood (ML) with an HYKþGþI model and maximum parsimony (MP) analysis with a TBR model were performed using MEGA7 (Kumar et al. 2016). Bayesian inference (BI) was constructed using MrBayes 3.2.5 (Ronquist et al. 2012) with a GTRþIþG model and 5,000,000 generations, two independent runs, and four chains. The other parameters were set as the default. We discarded 25% of these trees, with the remainder used to compute a 50% majority rule consensus tree to estimate posterior probabilities. A bootstrap analysis with 1000 replicates was performed to estimate the confidence of the tree nodes and a bootstrap percentage (BP) of 50% or Bayesian posterior probability (BPP) of 0.9 was considered supportive in all constructed trees in this study.
Table 1 e List of the Cystobasidium strains examined in this study. Strains
Isolation source
Cystobasidium tubakii JCM 31526T ¼ HUT7413T ¼ NBRC 112503T
Soil
JCM 31529 ¼ HUT7416 ¼ NBRC 112504
Soil
Cystobasidium ongulense JCM 31527T ¼ HUT7414T ¼ NBRC 112505T
Soil
JCM 31528 ¼ HUT7415 ¼ NBRC 112506
Soil
Locality
Accession numbers ITSþLSU rDNA
SSU rDNA
TEF1
CYTB
East Ongul Island, Antarctica East Ongul Island, Antarctica
LC155913
LC158350
LC158352
LC158354
East Ongul Island, Antarctica East Ongul Island, Antarctica
LC155915
LC158351
LC158353
LC158355
LC155914
LC155916
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We also determined the sequence similarity and nucleotide variation in the ITS region and LSU D1/D2 domain among the species most closely related with C. tubakii and C. ongulense, using the EMBOSS water alignment tool (http://www.ebi. ac.uk/Tools/psa/emboss_water/nucleotide.html).
2.4.
Physiological characteristics
The effect of temperature on the growth of fungi was determined on PDA plates in the range from 3 C to 37 C. The assessment of carbon assimilation was performed in glass vials with yeast nitrogen base liquid media for carbon assimilation tests according to following standard methods (Kurtzman et al. 2011), with incubation for 2 wk at 17 C. Assimilation of nitrogen and other physiological tests were also carried out in glass vials according to the protocols described by Kurtzman et al. (2011). Strains were examined for a sexual state after growth on the following media, which were incubated at 17e20 C: YM agar (3 g/L yeast extract, 3 g/L malt extract, 5 g/L peptone, 10 g/L glucose, and 20 g/L agar), 5% malt extract agar (50 g/L malt extract and 30 g agar/L), and corn meal agar (Difco).
3.
Results and discussion
A total of 293 fungal strains were isolated from the soil samples collected on East Ongul Island, East Antarctica. Among
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these strains, four were classified as genus Cystobasidium (Phylum Basidiomycota, subphylum Pucciniomycotina) according to sequence similarity of the ITS region and the LSU D1/D2 domain. Based on phylogenetic analysis of LSU D1/D2 domain sequences, strains JCM 31526T and JCM 31529 are branched from C. pallidum (Lodder) A. Yurkov, A. Kachalkin, H.M. Daniel, M. Groenewald, D. Libkind, V. de Garcia, P. Zalar, D. Gouliamova, T. Boekhout & D. Begerow; this branch is supported with 85% BP, 82% BP, and 0.96 BPP by ML, MP, and BI analyses, respectively (Fig. 1). Strains JCM 31527 and JCM 31528 are also € l) A. Yurkov, A. Kachalkin, divided from C. laryngis (Reierso H.M. Daniel, M. Groenewald, D. Libkind, V. de Garcia, P. Zalar, D. Gouliamova, T. Boekhout & D. Begerow. The clade of JCM 31527 and JCM 31528 exhibited 59% BP, 70% BP and 0.99 BPP scores by ML, MP, and BI analyses, respectively (Fig. 1). As they are considered to represent two new basidiomycetous yeast species in the genus Cystobasidium, strains JCM 31526T and JCM 31529 were named C. tubakii sp. nov., and strains JCM 31527 and JCM 31528 were named C. ongulense sp. nov. In addition, according to the LSU D1/D2 phylogenetic analysis, C. pallidum and C. benthicum (Nagah., Hamam., Nakase & Horikoshi) A. Yurkov, A. Kachalkin, H.M. Daniel, M. Groenewald, D. Libkind, V. de Garcia, P. Zalar, D. Gouliamova, T. Boekhout & D. Begerow were determined as the species most closely related to C. tubakii and C. laryngis, C. pinicola (F.Y. Bai, L.D. Guo & J.H. Zhao) A. Yurkov, A. Kachalkin, H.M. Daniel, M. Groenewald, D. Libkind, V. de Garcia, P. Zalar, D. Gouliamova, T. Boekhout & D. Begerow, and C. ritchiei A.M. Yurkov,
Fig. 1 e Phylogenetic tree based on the LSU D1/D2 domain. Maximum likelihood analysis of the LSU D1/D2 domain sequences of all Cystobasidium tubakii sp. nov., C. ongulense sp. nov., and closely related species. Cystobasidium tubakii strains and C. ongulense strains investigated in this study are highlighted in bold font. Sakaguchia dacryoidea CBS 6353 was designated as the outgroup. The tree backbone was constructed by maximum likelihood analysis with MEGA7. Bootstrap percentages of maximum likelihood and maximum parsimony analyses over 50% from 1000 bootstrap replicates and posterior probabilities of Bayesian inference above 0.9 are shown from left on the branches. The scale bar represents 0.02 substitutions per nucleotide position. ns, not supported (bootstrap percentages < 50% or Bayesian inference < 0.9).
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Kachalkin, H.M. Daniel, M. Groenew., T. Boekhout & D. Begerow were identified as the closest species to C. ongulense. Therefore, the LSU D1/D2 and ITS regions sequences of two new Cystobasidium species were compared with those of the closest related species. The interspecific nucleotide substitution in the LSU D1/D2 domain comprised 9 or 10 nucleotide substitutions between C. tubakii and C. benthicum or C. pallidum, respectively. Cystobasidium ongulense exhibited 5 and 9 nucleotide variations compared with C. laryngis and C. pinicola, respectively. Furthermore, C. pallidum was shown to differ by 6 nucleotides with C. benthicum and exhibited 5 nucleotides differences compared with C. ritchiei (Table 2). With respect to the nucleotide similarities and substitutions in the ITS region sequences, C. tubakii demonstrated 17 nucleotides changes and 96.9% sequence similarity with C. benthicum, as well as 118 nucleotide substitutions and 80.4% sequence similarity with C. pallidum. The ITS region sequence of C. ongulense identified 7 and 11 nucleotides substitutions in addition to 98.7% and 97.9% sequence similarity compared with those of C. laryngis and C. pinicola, respectively. In addition, C. laryngis showed 6 nucleotides differences and 98.9% similarity with C. pinicola in the ITS region sequence (Table 3). Previously, Yurkov et al. (2015) attempted phylogenetic analysis within the genus Cystobasidium using the combined sequences of SSU rDNA, ITS region, LSU D1/D2 domain, and TEF1 according to the ML method. This analytic method strongly supported classification of the genus Cystobasidium. Therefore, in the current study, we amplified and obtained SSU rDNA and TEF1 fragment sequences of the two new Cystobasidium species, as well for use in calculating their phylogenetic placement. From this, C. tubakii was supported with 76% BP, 68% BP and 0.96 BPP by ML, MP and BI analyses,
respectively, and C. ongulense was indicated by 97% BP, 97% BP, and 1.0 BI (Fig. 2). Therefore, considering the results of the phylogenetic analysis of LSU D1/D2 domain, the combined sequences of SSU rDNA, ITS region, LSU D1/D2 domain and TEF1, and nucleotide variations of LSU D1/D2 domain and ITS region sequences, C. tubakii and C. ongulense should be considered as representing novel species. The genus Cystobasidium is characterized by the following features: yeast cells are ovoid to elongated. The streak cultures are often pink to orange in color. D-Glucuronate is assimilated, whereas nitrate is not. Starch-like compounds are not produced. Fermentation abilities are absent (Sampaio and Oberwinkler 2011). As C. tubakii and C. ongulense, which were isolated from the soil of East Ongul Island, exhibited these characteristics, they were confirmed to belong to the genus Cystobasidium. Members of C. tubakii were able to utilize sucrose, melezitose, starch, and salicin, but were not able to assimilate sorbose. In contrast, C. pallidum could not assimilate sucrose, melezitose, and salicin. Comparison of the physiological characterization between C. tubakii and C. benthicum, indicated that they showed similar carbon assimilation patterns, but C. benthicum could not assimilate salicin. The carbon assimilation patterns of members of C. ongulense were highly similar to that of C. laryngis. However, starch was weakly assimilated and cellobiose and DL-lactate were not utilized by members of C. ongulense (Table 4). The optimum growth temperature of C. tubakii and C. ongulense was 15e17 C and 20 C, respectively. Cystobasidium tubakii could not grow at 30 C, whereas C. pallidum was able to grow at 30 C and C. benthicum could grow at 37 C (Table 5). The maximum growth temperature of C. ongulense was 30 C which is the same maximum growth temperature with C. laryngis and C. pinicola
Table 2 e Number of nucleotide substitutions in the LSU D1/D2 region sequence among the species most closely related to Cytobasidium tubakii and C. ongulense.
C. C. C. C. C. C. C.
tubakii ongulense benthicum laryngis pallidum pinicola ritchiei
C. tubakii
C. ongulense
C. benthicum
C. laryngis
C. pallidum
C. pinicola
C. ritchiei
e 21 10 17 9 15 19
21 e 15 5 17 8 16
10 15 e 10 6 9 13
17 5 10 e 12 7 11
9 17 6 12 e 11 5
15 8 9 7 11 e 12
19 16 13 11 5 12 e
Each value indicates the number of nucleotide substitutions (nt) between the indicated species pairs.
Table 3 e Nucleotide substitution and sequence similarity in the ITS region sequence among the species most closely related to Cytobasidium tubakii and C. ongulense.
C. C. C. C. C. C. C.
tubakii ongulense benthicum laryngis pallidum pinicola ritchiei
C. tubakii
C. ongulense
C. benthicum
C. laryngis
e 28 (94.8) 17 (96.9) 28 (94.8) 118 (80.4) 24 (95.4) 27 (95.0)
28 (94.8) e 23 (95.8) 7 (98.7) 129 (78.7) 11 (97.9) 11 (98.0)
17 (96.9) 23 (95.8) e 18 (96.9) 115 (80.5) 13 (97.4) 18 (96.5)
28 (94.8) 7 (98.7) 18 (96.9) e 124 (79.6) 6 (98.9) 12 (98.0)
C. pallidum 118 129 115 124 e 122 125
(80.4) (78.7) (80.5) (79.6) (79.9) (79.5)
C. pinicola
C. ritchiei
24 (95.4) 11 (97.9) 13 (97.4) 6 (98.9) 122 (79.9) e 10 (98.1)
27 (95.0) 11 (98.0) 18 (96.5) 12 (98.0) 125 (79.5) 10 (98.1) e
Each value indicates that the number of nucleotide substitutions (nt) between the indicated species pairs. Values in parentheses indicate the sequence similarity (%) between pairs of species.
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Fig. 2 e Phylogenetic reconstruction of Cystobasidium tubakii and C. ongulense using 18S rDNA, ITS region, LSU D1/D2 domain, and the TEF1 gene. Maximum likelihood analysis of the 18S rDNA, ITS, LSU D1/D2 domain and TEF1 sequences of all strains of C. tubakii, C. ongulense and their closely related species. Strains of C. tubakii and C. ongulense investigated in this study are highlighted in bold font. Sakaguchia dacryoidea CBS 6353 was designated as the outgroup. The tree backbone was constructed by maximum likelihood analysis using MEGA7. Bootstrap percentages of maximum likelihood and maximum parsimony analyses over 50% from 1000 bootstrap replicates and posterior probabilities of Bayesian inference above 0.9 are shown to left on the branches. The scale bar represents 0.02 substitutions per nucleotide position. ns, not supported (bootstrap percentages < 50% or Bayesian inference < 0.9).
Table 4 e Comparison of physiological characterizations among species of the genus Cytobasidium.
Cystobasidium benthicum C. calyptogenae C. fimetarium C. laryngis C. lysinophilum C. minutum C. oligophagum C. ongulense C. pallidum C. pinicola C. psychroaquaticum C. ritchiei C. slooffiae C. tubakii
Nitrate
D-glucuronate
Sucrose
Melezitose
Starch
Cellobiose
Salicin
L-Sorbose
DL-Lactate
¡ ¡
þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ v þ w þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ
þ w w þ w w
w þ þ þ w þ ¡ w v þ þ ¡
þ þ þ s þ w þ þ v þ
w v þ ¡ þ v w s ¡
þ þ v þ ¡ d v w s w
Main physiology test results for the characteristics of C. ongulense, C. tubakii, and related species are shown. Physiological data of related species were taken from Sampaio (2011), Sampaio and Oberwinkler (2011), Yurkov et al. (2015) and this study. þ, positive; w, weak; s, slow; , negative; v, variable; n, no data. Cystobasidium tubakii and C. ongulense investigated in this study are highlighted in bold font.
(Table 5). Moreover, we investigated the extracellular enzyme secretion by C. tubakii and C. ongulense using API zym (Biomerieux, Tokyo, Japan) and found that both species also have esterase, lipase, and b-glucosidase (Supplementary Table S1). After incubation for 10 d on YM agar at 17e20 C, yeast cells of C. tubakii were ovoid to elongated (3e4 mm 4e5 mm) and divided by polar budding (Fig. 3A). The cell morphology of C. ongulense also showed ovoid to elongated and the cell size was 3e3.5 mm 4e6 mm, which proliferation by polar budding after 10 d on YM agar at 17e20 C (Fig. 3B). Furthermore, neither species exhibited sexual activity, or produced ballistoconidia and pseudohyphae. Additionally, the results of a BLAST homology search, indicated that Cystobasidium sp. CBS 9086 and Basidiomycota sp. 3 HFP-2013 isolate A25M013 exhibited 100% matching over the LSU D1/D2 domain sequence with that of C. tubakii. CBS
9086 was isolated from Antarctic water and A25M013 was collected from King George Island, Antarctica (Scorzetti et al. 2002). These results suggest that C. tubakii may be distributed over a wide area in Antarctica. Conversely, C. ongulense exhibited the same LSU D1/D2 domain sequence as that of Cystobasidium sp. CCFEE 5633, Cystobasidium CCFEE 5628, Cystobasidium sp. CBS 8923, C. laryngis TP-Snow-Y131 and C. cf. laryngis DBVPG 10053. CCFEE 5628 and 5633 were collected from Mount Gran Sasso (Italy), CBS 8923 was isolated from Antarctica, TP-Snow-Y131 was obtained from snow in Tibet (China), and DBVPG 10053 was collected from the Italian alps (Scorzetti et al. 2002; Selbmann et al. 2014). Consequently, C. ongulense was considered to inhabit various cold region. All strains investigated in this study exhibited good growth in the vitamin-free medium, whereas other known type strains of Cystobasidium spp. could not grow in the medium.
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Table 5 e Comparison of additional growth tests and other characterizations among species of the genus Cytobasidium. Growth on vitaminfree
Optimal growth Growth at Growth at Growth at Growth at Fermentation 25 C 30 C 37 C ability temperature 3 C
Cell size (mm)
Cystobasidium benthicum C. calyptogenae C. fimetarium C. laryngis
n n n n
n n n n
þ þ þ þ
þ þ þ v
þ þ
2e3.5 3e5 2e3.5 3e6 2e3 5e7 2.5e4 3e8
C. lysinophilum C. minutum C. oligophagum C. ongulense C. pallidum C. pinicola C. psychroaquaticum
þ
n n 28e30 C 20 C n n n
n n n þ n n n
þ þ þ þ þ þ þ
þ þ þ w þ þ
þ ¡
¡
2e3 4e5 1.5e3 4e6 2e4 3e5 3e3.5 £ 4e6 2e4 4e6.5 2e3.5 2.5e7.5 4e5 6e9
C. ritchiei C. slooffiae C. tubakii
þ
n n 15e17 C
n n þ
þ þ þ
þ ¡
¡
¡
2.5e4 5e7 2e3.5 3e5 3e4 £ 4e5
Main characteristics of C. ongulense, C. tubakii, and related species are shown. Data in bold were collected in this study. Other physiological data were taken from Sampaio (2011), Sampaio and Oberwinkler (2011), Yurkov et al. (2015), and this study. þ, positive; w, weak; s, slow; , negative; v, variable; n, no data. Cystobasidium tubakii and C. ongulense investigated in this study are highlighted in bold font.
Fig. 3 e Morphology of Cystobasidium tubakii and C. ongulense. Cystobasidium tubakii (A) and C. ongulense (B) grown on YM agar for 10 d at 17e20 C. Bars 10 mm.
Cystobasidium tubakii and C. ongulense were found to grow under subzero temperature conditions. Based on the average total carbon and nitrogen concentrations in the soil samples, East Ongul Island constituted an oligotrophic environment. Thus, in comparison to related species, members of C. tubakii and C. ongulense were considered to have obtained these growth abilities to survive in an oligotrophic environments such as that of East Ongul Island. This is the first report of new fungal species isolated from near the Syowa station in the 60-y history of JARE.
3.1.
Taxonomy
Cystobasidium tubakii M. Tsuji, Tsujimoto & S. Imura, sp. nov. Fig. 3A. Mycobank no.: MB817702. Etymology: “tubakii” refers to the late mycologist Keisuke Tubaki, who was the first to report fungi from near the Syowa station, East Antarctica, to pay honor to his pioneering work.
Type: ANTARCTICA, East Ongul Island, 22 Dec 2007, isolated from soil, East Ongul Island, East Antarctica by M. Tsujimoto & S. Imura (holotype: strain JCM 31526T preserved in a metabolically inactive state at Japan Collection of Microorganisms, Riken, Japan; ex-type culture: HUT7413T and NBRC 112503T; paratype: JCM 31529, HUT7416, NBRC 112504); ITS and LSU D1/D2 domain: LC155913 (JCM 31526T), LC155914 (JCM 31529), SSU rDNA: LC158350 (JCM 31526T), TEF1: LC158352 (JCM 31526T), CYTB: LC158354 (JCM 31526T). Streak culture after 1 wk on 5% malt extract agar at 15e17 C: shiny, mucilaginous, smooth, entire margin. Yeast cells after 10 d on YM agar ovoid to elongated, 3e4 mm 4e5 mm, proliferating by polar budding. Sexual activity was not observed. Ballistoconidia and pseudohyphae not produced. Sugars not fermented. Assimilation of carbon compounds: D-glucose, sucrose, galactose (weak), trehalose (weak), melezitose, starch (weak), cellobiose (weak), salicin, D-xylose, ethanol, glycerol, ribitol, DL-lactate (weak), succinic acid, D-gluconate, Dglucuronate, N-acetyl-D-glucosamine, D-xylitol. Inulin, raffinose, melibiose, lactose, maltose, methyl-a-D-glucoside, L-
m y c o s c i e n c e 5 8 ( 2 0 1 7 ) 1 0 3 e1 1 0
sorbose, L-rhamnose, L-arabinose, D-arabinose, D-ribose, methanol, erythritol, galactitol, D-mannitol, D-glucitol, myoinositol, D-glucosamine, citric acid, potassium nitrate, and sodium nitrate are not assimilated. Growth on 5% glucose medium with 10% NaCl (w/v), 50% (w/v) glucose medium and in vitamin-free medium. Growth absent on 0.01% cyclohexamide. Maximum growth temperature at 25 C and optimal growth at 15e17 C. Growth on PDA at 3 C. Habitat: soil of East Ongul Island, East Antarctica in the vicinity of Syowa station, JARE. Cystobasidium ongulense M. Tsuji, Tsujimoto & S. Imura, sp. nov. Fig. 3B. Mycobank no.: MB817703. Etymology: “ongulense” refers to the geographic origin of this species. Type: ANTARCTICA, East Ongul Island, 22 Dec 2007, isolated from soil, East Ongul Island, East Antarctica by M. Tsujimoto & S. Imura (holotype: strain JCM 31527T preserved in a metabolically inactive state at Japan Collection of Microorganisms, Riken, Japan; ex-type culture: HUT7414T and NBRC 112505T; paratype: JCM 31528, HUT7415, NBRC 112506); ITS and LSU D1/D2: LC155915 (JCM 31527T), LC155916 (JCM 31528), SSU rDNA: LC158351 (JCM 31527T), TEF1: LC158353 (JCM 31527T), CYTB: LC158355 (JCM 31527T). Streak culture after 1 wk on YM agar at 17e20 C: shiny, mucilaginous, smooth, entire margin. Yeast cells after 10 d on YM agar ovoid to elongated, 3e3.5 mm 4e6 mm, proliferating by polar budding. Sexual activity was not observed. Ballistoconidia and pseudohyphae not produced. Sugars not fermented. Assimilation of carbon compounds: D-glucose, sucrose, trehalose, melezitose, starch (weak), salicin, D-xylose, ethanol, glycerol, ribitol, succinic acid (weak), D-gluconate, D-glucuronate, D-xylitol, L-arabinose, D-arabinose, Dmannitol, D-glucitol. No growth on inulin, raffinose, melibiose, galactose, lactose, maltose, methyl-a-D-glucoside, cellobiose, Lsorbose, L-rhamnose, D-ribose, methanol, erythritol, galactitol, DL-lactate, N-acetyl-D-glucosamine, myo-inositol, D-glucosamine, citric acid, potassium nitrate, or sodium nitrate. Growth on 5% glucose medium with 10% NaCl (w/v), 50% (w/v) glucose medium and in vitamin-free medium. Growth absent on 0.01% cyclohexamide. Maximum growth temperature at 30 C and optimal growth at 20 C. Growth on PDA at 3 C. Habitat: soil of East Ongul Island, East Antarctica in the vicinity of Syowa station, JARE.
Disclosure All necessary permits were obtained for the described field studies. Permission to undertake field studies and collect samples was granted by the Ministry of the Environment of Japan.
Acknowledgments This work was carried out as part of the Science Program of the 49th JARE. It was supported by NIPR under MEXT, Japan
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and SOKENDAI. This work was supported by a JSPS Grant-inAid for Challenging Exploratory Research (No. 16K12643), Young Scientists (A) (No. 16H06211) to M. Tsuji and Scientific Research (B) (No. 18310024) to S. Imura. The preparation of this paper was supported by an NIPR publication subsidy. We are grateful to Kenichi Watanabe for his technical support.
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.myc.2016.11.002.
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