Three novel lipomycetaceous yeasts, Lipomyces maratuensis sp. nov., Lipomyces tropicalis sp. nov., and Lipomyces kalimantanensis f.a., sp. nov. isolated from soil from the Maratua and Kalimantan Islands, Indonesia

Three novel lipomycetaceous yeasts, Lipomyces maratuensis sp. nov., Lipomyces tropicalis sp. nov., and Lipomyces kalimantanensis f.a., sp. nov. isolated from soil from the Maratua and Kalimantan Islands, Indonesia

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Three novel lipomycetaceous yeasts, Lipomyces maratuensis sp. nov., Lipomyces tropicalis sp. nov., and Lipomyces kalimantanensis f.a., sp. nov. isolated from soil from the Maratua and Kalimantan Islands, Indonesia Atsushi Yamazaki a,*, Atit Kanti b, Hiroko Kawasaki a a

NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE), 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan b Indonesian Culture Collection (InaCC), Research Center for Biology (RCB), Indonesian Institute of Science (LIPI), Jl. Raya Jakarta-Bogor Km. 46, Cibinong Science Center, Cibinong, West Java 16911, Indonesia

article info

abstract

Article history:

Three novel oleaginous yeasts were isolated from soil collected around Kalimantan Island,

Received 27 February 2017

Indonesia. Based on their morphological and biochemical characteristics and sequence

Received in revised form

typing using the D1/D2 domain of the large subunit (LSU) rRNA and translation elongation

13 June 2017

factor 1 alpha gene (EF-1a), the eight strains were shown to be novel species and were

Accepted 14 June 2017

named Lipomyces maratuensis sp. nov. (type strain: NBRC 110264T ¼ InaCC Y720T ¼ JSAT12-

Available online 5 October 2017

2-Y011T; MycoBank no. MB 816185), Lipomyces tropicalis sp. nov. (type strain: NBRC 110265T ¼ InaCC Y730T ¼ JSAT12-2-Y012T; MycoBank no. MB 816186), and Lipomyces kali-

Keywords:

mantanensis f.a., sp. nov. (type strain: NBRC 110267T ¼ InaCC Y721T ¼ JSAT12-2-Y029T;

Lipid production

MycoBank no. MB 816187). Strain NBRC 110264 exhibited high lipid production.

Oleaginous yeast

1.

© 2017 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

Introduction

Lipomyces Lodder & Kreger-van Rij was first reported as a lipidproducing ascosporic yeasts isolated from soil (Starkey 1946; Lodder and Kreger-van Rij 1952). The main habitat of these yeasts was thought to be soil; however, Lipomyces oligophaga (Van der Walt & Arx) Kurtzman et al. and Lipomyces smithiae (Van der Walt et al.) Kurtzman et al. were isolated from insect

frass. The taxonomical phenotypes of Lipomyces are the ability to produce starch-like polysaccharides and the inability to ferment D-glucose, assimilate nitrate, and form pseudohyphae (Smith and Kurtzman 2011). Many yeasts of the genus Lipomyces can accumulate lipids, and yeasts displaying this characteristic are known as oleaginous yeasts. Such oleaginous yeasts are valuable tools as they can be used for the industrial production of biofuels (Starkey 1946; Naganuma et al. 1985, 1986; Zhao et al. 2008; Meng et al. 2009; Oguri et al. 2012).

* Corresponding author. Fax: þ81 438 52 2329. E-mail address: [email protected] (A. Yamazaki). http://dx.doi.org/10.1016/j.myc.2017.06.002 1340-3540/© 2017 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

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In addition, the genome of Lipomyces starkeyi Lodder & Kregervan Rij NRRL Y-11557 has been analyzed and published (http:// genome.jgi-psf.org/Lipst1_1/Lipst1_1.home.html) by the Joint Genome Institute (JGI), and genetic engineering techniques have been applied to this strain (Calvey et al. 2014; Oguro et al. 2014). Therefore, novel Lipomyces species isolated from natural habitats with capacity for high lipid production are of increasing interest because of the possibility that the genetic techniques applied to L. starkeyi can also be used in these species to enhance their lipid production and thus their potential industrial utility. Currently, this genus is composed of 17 known species. Typical taxonomic criteria for yeasts, such as the gene sequence of the D1/D2 domain of the large subunit (LSU) rRNA, ascospore morphology, and assimilation profiles, cannot clearly differentiate among species of the genus Lipomyces. However, Kurtzman et al. (2007) conducted reclassification of Lipomycetaceae family yeasts using four gene sequences namely small subunit (SSU) rRNA, LSU rRNA, translation elongation factor 1 alpha (EF-1a) and mitochondrial SSU rRNA. And the work indicated that single gene sequence of EF-1a showed high resolution of Lipomyces species. Previous work from our laboratory showed that the sequence of EF-1a can be effectively used for species identification and taxonomic studies of the genus Lipomyces although LSU rRNA and the Internal Transcribed Spacer (ITS) region including 5.8S rRNA gene sequences were not because only 1e2 base pairs differences could be detected in ITS and LSU rRNA sequences of several species (Yamazaki and Kawasaki 2014). The genus Myxozyma Van der Walt et al. which was initially described as an anamorphic state of the genus Lipomyces, is composed of 12 species. The taxonomic phenotype of Myxozyma is the formation of extracellular starch-like compounds and growth on imidazole as the sole nitrogen source. Species of Lipomyces also utilize imidazole as a sole source of nitrogen, which means that growth on this compound cannot distinguish Lipomyces from Myxozyma. Although species of Myxozyma are mainly isolated from soil, species show a greater diversity in habitats than the genus Lipomyces, including tree-inhabiting lichen (M. lipomycoides Van der Walt et al.; van der Walt et al. 1987), sap flux on oak tree and soil (M. monticola Pretorius & Spaaij; Pretorius et al. 1993), cladode of prickly pear cactus (M. neglecta Spaaij et al.; Spaaij et al. 1998), necrotic tissue of cactus (M. mucilagina (Phaff et al.) Van der Walt et al.; van der Walt et al. 1981), beetle (M. melibiosi (Shifrine & Phaff) Van der Walt et al.; van der Walt et al. 1981), beetle frass (M. nipponensis Spaaij & G. Weber; Spaaij et al. 1993), and wood wasp frass (M. siricis (as M. sirexii) Spaaij et al.; Spaaij et al. 1992). The International Code of Nomenclature for algae, fungi, and plants (Melbourne Code) decided to discontinue the principal of dual scientific names (McNeill et al. 2011). Thus, the novel species belonging to the family Lipomycetaceae should be described with one single genus name having priority, although teleomorphic characters are not detected. In this study, we sought to examine the biodiversity of novel oleaginous yeasts and their lipid production, and we isolated three yeast species belonging to the family Lipomycetaceae from soil collected on the Kalimantan and Maratua Islands in Indonesia. Based on the taxonomic analysis and the Melbourne Cord, we proposed three novel Lipomyces species. We also

determined the fatty acid (FA) composition of these species and compared it to that of known species. One of the new isolates produced FA levels similar to that of the high lipid-producing strain L. starkeyi NBRC 10381, demonstrating the potential of this new strain for industrial use and the value of novel Lipomycetaceae environmental isolates.

2.

Materials and methods

2.1.

Strain isolation

Soil samples for isolating yeasts was collected on Maratua Island (Berau District, East Kalimantan, Indonesia, Latitude: N2 12ʹ21, Longitude: E118 35ʹ27 and Latitude: N2 12ʹ8.3, Longitude: E118 35ʹ33.1) on 5 Jun 2012 and near the Wain river, Balikpapan, East Kalimantan (Latitude: S1 8ʹ44, Longitude: E116 50ʹ11) on 7 Jun 2012. Those samples were taken in a depth of 5e10 cm from moist soil. One gram of the soil was suspended in 10 mL of saline (0.85% NaCl). A 100-mL aliquot of the suspension was spread on nitrogen-depleted agar medium (20 g/L glucose, 20 g/L agar, 0.85 g/L KH2PO4, 0.15 g/L K2HPO4, 0.5 g/L MgSO4$7H2O, 0.1 g/L NaCl, 0.1 g/L CaCl2$6H2O, 0.5 mg/L H3BO3, 0.04 mg/L CuSO4$H2O, 0.1 mg/L KI, 0.2 mg/L FeCl3$6H2O, 0.4 mg/L MnSO4$H2O, 0.2 mg/L Na2MoO4$2H2O, 0.4 mg/L ZnSO4$7H2O) (Thanh 2006), and the plates were incubated at 25  C for 3 wk. Yeast colonies were picked and streak purified on YM agar medium (10 g/L glucose, 15 g/L agar, 5 g/L peptone, 3 g/L yeast extract, 3 g/L malt extract, pH 5.6). Eight yeast isolates were selected and analyzed in this study.

2.2.

Cell staining

Yeasts were grown on 5% malt extract agar to assess lipid accumulation by Nile red (MP Biomedicals, Solon, OH, USA) staining. Cells were suspended in 1 mL of saline containing 3.3 mg of Nile red. Images were acquired using an Axioplan 2 microscope (Carl Zeiss, Oberkochen, Germany) with laser excitation at 530e560 nm and emission at 572.5e647.5 nm.

2.3.

Molecular phylogeny

2.3.1.

DNA isolation for phylogenetic studies

Yeast cells cultivated on YM agar medium for 3e4 d at 25  C were harvested using a loop, and DNA was isolated and purified using a Maxwell 16™ system (Promega, Madison, WI, USA) or a PrepMan Ultra (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.

2.3.2.

Sequencing of LSU rRNA and EF-1a

The 50 end of the LSU rRNA, including the D1/D2 domains, and translation elongation factor 1a gene (EF-1a) was amplified by polymerase chain reaction (PCR) using the EX Taq kit (Takara Bio Inc., Kusatsu, Shiga, Japan) and a Mastercycler ep Gradient S thermal cycler (Eppendorf, Hamburg, Germany). The standard primer pairs used for amplification and sequencing were: NL1 (50 -GCATATCAATAAGCGGAGGAAAAG-30 ) and NL4 (50 GGTCCGTGTTTCAAGACGG-30 ) for LSU rRNA (O'Donnell 1993), and EF1-983F (50 -GCYCCYGGHCAYCGTGAYTTYAT-30 )

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and EF1-2218R (50 -ATGACACCRACRGCRACRGTYTG-30 ) for EF1a (Kurtzman et al. 2007). PCR products were purified using the Agencourt AMPure purification system (Beckman Coulter Inc., Brea, CA, USA), and the sequencing reaction was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). DNA fragments from the sequencing reactions were purified with the Agencourt CleanSEQ system (Beckman Coulter), and the sequences were obtained either using an ABI PRISM 3130 or 3730xl Genetic Analyzer (Applied Biosystems) according to the manufacturer's instructions. The obtained nucleotide sequences were deposited into DDBJ/EMBL/GenBank, and the accession numbers are shown in Figs. 1, 2.

2.3.3.

Phylogenetic analysis

Molecular phylogenetic analysis based on the LSU rRNA and EF1a sequences was performed by three methods, the neighborjoining (NJ; Saitou and Nei 1987), maximum likelihood (ML; Felsenstein 1981) and maximum parsimony (MP; Fitch 1971). Initially, eight strains, and 33 and 34 type or authentic strains of species belonging to the family Lipomycetaceae were analyzed using the LSU rRNA D1/D2 domain sequences (Fig. 1) and the EF1a sequences (Fig. 2) respectively to establish their identity. MEGA 5.0 software (Tamura et al. 2011) was used to determine evolutionary distances (the Knuc value; Kimura 1980), assess similarities, and construct phylogenetic trees by the NJ method. The Knuc values and bootstrap resampling method (Felsenstein 1985) involving 1000 replicates were applied to evaluate the topology of the phylogenetic trees.

2.4.

Physiological and morphological tests

The physiological properties and morphological characteristics were examined by using methods currently used for yeast taxonomy (Kurtzman et al. 2011).

2.5.

DNA isolation

DNA was isolated according to the method of Holm et al. (1986). Yeast cells grown in 200 mL of YM broth were collected, washed twice with EDTA buffer (50 mM, pH 8.0), and treated with Zymolyase-100T (Seikagaku Co., Tokyo, Japan) and Proteinase K (Wako Pure Chemical Industries Ltd., Osaka, Japan) to disrupt the cells and extract the DNA. After RNase treatment (RNase A and T1; SigmaeAldrich, St. Louis, MO, USA), DNA was extracted with phenol-chloroform. The isolated DNA was purified by CsCl gradient ultracentrifugation according to the method of Yamazaki and Kawasaki (2014).

2.6.

Scanning electron microscope (SEM) observation

Samples for scanning electron microscopy (SEM) were washed twice with phosphate buffer (pH 7.4, 2.13 g/L sodium phosphate, 11.50 g/L disodium phosphate) by centrifugation. For ascospore observation, samples were incubated in 500 mL of 1 mg/mL Zymolyase 100T (Seikagaku Co.) for 1 h. Samples were fixed with 10% glutaraldehyde for 30 min and with 1% osmium tetroxide for 1 h. Following complete dehydration in a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and 100%) and substitution with isoamyl acetate (IAA) in a graded IAAeethanol series (33%, 50%, 66%, and 100%), the materials

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were critical point-dried in a Critical Point Dryer EM CPD300 (Leica microsystems, Wetzlar, Germany), coated with platinum and palladium with a Magnetron Sputter JUC-5000 (JEOL Ltd., Tokyo, Japan), and observed under a scanning electron microscope JSM-6060 (JEOL) operated at 15 kV.

2.7.

Lipid isolation and analysis

The lipids were isolated from the cells according to the method of Bligh and Dyer (1959). Yeast cells grown in 5 mL of YM (10 g/L glucose, 5 g/L peptone, 3 g/L yeast extract, 3 g/L malt extract) or 5G5M (50 g/L glucose, 50 g/L malt extract) medium at 28  C with shaking at 280 rpm for 10 d were collected by centrifugation. The cells were suspended in 1 mL of water containing 0.05% (w/v) 2,6-di-tert-butyl-p-cresol (Tokyo Chemical Industry Co. Ltd., Tokyo, Japan), which was added as antioxidant. The cell solution was transferred to new tubes containing zirconia beads with diameters of 0.5 mm and 5 mm (ZB-05 and ZB-50, respectively; TOMY, Tokyo, Japan), and the cells were disrupted by shaking at 4000 rpm twice for 5 min each with a cell disrupter Micro Smath™ MS-100R (TOMY). The disrupted cells in solution were transferred to a screw-capped glass test tube (16.5  105 mm). Next, 2.5 mL of chloroform and 1.25 mL each of methanol, chloroform, and water were added, and the tube was vortexed after adding each reagent. Then, the tube was centrifuged at 1500  g for 10 min, and the lower layer was transferred to a new glass test tube. Chloroform (1 mL) was added to the tube containing the lower layer for re-extraction. The tube was vortexed, centrifuged, and the lower layer was transferred to a new glass test tube. The solution transferred to the glass test tube was dried for use in the methanolysis/methylation reaction. The lipids were subjected to mild methanolysis/methylation using concentrated HCl according to the method reported by Ichihara and Fukubayashi (2010) to produce fatty acid methyl ester (FAME). A lipid sample was dissolved in 0.20 mL of toluene. Then, 1.50 mL of methanol and 0.30 mL of 8% HCl solution (9.7 mL of 35% HCl, 41.5 mL of methanol) were added. The sample was vortexed and incubated at 45  C overnight (14 h or longer) for mild methanolysis/methylation. After cooling to room temperature, 1 mL of hexane and 1 mL of water were added to the tube and vortexed. The hexane layer was transferred to a glass vial, and methyl heptadecanoate (SigmaeAldrich) was added as an internal standard. The samples were analyzed by gas chromatography (GC) by using a 6890N/G1530N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a column of Ultra 2 5% Phenyl Methyl Siloxane (0.20 mm  25 m) at a column temperature of 170e310  C. A commercial mixture of FAMEs, F.A.M.E Mix C8-C24 (SUPELCO, Bellefonte, PA, USA), was also analyzed to generate a calibration curve for quantitative calculation of the FAMEs in the lipid sample.

3.

Results and discussion

3.1.

Isolation of novel Lipomycetaceae species

Strains in this study were isolated from soil collected on Maratua Island, East of Kalimantan and around the Wain river in Balikpapan on Kalimantan Island.

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Strains NBRC 110264 (¼ InaCC Y723 ¼ JSAT12-2-Y011), NBRC 110261 (¼ InaCC Y723 ¼ JSAT12-2-Y003), NBRC 110262 (¼ InaCC Y724 ¼ JSAT12-2-Y004), NBRC 110263 (¼ InaCC Y725 ¼ JSAT12-2-Y005), NBRC 110265 (¼ InaCC Y730 ¼ JSAT122-Y012) and NBRC 110266 (¼ InaCC Y731 ¼ JSAT12-2-Y013) showed typical Lipomyces characteristics, including the ability to produce starch-like polysaccharides, 4e30 spherical ascospores per ascus (Figs. 3, 4). Strains NBRC 110267 (¼ InaCC Y721 ¼ JSAT12-2-Y029) and NBRC 110268 (¼ InaCC Y722 ¼ JSAT12-2-Y038) showed typical Myxozyma characteristics, including the formation of extracellular starch-like compounds. Ascospore formation was not observed on corn meal agar, 5% malt extract agar, V8 agar, potassium acetate agar, sodium acetate agar, or AF agar, which are used for inducing sporulation in Lipomycetaceae (Kurtzman et al. 2011) for up to 3 mo in 25  C.

3.2. Phylogenetic analysis of LSU rRNA and EF-1a, and comparison of physiological characteristics In the LSU rRNA tree, the eight isolates formed three clusters among the Lipomycetaceae species. Strains NBRC 110264 clustered separately from strains NBRC 110261, NBRC 110262, NBRC 110263, NBRC 110265 and NBRC 110266 in the region of the Lipomyces kononenkoae group, which consists of related four species such as L. kononenkoae Nieuwdorp et al., L. spencermartinsiae (Van der Walt & M.T. Sm.) Van der Walt & M.T. Sm., L. yarrowii M.T. Sm. & Van der Walt and L. yamadae Van der Walt & M.T. Sm., and L. starkeyi group, which consists of such as L. starkeyi, L. kockii M.T. Sm. & Van der Walt, L. mesembrius Botha et al. and L. doorenjongii Van der Walt & M.T. Sm. (Fig. 1). These two clusters are related to each other, but differ by 6e7 bp and two gaps in a stretch of 569 bp (1.41e1.58% of the sequence). Strain NBRC 110264 showed 6 bp differences in 567 bp (1.05% of the sequence) compared to L. starkeyi. The cluster comprising strains NBRC 110261, NBRC 110262, NBRC 110263, NBRC 110265 and NBRC 110266 was also related to L. starkeyi, with 3e4 bp differences and 2 gaps in 569 bp (0.88e1.05% of the sequence). Strains NBRC 110267 and NBRC 110268 have the exact same sequence across 568 bp, and these strains clustered near Myxozyma lipomycoides Van der Walt et al. These two strains showed a 5 bp difference and three gaps in a 568 bp stretch (1.41% of the sequence) when compared with M. lipomycoides. In the EF-1a tree, strains NBRC 110264, and NBRC 110261, NBRC 110262, NBRC 110263, NBRC 110265 and NBRC 110266 clustered separately (Fig. 2), and 83e85 bp differences in the 990 bp sequence (8.38e8.59% of the sequence) were detected among these strains. Strain NBRC 110264 is related to Lipomyces spencermartinsiae, but its sequence has a number of differences, 46 bp differences in the 957 bp sequence (4.81% of the sequence). In addition, tree of concatenated sequence of D1/D2 domain of LSU rRNA and EF-1a was shown in Supplementary Fig. S1. The tree showed that the strains made three isolated clade same as to the LSU rRNA and EF-1a trees. Strain NBRC 110264 has the ability to assimilate D-xylose (weak), galactitol (dulcitol), and succinate (weak or delayed), whereas L. spencermartinsiae does not. Conversely, L. spencermartinsiae has the ability to assimilate glycerol and grow at 37  C, whereas strain NBRC 110264 does not. Five strains,

NBRC 110261, NBRC 110262, NBRC 110263, NBRC 110265 and NBRC 110266, formed a separate clade that was most closely related to Lipomyces chichibuensis A. Yamaz. & H. Kawas., 31e33 bp differences and 1 gap in the 954 sequence (3.35e3.56% of the sequence) and Lipomyces kockii, 39e41 bp difference in the 962 sequence (4.05e4.26% of the sequence). Lipomyces chichibuensis has the ability to assimilate raffinose, D-ribose (weak), D-glucitol (D-sorbitol; may be delayed), methyl a-D-glucoside (may be delayed), and D-gluconate (weak), whereas the five isolated strains do not. In addition, L. kockii has the ability to assimilate galactose, maltose, melibiose (delayed), raffinose, galactitol, and methyl a-D-glucoside, whereas the five isolated strains do not. In contrast, the five strains assimilate trehalose and 2-keto-D-gluconate, whereas L. kockii does not. The clade comprising strains NBRC 110267 and NBRC 110268 was related to M. lipomycoides, 31 bp difference in the 873 sequence (3.55% of the sequence). Strains NBRC 110267 and NBRC 110268 have the ability to assimilate sucrose (weak), maltose, melezitose (weak), galactitol (dulcitol), methyl a-D-glucoside (may be delayed), salicin (may be delayed), inositol, D-gluconate, and 5-keto-D-gluconate, whereas M. lipomycoides does not. In contrast, M. lipomycoides has the ability to assimilate succinate (slow positive), whereas strains NBRC 110267 and NBRC 110268 do not. The genus Dipodascopsis L.R. Batra & Millner, which is composed of three species, was shown to be located on two separate branches in the phylogenetic trees shown in Figs. 1, 2, despite the shared morphological characteristics such as forming true hyphae and acicular asci (Kurtzman et al. 2007). Although the strain Dipodascopsis tothii (Zsolt) L.R. Batra & Millner NRRL Y-12690T and D. anomala (Babeva & Gorin) Kurtzman et al. NRRL Y-7931T formed a clade in the LSU rRNA tree, these two species were separately located in the EF-1a tree. In addition, the ascospores produced by the two groups such as the strains NBRC 110264, and NBRC 110261, NBRC 110262, NBRC 110263, NBRC 110265 and NBRC 110266 have pleat-like protuberances on their surfaces (Figs. 3E, F, 4E, F). However, the surface of the pleats differs between the two groups. The group including strain NBRC 110264 has a smooth surface of pleats, whereas the other group (five strains) displayed pleats with an angulated surface. The phylogenetic analysis based on the LSU rRNA and EF1a sequences of Lipomycetaceae species and the comparison of physiological properties suggested that there are three novel Lipomycetaceae species among our isolates. We propose three novel species of the genus Lipomyces, Lipomyces maratuensis, Lipomyces tropicalis and Lipomyces kalimantanensis Because L. kalimantanensis was not observed to have a teleomorphic state, it is designated as forma asexualis (f.a.), according to the recommendation of Lachance (2012).

3.3.

Taxonomy

Lipomyces maratuensis Atit Kanti, Yamazaki & Kawasaki, sp. nov. Fig. 3. MycoBank no.: MB 816185. Type: INDONESIA, Maratua Island (Latitude: 2 120 21N, Longitude: 118 350 27E), soil, isolated by A. Yamazaki on Jun 25,

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LSU rRNA D1/D2 MP/ML/NJ

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T -/35/28 Lipomyces starkeyi NBRC 10381 (AB747660) T -/-/29 Lipomyces kockii NBRC 107656 (AB747652)

Lipomyces mesembrius NBRC 107654T (AB747656)

79/67/68 Lipomyces doorenjongii NBRC 107655T (AB747650)

Lipomyces tropicalis NBRC 110265T (= InaCC Y730T = JSAT12-2-Y012T)(LC061900) Lipomyces tropicalis NBRC 110266 (= InaCC Y731 = JSAT12-2-Y013)(LC061901) -/-/25 Lipomyces tropicalis NBRC 110263 (= InaCC Y725 = JSAT12-2-Y005)(LC061898) 48/39/42 Lipomyces tropicalis NBRC 110262(= InaCC Y724 = JSAT12-2-Y004)(LC061897) 26/22/18 Lipomyces tropicalis NBRC 110261 (= InaCC Y723 = JSAT12-2-Y003)(LC061896) Lipomyces maratuensis NBRC 110264T (= InaCC Y720T = JSAT12-2-Y011T)(LC061899) Lipomyces chichibuensis NBRC 109582T (AB828721) 14/-/30 -/-/10 Lipomyces yamadae NBRC 107657T (AB747662) 97/92/94 27/26/26 Lipomyces yarrowii NBRC 107658T (AB747663) 24/-/47 Lipomyces kononenkoae NBRC 107661T (AB747653) 57/41/46 Lipomyces spencermartinsiae NBRC 10376T (AB747659) -/-/46 Lipomyces tetrasporus NBRC 10391T (AB747661) Lipomyces orientalis NBRC 107659T (AB747657) -/-/20 Lipomyces arxii NRRL Y-17921T (DQ518997) Lipomyces lipofer NBRC 1288T (AB747655) 28/26/36 Dipodascopsis anomala NRRL Y-7931T (DQ518970) -/-/-4 100/100/100 Dipodascopsis tothii NRRL Y-12690T (DQ518971) Lipomyces japonicus NBRC 10767T (AB747651) Dipodascopsis uninucleata var. uninucleata NRRL Y-17583T (DQ518972) -/-/14 -/-18 Dipodascopsis uninucleata var. wickerhamii NRRL Y-2181T (DQ518973) -/-/36 Myxozyma kluyveri NRRL Y-17277T (DQ518986) 96/96/98 Myxozyma udenii NRRL Y-17387T (DQ518995) Myxozyma nipponensis NRRL Y-27625A (DQ518993) -/-37 Myxozyma lipomycoides NRRL Y-17253T (DQ518987) 82/85/88 94/92/99 Lipomyces kalimantanensis NBRC 110267T (= InaCC Y721T = JSAT12-2-Y029T)(LC061902) Lipomyces kalimantanensis NBRC 110268 (= InaCC Y722 = JSAT12-2-Y038)(LC061903) Lipomyces smithiae NRRL Y-17922T (DQ518999) -/-/99 Myxozyma monticola NRRL Y-17726T (DQ518989) 91/88/88 Lipomyces oligophaga NRRL Y-17247T (DQ518998) Myxozyma sirexii NRRL Y-27626A (DQ518994) 95/93/92 98/92/73 Lipomyces suomiensis NRRL Y-17356T (DQ519000) 97/94/98 90/82/66 Myxozyma geophila NRRL Y-17252T (DQ518985) Myxozyma melibiosi NRRL Y-11781T (DQ518988) Myxozyma mucilagina NRRL Y-11823T (DQ518990) 99/99/100 99/98/100 Myxozyma neglecta NRRL Y-27508T (DQ518991) 60/45/56 Myxozyma neotropica NRRL Y-17859A (DQ518992) 69/62/67 Myxozyma vanderwaltii NRRL Y-17727A (DQ518996) Saccharomyces cerevisiae NRRL Y-12632T (AY048154) Schizosaccharomyces pombe NRRL Y-12796T (DQ442711) 0.02 Knuc

Fig. 1 e Neighbor-joining analysis showing the extent of divergence in the LSU rRNA sequences among the eight isolates and 33 yeast strains belonging to the family Lipomycetaceae. Values of neighbor-joining bootstrap probabilities (NJ), maximum-likelihood bootstrap probabilities (ML), and maximum parsimony bootstrap probabilities (MP) are shown on each branch as “MP/ML/NJ” The values indicating the branch, which is not found in the ML and/or MP tree, are shown as “¡”.

2012 (holotype, strain NBRC 110264T preserved in glass ampoule as metabolically inactivated state by liquid-drying method at NITE Biological Resource Center (NBRC), Chiba Prefecture, Japan). Ex-type culture, strain InaCC Y720T preserved at Indonesian Culture Collection (InaCC), Cibinong, Indonesia. These ex-type cultures will be available without any restriction for research from the two culture collections. Gene sequences ex-holotype: LC061899 (D1/D2 LSU rRNA gene), LC061907 (EF-1a). Etymology: ma-ra-tu-en'sis N.L. masc. adj. maratuensis, referring to the site the species was first isolated from soil on Maratua Island, Indonesia.

After 3 d of culture on YMA at 25  C, cells are ovoid, ellipsoid, 3.6e6.8  4.1e7.6 mm in size, and occur singly, in pairs, or in short chains (Fig. 3A) with multilateral budding. The streak culture on YMA is mucoid, partly hyaline, partly creamishopaque, smooth, and glistening, with an entire margin. In Dalmau plate cultures on corn meal agar after 10 d at 25  C, neither hyphae nor pseudohyphae are formed. In YM broth after 1 mo at 25  C, sediment is not present. As a rule, asci are attached, saccate to irregularly tubular or contorted, with slowly deliquescent walls. Ascospores are globose, pigmented amber, smooth surface of pleats, 1.8e2.4 mm diam, with 4e30 per ascus (Fig. 3BeF).

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EF-1α MP/ML/NJ

Lipomyces doorenjongii NBRC 107655T (AB747664) Lipomyces starkeyi NBRC 10381T (AB747675) Lipomyces kockii NBRC 107656T (AB747666) 56/47/46 66/64/48 Lipomyces mesembrius NBRC 107654T (AB747670) Lipomyces chichibuensis NBRC 109582T (AB828725) 96/96/99 Lipomyces tropicalis NBRC 110266 (= InaCC Y731 = JSAT12-2-Y013)(LC061909) Lipomyces tropicalis NBRC 110265T (= InaCC Y730T = JSAT12-2-Y012T)(LC061908) 100/99/100 65/75/69 Lipomyces tropicalis NBRC 110261 (= InaCC Y723 = JSAT12-2-Y003)(LC061904) Lipomyces tropicalis NBRC 110262 (= InaCC Y724 = JSAT12-2-Y004)(LC061905) Lipomyces tropicalis NBRC 110263 (= InaCC Y725 = JSAT12-2-Y005)(LC061906) Lipomyces tetrasporus NBRC 10391T (AB747674) -/97/98 98/99/99 Lipomyces orientalis NBRC 107659T (AB747671) Lipomyces yarrowii NBRC 107658T (AB747677) -/-/99 Lipomyces yamadae NBRC 107657T (AB747676) Lipomyces kononenkoae NBRC 107661T (AB747667) -/-/16 -/-/70 Lipomyces maratuensis NBRC 110264T (= InaCC Y720T = JSAT12-2-Y011T)(LC061907) 75/78/89 52/43/51 Lipomyces spencermartinsiae NBRC 10376T (AB747673) Lipomyces lipofer NBRC 1288T (AB747669) Myxozyma monticola CBS 7806T (DQ496144) -/-/22 -/-/25 Lipomyces sp. NRRL Y-27488 (DQ496136) -/-/36 Lipomyces smithiae CBS 7407T (DQ496119) -/21/24 Dipodascopsis tothii CBS 759.85T (DQ496121) Myxozyma kluyveri CBS 7332T (DQ496124) 27/23/21 39/45/55 Myxozyma udenii CBS 7439T (DQ496125) 30/20/19 Myxozyma geophila CBS 7219T (DQ496142) 24/31/32 Myxozyma sirexii NRRL Y-27626A (DQ496141) 85/87/87 Myxozyma melibiosi CBS 2102T (DQ496143) 70/66/51 Lipomyces suomiensis CBS 7251T (DQ496120) 30/37/40 Myxozyma neotropica NRRL Y-17859A (DQ496137) -/-/59 Myxozyma vanderwaltii CBS 7793A (DQ496138) -/-/36 39/39/29 Myxozyma mucilagina CBS 7071T (DQ496139) 56/51/44 98/99/97 Myxozyma neglecta CBS 7058T (DQ496140) Myxozyma nipponensis NRRL Y-27625A (DQ496146) Myxozyma lipomycoides CBS 7038T (DQ496145) 36/52/55 58/50/48 100/99/100 Lipomyces kalimantanensis NBRC 110267T (= InaCC Y721T = JSAT12-2-Y029T)(LC061910) 99/99/100 Lipomyces kalimantanensis NBRC 110268 (= InaCC Y722 = JSAT12-2-Y038)(LC061911) Lipomyces japonicus NBRC 10767T (AB747665) 70/73/87 34/25/28 Lipomyces arxii CBS 7333T (DQ496117) Lipomyces oligophaga CBS 7107T (DQ496118) 97/90/87 Dipodascopsis anomala CBS 6740T (DQ496147) Dipodascopsis uninucleata var. uninucleata NRRL Y-17583T (DQ496122) 100/100/100 Dipodascopsis uninucleata var. wickerhamii CBS 741.74T (DQ496123) Saccharomyces cerevisiae NRRL Y-12632T (AF402004) Schizosaccharomyces pombe NRRL Y-12796T (AF402093) -/-/23

-/-/72

0.05 Knuc

Fig. 2 e Neighbor-joining analysis showing the extent of divergence in the EF-1a sequences among the eight isolates and the 34 yeast strains belonging to the family Lipomycetaceae. Values of neighbor-joining bootstrap probabilities (NJ), maximum-likelihood bootstrap probabilities (ML), and maximum parsimony bootstrap probabilities (MP) are shown on each branch as “MP/ML/NJ” The values indicating the branch, which is not found in the ML and/or MP tree, are shown as “¡”.

Does not ferment D-glucose. Assimilates D-glucose, Dgalactose, L-sorbose (delayed), sucrose, maltose, cellobiose (weak), a,a-trehalose, methyl a-D-glucoside, melibiose (may be delayed), raffinose (may be weak), melezitose (may be delayed), inulin (delayed), starch (weak), D-xylose (weak), Dribose (weak or not at all), ethanol (weak), galactitol, xylitol (may be delayed), D-glucitol, D-mannitol (may be delayed), Dglucono-1,5-lactone (delayed or weak), 2-keto-D-gluconate, 5keto-D-gluconate, succinate (delayed or weak), and citrate (may be weak). Does not assimilate L-arabinose, D-arabinose, L-rhamnose, salicin, arbutin, lactose, erythritol, ribitol, glycerol, myo-inositol, D-galacturonate, DL-lactate, methanol,

propane-1,2-diol, butane-2,3-diol, D-gluconate, D-glucosamine, N-acetyl-D-glucosamine, hexadecane, D-glucuronate, or arabinitol. Assimilates ammonium sulfate, ethylamine hydrochloride, L-lysine, cadaverine dihydrochloride, and imidazole. Does not assimilate potassium nitrate or sodium nitrite. Produces starch-like substances. Urease reaction is weak positive. Does not grow in YNB medium (Difco) containing 10% NaCl and 5% glucose. Grows in vitamin-free medium. Does not grow at 35  C, but grows at 30  C. Growth in medium containing 0.1% cycloheximide is positive. The G þ C content of nuclear DNA is 47.9e48.2 mol%. The major ubiquinone is Q-9.

m y c o s c i e n c e 5 8 ( 2 0 1 7 ) 4 1 3 e4 2 3

419

Fig. 3 e Lipomyces maratuensis (NBRC 110264). A: Budding cells grown on YMA for 3 d at 25  C. BeD: Ascus and ascospores produced on V8 agar for 40 d at 25  C (LM). E, F: Ascospores (SEM). G, H: Lipid-accumulating cells of NBRC 110264 stained with Nile red after growth on 5% malt extract agar for 1 wk at 25  C (G: LM, H: FM). Bars: AeD, G, H 5 mm; E, F 1 mm, FM: fluorescence microscope, LM: light microscope, SEM: scanning electron microscope (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

420

m y c o s c i e n c e 5 8 ( 2 0 1 7 ) 4 1 3 e4 2 3

Fig. 4 e Lipomyces tropicalis. A: Budding cells of strain NBRC 110265 grown on YMA for 3 d at 25  C. BeD: Ascus and ascospores produced by NBRC 110264 grown on V8 agar for 40 d at 25  C (LM). E: Ascospores of strain NBRC 110262 (SEM). F: Ascospore of strain NBRC 110265 (SEM). G, H: Lipid-accumulating cells of strain NBRC 110265 stained with Nile red after growth on 5% malt extract agar for 1 wk at 25  C (G: LM, H: FM). Bars: AeD, G, H, 5 mm; E, F 1 mm, FM: fluorescence microscope,

m y c o s c i e n c e 5 8 ( 2 0 1 7 ) 4 1 3 e4 2 3

421

Lipomyces tropicalis Atit Kanti, Yamazaki & Kawasaki, sp. nov. Fig. 4. MycoBank no.: MB 816186. Type: INDONESIA, Maratua Island (Latitude: 2 120 21N, Longitude: 118 350 27E), soil, isolated by A. Yamazaki on Jun 25, 2012 (holotype, strain NBRC 110265T preserved in glass ampoule as metabolically inactivated state by liquid-drying method at NITE Biological Resource Center (NBRC), Chiba Prefecture, Japan). Ex-type culture, strain InaCC Y730T preserved at Indonesian Culture Collection (InaCC), Cibinong, Indonesia. These ex-type cultures will be available without any restriction for research from the two culture collections. Gene sequences ex-holotype: LC061900 (D1/D2 LSU rRNA gene), LC061908 (EF-1a). Etymology: trop-i-ca'lis L. masc. adj. tropicalis, referring to the site the species was first isolated, from soil in a tropical zone. After 3 d of culture on YMA at 25  C, cells are ovoid, ellipsoid, 4.3e7.5  5.1e8.5 mm in size, and occur singly, in pairs, or in short chains (Fig. 4A), with multilateral budding. The streak culture on YMA is mucoid, partly hyaline, partly creamishopaque, smooth and glistening, with an entire margin. In Dalmau plate cultures on corn meal agar after 10 d at 25  C, neither hyphae nor pseudohyphae are formed. In YM broth after 1 mo at 25  C, sediment is not present. As a rule, asci are attached, saccate to irregularly tubular or contorted, with slowly deliquescent walls. Ascospores are globose, pigmented amber, angulated surface of pleats, 2.1e2.4 mm diam, with 4e28 per ascus (Fig. 4BeF). Does not ferment D-glucose. Assimilates D-glucose, L-sorbose (delayed or not at all), sucrose, cellobiose (may be weak), a,a-trehalose, melezitose (variable), inulin (may be weak), starch (may be weak), D-xylose (may be weak), ethanol (variable), xylitol (variable), D-mannitol (variable), salicin (may be weak), D-glucono-1,5-lactone (may be weak), 2-keto-D-gluconate, 5-keto-D-gluconate, propane-1,2-diol (may be weak), succinate (variable), citrate (variable), and arbutin (may be weak). Does not assimilate D-galactose, maltose, L-arabinose, D-arabinose, D-ribose, L-rhamnose, melibiose, raffinose, methyl a-D-glucoside, lactose, erythritol, ribitol, D-glucitol, galactitol, glycerol, myo-inositol, D-galacturonate, DL-lactate, methanol, butane-2,3-diol, D-gluconate, D-glucosamine, Nacetyl-D-glucosamine, hexadecane, D-glucuronate, or arabinitol. Assimilates ammonium sulfate, ethylamine hydrochloride, L-lysine, cadaverine dihydrochloride, and imidazole. Does not assimilate potassium nitrate or sodium nitrite. Produces starch-like substances. Urease reaction is weak positive. Does not grow in YNB medium (Difco) containing 10% NaCl and 5% glucose. Grows in vitamin-free medium. Does not grow at 35  C, but grows at 30  C. Growth in medium containing 0.1% cycloheximide is positive. The G þ C content of nuclear DNA is 45.9e46.8 mol%. The major ubiquinone is Q-9. Other cultures examined: INDONESIA, Maratua Island (Latitude: 2 120 .8.3N, Longitude: 118 350 33.1), soil, isolated by A. Yamazaki on Jun 25, 2012 (NBRC 110261, InaCC Y723, NBRC 110262, InaCC Y724, NBRC 110263, InaCC Y725, NBRC 110266,

Fig. 5 e Lipomyces kalimantanensis (NBRC 110267). Budding cells grown on YMA for 3 d at 25  C. Bar: 5 mm.

InaCC Y731). These strains are preserved in glass ampoule as metabolically inactivated at NBRC and InaCC. Lipomyces kalimantanensis Atit Kanti, Yamazaki & Kawasaki f.a., sp. nov. Fig. 5. MycoBank no.: MB 816187. Type: INDONESIA, around the Wain river on Kalimantan Island (Latitude: 1 80 44S, Longitude: 116 500 11E), soil, isolated by A. Yamazaki on Jun 25, 2012 (holotype, strain NBRC 110267T preserved in glass ampoule as metabolically inactivated state by liquid-drying method at NITE Biological Resource Center (NBRC), Chiba Prefecture, Japan). Ex-type culture, strain InaCC Y720T preserved at Indonesian Culture Collection (InaCC), Cibinong, Indonesia. These ex-type cultures will be available without any restriction for research from the two culture collections. Gene sequences ex-holotype: LC061902 (D1/D2 LSU rRNA gene), LC061910 (EF-1a). Etymology: ka-li-man-tan-en'sis N.L. masc. adj. kalimantanensis, referring to the site the species was first isolated, from soil on Kalimantan Island, Indonesia. After 3 d of culture on YMA at 25  C, cells are ovoid, ellipsoid, 3.0e6.3  3.2e6.5 mm, and occur singly, in pairs, or in short chains (Fig. 5) with multilateral budding. The streak culture on YMA is mucoid, partly hyaline, partly creamishopaque, smooth and glistening, with an entire margin. In Dalmau plate cultures on corn meal agar after 10 d at 25  C, neither hyphae nor pseudohyphae are formed. In YM broth after 1 mo at 25  C, sediment is not present. Does not ferment D-glucose. Assimilates D-glucose, Dgalactose, L-sorbose (delayed), sucrose (weak), maltose, cellobiose, a,a-trehalose, lactose, melibiose (weak or not at all), raffinose (weak or not at all), methyl a-D-glucoside (may be delayed), salicin (may be delayed), DL-lactate (weak or not at all), arbutin (may be delayed), melezitose (weak or delayed), inulin (weak or not at all), starch, D-xylose, L-arabinose, D-

LM: light microscope, SEM: scanning electron microscope (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

422

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Table 1 e Fatty acids production by the isolates cultivated in YM and 5G5M medium at 28  C for 10 d. Medium

YM

5G5M

a b

Fatty acids/ strain no.

Palmitoleate (C16:1) Palmitate (C16) Linoleate (C18:2) Oleate (C18:1) Stearate (C18) Total FAs SDb Palmitoleate (C16:1) Palmitate (C16) Linoleate (C18:2) Oleate (C18:1) Stearate (C18) Total FAs SDb

L. maratuensisa

L. starkeyia

L. tropicalisa

L. kalimantanensisa

NBRC 10381

NBRC 110264

NBRC 110261

NBRC 110262

NBRC 110263

NBRC 110265

NBRC 110266

NBRC 110267

NBRC 110268

0.028 0.114 0.038 0.147 0.004 0.330 0.067 0.247 2.442 0.210 1.985 0.171 5.054 0.167

0.009 0.051 0.021 0.058 0.002 0.140 0.013 0.101 1.958 0.136 1.230 0.271 3.696 0.097

0.006 0.054 0.022 0.043 0.004 0.128 0.018 0.033 0.318 0.030 0.093 0.024 0.497 0.292

0.013 0.044 0.019 0.037 0.006 0.119 0.087 0.025 0.394 0.068 0.345 0.074 0.905 0.100

0.012 0.044 0.043 0.038 0.004 0.142 0.001 0.030 0.159 0.029 0.171 0.016 0.405 0.171

0.006 0.020 0.014 0.018 0.001 0.059 0.013 0.017 0.315 0.047 0.276 0.065 0.719 0.158

0.006 0.059 0.042 0.031 0.009 0.147 0.037 0.063 1.050 0.088 0.872 0.195 2.267 0.146

0.013 0.012 0.012 0.023 0.000 0.061 0.007 0.041 0.064 0.038 0.054 0.004 0.201 0.025

0.019 0.015 0.014 0.030 0.000 0.077 0.008 0.050 0.076 0.040 0.064 0.005 0.235 0.042

L., Lipomyces. SD, standard deviation of the total FAs. Each value shows the average or SD of three replicate experiments.

arabinose (may be delayed), D-ribose (may be weak or delayed), L-rhamnose, ethanol (may be delayed), erythritol (may be weak or not at all), ribitol, glycerol, galactitol, xylitol, D-glucitol (weak or delayed), D-mannitol (weak or not at all), myo-inositol, propane-1,2-diol (weak or delayed), butane-2,3diol (weak or delayed), D-glucono-1,5-lactone, D-gluconate (weak or delayed), 2-keto-D-gluconate, 5-keto-D-gluconate, citrate (may be delayed), D-glucuronate, D-galacturonate and arabinitol. Does not assimilate methanol, succinate, Dglucosamine, N-acetyl-D-glucosamine, or hexadecane. Assimilates ammonium sulfate, ethylamine hydrochloride, Llysine, cadaverine dihydrochloride, and imidazole. Does not assimilate potassium nitrate or sodium nitrite. Produces starch-like substances. Urease reaction is negative. Does not grow in YNB medium (Difco) containing 10% NaCl and 5% glucose. Grows in vitamin-free medium. Does not grow at 35  C, but grows at 30  C. Growth in medium containing 0.1% cycloheximide is positive. The G þ C content of nuclear DNA is 45.7e46.1 mol%. The major ubiquinone is Q-9. Other cultures examined: INDONESIA, isolated from the same soil sample as in the type strain (NBRC 110268, InaCC Y722). These strains are preserved in glass ampoule as metabolically inactivated at NBRC and InaCC.

3.4.

Lipid analysis

Many yeasts belonging to the genus Lipomyces accumulate triacylglycerol in their cells as lipid droplets. We analyzed and compared the FAs derived from the lipids produced by the eight isolates cultivated in YM or 5G5M medium, including the total amounts and composition of the FAs produced. Data are shown in Table 1. The major FA constituents of the lipids produced, which were same in each strains, were palmitate (hexadecanoate, C16), palmitoleate (hexadecenoate, C16:1), stearate (octadecanoate, C18), oleate (cis-9-octadecenoate, C18:1), and linoleate (cis-9-cis-12-octadecadienoate, C18:2). The composition

of the FAs of strains cultivated in YM and 5G5M medium differed; strains cultivated in YM displayed higher percentages of linoleate (C18:2) than strains grown in 5G5M, except for strains NBRC 110267 and NBRC 110268 which were unchanged. The total FA content in the strains cultivated in 5G5M was also higher than that of the strains grown in YM. Among the tested strains, L. starkeyi NBRC 10381, which is known for its high lipid production, showed the highest production of total FAs (5.05 g/L) after growth in 5G5M medium for 10 d, followed by NBRC 110264 (3.70 g/L), NBRC 110266 (2.27 g/ L), NBRC 110262 (0.91 g/L), NBRC 110265 (0.72 g/L), NBRC 110261 (0.50 g/L), NBRC 110263 (0.40 g/L), NBRC 110268 (0.24 g/L), and NBRC 110267 (0.20 g/L) (Table 1). Among the isolated strains, NBRC 110264 produced the highest amount of total FAs, and the amount of FAs produced by this strain was around 70% of that produced by L. starkeyi NBRC 10381T. Thus, NBRC 110264 is a candidate strain for lipid production.

Disclosure The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.

Acknowledgments The authors would like to thank Ms. Ikuko Hatakeyama, Ms. Ayumi Fujita, and Mr. Fumihiko Tanaka for technical support. This work was supported by the Science and Technology Research Partnership for Sustainable Development (SATREPS), which is a collaborative research program of the Japan Science and Technology Agency (JST) and the Japan International Cooperation Agency (JICA).

m y c o s c i e n c e 5 8 ( 2 0 1 7 ) 4 1 3 e4 2 3

Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.myc.2017.06.002.

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