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Kodamaea hongheensis f.a., sp. nov., Kodamaea ovata f.a., sp. nov. and Kodamaea yamadae f.a., sp. nov., three new yeast species of Kodamaea (Saccharomycetales, Debaryomycetacae) from China
expand article infoChun-Yue Chai‡§, Wan-Li Gao‡, Ying Li‡, Zhen-Li Yan|, Feng-Li Hui‡§
‡ Nanyang Normal University, Nanyang, China
§ Research Center of Henan Provincial Agricultural Biomass Resource Engineering and Technology, Nanyang, China
| State Key Laboratory of Motor Vehicle Biofuel Technology, Nanyang, China

Corresponding author: Feng-Li Hui ( fenglihui@yeah.net )

Academic editor: Xinlei Fan
© 2022 Chun-Yue Chai, Wan-Li Gao, Ying Li, Zhen-Li Yan, Feng-Li Hui.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Chai C-Y, Gao W-L, Li Y, Yan Z-L, Hui F-L (2022) Kodamaea hongheensis f.a., sp. nov., Kodamaea ovata f.a., sp. nov. and Kodamaea yamadae f.a., sp. nov., three new yeast species of Kodamaea (Saccharomycetales, Debaryomycetacae) from China. MycoKeys 89: 89-137. https://doi.org/10.3897/mycokeys.89.81119
Open Access

Abstract

Kodamaea includes a growing number of interesting yeasts of the family Debaryomycetacae that are widely distributed in temperate, subtropical and tropical regions of different continents. During recent yeast collections in Henan and Yunnan Province in China, several isolates of Kodamaea were obtained from rotting wood, all of which represent undescribed taxa. Based on morphological and phylogenetic analyses (ITS and LSU rDNA), three new species are proposed: K. hongheensis f.a., sp. nov., K. ovata f.a., sp. nov. and K. yamadae f.a., sp. nov. In addition, sixteen Candida species, which are members of the Kodamaea clade based on phylogenetic analysis, are transferred to Kodamaea as new combinations. Our results indicate high species diversity of Kodamaea waiting to be discovered in rotting wood from tropical and subtropical China.

Keywords

Debaryomycetacae, 16 new combinations, Saccharomycetales, three new species, yeast taxonomy

Introduction

Kodamaea was proposed by Yamada et al. (1995a, b) to accommodate a single species, K. ohmeri, which was initially classified in the genus Pichia. Kurtzman (1998) did not accept this proposal because the entire genus Pichia was clearly polyphyletic and in need of careful revision. However, the discovery of four new ascosporogenous relatives of K. ohmeri, namely K. anthophila, K. kakaduensis, K. laetipori and K. nitidulidarum, added further justification to the recognition of Kodamaea as a separate genus (Lachance et al. 1999; Rosa et al. 1999; Suh and Blackwell 2005). Subsequently, four new anamorphic species of Kodamaea, K. jinghongensis, K. meredithiae, K. neixiangensis and K. transpacifica have been proposed as part of the genus based on their phylogenetic placement (Freitas et al. 2013; Sylvester et al. 2015; Gao et al. 2017). In addition, more than 16 species of Candida are recognized as members of the Kodamaea clade based on phylogenetic analysis of rDNA sequences (Hsieh et al. 2010; Lachance et al. 2011; Nakase et al. 2011; Daniel et al. 2014). The Kodamaea clade currently consists of nine species of the genus Kodamaea and 16 asexual species still assigned to the genus Candida, making it one of the growing numbers of interesting genera tentatively assigned to the family Metschnikowiaceae (Lachance and Kurtzman 2011; Nakase et al. 2011; Freitas et al. 2013; Daniel et al. 2014; Sylvester et al. 2015; Gao et al. 2017). On the basis of multigene phylogenetic analysis of nearly entire LSU rDNA, SSU rDNA, translation elongation factor-1a (EF-1a), two RNA polymerase II subunits gene (RPB1 and RPB2) and whole genome, the Kodamaea clade has been found to be closely related to the Aciculoconidium clade and should be allocated to the family Debaryomycetacae (Kurtzman and Robnett 2013; Shen et al. 2018).

Species in Kodamaea are very common and inhabit a wide range of habitats, such as plant-related substrates, insects, insect frass, soil and seawater (Lachance and Kurtzman 2011; Lachance et al. 2011; Nakase et al. 2011; Freitas et al. 2013; Sylvester et al. 2015; Gao et al. 2017). Many of these species are associated with insects that occupy this ecological niche (Freitas et al. 2013; Suh and Blackwell 2005). They were isolated either directly from insects and insect frass or from common insect habitats, such as rotting wood, forest soil, mushrooms or flowers (Suh and Blackwell 2005; Hsieh et al. 2010; Lachance et al. 2011; Freitas et al. 2013; Gao et al. 2017). Significantly, K. mesenterica and K. ohmeri are also found in clinical specimens; it is thus possible that these species might act as emerging opportunistic pathogens (Lachance and Kurtzman 2011; Lachance et al. 2011).

Species of Kodamaea are widely distributed in temperate, subtropical and tropical regions of different continents, but most known species appear to exist in Asia, North America and South America (Suh and Blackwell 2005; Hsieh et al. 2010; Lachance et al. 2011; Nakase et al. 2011; Gao et al. 2017). The genus Kodamaea has been heavily studied in Asia, and 11 species of this clade were previously reported in Thailand, Japan and China. Among them, K. loeiensis is from Thailand (Nakase et al. 2011), while K. fukazawae, K. fungicola and K. sagamina are from Japan (Lachance et al. 2011). In China, K. alishanica, K. hsintzibuensis, K. kaohsiungensis, K. lidongshanica and K. smagusa are described from Taiwan Provinces (Hsieh et al. 2010), and K. jinghongensis and K. neixiangensis are from Henan and Yunnan Provinces (Gao et al. 2017). Our studies suggest the existence of other potentially new species of the genus in China. In this study, we collected rotting wood samples from Henan and Yunnan Provinces in China. After isolation and examination, three new species of Kodamaea were identified based on phenotypic characteristics and phylogenetic analysis, increasing the species diversity of Kodamaea in China.

Materials and methods

Sample collection and isolation

Samples of rotting wood were collected in the Xishuangbanna Primeval Forest Park (Yunnan Province, China) and the Baotianman Nature Reserve (Henan Province, China). The Xishuangbanna Primeval Forest Park (21°98'N, 100°88'E) is 1355 m above sea level (MASL), with a hot and humid climate. The average annual temperature is between 16 °C and 28 °C, and the average annual rainfall is above 1,100 mm. The Baotianman Nature Reserve (33°30'44"N, 111°55'47"E) is at 1830 (MASL), with a transitional climate from a northern subtropical zone to a warm temperate zone, average annual temperature of 14–16 °C, and average annual rainfall between 800 mm and 900 mm. Forty rotting wood samples were collected, twenty from each area, during July to August in 2016 and 2017. The samples were stored in sterile plastic bags and transported under refrigeration to the laboratory over a period of no more than 24 h. Yeast strains were isolated from rotting wood samples in accordance with the methods described by Gao et al. (2017) and Zheng et al. (2017). Each sample (1 g) was added to 20 ml sterile yeast extract-malt extract (YM) broth (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, pH 5.0 ± 0.2) supplemented with 0.025% sodium propionate and 200 mg/L chloramphenicol in a 150 ml Erlenmeyer flask and then cultured for 3–10 days on a rotary shaker. Subsequently, 0.1 ml aliquots of the enrichment culture and appropriate decimal dilutions were spread on YM agar plates and then incubated at 25 °C for 3–4 days. Different yeast colony morphotypes were then isolated by repeated plating on YM agar. All isolates were stored in Microbiology Lab of Nanyang Normal University (NYNU; Nanyang, China), and ex-type cultures of novel yeast were deposited in the fungal collection at Westerdijk Fungal Biodiversity Institute (CBS; Utrecht, The Netherlands) and China Centre of Industrial Culture Collection (CICC; Beijing, China).

Morphological, physiological and biochemical studies

Morphological and physiological properties were determined according to Kurtzman et al. (2011). Carbon and nitrogen assimilation tests were performed using liquid media, and growth was observed for up to 4 weeks. Carbon fermentation was tested in a yeast extract peptone (YP) base media (1% yeast extract and 2% peptone, pH 5.0 ± 0.2), and Durham tubes were used to visualize carbon dioxide production. Growth at various temperatures (30 °C, 35 °C, 37 °C and 40 °C) was assessed by streaking cells onto yeast extract peptone glucose (YPD) agar (1% yeast extract, 2% peptone, 2% glucose, 2% agar, pH 5.0 ± 0.2) plates and incubating them for ~2 weeks. Formation of true hyphae and pseudohyphae was investigated using the Dalmau plate method on both cornmeal (CM) and 5% malt extract (ME) agar plates. Induction of the sexual stage was tested by incubating single or mixed cultures of the each of the two strains on YM agar, cornmeal (CM) agar, 5% malt extract (ME) agar, V8 agar, Gorodkowa agar, McClary’s acetate agar or yeast carbon base plus 0.01% ammonium sulphate (YCBAS) agar at 25 °C for 2 months (Lachance and Kurtzman 2011; Sylvester et al. 2015; Gao et al. 2017).

DNA extraction, PCR amplification and nucleotide sequencing

Genomic DNA was extracted from the yeasts using the Ezup Column Yeast Genomic DNA Purification Kit according to the manufacturer’s protocol (Sangon Biotech, China). The nuclear rDNA ITS1-5.8S-ITS2 (ITS) region was amplified using the primer pair ITS1/ITS4 (White et al. 1990). The D1/D2 domain of LSU rDNA was amplified using the primer pair NL1/NL4 (Kurtzman and Robnett 1998). The following thermal profile was used to amplify the ITS and LSU rDNA regions: an initial denaturation step of 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 51 °C, and 40 s at 72 °C, with a final extension of 10 min at 72 °C (Lv et al. 2020). PCR products were directly purified and sequenced by Sangon Biotech Inc. (Shanghai, China). We determined the identity and accuracy of the newly obtained sequences by comparing them to sequences in GenBank and assembled them using BioEdit (Hall 1999). Newly obtained sequences were then submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/; Table 1).

Table 1.
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DNA sequences used in the molecular phylogenetic analysis. Entries in bold are newly generated in this study.

Species Strain Locality Sample ITS D1/D2
Kodamaea arcana CBS 9883T USA Beetle N/A AY242347
K. alishanica CBS 11429T China Soil NR_159556 NG_063941
K. derodonti CBS 9882T USA Beetle NR_111388 AY242346
K. fukazawae CBS 9137T Japan Mushroom AB028033 AY313957
K. fungicola CBS 9138T Japan Mushroom AB028031 AY313958
K. hsintzibuensis CBS 11427T China Soil NR_160557 HQ999957
K. kaohsiungensis CBS 11435T China Mushroom NR_159557 HQ999958
K. leandrae CBS 9735T Brazil Decaying fruit NR_155222 AY449659
K. lidongshanica CBS 11426T China Fruiting body GU126451 HQ999959
K. loeiensis CBS 11899T Thailand Insect frass NR_155223 NG_073574
K. mesenterica CBS 602T Germany Beer NR_111297 U45720
K. plutei CBS 9885T USA Beetle NR_111389 AY520388
K. restingae CBS 8493T Brazil Flowers Nitidulid beetles NR_155225 AF059667
K. sagamina CBS 9140T Japan Mushroom AB028032 AY313959
K. smagusa CBS 11430T China Mushroom NR_111611 FJ873476
K. suecica CBS 5724T Sweden Seawater N/A U45732
K. anthophila CBS 8494T Australia Flowers, Nitidulid beetle NR_155239 AF059668
K. transpacifica CBS 12823T Ecuador Flowers NR_173358 KF002564
K. nitidulidarum CBS 8491T Brazil Flower Nitidulid beetle NR_155241 AF059665
K. ovata NYNU 167144T China Rotting wood OK381035 OK381037
K. ovata NYNU 1685 China Rotting wood OM327522 OM327519
K. ohmeri CBS 5367T USA Cucumber brines NR_121464 U45702
K. jinghongensis CBS 14700T China Rotting wood KY213814 KY213807
K. hongheensis NYNU 17423T China Rotting wood MG255723 MG255704
K. hongheensis NYNU 17409 China Rotting wood OM327517 OM327518
K. kakaduensis CBS 8611T Australian Flower NR_155240 AF092279
K. laetipori CBS 9884T USA Beetle N/A AY520398
K. meredithiae CBS 13899T USA soil OK050648 KM408122
K. neixiangensis CBS 14699T China Rotting wood KY213808 KY213820
K. yamadae NYNU 168114T China Rotting wood OK381036 OK381034
K. yamadae NYNU 16858 China Rotting wood OM327521 OM327516
Metschnikowia lochheadii CBS 8807T USA flowers NR_164507 NG_058341
M. cubensis MUCL 45753T Cuba flowers N/A EU143316
M. matae var. maris CBS 13986T Brazilian flowers N/A KP241777
M. cerradonensis CBS 10409T Brazil flowers nitidulid beetles N/A DQ641237
M. continentalis CBS 8430T Germany flowers N/A DQ641238
M. santaceciliae CBS 9149T Costa Rica nitidulid beetles N/A DQ641242
M. borealis CBS 8431T USA beetles N/A DQ641243
Aciculoconidium aculeatum NRRL YB-4298T USA Drosophila pinicola N/A JQ689029
Schizosaccharomyces pombe NRRL Y-12796T Jamaica, South Africa, Poland Apple, Molass KY105378 KY109602

Notes: Type strains are marked with T. N/A: sequences not available.

Phylogenetic analyses

Species in the Kodamaea clade with high similarity to our new species were selected for phylogenetic analyses. Schizosaccharomyces pombe NRRL Y-12796T was used as an outgroup, based on Kuramae et al. (2006a). NCBI accession numbers of sequences used in the phylogenetic tree are listed in Table 1. Initial alignment of the combined ITS and partial LSU rDNA dataset was performed using the online version of MAFFT 6.0 (Katoh and Toh 2010) with manual evaluations and adjustments in BioEdit when necessary to obtain reliable and reasonable results (Hall 1999). The best-fit nucleotide substitution models for each gene were selected using jModelTest v2.1.7 (Darriba et al. 2012) according to the Akaike information criterion.

Neighbour-joining (NJ) and Maximum parsimony (MP) analyses were implemented for inferring the phylogenetic analyses by using MEGA software version 7.0 (Kumar et al. 2016). The NJ analysis was carried out using Kimura’s two parameter model (Kimura 1980) in the neighbour-joining method (Saitou and Nei 1987). Bootstrapping with 1,000 replicates was performed to determine branch support (Felsenstein 1985). The MP analysis was run using a heuristic search option of 1,000 search replicates with random-addition of sequences and tree bisection and reconnection (TBR) as the branch-swapping algorithm. NJ and MP bootstrap support values above 50% are shown as first and second positions above nodes, respectively.

Results

Phylogenetic analyses

The combined ITS and LSU rDNA sequences dataset was analysed to infer the interspecific relationships within the Kodamaea clade of the family Debaryomycetacae. The dataset consisted of 40 sequences including the outgroup, Schizosaccharomyces pombe NRRL Y-12796T. A total of 896 characters including gaps (372 for ITS and 524 for LSU rDNA) were included in the phylogenetic analysis. Both NJ and MP analyses resulted in similar tree topologies, and only the NJ tree is shown in Fig. 1.

Figure 1. 

The NJ tree based on an analysis of a combined ITS and LSU rDNA dataset from the genus Kodamaea and related taxa from the family Debaryomycetacae. Schizosaccharomyces pombe NRRL Y-12796T was used as outgroup. Bootstrap support values (BS) for the neighbour-joining and maximum parsimony programs of above 50% are given at nodes based on 1000 replications, a dash (“-”) indicates a value < 50% (BS). Bar, 2% sequence difference. The strain number is indicated after the species name. The strains in this study are in bold. T, type strains.

In the NJ phylogenetic tree (Fig. 1), the genus Kodamaea formed a monophyletic clade distant from its related taxa of the family Debaryomycetacae. The samples of the three new species of Kodamaea, Kodamaea hongheensis, Kodamaea ovata and Kodamaea yamadae, formed each a strongly supported sub-clade and were clearly distinct from other known species of Kodamaea. Two strains of K. hongheensis formed a unique lineage with K. laetipori, but with low support (NJ 57%, MP 60%). K. ovata clustered with K. neixiangensis with high support (NJ 94%, MP 90%), while K. yamadae clustered with K. jinghongensis and K. fukazawae with evident statistic support (NJ 87%, MP 80%).

Taxonomy

Kodamaea hongheensis C.Y. Chai & F.L. Hui, sp. nov.

MycoBank No: 842625
Fig. 2

Holotype

China, Yunnan Province, Honghe Prefecture, Luxi County, in rotting wood in Jiuxi Mountain Forest Park, April 2017, K.F. Liu & Z.W. Xi (holotype NYNU 17423T, ex-holotype CICC 33265).

Etymology

The specific epithet hongheensis refers to the geographic origin of the type strain, Honghe Prefecture, Yunnan.

Description

In YM broth, after 3 days at 25 °C, cells are ovoid to elongate (3–5 × 3–7 μm) and occur singly or in pairs (Fig. 2a). Sediment is formed after a month, but a pellicle is not observed. On YM agar, after 3 days at 25 °C, colonies are white to cream-colored, butyrous and smooth with entire margins. In Dalmau plate culture on CM agar, pseudohyphae are formed but not true mycelia (Fig. 2b). Asci or signs of conjugation are not seen on sporulation media. Glucose, maltose and trehalose are fermented but not galactose, sucrose, melibiose, lactose, cellobiose, melezitose, raffinose, inulin, or xylose. Glucose, glucosamine, d-xylose, sucrose, maltose, trehalose, methyl α-d-glucoside, cellobiose, salicin, melezitose, glycerol, ribitol, d-glucitol, d-mannitol, d-glucono-1, 5-lactone, 2-keto-d-gluconate, succinate, citrate and ethanol are assimilated as sole carbon sources. Galactose, l-sorbose, d-ribose, d-arabinose, l-arabinose, l-rhamnose, melibiose, lactose, raffinose, inulin, erythritol, xylitol, galactitol, myo-inositol, d-gluconate, d-glucuronate, dl-lactate and methanol are not assimilated. l-lysine, glucosamine and d-tryptophan are assimilated as sole nitrogen sources. Nitrate, nitrite, ethylamine, cadaverine, creatine, creatinine and imidazole are not assimilated. Growth is observed at 35 °C but not at 37 °C. Growth in the presence of 0.01% cycloheximide is positive, but growth in the presence of 10% NaCl plus 5% glucose and 1% acetic acid is negative. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are negative.

Figure 2. 

Morphology of Kodamaea hongheensis (NYNU 17423, holotype) a budding cells in YM broth after 3 d b simple pseudohyphae on CM agar after 14 d. Scale bars: 10 μm.

Additional isolate examined

China, Yunnan Province, Honghe Prefecture, Luxi County, in rotting wood in Jiuxi Mountain Forest Park, April 2017, K.F. Liu & Z.W. Xi (NYNU 17409).

Notes

Two strains NYNU 17409 and NYNU 17423, both representing K. hongheensis, were grouped in an independent lineage and are related to K. laetipori. K. hongheensis differed from its closest relative K. laetipori by 2.9% substitutions in the LSU rDNA sequence. However, the ITS sequence of K. hongheensis could not be successfully aligned with the type strain of K. laetipori because its ITS sequences are not currently available from either the NCBI GenBank database or the CBS database. Physiologically, K. hongheensis can be differentiated from K. laetipori (Lachance and Kurtzman 2011) based on growth in l-sorbose, d-arabinose, d-gluconate, and dl-lactate, which are positive for K. laetipori and negative for the new species. Moreover, K. laetipori grows in the presence of 0.1% cycloheximide and 10% NaCl plus 5% glucose, but K. hongheensis does not.

Kodamaea ovata C.Y. Chai & F.L. Hui, sp. nov.

MycoBank No: 842623
Fig. 3

Holotype

China, Henan Province, Nanyang City, the Baotianman Nature Reserve, in rotting wood under a mixed forest, July 2016, K.F. Liu & Z.W. Xi (holotype NYNU 167144T, ex-holotype CBS 14702).

Etymology

The specific epithet ovata refers to the ovoid cell morphology of the type strain.

Description

In YM broth, after 3 days at 25 °C, cells are ovoid (2–4 × 3–5 μm) and occur singly or in pairs (Fig. 3a). Sediment is formed after a month, but a pellicle is not observed. On YM agar, after 3 days at 25 °C, colonies are white to cream-colored, butyrous and smooth with entire margins. In Dalmau plate culture on CM agar, a rudimentary pseudomycelium is formed (Fig. 3b). Asci or signs of conjugation are not seen on sporulation media. Glucose, galactose, maltose and trehalose are fermented but sucrose, melibiose, lactose, cellobiose, melezitose, raffinose, inulin and xylose are not. Glucose, galactose, glucosamine, d-xylose, d-arabinose, l-arabinose, sucrose, maltose, trehalose, methyl α-d-glucoside, cellobiose, salicin, arbutin, melezitose, inulin, glycerol, ribitol, xylitol, d-glucitol, d-mannitol, galactitol, d-glucono-1, 5-lactone, 2-keto-d-gluconate, dl-lactate, succinate, citrate and ethanol are assimilated as sole carbon sources. l-sorbose, d-ribose, l-rhamnose, melibiose, lactose, raffinose, erythritol, myo-inositol, d-gluconate, d-glucuronate and methanol are not assimilated. Ethylamine, l-lysine and creatine are assimilated as sole nitrogen sources. Nitrate, nitrite, cadaverine, creatinine, glucosamine, imidazole and d-tryptophan are not assimilated. Growth is observed at 42 °C but not at 45 °C. Growth in the presence of 0.1% cycloheximide and 16% NaCl plus 5% glucose is positive, but growth in the presence of 1% acetic acid is negative. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are negative.

Figure 3. 

Morphology of Kodamaea ovata (NYNU 167144, holotype) a budding cells in YM broth after 3 d b pseudohyphae on CM agar after 14 d. Scale bars: 10 μm.

Additional isolate examined

China, Henan Province, Nanyang City, the Baotianman Nature Reserve, in rotting wood under a mixed forest, July 2016, K.F. Liu & Z.W. Xi (NYNU 1685).

Notes

Two strains NYNU 1685 and NYNU 167144 representing K. ovata grouped in a well-supported clade and appear to be most closely related to K. neixiangensis (Gao et al. 2017). The nucleotide differences between the new species and the close relative K. neixiangensis are 1% substitutions in the LSU rDNA sequence and 4.8% substitutions in the ITS region, respectively. Physiologically, K. ovata can be differentiated from K. neixiangensis based on growth in l-arabinose, d-arabinose, dl-lactate and 16% NaCl plus 5% glucose, all of which were positive for K. ovata and negative for K. neixiangensis. Additionally, the new species ferments galactose and maltose and grows at 35 °C, but K. neixiangensis does not have these characteristics.

Kodamaea yamadae C.Y. Chai & F.L. Hui, sp. nov.

MycoBank No: 842626
Fig. 4

Holotype

China, Henan Province, Nanyang City, the Baotianman Nature Reserve, in rotting wood under a mixed forest, August 2016, K.F. Liu & Z.W. Xi (holotype NYNU 168114T, ex-holotype CBS 14703).

Etymology

The specific epithet yamadae is used in honour of Y. Yamada for his proposal of the genus Kodamaea.

Description

In YM broth, after three days at 25 °C, cells are ellipsoidal to elongate (2–3 × 4.5–10 μm) and occur singly or in pairs (Fig. 4a). Sediment is formed after a month, but a pellicle is not observed. On YM agar, after 3 days at 25 °C, colonies are white, convex, sometimes fringed, glabrous or membranous, smooth or rugose and butyrous to tough due to filamentous growth. On Dalmau plate culture on CM agar, a rudimentary pseudomycelium is formed (Fig. 4b). Asci or signs of conjugation are not seen on sporulation media. Glucose, maltose, sucrose, trehalose and cellobiose are fermented but not galactose, melibiose, lactose, melezitose, raffinose, inulin or xylose. Glucose, galactose, glucosamine, d-ribose, d-xylose, sucrose, maltose, trehalose, methyl α-d-glucoside, cellobiose, salicin, inulin, glycerol, erythritol, ribitol, d-glucitol, d-mannitol, d-glucono-1, 5-lactone, 2-keto-d-gluconate, succinate, citrate and ethanol are assimilated as sole carbon sources. l-sorbose, d-arabinose, l-arabinose, l-rhamnose, arbutin, melibiose, lactose, raffinose, melezitose, xylitol, galactitol, myo-inositol, 5-keto-d-gluconate, d-gluconate, d-glucuronate, dl-lactate and methanol are not assimilated. Ethylamine, l-lysine, creatine, glucosamine and d-tryptophan are assimilated as sole nitrogen sources. Nitrate, nitrite, cadaverine, creatinine and imidazole are not assimilated. Growth is observed at 30 °C but not at 35 °C. Growth in the presence of 0.1% cycloheximide is positive, but growth in the presence of 10% NaCl plus 5% glucose and 1% acetic acid is negative. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are negative.

Figure 4. 

Morphology of Kodamaea yamadae (NYNU 168114, holotype) a budding cells in YM broth after 3 d b pseudohyphae on CM agar after 14 d. Scale bars: 10 μm.

Additional isolates examined

China, Henan Province, Nanyang City, the Baotianman Nature Reserve, in rotting wood under an oak forest, August 2016, K.F. Liu & Z.W. Xi (NYNU 16858).

Notes

Two strains NYNU 16858 and NYNU 168114, representing K. yamadae clustered in a well-supported clade that is closely related to K. jinghongensis (Gao et al. 2017) and K. fukazawae (Nakase et al. 1999). The nucleotide differences between K. yamadae and K. jinghongensis were 2.8% substitutions in the LSU rDNA sequences and 3.9% substitutions in the ITS region. Similarly, K. yamadae and K. fukazawae showed differences of 2.6% substitutions in the LSU rDNA sequences and 4.7% substitutions in the ITS region. Physiologically, the novel species differed from K. jinghongensis by its ability to ferment cellobiose and its inability to assimilate arbutin. Unlike K. fukazawae, the novel species was able to assimilate d-galactose, l-sorbose, inulin, d-arabinose, l-arabinose, l-rhamnose, and methanol, and was not able to grow in the presence of 0.1% cycloheximide. In all cases, identification by sequencing was the best approach.

Sixteen new combinations

In addition to the previously described taxa, we propose sixteen new combinations in the genus Kodamaea by including clade members that previously were described as species of the polyphyletic asexual genus Candida based on the combined ITS and LSU rDNA sequences from type strains of the genus Kodamaea and related taxa of the family Debaryomycetacae.

Kodamaea alishanica (C.W. Hsieh) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 843566

Basionym

Candida alishanica C.W. Hsieh, FEMS Yeast Research 10 (7): 948 (2010).

Kodamaea arcana (S.-O. Suh & M. Blackw) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 843565

Basionym

Candida arcana S.-O. Suh & M. Blackw, Mycologia 97 (1): 172 (2005).

Kodamaea derodonti (S.-O. Suh & M. Blackw) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 843567

Basionym

Candida derodonti S.-O. Suh & M. Blackw, Mycologia 97 (1): 172 (2005).

Kodamaea fukazawae (Nakase, M. Suzuki, Sugita, S.O. Suh & Komag) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 843579

Basionym

Candida fukazawae Nakase, M. Suzuki, Sugita, S.O. Suh & Komag, Mycoscience 40 (6): 473 (1999).

Kodamaea fungicola (Nakase, M. Suzuki, Sugita, S.O. Suh & Komag) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 843568

Basionym

Candida fungicola Nakase, M. Suzuki, Sugita, S.O. Suh & Komag, Mycoscience 40 (6): 470 (1999).

Kodamaea hsintzibuensis (C.W. Hsieh) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 843569

Basionym

Candida hsintzibuensis C.W. Hsieh, FEMS Yeast Research 10 (7): 948 (2010).

Kodamaea kaohsiungensis (C.W. Hsieh) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 843570

Basionym

Candida kaohsiungensis C.W. Hsieh, FEMS Yeast Research 10 (7): 948 (2010).

Kodamaea leandrae (Ruivo, Pagnocca, Lachance & Rosa) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 842628

Basionym

Candida leandrae Ruivo, Pagnocca, Lachance & Rosa, International Journal of Systematic and Evolutionary Microbiology 54(6): 62407 (2004).

Kodamaea lidongshanica (C.W. Hsieh) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 843571

Basionym

Candida lidongshanica C.W. Hsieh, FEMS Yeast Research 10 (7): 948 (2010).

Kodamaea loeiensis (Nakase, Jindamorakot, Am-In, Ninomiya & Kawasaki) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 842627

Basionym

Candida loeiensis Nakase, Jindamorakot, Am-In, Ninomiya & Kawasaki, Journal of General and Applied Microbiology 57(6): 2011(388).

Kodamaea mesenterica ((A. Geiger) Diddens & Lodder) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 843572

Basionym

Candida mesenterica (A. Geiger) Diddens & Lodder, Die anaskosporogenen Hefen, II Hälfte: 196 (1942).

Kodamaea plutei (S.-O. Suh & M. Blackw) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 843573

Basionym

Candida plutei S.-O. Suh & M. Blackw, Mycologia 97 (1): 173 (2005)

Kodamaea restingae (Rosa, Lachance, Starmer, Barker, Bowles & Schlag-Edler) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 842629

Basionym

Candida restingae Rosa, Lachance, Starmer, Barker, Bowles & Schlag-Edler, International Journal of Systematic Bacteriology 49(1):313 (1999).

Kodamaea sagamina (Nakase, M. Suzuki, Sugita, S.O. Suh & Komag) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 843575

Basionym

Candida sagamina Nakase, M. Suzuki, Sugita, S.O. Suh & Komag, Mycoscience 40 (6): 471 (1999).

Kodamaea smagusa (C.W. Hsieh) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 843576

Basionym

Candida smagusa C.W. Hsieh, FEMS Yeast Research 10 (7): 948 (2010).

Kodamaea suecica (Rodr. Mir. & Norkrans) C.Y. Chai & F.L. Hui, comb. nov.

MycoBank No: 843577

Basionym

Candida suecica Rodr. Mir. & Norkrans, Antonie van Leeuwenhoek 34: 115 (1968).

Discussion

In this study, three new species of Kodamaea, namely Kodamaea hongheensis f.a., sp. nov., Kodamaea ovata f.a., sp. nov. and Kodamaea yamadae f.a., sp. nov., from Henan and Yunnan Province in China are described and compared with similar species based on morphological and molecular data. A more comprehensive phylogenetic placement of the genus Kodamaea based on the combined ITS and LSU rDNA sequences is provided, including almost all representatives from GenBank database and newly generated sequences. This study provides some ideas on the species delimitation within Kodamaea based on morphological and phylogenetic placement evidence.

The phylogenetic relationships in Kodamaea have been unclear, mainly due to lacking a multigene phylogeny (Daniel et al. 2014). In this article, we used all currently known species and the new species to revise this genus, based on a phylogenetic analysis of the combined ITS and LSU rDNA sequences. As shown in Fig. 1, the genus Kodamaea formed a monophyletic clade with well support (NJ 99%, MP 100%). This result is similar to the results of previous phylogenetic analyses based on the D1/D2 domain of LSU rDNA sequences (Freitas et al. 2013; Gao et al. 2017). According to the nomenclature of “one fungus, one name”, sixteen asexual Candida species, which are members of the Kodamaea clade based on phylogenetic analysis, are transferred to Kodamaea as K. alishanica comb. nov., K. arcana comb. nov., K. derodonti comb. nov., K. fukazawae comb. nov., K. fungicola comb. nov., K. hsintzibuensis comb. nov., K. kaohsiungensis comb. nov., K. leandrae comb. nov., K. lidongshanica comb. nov., K. loeiensis comb. nov., K. mesenterica comb. nov., K. plutei comb. nov., K. restingae comb. nov., K. sagamina comb. nov., K. smagusa comb. nov. and K. suecica comb. nov.

In recent years, many new yeast species have been identified from rotting wood in China (Gao et al. 2017; Zheng et al. 2017; Lv et al. 2020). However, there is still a large number of undescribed yeast taxa in China. This study indicates that there are at least five species of Kodamaea isolated from rotting wood in China, including two species known previously to occur in China (K. jinghongensis and K. neixiangensis), and three novel species (K. hongheensis, K. ovata and K. yamadae). In China, there are still some species that need to be discovered, such as those listed under GenBank accessions KM598654 and HQ623482. Our study indicates that there is high species diversity of Kodamaea waiting to be discovered in rotting wood in tropical and subtropical China and nearby areas as with other genera (Lv et al. 2020).

Acknowledgements

We sincerely thank Dr Kai-Fang Liu and Dr Zhi-Wen Xi for their kind help with collecting specimens. This project was supported by Grant No. 31570021 from the National Natural Science Foundation of China (NSFC), P. R. China, Grant No. 2018001 from the State Key Laboratory of Motor Vehicle Biofuel Technology, Henan Tianguan Enterprise Group Co., Ltd., China, and Grant No. 212102110261 Key specialized research and development breakthrough program in Henan province, China.

References

  • Daniel HM, Lachance MA, Kurtzman CP (2014) On the reclassification of species assigned to Candida and other anamorphic ascomycetous yeast genera based on phylogenetic circumscription. Antonie van Leeuwenhoek International Journal of General and Molecular Microbiology 106(1): 67–84. https://doi.org/10.1007/s10482-014-0170-z
  • Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: More models, new heuristics and parallel computing. Nature Methods 9(8): 772–772. https://doi.org/10.1038/nmeth.2109
  • Freitas LF, Carvajal Barriga EJ, Barahona PP, Lachance MA, Rosa CA (2013) Kodamaea transpacifica f.a., sp. nov., a yeast species isolated from ephemeral flowers and insects in the Galapagos Islands and Malaysia: Further evidence for ancient human transpacific contacts. International Journal of Systematic and Evolutionary Microbiology 63(Pt_11): 4324–4329. https://doi.org/10.1099/ijs.0.052282-0
  • Gao WL, Liu TT, Zheng J, Hui FL (2017) Kodamaea neixiangensis f.a., sp. nov. and Kodamaea jinghongensis f.a., sp. nov., two yeast species isolated from rotting wood. International Journal of Systematic and Evolutionary Microbiology 67(9): 3358–3362. https://doi.org/10.1099/ijsem.0.002117
  • Hall TA (1999) Bioedit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
  • Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16(2): 111–120. https://doi.org/10.1007/BF01731581
  • Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary genetics analysis version 7.0 bigger datasets. Molecular Biology and Evolution 33(7): 1870–1874. https://doi.org/10.1093/molbev/msw054
  • Kurtzman CP (1998) Discussion of teleomorphic and anamorphic ascomycetous yeasts and a key to genera. In: Kurtzman CP, Fell JW (Eds) The Yeasts, A Taxonomic Study 4th edn. Elsevier, Amsterdam, 111–121. https://doi.org/10.1016/B978-044481312-1/50018-6
  • Kurtzman CP, Robnett CJ (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek International Journal of General and Molecular Microbiology 73(4): 331–371. https://doi.org/10.1023/A:1001761008817
  • Kurtzman CP, Robnett CJ (2013) Relationships among genera of the Saccharomycotina (Ascomycota) from multigene phylogenetic analysis of type species. FEMS Yeast Research 13(1): 23–33. https://doi.org/10.1111/1567-1364.12006
  • Kurtzman CP, Fell JW, Boekhout T, Robert V (2011) Methods for isolation, phenotypic characterization and maintenance of yeasts. In: Kurtzman CP, Fell JW, Boekhout T (Eds) The Yeasts – a Taxonomic Study, 5th edn. , vol. 1. Amsterdam, Elsevier, 87–110. https://doi.org/10.1016/B978-0-444-52149-1.00007-0
  • Lachance MA, Kurtzman CP (2011) Kodamaea Yamada Y, Suzuki T, Matsuda, Mikata emend. Rosa, Lachance, Starmer, Barker, Bowles and Schlag-Edler (1999). In: Kurtzman CP, Fell JW, Boekhout T (Eds) The Yeasts – A Taxonomic Study, 5th edn. , vol. 2. Amsterdam, Elsevier, 483–490. https://doi.org/10.1016/B978-0-444-52149-1.00036-7
  • Lachance MA, Bowles JM, Starmer WT, Barker JSF (1999) Kodamaea kakaduensis and Candida tolerans, two new ascomycetous yeast species from Australian Hibiscus flowers. Canadian Journal of Microbiology 45(2): 172–177. https://doi.org/10.1139/w98-225
  • Lachance MA, Boekhout T, Scorzetti G, Fell JW, Kurtzman CP (2011) Candida Berkhout (1923). In: Kurtzman CP, Fell JW, Boekhout T (Eds) The Yeasts – a Taxonomic Study, 5th edn. , vol. 2. Amsterdam, Elsevier, 987–1278. https://doi.org/10.1016/B978-0-444-52149-1.00090-2
  • Lv SL, Chai CY, Wang Y, Yan ZL, Hui F-L (2020) Five new additions to the genus Spathaspora (Saccharomycetales, Debaryomycetaceae) from southwest China. MycoKeys 75: 31–49. https://doi.org/10.3897/mycokeys.75.57192
  • Nakase T, Suzuki M, Sugita T, Suh SO, Komagata K (1999) Three new species of anamorphic yeasts phenotypically and phylogenetically related to Candida mesenterica: The description of Candida fungicola sp. nov., Candida sagamina sp. nov., and Candida fukazawae sp. nov. isolated from fruit bodies of mushrooms. Mycoscience 40(6): 465–476. https://doi.org/10.1007/BF02461023
  • Nakase T, Jindamorakot S, Am-In S, Ninomiya S, Kawasaki H (2011) Candida loeiensis sp. nov., a novel anamorphic yeast species found in Thailand. The Journal of General and Applied Microbiology 257(6): 387–391. https://doi.org/10.2323/jgam.57.387
  • Rosa CA, Lachance MA, Starmer WT, Barkert JSF, Bowles JM, Schlag-Edler B (1999) Kodarnaea nitidulidarurn, Candida restingae and Kodarnaea anthophila, three new related yeast species from ephemeral flowers. International Journal of Systematic Bacteriology 49: 309–318. https://doi.org/10.1099/00207713-49-1-309
  • Shen XX, Opulente DA, Kominek J, Zhou X, Steenwyk JL, Buh KV, Haase MAB, Wisecaver JH, Wang M, Doering DT, Boudouris JT, Schneider RM, Langdon QK, Ohkuma M, Endoh R, Takashima M, Manabe R, Čadež N, Libkind D, Rosa CA, DeVirgilio J, Hulfachor AB, Groenewald M, Kurtzman CP, Hittinger CT, Rokas A (2018) Tempo and Mode of Genome Evolution in the Budding Yeast Subphylum. Cell 175(6): 1–13. https://doi.org/10.1016/j.cell.2018.10.023
  • Sylvester K, Wang QW, James B, Mendez R, Hulfachor AB, Hittinger CT (2015) Temperature and host preferences drive the diversification of Saccharomyces and other yeasts: A survey and the discovery of eight new yeast species. FEMS Yeast Research 15(3): fov002. https://doi.org/10.1093/femsyr/fov002
  • White TJ, Bruns TD, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR protocols, a guide to methods and applications. Academic Press.
  • Yamada Y, Suzuki T, Matsuda M, Mikata K (1995a) The phylogenetic relationships of the Q9-equipped, hat-shaped ascospore-forming species of the genus Yamadazyma BillonGrand (Saccharomycetaceae) based on the partial sequences of 18S and 26S rRNAs. Bioscience, Biotechnology, and Biochemistry 59(3): 445–450. https://doi.org/10.1271/bbb.59.445
  • Yamada Y, Suzuki T, Matsuda M, Mikata K (1995b) The phylogeny of Yamadazyma ohmeri (Etchells et Bell) Billon Grand based on the partial sequences of 18S and 26S rRNAs: The proposal of Kodamaea gen. nov. (Saccharomycetaceae). Bioscience, Biotechnology, and Biochemistry 59(6): 1172–1174. https://doi.org/10.1271/bbb.59.1172
  • Zheng J, Liu KF, Liu XJ, Zhang L, Hui FL (2017) Deakozyma yunnanensis sp. nov., a novel yeast species isolated from rotten wood. International Journal of Systematic and Evolutionary Microbiology 67(7): 2436–2439. https://doi.org/10.1099/ijsem.0.001978
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