Introduction

The genus Yamadazyma was described by Billon-Grand (1989) to accommodate 16 species that had previously been assigned to the genus Pichia and formed CoQ-9 as their major ubiquinone, as well as hat-shaped ascospores [1, 2]. Unfortunately, the genus has not been generally accepted as the polyphyletic nature of Yamadazyma became evident from the D1/D2 domains of the large subunit (LSU) rRNA gene sequence analysis after a few years [2]. In 2010, some of the species that had been assigned to this genus by Billon-Grand (1989) were transferred to the newly described genera Babjeviella, Meyerozyma, Millerozyma and Priceomyces based on the phylogenetic analysis of the D1/D2 domains of the LSU rRNA and the nearly complete small subunit (SSU) rRNA gene [3]. Thereafter, the genus Yamadazyma became a well-supported clade and a generally accepted genus in the family Debaryomycetaceae of the order Saccharomycetales [3, 4]. In the fifth edition of “The Yeasts, A Taxonomic Study”, Yamadazyma philogaea, the type species, as well as Yamadazyma akitaensis, Yamadazyma mexicana, Yamadazyma nakazawae, Yamadazyma scolyti, Yamadazyma triangularis and 23 Candida species are placed in the Yamadazyma clade [4, 5]. Since then, a few novel Candida species in this clade have been described, including Candida kanchanaburiensis [6], Candida khaothaluensis, Candida vaughaniae, Candida tallmaniae [7] and Candida oceani [8]. Recently, several additional members of the genera Yamadazyma have been proposed, namely, Yamadazyma terventina [9], Yamadazyma siamensis, Yamadazyma phyllophila, Yamadazyma paraphyllophila [10] and Yamadazyma ubonensis [11].

During an investigation of the yeast species diversity associated with rotten wood in Central China, three asexual cellobiose-fermenting yeast strains have been obtained. The sequences of the D1/D2 domains of the LSU rRNA gene have shown that these isolates are closely related to Y. mexicana, Y. terventina and Candida trypodendroni, belonging to the Yamadazyma clade. However, the internal transcribed spacers (ITS) regions sequences of the isolates are quite divergent from these three known species, which represent a new species of the genus Yamadazyma. In this study, we propose the description of this new species as Yamadazyma dushanensis f.a., sp. nov. based on the morphology, physiology and phylogenetic analysis of the D1/D2 domains of the LSU rRNA gene and the ITS regions.

Materials and Methods

Yeast Isolation and Culture

The strains belonging to the proposed novel species, NYNU 14656, NYNU14668T and NYNU14669, were isolated from three different rotten wood samples collected in the Dushan Forest Park located near Nanyang (33°3′38″N and 112°34′44″E) in Henan Province, Central China. This area is a warm, temperate zone, with monsoon-influenced semi humid continental climate. All necessary permits were obtained from Dushan Forest Park Administration of Nanyang, Henan Province, China.

The methods for yeast isolation from decayed wood samples were detailed by Cadete et al. [12]. Approximately 1 g of each sample was added to 20 mL of sterile yeast extract-malt extract (YM) broth (0.3 % yeast extract, 0.3 % malt extract, 0.5 % peptone and 1 % glucose; adjusted to pH 4.0–4.5 with 1 M HCl) supplemented with 0.025 % sodium propionate and 200 mg/L chloramphenicol in a 150 ml Erlenmeyer flask and incubated at 25 °C for 3 days on a rotary shaker. The enrichment culture was spread out on YM agar supplemented with 0.025 % sodium propionate and 200 mg/L chloramphenicol, and then incubated at 25 °C for 3–4 days. The different yeast morphotypes were purified at least twice, and then stored on YM agar slants at 4 °C and in 15 % glycerol at −80 °C.

Morphological, Physiological and Biochemical Characteristics

The methods used to determine the morphological, physiological and biochemical properties were performed according to established methods [13, 14]. All assimilation tests were performed twice in liquid media, and the results were read after 5 and 21 days of incubation. Starved inocula were used in the nitrogen assimilation tests. Sporulation tests were performed on YM agar, 5 % malt extract agar, corn meal agar and yeast carbon base supplemented with 0.01 % ammonium sulphate (YCBAS) agar (1.1 % yeast carbon base, 0.01 % ammonium sulphate and 1.8 % agar) in pure and mixed cultures. The cultures were observed for ascospore production every week for a month at 15 and 25 °C.

DNA Extraction, PCR Amplification and Sequencing

Genomic DNA was extracted using the Ezup Column Yeast Genomic DNA Purification Kit according to the protocol of the manufacturer (Sangon Biotech, Shanghai, China). The D1/D2 domains of the LSU rRNA gene and ITS regions were amplified by PCR, and sequenced using primers NL1 and NL4 [2], and ITS1 and ITS4 [15], respectively. Both DNA strands were sequenced, and the reactions were carried out using a Dye Terminator cycle sequencing kit (Applied Biosystems, Warrington).

Phylogenetic Analysis

The sequences were compared pairwise using BLAST search [16] and then aligned with type strain sequences of the related species acquired from GenBank using the multiple alignment program CLUSTAL_X version 1.81 [17]. The alignment covered 1096 characters, of which 694 were constant, 125 were phylogenetically uninformative and 277 were informative. Phylogenetic and molecular evolutionary analyses were conducted using the MEGA software version 5.0 [18]. The evolutionary distance data were calculated from Kimura’s two-parameter model in the neighbour-joining analyses [19, 20]. The heuristic search (close-neighbour-interchange) was used in the maximum parsimony analyses. Gaps and missing data were treated as complete deletion. Yamadazyma triangularis NRRL Y-5714 T was set as the outgroup. Confidence limits were estimated from bootstrap analysis (1000 replicates) [21], and only values above 50 % were recorded on the resulting tree. Reference sequences were retrieved from GenBank under the accession numbers indicated in the tree.

Results and Discussion

Yeast Isolation and Diversity

In this work, 53 yeast strains were isolated from 17 rotten wood samples collected in the Dushan Forest Park. By comparison of the D1/D2 domains of the LSU rRNA gene and ITS sequences for all isolates for rapid identification, 50 isolates present in the samples were identified as 13 known species, wherein Candida chauliodes, Candida fructus, Candida maltosa, Hanseniaspora uvarum, Kluyveromyces lactis, Lachancea kluyveri, Pichia membranifaciens, Prototheca zopfii, Saccharomyces cerevisiae, Saccharomycopsis selenospora, Scheffersomyces lignicola, Torulaspora delbruecki, and Trichosporon siamense. The remaining three strains, including NYNU 14656, NYNU14668T and NYNU14669, which could ferment cellobiose, were distinct from any previously described species based on the sequence comparisons of the D1/D2 domains of the LSU rRNA gene and the ITS regions.

Novel Species Delineation, Identification and Ecology

The three strains of Y. dushanensis sp. nov. shared identical sequences in both D1/D2 and ITS regions. Sequence analyses of the D1/D2 domains of the LSU rRNA gene revealed that this new species was closely related to species in the Yamadazyma clade (Debaryomycetaceae and Saccharomycetales). In terms of pairwise sequence similarity, the close relatives of Y. dushanensis sp. nov. include Y. terventina, Y. mexicana and C. trypodendroni. The D1/D2 sequences of the novel species differed by five nt substitutions and two gaps from Y. terventina, by six nt substitutions from Y. mexicana, and by five nt substitutions and one gap from the later species. In general, the strains of a species show no more than 0–3 nt differences (0–0.5 %). Strains showing six (1 %) or greater substitutions are usually separate species, whereas strains with an intermediate number of nucleotide substitutions are also generally considered as separate species [2, 22–24]. For further assessment of genetic separation, the ITS sequences of the three strains of novel species were analysed. In the ITS regions, the sequence of this new species significantly differed with 12 nt substitutions and three gaps from Y. terventina, 17 nt substitutions and six gaps from Y. mexicana, and 18 nt substitutions and 10 gaps from C. trypodendroni, respectively. Groenewald et al. also observed that only 2 nt substitutions in the D1/D2 domain may represent different taxa of the Yamadazyma clade, where 9 or more nucleotide differences can be found between the ITS regions of these species [7]. This study clearly indicated that the D1/D2 sequence alone is insufficient for species delimitation in the Yamadazyma clade, and that the ITS sequence is a good alternative marker to obtain better understanding of the relatedness of the different Yamadazyma species, including its anamorphs.

In the neighbour-joining phylogenetic tree constructed from the combined sequences of the D1/D2 domains of the LSU rRNA gene and ITS regions, Y. dushanensis sp. nov. occupies a basal position with respect to Y. terventina, Y. mexicana and C. trypodendroni (Fig. 1). Similar result was obtained from the Maximum Parsimony analysis (results not shown). These results lend further support to the conclusion that Y. dushanensis sp. nov., Y. terventina, Y. mexicana and C. trypodendroni are regarded as closely related but different species.

Fig. 1
figure 1

Phylogenetic tree derived from neighbour-joining analysis based on the combined sequences of the D1/D2 domains of the LSU rRNA gene and ITS regions, depicting the relationships of Yamadazyma dushanensis sp. nov. with its nearest phylogenetic relatives. Yamadazyma triangularis NRRL Y-5714 T was used as an outgroup. Bootstrap values of above 50 % are given at nodes based on 1000 replications. The bar represents two substitution per 100 nucleotides

Full size image

Cells of Y. dushanensis sp. nov. were ovoid (Fig. 2a) and proliferated by multilateral budding. Pseudohyphae were formed, but true hyphae were not (Fig. 2b). The new species is not able to ferment D-xylose, but weak fermentation of cellobiose in Durham tubes was observed after 3 days. However, the strains of the new species have not been observed to produce ascospores or exhibit conjugation on the most common sporulation media (YM agar, 5 % malt extract agar, corn meal agar and YCBAS agar), either alone or in pairwise mixtures, at 15 and 25 °C for 1–4 weeks. In spite of this result, the novel species has been named Y. dushanensis and not C. dushanensis, according to the ‘one fungus—one name’ recommendation [25].

Fig. 2
figure 2

Photomicrographs of Yamadazyma dushanensis sp. nov. NYNU 14668T. a Budding cells grown on YM broth for 3 days at 25 °C. b Pseudohyphae formed on cornmeal agar after 12 days at 25 °C. Bar 10 μm

Full size image

Yamadazyma dushanensis sp. nov. could be distinguished physiologically from Y. terventina with respect to its ability to assimilate d-arabinose, melibiose and raffinose, and grow at 37 °C, as well as its inability to assimilate dl-lactate. The new species also differed from Y. mexicana by positive assimilation of l-sorbose, growth in 10 % NaCl plus 5 % glucose, and lack of lactose assimilation. The new species was easily separated from Candida sergipensis based on its ability to assimilate L-sorbose and melibiose and grow at 37 °C (Table 1).

Table 1 Physiological characteristics that differentiate Yamadazyma dushanensis sp. nov. from closely related taxa
Full size table

Species in the Yamadazyma clade are very common and could be isolated from diverse habitats, such as water, plants, animals and guts of insects and termites [4–10, 26, 27]. Many of these species have been isolated from plants, such as Y. akitaensis, Y. mexicana, Candida diospyri, Candida buinensis; whereas Candida michaelii, Candida gorgasii, Candida lessepsii, Candida cerambycidarum, Candida endomychidarum, Candida amphixiae, Candida diddensiae, Candida naeodendra and Candida dendronema exhibited an association with the gut of beetles and other insects [4, 5]. Until the present, no members of this clade have been reported to have been isolated from rotten wood. The presence of this novel species from rotting wood in this study may be attributed to the idea that these yeasts are being carried to rotten wood by visiting insects. Therefore, the rotten wood may be an interesting subject for further investigation of yeast in this clade.

Description of Yamadazyma dushanensis Hui, Wang, Ren and Li sp. nov

In YM broth after three days at 25 °C, cells are ovoid and variable in size (2–5 × 2.5–7 μm) and occur singly or in pairs (Fig. 2a). Budding is multilateral. Sediment formed after 1 month, but no pellicle was observed. On the YM agar after 3 days at 25 °C, the streak culture was butyrous, white, raised with a smooth surface and had an entire margin. In Dalmau plates after 12 days on cornmeal agar at 25 °C, pseudohyphae were formed, but true hyphae were not (Fig. 2b). No asci or signs of conjugation were observed after growth on the most common sporulation media. A summary of the physiological and other growth characteristics of Y. dushanensis is given in Table 2. The GenBank/EMBL/DDBJ accession numbers for the sequences of the D1/D2 domains of the LUS rRNA gene and the ITS regions of Y. dushanensis sp. nov. NYNU 14668T are KM272248 and KM272249, respectively. The Mycobank deposit number is MB 811446.

Table 2 Physiological characteristics of Yamadazyma dushanensis sp. nov.
Full size table

The type strain NYNU 14668T was isolated from rotten wood collected in June 2014 from Dushan Forest Park in Nanyang, Henan Province, Central China. The living culture from type was deposited at China Center of Industrial Culture Collection (CICC), Beijing, China as CICC 33051T and Centraalbureau voor Schimmelcultures (CBS), Utrecht, the Netherlands as CBS 13914 T.

The species name dushanensis (du.shan.en′sis. N.L. fem. adj.) refers to Dushan, Nanyang, Henan Province, central China, the geographical origin of the species.