Introduction

The genus Saccharomycodes contains previously two species, namely S. ludwigii, the type species of Saccharomycodes, and S. sinensis (Kurtzman and Robnett 1998; Boundy-Mills et al. 2011). As S. sinensis was reclassified as Yueomyces sinensis based on a multi-gene sequence analysis (Wang et al. 2015b, c, a), currently it is not assigned to Saccharomycodes, and the genus Saccharomycodes includes only one species, S. ludwigii. Saccharomycodes vini (Kroemer and Heinrich) ex Kudryavtsev (1954) [MB#338772] (= Saccharomycodes ludwigii var. vini Kroemer and Heinrich (1922) [MB#456537]), Saccharomycodes bisporus Castelli (1941) [MB#290745] (= Saënkia bispora Castelli ex Kudryavtsev (1960) [MB#456541] = Saccharomycodes ludwigii var. bisporus Castelli ex Hjort (1954) [MB#352485]) and Saccharomycodes lipophora Bachinskaya (1941) [MB#456536] were assigned as synonyms of S. ludwigii as determined from phenotypic characters (Miller and Phaff 1998; Boundy-Mills et al. 2011). However, the genetic diversity within these synonymized species is unclear. Nine strains of S. ludwigii deposited in the CBS yeast collection, Westerdijk Fungal Biodiversity Institute, CBS 820 (type of S. vini), CBS 821 (type of S. ludwigii), CBS 2624 (type of S. bisporus), CBS 1168, CBS 1169, CBS 2625, CBS 5929, CBS 7780 and CBS 8338, were phylogenetically analyzed based on five loci, including the 18S rRNA and 26/28S rRNA ( D1/D2 domains) genes, and fragments of genes encoding the first and second large subunits of RNA polymerase II (RPB1 and RPB2) and translation elongation factor 1-α (TEF1) in this study. Additionally, a new species of Saccharomycodes, S. pseudoludwigii, represented by three strains isolated from fruit and tree bark in China, is proposed.

Materials and methods

Strains and phenotypic characteristics

The S. ludwigii strains, CBS 820 (type of S. vini), CBS 821 (type of S. ludwigii), CBS 1168, CBS1169, CBS 2624 (type of S. bisporus), CBS 2625, CBS 5929, CBS 7780 and CBS 8338, obtained from the CBS yeast collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands, were used in this study (Table 1). Strain CGMCC 2.4525 (= SD 169 = TAli83-3B = CBS 12,643) was isolated from a fruit of Pyrus sorotina, collected from Tai’an, Shandong province in China (Coordinate: 117°08′40′′N 36°18′32′′E). CGMCC 2.4526 T (= SD 139 = AQSZP6L-3 = CBS 12,644) and CGMCC 2.4527 (= SD 200 = AQSZP6L-1 = CBS 12,645) were isolated from the bark of Diospyros kaki, collected from Anqiu, Shandong province in China (Coordinate: 119°02′13′′N 36°43′20′′E). The bark samples were obtained from around the bases of the trees using a sterile scalpel and deposited in sterile 15-ml conical tubes. The fruit samples were collected using sterile sampling bags in the field, cut into small pieces and filled with enrichment medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, 8% v/v ethanol, 200 µg chloramphenicol ml–1 and 1 ml 1 M HCl per liter) described by Wang et al. (2012) and incubated at 25 °C for one or two weeks. The cultured liquid was gradiently diluted and spread on YM plates (1.0% glucose, 0.5% peptone, 0.3% malt extract, 0.3% yeast, 2.0% agar, pH 6.0 with chloramphenicol 100 µg/ml) and incubated at 25 °C for 2–5 days. Single yeast colonies were picked and purified on YM agar plates. The phenotypic and biochemical characters were examined according to standard methods for yeast classification (Kurtzman et al. 2011). The assimilation of carbon and nitrogen compounds were examined in liquid medium at 25 °C. The sexual reproduction of these strains were examined using YPD and McClary acetate agars. A loopful of cells of individual strains were spotted in the agar plate and incubated at 25 °C for four weeks and examined weekly under a microscope for ascosporulation.

Table 1 Yeast strains studied
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Molecular phylogenetic analysis

Genomic DNA was extracted using the method as described by Wang and Bai (2008). Sequences of the ribosomal cistron, including ITS (ITS1 + 5.8S + ITS2), the D1/D2 domains of 26S rRNA, 18S rRNA, and RPB1, RPB2 and TEF1 gene sequences were determined as described by Wang et al. (2014, 2015a, b). Purified PCR products of the ITS region from strain CGMCC 2.4525 were cloned using the pUCm-T Vector system (BBI) and transformed into E. coli DH5α competent cells. White clones were randomly picked and sequenced using the procedure described by Wang et al. (2014). Sequences were aligned with the MUSCLE program in MEGA 7 (Kumar et al. 2016). A general time-reversible model of DNA substitution that assumes a percentage of invariable sites and Γ-distributed substitution rates at the remaining sites (GTR + I + G) was selected for Maximum likelihood (ML) analyses conducted in MEGA 7 using 1000 bootstrap replicates analysis. All sites including the gaps in the alignments were used to construct the ML trees. A phylogenetic network was constructed in SplitsTree4 (Huson and Bryant 2006) using the Consensus Network algorithm with default parameter settings based on the five genes studied i.e. 18S rRNA, 26S rRNA D1/D2, ITS, RPB2 and TEF1, to infer the phylogenetic relationships between the isolates and Saccharomycodes species. The GenBank accession numbers obtained during this study are listed in Table 1.

Results and discussion

Saccharomycodes vini (= S. ludwigii var. vini), S. bisporus (= Saënkia bispora = S. ludwigii var. bisporus) and S. lipophora are current synonyms of S. ludwigii. S. lipophora was described without a Latin description by Bachinskaya (1941), which is invalid according to Art. 39.1 of the International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code, Turland et al. 2018). The type strain of S. lipophora is not available in the Russian Collection of Microorganisms (VKM) nor the CBS Collections. It is unclear whether the type of S. lipophora was lost in other collections, therefore it was not included in this study. Nine strains of S. ludwigii, CBS 820 (type of S. vini), CBS 821 (type of S. ludwigii), CBS 2624 (type of S. bisporus), CBS 1168, CBS 1169, CBS 2625, CBS 5929, CBS 7780 and CBS 8338, have identical D1/D2, 18S and RPB2 sequences, and differed from each other by 5 (including 3 substitutions and 2 inserts/deletions), 4 synonymous substitutions and 5 nucleotides (nt) (1 non-synonymous and 4 synonymous substitutions) in the ITS region, RPB1 and TEF1 genes, respectively. The above results indicated that the nine CBS strains are conspecific as indicated before by phenotypic data (Boundy-Mills et al. 2011).

Three strains isolated from bark and fruit, namely CGMCC 2.4525, CGMCC 2.4526 T and CGMCC 2.4527, are characterized by lemon-shape cells with bipolar budding. These three strains have identical 18S rRNA and D1/D2 sequences, but CGMCC 2.4526 T and CGMCC 2.4527 with identical ITS sequences were found to differ from the nine clone sequences of CGMCC 2.4525 by 0–4 nt mismatches (3 substitutions and 1 insert/deletion) in the ITS region. The ITS sequences of CGMCC 2.4525 is polymorphic and have 0–4 nt differences (3 substitutions including 2 transitions and 1 transversion, and 1 insert/deletion) in the ITS1 region among 9 clones (GenBank numbers KP866238-KP866246). A BLAST search in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using these sequences revealed that these strains were close relatives to S. ludwigii. CGMCC 2.4526 T differs from the type strain of S. ludwigii by 4 substitutions and 1 inserts/deletions in the D1/D2 domains, and by 21 nt (including 12 substitutions and 9 inserts/deletions, 3%) in the ITS regions. Forty three nt (6%, 39 synonymous and 4 non-synonymous substitutions) and 21 nt (2%, 18 synonymous and 3 non-synonymous substitutions) differences were observed between these strains and the type strain of S. ludwigii in the RPB2 and TEF1 genes, respectively.

The 18S, D1/D2, ITS, RPB2 and TEF1 were aligned using the MUSCLE program, resulting in alignments of 1 583, 538, 801, 712 and 866 nucleotide lengths, respectively. 2, 5, 22, 47 and 24 variable sites were found between all strains of Saccharomycodes in the 18S, D1/D2, ITS, RPB2 and TEF1 alignments, respectively. Our multi-locus sequence analysis showed that the strains CGMCC 2.4525, CGMCC 2.4526 T and CGMCC 2.4527 form a clade distinct from S. ludwigii (Fig. 1). The ITS tree including all polymorphic sequences of CGMCC 2.4525 showed that those three strains formed a separate clade from the S. ludwigii clade (Fig. 2). The five-gene phylogenetic network supported that those three strains and S. ludwigii strains formed separate lineage (Fig. 3). The single gene trees of 18S, D1/D2, RPB2 and TEF1 showed a topology in agreement with the five-gene tree (Fig. S1). Thus, a new species of Saccharomycodes, namely Saccharomycodes pseudoludwigii sp. nov., is proposed to accommodate these three strains.

Fig. 1
figure 1

Multi-locus ML tree depicting relationships of S. pseudoludwigii sp. nov. and S. ludwigii. Phylogenetic tree constructed from ML analysis of the combined sequences of the18S rRNA, D1/D2 domains of the 26S rRNA, the ITS region (including the 5.8S rRNA), the RPB2 and TEF1, depicting S. pseudoludwigii sp. nov. and S. ludwigii relationships. Bootstrap percentages over 50% from 1000 bootstrap replicates are shown. The tree is rooted with Saccharomyces cerevisiaae. Bar = 0.02 indicates 2% sequence divergence

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Fig. 2
figure 2

The ITS ML tree depicting relationships of S. pseudoludwigii sp. nov. and S. ludwigii. Phylogenetic tree constructed from ML analysis of the combined sequences of the ITS region (including the 5.8S rRNA), depicting S. pseudoludwigii sp. nov. and S. ludwigii relationships. Bootstrap percentages over 50% from 1000 bootstrap replicates are shown. The tree is rooted with Saccharomyces cerevisiaae. Bar = 0.05 indicates 5% sequence divergence

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Fig. 3
figure 3

The phylogenetic network tree depicting relationships of S. pseudoludwigii sp. nov. and S. ludwigii. Phylogenetic network of the strains of Saccharomycodes. Single gene ML trees inferred with MEGA 7 were investigated in Splitstree 4.13.1 using the ConsensusNetwork algorithm under default settings. Bar = 0.001 substitutions per nucleotide position

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Description of Saccharomycodes pseudoludwigii Q.M. Wang and T. Boekhout sp. nov. MycoBank MB811650

Etymology: The specific epithet pseudoludwigii refers to the morphology of this species being similar to Saccharomycodes ludwigii.

In YM broth, after 3 days at 25 °C, the cells are lemon-shaped (apiculate), 4.0–6.5 × 8.0–24 µm, and occur singly or in pairs. Budding is bipolar. After 1 month at 25 °C, a sediment and ring are present. After 1 month at 25 °C, the streak culture is cream-colored to light yellow, butyrous, smooth, and the margins are entire or somewhat curved. Pseudohyphae are not observed on corn-meal agar. Sporulation is observed on YPD and McClary acetate agars after 5 days at 25 °C. Sausage or lemon-shaped asci are unconjugated and contain two or four globose ascospores (Fig. 4). Ascus walls do not lyse when the spores are mature.

Fig. 4
figure 4

Picture of the cells and asci. Saccharomycodes pseudoludwigii sp. nov. CGMCC 2.4526 asexual cells grown in YM broth for 3 days at 25 °C a and asci and ascospores formed on McClary acetate agar after 5 days at 25 °C b. Bars, 10 μm

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Glucose, sucrose and raffinose are fermented, but galactose, maltose and lactose are not. Glucose, sucrose, raffinose, cellobiose, ethanol, glycerol and salicin are assimilated; whereas galactose, L-sorbose, maltose, lactose, trehalose, D-ribose, inulin, melibiose, melezitose, soluble starch, D-xylose, L-arabinose, D-arabinose, L-rhamnose, D-glucosamine, methanol, erythritol, ribitol, galactitol, D-mannitol, α-methyl-D-glucoside, DL-lactic acid, succinic acid, citric acid, inositol and hexadecane are not. Ammonium sulfate, ethylamine hydrochloride, cadaverine hydrochloride and L-lysine are assimilated, whereas potassium nitrate and sodium nitrite are not. Starch-like compounds are not produced. Growth in vitamin-free medium is absent. Maximum growth temperature is 32 ºC. Growth occurred in 0.0001% cycloheximide, but not in media containing 0.001% cycloheximide or 50% glucose. Urease activity and diazonium blue B reaction are negative.

Physiologically, S. pseudoludwigii differs from S. pseudoludwigii in the inability to assimilate DL-lactic acid.

The holotype CGMCC 2.4526 T was isolated from the bark of Diospyros kaki in Anqiu, Shandong province, China, in August 2008, and is deposited in a metabolically inactive state in the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. The ex-type culture has been deposited in the CBS yeast collection of the Westerdijk Institute, Utrecht, The Netherlands, as CBS 12,644 (= SD 139 = AQSZP6L-3). The paratypes SD 169 = (TAli83-3B) isolated from the fruit of Pyrus sorotina and SD 200 = (AQSZP6L-1) isolated from the bark of Diospyros kaki, have been deposited in the above two collections, as CGMCC 2.4525 (= CBS 12,643), CGMCC 2.4527 (= CBS 12,645), respectively.