International Journal of Systematic and Evolutionary Microbiology (2013), 63, METHODS

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1 International Journal of Systematic and Evolutionary Microbiology (2013), 63, DOI /ijs Proposal of Zygosaccharomyces parabailii sp. nov. and Zygosaccharomyces pseudobailii sp. nov., novel species closely related to Zygosaccharomyces bailii Sung-Oui Suh, 1 Pushpa Gujjari, 1 Carolyn Beres, 2 Brian Beck 1 and Jianlong Zhou 1 Correspondence Sung-Oui Suh ssuh@atcc.org 1 Mycology and Botany Program, American Type Culture Collection (ATCC), University Blvd, Manassas, VA 20110, USA 2 biomérieux, Inc., 595 Anglum Road, Hazelwood, MO 63042, USA Twenty-three yeast strains traditionally identified as Zygosaccharomyces bailii were studied in order to clarify their taxonomy and phylogenetic relationships. The molecular phylogeny from rrna gene sequences showed that these yeasts were well divided into three major groups, and two of the groups could be clearly distinguished from the type strain of Z. bailii at the species level. Therefore, we propose Zygosaccharomyces parabailii sp. nov. (type strain ATCC T 5NBRC 1047 T 5NCYC 128 T 5CBS T ) and Zygosaccharomyces pseudobailii sp. nov. (type strain ATCC T 5NBRC 0488 T 5CBS 2856 T ) to accommodate the yeasts belonging to the two groups. By conventional physiological tests, Z. bailii and the two novel species are not clearly distinguished from one another, as variations exist more frequently between individual strains and are not species-specific. However, the conclusions from rrna gene sequence analyses are well supported by genome fingerprinting patterns as well as other protein-coding gene sequence comparisons. INTRODUCTION Zygosaccharomyces bailii is a widely distributed yeast species that is often associated with food spoilage, particularly of acidified, preserved foods containing high concentrations of fermentable sugars (Thomas & Davenport, 1985; Cole & Keenan, 1987; Makdesi & Beuchat, 1996; James & Stratford, 2011). The yeast has been proposed as a new host for several biotechnological processes (e.g. Branduardi et al., 2004) due to its ability to tolerate such environments at relatively high temperatures, which could improve the efficiency of these processes under restrictive conditions. Moreover, the high growth rate of Z. bailii and its high biomass yield make this yeast particularly attractive for heterologous protein and metabolite production (e.g. Sousa et al., 1996, 1998). Abbreviations: ITS, internal transcribed spacer; LSU, large subunit; SSU, small subunit. The GenBank/EMBL/DDBJ accession numbers for the sequences generated in this study are FJ914883, FJ914902, JX JX and KC KC678108, as detailed in Table S1. The Mycobank numbers for Zygosaccharomyces parabailii and Zygosaccharomyces pseudobailii are MB and MB , respectively. Three supplementary figures and a supplementary table are available with the online version of this paper. Despite their well-known role in food/beverage spoilage, accurate identification of Z. bailii and related yeasts to the species level using conventional taxonomic tests remains problematic. An inability to ferment and assimilate many of the carbon compounds typically used in yeast identification, as well as ambiguous tests results due to strain variability, often hampers identification (James & Stratford, 2011). Furthermore, significant intraspecific variation in internal transcribed spacer (ITS) sequences was also reported among some strains of the species (James et al., 1996), which may cause difficulties for the use of this barcode region for identifying the species (Schoch et al., 2012). We hypothesized that polyphasic analyses of the yeasts encompassed by Zygosaccharomyces bailii sensu lato may lead to a more accurate understanding of their phylogenetic relationship and taxonomic status. Here we report the molecular, physiological and morphological characterization of these yeasts, and propose two novel species near Z. bailii. METHODS Yeast strains and characterization. Strains of Z. bailii sensu lato and related taxa were selected from the ATCC Mycology Collection or were provided by biomérieux, Inc. (Table 1). Morphological observations and metabolic tests comprising the yeast standard G 2013 IUMS Printed in Great Britain

2 Two novel species near Zygosaccharomyces bailii Table 1. Strains of Z. bailii and related yeasts investigated in this study The genotypes were designated LSU1 LSU7 for the D1/D2 region of the LSU rrna gene and ITSa ITSn for the ITS region including the 5.8S rrna gene. The identification test with the VITEK 2 system was performed at least twice for each strain. If the result was not consistent for a strain, all the identifications from each test are listed. Culture collections: ATCC, American Type Culture Collection, Manassas, VA, USA; CBS, Centraalbureau voor Schimmelcultures, Utrecht, Netherlands; MUCL, BCCM/MUCL (Agro)Industrial Fungi & Yeasts Collection, Université Catholique de Louvain, Belgium; NBRC, Biological Resource Center, National Institute of Technology and Evaluation, Japan; NCYC, National Collection of Yeast Cultures, Institute of Food Research, UK; NRRL, Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, IL, USA; PYCC, Portuguese Yeast Culture Collection, New University of Lisbon, Portugal. Strain Source and comments Genotype from DNA sequences VITEK 2 result with YST identification card (P) Z. bailii ATCC T (5CBS 680 T 5NCYC 1416 T 5NRRL Y-2227 T ) ATCC ATCC 8766 (5CBS 749 5NCYC 573 5NRRL Y-1011) ATCC (5CBS NRRL Y-1404) Brewery, Tokyo; type strain of Z. bailii Deposited at ATCC as Saccharomyces acidifaciens Domestic wine; type strain of Zygosaccharomyces acidifaciens Vinegar, Spain; deposited as Saccharomycodes mestris LSU1/ITSa Unidentified organism; low discrimination LSU1/ITSb Z. bailii (93 %); Rhodotorula glutinis/r. mucilaginosa (91 %) LSU1/ITSc R. glutinis/r. mucilaginosa (94 95 %); low discrimination LSU1/ITSc Z. bailii (99 %) ATCC (5PYCC 4267) Bottled white wine, Portugal LSU1/ITSf Z. bailii (99 %) ATCC Used for degradation of malic acid in grape must LSU1/ITSe Unidentified organism/candida sphaerica (86 %) ATCC Used for production of sherry wine LSU2/ITSd R. glutinis/r. mucilaginosa (98 %); low discrimination ATCC Used for production of sherry wine LSU2/ITSd Candida sake (96 %); R. glutinis/r. mucilaginosa (98 %); low discrimination Zygosaccharomyces parabailii sp. nov. ATCC T (5NBRC 1047 T 5NCYC 128 T 5CBS T ) Wild-type isolate associated with fermentation LSU4/ITSj C. sake (91 %); low discrimination ATCC MYA-4549 (5MUCL 51627) Quality control strain for VITEK 2 LSU3/ITSh Z. bailii (96 97 %) yeast identification card for Z. bailii ATCC (5NCYC 1520) Netherlands LSU4/ITSi Z. bailii (96 %) ATCC Imported citrus concentrate, LSU4/ITSh Z. bailii (96 %) Netherlands ATCC Citrus paste, Netherlands LSU4/ITSk Z. bailii (96 %) ATCC Salad dressing, USA; caused spoilage LSU5/ITSi Z. bailii (92 93 %) outbreak of salad dressing ATCC 8099 (5NCYC 580) Not known LSU6/ITSg Z. bailii (97 %); R. glutinis/r. mucilaginosa (95 %) Provided by biomérieux Inc. LSU3/ITSh Z. bailii (99 %) Provided by biomérieux Inc. LSU4/ITSi Z. bailii (99 %) Provided by biomérieux Inc. LSU3/ITSh Z. bailii (99 %) Zygosaccharomyces pseudobailii sp. nov. ATCC T (5CBS 2856 T 5NBRC 0488 T ) Worcestershire sauce, Japan LSU7/ITSl Z. bailii (99 %) ATCC 2602 (5NRRL Y-54) Deposited as Zygosaccharomyces mandshuricus LSU7/ITSm Z. bailii (96 %); R. glutinis/r. mucilaginosa (98 %) ATCC 2333 (5NRRL Y-125) Deposited as Z. mandshuricus LSU7/ITSm Z. bailii (97 %); R. glutinis/r. mucilaginosa (97 %) ATCC Pickle relish; deposited as S. LSU7/ITSn Z. bailii (96 %) acidifaciens ATCC 13 Deposited as Zygosaccharomyces LSU7/ITSl Z. bailii (96 97 %) naniwaensis Z. bisporus Provided by biomérieux Inc

3 S.-O. Suh and others description were performed according to established methods (Barnett et al., 2000; Kurtzman et al., 2011). Assimilation tests for carbon and nitrogen compounds were conducted in liquid media. Automated identification by the VITEK 2 system was performed with the YST card (biomérieux) by following the manufacturer s instructions. Ascospore formation was tested on YM agar, 2 % malt agar, cornmeal agar, V8 juice agar and V8 Ray agar (ATCC medium 1970; 0.2 g CaCO 3, 50 ml V-8 juice and 20 g agar in 1 l Ray broth) at 25 uc for up to 4 weeks. DNA sequencing and molecular phylogenetic analyses. Nucleic acids were extracted and purified following the procedures of Lee & Taylor (1990). The primer sets ITS5/ITS4 and LR0R/LR3 were used for PCR amplification of nuclear rrna gene repeats (White et al., 1990; Hausner et al., 1993). Six additional genes were also sequenced for representatives of each major group from the rrna gene analyses. These genes and the primers used for PCR and sequencing were: the cytochrome oxidase subunit 2 gene (coxii) with primers COII-3 and COII-5 (Belloch et al., 2000); the mitochondrial small-subunit (mssu) rrna gene with MS-1A and MS-2 (Kurtzman & Robnett, 2003); the b-tubulin gene with btub3 and btub4r (Einax & Voigt, 2003); the translation elongation factor 1-a gene (EF-1a) with primers EF1-983F and EF1-2218R (Rehner & Buckley, 2005); the RNA polymerase II largest subunit gene (RPB1) with primers RPB1-Af and RPB1-Cr (Stiller & Hall, 1997; Matheny et al., 2002); and the RNA polymerase II second-largest subunit gene (RPB2) with primers frpb2-5f and frpb2-7cr (Kurtzman & Robnett, 2003). PCR conditions for the additional genes were those recommended in the references cited for each primer. PCR products were purified using Quantum Prep PCR Kleen spin columns (Bio-Rad Laboratories), and the purified PCR products were used as templates for sequencing with an ABI PRISM BigDye Terminator cycle sequencing kit version 3.1 (PE Applied Biosystems) by using an ABI 3130xl automated DNA sequencer. GenBank accession numbers for the DNA sequences generated from this study are detailed in Fig. 1 and Table S1, available in IJSEM Online. rrna gene sequences were aligned by using the multiple-alignment program CLUSTAL_X (Thompson et al., 1997) and the alignment was optimized visually. Maximum-parsimony analyses were performed using PAUP 4.0b10 (Swofford, 2002). Heuristic tree searches were executed using the tree bisection-reconnection branchswapping algorithm with random sequence analysis. Bootstrap values of the most parsimonious tree were obtained from 0 replications. Bayesian Markov chain Monte Carlo analysis was performed with MRBAYES version 3.0b4 (Ronquist & Huelsenbeck, 2003) to estimate the probability of nodes. The analysis consisted of generations of four chains sampling every 10 generations, and the first 000 generations were discarded as burn-in. The remaining trees were imported into PAUP to estimate the posterior probability. Base-pair differences were counted using BLAST 2 sequences (Tatusova & Madden, 1999) or from a manually aligned sequence database. PCR fingerprinting and DiversiLab. For microsatellite-primed PCR, methods and conditions for DNA extraction, PCR and electrophoresis followed Sampaio et al. (2001). Two primers, (GTG) 5 and (GAG) 5, were used for the analysis. For DNA fingerprinting by DiversiLab (biomérieux), all strains were cultured on Saboraud dextrose agar (Remel) at uc for h prior to handling. DNA was extracted using the Mo Bio UltraClean Microbial DNA isolation kit (biomérieux), and was diluted to approximately 25 ng ml 21. Amplification of repetitive sequences was carried out using the DiversiLab Saccharomyces kit (biomérieux) and an Applied Biosystems 9700 Thermocycler programmed with rep- PCR protocol reppcr50v3 (initial denaturation at 92 uc for 30 s; 35 cycles of denaturation at 92 uc for 30 s, annealing at 50 uc for 45 s and extension at 72 uc for 60 s; and final extension at 72 uc for 5 min) as recommended in the Saccharomyces kit. Amplified DNA was run on DiversiLab chips using an Agilent Bioanalyser (bio- Mérieux) and compared using Kullback Leibler analysis. RESULTS AND DISCUSSION A total of 23 yeast strains that have been identified as Z. bailii were sequenced for the D1/D2 region of the largesubunit (LSU) rrna gene and the ITS region, including the 5.8S rrna gene. As a result, the yeasts were divided into seven genotypes by their D1/D2 sequences (LSU1 LSU7; 606 nt in total), which showed one to five nucleotide variations between different genotypes (Table 1; Fig. S1). Strain ATCC MYA-4549 and two biomérieux strains, and , in genotype LSU3 were the most distinct group from the type strain of Z. bailii (ATCC T ; genotype LSU1), with four substitutions and gaps in the D1/D2 sequence. Strains of LSU2 and two other genotypes, LSU3 and LSU5, were distinct from each other by five nucleotide substitutions. On the other hand, the ITS region showed more significant variations among the test strains. Fourteen genotypes (ITSa ITSn) were recognized from the 23 strains, which could be distinguished from each other by 1 to 71 nucleotide substitutions and gaps in the 806-bp aligned sequences (Table 1; Fig. S2). ATCC and ATCC 13 in genotype ITSl were the most distinct strains from the type strain of Z. bailii, ATCC T (ITSa), which showed 71 nucleotide differences including gaps. Genotypes ITSl, ITSm and ITSn were highly divergent from all other ITS types based on the sequence comparison (Fig. S2). The pattern of ITS genotyping in this study was somewhat similar to that of James et al. (1996), which was performed with only seven Z. bailii strains stored in the National Collection of Yeast Cultures (NCYC). They found three ITS types, designated A, B and C, from ITS1 and ITS2 region sequences. The sequence of ITS type A is similar to those of ITSa f in this study (James et al., 1996; Fig. S2). Their ITS types B and C correspond to our groups ITSg k and ITSl n, respectively, based on sequence similarity (James et al., 1996; Fig. S2). Both studies clearly indicate that there are at least three major ITS groups among the yeasts that have been identified as Z. bailii by traditional taxonomic criteria. Sequence variations in the ITS region among the three major groups in this study were bp between ITSa f and ITSg k, bp between ITSa f and ITSl n and bp between ITSg k and ITSl n. Genotypes from the D1/ D2 region and the ITS region were correlated, i.e. the strains in LSU1 and LSU2 have ITS sequences of ITSa f, while those in LSU3 6 share the sequences of ITSg k. Strains in LSU7 showed three ITS genotypes, ITSl n (Table 1). This close genotype correlation between the two regions implies that these 23 Z. bailii sensu lato strains have evolved distinctly along three lineages since they diverged from a common ancestor. As the ITS and D1/D2 regions have been widely accepted as fungal DNA barcodes (e.g. Kurtzman, 2006; Kurtzman & Robnett, 1998; Scorzetti et al., 2002; Schoch et al., 2012), the significant variations found in these two genetic loci are strong evidence of the 1924 International Journal of Systematic and Evolutionary Microbiology 63

4 Two novel species near Zygosaccharomyces bailii changes Lachancea thermotolerans NRRL Y-8284 T (FJ153217/U69581) Zygosaccharomyces mellis NRRL Y T (AY046190/U72164) 81 Z. siamensis JCM T (AB565768/AB565756) 73 Zygosaccharomyces sapae CBS ITS copy 3 (AM279466/AJ966342) Zygosaccharomyces sapae CBS ITS copy 2 (AM279464/AJ966342) 60/ Zygosaccharomyces rouxii NRRL Y-229 T (AY046189/U72163) Zygosaccharomyces machadoi CBS T ( /AF432228) 62/ 99/ Zygosaccharomyces gambellarensis CBS T ( /FR725931) Zygosaccharomyces sapae CBS ITS copy 1 (AM279465/AJ966342) Zygosaccharomyces bisporus NRRL Y T (AY046192/U72162) ATCC MYA-4549 (FJ914883/FJ914902) (JX458129/JX458132) (JX458130/JX458133) ATCC (JX458091/JX458110) ATCC 8099 (JX458095/JX458114) ATCC (JX458092/JX458111) Zygosaccharomyces parabailii 95/ (JX458131/JX458134) 99 ATCC (JX458093/JX458112) 97/96 ATCC T (JX458094/JX458113) ATCC (JX458096/JX458115) ATCC (JX458097/JX458116) ATCC T (JX458098/JX458117) ATCC (JX458099/JX458118) ATCC 8766 (JX458/JX458119) ATCC (JX458101/JX458120) Zygosaccharomyces bailii 99 ATCC (JX458102/JX458121) ATCC (JX458103/JX458122) ATCC (JX458104/JX458123) ATCC 2333 (JX458105/JX458124) ATCC 2602 (JX458106/JX458125) / ATCC (JX458107/JX458126) ATCC 13 (JX458108/JX458127) Zygosaccharomyces pseudobailii ATCC T (JX458109/JX458128) / Zygosaccharomyces kombuchaensis NRRL YB-4811 T (AY046193/AF339904) Zygosaccharomyces lentus NRRL Y T (AY046194/AF339888) Fig. 1. Consensus of parsimonious trees obtained from sequence data of the ITS region and the D1/D2 region of the LSU rrna gene from Z. bailii and related yeasts. Lachancea thermotolerans NRRL Y-8284 T was used as the outgroup taxon. GenBank accession numbers after the names of yeast species are for the sequences of ITS and D1/D2 regions, respectively, used in the analyses. Numbers on tree branches indicate percentages of bootstrap support derived from 0 samples (upper) and the probability of nodes from Bayesian Markov chain Monte Carlo analysis (lower). need for taxonomic revision among traditionally recognized Z. bailii strains. Similar sequence variations were also reported in the two loci among strains of Zygosaccharomyces rouxii isolated from miso and soy sauce (Suezawa et al., 2008). However, as some of the genotypes in Z. rouxii are homologous in one or other of the two loci, it is difficult to distinguish them clearly as two or more species based on sequence comparison. A parsimonious tree was generated from the combined dataset of ITS and D1/D2 sequences from the Z. bailii sensu lato strains and other Zygosaccharomyces species using Lachancea thermotolerans NRRL Y-8284 T as an outgroup (Fig. 1). Z. bailii and related yeasts were well divided into three major clades on the tree: (i) the type strain ATCC T and seven other strains belong to genotypes LSU1 2/ITSa f, (ii) ten strains belong to genotypes LSU3 6/ ITSg k and (iii) five strains belong to LSU7/ITSl n (Fig. 1). These three groups could be distinguished from each other by approximately 3 5 % sequence variation in the ITS and D1/D2 regions, while the variation among strains within each group was less than 0.5 % in the same region. The degree of sequence variation in the two loci among the groups is significant enough to separate them at the species level, as concluded in many studies (e.g. Kurtzman, 2006; Kurtzman & Robnett, 1998; Scorzetti et al., 2002). Therefore, we propose two novel species near Z. bailii, Zygosaccharomyces parabailii sp. nov. and Zygosaccharomyces pseudobailii sp. nov., to accommodate the yeast strains that belong to the two major groups distinct from the type strain of Z. bailii (Fig. 1). Zygosaccharomyces bisporus is the taxon closest to Z. bailii and the two novel species in the tree reconstructed from the combined ITS and D1/D2 region sequences (Fig. 1), as well as in those reconstructed from the individual sequences (Fig. S3; Kurtzman et al., 2001; Rosa & Lachance, 2005; Saksinchai et al., 2012; James & Stratford, 2011; Solieri et al., 2013). Interestingly, some intragenomic polymorphism in the ITS region has been reported within individual strains of Zygosaccharomyces species (Gordon & Wolfe, 2008; Solieri et al., 2007, 2013). Zygosaccharomyces sapae CBS T is a good example, which has three different ITS types in the genome (Figs 1 and S3). Among the 23 strains of Z. bailii and the two novel species tested in this study, however, we did not find any intragenomic polymorphism in the region, although some poly(a) or poly(t) repeats often caused difficulties in sequencing the gene (Fig. S2)

5 S.-O. Suh and others Z. bailii and the two novel species were also well distinguished from one another by the sequences of additional genes other than those in the nuclear rrna gene repeat (Table S1). Although the two mitochondrial genes, coxii and the mssu rrna gene, were not variable enough to distinguish the three species, the other four protein-coding genes showed significant sequence variations among the species: i.e (4 4.8 %) nucleotide mismatches in the b-tubulin gene, (2 3.3 %) in EF- 1a, ( %) in RPB1 and (4 7.2 %) in RPB2. Intraspecific variations of RPB1 and RPB2 were less than 0.6 % in both genes (Table S1). The results from genome-wide DNA fingerprinting were consistent with the genetic heterogeneity in the Z. bailii sensu lato strains and the existence of three major clades revealed by rrna gene sequence analyses. The PCR amplification patterns with DiversiLab clearly grouped the 23 strains into three clusters with significant distances in similarity scores on the dendrogram (Fig. 2): Z. bailii and Z. parabailii were separated from each other at approximately 80 %, while Z. pseudobailii was distinguished from the other two species at a similarity of 60 % (Fig. 2). The result from microsatellite-primed PCR was similar to that for the DiversiLab test, and clearly showed three distinct PCR amplification patterns (data not shown). The ecology of the three species could be similar, based on information from isolation sources, often being associated with food spoilage, especially in acidified preserved foods with high concentrations of sugars (Table 1). Interestingly, the type strains of the two novel species, ATCC T and ATCC T, are known to have episomal plasmids, psb1 and psb2, that resemble the 2 mm DNA of Saccharomyces cerevisiae (Toh-e et al., 1984). It appears that these plasmids are common among strains of Z. bailii and related species, which may help exchange of genetic information across these yeasts. It is difficult to distinguish Z. bailii and the two novel species from one another with conventional physiological tests, because many assimilation or fermentation abilities of the yeasts are variable at the strain level, e.g. those for sucrose, inulin or xylitol. Phenotypic variations were confirmed among Z. bailii and related yeasts from automated identification tests by the VITEK 2 system, although variation was more frequent among individual strains and was not species-specific (Table 1). As such, some of the Z. bailii strains, including the type strain ATCC T, were not correctly identified as the species by the current VITEK 2 system, while most strains in Z. parabailii and Z. pseudobailii were identified as Z. bailii. In light of our new understanding of the taxonomic and phylogenetic relationships among strains of Z. bailii sensu lato, an updated VITEK system profile that reflects this taxonomic revision is desirable. Strain ATCC MYA-4549 (5MUCL 51627) has been designated the quality control strain for Z. bailii for the VITEK 2 system based on its biochemical profile. Taxonomically, the strain was clearly distinguished from Z. bailii Z. parabailii Z. pseudobailii Z. bisporus Similarity (%) Fig. 2. Dendrogram and genome fingerprinting profiles of strains in Z. bailii and the two novel species generated by DiversiLab. Similarity scores on the dendrogram were calculated by Kullback Leibler analysis. Z. bisporus was used as a reference strain. the type strain of Z. bailii by molecular analyses and was reclassified as a strain of Z. parabailii in this study (Table 1, Fig. 1). Data from this study may be used to update the nomenclature of the Z. bailii quality control strain for the VITEK 2 system. Description of Zygosaccharomyces parabailii sp. nov. Suh, Gujjari, Beres, Beck & Zhou Zygosaccharomyces parabailii (pa.ra.bai9li.i. Gr. prep. para alongside of, resembling, like, beside; N.L. gen. n. bailii a specific epithet; N.L. gen. n. parabailii bailii-like, referring to its phylogenetic closeness to Z. bailii). After 7 days of growth on YM agar at 25 uc, cells are ellipsoidal to ovoid ( mm) and occur singly, in pairs or in small clusters (Fig. 3a). Pseudohyphae and true T T T 1926 International Journal of Systematic and Evolutionary Microbiology 63

6 Two novel species near Zygosaccharomyces bailii hyphae are not present. Colonies are cream-coloured, butyrous and smooth and have a somewhat uneven margin. After 11 days of growth in Dalmau plate culture on cornmeal agar at 25 uc, pseudohyphae and true hyphae are not present. Aerobic growth is white to cream-coloured and smooth. Sporulation occurs on 2 % malt extract agar and V8 Ray agar after 7 10 days at 25 uc. Vegetative cells may differentiate after conjugation between individual cells into persistent asci containing up to two ascospores, which are spherical to ovoid ( mm; Fig. 3b). Glucose and sucrose (variable) are fermented; galactose, maltose, methyl a-d-glucoside, trehalose, melibiose, lactose, cellobiose, melezitose, raffinose, inulin, starch and D-xylose are not fermented. Glucose, galactose (variable), L-sorbose (variable), sucrose (variable), trehalose, glycerol, ribitol, xylitol (variable), D-glucitol, D-mannitol, D-glucono-1,5- lactone (variable), 2-keto-D-gluconate (variable) and ethanol (variable) are assimilated; D-glucosamine, D-ribose, D-xylose, L- and D-arabinose, L-rhamnose, maltose, methyl a-d-glucoside, cellobiose, salicin, arbutin, melibiose, lactose, raffinose, melezitose, inulin, soluble starch, erythritol, L-arabinitol, galactitol, myo-inositol, D-gluconate, D- glucuronate, D-galacturonic acid, succinate, DL-lactate, citrate, methanol, propane-1,2-diol, butane-2,3-diol and quinic acid are not assimilated. L-Lysine (variable), ethylamine, cadaverine and D-tryptophan (variable) are assimilated; potassium nitrate, sodium nitrite, creatine, creatinine, D-glucosamine (as nitrogen source) and imidazole are not assimilated. Growth without vitamins is negative. Growth in 0.01 % cycloheximide is negative. Growth on 1 % acetic acid, 50 % D-glucose, 60 % D-glucose (variable) and 10 % NaCl is positive, but growth on 16 % NaCl is negative. Growth at 30 uc is positive, while growth at 37 uc is negative. Starch-like compounds are not produced. Acetic acid production, the diazonium blue B reaction and urease activity are absent. The type strain is ATCC T (5NBRC 1047 T 5NCYC 128 T 5CBS T ), a wild-type isolate associated with fermentation. GenBank accession numbers for the DNA barcode sequences of the type strain are JX and JX (ITS and D1/D2 region of LSU rrna gene, respectively). The Mycobank number for Zygosaccharomyces parabailii is MB Description of Zygosaccharomyces pseudobailii sp. nov. Suh, Gujjari, Beres, Beck & Zhou Zygosaccharomyces pseudobailii (pseu.do.bai9li.i. Gr. adj. pseudês false; N.L. gen. n. bailii a specific epithet; N.L. gen. n. pseudobailii false bailii, referring to its phylogenetic closeness to Z. bailii). After 7 days of growth on YM agar at 25 uc, cells are ellipsoidal to ovoid ( mm) and occur singly, in pairs or in small clusters (Fig. 3c). Pseudohyphae and true hyphae are not present. Colonies are cream-coloured, butyrous and smooth and have a somewhat clear margin. Colonies on 2 % malt extract agar under the Fig. 3. Cells of Z. parabailii sp. nov. ATCC T (a, b) and Z. pseudobailii sp. nov. ATCC T (c, d). Vegetative yeast cells (a, c) after 7 days on YM agar at 25 6C, and asci with two ascospores (arrows) on V8 Ray agar (b) or 2 % malt extract agar (d) after 9 days at 25 6C. Bar, 10 mm. same conditions are slightly wrinkled on top. After 11 days of growth in Dalmau plate culture on cornmeal agar at 25 uc, pseudohyphae and true hyphae are not present. Aerobic growth is white to cream-coloured and smooth. Sporulation occurs on 2 % malt extract agar and V8 Ray agar after 9 days at 25 uc. Vegetative cells may differentiate after conjugation between individual cells into persistent asci containing up to two ascospores that are spherical to ovoid ( mm; Fig. 3d). Glucose is fermented; galactose, maltose, methyl a-d-glucoside, sucrose, trehalose, melibiose, lactose, cellobiose, melezitose, raffinose, inulin, starch and D-xylose are not fermented. Glucose, galactose (variable), L-sorbose (variable), trehalose, glycerol, ribitol, D-glucitol, D-mannitol, D-glucono-1,5-lactone (variable), 2-keto-D-gluconate (weak), DL-lactate (weak) and ethanol are assimilated; sucrose, D-glucosamine, D-ribose, D-xylose, L- and D-arabinose, L-rhamnose, maltose, methyl a-dglucoside, cellobiose, salicin, arbutin, melibiose, lactose, raffinose, melezitose, inulin, soluble starch, erythritol, xylitol, L-arabinitol, galactitol, myo-inositol, D-gluconate, D-glucuronate, D-galacturonic acid, succinate, citrate, methanol, propane-1,2-diol, butane-2,3-diol and quinic acid are not assimilated. L-Lysine, ethylamine, cadaverine and D- tryptophan (variable) are assimilated; potassium nitrate, sodium nitrite, creatine, creatinine, D-glucosamine (as nitrogen source) and imidazole are not assimilated. Growth without vitamins is negative. Growth in 0.01 % cycloheximide is negative. Growth on 1 % acetic acid, 50 % D-glucose, 60 % D-glucose and 10 % NaCl (weak) is positive, but growth on 16 % NaCl is negative. Growth at 37 ucisvariable. Starch-like compounds are not produced. Acetic acid

7 S.-O. Suh and others production, the diazonium blue B reaction and urease activity are absent. The type strain is ATCC T (5NBRC 0488 T 5CBS 2856 T ), isolated from Worcestershire sauce in Japan. Gen- Bank accession numbers for the DNA barcode sequences of the type strain are JX and JX (ITS and D1/D2 region of LSU rrna gene, respectively). The Mycobank number for Zygosaccharomyces pseudobailii is MB ACKNOWLEDGEMENTS We thank Jamin Letcher for performing VITEK 2 analyses and David Creely for his assistance with DiversiLab data interpretation. REFERENCES Barnett, J. A., Payne, R. W. & Yarrow, D. (2000). Yeasts: Characteristics and Identification, 3rd edn. Cambridge: Cambridge University Press. Belloch, C., Querol, A., García, M. D. & Barrio, E. (2000). Phylogeny of the genus Kluyveromyces inferred from the mitochondrial cytochrome-c oxidase II gene. Int J Syst Evol Microbiol 50, Branduardi, P., Valli, M., Brambilla, L., Sauer, M., Alberghina, L. & Porro, D. (2004). The yeast Zygosaccharomyces bailii: a new host for heterologous protein production, secretion and for metabolic engineering applications. FEMS Yeast Res 4, Cole, M. B. & Keenan, M. H. J. (1987). Effects of weak acids and external ph on the intracellular ph of Zygosaccharomyces bailii, and its implications in weak-acid resistance. Yeast 3, Einax, E. & Voigt, K. (2003). Oligonucleotide primers for the universal amplification of b-tubulin genes facilitate phylogenetic analyses in the regnum Fungi. Org Divers Evol 3, Gordon, J. L. & Wolfe, K. H. (2008). Recent allopolyploid origin of Zygosaccharomyces rouxii strain ATCC Yeast 25, Hausner, G., Reid, J. & Klassen, G. R. (1993). On the subdivision of Ceratocystis s.l., based on partial ribosomal DNA sequences. Can J Bot 71, James, S. A. & Stratford, M. (2011). Zygosaccharomyces Barker (1901). In The Yeasts, a Taxonomic Study, 5th edn, pp Edited by C. P. Kurtzman, J. W. Fell & T. Boekhout. London: Elsevier. James, S. A., Collins, M. D. & Roberts, I. N. (1996). Use of an rrna internal transcribed spacer region to distinguish phylogenetically closely related species of the genera Zygosaccharomyces and Torulaspora. Int J Syst Bacteriol 46, Kurtzman, C. P. (2006). Yeast species recognition from gene sequence analyses and other molecular methods. Mycoscience 47, Kurtzman, C. P. & Robnett, C. J. (1998). Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73, Kurtzman, C. P. & Robnett, C. J. (2003). Phylogenetic relationships among yeasts of the Saccharomyces complex determined from multigene sequence analyses. FEMS Yeast Res 3, Kurtzman, C. P., Robnett, C. J. & Basehoar-Powers, E. (2001). Zygosaccharomyces kombuchaensis, a new ascosporogenous yeast from Kombucha tea. FEMS Yeast Res 1, Kurtzman, C. P., Fell, J. W., Boekhout, T. & Robert, V. (2011). Methods for isolation, phenotypic characterization and maintenance of yeasts. In The Yeasts, a Taxonomic Study, 5th edn, pp Edited by C. P. Kurtzman, J. W. Fell & T. Boekhout. London: Elsevier. Lee, S. B. & Taylor, J. W. (1990). Isolation of DNA from fungal mycelia and single spores. In PCR Protocols: a Guide to Methods and Applications, pp Edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White. San Diego: Academic Press. Makdesi, A. K. & Beuchat, L. R. (1996). Evaluation of media for enumerating heat-stressed, benzoate-resistant Zygosaccharomyces bailii. Int J Food Microbiol 33, Matheny, P. B., Liu, Y. J., Ammirati, J. F. & Hall, B. D. (2002). Using RPB1 sequences to improve phylogenetic inference among mushrooms (Inocybe, Agaricales). Am J Bot 89, Rehner, S. A. & Buckley, E. (2005). A Beauveria phylogeny inferred from nuclear ITS and EF1-a sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97, Ronquist, F. & Huelsenbeck, J. P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, Rosa, C. A. & Lachance, M.-A. (2005). Zygosaccharomyces machadoi sp. n., a yeast species isolated from a nest of the stingless bee Tetragonisca angustula. Lundiana 6 (Suppl., ), Saksinchai, S., Suzuki, M., Chantawannakul, P., Ohkuma, M. & Lumyong, S. (2012). A novel ascosporogenous yeast species, Zygosaccharomyces siamensis, and the sugar tolerant yeasts associated with raw honey collected in Thailand. Fungal Divers 52, Sampaio, J. P., Gadanho, M., Santos, S., Duarte, F., Pais, C., Fonseca, A. & Fell, J. W. (2001). Polyphasic taxonomy of the genus Rhodosporidium: R. kratochvilovae and related anamorphic species. Int J Syst Evol Microbiol 51, Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C. A., Chen, W., Bolchacova, E., Voigt, K. & other authors (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci U S A 109, Scorzetti, G., Fell, J. W., Fonseca, A. & Statzell-Tallman, A. (2002). Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rdna regions. FEMS Yeast Res 2, Solieri, L., Cassanelli, S. & Giudici, P. (2007). A new putative Zygosaccharomyces yeast species isolated from traditional balsamic vinegar. Yeast 24, Solieri, L., Chand Dakal, T. & Giudici, P. (2013). Zygosaccharomyces sapae sp. nov., isolated from Italian traditional balsamic vinegar. Int J Syst Evol Microbiol 63, Sousa, M. J., Miranda, L., Côrte-Real, M. & Leão, C. (1996). Transport of acetic acid in Zygosaccharomyces bailii: effects of ethanol and their implications on the resistance of the yeast to acidic environments. Appl Environ Microbiol 62, Sousa, M. J., Rodrigues, F., Côrte-Real, M. & Leão, C. (1998). Mechanisms underlying the transport and intracellular metabolism of acetic acid in the presence of glucose in the yeast Zygosaccharomyces bailii. Microbiology 144, Stiller, J. W. & Hall, B. D. (1997). The origin of red algae: implications for plastid evolution. Proc Natl Acad Sci U S A 94, Suezawa, Y., Suzuki, M. & Mori, H. (2008). Genotyping of a miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii, based on sequence analysis of the partial 26S ribosomal RNA gene and two internal transcribed spacers. Biosci Biotechnol Biochem 72, Swofford, D. L. (2002). PAUP*: Phylogenetic analysis using parsimony (and other methods), version 4. Sunderland, MA: Sinauer Associates International Journal of Systematic and Evolutionary Microbiology 63

8 Two novel species near Zygosaccharomyces bailii Tatusova, T. A. & Madden, T. L. (1999). BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences. FEMS Microbiol Lett 174, Thomas, D. S. & Davenport, R. R. (1985). Zygosaccharomyces bailii a profile of characteristics and spoilage activities. Food Microbiol 2, Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, Toh-e, A., Araki, H., Utatsu, I. & Oshima, Y. (1984). Plasmids resembling 2-mm DNA in the osmotolerant yeasts Saccharomyces bailii and Saccharomyces bisporus. J Gen Microbiol 130, White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: a Guide to Methods and Applications, pp Edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White. San Diego: Academic Press