DOI: 10.15468/te747s
title: Wickerhamomyces menglaensis f. a., sp. nov., a yeast species isolated from rotten wood

description: This dataset contains the digitized treatments in Plazi based on the original journal article Chai, Chun-Yue, Huang, Lin-Na, Cheng, Han, Liu, Wen-Jing, Hui, Feng-Li (2019): Wickerhamomyces menglaensis f. a., sp. nov., a yeast species isolated from rotten wood. International Journal of Systematic and Evolutionary Microbiology 69 (5): 1509-1514, DOI: 10.1099/ijsem.0.003350, URL: http://dx.doi.org/10.1099/ijsem.0.003350AbstractFive strains, NUNU 16637, NYNU 16645, NYNU 1673, NYNU 1680 and NYNU 1689, of a novel ascomycetous yeast were isolated from the Xishuangbanna tropical rainforest, Yunnan Province, PR China. The five strains shared identical sequences in both of the D1/D2 domains of the large subunit rRNA gene and the internal transcribed spacer (ITS) regions. Sequence analysis showed that they represent undescribed yeast species belonging to the genus Wickerhamomyces. They differed from their closest known species, Wickerhamomyces xylosivorus NBRC 111553 T, by 3.4 % sequence divergence (14 substitutions and six gaps out of 584 bp) in the D1/D2 domains and by 9.6 % sequence divergence (28 substitutions and 24 gaps over 543 bp) in the ITS regions, respectively. The five strains of novel species reproduced asexually; no sexual reproduction could be found. In contrast to W. xylosivorus, the novel yeast species were able to assimilate L- arabinose, inulin, soluble starch, D- mannitol and citrate, and unable to assimilate trehalose, raffinose, 5 -keto-D- gluconate, D- gluconate, ethanol, ethylamine and cadaverine. Growth was observed at 35 Ǫ C. The name Wickerhamomyces menglaensis f.a., sp. nov. is proposed to accommodate these strains, with NYNU 1673 as the holotype.The genus Wickerhamomyces was proposed by Kurtzman et al. with Wickerhamomyces canadensis as the type species, this type species was originally transferred from the genus Pichia [1]. At the time of writing, 17 species of the genus Wickerhamomyces were originally transferred from the genera Pichia, Williopsis and Hansenula following a recent detailed phylogenetic analysis of nucleotide divergence in the genes coding for the large-subunit (LSU) and small-subunit rRNA genes and for elongation factor 1a [1, 2], and 16 species have subsequently been included [3 – 18]. Additionally, eight species of the genus Candida are known to be members of the Wickerhamomyces clade [2]. Species of the genus Wickerhamomyces produce persistent or deliquescent, hat- or Saturn-shaped, spherical ascospores. They are characterized by a negative diazonium blue B colour reaction, the inability to assimilate methanol and the presence of Q-7 as the predominant ubiquinone [1, 2].During a study on yeasts associated with rotten wood in Xishuangbanna tropical rainforest, more than 90 % of the samples used in this study contained yeasts and 145 yeast strains were obtained. Based on the D1/D2 domains of the large subunit (LSU) rRNA gene sequence comparisons, the majority of the yeasts belonged to several major clades in the subphylum Saccharomycotina; some of these species have been identified as novel species in earlier papers, for example Cyberlindnera xishuangbannaensis CBS 14692 [19], Deakozyma yunnanensis CBS 14688 [20] and Vanrija jinghongensis CBS 15229 [21]. Amongst these associates, five novel yeast strains could not be ascribed to any validly known species. Further sequence analysis of the D1/D2 domains of the LSU rRNA gene and the internal transcribed spacer (ITS) regions showed that these strains represent a novel species belonging to the genus Wickerhamomyces. In this report, a novel species, Wickerhamomyces menglaensis f.a., sp. nov., is described.More than 50 rotten wood samples were collected from the different locations in the Xishuangbanna tropical rainforest, Yunnan Province, PR China. Three strains, NYNU 16637, NYNU 16645 and NYNU 1673, were isolated from three samples of rotting wood collected in in Menglun, Mengla (approximate GPS coordinates: 21 Ǫ 41′ N 101 Ǫ 25′ E). The other two strains, NYNU 1680 and NYNU 1689, were isolated from two samples of rotting wood collected in Galan, Jinghong (approximate GPS coordinates: 21 Ǫ 27′ N 100 Ǫ 25′ E). The yeast strains were isolated from rotting wood samples in accordance with the methods described by Hui et al. [22]. Each sample (1 g) was added to 20 ml sterile yeast extract–malt extract (YM) broth [1 % glucose (w/v), 0.5 % peptone (w/v), 0.3 % yeast extract (w/v) and 0.3 % malt extract (w/v); (pH 5.4) supplemented with 0.02 % chloramphenicol (w/v)] in a 150 ml Erlenmeyer flask and then incubated at 25 Ǫ C for 3 days on a rotary shaker. Subsequently, 0.1 ml enrichment culture and appropriate decimal dilutions were spread on YM agar plates supplemented with 0.02 % chloramphenicol and then incubated at 25 Ǫ C for 3–4 days. Different yeast colony morphotypes were then purified at least twice and then stored on YM agar slants at 4 Ǫ C or in 15 % glycerol at ‒80 Ǫ C.Fig. 1. Phylogenetic tree derived from the neighbour-joining analysys based on sequences of the D1/D2 domains of the LSU rRNA gene, showing the placement of Wickerhamomyces menglaensis f.a., sp. nov. in the Wickerhamomyces clade. Saccharomyces cerevisiae NRRL Y-12632 was used as an outgroup. Sequences were retrieved from the GenBank and CBS (*) databases. Bootstrap values of above 50 % are given at nodes based on 1000 replications. Bar, 0.02 substitutions per site.Morphological, physiological and biochemical characteristics were examined according to standard methods by Kurtzman et al. [2]. Assimilation tests were performed by replica plating on solid and in liquid media twice, and the results were recorded after 5 and 21 days of incubation. Mating and ascus formation were assessed on YM, 5 % malt extract (MEA), corn meal agar (CEA), V8 and diluted V8, yeast carbon base supplemented with 0.01 % ammonium sulphate (YCBAS) agar (1.1 % yeast carbon base, 0.01 % ammonium sulphate and 1.8 % agar) and Gorodkowa agars (0.5 % sodium chloride, 0.1 % glucose, 2 % agar) individually or by mixing strains in pairs and incubating for up to 4 weeks at 15 and 25 Ǫ C.Genomic DNA was extracted using the Ezup Column Yeast Genomic DNA Purification Kit according to the manufacturer’ s protocol (Sangon Biotech). The ITS region was amplified with the primers forward ITS1 (5′-TCCGTAGG TGAACCTGCGG-3′) and reverse ITS4 (5′-TCCTCCGC TTATTGATATGC-3′) [23] and the D1/D2 domains of the LSU rRNA gene were amplified using the pair primers forward NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and reverse NL4 (5′ -GGTCCGTGTTTCAAGACGG-3′) [24]. Each 50 µl PCR mixture included 21 µl PCR-grade water, 1 µl DNA template, 1.5 µM of each primer and 1 µl PCR Master Mix (2×; 0.05 u µl ‒1 de Taq DNA polymerase, reaction buffer, 4 mM MgCl2, 0.4 mM of each dNTP; Sangon Biotech). PCR reactions were carried out in an S1000 thermal cycler (Bio-Rad Laboratories). The amplified products were purified with a QIAquick purification kit (Sangon Biotech) according to the manufacturer’ s instructions. Both DNA strands were sequenced using the ABI BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and run on an ABI 3730 automated DNA sequencer according to the manufacturer’ s instructions.Comparisons with sequences from the GenBank database (www.ncbi.nlm.nih.gov/) were done using a BLASTN search [25]. The sequences of related strains retrieved from GenBank were initially aligned using the multiple alignment program CLUSTAL_X 1.83 [26]. The phylogenetic trees were reconstructed based on the D1/D2 domains of the LSU rRNA gene sequences with MEGA software version 5.0 [27]. The evolutionary distances were calculated by using Kimura’ s two-parameter model [28] for the neighbour-joining and the maximum likelihood analyses, respectively [27, 29]. Saccharomyces cerevisiae NRRL Y-12632N T was used as an outgroup. Bootstrap analyses were performed based on 1000 random resamplings [30], and only values above 50 % were recorded on the resulting trees. Reference sequences were retrieved from GenBank or from the CBS database under the accession numbers indicated in the tree.