An Updated Global Species Diversity and Phylogeny in the Genus Wickerhamomyces with Addition of Two New Species from Thailand

Ascomycetous yeast species in the genus Wickerhamomyces (Saccharomycetales, Wickerhamomycetaceae) are isolated from various habitats and distributed throughout the world. Prior to this study, 35 species had been validly published and accepted into this genus. Beneficially, Wickerhamomyces species have been used in a number of biotechnologically applications of environment, food, beverage industries, biofuel, medicine and agriculture. However, in some studies, Wickerhamomyces species have been identified as an opportunistic human pathogen. Through an overview of diversity, taxonomy and recently published literature, we have updated a brief review of Wickerhamomyces. Moreover, two new Wickerhamomyces species were isolated from the soil samples of Assam tea (Camellia sinensis var. assamica) that were collected from plantations in northern Thailand. Herein, we have identified these species as W. lannaensis and W. nanensis. The identification of these species was based on phenotypic (morphological, biochemical and physiological characteristics) and molecular analyses. Phylogenetic analyses of a combination of the internal transcribed spacer (ITS) region and the D1/D2 domains of the large subunit (LSU) of ribosomal DNA genes support that W. lannaensis and W. nanensis are distinct from other species within the genus Wickerhamomyces. A full description, illustrations and a phylogenetic tree showing the position of both new species have been provided. Accordingly, a new combination species, W. myanmarensis has been proposed based on the phylogenetic results. A new key for species identification is provided.


Introduction
The genus Wickerhamomyces was first proposed by Kurtzman et al. [1] in 2008 with W. canadensis (basionym Hansenula canadensis) as the type species. This genus belongs to the family Wickerhamomycetaceae of the order Saccharomycetales [1]. Wickerhamomyces species can reproduce both asexually and sexually. Through asexual reproduction, the species reproduce by budding and some species produce pseudohyphae and/or true hyphae. Alternatively, in sexual reproduction they produce hat-shaped or spherical ascospores with an equatorial ledge for sexual reproduction [1,2]. Most of the known Wickerhamomyces species can utilize various carbon sources, but not methanol or hexadecane. Nitrate utilization was observed in some species, while the diazonium blue B reaction was W. anomalus has been labeled a biosafety level 1 organism by the European Food Safety Authority [112] and is considered safe for consumption by healthy individuals.
Currently, only eight Wickerhamomyces species, namely W. anomalus, W. ciferrii, W. edaphicus, W. rabaulensis, W. siamensis, W. sydowiorum, W. tratensis and W. xylosicus, have been reported in Thailand [4,8,16,17,19,20,46,70,78]. Accordingly, Thailand has been identified as a hotspot for unexpected novel species and the newly recorded discovery of many microorganisms [113,114]. In our previous investigation on yeasts in soil samples collected from Assam tea (Camellia sinensis var. assamica) plantations in northern Thailand [16], we obtained five yeast strains belonged to the genus Wickerhamomyces that represent potentially new species. In our present study, we have described them into two novel species. These two novel species are introduced based on their phenotypic (morphological, biochemical and physiological data) and molecular characteristics. To confirm their taxonomic status, phylogenetic relationship was determined by analysis of the combined sequence dataset of the D1/D2 domains of LSU and ITS sequences.

Morphological Study
The morphological characteristics of yeast strains were determined according to established methods by Kurtzman et al. [2], Yarrow [3] and Limtong et al. [10]. Colony characters were observed on yeast extract-malt extract agar (YMA) after two days of incubation in darkness at 30 • C. Ascospore formation was investigated on YMA, 5% malt extract agar (MEA), potato dextrose agar (PDA) and V8 agar after incubation at 25 • C in the dark for four weeks. Micromorphological characteristics were examined under a light microscope (Nikon Eclipse Ni U, Tokyo, Japan). Size data of the anatomical structure (e.g., cells, pseudohyphae, asci and ascospores) were based on at least 50 measurements of each structure using the Tarosoft (R) Image Frame Work program.

Molecular Study
Each yeast strain was grown in 5 mL of yeast extract-malt extract broth in 18 × 180 mm test tubes with shaking at 150 rpm on an orbital shaker in the dark for two days. Yeast cells were harvested by centrifugation at 11,000 rpm and washed three times with sterile distilled water. Genomic DNA was extracted from yeast cells using DNA Extraction Mini Kit (FAVORGEN, Taiwan) following the manufacturer's protocol. The ITS region and D1/D2 domains of LSU gene were amplified by polymerase chain reactions (PCR) using ITS1/ITS4 primers [117] and NL1/NL4 primers [118], respectively. The amplification of both D1/D2 domains and ITS region process consisted of an initial denaturation at 95 • C for 5 min, followed by 35 cycles of denaturation at 95 • C for 30 s, annealing at 52 • C for 45 s, an extension at 72 • C for 1 min and 72 • C for 10 min on a peqSTAR thermal cycler (PEQLAB Ltd., UK). PCR products were checked and purified by a PCR clean up Gel Extraction NucleoSpin ® Gel and PCR Clean-up Kit (Macherey-Nagel, Germany). Final PCR products were sent to 1st Base Company Co., Ltd., (Kembangan, Malaysia) for sequencing. The obtained sequences were used to query GenBank via BLAST (http://blast.ddbj.nig.ac.jp/top-e.html, accessed on 25 August 2021).
Phylogenetic analysis was carried out based on the combined dataset of ITS and D1/D2 domains of LSU sequences. Sequences from this study along with those obtained from previous studies and the GenBank database were selected and provided in Table 2. Multiple sequence alignment was performed using MUSCLE [119]. A combination of D1/D2 domains of LSU and ITS alignment was deposited in TreeBASE under the study ID number 28785. A phylogenetic tree was constructed under maximum likelihood (ML) and Bayesian inference (BI) methods. The ML analysis was carried out using RAxML-HPC2 on XSEDE (8.2.10) in CIPRES Science Gateway V. 3.3 [120] using GTRCAT model with 25 categories and 1000 bootstrap (BS) replications. The optimum nucleotide substitution model was obtained using jModeltest v.2.3 [121] under the Akaike information criterion (AIC) method. The BI analysis was performed using MrBayes 3.2.6 software for Windows [122]. The selected optimal model of each gene is similar as GTR + I + G model. Six simultaneous Markov chains were run with one million generations and starting from random trees and keeping one tree every 100th generation until the average standard deviation of split frequencies was below 0.01. The value of burn-in was set to discard 25% of trees when calculating the posterior probabilities. Bayesian posterior probabilities (PP) were obtained from the 50% majority rule consensus of the trees kept. The tree topologies were visualized in FigTree v1.4.0 [123].   Saccharomyces cerevisiae NRRL Y-12632 T AY046146 JQ689017 [128] Spathaspora allomyrinae CBS 13924 T KP054268 KP054267 [129] Note: sepecies obtained in this study are in bold.

Phylogenetic Results
The sequences of five yeast strains were deposited in the GenBank database ( Table 2). The alignment of a combination of ITS and D1/D2 domains of the LSU genes contained 1544 characters including gaps (ITS: 1−823 and D1/D2 domains of LSU: 824−1544). RAxML analysis of the combined dataset yielded a best scoring tree with a final ML optimization likelihood value of −12,120.4323. The matrix contained 776 distinct alignment patterns with 42.33% undetermined characters or gaps. Estimated base frequencies were recorded as follows: A = 0.2730, C = 0.1821, G = 0.2603, T = 0.2844; substitution rates AC = 1.0574, AG = 2.0209, AT = 1.4684, CG = 0.6712, CT = 4.4165, GT = 1.0000. The gamma distribution shape parameter alpha was equal to 0.2698 and the Tree-Length was equal to 4.4075. In addition, the final average standard deviation of the split frequencies at the end of the total MCMC generations was calculated as 0.00638 through BI analysis. Phylograms of the ML and BI analyses were similar in terms of topology (data not shown). Therefore, the phylogram obtained from the ML analysis was selected and presented for this study. The phylogram was comprised of 67 sequences of Wickerhamomyces strains (including 37 type strains obtained from either previous studies or the present study) and two sequences (Saccharomyces cerevisiae NRRL 12632 and Spathaspora allomyrinae CBS 13924) of the outgroup (Figure 3). Our phylogenetic analysis separated Wickerhamomyces by different species based on different topologies. Our analysis confirmed that W. myanmarensis (previously known as P. myanmarensis) belonged to the genus Wickerhamomyces according to the phylogenetic results of Arastehfar et al. [8] and Shimizu et al. [65]. Moreover, a phylogram clearly separated our yeast strains into two monophyletic clades with high support values (BS = 100% and PP = 1.0). The results indicated that our two yeast strains, SDBR-CMU-S2-17 and SDBR-CMU-S2-14 (introduced as W. nanensis), were clearly distinguished from the previously known species of Wickerhamomyces. Moreover, three yeast strains in this study, SDBR-CMU-S2-02, SDBR-CMU-S2-15, and CMU-S3-06 (described here as W. lannaensis) formed a sister clade to W. ochangensis with high support (BS = 100% and PP = 1.0).   Description: The streak culture on YMA after two days at 30 • C is circular from (1-2 mm in diameter), white to cream color, smooth surface, dull-shining, entire margin, and raised elevation. After growth on YMA at 30 • C for two days, the cells are spheroidal to short ovoidal (3.6-3.8 × 2.4-2.6 µm), occur singly or in budding pairs. Pseudohyphae and true hyphae were absent. Ascospores were not obtained for individual strains and strain pairs on YMA, 5% MEA, PDA and V8 agar after incubation at 30 • C for one month. Urea hydrolysis and diazonium blue B reactions are negative. Fermentation tests, glucose is delayed positive, but galactose, maltose, sucrose, trehalose, melibiose, lactose, raffinose, and xylose are negative. D-glucose, D-xylose, rhamnose, cellobiose, salicin, inulin (weak), glycerol, D-glucitol, D-mannitol, D-glucono-1,5-lactone, D-gluconate, DL-lactate (weak), succinate, and ethanol are assimilated. No growth was observed in L-sorbose, N-acetyl glucosamine, D-ribose, L-arabinose, D-arabinose, sucrose, maltose, α,α-trehalose, α-methyl-D-glucoside, melibiose, lactose, raffinose, melezitose, soluble starch, erythritol, ribitol, galactitol, myo-inositol, 2-ketogluconic acid, 5-ketogluconic acid, D-glucuronate, D-galacturonic acid, citrate, methanol, and xylitol. For the assimilation of nitrogen com-pounds, growth on ammonium sulfate, potassium nitrate, sodium nitrite, ethylamine HCl, cadaverine, and creatine (weak) are positive and on L-lysine is latent positive.
Growth in the vitamin-free medium is weak positive. Growth was observed at 15 • C and 30 • C, but not at 35, 37, 40, 42 and 45 • C. Growth in the presence of 50% glucose is positive, but growth in the presence of 0.01% cycloheximide, 0.1% cycloheximide, 60% glucose, 10% NaCl with 5% glucose and 15% NaCl with 5% glucose are negative. Acid formation is negative.
Note: Several morphological and biochemical characteristics of W. nanensis were similar to W. chambardii. However, W. chambardii differed from W. nanensis by its ascospore formation and could not assimilate D-mannitol [2]. Phylogenetic analyses clearly separated W. nanensis and W. chambardii as different species. Moreover, W. nanensis formed a monophyletic clade in a well-supported clade and was separated from other Wickerhamomyces species (Figure 3).

New Combination
Wickerhamomyces myanmarensis (Nagats., H. Kawas. and T. Seki) J. Kumla Note: The combined ITS and D1/D2 phylogenetic analyses indicated that the type species, P. myanmarensis, belongs to the genus Wickerhamomyces and has a close phylogenetic relationship with W. anomalus (Figure 3). Accordingly, the phylogenetic results of Arastehfar et al. [8] and Shimizu et al. [65] found that P. myanmarensis was placed within the genus Wickerhamomyces.

Key to Species of Wickerhamomyces
A key to the identification of the Wickerhamomyces species introduced in the present study was derived from the key described by Kurtzman et al. [2]. Key characteristics are shown in Table 3     "+" = strong growth or produce, "-" = absence of growth or not produce, "w" = weak growth, "v" = strain variable response, "l" = latent positive, "s" = slow positive, and "n" = no data.

Discussion
Traditional methods of identification and characterization for the Wickerhamomyces species are based primarily on phenotypical characteristics. These are further recognized as relevant morphological, biochemical, and physiological characteristics [41,128]. However, identification can be difficult because some species have similar appearances, and some biochemical characteristics are consistent across a number of species. In accordance with this evidence, previous species of Wickerhamomyces were originally classified into various yeast genera [1,2,4,5,50,70,75]. In 2008, the genus Wickerhamomyces was proposed by Kurtzman et al. [1], wherein this genus was clearly separated from other yeast genera based on phylogenetic evidence. Subsequently, some previously identified species were then transferred from the genera Candida, Hansenula, Pichia, and Williopsis [1,4,5,50,54,70]. Therefore, molecular phylogenetic analysis is necessary to concretely identify the Wickerhamomyces species. Species of the genus Wickerhamomyces are known to be widely distributed throughout the world and have been isolated from various habitats as shown in Table 1. Prior to conducting our study, Wickerhamomyces consisted of 35 accepted and published species according to molecular phylogenetic analysis. Our phylogenetic results were similar to those of Arastehfar et al. [8] and Shimizu et al. [65] who indicated that P. myanmarensis should be placed in the genus Wickerhamomyces. Consequently, we have proposed that this yeast species be named W. myanmarensis.
Yeast diversity has been investigated in various habitats throughout different regions of Thailand [5,10,15,16,18,19,46,70,78]. Wickerhamomyces anomalus was first species reported in Thailand in 2002 [20]. In 2009, the first new species, W. edaphicus has been discovered in Thailand [10]. Until now, a total of eight Wickerhamomyces species have been found [5,10,20,46,68,70,78,82]. However, W. siamensis, W. tratensis, and W. xylosicus were only known to be from Thailand [5,70,82]. In this study, two new Wickerhamomyces species, namely W. lannaensis and W. nanensis, that were isolated from soil collected from Assam tea plantations in northern Thailand were proposed based on identification through molecular phylogenetic and phenotypic (morphological, biochemical, and physiological characteristics) analyses. Therefore, effective identification of the Wickerhamomyces species has increased the number of species found in Thailand to 10 species and has led to 38 global species. This present discovery has increased the number of species of yeast known to be from Thailand and is considered important in terms of stimulating deeper investigations of yeast varieties in Thailand. Ultimately, these findings will help researchers gain a better understanding of the distribution and ecology of Wickerhamomyces.
Many species of the genus Wickerhamomyces have been investigated, and some strains have been used in a variety of biotechnology, food, and beverage industries, as well as in medical and agricultural fields [83][84][85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102]. Despite the fact that many Wickerhamomyces species can survive in a variety of environments, climate change has had an impact on both the terrestrial biome and the aquatic environment. These environments are known to serve as habitats for a number of microorganisms [130][131][132][133][134] and may have an impact on the global diversity and distribution of Wickerhamomyces. Therefore, in addition to studying the diversity and distribution of newly identified species, future research should focus on the effects of climate change on Wickerhamomyces.