Diversity of Wickerhamomyces (Wickerhamomycetaceae, Saccharomycetales) in China with the description of four new species

Wickerhamomyces is a well-known genus of the family Wickerhamomycetaceae in the class Ascomycetes. These fungi can survive in a variety of substrates and environments and perform many valuable roles in both industrial processes and the natural ecosystems. During our investigation of yeast diversity associated with plant materials, 53 Wickerhamomyces isolates were obtained from rotting wood and plant leaves collected in Fujian, Guizhou, Henan, and Yunnan Provinces of China. Isolates were identified as 14 Wickerhamomyces species, including 1 species known previously to occur in China (W. anomalus), 9 new record species in China (W. arborarius, W. ciferrii, W. edaphicus, W. lynferdii, W. pijperi, W. subpelliculosa, W. xylosica, W. strasburgensis, and W. sydowiorum), and 4 novel species (W. guiyangensis sp. nov., W. paramyanmarensis sp. nov., W. quanzhouensis sp. nov., and W. phyllophilus sp. nov.). This study presents a detailed account of these new species, illustrating their morphology and analyzing their phylogenetic relationships with other Wickerhamomyces species. Our study is the first comprehensive study on Wickerhamomyces species associated with plant materials from tropical and subtropical China. The results of this study update our understanding of the phylogenetic relationships, systematics, and ecology of Wickerhamomyces.


Introduction
The genus Wickerhamomyces was first established by Kurtzman et al. (2008) with the type species Wickerhamomyces canadensis, which formerly belonged to the genus Pichia (Kurtzman, 2011a).Based on the phylogenetic analysis of concatenated sequences of LSU, SSU, and EF-1α, this genus was placed in the family Wickerhamomycetaceae of the order Saccharomycetales (Kurtzman et al., 2008), which was further confirmed by using a genomescale phylogeny (Groenewald et al., 2023).With the progress of yeast isolation technology and molecular biology, a number of Wickerhamomyces species have been discovered in different regions of the world.Currently, Wickerhamomyces consists of 38 defined species, including the most recent additions of W. lannaensis, W. nanensis, and W. sinyiensis (Nundaeng et al., 2021;Chang et al., 2022).Among the 38 valid species, 12 are asexual morphs and 26 have known for their ascosporic states (Kurtzman, 2011b;Shimizu et al., 2020;Nundaeng et al., 2021;Chang et al., 2022).In addition, 10 anamorphic Candida species, including C. mycetangii, C. namnaoensis, C. nitrativorans, C. odintsovae, C. peoriensis, C. ponderosae, C. quercuum, C. silvicultrix, C. solani, and C. ulmi, belong to the Wickerhamomyces clade based on phylogenetic analysis (Kurtzman, 2011b;Lachance et al., 2011;Shimizu et al., 2020), which will be transferred to the genus Wickerhamomyces as new combinations in the future.Phenotypically, the asexual morphs of Wickerhamomyces reproduced by multilateral budding on a narrow base, and some species produce pseudohyphae and/or true hyphae (Shimizu et al., 2020;Nundaeng et al., 2021;Chang et al., 2022).The sexual morphs of Wickerhamomyces are characterized by the production of hat-shaped or spherical ascospores with equatorial ledges (Kurtzman et al., 2008;Kurtzman, 2011b).Members of the genus Wickerhamomyces ferment glucose and possess Q-7 as a predominant ubiquinone.They can utilize various carbon sources but not methanol or hexadecane (Kurtzman et al., 2008;Kurtzman, 2011b).Due to the similarity in phenotypic characteristics within the genus, a combination of phenotypical characteristics and phylogenetic analysis has been adopted as the standard method for concretely identifying Wickerhamomyces species (Kurtzman et al., 2008;Nundaeng et al., 2021).
The presence of Wickerhamomyces in China has not been extensively documented.Current members of the genus have been reported only in Sichuan Province (W.anomalus) (You et al., 2016), the Inner Mongolia Autonomous Region (W.kurtzmanii) (Zhou et al., 2019), Yunnan Province (W.menglaensis) (Chai et al., 2019), and Henan Province (W.mori) (Hui et al., 2013).China is located in the northern hemisphere, providing a temperate environment suitable for hosting Wickerhamomyces species.To investigate the diversity of Wickerhamomyces in China, we isolated yeast strains from plant materials collected in Fujian, Guizhou, Henan, and Yunnan Provinces using a culture-based approach.A total of 14 species, including 4 new species, were identified from the isolated yeast strains.This study aims to characterize the diversity of Wickerhamomyces species inhabiting these substrates and describe four new species based on morphological characteristics and phylogenetic analyses of ITS and LSU.

Sampling and yeast isolation
Thirty-six strains used in this study were isolated from rotting wood by an enrichment method described by Shi et al. (2020).An additional 17 strains were isolated from plant leaves by the ballistospore-fall method as previously described (Nakase and Takashima, 1993;Hu et al., 2022).Vaseline was employed to adhere semi-withered leaf samples inside the lids of Petri plates containing yeast malt (YM) agar (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, and 2% agar).The plates were then incubated at 25°C for 7 days until colonies gradually formed.Different yeast morphotypes were picked and purified by streaking onto fresh YM agar.All purified yeast strains were then suspended in YM broth supplemented with 20% (v/v) glycerol and stored at −80°C.All isolates used in this study and their origins are presented in Table 1.

Morphological and physiological characterization
The morphological and physiological characteristics of each yeast strain were examined according to the methods established by Kurtzman et al. (2011).Colony characteristics were observed on YM agar after 2 weeks of incubation at 25°C.Mycelium formation was investigated by cultivation on corn meal (CM) agar (2% cornmeal infusion and 2% agar) in slide culture at 25°C for 2 weeks.Sexual processes of all strains were studied individually and in strain pairs on YM agar, CM agar, 5% malt extract (ME) agar, and V8 agar at 20°C and 25°C for 2 months with weekly observation (Kurtzman, 2011b).Fermentation of glucose was carried out in liquid medium using

DNA extraction, PCR amplification, and sequencing
Genomic DNA was extracted from each yeast isolate using the Ezup Column Yeast Genomic DNA Purification Kit, according to the manufacturer's protocol (Sangon Biotech, Shanghai, China).Sequenced data were generated from the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit (LSU) rRNA gene using primer pairs ITS1/ITS4 (White et al., 1990) and NL1/NL4 (Kurtzman and Robnett, 1998).PCR amplification was performed in a 25 μL reaction volume containing 9.5 μL of ddH 2 O, 12.5 μL of 2 × Taq PCR Master Mix with blue dye (Sangon Biotech, Shanghai, China), 1 μL of DNA template, and 1 μL of each primer.PCR reactions were carried out according to the following conditions: initial denaturation step at 95°C for 2 min, followed by 35 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 40 s, and a final extension at 72°C for 10 min.PCR products were checked and purified in 1% agarose gels before being sequenced by Sangon Biotech (Shanghai) Co., Ltd.The identity and accuracy of each sequence were determined by GenBank sequences and assembled using BioEdit 7.1.3.0 (Hall, 1999).Newly obtained sequences were then submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/; Table 2).

Phylogenetic analysis
A total of 116 nucleotide sequences that belonged to 54 taxa were included in the phylogenetic analyses.Except for 20 sequences recognized in this study, the other sequences were obtained from previous studies (Shimizu et al., 2020;Nundaeng et al., 2021;Chang et al., 2022) and GenBank (Table 2).Spathaspora allomyrinae CBS 13924 T and Saccharomyces cerevisiae CBS 1171 T were used as the outgroup.The phylogenetic relationships between Wickerhamomyces species and related species were determined through the analysis of combined ITS and LSU datasets.Single gene sequence alignments were generated using CLUSTAL_X v.1.83(Thompson et al., 1997) or MAFFT 7.110 (Katoh and Standley, 2013) before being manually adjusted with BioEdit v. 7.1.3.0 (Hall, 1999).Multiple genes were concatenated using SequenceMatrix v. 1.7.8 (Vaidya et al., 2011).Gblocks 0.91b was used to detect and remove ambiguously aligned regions from each sequence before phylogenetic analysis (Castresana, 2000).The best-fit evolutionary model was estimated by using Modelfinder (Kalyaanamoorthy et al., 2017).
Phylogenetic analyses were performed using both maximum likelihood (ML) and Bayesian (BI) analyses.ML analysis was performed using RAxML v. 8.2.10 (Stamatakis, 2006) with PHY files generated by CLUSTAL_X v.1.83(Thompson et al., 1997).The best scoring tree was selected among suboptimal trees from each run by comparing likelihood scores under the GTRGAMMA substitution model.The resulting replicates were plotted on the best scoring tree, which was obtained previously.Statistical support values (BS) were obtained using non-parametric bootstrapping with 1,000 replicates.BI analysis was conducted by MrBayes v. 3.2.7a(Ronquist et al., 2012) in the CIPRES Science Gateway version 3.3.GTR + I + G was selected as the best-fit model for the concatenated dataset.Posterior probabilities (PP) were determined by Markov chain Monte Carlo sampling (MCMC) (Rannala and Yang, 1996).Six simultaneous Markov chains were run for 50 million generations, and the trees were sampled every 1000th generation; thus, 50,000 trees were obtained.The first 25% of the saved trees, representing the burn-in phase of the analysis, were discarded.The remaining trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree.
The phylogenetic trees were viewed with FigTree v. 1.4.3 (Andrew, 2016) and processed with Adobe Illustrator CS5.ML bootstrap 3 Results

Diversity of Wickerhamomyces species
During this investigation, we isolated 36 and 17 yeast strains from rotting wood and plant leaf samples, respectively.Each strain represents the morphology in an individual sample (Table 1).All isolates were identified at the species level based on the threshold of >99% sequence identity with the type strain of described species in the D1/D2 domain or ITS region (Kurtzman and Robnett, 1998;Fell et al., 2000;Vu et al., 2016).In total, we identified 14 distinct species belonging to Wickerhamomyces.Among the wood samples, eight known species were represented (W.anomalus, W. arborarius, W. ciferrii, W. edaphicus, W. lynferdii, W. pijperi, W. subpelliculosa, and W. xylosica) as well as one potentially novel (Wickerhamomyces sp. 1).W. anomalus and W. pijperi were the most dominant, occurring in seven to 10 samples collected across the collection locations.W. arborarius, W. ciferrii, and W. subpelliculosa were the most scarce, occurring only in one sample or location (Table 1).The 17 Wickerhamomyces strains collected from plant leaf samples were identified as belong to two known species (W.strasburgensis and W. sydowiorum) and three potentially new species (Wickerhamomyces sp. 2, Wickerhamomyces sp. 3, and Wickerhamomyces sp. 4).W. sydowiorum was the most abundant yeast species among the leaf samples, followed by Wickerhamomyces sp. 3, which occurred in four to seven samples (Table 1).

Phylogeny of new yeast species
Among the 53 yeast strains isolated, 10 strains represented 4 new species in the genus Wickerhamomyces.To reveal the phylogenetic position of the new species, phylogenetic analyses were performed with a combination of ITS and LSU datasets.A total of 1,276 characteristics, including gaps (ITS: 1-666 and LSU: 667-1,276), were included in the sequence dataset for the phylogenetic analysis.Of these characteristics, 517 were constant, 156 were parsimonyuninformative, and 603 were parsimony-informative.The topology of the ML and Bayesian trees was consistent, therefore only the ML analysis is displayed with BS (≥50%) and PP (≥0.95) at the nodes (Figure 1).In our phylogenetic tree, 10 newly isolated strains were formed into four separate groups within the Wickerhamomyces clade, all of which were well supported (100% BS/1 PP) and distinct from other known species.
Four strains isolated from the phylloplane, NYNU 2286 T , NYNU 2288, NYNU 22815, and NYNU 22852, formed a well-supported clade that clustered with W. hampshirensis, W. scolytoplatypi, and W. strasburgensis with high statistical support (99% BS/1 PP; Figure 1).The four strains of the NYNU 2286 T group possess similar sequences with zero to six nt substitutions in the D1/D2 domain and ITS region, indicating that they belong to the same species.BLASTn searches of the D1/D2 and ITS sequences indicate that the NYNU 2286 T group is most closely related to type strain of W. strasburgensis, differing by 19 nt (~3.3%) substitutions in the D1D2 domain and by 21-29 nt (~3.4%-4.7%)mismatches in the ITS region, respectively.Therefore, the NYNU 2286 T group represents a novel Wickerhamomyces species, for which the name W. guiyangensis sp.nov. is proposed.
Strain NYUN 22853 T , isolated from the phylloplane, was found to be closely related to W. myanmarensis CBS 9786 T with strong statistical support (89% BS/0.96PP; Figure 1).The two strains differed by 7 nt (~1.2%) substitutions in the D1/D2 domain and 19 nt (~3%) mismatches in the ITS region, respectively.These findings suggest that NYUN 22853 T represents a novel species in the genus Wickerhamomyces, for which the name W. paramyanmarensis sp.nov. is proposed.
Three isolates collected from rotting wood, NYNU 22496 T , NYNU 224139, and NYNU 224203, formed a separate branch, clustering with C. solani, W. xylosica, W. pijperi, and W. chaumierensis with good statistical support (98% BS/1 PP; Figure 1).Three isolates of the NYNU 22496 T group shared 100% nucleotide identity based on D1/ D2 and ITS sequences, indicating that they are conspecific.This group differed from these four known species by 44-53 nt (~7.7-9.3%)substitutions in the D1/D2 domain and more than 26-74 nt (~7.9-11.7%)mismatches in the ITS region.Hence, the NYNU 22496 T group represents a novel Wickerhamomyces species, for which the name W. quanzhouensis sp.nov. is proposed.
Two strains isolated from the phylloplane, NYNU 2285 T and NYNU 2289, were located at a basal branch related to C. quercuum CBS 6422 T but without support (Figure 1).The two strains of the NYNU 2285 T group had identical sequences in both the D1/D2 domain and ITS region.This group differed from the closest relative C. quercuum CBS 6422 T by 20 nt (~3.4%) substitutions in the D1/D2 domain and 18 nt (~5.9%) mismatches in the ITS region, respectively, suggesting that the NYNU 2285 T group represents a new species in Wickerhamomyces, for which the name W. phyllophilus sp.nov. is proposed.
MycoBank: MB 850882.Etymology: the specific epithet "guiyangensis" refers to the geographic origin of the type strain, Guiyang city, Guizhou.
Etymology: the specific epithet "paramyanmarensis" refers to its phylogenetic closeness to W. myanmarensis.
MycoBank: MB 850884.Etymology: the specific epithet "quanzhouensis" refers to the geographic origin of the type strain, Quanzhou city, Fujian.
MycoBank: MB 850885.Etymology: the specific epithet "phyllophilus" refers to leaves, the substrate origin of the type strain.

Discussion
Before advances in gene sequencing, the identification of Wickerhamomyces species was primarily based on phenotypic characteristics.However, due to the presence of many shared polymorphic characteristics and similar appearances across different species, this method is often inaccurate (Nundaeng et al., 2021).Now, ribosomal DNA gene sequence analysis has become the dominant form of identification as it has proven to be very effective (Vu et al., 2016).Molecular taxonomic studies have deeply improved our understanding of the phylogenetic relationships, systematics, and ecology of yeasts.As a result, a combination of phenotypic and phylogenetic data is used to concretely identify the Wickerhamomyces species (Kurtzman and Robnett, 1998;Nundaeng et al., 2021).In this study, we introduced four new Wickerhamomyces species, consisting of W. guiyangensis sp.nov., W. paramyanmarensis sp.nov., W. quanzhouensis sp.nov., and W. phyllophilus sp.nov., and describe them in terms of both phenotype and phylogeny.These new species formed four well-separated clades in the resultant phylogram, which indicated the distinct phylogenetic positions of each species within the genus Wickerhamomyces.Pairwise sequence comparisons of the D1/D2 domain and the ITS region between these four species and closely related species showed that they had lower similarity values than the common threshold for species demarcation in ascomycetous yeasts (Fell et al., 2000;Kurtzman et al., 2008;Vu et al., 2016).However, they were found to be highly similar in cell shape, colony morphology, and color, but they differed from the closest related species in terms of their physiological and biochemical characteristics.Therefore, a combination of phenotypic characteristics and molecular phylogenetic analyses conducted in our study confirmed the existence of these new species in China.Before our study, only four Chinese species, W. anomalus, W. kurtzmanii, W. menglaensis, and W. mori, were known from China (Hui et al., 2013;You et al., 2016;Chai et al., 2019;Zhou et al., 2019).This study provides 14 additional species, including 1 previously known species (W.anomalus), 9 new recorded species (W.arborarius, W. ciferrii, W. edaphicus, W. lynferdii, W. pijperi, W. subpelliculosa, W. xylosica, W. strasburgensis, and W. sydowiorum), and 4 novel species (W.guiyangensis sp.nov., W. paramyanmarensis sp.nov., W. quanzhouensis sp.nov., and W. phyllophilus sp.nov.), increasing the number of Wickerhamomyces species from 4 to 17.In China, there are likely species that need to be identified, such as GenBank accession JQ901898.These studies suggest that there are likely even more Wickerhamomyces species waiting to be discovered.So far, almost no Wickerhamomyces species have been reported from eastern China, which also have abundant forest resources, so it is necessary to investigate yeast resources in these regions in the future studies.

Conclusion
In this study, the species diversity of the genus Wickerhamomyces in China was studied.A total of 14 species were obtained and circumscribed as four novel species, namely, W. guiyangensis sp.nov., W. paramyanmarensis sp.nov., W. quanzhouensis sp.nov., and W. phyllophilus sp.nov., nine newly recorded species W. arborarius, W. ciferrii, W. edaphicus, W. lynferdii, W. pijperi, W. subpelliculosa, W. xylosica, W. strasburgensis, and W. sydowiorum in China, and a known species W. anomalus.All the studied species were identified by morphological characteristics and phylogenetic analysis of combined ITS and LSU sequences.Four new species are described based on their asexual and/or sexual states, and their differences with the close relatives were compared and discussed.

FIGURE 1
FIGURE 1 Maximum likelihood phylogram of Wickerhamomyces based on the combined ITS and LSU sequence data.Spathaspora allomyrinae CBS 13924 T and Saccharomyces cerevisiae CBS 1171 T were used as outgroups.Branches are labeled with BS >50% and PP >0.95, respectively.Strains obtained in the present study are shown in bold.

TABLE 1
Yeast strains and isolation sources investigated in this study.

TABLE 1 (
Continued) ≥50% and Bayesian posterior probabilities (PP) ≥0.95 are displayed on the edited phylogenetic tree in the first and second positions, respectively.
Taxa, strains, sources, locations, and corresponding GenBank numbers of the taxa used in this study.