﻿Four new species of Trichomonascaceae (Saccharomycetales, Saccharomycetes) from Central China

﻿Abstract Trichomonascaceae is the largest family of ascomycetous yeast in the order Saccharomycetales. In spite of the extensive body of research on Trichomonascaceae in China, there remain new species to be discovered. Here, we describe four new species isolated from several rotting wood samples from Henan Province, Central China. Phylogenetic analysis of a combined ITS and nrLSU dataset with morphological studies revealed four new species in the Trichomonascaceae: Diddensiellaluoyangensis, Sugiyamaellacylindrica, Su.robnettiae, and Zygoascusdetingensis. Clustering in the Diddensiella clade, D.luoyangensis’ closest neighbour was D.transvaalensis. Meanwhile, Su.cylindrica clustered in the Sugiyamaella clade closest to Su.marilandica and Su.qingdaonensis. Also clustering in the Sugiyamaella clade, Su.robnettiae was most closely related to Su.chuxiongensis. Finally, Z.detingensis occupied a distinct and separated basal branch from the other species of the genus Zygoascus. These results indicate a high species diversity of Trichomonascaceae.


Introduction
The family of Trichomonascaceae was described by Kurtzman and Robnett (2007) to accommodate the genera Sugiyamaella Kurtzman and Robnett, Trichomonascus known species (Wang et al. 2010;Guo et al. 2012;Huang et al. 2018;Chai et al. 2020;Shi et al. 2021). Although the samples of rotting wood were collected in a relatively small geographical area in China, the Trichomonascaceae species are diverse in this rich ecological environment.
During extensive investigations on the diversity of yeast inhabiting rotting wood from China, several unknown yeast strains were collected from Henan Province, and their morphology suggested species of Diddensiella, Sugiyamaella, and Zygoascus. To investigate their taxonomy further, phylogenetic analyses, based on combined ITS and nrLSU sequences, were carried out. Both morphological characteristics and molecular evidence demonstrate that these yeasts represent four new species of Trichomonascaceae, which are described here.

Sample collection and yeast isolation
Samples of rotting wood were collected in the Tianchi Mountain National Forest Park (34°33'N, 112°28'E) located near Luoyang City, Henan Province, China. The national forest park is at 850 m above sea level (MASL) and has a continental monsoon climate. The average annual temperature is between 14 °C and 16 °C, and the average annual rainfall is greater than 800 mm. Forty samples of decaying wood were collected between September and October in 2020. Samples were stored in sterile plastic bags and transported under refrigeration to the laboratory within 24 hours. Yeast strains were isolated from rotting wood samples according to previously described methods (Huang et al. (2018) and Shi et al. (2021). One gram of each sample was added to 20 mL sterile yeast extract-malt extract (YM) broth (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, pH 5.0 ± 0.2), supplemented with 0.025% sodium propionate and 200 mg/L chloramphenicol in a 150 mL Erlenmeyer flask, and then cultured for 3-10 days at 180 rpm on a rotary shaker. Subsequently, 0.1 mL aliquots of the enrichment culture and appropriate decimal serial dilutions were plated on YM agar plates and incubated at 25 °C for 3-4 days. Different yeast colony morphotypes were then isolated via repeated plating on YM agar. Isolates were stored on YM agar slants at 4 °C or in 15% glycerol at −80 °C. All isolates were stored in Microbiology Lab of Nanyang Normal University (NYNU; Nanyang, China), and ex-type cultures of novel yeast were deposited in the fungal collection at Westerdijk Fungal Biodiversity Institute (CBS; Utrecht, The Netherlands). Species nomenclature and descriptions were registered in MycoBank (www.mycobank.org, accessed on February 9, 2022).

Morphological and physiological investigation
Morphological and physiological properties were determined according to methods previously described in Kurtzman et al. (2011). Carbon and nitrogen assimilation tests were performed using liquid media and growth was observed for up to 4 weeks. Carbon fermentation was tested in yeast extract peptone (YP) base media (1% yeast extract and 2% peptone, pH 5.0 ± 0.2), and Durham tubes were used to visualise carbon dioxide production. Growth rates at a range of temperatures (30 °C, 35 °C, 37 °C, and 40 °C) were assessed by streaking cells on to yeast extract peptone glucose (YPD) agar (1% yeast extract, 2% peptone, 2% glucose, 2% agar, pH 5.0 ± 0.2) plates and incubating them for~2 weeks. Formation of true hyphae and pseudohyphae were investigated using the Dalmau plate method on both cornmeal (CM) and 5% malt extract (ME) agar plates. Induction of the sexual stage was tested by incubating single or mixed cultures of the each of the two strains on PDA agar, cornmeal (CM) agar, 5% malt extract (ME) agar, V8 (1:9) agar, Gorodkowa agar, or yeast carbon base plus 0.01% ammonium sulfate (YCBAS) agar at 25 °C for 2 months (Kurtzman 2011b;Péter et al. 2012;Nagatsuka et al. 2016).

DNA amplification and sequencing
Genomic DNA was extracted from each of the yeasts using the Ezup Column Yeast Genomic DNA Purification Kit according to the manufacturer's protocol (Sangon Biotech, China). The rDNA ITS1-5.8S-ITS2 (ITS) region was amplified using the primer pair ITS1/ITS4 (White et al. 1990). The D1/D2 domain of nrLSU rDNA (nrLSU) was amplified using the primer pair NL1/NL4 (Kurtzman and Robnett 1998). The following parameters were used to amplify the ITS and nrLSU regions: an initial denaturation step of 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 51 °C, and 40 s at 72 °C, and a final extension of 10 min at 72 °C (Shi et al. 2021). PCR products were directly purified and sequenced by Sangon Biotech Inc. (Shanghai, China). The identity and quality of the newly-obtained sequences were assessed by comparing them to sequences in GenBank and assembling them with BioEdit (Hall 1999). Sequences were then submitted to GenBank (https://www. ncbi.nlm.nih.gov/ genbank/; Table 1).

Phylogenetic analyses
Species in the family Trichomonascaceae with high similarity to the new species described here were selected as references in the phylogenetic analyses. Tortispora caseinolytica CBS 7781 T and Tor. ganteri CBS 12581 T were used as outgroup. NCBI accession numbers of sequences used in the phylogenetic tree are listed in Table 1. Initial alignment of the combined ITS + nrLSU dataset was performed using the online version MAFFT 6.0 (Katoh and Toh 2010) followed by manual evaluations and adjustments in BioEdit as needed to obtain reliable and high quality results (Hall 1999). The best-fit nucleotide substitution models for separate and combined nucleotide sequences were selected using jModelTest v2.1.7 (Darriba et al. 2012) according to the Akaike Information Criterion (AIC). The final concatenated sequence alignment was deposited in TreeBase (http://www.treebase.org; submission ID S29358). Maximum likelihood (ML) and Bayesian inference (BI) analyses were used for the phylogenetic analyses. The ML analysis was carried out using RAxmL v.7.2.8 with a GTR + G + I, model of site substitution including estimation of Gammadistributed rate heterogeneity and a proportion of invariant sites (Stamatakis 2006). Branch support was evaluated using bootstrapping with 1000 replicates (Hillis and Bull 1993). The BI analysis was performed using MrBayes v3.2 (Ronquist et al. 2012), for two independent runs, each with four Markov chains Monte Carlo (MCMC) independent runs for 5 ×10 6 generations (split frequencies = 0.011). The first 25% of trees were discarded as "burn-in" of each analysis and the remaining 75% were then used to calculate Bayesian posterior probabilities of the majority rule consensus tree.
Phylogenetic trees from the ML and BI analyses were visualised with FigTree v1.4.3 (Rambaut 2016) and edited in Adobe Illustrator CS6. Branches that received bootstrap support for maximum likelihood (BS) and Bayesian posterior probabilities (BPP) greater than or equal to 50% (BS) and 0.95 (BPP) were considered to be significantly supported.

Molecular phylogenetic analysis
The combined ITS and nrLSU dataset was analysed to infer the phylogenetic relationships of the family Trichomonascaceae and the new Chinese isolates. The dataset consisted of 59 sequences including the outgroup, Tortispora caseinolytica CBS 7781 T and Tor. ganteri CBS 12581 T . A total of 943 characters including gaps (376 for ITS and 567 for nrLSU) were included in the phylogenetic analysis. GTR + I + G was inferred as the best-fit model for the combined nrLSU and ITS nucleotide sequences according to the AIC in jModelTest v2.1.7 (Darriba et al. 2012). The topologies of the phylogenetic tree of ML and BI analyses are identical, and only the ML tree with a final optimisation likelihood value of -12097.50 is shown in Fig. 1 Notes: Metabolically inactive ex-type strains are indicated by "T" after the species name; "N/A" means that sequences were not available; Bold indicates strains that were isolated in this study. In the phylogeny (Fig. 1), newly generated strains in this study nested in the genera Diddensiella, Sugiyamaella, and Zygoascus within the Trichomonascaceae. Description. In YM broth after 3 days at 25 °C, cells are ovoid (2-3 × 3-5 μm) and occur singly or in pairs. Budding is multilateral. Sediment is formed after a month, but a pellicle is not observed. On YM agar after 3 days at 25 °C, colonies are white to cream-coloured, convex, butyrous, and smooth with entire margins. In Dalmau plate culture on corn meal agar, pseudohyphae and true hyphae are formed. Asci or signs of conjugation are not observed on sporulation media. Fermentation of sugars is absent. Glucose, galactose, l-sorbose, glucosamine, d-ribose, d-xylose, l-arabinose, d-arabinose, l-rhamnose, sucrose, maltose, trehalose, methyl α-d-glucoside, cellobiose, salicin, melibiose, lactose, raffinose, melezitose, inulin, glycerol, erythritol, ribitol, d-glucitol, d-mannitol, galactitol, myo-inositol, d-glucono-1, 5-lactone, 2-keto-d-gluconate, 5-keto-d-gluconate, d-gluconate, d-glucuronate, dl-lactate succinate, citrate, and ethanol are assimilated as sole carbon sources. Methanol is not assimilated. l-lysine, creatine, glucosamine, and d-tryptophan are assimilated as sole nitrogen sources, while nitrate, nitrite, ethylamine, cadaverine, creatinine, and imidazole are not assimilated. Minimum growth temperature is 15 °C, and maximum growth temperature is 37 °C. Growth in the presence of 0.1% cycloheximide is present, but growth in the presence of 10% NaCl plus 5% glucose and 1% acetic acid is absent. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are negative.
Notes. Two strains were collected from two different substrates, representing D. luoyangensis, clustered in the Diddensiella clade which is sister to species D. transvaalensis. D. luoyangensis differed from D. transvaalensis by 1.6% substitutions in the D1/D2 domain. Furthermore, we were unable to align the ITS sequence of D. luoyangensis with the D. transvaalensis type strain, because the ITS sequence of D. transvaalensis is not currently available from either the NCBI GenBank or CBS databases. Physiologically, D. luoyangensis differs from its closely related species, D. transvaalensis , based on growth in l-rhamnose, lactose, inulin, d-gluconate and growth at 37 °C, which are present for D. luoyangensis and absent for the latter species. Moreover, D. transvaalensis ferments glucose and galactose, while this new species does not.  (holotype NYNU 201067 T , ex-type CBS 16662 = CICC 33514, holotype and ex-type are preserved in a metabolically inactive state).
Description. In YM broth after 3 days at 25 °C, cells are cylindrical (2-3 × 5-7 μm) and occur singly or in pairs. Budding is multilateral. Sediment is formed after a month, but a pellicle is not observed. On YM agar after 3 days at 25 °C, colonies are white to cream-coloured, butyrous, convex and smooth with entire margins. In Dalmau plate culture on corn meal agar, rudimentary pseudohyphae are formed. Asci or signs of conjugation are not observed on sporulation media. Glucose and trehalose are weakly fermented, but, galactose, maltose sucrose, melibiose, lactose, cellobiose, melezitose, raffinose, inulin and xylose are not fermented. Glucose,galactose,glucosamine,dxylose,sucrose,maltose,trehalose,cellobiose,salicin,melibiose,raffinose,melezitose,inulin,glycerol,erythritol,ribitol,galactitol,and ethanol are assimilated as sole carbon sources. Lactose, d-gluconate, citrate and methanol are not assimilated. Nitrate, nitrite, l-lysine, creatine, glucosamine, and d-tryptophan are assimilated as sole nitrogen sources. Ethylamine, cadaverine, creatinine, and imidazole are not assimilated. Minimum growth temperature is 15 °C, and maximum growth temperature is 35 °C. Growth in the presence of 0.1% cycloheximide is present, but growth in the presence of 1% acetic acid and 10% NaCl plus 5% glucose is absent. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are negative.
Notes. Two strains were collected from two different substrates, representing Su. cylindrica, clustered in the Sugiyamaella clade and are closely related to Su. marilandica and Su. qingdaonensis. The nucleotide differences between the new species and the close relatives Su. marilandica and Su. qingdaonensis are 1.1-1.4% substitutions in the D1/D2 domain and 5.0-5.9% substitutions in the ITS region, respectively. Physiologically, Su. cylindrica differs from the closely related species Su. marilandica and Su. qingdaonensis (Wang et al. 2010;Kurtzman 2011b) in its ability to assimilate glycerol and dl-lactate and to grow at 35 °C. Additionally, the new species ferments trehalose, while Su. marilandica and Su.qingdaonensis do not. Description. In YM broths after 3 days at 25 °C, the cells are ellipsoidal to elongate (2-4 × 2-8 μm) and occur singly or in pairs. Budding is multilateral. Sediment is formed after a month, but a pellicle is not observed. On YM agar after 3 days at 25 °C, colonies are white to cream-coloured, convex, buttery and smooth with entire margins. In Dalmau plate culture on corn meal agar, pseudohyphae and true hyphae are formed. Asci or signs of conjugation are not observed on sporulation media. Fermentation of sugars is absent. Glucose, galactose, l-sorbose, glucosamine, d-xylose, l-arabinose, d-arabinose, l-rhamnose, sucrose, maltose, trehalose, methyl α-d-glucoside, cellobiose, salicin, arbutin, lactose, inulin, glycerol, erythritol, ribitol, xylitol, d-glucitol, dmannitol, galactitol, d-glucono-1, 5-lactone, 2-keto-d-gluconate, 5-keto-d-gluconate, succinate, citrate, and ethanol are assimilated as sole carbon sources. d-ribose, melibiose, raffinose, melezitose, myo-inositol, d-gluconate, dl-lactate, and methanol are not assimilated. Nitrate, nitrite, creatine, glucosamine, and d-tryptophan are assimilated as sole nitrogen sources. Ethylamine, l-lysine, creatinine, and imidazole are not assimilated. Minimum growth temperature is 15 °C, and maximum growth temperature is 35 °C. Growth in the presence of 0.1% cycloheximide is present, but growth in the presence of 10% NaCl plus 5% glucose and 1% acetic acid is absent. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are negative.
Notes. Two strains were collected from two different substrates, formed a wellsupported group related to Su. chuxiongensis, representing a new species, Su. robnettiae. Su. robnettiae differs from Su. chuxiongensis by 1.9% substitutions in the D1/D2 domain and 6.4% substitutions in the ITS region. Physiologically, unlike Su. chuxiongensis (Shi et al., 2021), Su. robnettiae is unable to assimilate d-ribose, melibiose, raffinose, or melezitose but is able to assimilate glycerol and lactose. Etymology. The specific epithet detingensis refers to the geographic origin of the type strain, Deting Town, Henan.

Discussion
In the present study, we collected rotting wood from the Tianchi Mountain National Forest Park located near Luoyang City in Henan Province of China. From these samples, we isolated several yeast strains. Some of these yeasts are known species, such as Metschnikowia henanensis, Saturnispora galanensis, Wickerhamomyces menglaensis and Deakozyma yunnanensis. Here, we recovered eight isolates from eight rotting woods of Trichomonascaceae yeast representing four new species belonging to the genera Diddensiella, Sugiyamaella, and Zygoascus. We described these new species as D. luoyangensis, Su. cylindrica, Su. Robnettiae, and Z. detingensis based on molecular phylogenetic and morphological evidence. A thorough and comprehensive phylogenetic analysis of the family Trichomonascaceae based on the combined ITS and the D1/D2 domains of the LSU rRNA gene sequences is provided, including almost all GenBank representatives and newly generated sequences, which may serve as a reference for the field. This study provides information on the species delimitation of the family Trichomonascaceae based on morphological and phylogenetic evidence.
Our phylogenetic analyses, based on ITS and the D1/D2 domains of the LSU rRNA gene sequences, are in concordance with previous studies (Morais et al. 2013b;Sena et al. 2017;Shi et al. 2021). However, the genus Sugiyamaella of Trichomonascaceae is not a monophyletic group. Morais et al. (2013b) indicated that Sugiyamaella is polyphyletic, where the species are intertwined with representatives of the genera Diddensiella and Spencermartinsiella. From the latter study, the genus could be divided into two main clades, which were later supported by Sena et al. (2017) and Shi et al. (2021). In this study, all species of Sugiyamaella and related genera were used to refine our understanding of the evolutionary relationships of this family, based on the ITS and nrLSU dataset. As shown in Fig. 1, all genera of Trichomonascaceae formed monophyletic groups with the exception of Sugiyamaella in which two main clades were reconstructed: (i) Su. smithiae (the type species), Su. lignohabitans, and In recent years, more than 40 yeast species have been identified from rotting wood in China (Wang et al. 2010;Guo et al. 2012;Gao et al. 2017;Zheng et al. 2017;Huang et al. 2018;Chai et al. 2020;Lv et al. 2020;Shi et al. 2021). Among them, at least 16 species of Trichomonascaceae have been isolated from rotting wood in China, including six new species previously obtained from China (Bla. xishuangbannaensis, Sp. cellulosicola, Su. qingdaonensis, Su. xiaguanensis, Su. Chuxiong, and Su. yunanensis) (Wang et al. 2010;Guo et al. 2012;Huang et al. 2018;Chai et al. 2020;Shi et al. 2021), new records of six species not known to occur in China (Su. americana, Su. ayubii, Su. novakii, Su. paludigena, Su. Valenteae, and Su. valdiviana) (Shi et al. 2021), and four novel species identified in this study (D. luoyangensis, Su. cylindrica, Su. robnettiae, and Z. detingensis). In China, there remain species to be discovered, such as those sequences of the D1/D2 domains of the LSU rRNA gene listed under GenBank accessions JN581115 and JN581116. To date, including the four new species described in this study, there are more than 100 species of Trichomonascaceae worldwide (www.mycobank.org). Although the taxonomy of Trichomonascaceae has been a focus of research in the past, many regions are under-sampled and more novel indigenous Trichomonascaceae species will undoubtedly be discovered in the future.