Three new species of Teunia (Cryptococcaceae, Tremellales) identified through phenotypic and phylogenetic analyses

Qi-Chao Guo; Shan Liu; Ya-Zhuo Qiao; Feng-Li Hui

doi:10.3897/mycokeys.105.120534

Abstract

Teunia, belonging to the family Cryptococcaceae of the order Tremellales, is a genus of plant-inhabiting fungi distributed across the globe. Its members form associations with different plant parts, including flowers, fruits, leaves, seeds, and twigs. Recent efforts have aimed to explore the diversity of Teunia in China, however, many geographical regions have not yet been explored. In this study, we included results of five Teunia yeast strains that were isolated from plant materials collected in Fujian, Guizhou and Henan provinces, with descriptions, illustrations, and phylogenetic analyses of three new species: T. acericola, T. mussaendrae isolated from leaf surfaces in Fujian, Guizhou and Henan Provinces, and T. qingyuanensis obtained from rotting wood in Fujian Province.

Key words

Basidiomycota, fungal diversity, new species, plant, taxonomy

Introduction

Teunia is a recently established genus by Li et al. (2020), based on the phylogenetic analysis of a seven-gene dataset consisting of SSU rRNA, D1/D2 LSU rRNA domain, ITS region, RPB1, RPB2, TEF1-α, and CYTB. This analysis revealed a well-supported clade encompassing Cryptococcus cuniculi K.S. Shin & Y.H. Park, Fonsecazyma tronadorensis Yurkov (= Cryptococcus tronadorensis V. de García, Zalar, Brizzio, Gunde-Cim & Van Broock), Fonsecazyma betulae Yurkov, Kachalkin & Boekhout (= Kwoniella betulae K. Sylvester, Q.M. Wang & Hittinger), along with three new species T. globosa Q.M. Wang, F.Y. Bai & A.H. Li, T. helanensis Q.M. Wang, F.Y. Bai & A.H. Li, and T. korlaensis Q.M. Wang, F.Y. Bai & A.H. Li that was designated as the type species of the genus (Li et al. 2020). Since then, the increasing accessibility of sequencing services and a large quantity of available molecular data have led to a rapid expansion in the knowledge of the genus, and seven new species have been described: T. rosae Q.M. Wang, A.H. Li, G.S. Wang & Wangmu, T. rudbeckiae Q.M. Wang, A.H. Li, G.S. Wang & Wangmu (Wang et al. 2020), T. siamensis Khanam & Limtong (Khunnamwong et al. 2020), T. lichenophila Kachalkin, M.A. Tomashevskaya & T.A. Pankrato (Crous et al. 2021), T. nitrariae X.Z. Liu, F.Y. Bai & X.Y. Wei (Wei et al. 2022), and T. virginiahalliae Y.P. Tan & G.S. Pegg (Tan and Pegg 2023). In the case of T. virginiahalliae, which has been proposed based only on the ITS sequence, a representative reference culture has not been deposited in a culture collection, which hampers further studies on this species.

Until now, 12 species have been accepted in Teunia (www.indexfungorum.org/; www.mycobank.org). They all share cream to yellow-colored colonies, polar budding, non-fermentative nature, and inability to form pseudohyphae, hyphae, and ballistoconidia (Li et al. 2020). The members of Teunia have been found in diverse habitats and are frequently isolated as epiphytes from flowers (Wang et al. 2020), leaves (Sylvester al. 2015; Li et al. 2020), and tree barks (Sylvester et al. 2015), T. lichenophila was isolated as endophyte from Cladonia rangiferina and C. stellaris (Crous et al. 2021). Species of Teunia have also been isolated from soil (Khunnamwong et al. 2020; Li et al. 2020), barley from wild rabbit feces (Shin et al. 2006) and glacial biomes (de Garcia et al. 2012). Furthermore, it is hypothesized that an excess of 30 undescribed or erroneously identified strains may represent an additional 20 Teunia species (Wang et al. 2020). These potential members originate from various diverse substrates, including plant materials such as flowers (Herzberg et al. 2002; Mittelbach et al. 2015), floral nectars (Alvarez-Pérez and Herrera 2013), seeds (Fernández et al. 2012), fruits, leaves, and twigs. Others have been collected from soil (Takashima et al. 2012; Yurkov et al. 2016), coastal seawater, and extreme acidic environments (Gadanho et al. 2006). Taken together, these previous findings could be an indication that the habitat of these fungi is different plant parts.

Currently, half of the accepted species in Teunia were described from China, T. globosa, T. helanensi, T. korlaensis (Li et al. 2020), T. rosae, T. rudbeckiae (Wang et al. 2020), and T. nitrariae (Wei et al. 2022). However, these species have been collected from limited geographical ranges, and it is hoped that broader field investigations will reveal additional members of the genus.

During our investigation, we isolated five strains of Teunia from various substrates across different regions of China. Our phylogenetic analyses and examination of phenotypic features determined that the isolates represent three new species. The objective of this paper is to describe these species with morphological and molecular characters and contribute to knowledge of the diversity of Teunia in China.

Materials and methods

Sample collection and yeast isolation

Materials were collected from the Fujian, Guizhou, and Henan Provinces of China. One of the yeast strains was isolated from rotting wood through the enrichment method described by Shi et al. (2021). Four additional strains were harvested from leaf surfaces using the improved ballistospore-fall method described by Nakase and Takashima (1993). Based on this method, fresh leaves were cut into small pieces and adhered with a thin layer of petroleum jelly to the inner lid of a Petri dish containing yeast extract-malt extract (YM) agar (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, and 2% agar). The mixture was supplemented with 0.01% chloramphenicol to avoid bacterial growth. Plates were incubated at 20 °C and monitored daily for colony formation. Selected colonies were streaked onto separate YM agar plates for purification. Following purification, strains were suspended in YM broth supplemented with 20% (v/v) glycerol and stored at –80 °C for future use. Cultures of all obtained isolates were preserved at the Microbiology Lab, Nanyang Normal University, Henan, China.

Phenotypic characterization

Morphological, physiological, and biochemical analyses were performed according to the standard methods described by Kurtzman et al. (2011). To examine the inducibility of the sexual state in each isolate, single or double strains were mixed on corn meal agar (CMA), potato dextrose agar (PDA), and V8 agar (10% V8 juice, 2% agar) at 20 °C for up to 8 weeks (Wang et al. 2020). Glucose fermentation was tested in a liquid medium with Durham fermentation tubes. Carbon and nitrogen assimilation capabilities were examined in a liquid medium, with starved inoculum used for nitrogen testing (Kurtzman et al. 2011). Growth at various temperatures (15, 20, 25, 30, 35, and 37 °C) was assessed through cultivation on YM agar plates. Cell morphology was examined with LEICA DM2500 cameras (LECIA Co, Wetzlar, Germany) and LASV4.13 software. All proposed names and descriptions were deposited in the MycoBank database (http://www.mycobank.org; 8 February 2024).

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from each strain using the Ezup Column Yeast Genomic DNA Purification Kit, according to the manufacturer’s instructions (Sangon Biotech Co., Shanghai, China). The ITS region, D1/D2 domain of the LSU rRNA, and a partial segment RPB1 were amplified with primers ITS1/ITS4 (White et al. 1990), NL1/NL4 (Kurtzman and Robnett 1998), and RPB1-Af and RPB1-Cr (Kurtzman and Robnett 2003), respectively. Amplifications were performed in a 25 µL reaction-volume tube containing 9.5 µL ddH₂O, 12.5 µL Taq 2X PCR Master Mix with blue dye (Sangon Biotech Co., Shanghai, China), 1 µL DNA template, and 1 µL of each primer. The ITS region and D1/D2 domain were amplified with 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, 40 s at 72 °C, and a final extension of 10 min at 72 °C (Toome et al. 2013). Amplification of the partial RPB1 gene was conducted using a touchdown PCR protocol as described by Wang et al. (2014). PCR products were then purified and sequenced by Sangon Biotech Co., Ltd (Shanghai, China) using the same primers. The identity and accuracy of each sequence were determined by comparison to sequences in GenBank. Assembly was performed with BioEdit v.7.1.3.0 (Hall 1999). All newly generated sequences were deposited in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/).

Phylogenetic analysis

Phylogenetic analyses were conducted based on LSU sequences alone and a combination of the ITS, LSU, and RPB1 dataset. Cryptococcus amylolentus CBS 6039^T and Cryptococcus neoformans CBS 8710^T were designated as outgroups (Crous et al. 2021). Individual loci sequences were aligned using MAFFT v.7.110 (Katoh and Standley 2013) under the G-INI-I option. Poorly aligned regions were removed and adjusted manually using MEGA v.11 (Tamura et al. 2021). Aligned sequences of the different loci were concatenated with Phylosuit v.1.2.2 (Zhang et al. 2020).

Maximum likelihood (ML) analysis was conducted using RAxML v.8.2.3 with the GTRGAMMA model (Stamatakis 2014). Node ML bootstrap values (MLBS) were evaluated using 1,000 rapid replicates. The Best-fit evolution model for Bayesian inference (BI) was determined with ModelFinder (Kalyaanamoorthy et al. 2017). BI analysis was performed using MrBayes v.3.2.7a (Ronquist et al. 2012) through the CIPRES Science Gateway. Six simultaneous Markov chains were run for 50 million generations, with trees sampled every 1,000^th generation. The first 25% of trees were discarded, representing the burn-in phase. Remaining trees were used to calculate the Bayesian posterior probabilities (BPP) of each clade. Trees were examined using FigTree v.1.4.3 (Andrew 2016). Branches exhibiting MLBS values ≥50% and BPP values ≥0.95 were shown at the nodes.

Results

Molecular phylogeny

A total of five yeast strains preliminarily identified as Teunia were studied further (Table 1). Besides the newly generated sequences, additional related sequences were also downloaded from GenBank (Table 2) for inclusion in the phylogenetic analyses.

Table 1.

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Yeast strains and origins investigated in this study.

Strain	Source	Location
Teunia acericola Y.Z. Qiao & F.L. Hui
NYNU 2111141^T	Leaf of Acer palmatum	Baotianman Nature Reserve, Neixiang County, Henan Province, China
NYNU 2111157	Leaf of Rhus chinensis	Baotianman Nature Reserve, Neixiang County, Henan Province, China
Teunia qingyuanensis Y.Z. Qiao & F.L. Hui
NYNU 22475^T	Rotting wood	Qingyuan Mountain, Quanzhou City, Fujian Province, China
Teunia mussaendrae Y.Z. Qiao & F.L. Hui
NYNU 23232^T	Leaf of Mussaenda pubescens	Sifangjing Village, Pingtang County, Guizhou Province, China
NYNU 23257	Leaf of Viburnum sp.	Sifangjing Village, Pingtang County, Guizhou Province, China

Table 2.

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Species name, strain/clone numbers, and GenBank accession numbers included in phylogenetic analyses. Entries in bold represent newly generated materials.

Species name	Strain/clone number	GenBank accession numbers
Species name	Strain/clone number	ITS	LSU D1/D2	RPB1
Cryptococcus amylolentus	CBS 6039^T	NR_111372	KY106966	KF036342
Cryptococcus neoformans	CBS 8710^T	NR_171785	NG_058766	KF036472
Kwoniella bestiolae	CBS 10118^T	NR_111373	NG_042482	KF036351
Kwoniella dejecticola	CBS 10117^T	NR_111374	NG_042483	KF036362
Kwoniella dendrophila	CBS 6074^T	NR_073257	NG_058326	KF036320
Kwoniella endophytica	CBS 15359^T	MH237945	MH237945	LS992197
Kwoniella heveanensis	CBS 569^T	NR_073210	AF075467	FJ534921
Kwoniella mangrovensis	CBS 8507^T	NR_073332	AF444742	KF036498
Kwoniella ovata	CGMCC 2.3439^T	NR_174734	MK050289	MK849160
Kwoniella pini	CBS 10737^T	NR_111269	KY108203	KF036395
Kwoniella shivajii	CBS 11374^T	NR_165977	NG_042515	KF036401
*Teunia acericola*	NYNU 2111141^T	OM017172	OM017170	PP236726
*Teunia acericola*	NYNU 2111157	PP239073	PP239062	PP236727
Teunia acericola	BI226	–	EU678944	–
Teunia acericola	HB31-3	–	KJ507251	–
Teunia acericola	MUCC1912	–	LC715712	–
Teunia acericola	MUCC2071	–	LC715721	–
Teunia acericola	F3-5	–	AB618905	–
Teunia betulae	NRRL Y-63732^T	KM384102	KM408130	–
Teunia cuniculi	CBS 10309^T	NR_137887	KY106982	MN014082
Teunia globosa	CGMCC 2.5648^T	NR_174733	MK050288	MK849235
Teunia helanensis	CGMCC 2.4450^T	NR_174732	MK050287	MK849208
Teunia korlaensis	CGMCC 2.3835^T	NR_174731	MK050286	MK849194
Teunia lichenophila	CBS 16716^T	MN128421	MN128421	HG992858
*Teunia mussaendrae*	NYNU 23232^T	OQ851888	OQ851887	PP236729
*Teunia mussaendrae*	NYNU 23257	PP239074	PP239072	PP236730
Teunia nitrariae	CGMCC 2.6797^T	OM417183	OM417183	–
*Teunia qingyuanensis*	NYNU 22475^T	OP269841	OP269842	PP236728
Teunia rosae	CGMCC 2.5830^T	MK942578	MK942560	MT268696
Teunia rudbeckiae	CGMCC 2.5840^T	MK942577	MK9425595	MT268698
Teunia siamensis	DMKU-XD44^T	LC440108	LC420623	–
Teunia tronadorensis	DSM 26994^T	MF959620	MF959620	–
Teunia virginiahalliae	BRIP 64084e^T	OR660683	–	–

The LSU dataset consisted of 32 sequences representing 25 species. The aligned set had a length of 603 characters, of which 480 were constant, 34 were variable and parsimony-uninformative, and 89 were parsimony-informative. The BI yielded a topology similar to the ML analysis, with an average standard deviation of split frequencies equal to 0.009938. In the LSU based phylogenetic tree (Fig. 1), five newly isolated strains formed three distinct and well-supported lineages that are distant from other Teunia species. Since T. virginiahalliae only has ITS sequence data, the phylogenetic analysis based on the ITS dataset was also performed. The phylogenetic tree (Suppl. material 1) recovered 12 known species of Teunia, while the newly isolated strains formed three independent lineages as in the phylogeny inferred from the LSU dataset.

Figure 1.

Maximum likelihood phylogenetic tree of Teunia generated from the LSU sequence data. The tree is rooted with Cryptococcus amylolentus CBS 6039^T and Cryptococcus neoformans CBS 8710^T. Bootstrap values (MLBS ≥ 50% and BPP ≥ 0.95) are displayed near branches. Type strain sequences are marked with (T).

The combined ITS, LSU, and RPB1 dataset encompassed sequences from 28 yeast strains representing 26 species. Including gaps, the dataset had an aligned length of 1,978 characters (549, 603, and 826 characters for ITS, LSU, and RPB1, respectively), of which 873 were constant, 381 were variable and parsimony-uninformative, and 724 were parsimony-informative. The best-fit model of the combined dataset for BI analysis was determined to be GTR+I+G, with equal nucleotide frequencies. The BI yielded a topology similar to the ML analysis, with an average standard deviation of split frequencies equal to 0.009550. The ITS, LSU, and RPB1 based phylogenetic tree (Fig. 2) produced a topology similar to that generated by the LSU based phylogenetic tree, and further confirmed the groupings of the three new species within Teunia.

Figure 2.

Maximum likelihood phylogenetic tree of Teunia generated from the combined ITS, LSU, and RPB1 sequence data. The tree is rooted with Cryptococcus amylolentus CBS 6039^T and Cryptococcus neoformans CBS 8710^T. Bootstrap values (MLBS ≥ 50% and BPP ≥ 0.95) are displayed near branches. Type strain sequences are marked with (T).

Strains NYNU 2111141 and NYNU 2111157 were isolated from different leaves, but possess identical D1/D2 and ITS sequences. Both phylogenetic trees (Figs 1, 2) revealed that these two strains clustered with T. korlaensis, T. nitrariae, T. rosae, and T. rudbeckiae, with variations of eight to10 nt (~1.3–1.7%) substitutions in the D1/D2 domain and more than 19 nt (~3.7%) mismatches in the ITS region. This suggests that the strains represent a novel Teunia species. A search of GenBank for entries associated with our test isolates unveiled the sequences of four unpublished strains: ‘Cryptococcus’ sp. BI226 (EU678944), ‘Kwoniella’ sp. HB31-3 (KJ507251), Teunia sp. MUCC1912 (LC715712), and Teunia sp. MUCC2071 (LC715721), along with an uncultured fungus clone F3-5 (AB618905) (Fig. 1). These sequences exhibit highly similar D1/D2 domain (0–2 nt differences) when compared with NYNU 2111141 and NYNU 2111157. This suggests they may all belong to the same novel species, for which we propose the name Teunia acericola sp. nov.

Isolated from rotting wood, strain NYNU 22475 formed a branch distant from the other Teunia species in the D1/D2 phylogenetic tree (Fig. 1). However, the tree based on the combined ITS, LSU, and RPB1 dataset weakly supported a cluster with T. cuniculi CBS 10309 (Fig. 2). The two strains differed by 16 nt (~2.9%) substitutions in the D1/D2 domain and 22 nt (~4.3%) mismatches in the ITS region, suggesting they are closely related but do not belong to the same species. Taken together, these findings indicate that NYNU 22475 represents a novel Teunia species, for which we propose the name Teunia qingyuanensis sp. nov.

Finally, isolated from separate leaves, strains NYNU 23232 and NYNU 23257 were found to possess identical sequences and formed an independent single-species lineage in the D1/D2 phylogenetic tree (Fig. 1). The ITS, LSU, and RPB1 combined tree presented a non-supported cluster with T. cuniculi and the newly discovered T. qingyuanensis sp. nov. (Fig. 2). BLASTn searches using D1/D2 sequences indicated that novel strains were most closely related to T. globosa, with variations of eight nt (~1.4%) substitutions in the D1/D2 domain and 28 nt (~5%) mismatches in the ITS region. Based on the ITS region, T. virginiahalliae represented the closest relative, differing by 19 nt (~3.4%) substitutions. The D1/D2 sequence of T. virginiahalliae was not available for comparison. Thus, it was determined that NYNU 23232 and NYNU 23257 represent a novel Teunia species, for which we propose the name Teunia mussaendrae sp. nov.

Taxonomy

Teunia acericola Y.Z. Qiao & F.L. Hui, sp. nov.

MycoBank No: 852101

Fig. 3A

Etymology

Referring to Acer, the genus of the plant where the type strain was isolated.

Typus

China. Henan Prov. Neixiang Co., Baotianman Nature Reserve; in the phylloplane of Acer palmatum; Nov 2021; R.R.Jia & W.T.Hu; NYNU 2111141 (holotype CICC 33544^T, culture ex-type JCM 35732; GenBank Nos: OM017172, OM017170, PP236726).

Figure 3.

Vegetative cells of A T. acericola sp. nov. NYNU 2111141^T B T. qingyuanensis sp. nov. NYNU 22475^T and C T. mussaendrae sp. nov. NYNU 23232^T, following 7 days of growth in YM broth at 20 °C. Scale bars: 10 μm.

Description

On YM agar after seven days at 20 °C, the streak culture was cream, mucoid, smooth, with entire margin. After seven days in YM broth at 20 °C, single cells were globose to ovoid, 2.5–5.5 × 4–6 μm, budding polar. After one month at 20 °C, sediment was present. In Dalmau plate culture on CMA, no hyphae or pseudohyphae were formed. Sexual structures were not observed in any of the strains or when strains are paired on PDA, CMA or V8 agar. Glucose fermentation was absent. Glucose, inulin, sucrose, raffinose, melibiose, galactose, lactose, trehalose, maltose, melezitose, cellobiose, salicin, L-sorbose, L-rhamnose, D-xylose, L-arabinose, D-arabinose (weak), 5-keto-D-gluconate, D-ribose, ethanol (weak), glycerol, ribitol, galactitol, D-mannitol, D-glucitol, myo-inositol, DL-lactate, succinate, D-gluconate, D-glucosamine (weak), 2-keto-D-gluconate, D-glucuronate, and glucono-1,5-lactone were assimilated as carbon sources; methanol, erythritol, and N-acetyl-D-glucosamine were not assimilated. Ethylamine and L-lysine were assimilated as nitrogen sources, nitrate, nitrite, and cadaverine were not assimilated. Maximum growth temperature was 35 °C. Growth in vitamin-free medium was negative. Growth on 50% (w/w) glucose-yeast extract agar was negative. Starch-like substances were not produced. Urease activity and Diazonium Blue B reaction were positive.

Additional strain examined

China. Henan Prov. Neixiang Co., Baotianman Nature Reserve; in the phylloplane of Rhus chinensis; Nov 2021; R.R.Jia & W.T.Hu; NYNU 2111157 (GenBank No: PP239073, PP239062, PP236727).

Note

In the molecular analysis (Figs 1, 2), T. acericola sp. nov. was clustered with T. korlaensis, T. nitrariae, T. rosae, and T. rudbeckiae. T. acericola sp. nov. can be differentiated from those four species by its ability to assimilate raffinose and its growth capacity at 35 °C.

Teunia qingyuanensis Y.Z. Qiao & F.L. Hui, sp. nov.

MycoBank No: 852102

Fig. 3B

Etymology

Referring to the locality, Qingyuan Mountain, where the type strain was collected.

Typus

China. Fujian Prov. Quanzhou City, Qingyuan Mountain; in rotting wood; Mar 2022; W.T.Hu & S.B.Chu; NYNU 22475 (holotype GDMCC 2.294^T, culture ex-type PYCC 9929; GenBank Nos: OP269841, OP269842, PP236728).

Description

On YM agar after seven days at 20 °C, the streak culture was cream, mucoid and smooth, with an entire margin. After seven days in YM broth at 20 °C, single cells were ovoid to ellipsoidal, 3–7 × 4–7.5 μm, budding polar. After one month at 20 °C, sediment was present. In Dalmau plate culture on CMA, no hyphae or pseudohyphae were formed. Sexual structures were not observed on PDA, CMA or V8 agar. Glucose fermentation was absent. Glucose, inulin, sucrose, raffinose, melibiose, galactose, lactose, trehalose, maltose, melezitose, cellobiose, salicin, L-sorbose (weak), L-rhamnose, D-xylose, L-arabinose, D-arabinose, 5-keto-D-gluconate, D-ribose, ethanol, glycerol, ribitol, galactitol, D-mannitol, D-glucitol, myo-inositol, DL-lactate, succinate, D-gluconate, 2-keto-D-gluconate, D-glucuronate, and glucono-1,5-lactone were assimilated as carbon sources; methanol, erythritol, and D-glucosamine were not assimilated. Ethylamine and L-lysine were assimilated as nitrogen sources; nitrate, nitrite, and cadaverine were not assimilated. Maximum growth temperature was 30 °C. Growth in vitamin-free medium was positive. Growth on 50% (w/w) glucose-yeast extract agar was negative. Starch-like substances were not produced. Urease activity and Diazonium Blue B reaction were positive.

Note

In the molecular analysis (Fig. 2), T. qingyuanensis sp. nov. formed a distinct clade together with T. cuniculi. They can be differentiated by the ability of T. qingyuanensis sp. nov. to grow in vitamin-free medium and to assimilate raffinose, melibiose, and DL-Lactate.

Teunia mussaendrae Y.Z. Qiao & F.L. Hui, sp. nov.

MycoBank No: 852103

Fig. 3C

Etymology

Referring to Mussaenda, the genus of the plant where the type strain was isolated.

Typus

China. Guizhou Prov. Pingtang Co., Sifangjing Vil.; in the phylloplane of Mussaenda pubescens; Feb 2023; D.Lu; NYNU 23232 (holotype CICC 33594^T, culture ex-type PYCC 9974; GenBank Nos OQ851888, OQ851887, PP236729).

Description

On YM agar after seven days at 20 °C, the streak culture was yellowish-cream, mucoid and smooth, entire margin. After seven days in YM broth at 20 °C, cells isolated were globose to ovoid, 3.5–5 × 4.5–6 μm, budding polar. After one month at 20 °C, a ring and sediment was present. In Dalmau plate culture on CMA, no hyphae or pseudohyphae were formed. Sexual structures were not observed in any of the strains or when strains were paired on PDA, CMA or V8 agar. Glucose fermentation was absent. Glucose, inulin, sucrose, galactose, lactose, trehalose, maltose, melezitose, cellobiose, salicin, L-sorbose, L-rhamnose, D-xylose, L-arabinose, D-arabinose, 5-keto-D-gluconate, D-ribose, ethanol (weak), glycerol (weak), ribitol, galactitol, D-mannitol, D-glucitol, myo-inositol, DL-lactate, succinate, D-gluconate, D-glucosamine (weak), 2-keto-D-gluconate, D-glucuronate, and glucono-1,5-lactone were assimilated as carbon sources; raffinose, melibiose, methanol, erythritol, and N-acetyl-D-glucosamine were not assimilated. Ethylamine (delayed), L-lysine, and cadaverine (delayed) were assimilated as nitrogen sources; nitrate and nitrite were not assimilated. Maximum growth temperature was 25 °C. Starch-like substances were not produced. Urease activity and Diazonium Blue B reaction were positive.

Additional strain examined

China. Guizhou Prov. Pingtang Co., Sifangjing Vil.; in the phylloplane of Viburnum sp.; Feb 2023; D.Lu; NYNU 23257 (GenBank Nos: PP239074, PP239072, PP236730).

Note

Based on the D1/D2 sequences, T. mussaendrae sp. nov. was most closely related to T. globosa. It can be differentiated from T. globosa by the ability to assimilate L-sorbose, L-arabinose, D-arabinose, ribitol, galactitol, D-glucitol, and D-gluconate. Additionally, T. mussaendrae sp. nov. can grow in vitamin-free medium at 25 °C, while T. globosa cannot.

Discussion

Our study confirms that three species with similar colors, colony morphology, and cell shapes, can be distinguished from previously described species using the polyphasic approach recommended by Li et al. (2020) and Wang et al. (2020). In this case we use physiological and biochemical characters as well as morphological and phylogenetic ones.

The genus Teunia is widely distributed in China, but knowledge about it is still in its infancy. The six species previously reported, come mainly from the northern regions (Li et al. 2020; Wang et al. 2020; Wei et al. 2022). The exploration of new territories, such as that carried out in the provinces of Fujian, Guizhou and Henan, is necessary to have a more exact knowledge of their distribution and ecology. The results presented in this paper increase the total number of Teunia species from six to nine.

Furthermore, four unpublished strains, BI226 from Brazil, HB31-3 from South Korea, MUCC1912 and MUCC2071 from Japan, as well as an uncultured fungus clone F3-5 from Japan, are conspecific with T. acerica sp. nov. These observations suggest that this species can have a wide distribution area. Therefore, a broader taxon sampling effort, coupled with molecular, phenotypic, physiological and biochemical data, is needed to fully understand the species diversity of Torula in the world.

The species of Teunia are frequently isolated as epiphytes from different parts of herbaceous plants, more rarely from tree barks or lichens; in this case, we isolated five yeast strains, which led to the discovery of three new species: T. acericola sp. nov., T. mussaendrae sp. nov. isolated from leaf surfaces, and T. qingyuanensis sp. nov. from rotting wood. We have found no previous reports of the presence of Teunia in rotting wood in China, hence our study is the first to report the presence of Teunia in rotten wood in China.

Teunia korlaensis and T. nitrariae are versatile extremophilic species that have been frequently found in plants inhabiting dry and alkaline environments (Wei et al. 2022), implying that these species may help plants survive in dry areas. We also isolated four strains of two novel Teunia species - T. acericola sp. nov. and T. mussaendrae sp. nov. - from plant leaves, and it is possible that these species provide similar ecological functions’ benefits to their hosts as do T. korlaensis and T. nitrariae.

Acknowledgments

The authors express their immense gratitude to their colleagues at the School of Life Science and Agricultural Engineering, Nanyang Normal University. Special thanks to Dr. Jing-Zhao Li and Lin Zhang for their help in specimen collection and Ting Lei for assistance with phylogenetic analysis.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research was funded by the National Natural Science Foundation of China (Grant No. 31570021) and Agricultural Biomass Green Conversion Technology University Scientific Innovation Team in Henan Province, China (Grant No. 24IRTSTHN036).

Author contributions

Data curation: QCG. Methodology: SL, QCG. Molecular phylogeny: QCG, YZQ. Writing - original draft: QCG. Writing - review and editing: FLH. All authors read and approved the final manuscript.

Author ORCIDs

Qi-Chao Guo https://orcid.org/0009-0002-9245-479X

Shan Liu https://orcid.org/0009-0003-2845-1495

Ya-Zhuo Qiao https://orcid.org/0009-0000-9074-2443

Feng-Li Hui https://orcid.org/0000-0001-7928-3055

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

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Supplementary material

Supplementary material 1

Supplementary data

Qi-Chao Guo, Shan Liu, Ya-Zhuo Qiao, Feng-Li Hui

Data type: pdf

Explanation note: fig. S1. Maximum likelihood phylogenetic tree of Teunia generated from the ITS sequence data.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

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﻿Abstract

Key words

﻿Introduction

﻿Materials and methods

﻿Sample collection and yeast isolation

﻿Phenotypic characterization

﻿DNA extraction, PCR amplification, and sequencing

﻿Phylogenetic analysis

﻿Results

﻿Molecular phylogeny

﻿Taxonomy

﻿ Teunia acericola Y.Z. Qiao & F.L. Hui, sp. nov.

Etymology

Typus

Description

Additional strain examined

Note

﻿ Teunia qingyuanensis Y.Z. Qiao & F.L. Hui, sp. nov.

Etymology

Typus

Description

Note

﻿ Teunia mussaendrae Y.Z. Qiao & F.L. Hui, sp. nov.

Etymology

Typus

Description

Additional strain examined

Note

﻿Discussion

﻿Acknowledgments

﻿Additional information

Conflict of interest

Ethical statement

Funding

Author contributions

Author ORCIDs

Data availability

References

Supplementary material

Abstract

Introduction

Materials and methods

Sample collection and yeast isolation

Phenotypic characterization

DNA extraction, PCR amplification, and sequencing

Phylogenetic analysis

Results

Molecular phylogeny

Taxonomy

Teunia acericola Y.Z. Qiao & F.L. Hui, sp. nov.

Teunia qingyuanensis Y.Z. Qiao & F.L. Hui, sp. nov.

Teunia mussaendrae Y.Z. Qiao & F.L. Hui, sp. nov.

Discussion

Acknowledgments

Additional information