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

﻿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.


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  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 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.

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 LEI-CA 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 ddH 2 O, 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.

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.
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.
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.
Strains NYNU 2111141 and NYNU 2111157 were isolated from different leaves, but possess identical D1/D2 and ITS sequences.Both phylogenetic    '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.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.
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.

Figure 1 .
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).

Figure 2 .
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).

Table 1 .
Yeast strains and origins investigated in this study.

Table 2 .
Species name, strain/clone numbers, and GenBank accession numbers included in phylogenetic analyses.Entries in bold represent newly generated materials.
T, type strain.Species obtained in this study are in bold.