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Research Article
Three new Dioszegia species (Bulleribasidiaceae, Tremellales) discovered in the phylloplane in China
expand article infoYa-Zhuo Qiao‡, Shan Liu‡, Qiu-Hong Niu‡, Feng-Li Hui‡
‡ Nanyang Normal University, Nanyang, China

Corresponding author: Qiu-Hong Niu ( qiuhongniu723@163.com )

Corresponding author: Feng-Li Hui ( fenglihui@yeah.net )

Academic editor: R. Henrik Nilsson
© 2024 Ya-Zhuo Qiao, Shan Liu, Qiu-Hong Niu, Feng-Li Hui.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Qiao Y-Z, Liu S, Niu Q-H, Hui F-L (2024) Three new Dioszegia species (Bulleribasidiaceae, Tremellales) discovered in the phylloplane in China. MycoKeys 101: 313-328. https://doi.org/10.3897/mycokeys.101.117174
Open Access

Abstract

The genus Dioszegia is comprised of anamorphic basidiomycetous yeasts and is classified in the family Bulleribasidiaceae of the order Tremellales. Currently, 24 species have been described and accepted as members of the genus, although its diversity and global distribution have not been thoroughly investigated. In this study, yeasts were isolated from plant leaves collected in the Guizhou and Henan Provinces of China and identified through a combination of morphological and molecular methods. Phylogenetic analyses of the combined ITS and LSU sequences coupled with morphological studies revealed three novel species, D. guizhouensis sp. nov., D. foliicola sp. nov., and D. aurantia sp. nov., proposed here. Additionally, our phylogenetic analyses suggest that the recently discovered species D. terrae is a synonym of D. maotaiensis. This study presents detailed descriptions and illustrations of three new Dioszegia species and highlights distinctions between them and their close relatives. The findings of this study contribute to our knowledge of the biodiversity of Dioszegia, offering a foundation for future research.

Key words

Basidiomycota, leaf, phylogenetic analysis, taxonomy, Tremellomycetes

Introduction

The genus Dioszegia encompasses a group of epiphytic basidiomycetes that inhabit the phylloplane. It was first proposed by Zsolt (1957) based on the single species Dioszegia hungarica. Roughly a decade later, the presence of sterigmata or ‘neck-likeconnections’ and lack of ballistoconidia in the species led to its reclassification as a member of the genus Cryptococcus (Phaff and Fell 1970). This was later disputed based on new molecular phylogenetic analyses which indicated a great distance between the species and other members of Cryptococcus (Takashima and Nakase 1999). In 2001, Dioszegia was reinstated and confirmed as a distinct genus based on phylogenetic analysis of the small subunit (SSU) rRNA genes. This finding allowed D. hungarica to re-join the genus along with two new combinations, D. aurantiaca and D. crocea (Takashima et al. 2001). Since then, the genus has expanded and now accommodates a total of 24 described species (Bai et al. 2002; Wang et al. 2003, 2008; Inácio et al. 2005; Connell et al. 2010; Takashima and Nakase 2011; Takashima et al. 2011; Trochine et al. 2017; Li et al. 2020; Maeng et al. 2022). A multi-gene phylogeny placed the genus Dioszegia within the newly proposed family Bulleribasidiaceae of the order Tremellales (Liu et al. 2015).

Members of the genus Dioszegia share several characteristics that are helpful for phenotypic identification. They exhibit orange or orange-red colonies, polar budding, a non-fermentative nature, and possess co-enzyme Q-10 (Takashima et al. 2001; Takashima and Nakase 2011). Additionally, all known species have thus far only been documented in an asexual stage (Takashima et al. 2001; Wang et al. 2008; Takashima and Nakase 2011). Some species may also form ballistoconidia, hyphae, and poorly developed pseudohyphae (Connell et al. 2010; Li et al. 2020).

Members of Dioszegia have been increasingly studied for a wide array of biotechnological applications. The carotenoid-producing abilities of species such as D. patagonica and D. takashimae offer commercial potential for products such as pigments, nutritional supplements, and pharmaceuticals (Mannazzu et al. 2015). At low temperatures, D. fristingensis and D. patagonica can secrete extracellular enzymes such as amylase, esterase, pectinase, cellulase, and lipase, making them potential sources of industrially relevant cold-active enzymes (Carrasco et al. 2012; Trochine et al. 2017)

In the past two decades, there has been a flurry of taxonomic research elucidating the diversity of Dioszegia species in China. At present, 18 of the 24 accepted Dioszegia species have been reported in China, 10 of which were initially described in the country (D. athyri, D. butyracea, D. changbaiensis, D. heilongjiangensis, D. kandeliae, D. maotaiensis, D. milinica, D. ovata, D. xingshanensis, and D. zsoltii). The remaining eight species were first documented in other countries (D. thyrium, D. aurantiaca, D. butyracea, D. cream, D. fristingensis, D. hungarica, D. statzelliae, D. takashimae, and D. zsoltii) (Bai et al. 2002; Wang et al. 2003, 2008; Li et al. 2020). There is still much to learn about the Dioszegia diversity and distribution in China and beyond. Our recent investigations revealed three new species over two years. This paper aims to employ an integrative taxonomic approach for the delimitation and description of these new taxa, providing a foundation for future investigations of Dioszegia.

Materials and methods

Sample collection and yeast isolation

Leaf samples were collected in the Guiyang Medicinal Botanical Garden (26°53'72"N, 106°70'52"E) and Baotianman Nature Reserve (33°30'44"N, 111°55'47"E) in China. The Guiyang Medicinal Botanical Garden is located in the city of Guiyang in the Yunnan Province of southwest China. With more than 1200 kinds of medicinal plants, it is known as the natural medicine valley. The local climate in this botanical garden is warm winters and fresh and cool summers, with annual mean temperatures around 15.3 °C. The Baotianman Nature Reserve, located in the Henan Province of central China, measures 4,285 ha. With a forest coverage rate of 98%, it is classified as World Biosphere Reserve by the United Nations Educational, Scientific and Cultural Organization (UNESCO). The reserve encompasses a virgin forest with more than 2000 species of vascular plants. The local climate is typical of a transitional climate from northern subtropical zone to warm temperate zone, with cold dry winters, and fresh rainy summers. The annual mean temperature is 15.1 °C.

Yeast strains were isolated from leaf surfaces using the improved ballistospore-fall method as described by Nakase and Takashima (1993). In brief, vaseline was employed to affix fresh and healthy leaves to the inside lids of Petri dishes containing yeast extract-malt extract (YM) agar (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, and 2% agar). Plates were then incubated at 20 °C until visible colonies had formed. Colonies with different morphotypes were selected and streaked onto additional YM agar plates for purification. After purification, strains were suspended in YM broth supplemented with 20% (v/v) glycerol and stored at −80 °C for future use. All obtained isolates were preserved at the Microbiology Lab, Nanyang Normal University, Henan, China.

Morphological and physiological characterization

Phenotypic and physiological characteristics of each yeast isolate were examined using the methods established by Kurtzman et al. (2011). Cell morphology was examined using a Leica DM2500 microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a Leica DFC295 digital microscope color camera under bright field, phase contrast, and differential interference contrast (DIC) conditions. Sexual cycles were investigated for both individual and paired strains on potato dextrose agar (PDA) (20% potato infusion, 2% glucose, and 1.5% agar), corn meal (CM) agar, and yeast carbon base plus 0.01% ammonium sulphate (YCBS) agar for two months and observed at weekly intervals (Li et al. 2020). Ballistoconidium-forming activity was investigated using the inverted-plate method (do Carmo-Sousa and Phaff 1962) after two weeks of incubation on CM agar at 20 °C. Glucose fermentation was observed using Durham fermentation tubes with a liquid medium. Carbon and nitrogen assimilation tests were conducted in a liquid medium, with starved inoculum employed for the latter (Kurtzman et al. 2011). Growth at various temperatures (15, 20, 25, 30, 35, and 37 °C) was determined by cultivation on YM agar. All novel taxonomic descriptions and proposed names were deposited in the MycoBank database (Robert et al. 2013).

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from each yeast strain using the Ezup Column Yeast Genomic DNA Purification Kit according to the manufacturer’s instructions (Sangon Biotech Co., Shanghai, China). The ITS region and the D1/D2 domain of the LSU rRNA gene were amplified using primer sets ITS1/ITS4 (White et al. 1990) and NL1/NL4 (Kurtzman and Robnett 1998), respectively. Amplifications were performed in a 25 µL reaction- tube containing 9.5 µL ddH2O, 12.5 µL 2× Taq PCR Master Mix with blue dye (Sangon Biotech Co., Shanghai, China), 1 µL DNA template, and 1 µL of each primer. Amplifications were conducted with the following parameters: initial denaturation at 95 °C for 2 min, followed by 35 cycles of 95 ° for C 30 s, 51 °C for 30 s, 72 °C for 40 s, and a final extension at 72 °C for 10 min (Wang et al. 2014). PCR products were purified and sequenced using the same primers by Sangon Biotech Co., Ltd (Shanghai, China). The identity and accuracy of the newly obtained sequences were determined by comparison to GenBank (Sayers et al. 2022) entries. Sequence assembly was conducted using BioEdit v. 7.1.3.0 (Hall 1999). All generated sequences were submitted to GenBank and their corresponding accession numbers are listed in Table 1.

Table 1.
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Taxon names, strain numbers, and GenBank accession numbers used for phylogenetic analyses. Entries in bold were newly generated for this study.

Taxa name Strain number GenBank accession numbers
ITS LSU D1/D2
Bulleribasidium begoniae CBS 10762T NR_154878 NG_058707
Bulleribasidium foliicola CBS 11407T KY101801 NG_058708
Bulleribasidium hainanense CBS 11409T NR_154879 NG_058709
Bulleribasidium oberjochense CBS 9110T NR_121467 NG_042388
Bulleribasidium panici CBS 9932T NR_121293 NG_058710
Bulleribasidium pseudovariabile CBS 9609T NR_111085 NG_042393
Bulleribasidium sanyaense CBS 11408T NR_159742 GQ438831
Bulleribasidium setariae CBS 10763T NR_154880 NG_058610
Bulleribasidium siamensis CBS 9933T NR_144773 AY188388
Bulleribasidium variabile CBS 7347T NR_111058 AF189855
Bulleribasidium wuzhishanense CBS 11411T NR_153643 GQ438830
Dioszegia aurantia sp. nov. NYNU 229189T OP566892 OP566893
Dioszegia aurantia sp. nov. G.M. 2006-09-03.6 951 OP419710 OP419710
Dioszegia antarctica CBS 10920 T NR_159813 FJ640575
Dioszegia athyri CBS 10119T EU070926 EU070931
Dioszegia aurantiaca CBS 6980 T NR_155060 NG_059153
Dioszegia buhagiarii CBS 10054T NR_073346 NG_059154
Dioszegia butyracea CBS 10122 T KY103348 KY107637
Dioszegia catarinonii CBS 10051T NR_155061 NG_059155
Dioszegia changbaiensis CBS 9608T NR_136964 NG_059069
Dioszegia crocea CBS 6714T NR_155062 KY107640
Dioszegia cryoxerica CBS 10919T FJ640565 FJ640562
Dioszegia dumuzii CBS 12501T LT548261 LT548261
Dioszegia foliicola sp. nov. NYUN 229182T OP566887 OP566888
Dioszegia foliicola sp. nov. NYNU 229188 OP566890 OP566889
Dioszegia foliicola sp. nov. NYNU 2211140 OR863956 OR863957
Dioszegia fristingensis CBS 10052 T NR_136970 NG_070549
Dioszegia guizhouensis sp. nov. NYNU 22985T OP566883 OP566880
Dioszegia guizhouensis sp. nov. NYUN 229195 OP566896 OP581919
Dioszegia heilongjiangensis CGMCC 2.5674T NR_174736 MK050291
Dioszegia hungarica CBS 4214T NR_073227 NG_042350
Dioszegia kandeliae CGMCC 2.5658T NR_174739 MK050296
Dioszegia maotaiensis CGMCC 2.4537T NR_174738 MK050295
Dioszegia milinica CGMCC2.5628T MK050290 NR_174735
Dioszegia ovata CGMCC 2.3625T NR_174737 MK050294
Dioszegia patagonica CBS 14901T NR_158412 NG_088008
Dioszegia rishiriensis CBS 11844T NR_157461 NG_059156
Dioszegia statzelliae CBS 8925T AY029342 AY029341
Dioszegia takashimae CBS 10053 T NR_136971 AY562149
Dioszegia terrae KCTC 27998 T MZ734406 MZ734403
Dioszegia xingshanensis CBS 10120T KY103359 KY107649
Dioszegia zsoltii var. yunnanensis CBS 9128T NR_156190 NG_070550
Dioszegia zsoltii var. zsoltii CBS 9127T AF385445 NG_059157
Nielozyma formosana CBS 10306 T NR_154221 NG_058356
Nielozyma melastomae CBS 10305T NR_154221 AB119464
Sugitazyma miyagiana CBS 7526T NR_073237 AF189858

CBS, CBS-KNAW Collections, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CGMCC, China General Microbiological Culture Collection Center, Beijing, China; KCTC, Korea Collection for Type Cultures, KRIBB, Korea; NYNU, Microbiology Lab, Nanyang Normal University, Henan, China; T, type strain. Species obtained in this study are in bold.

Phylogenetic analysis

Phylogenetic analyses employed a total of 92 nucleotide sequences, including 12 novel sequences generated in this study. The remaining sequences were obtained from previous studies (Li et al. 2020; Maeng et al. 2022) and GenBank (Table 1). Sugitazyma miyagiana CBS 7526T was used as the outgroup. Phylogenetic relationships between the new Dioszegia species and their close relatives were determined using a combined ITS and LSU sequence dataset. Sequences of individual markers were aligned with either Clustal X v. 1.83 (Thompson et al. 1997) or MAFFT v. 7.110 (Katoh and Standley 2013) using default settings. Aligned sequences of the different markers were concatenated with PhyloSuite v. 1.2.2 (Zhang et al. 2020). Alignments were improved through manual gap adjustments. Ambiguously aligned regions were excluded prior to analysis.

Phylogenetic analyses were conducted employing both maximum likelihood (ML) and Bayesian inference (BI). ML was determined with 1,000 searches on RAxML v. 8.2.3 (Stamatakis 2014) and ML bootstrap values (MLBS) were assessed through 1,000 rapid bootstrap replicates using the GTRCAT model. For BI, ModelFinder (Kalyaanamoorthy et al. 2017) was used to determine the optimal substitution model to fit the DNA evolution. BI data was analysed with MrBayes v. 3.2.7a (Ronquist et al. 2012) through the CIPRES Science Gateway version 3.3. Best-fit evolution models for the ITS and LSU partitions were GTR+I+G. Six simultaneous Markov chains were run for 50 million generations with trees being sampled every 1,000th generation. The first 25% of created sample trees were discarded as the burn-in phase of analysis. The remaining trees were used to infer Bayesian posterior probabilities (BPP) for the clades.

The resulting trees were viewed in FigTree v. 1.4.3 (Andrew 2016) and processed with Adobe Illustrator CS5. Branches that received MLBS ≥ 50% and BPP ≥ 0.95 were considered significantly supported.

Results

Molecular phylogeny

This study presents the discovery of three novel Dioszegia species represented by six strains isolated from leaf samples in the provinces of Guizhou and Henan (Table 2). The combined ITS and LSU sequence data was utilized to elucidate the phylogenetic positions of the new species. 120 aligned positions were excluded from the alignment due to problematic homology assessment. This final dataset consisted of 997 characters, 588 from ITS and 409 from LSU. Among these, 604 were constant and 393 were variable, out of which 292 were parsimony-informative. Finally, 101 were singletons. The topology of the ML and Bayesian trees was consistent with each other, and only the ML tree is shown (Fig. 1). The five strains isolated in this study formed three strongly supported groups (100% MLBS/1 BPP), distinct from other known species of Dioszegia.

Table 2.
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Strains representing the novel species described in this study and relevant information associated to them.

Strain Source Location
Dioszegia guizhouensis sp. nov.
NYNU 22985T Leaf of Schisandra sp. Guiyang Medicinal Botanical Garden, Guiyang, Guizhou Province, China
NYUN 229195 Leaf of Mussaendae sp. Guiyang Medicinal Botanical Garden, Guiyang, Guizhou Province, China
Dioszegia foliicola sp. nov.
NYUN 229182T Leaf of Salvia sp. Guiyang Medicinal Botanical Garden, Guiyang, Guizhou Province, China
NYNU 229188 Leaf of Broussonetia papyrifera Guiyang Medicinal Botanical Garden, Guiyang, Guizhou Province, China
NYNU 2211140 Leaf from an unidentified tree Baotianman Nature Reserve, Nanyang, Henan Province, China
Dioszegia aurantia sp. nov.
NYNU 229189T Leaf of Cornus officinalis Guiyang Medicinal Botanical Garden, Guiyang, Guizhou Province, China
Figure 1. 

Maximum likelihood (ML) phylogram of Dioszegia species and close relatives based on combined ITS and LSU sequence data. Sugitazyma miyagiana CBS 7526T serves as the outgroup. Branches are labelled with MLBS ≥ 50% and BPP ≥ 0.95. Novel strains are highlighted in bold.

The strains NYUN 22985 and NYUN 229195 had similar sequences with only one nt difference in the ITS region, suggesting that they belong to the same species. Two strains in the NYUN 22985 group formed a separate branch on the phylogenetic tree (Fig. 1), forming a clade with D. hungarica, the Dioszegia type species, and 15 other known species with strong support (100 MLBS/1 BPP). BLASTn searches of the D1/D2 and ITS sequences indicated that D. hungarica is the closet relative, differing by four nt (~0.7%) substitutions in the D1D2 domain and 14–15 nt (~2.9–3.1%) mismatches in the ITS region. The NYUN 22985 group is considered a distinct Dioszegia species based on the basidiomycetous yeast species threshold (Fell et al. 2000; Vu et al. 2016), which suggests that strains differing by two or more nucleotide substitutions in the D1/D2 domains or exhibiting 1–2% nucleotide differences in the ITS regions may represent different taxa. Therefore, D. guizhouensis sp. nov. is proposed as a novel Dioszegia species to accommodate the strains.

Three strains, viz. NYNU 229182, NYNU 229188, and 2211140, possessed mutually similar sequences with three nt differences in the D1/D2 region and one in the ITS region, indicating conspecificity. Additionally, the NYNU 229182 group shared similar D1/D2 sequences (one to two nt differences) with the GenBank isolate WOct07D (2)-Y3 (GQ352531) identified as ‘Dioszegia zsoltii’, suggesting another conspecific relationship. BLASTn searches of the D1/D2 sequences indicated that this group was most closely related to D. maotaiensis and D. terrae, differing by 10–11 nt (~1.7–1.8%) substitutions in the D1/D2 domain and more than 27 nt (5.4%) mismatches in ITS region. Thus, the group represents a novel Dioszegia species, for which the name D. foliicola sp. nov. is proposed.

Strain NYNU 229189 grouped with G.M.2006-09-03.6951 (OP419710), an unpublished strain obtained from the bark of rotting branches collected in Australia, which jointly were placed as a separate branch as the sister clade to the remaining part of of Dioszegia (Fig. 1). The two strains differed by only two and four nt differences in the D1/D2 and ITS region, respectively, suggesting conspecificity. NYNU 229189 is closely related to D. maotaiensis and D. terrae, differing from the latter two by 16 nt (~2.7%) substitutions in the D1/D2 domain and more than 23 nt (~5.7%) mismatches in the ITS region. This suggests that NYNU 229189 represents a new Dioszegia species, for which the name D. aurantia sp. nov. is proposed.

Taxonomy

Dioszegia guizhouensis Y.Z. Qiao & F.L. Hui, sp. nov.

MycoBank No: MycoBank No: 851291
Fig. 2A

Etymology

The specific epithet guizhouensis refers to the geographic origin of the type strain, Guizhou province.

Typus

China, Guizhou Province, Guiyang City, Guiyang Botanical Garden, in the phylloplane of Schisandra sp., September 2022, L. Zhang and F.L. Hui, NYUN 22985 (holotype GDMCC 2.311T preserved as a metabolically inactive state, culture ex-type PYCC 9938).

Figure 2. 

Vegetative cells of Dioszegia guizhouensis sp. nov. NYNU 22985T (A), Dioszegia foliicola sp. nov. NYUN 229182T (B), and Dioszegia aurantia sp. nov. NYNU 229189T (C) following growth in YM broth for 7 days at 20 °C. Scale bars: 10 μm.

Description

On YM agar, after 7 days at 20 °C, the streak culture is pink to orange, butyrous, smooth. The margin is entire. On YM agar, after 7 days at 20 °C, cells are ovoid and ellipsoidal, 2.8–4.6 × 4.1–6.8 μm and single, budding is polar. After 1 month at 20 °C, a ring and sediment are present. In Dalmau plate culture on corn meal agar, hyphae and pseudohyphae are not formed. Sexual structures are not observed for individual strains and strain pairs on PDA, CM agar, and YCBS agar for two months. Ballistoconidia are not produced on CM agar after two weeks at 20 °C. Glucose fermentation is absent. Glucose, sucrose, raffinose, melibiose, galactose, trehalose, maltose, melezitose, cellobiose, salicin, L-sorbose (delayed), L-rhamnose, D-xylose, L-arabinose, D-arabinose, 5-keto-D-gluconate (weak), D-ribose, galactitol, D-mannitol, D-glucitol, succinate (weak), citrate, D-gluconate, N-acetyl-D-glucosamine, 2-keto-D-gluconate, D-glucuronate, and glucono-1,5-lactone are assimilated as carbon sources. Inulin, lactose, methyl-α-D-glucoside, methanol, ethanol, glycerol, erythritol, ribitol, myo-inositol, DL-lactate, and D-glucosamine are not assimilated. Nitrite is assimilated as the sole nitrogen source. Nitrate, ethylamine, L-lysine, and cadaverine are not assimilated. Maximum growth temperature is 30 °C. Growth in vitamin-free medium is positive. Starch-like substances are produced. Urease activity is positive. Diazonium Blue B reaction is positive.

Additional strain examined

China, Guizhou Province, Guiyang City, Guiyang Botanical Garden, in the phylloplane of Mussaendae sp., September 2022, L. Zhang and F.L. Hui, NYUN 229195.

GenBank accession numbers

Holotype NYUN 22985T (ITS: OP566883, D1/D2: OP566880); additional strain 229195 (ITS: OP566896, D1/D2: OP581919).

Note

Dioszegia guizhouensis sp. nov. can be physiologically differentiated from its closest known species D. hungarica (Takashima and Nakase 2011) by its inability to assimilate D-glucosamine, its ability to assimilate melibiose and L-sorbose, and its capacity to grow in vitamin-free medium and at 30 °C.

Dioszegia foliicola Y.Z. Qiao & F.L. Hui, sp. nov.

MycoBank No: MycoBank No: 851294
Fig. 2B

Etymology

The specific epithet foliicola refers to the type strain isolated from a leaf.

Typus

China, Guizhou Province, Guiyang City, Guiyang Botanical Garden, in the phylloplane of Salvia sp., September 2022, L. Zhang and F.L. Hui, NYUN 229182 (holotype GDMCC 2.316T preserved as a metabolically inactive state, culture ex-type PYCC 9939 and CICC 33571).

Description

On YM agar, after 7 days at 20 °C, the streak culture is orange, butyrous, smooth. The margin is entire. On YM agar, after 7 days at 20 °C, cells are ovoid and ellipsoidal, 3.9–4.8 × 4.8–7.9 μm and single, budding is polar. After 1 month at 20 °C, a ring and sediment are present. In Dalmau plate culture on corn meal agar, hyphae and pseudohyphae are not formed. Sexual structures are not observed for individual strains and strain pairs on PDA, CM agar and YCBS agar for two months. Ballistoconidia are not produced on CM agar after two weeks at 20 °C. Glucose fermentation is absent. Glucose, sucrose, raffinose, melibiose, galactose, trehalose, maltose, melezitose, methyl-α-D-glucoside, cellobiose, salicin, L-sorbose, L-rhamnose, D-xylose, L-arabinose, D-arabinose, 5-keto-D-gluconate, D-ribose, galactitol, D-mannitol, succinate, D-gluconate, N-acetyl-D-glucosamine, 2-keto-D-gluconate and D-glucuronate are assimilated as carbon sources. Inulin, lactose, methanol, ethanol, glycerol, erythritol, ribitol, D-glucitol, myo-inositol, DL-lactate, citrate, D-glucosamine, and glucono-1,5-lactone are not assimilated. Nitrite and L-lysine are assimilated as nitrogen sources. Nitrate, ethylamine, and cadaverine are not assimilated. Maximum growth temperature is 30 °C. Growth in vitamin-free medium is positive. Starch-like substances are produced. Urease activity is positive. Diazonium Blue B reaction is positive.

Additional strain examined

China, Guizhou Province, Guiyang City, Guiyang Botanical Garden, in the phylloplane of Broussonetia papyrifera, September 2022, L. Zhang and F.L. Hui, NYUN 229188 and China, Henan Province, Nanyang City, Baotianman Nature Reserve, in the phylloplane from an unidentified tree, October 2022, J.Z. Li, NYUN 2211140.

GenBank accession numbers

Holotype GDMCC 2.316T (ITS: OP566887, D1/D2: OP566888); additional strains NYUN 229188 (ITS: OP566890, D1/D2: OP566889) and NYUN 2211140 (ITS: OR863956, D1/D2: OR863957).

Note

Dioszegia foliicola sp. nov. can be physiologically differentiated from its closest known species D. maotaiensis (Li et al. 2020) by its inability to assimilate inulin and citrate, its ability to assimilate methyl-α-D-glucoside, salicin, L-sorbose, D-ribose, galactitol, and D-mannitol, and its capacity to grow at 30 °C.

Dioszegia aurantia Y.Z. Qiao & F.L. Hui, sp. nov.

MycoBank No: MycoBank No: 851296
Fig. 2C

Etymology

The specific epithet aurantia refers to the aurantiaca colony morphology.

Typus

China, Guizhou Province, Guiyang City, Guiyang Botanical Garden, in the phylloplane of Cornus officinalis, September 2022, L. Zhang and F.L. Hui, NYUN 229189 (holotype GDMCC 2.335T preserved as a metabolically inactive state, culture ex-type PYCC 9937 and CICC 33572).

Description

On YM agar, after 7 days at 20 °C, the streak culture is orange, butyrous, smooth. The margin is entire. On YM agar, after 7 days at 20 °C, cells are ovoid and ellipsoidal, 4.6–5.0 × 5.0–8.2 μm and single, budding is polar. After 1 month at 20 °C, a ring and sediment are present. In Dalmau plate culture on corn meal agar, hyphae and pseudohyphae are not formed. Sexual structures are not observed for individual strains and strain pairs on PDA, CM agar, and YCBS agar for two months. Ballistoconidia are not produced on CM agar after two weeks at 20 °C. Glucose fermentation is absent. Glucose, inulin, sucrose, raffinose, melibiose, galactose, trehalose, maltose, melezitose, methyl-α-D-glucoside (delayed), cellobiose, salicin (weak), L-sorbose (delayed), L-rhamnose (delayed and weak), D-xylose, L-arabinose, D-arabinose (weak), 5-keto-D-gluconate, D-ribose, galactitol, D-mannitol, D-glucitol, succinate (weak), N-acetyl-D-glucosamine, 2-keto-D-gluconate (delayed and weak), and D-glucuronate are assimilated as carbon sources. Lactose, methanol, ethanol, glycerol, erythritol, ribitol, myo-inositol, DL-lactate, citrate, D-gluconate, D-glucosamine, and glucono-1,5-lactone are not assimilated. Nitrite (delayed) and L-lysine (delayed and weak) are assimilated as nitrogen sources. Nitrate, ethylamine, and cadaverine are not assimilated. Maximum growth temperature is 25 °C. Growth in vitamin-free medium is negative. Starch-like substances are produced. Urease activity is positive. Diazonium Blue B reaction is positive.

GenBank accession numbers

Holotype GDMCC 2.335T (ITS: OP566892, D1/D2: OP566893).

Note

Dioszegia aurantia sp. nov. can be physiologically differentiated from its closest known species D. maotaiensis (Li et al. 2020) by its inability to assimilate citrate, its ability to assimilate methyl-α-D-glucoside, salicin, L-sorbose, D-ribose, D-mannitol, D-glucitol, and N-acetyl-D-glucosamine, and its capacity to grow in vitamin-free medium and at 30 °C.

Discussion

In this study, we present three novel Dioszegia species discovered in China: D. guizhouensis sp. nov., D. foliicola sp. nov., and D. aurantia sp. nov. This work provides a comprehensive description of each species based on molecular analyses and morphological examinations. Moreover, our phylogenetic analyses illustrate clear distinctions between each new species and other members of Dioszegia, which was confirmed as a monophyletic genus in a strongly supported clade (Fig. 1). Pairwise sequence comparisons of the D1/D2 domain and the ITS region of the novel species and their close relatives support species differentiation based on the common threshold applied to basidiomycetous yeasts (Fell et al. 2000; Vu et al. 2016). The new species were highly similar in cell shape, colony morphology, and color, but differed from closely related species in terms of physiological and biochemical characteristics. Therefore, the results of our molecular phylogenetic analyses and phenotypic examinations support the description of three new Dioszegia species.

Several new species have been added to Dioszegia recently (Li et al. 2020; Maeng et al. 2022). Notably, our phylogenetic analyses revealed that the recently described species D. terrae clustered with D. maotaiensis in a well-supported clade within Dioszegia (Fig. 1). D. maotaiensis was described first and the description of D. terrae seminly overlooked the previously validly described species D. maotaiensis. These two species had only one nt difference in the ITS region, suggesting that D. terrae is a synonym of D. maotaiensis. Consequently, 26 species, including three new species described in the present study, are currently included in the genus Dioszegia.

Members of the genus Dioszegia are widely distributed across a variety of habitats. Although isolates are commonly obtained as epiphytic phylloplane fungi in temperate and subtropical climate regions (Inácio et al. 2005; Wang et al. 2008; Li et al. 2020), previous studies have also collected samples from roots (Renker et al. 2004) and soil (Takashima et al. 2011; Maeng et al. 2022). Additionally, isolates have also been collected from cold substrates such as snow (Trochine et al. 2017), glacial melt (de García et al. 2007; Trochine et al. 2017), and polar desert soil (Connell et al. 2010). In this study, six strains of three new Dioszegia species share with most other species in the genus association with plant leaves. The results further confirm that the natural distribution of Dioszegia species in the phylloplane is common. Furthermore, strain WOct07D (2)-Y3 (GQ352531), identified as ‘Dioszegia zsoltii’ from USA, is conspecific with D. foliicola sp. nov., while strain G.M.2006-09-03.6951 (OP419710) from Australia is conspecific with D. aurantia sp. nov. These observations suggests that the two new species D. foliicola sp. nov. and D. aurantia sp. nov. may be broadly distributed outside of China. Indeed, further large-scale studies are needed to explore the diversity and distribution of Dioszegia species worldwide. D. fristingensis is a versatile extremophilic species that has been frequently found in plants inhabiting hyper-arid, alkaline, and hypersaline environments (Abu-Ghosh et al. 2014; Wei et al. 2022), implying that this species may help plants survive in dry areas. We also isolated six strains of three novel Dioszegia species—D. guizhouensis sp. nov., D. foliicola sp. nov., and D. aurantia sp. nov.—from plant leaves, and it is possible that these species provide similar ecological functions benefits to their hosts as does D. fristingensis.

Many Dioszegia species have adapted to tolerate challenges presented by their environments. Notably, more than 10 Dioszegia species are known to accumulate mycosporin-glutamine-glucoside (MGG), a UVB-absorbing molecule that acts in response to photostimulation (Trochine et al. 2017). D. patagonica even contains higher levels of MGG than Phaffia rhodozyma, which is recognized for its ability to endure UV-B radiation (Madhour et al. 2005; Libkind et al. 2009). Further exploration of Dioszegia diversity is necessary to determine whether MGG is associated with other taxonomic traits or influences UV radiation tolerance (Libkind et al. 2009).

Acknowledgments

The authors express deep 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 providing specimens and Dr. 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 (Project No. 31570021 and 3217010010) and Agricultural Biomass Green Conversion Technology University Scientific Innovation Team in Henan Province, China (Project No. 24IRTSTHN036).

Author contributions

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

Author ORCIDs

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

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

Qiu-Hong Niu https://orcid.org/0000-0003-1695-7117

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.

References

  • Abu-Ghosh S, Droby S, Korine C (2014) Seasonal and plant-dependent variations in diversity, abundance and stress tolerance of epiphytic yeasts in desert habitats. Environmental Microbiology Reports 6(4): 373–382. https://doi.org/10.1111/1758-2229.12161
  • Andrew R (2016) FigTree: Tree figure drawing tool Version 1.4.3. Institute of Evolutionary Biology, University of Edinburgh Press, United Kingdom.
  • Bai FY, Takashima M, Jia JH, Nakase T (2002) Dioszegia zsoltii sp. nov., a new ballistoconidium-forming yeast species with two varieties. The Journal of General and Applied Microbiology 48(1): 17–23. https://doi.org/10.2323/jgam.48.17
  • Carrasco M, Rozas JM, Barahona S, Alcaíno J, Cifuentes V, Baeza M (2012) Diversity and extracellular enzymatic activities of yeasts isolated from King George Island, the sub-Antarctic region. BMC Microbiology 12(1): е251. https://doi.org/10.1186/1471-2180-12-251
  • Connell LB, Redman R, Rodriguez R, Barrett A, Iszard M, Fonseca A (2010) Dioszegia antarctica sp. nov. and Dioszegia cryoxerica sp. nov., psychrophilic basidiomycetous yeasts from polar desert soils in Antarctica. International Journal of Systematic and Evolutionary Microbiology 60(Pt 6): 1466–1472. https://doi.org/10.1099/ijs.0.015412-0
  • de García V, Brizzio S, Libkind D, Buzzini P, van Broock M (2007) Biodiversity of cold-adapted yeasts from glacial meltwater rivers in Patagonia, Argentina. FEMS Microbiology Ecology 59(2): 331–241. https://doi.org/10.1111/j.1574-6941.2006.00239.x
  • Fell JW, Boekhout T, Fonseca A, Scorzetti G, Statzell-Tallman A (2000) Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. International Journal of Systematic and Evolutionary Microbiology 50(Pt 3): 1351–1371. https://doi.org/10.1099/00207713-50-3-1351
  • Hall TA (1999) Bioedit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
  • Inácio J, Portugal L, Spencer-Martins I, Fonseca A (2005) Phylloplane yeasts from Portugal: Seven novel anamorphic species in the Tremellales lineage of the Hymenomycetes (Basidiomycota) producing orange-coloured colonies. FEMS Yeast Research 5(12): 1167–1183. https://doi.org/10.1016/j.femsyr.2005.05.007
  • Kalyaanamoorthy S, Minh BQ, Wong TKF, Von Haeseler A, Jermiin LS (2017) Modelfinder: Fast model selection for accurate phylogenetic estimates. Nature Methods 14(6): 587–589. https://doi.org/10.1038/nmeth.4285
  • Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30(4): 772–780. https://doi.org/10.1093/molbev/mst010
  • Kurtzman CP, Robnett CJ (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73(4): 331–371. https://doi.org/10.1023/A:1001761008817
  • Kurtzman CP, Fell JW, Boekhout T (2011) Methods for isolation, phenotypic characterization and maintenance of yeasts. In: Kurtzman CP, Fell JW, Boekhout T (Eds) The Yeasts – A Taxonomic Study (5th edn. , Vol. 2). Amsterdam, Elsevier, 87–110. https://doi.org/10.1016/B978-0-444-52149-1.00007-0
  • Li AH, Yuan FX, Groenewald M, Bensch K, Yurkov AM, Li K, Han PJ, Guo LD, Aime MC, Sampaio JP, Jindamorakot S, Turchetti B, Inacio J, Fungsin B, Wang QM, Bai FY (2020) Diversity and phylogeny of basidiomycetous yeasts from plant leaves and soil: Proposal of two new orders, three new families, eight new genera and one hundred and seven new species. Studies in Mycology 96: 17–140. https://doi.org/10.1016/j.simyco.2020.01.002
  • Liu XZ, Wang QM, Theelen B, Groenewald M, Bai FY, Boekhout T (2015) Phylogeny of tremellomycetous yeasts and related dimorphic and filamentous basidiomycetes reconstructed from multiple gene sequence analyses. Studies in Mycology 81(1): 1–26. https://doi.org/10.1016/j.simyco.2015.08.001
  • Madhour A, Anke H, Mucci A, Davoli P, Weber RW (2005) Biosynthesis of the xanthophyll plectaniaxanthin as a stress response in the red yeast Dioszegia (Tremellales, Heterobasidiomycetes, Fungi). Phytochemistry 66(22): 617–626. https://doi.org/10.1016/j.phytochem.2005.09.010
  • Maeng S, Park Y, Sung GH, Lee HB, Kim MK, Srinivasan S (2022) Description of Vishniacozyma terrae sp. nov. and Dioszegia terrae sp. nov., two novel basidiomycetous yeast species isolated from soil in Korea. Mycobiology 50(6): 439–447. https://doi.org/10.1080/12298093.2022.2147135
  • Mannazzu I, Landolfo S, da Silva TL, Buzzini P (2015) Red yeasts and carotenoid production: Outlining a future for nonconventional yeasts of biotechnological interest. World Journal of Microbiology & Biotechnology 31(11): 1665–1673. https://doi.org/10.1007/s11274-015-1927-x
  • Nakase T, Takashima M (1993) A simple procedure for the high frequency isolation of new taxa of ballistosporous yeasts living on the surfaces of plants. RIKEN Review 3: 33–34.
  • Phaff HJ, Fell JW (1970) Cryptococcus Kutzing emend. Phaff et Spencer. In: Lodder J (Ed.) The Yeasts, A Taxonomic Study (2nd edn. ). North-Holland, Amsterdam, 1088–1145.
  • Renker C, Blanke V, Börstler B, Heinrichs J, Buscot F (2004) Diversity of Cryptococcus and Dioszegia yeasts (Basidiomycota) inhabiting arbuscular mycorrhizal roots or spores. FEMS Yeast Research 4(6): 597–603. https://doi.org/10.1016/j.femsyr.2004.01.001
  • Robert V, Vu D, Amor AB, van de Wiele N, Brouwer C, Jabas B, Szoke S, Dridi A, Triki M, Ben Daoud S, Chouchen O, Vaas L, de Cock A, Stalpers JA, Stalpers D, Verkley GJ, Groenewald M, Dos Santos FB, Stegehuis G, Li W, Wu L, Zhang R, Ma J, Zhou M, Gorjón SP, Eurwilaichitr L, Ingsriswang S, Hansen K, Schoch C, Robbertse B, Irinyi L, Meyer W, Cardinali G, Hawksworth DL, Taylor JW, Crous PW (2013) MycoBank gearing up for new horizons. IMA Fungus 4(2): 371–379. https://doi.org/10.5598/imafungus.2013.04.02.16
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice, across a large model space. Systematic Biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029
  • Takashima M, Nakase T (1999) Molecular phylogeny of the genus Cryptococcus and related species based on the sequences of 18S rDNA and internal transcribed spacer regions. Microbiology and Culture Collections 15: 35–47.
  • Takashima M, Nakase T (2011) Dioszegia Zsolt emend. Takashima, Deák & Nakase (2001). In: Kurtzman CP, Fell JW, Boekhout T (Eds) The Yeasts – A Taxonomic Study (5th edn. , Vol. 2). Elsevier, Amsterdam, 1747–1757. https://doi.org/10.1016/B978-0-444-52149-1.00141-5
  • Takashima M, Deak T, Nakase T (2001) Emendation of Dioszegia with redescription of Dioszegia hungarica and two new combinations, Dioszegia aurantiaca and Dioszegia crocea. The Journal of General and Applied Microbiology 47(2): 75–84. https://doi.org/10.2323/jgam.47.75
  • Takashima M, Van BH, An KD, Ohkuma M (2011) Dioszegia rishiriensis sp. nov., a novel yeast species from soil collected on Rishiri Island, Hokkaido, Japan. International Journal of Systematic and Evolutionary Microbiology 61(Pt 7): 1736–1739. https://doi.org/10.1099/ijs.0.025254-0
  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25(24): 4876–4882. https://doi.org/10.1093/nar/25.24.4876
  • Trochine A, Turchetti B, Vaz ABM, Brandao L, Rosa LH, Buzzini P, Rosa C, Libkind D (2017) Description of Dioszegia patagonica sp. nov., a novel carotenogenic yeast isolated from cold environments. International Journal of Systematic and Evolutionary Microbiology 67(Pt 11): 4332–4339. https://doi.org/10.1099/ijsem.0.002211
  • Vu D, Groenewald M, Szöke S, Cardinali G, Eberhardt U, Stielow B, de Vries M, Verkleij GJ, Crous PW, Boekhout T, Robert V (2016) DNA barcoding analysis of more than 9 000 yeast isolates contributes to quantitative thresholds for yeast species and genera delimitation. Studies in Mycology 85(1): 91–105. https://doi.org/10.1016/j.simyco.2016.11.007
  • Wang QM, Bai FY, Zhao JH, Jia JH (2003) Dioszegia changbaiensis sp. nov., a basidiomycetous yeast species isolated from northeast China. The Journal of General and Applied Microbiology 49(5): 295–299. https://doi.org/10.2323/jgam.49.295
  • Wang QM, Jia JH, Bai FY (2008) Diversity of basidiomycetous phylloplane yeasts belonging to the genus Dioszegia (Tremellales) and description of Dioszegia athyri sp. nov., Dioszegia butyracea sp. nov. and Dioszegia xingshanensis sp. nov. Antonie van Leeuwenhoek International Journal of General and Molecular Microbiology 93(4): 391–399. https://doi.org/10.1007/s10482-007-9216-9
  • Wang QM, Theelen B, Groenewald M, Bai FY, Boekhout T (2014) Moniliellomycetes and Malasseziomycetes, two new classes in Ustilaginomycotina. Persoonia 33(1): 41–47. https://doi.org/10.3767/003158514X682313
  • Wei XY, Zhu HY, Song L, Zhang RP, Li AH, Niu QH, Liu XZ, Bai FY (2022) Yeast diversity in the Qaidam Basin Desert in China with the description of five new yeast species. Journal of Fungi 8(8): е858. https://doi.org/10.3390/jof8080858
  • White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR Protocols: A Guide to Methods and Applications. Academic Press, New York, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
  • Zhang D, Gao F, Jakovlić I, Zou H, Zhang J, Li WX, Wang GT (2020) Phylosuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources 20(1): 348–355. https://doi.org/10.1111/1755-0998.13096
  • Zsolt J (1957) Egy új élesztö: Dioszegia hungarica nov. gen. et sp. Botanikai Közlemények 47(1–2): 63–66.

Supplementary material

Supplementary material 1 

Molecular data

Ya-Zhuo Qiao, Shan Liu, Qiu-Hong Niu, Feng-Li Hui

Data type: fas

Explanation note: A dataset of ITS and LSU for Fig. 1.

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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