﻿Three new Dioszegia species (Bulleribasidiaceae, Tremellales) discovered in the phylloplane in China

﻿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.guizhouensissp. nov., D.foliicolasp. nov., and D.aurantiasp. 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.


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. 2003Wang et al. , 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. 2003Wang et al. , 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.

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 ddH 2 O, 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 Gen-Bank and their corresponding accession numbers are listed in Table 1.

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 7526 T 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 Mr-Bayes 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 1,000 th 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.

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.
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.
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: 851294 Fig. 2B Etymology.The specific epithet foliicola refers to the type strain isolated from a leaf.
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.Etymology.The specific epithet aurantia refers to the aurantiaca colony morphology.
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).

Figure 1 .
Figure 1.Maximum likelihood (ML) phylogram of Dioszegia species and close relatives based on combined ITS and LSU sequence data.Sugitazyma miyagiana CBS 7526 T serves as the outgroup.Branches are labelled with MLBS ≥ 50% and BPP ≥ 0.95.Novel strains are highlighted in bold.

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

Table 2 .
Strains representing the novel species described in this study and relevant information associated to them.
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.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 2.311 T preserved as a metabolically inactive state, culture ex-type PYCC 9938).