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Research Article
Phylogeny of Phyllozyma (Spiculogloeaceae, Spiculogloeales) with description of four new species from China
expand article infoZhi-Wen Xi‡, Chun-Yue Chai‡, 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: Merje Toome
© 2025 Zhi-Wen Xi, Chun-Yue Chai, 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: Xi Z-W, Chai C-Y, Niu Q-H, Hui F-L (2025) Phylogeny of Phyllozyma (Spiculogloeaceae, Spiculogloeales) with description of four new species from China. MycoKeys 123: 235-251. https://doi.org/10.3897/mycokeys.123.161540
Open Access

Abstract

Phyllozyma, belonging to the family Spiculogloeaceae of the order Spiculogloeales, is a genus of blastoconidia-forming yeasts. Until now, nine Phyllozyma species have been described. During our investigation of yeast diversity in China, several Phyllozyma strains were isolated from the surface of plant leaves collected in Guizhou and Hainan provinces, which represent undescribed taxa. Based on multi-locus (ITS, LSU, TEF1, and RBP1) and single-locus (ITS) phylogenetic analyses, as well as phenotypic characteristics, these strains were identified as four new species of Phyllozyma: P. aucubae sp. nov. (holotype CICC 33627T), P. camelliae sp. nov. (holotype CICC 33625T), P. diaoluoensis sp. nov. (holotype CICC 33620T), and P. guizhouensis sp. nov. (holotype CICC 33628T). P. aucubae sp. nov. was identified as a nonballistoconidium-forming species. This phenomenon is extremely rare in the genus Phyllozyma, and prior to this report, only P. jiayinensis was reported to lack the ability to produce ballistoconidia.

Key words:

Basidiomycetes, phylogenetic analysis, phylloplane yeast, taxonomy

Introduction

Spiculogloeales was established by Bauer et al. (2006), originally comprising two teleomorphic genera, Spiculogloea and Mycogloea, along with one anamorphic genus, Sporobolomyces. Among them, Sporobolomyces represented the largest genus, encompassing over 50 species as of earlier reports (Hamamoto et al. 2011). However, molecular phylogenetic analyses based on the D1/D2 domain of the large subunit (LSU) rRNA gene (Fell et al. 2000; Scorzetti et al. 2002; Furuya et al. 2012), the small subunit (SSU) rRNA gene (Hamamoto and Nakase 2000), and the internal transcribed spacer (ITS) region (Scorzetti et al. 2002) demonstrated that Sporobolomyces is a polyphyletic taxon. To address this polyphyly, Wang et al. (2015) reclassified seven Sporobolomyces species from the subbrunneus clade into a newly proposed genus, Phyllozyma. These species—P. coprosmicola, P. corallina, P. dimennae, P. linderae, P. novozealandica, P. producta, and P. subbrunnea—were segregated based on multigene phylogenetic analyses involving seven loci: SSU, ITS, LSU, RPB1, RPB2, TEF1, and CYTB, along with a revised LSU dataset. Among these, P. subbrunnea was designated as the type species of the newly circumscribed genus (Wang et al. 2015). More recently, two additional species, P. aceris and P. jiayinensis, were described from phylloplane habitats—specifically, from Acer caudatum and an unidentified plant species collected in China, respectively (Li et al. 2020).

All currently known species of the genus Phyllozyma are represented solely by their asexual yeast forms, characterized morphologically by polar budding as the mode of propagation. Most species may form ballistoconidia, and some species may also form hyphae and pseudohyphae (Hamamoto et al. 2011; Wang et al. 2015). Physiologically, all members of the genus lack fermentative ability, possess Q-10 as a predominant ubiquinone, and assimilate various carbon sources, but not maltose, melezitose, L-arabinose, or myo-inositol (Hamamoto et al. 2011; Wang et al. 2015; Li et al. 2020). Phyllozyma species are associated with plant leaves (Nakase and Suzuki 1985; Hamamoto and Nakase 1995; Nakase et al. 1994; Furuya et al. 2012; Li et al. 2020) and are ecologically distinct from the teleomorphic species in the genera Spiculogloea and Mycogloea, which function as mycoparasites and are characterized by tremelloid haustorial cells (Roberts 1996; Bauer 2004; Weiß et al. 2004; Wang et al. 2015).

Until now, nine Phyllozyma species have been accepted, and they are mainly distributed in temperate and subtropical regions, especially in Asia (Nakase and Suzuki 1985; Nakase et al. 1994; Furuya et al. 2012; Li et al. 2020). In China, P. corallina and P. producta were first reported in Zhejiang Province in 2018 (Zang et al. 1998). Later, six additional species, including the newly identified P. aceris and P. jiayinensis, were discovered in Jilin Province and the Tibet Autonomous Region (Li et al. 2020). China’s vast temperate regions in the Northern Hemisphere likely host a diverse array of Phyllozyma species, yet they are poorly documented. In this study, we isolated nine Phyllozyma strains from Guizhou and Hainan provinces, China. Molecular phylogenetic analyses combined with phenotypic characterization revealed that they represent four previously undescribed species. The aim of this investigation is to apply an integrative taxonomic approach for the identification and description of these new taxa.

Materials and methods

Sample collection and yeast isolation

Leaf samples were collected in Guizhou and Hainan provinces, China. Yeast strains were isolated from the leaf surfaces using the improved ballistospore-fall method described by Nakase and Takashima (1993). Briefly, 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 malt (YM) agar. YM agar consisted of 0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, and 2% agar and was supplemented with 0.01% chloramphenicol to prevent bacterial growth. Plates were incubated at 20 °C and monitored daily for colony formation. Selected colonies were streaked back onto YM agar plates for purification. Following purification, strains were suspended in 20% (v/v) glycerol and stored at −80 °C for long-term preservation.

Phenotypic characterization

Morphological, physiological, and biochemical characterizations were conducted according to standardized methods established by Kurtzman et al. (2011). Glucose fermentation was tested in liquid medium using Durham fermentation tubes. Carbon and nitrogen assimilation capabilities were examined in liquid medium, with starved inoculum used for nitrogen assimilation testing (Kurtzman et al. 2011). Cell morphology was examined with a Leica DM2500 microscope (Leica, Wetzlar, Germany) and LAS v.4.13 software. Ballistoconidium-forming activity was investigated using the inverted-plate method (do Carmo-Sousa and Phaff 1962) after 2 weeks of incubation on cornmeal agar (CMA; 2.5% cornstarch and 2% agar) at 20 °C. Growth at various temperatures (15, 20, 25, 30, 35, and 37 °C) was assessed through cultivation on YM agar plates. The potential sexual cycle of each strain was investigated on CMA, potato dextrose agar (PDA; 20% potato infusion, 2% glucose, and 2% agar), and V8 agar (10% V8 juice and 2% agar). Each tested strain was inoculated on agar plates and incubated at 20 °C for up to 2 months, with observations made every 2 weeks (Li et al. 2020). All novel taxonomic descriptions and proposed names were deposited in the MycoBank database (http://www.mycobank.org; accessed 8 January 2025).

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 internal transcribed spacer (ITS) region, the D1/D2 domain of the large subunit (LSU) rRNA gene, the translation elongation factor 1-α gene (TEF1), and the RNA polymerase II largest subunit (RPB1) were amplified with primers ITS1/ITS4 (White et al. 1990), NL1/NL4 (Kurtzman and Robnett 1998), EF1-526F/EF1-1567R (Rehner and Buckley 2005), and RPB1-Af/RPB1-Cr (Matheny et al. 2002), respectively. The PCR products were checked in a 1% (w/v) agarose gel, purified using a SanPrep Column PCR Product Purification Kit (Sangon Biotech, Shanghai, China), and sequenced using an ABI 3730xl DNA analyzer with the same primers used for PCR amplification. The identity and accuracy of each sequence were verified by comparison with sequences in the GenBank database. 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

The sequences generated in this study, along with additional sequences downloaded from the GenBank database (Table 1), were used in phylogenetic analyses. Species of Spiculogloea were not included in the phylogenetic analysis, except for Spiculogloea sp. DB 1496, because sequence data for the type species of this genus are presently not available in public databases. Following Li et al. (2020), Mixia osmundae CBS 9802 was selected as the outgroup. The combined dataset of ITS, LSU, TEF1, and RPB1 was used to explore the phylogenetic positions of the newly isolated strains within Phyllozyma. The ITS dataset was then used to further differentiate species identities within this genus. Sequences from each locus were first aligned individually using MAFFT v.7.110 (Katoh and Standley 2013) with the G-INS-i option. Poorly aligned regions were manually removed using MEGA v.11 (Tamura et al. 2021). The aligned sequences from different loci were concatenated with PhyloSuite v.1.2.2 (Zhang et al. 2020).

Table 1.
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List of species, strains, and GenBank accession numbers of sequences used in this study.

Species Strain number Locality GenBank accession no. References
ITS LSU D1/D2 RPB1 TEF1
Meniscomyces layueensis CGMCC 2.5681T China MK050380 MK050380 MK849248 MK849112 Li et al. 2020
Meniscomyces senecionis PYCC 9960T China OR035763 OP954745 – – Li et al. 2020
Phyllozyma aceris CGMCC 2.2662T China NR_175625 MK050377 MK849136 MK849006 Li et al. 2020
Phyllozyma aceris CGMCC 2.2617T China MK050378 MK050378 MK849132 – Li et al. 2020
Phyllozyma aucubae NYNU 239180T China OR961460 OR958754 PX353021 PV654547 This study
Phyllozyma aucubae NYNU 239198 China PP660918 PP660917 – PV654548 This study
Phyllozyma camelliae NYNU 23731T China PP033661 PP033657 PX353019 PV654542 This study
Phyllozyma camelliae NYNU 24899 China PQ899973 PQ899972 PX353020 PV654543 This study
Phyllozyma coprosmicola CBS 7897T New Zealand NR_073316 NG_058371 – KJ707908 Hamamoto et al. 1995
Phyllozyma corallina MAFF 654003T Japan AB638335 AB638335 – – Furuya et al. 2012
Phyllozyma diaoluoensis NYNU 2377T China OR526726 OR511464 PX353016 PV654544 This study
Phyllozyma diaoluoensis NYNU 23732 China OR961462 OR958779 PX353017 PV654545 This study
Phyllozyma diaoluoensis NYNU 23718 China OR958777 OR958778 PX353018 PV654546 This study
Phyllozyma dimennae JCM 8762T New Zealand NR_144764 AB644404 KJ707991 KJ707907 Hamamotoet al. 1995
Phyllozyma guizhouensis NYNU 239199T China OR958770 OR958769 – PV654549 This study
Phyllozyma guizhouensis NYNU 248104 China PQ899975 PQ899974 – PV654550 This study
Phyllozyma jiayinensis CGMCC 2.5669T China MK050376 MK849108 – MK849108 Li et al. 2020
Phyllozyma linderae CBS 7893T Japan NR_073319 AF189989 – KJ707906 Nakase et al. 1994
Phyllozyma novozealandica JCM 8756 T New Zealand NR_144765 KJ708467 KJ708073 KJ707851 Hamamotoet al. 1995
Phyllozyma producta MAFF 654001T Japan AB638334 AB638334 – – Furuya et al. 2012
Phyllozyma subbrunnea CBS 7196T Japan NR_077094 AF189997 – KJ707909 Nakase and Suzuki 1985
Sporobolomyces sp. TY-285 Japan AY313080 AY313059 – – –
Spiculogloea sp. DB 1496 Germany – AY512885 – – –
Uncultured basidiomycete yeast TFL3-16 China AJ582959 – – – –
Mixia osmundae CBS 9802T Sezawa DQ831010 DQ831009 KJ708076 KJ707837 Sjamsuridzal et al. 2002

T , type strain. Species obtained in this study are in bold.

Maximum likelihood (ML) and Bayesian inference (BI) analyses were performed with RAxML v.8.2.3 (Stamatakis 2014) and MrBayes v.3.1.2 (Ronquist and Huelsenbeck 2003), respectively. The best nucleotide substitution model was estimated using Modeltest v.3.04 (Posada and Crandall 1998). In the ML analyses, bootstrap (BS) values were assessed through 1,000 rapid bootstrap replicates. For BI analyses, six Markov chain Monte Carlo (MCMC) chains were run simultaneously for 50 million generations, and trees were sampled every 1,000 generations. The first 25% of the generated trees were discarded as burn-in, and the remaining trees were used to estimate Bayesian posterior probabilities (BPPs) for the clades.

Results

Molecular phylogeny

Among the yeasts isolated from leaf samples collected across different regions of China, nine strains identified as Phyllozyma based on their rRNA gene sequences were selected for further phylogenetic studies.

The combined dataset of ITS, LSU, TEF1, and RPB1 from 60 sequences generated a concatenated alignment of 2,368 characters (543 characters from ITS, 638 characters from LSU, 504 characters from TEF1, and 693 characters from RPB1) with GTR+I+G as the best-fit evolutionary model. ML and BI methods generated similar topologies in the main lineages; therefore, only the topology generated by the ML method is presented, along with BS values and BPPs above 50% and 0.95, respectively, at the nodes (Fig. 1). The phylogeny generated from this dataset strongly supported Phyllozyma as a monophyletic genus (BS = 95%, BPP = 1.0). The nine newly isolated strains formed four well-supported groups distinct from other species of Phyllozyma.

Figure 1. 

Phylogenetic positions of the newly studied strains of Phyllozyma inferred from the combined dataset of ITS, LSU, TEF1, and RPB1. The topology generated by the maximum likelihood method is presented along with bootstrap values and Bayesian posterior probabilities above 50% and 0.95, respectively, at the nodes. The tree is rooted with Mixia osmundae CBS 9802. Type strain sequences are marked with superscript T. New species are highlighted in bold.

The ITS dataset of Phyllozyma, comprising 24 sequences, generated an alignment of 543 characters with GTR+I+G as the best-fit evolutionary model. ML and BI methods produced similar topologies in the main lineages; therefore, the topology inferred from the ML method is presented, along with BS values and BPPs above 50% and 0.95, respectively, at the nodes (Fig. 2). This tree recovered nine known species of Phyllozyma, while the newly studied strains formed four independent groups, consistent with the phylogeny inferred from the combined ITS, LSU, TEF1, and RPB1 dataset.

Figure 2. 

Species identities of Phyllozyma differentiated by ITS-based phylogeny. The tree generated by the maximum likelihood method is presented along with bootstrap values and Bayesian posterior probabilities above 50% and 0.95, respectively, at the nodes. The tree is rooted with Mixia osmundae CBS 9802. Type strain sequences are marked with superscript T. New species are highlighted in bold.

Groups NYNU 23731 and NYNU 239180, each containing two strains, clustered in the same clade as P. novozealandica in the trees constructed with all datasets (Figs 1, 2). Strains in the NYNU 23731 group had identical ITS and D1/D2 sequences, indicating that they are conspecific. Similarly, strains in the NYNU 239180 group shared identical ITS and D1/D2 sequences, which differed from those of the NYNU 23731 group by four nucleotides (nt) (~0.7%) in the D1/D2 domain and 12 nt (~2.4%) in the ITS region. In addition, these two groups differed from their closest known species, P. novozealandica, by 10–14 nt (~1.7–2.4%) in the D1/D2 domain and 25–29 nt (~5.0–5.9%) in the ITS region. These results suggest that groups NYNU 23731 and NYNU 239180 represent two novel species of Phyllozyma.

The group NYNU 2377, consisting of three strains, clustered together with an unpublished strain, ‘Sporobolomyces’ sp. TY-285 (Fig. 1), and clone TFL3-16, an unculturable basidiomycete (Fig. 2). These five strains possessed similar ITS and D1/D2 sequences, with no more than four nucleotide differences, suggesting that they are conspecific. The NYNU 2377 group differed from its closest related species, P. jiayinensis, by 31–32 nt (~5.2–5.5%) in the D1/D2 domain and 52–65 nt (~8.9–9.4%) in the ITS region. These results strongly suggest that the NYNU 2377 group represents another novel species of Phyllozyma.

The group NYNU 239199, containing two strains, had identical ITS and D1/D2 sequences, indicating that they are conspecific. Strains in the NYNU 239199 group formed a separate subclade from other Phyllozyma species in the tree of the multilocus dataset (Fig. 1) but clustered with P. subbrunnea with high support in the tree of the ITS dataset (Fig. 2). This group differed from its closest related species, P. subbrunnea, by 13 nt (~2.2%) in the D1/D2 domain and 7 nt (~1.4%) in the ITS region. These results indicate that the NYNU 239199 group belongs to a novel species of Phyllozyma.

Taxonomy

Phyllozyma aucubae Z.W. Xi & F.L. Hui, sp. nov.

MycoBank No: MB 857393
Fig. 3

Etymology.

The specific epithet “aucubae” refers to Aucuba, the plant genus from which the type strain was isolated.

Typus.

China • Guizhou Prov.: Guiyang City, East Mountain Park, in the phylloplane of Aucuba japonica, 15 Sept 2023, D. Lu, NYNU 239180 (holotype CICC 33627T preserved as a metabolically inactive state, culture ex-type PYCC 9993).

Description.

On YM agar after 7 days at 20 °C, the streak culture is cream, mucoid, smooth, and glistening, with an entire margin. After 3 days in YM broth at 20 °C, cells are cylindrical, 2.1–2.2 × 6.4–11.9 μm and single, budding is polar. After 1 month at 20 °C, a ring and sediment are present. In Dalmau plate culture on CMA, simple pseudohyphae and formed. Sexual structures are not observed on PDA, CMA, or V8 agar. Ballistoconidia are not produced. Glucose fermentation is absent. Glucose, inulin (weak), D-arabinose (delayed), glycerol (delayed), D-mannitol, D-glucitol (delayed), DL-lactate (delayed and weak), and succinate (delayed) are assimilated as sole carbon sources. Sucrose, raffinose, melibiose, galactose, lactose, trehalose, maltose, melezitose, methyl-α-D-glucoside, cellobiose, salicin, L-sorbose, L-rhamnose, D-xylose, L-arabinose, 5-keto-D-gluconate, D-ribose, methanol, ethanol, erythritol, ribitol, galactitol, myo-inositol, citrate, D-gluconate, D-glucosamine, N-acetyl-D-glucosamine, 2-keto-D-gluconate, D-glucuronate, and glucono-1,5-lactone are not assimilated. Nitrate (delayed and weak), nitrite (delayed and weak), ethylamine (delayed), and L-lysine are assimilated as sole nitrogen sources. Cadaverine is not assimilated. Maximum growth temperature is 25 °C. Growth on 50% (w/w) glucose-yeast extract agar is negative. Starch-like substances are not produced. Urease activity is positive. Diazonium Blue B reaction is positive.

Figure 3. 

Morphological characteristics of P. aucubae sp. nov. NYNU 239180T. A. The streak culture grown on YM agar after 7 d at 20 °C; B. Budding cells grown in YM broth for 3 d at 20 °C; C. Simple pseudohyphae produced on CMA after 7 d at 20 °C. Scale bars: 10 μm.

Additional strain examined.

China • Guizhou Prov.: Guiyang City, East Mountain Park, in the phylloplane of Aucuba japonica, 15 Sept 2023, D. Lu, NYNU 239198.

GenBank accession numbers.

Holotype CICC 33627T (ITS: OR961460, D1/D2: OR958754, RPB1: PX353021, TEF1: PV654547); additional strain NYNU 239198 (ITS: PP660918, D1/D2: PP660917, TEF1: PV654548).

Note.

Physiologically, P. aucubae sp. nov. differs from its closely related species, P. camelliae sp. nov., described in this study, by its inability to assimilate galactose and trehalose, as well as its ability to grow at 30 °C (Table 2).

Table 2.
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Physiological and biochemical characteristics that differentiate the new species and their closest related species.

Characteristics 1 2 3 4* 5 6*
Carbon assimilation
Inulin w d/w + – + –
Sucrose – – + – + +
Raffinose – – d – d +
Galactose – d + – – –
Trehalose – + d + + +
D-Arabinose d d w – d +
Glycerol d d + – + +
Ribitol – – w – d/w +
DL-Lactate d/w d/w – w + +
Succinate d d/w w – + +
Citrate – – w – – +
Nitrogen assimilation
Nitrite d/w d d – d/w +
Ethylamine d d – – – –
L-Lysine + + – – + w
Growth tests
Growth at 30 °C – + + – + –

Species: 1, P. aucubae sp. nov.; 2, P. camelliae sp. nov.; 3, P. diaoluoensis sp. nov.; 4, P. jiayinensis; 5, P. guizhouensis sp. nov.; 6, P. subbrunnea. +, positive reaction; –, negative reaction; d, delayed positive; w, weakly positive. All data were produced in this study, except where indicated with an asterisk (*), which were obtained from the original description (Hamamoto et al. 2011; Li et al. 2020).

Phyllozyma camelliae Z.W. Xi & F.L. Hui, sp. nov.

MycoBank No: MB 857394
Fig. 4

Etymology.

The specific epithet “camelliae” refers to Camellia, the plant genus from which the type strain was isolated.

Typus.

China • Hainan Prov.: Qiongzhong Li and Miao Autonomous County, Diaoluo Mountain, in the phylloplane of Camellia oleifera, 15 Jul 2023, X.M. Han, NYNU 23731 (holotype CICC 33625T preserved as a metabolically inactive state, culture ex-type PYCC 9991).

Description.

On YM agar after 7 days at 20 °C, the streak culture is pale-yellow, mucoid, smooth, and glistening, with an entire margin. After 3 days in YM broth at 20 °C, cells are cylindrical, 1.5–2.4 × 5.3–8.5 μm and single, budding is polar. After 1 month at 20 °C, a ring and sediment are present. In Dalmau plate culture on CMA, pseudohyphae and hyphae are not formed. Sexual structures are not observed on PDA, CMA, or V8 agar. Ballistoconidia are ellipsoidal, 2.0–2.7 × 2.7–4.3 μm. Glucose fermentation is absent. Glucose, inulin (delayed and weak), galactose (delayed), trehalose, D-arabinose (delayed), glycerol (delayed), D-mannitol, D-glucitol (delayed), DL-lactate (delayed and weak), and succinate (delayed and weak) are assimilated as sole carbon sources. Sucrose, raffinose, melibiose, lactose, maltose, melezitose, methyl-α-D-glucoside, cellobiose, salicin, L-sorbose, L-rhamnose, D-xylose, L-arabinose, 5-keto-D-gluconate, D-ribose, methanol, ethanol, erythritol, ribitol, galactitol, myo-inositol, citrate, D-gluconate, D-glucosamine, N-acetyl-D-glucosamine, 2-keto-D-gluconate, D-glucuronate, and glucono-1,5-lactone are not assimilated. Nitrate, nitrite (delayed), ethylamine (delayed), and L-lysine are assimilated as sole nitrogen sources. Cadaverine is not assimilated. Maximum growth temperature is 30 °C. Growth on 50% (w/w) glucose-yeast extract agar is negative. Starch-like substances are not produced. Urease activity is positive. Diazonium Blue B reaction is positive.

Figure 4. 

Morphological characteristics of P. camelliae sp. nov. NYNU 23731T. A. The streak culture grown on YM agar after 7 d at 20 °C; B. Budding cells grown in YM broth for 3 d at 20 °C; C. Ballistoconidia produced on CMA after 7 d at 20 °C. Scale bars: 10 μm.

Additional strain examined.

China • Hainan Prov.: Wuzhishan City, Wuzhi Mountain, in the phylloplane of Symplocos adenophylla, 12 Aug 2024, Y.F. Lu & F.L. Hui, NYNU 24899.

GenBank accession numbers.

Holotype CICC 33625T (ITS: PP033661, D1/D2: PP033657, RPB1: PX353019, TEF1: PV654542); additional strain NYNU 24899 (ITS: PP239073, D1/D2: PP239062, RPB1: PX353020, TEF1: PV654543).

Note.

See the “Notes” of the previous species.

Phyllozyma diaoluoensis Z.W. Xi & F.L. Hui, sp. nov.

MycoBank No: MB 857395
Fig. 5

Etymology.

The specific epithet “diaoluoensis” refers to the geographic origin of the type strain, Diaoluo Mountain, Qiongzhong Li and Miao Autonomous County, Hainan Province.

Typus.

China • Hainan Prov.: Qiongzhong Li and Miao Autonomous County, Diaoluo Mountain, in the phylloplane of Zanthoxylum avicennae, 15 Jul 2023, X.M. Han, NYNU 2377 (holotype CICC 33620T preserved as a metabolically inactive state, culture ex-type PYCC 9983).

Description.

On YM agar after 7 days at 20 °C, the streak culture is pale-yellow, tough, and somewhat wrinkled, with an entire margin. After 3 days in YM broth at 20 °C, cells are cylindrical, 2.0–2.8 × 4.3–6.8 μm and single, budding is polar. After 1 month at 20 °C, a ring and sediment are present. In Dalmau plate culture on CMA, pseudohyphae and hyphae are not formed. Sexual structures are not observed on PDA, CMA, or V8 agar. Ballistoconidia are falcate or cylindrical, 2.5–4.9 × 8.7–12.6 μm. Glucose fermentation is absent. Glucose, inulin, sucrose, raffinose (delayed), galactose, trehalose (delayed), D-arabinose (weak), glycerol, ribitol (weak), D-mannitol (weak), D-glucitol, succinate (weak), citrate (weak), and D-glucuronate (weak) are assimilated as sole carbon sources. Melibiose, lactose, maltose, melezitose, methyl-α-D-glucoside, cellobiose, salicin, L-sorbose, L-rhamnose, D-xylose, L-arabinose, 5-keto-D-gluconate, D-ribose, methanol, ethanol, erythritol, galactitol, myo-inositol, DL-lactate, D-gluconate, D-glucosamine, N-acetyl-D-glucosamine, 2-keto-D-gluconate, and D-glucono-1,5-lactone are not assimilated. Nitrate (delayed) and nitrite (delayed) are assimilated as sole nitrogen sources. Ethylamine, L-lysine, and cadaverine are not assimilated. Maximum growth temperature is 30 °C. Growth on 50% (w/w) glucose-yeast extract agar is negative. Starch-like substances are not produced. Urease activity is positive. Diazonium Blue B reaction is positive.

Figure 5. 

Morphological characteristics of P. diaoluoensis sp. nov. NYNU 2377T. A. The streak culture grown on YM agar after 7 d at 20 °C; B. Budding cells grown in YM broth for 3 d at 20 °C; C. Ballistoconidia produced on CMA after 7 d at 20 °C. Scale bars: 10 μm.

Additional strain examined.

China • Hainan Prov.: Qiongzhong Li and Miao Autonomous County, Diaoluo Mountain, in the phylloplane of Gordonia hainanensis, 15 Jul 2023, X.M. Han, NYNU 23732; in the phylloplane of Altingia obovata, 15 Jul 2023, X.M. Han, NYNU 23718.

GenBank accession numbers.

Holotype CICC 33620T (ITS: OR526726, D1/D2: OR511464, RPB1: PX353016, TEF1: PV654544); additional strains NYNU 23732 (ITS: OR961462, D1/D2: OR958779, RPB1: PX353017, TEF1: PV654545) and NYNU 23718 (ITS: OR958777, D1/D2: OR958778, RPB1: PX353018, TEF1: PV654546).

Note.

Physiologically, P. diaoluoensis sp. nov. differs from its closely related species P. jiayinensis in its ability to assimilate inulin, sucrose, raffinose, galactose, trehalose, D-arabinose, glycerol, ribitol, succinate, and citrate and its inability to assimilate DL-Lactate. Furthermore, P. diaoluoensis sp. nov. can grow at 30 °C, while P. jiayinensis cannot (Table 2).

Phyllozyma guizhouensis Z.W. Xi & F.L. Hui, sp. nov.

MycoBank No: MB 857396
Fig. 6

Etymology.

The specific epithet “guizhouensis” refers to the geographic origin of the type strain, East Mountain Park, Guiyang City, Guizhou Province.

Typus.

China • Guizhou Prov.: Guiyang City, East Mountain Park, in the phylloplane of Aucuba japonica, 15 Sept 2023, D. Lu, NYNU 239199 (holotype CICC 33628T preserved as a metabolically inactive state, culture ex-type PYCC 9994).

Description.

On YM agar after 7 days at 20 °C, the streak culture is pale-yellow, butyrous, smooth, and glossy, with an entire margin. After 3 days in YM broth at 20 °C, cells are cylindrical, 2.2–3.5 × 7.0–10.4 μm and single, budding is polar. After 1 month at 20 °C, a ring and sediment are present. In Dalmau plate culture on CMA, pseudohyphae and hyphae are not formed. Sexual structures are not observed on PDA, CMA, or V8 agar. Ballistoconidia are falcate or cylindrical, 2.8–3.4 × 7.9–9.6 μm. Glucose fermentation is absent. Glucose, inulin, sucrose, raffinose (delayed), trehalose, D-arabinose (delayed), glycerol, ribitol (delayed and weak), D-mannitol, D-glucitol (delayed), DL-lactate, and succinate are assimilated as sole carbon sources. Melibiose, galactose, lactose, maltose, melezitose, methyl-α-D-glucoside, cellobiose, salicin, L-sorbose, L-rhamnose, D-xylose, L-arabinose, 5-keto-D-gluconate, D-ribose, methanol, ethanol, erythritol, galactitol, myo-inositol, citrate, D-gluconate, D-glucosamine, N-acetyl-D-glucosamine, 2-keto-D-gluconate, D-glucuronate, and D-glucono-1,5-lactone are not assimilated. Nitrate, nitrite (delayed and weak), and L-lysine are assimilated as sole nitrogen sources. Ethylamine and cadaverine are not assimilated. Maximum growth temperature is 30 °C. Growth on 50% (w/w) glucose-yeast extract agar is negative. Starch-like substances are not produced. Urease activity is positive. Diazonium Blue B reaction is positive.

Figure 6. 

Morphological characteristics of P. guizhouensis sp. nov. NYNU 239199T. A. The streak culture grown on YM agar after 7 d at 20 °C; B. Budding cells grown in YM broth for 3 d at 20 °C; C. Ballistoconidia produced on corn meal agar after 7 d at 20 °C. Scale bars: 10 μm.

Additional strain examined.

China • Hainan Prov.: Wuzhishan City, Wuzhi Mountain, in the phylloplane of Ervatamia divaricata, 12 Aug 2024, Y.F. Lu & F.L. Hui, NYNU 248104.

GenBank accession numbers.

Holotype CICC 33628T (ITS: OR958770, D1/D2: OR958769, TEF1: PV654549); additional strain NYNU 248104 (ITS: OR961462, D1/D2: OR958779, TEF1: PV654550).

Note.

Physiologically, P. guizhouensis sp. nov. differs from its closely related species P. subbrunnea in its ability to assimilate inulin and its inability to assimilate citrate. Furthermore, P. guizhouensis sp. nov. can grow at 30 °C, while P. subbrunnea cannot (Table 2).

Discussion

In the present study, nine yeast strains were isolated from the surfaces of plant leaves collected across different regions of China during a yeast diversity survey conducted between 2023 and 2024. Four novel Phyllozyma species—P. aucubae sp. nov., P. camelliae sp. nov., P. diaoluoensis sp. nov., and P. guizhouensis sp. nov.—were discovered among these strains based on multi-locus (ITS, LSU, TEF1, and RPB1) and single-locus (ITS) analyses. Our phylogenetic results are consistent with previous observations (Li et al. 2020) and provide further insight into the taxonomy of Phyllozyma.

The genus Phyllozyma is known as a representative group of ballistosporous yeasts. Most species of the genus generally produce ballistoconidia, which are detected as an opaque mirror image of the culture formed by discharged spores on the lid of an inverted Petri dish (do Carmo-Sousa and Phaff 1962; Kurtzman et al. 2011). However, the production of ballistoconidia is influenced by cultivation methods and varies from clone to clone (Nakase et al. 1993; Nakase 2000). In this study, the two strains of the new species P. aucubae sp. nov. were identified as nonballistoconidium-forming yeasts. This phenomenon is extremely rare in the genus Phyllozyma, with only P. jiayinensis reported to lack the ability to produce ballistoconidia (Li et al. 2020).

Species of Phyllozyma are primarily associated with plant substrates and have thus far been isolated only in their yeast morphs, particularly from phyllospheric environments (Nakase and Suzuki 1985; Nakase et al. 1994; Furuya et al. 2012; Li et al. 2020). In this study, the nine strains, characterized as belonging to four new Phyllozyma species, were also associated with plant leaves, like most other species in the genus. P. aucubae sp. nov. was repeatedly recovered from the same plant, Aucuba japonica, collected at East Mountain Park. P. diaoluoensis sp. nov. was isolated from three different plants gathered on Diaoluo Mountain. P. camelliae sp. nov. and P. guizhouensis sp. nov. were found on different plants collected from two different mountains. These findings further support the hypothesis that Phyllozyma species are widely distributed in association with plant surfaces. Although the ecology of Phyllozyma remains largely uncharacterized, it has been speculated—based on habitat associations and morphological traits—that their propagules may originate from meiosporangia of unknown mycoparasitic basidiomycetes inhabiting leaves or neighboring twigs. The presence of ballistoconidia may facilitate aerial dispersal between fungal hosts and plant surfaces (Oberwinkler 2017). Furthermore, recent work by Schoutteten et al. (2023) demonstrated that Slooffia, previously known exclusively in its yeast state, possesses a mycoparasitic filamentous morph. This raises the possibility that Phyllozyma species may also be dimorphic, with an as-yet-undiscovered hyphal stage. Given that filamentous morphs in Basidiomycota are typically associated with basidium formation and sexual reproduction, mating assays involving compatible yeast strains may provide insights into their sexual cycle (Schoutteten et al. 2024). Additional efforts, including pure culture isolation and co-cultivation experiments, are warranted to uncover the putative filamentous stage and to elucidate the full life cycle of Phyllozyma.

As a result of this study, 13 species are currently assigned to the genus Phyllozyma. Among them, P. producta and P. corallina have been reported as pathogens causing pseudo-greasy spot of citrus (Furuya et al. 2012). Consequently, interest in these yeasts extends beyond their taxonomy to include their ecosystem function, pathogenicity, and potential agricultural, industrial, and medical applications of economic value.

Acknowledgements

We extend our sincere gratitude to Dan Lu and Xue-Mei Han for their invaluable assistance in collecting samples from Guizhou and Hainan provinces.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Use of AI

No use of AI was reported.

Funding

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

Author contributions

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

Author ORCIDs

Zhi-Wen Xi https://orcid.org/0000-0002-5814-5283

Chun-Yue Chai https://orcid.org/0000-0003-0284-5560

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.

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Supplementary materials

Supplementary material 1 

A dataset of ITS, LSU, TEF1, and RBP1 for Fig. 1

Zhi-Wen Xi, Chun-Yue Chai, Qiu-Hong Niu, Feng-Li Hui

Data type: fas

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.
Download file (53.59 kb)
Supplementary material 2 

A dataset of ITS for Fig. 2

Zhi-Wen Xi, Chun-Yue Chai, Qiu-Hong Niu, Feng-Li Hui

Data type: fas

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.
Download file (13.29 kb)
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