Phylogeny of Phyllozyma (Spiculogloeaceae, Spiculogloeales) with description of four new species from China

235 Phylogeny of Phyllozyma (Spiculogloeaceae, Spiculogloeales) with description of four new species from China Zhi-Wen Xi1,2 , Chun-Yue Chai1,2 , Qiu-Hong Niu1,2 , Feng-Li Hui1,2 1 School of Life Science, Nanyang Normal University, Nanyang 473061, China 2 Research Center of Henan Provincial Agricultural Biomass Resource Engineering and Technology, Nanyang Normal University, Nanyang 473061, China Corresponding authors: Qiu-Hong Niu ([email protected]); Feng-Li Hui ([email protected]) Copyright: © Zhi-Wen Xi et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article 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 Academic editor: Merje Toome Received: 9 June 2025 Accepted: 26 September 2025 Published: 15 October 2025 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 MycoKeys 123: 235–251 (2025) DOI: 10.3897/mycokeys.123.161540 236 MycoKeys 123: 235–251 (2025), DOI: 10.3897/mycokeys.123.161540 Zhi-Wen Xi et al.: Four new Phyllozyma species 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. 237 MycoKeys 123: 235–251 (2025), DOI: 10.3897/mycokeys.123.161540 Zhi-Wen Xi et al.: Four new Phyllozyma species 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 238 MycoKeys 123: 235–251 (2025), DOI: 10.3897/mycokeys.123.161540 Zhi-Wen Xi et al.: Four new Phyllozyma species 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). 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 burnin, and the remaining trees were used to estimate Bayesian posterior probabilities (BPPs) for the clades. Table 1. 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 2.5681TChina MK050380 MK050380 MK849248 MK849112 Li et al. 2020 Meniscomyces senecionis PYCC 9960TChina OR035763 OP954745 – – Li et al. 2020 Phyllozyma aceris CGMCC 2.2662TChina NR_175625 MK050377 MK849136 MK849006 Li et al. 2020 Phyllozyma aceris CGMCC 2.2617TChina MK050378 MK050378 MK849132 – Li et al. 2020 Phyllozyma aucubae NYNU 239180TChina OR961460 OR958754 PX353021 PV654547 This study Phyllozyma aucubae NYNU 239198 China PP660918 PP660917 –PV654548 This study Phyllozyma camelliae NYNU 23731TChina PP033661 PP033657 PX353019 PV654542 This study Phyllozyma camelliae NYNU 24899 China PQ899973 PQ899972 PX353020 PV654543 This study Phyllozyma coprosmicola CBS 7897TNew Zealand NR_073316 NG_058371 –KJ707908 Hamamoto et al. 1995 Phyllozyma corallina MAFF 654003TJapan AB638335 AB638335 – – Furuya et al. 2012 Phyllozyma diaoluoensis NYNU 2377TChina 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 8762TNew Zealand NR_144764 AB644404 KJ707991 KJ707907 Hamamotoet al. 1995 Phyllozyma guizhouensis NYNU 239199TChina OR958770 OR958769 –PV654549 This study Phyllozyma guizhouensis NYNU 248104 China PQ899975 PQ899974 –PV654550 This study Phyllozyma jiayinensis CGMCC 2.5669TChina MK050376 MK849108 –MK849108 Li et al. 2020 Phyllozyma linderae CBS 7893TJapan NR_073319 AF189989 –KJ707906 Nakase et al. 1994 Phyllozyma novozealandica JCM 8756 TNew Zealand NR_144765 KJ708467 KJ708073 KJ707851 Hamamotoet al. 1995 Phyllozyma producta MAFF 654001TJapan AB638334 AB638334 – – Furuya et al. 2012 Phyllozyma subbrunnea CBS 7196TJapan 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 9802TSezawa DQ831010 DQ831009 KJ708076 KJ707837 Sjamsuridzal et al. 2002 T, type strain. Species obtained in this study are in bold. 239 MycoKeys 123: 235–251 (2025), DOI: 10.3897/mycokeys.123.161540 Zhi-Wen Xi et al.: Four new Phyllozyma species 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. 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. 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 TFL316, 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 240 MycoKeys 123: 235–251 (2025), DOI: 10.3897/mycokeys.123.161540 Zhi-Wen Xi et al.: Four new Phyllozyma species 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). 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. 241 MycoKeys 123: 235–251 (2025), DOI: 10.3897/mycokeys.123.161540 Zhi-Wen Xi et al.: Four new Phyllozyma species 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 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. 242 MycoKeys 123: 235–251 (2025), DOI: 10.3897/mycokeys.123.161540 Zhi-Wen Xi et al.: Four new Phyllozyma species negative. Starch-like substances are not produced. Urease activity is positive. Diazonium Blue B reaction is positive. 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). 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, 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. 243 MycoKeys 123: 235–251 (2025), DOI: 10.3897/mycokeys.123.161540 Zhi-Wen Xi et al.: Four new Phyllozyma species 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. Table 2. 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). 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. 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Molecular Ecology Resources 20(1): 348–355. https://doi.org/10.1111/1755-0998.13096 251 MycoKeys 123: 235–251 (2025), DOI: 10.3897/mycokeys.123.161540 Zhi-Wen Xi et al.: Four new Phyllozyma species Supplementary material 1 A dataset of ITS, LSU, TEF1, and RBP1 for Fig. 1 Authors: Zhi-Wen Xi, Chun-Yue Chai, Qiu-Hong Niu, Feng-Li Hui Data type: fas Copyright notice: 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. Link: https://doi.org/10.3897/mycokeys.123.161540.suppl1 Supplementary material 2 A dataset of ITS for Fig. 2 Authors: Zhi-Wen Xi, Chun-Yue Chai, Qiu-Hong Niu, Feng-Li Hui Data type: fas Copyright notice: 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. Link: https://doi.org/10.3897/mycokeys.123.161540.suppl2