Two new species of Papiliotrema (Rhynchogastremataceae, Tremellales) from China

171 Two new species of Papiliotrema (Rhynchogastremataceae, Tremellales) from China Wan-Li Gao1, 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: © Wan-Li Gao 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 Papiliotrema species are commonly found in different habitats and many of them are reported as epiphytic yeasts associated with plants. In this study, several yeast strains were isolated from the surface of plants collected in the Chinese provinces of Guizhou and Hainan. Phylogenetic analyses of combined ITS and LSU sequence data placed them in Papiliotrema. Two new species, Papiliotrema millettiae sp. nov. (holotype CICC 33641) and P. mussaendae sp. nov. (holotype CICC 33595), are proposed on the basis of phylogenetic analyses and phenotypic characterisation. Detailed descriptions of both species are provided, allowing clear differentiation from other known species in the genus Papiliotrema. In addition, P. fudaokuniae is considered a synonym of P. fusca, based on our phylogenetic analyses. This study contributes to a better understanding of species diversity within the genus Papiliotrema and provides a framework for future taxonomic research in the group. Key words: Basidiomycetes, phylogeny, phylloplane yeasts, taxonomy Introduction Papiliotrema, a dimorphic yeast genus, was erected by Sampaio et al. (2002) to accommodate the sexual morph Papiliotrema bandonii J.P. Samp., Gadanho, M. Weiss & R. Bauer as the type species. Phylogenetically, P. bandonii is situated within the Bulleromyces clade of the order Tremellales, class Tremellomycetes (Sampaio et al. 2002). This clade comprises teleomorphic genera including Bulleromyces, Papiliotrema and Auriculibuller, as well as several species from the polyphyletic anamorphic genera Bullera and Cryptococcus (Boekhout 2011). In accordance with the International Code of Nomenclature for algae, fungi and plants and the ‘one fungus, one name’ principle (McNeill et al. 2012), the second species P. siamensis Suruss & Limtong was later described, based on two anamorphic strains (Surussawadee et al. 2014). In a major taxonomic revision of basidiomycetous yeasts, Liu et al. (2015) transferred 20 anamorphic species to Papiliotrema, including 17 previously classified in Cryptococcus and three in Bullera, all phylogenetically related to P. bandonii. This revision also included the transfer of the sexual species Auriculibuller fuscus J.P. Samp, J. Inácio, A. Fonseca & J.W. Fell to Papiliotrema. Consequently, the genus now inAcademic editor: Margarita Dueñas Received: 1 July 2025 Accepted: 4 December 2025 Published: 16 December 2025 Citation: Gao W-L, Chai C-Y, Niu Q-H, Hui F-L (2025) Two new species of Papiliotrema (Rhynchogastremataceae, Tremellales) from China. MycoKeys 126: 171–186. https://doi. org/10.3897/mycokeys.126.163715 MycoKeys 126: 171–186 (2025) DOI: 10.3897/mycokeys.126.163715 172 MycoKeys 126: 171–186 (2025), DOI: 10.3897/mycokeys.126.163715 Wan-Li Gao et al.: Two new Papiliotrema species cludes more than 20 species, classified in the family Rhynchogastremataceae within the order Tremellales (Liu et al. 2015). The genus has since expanded with the addition of several new species, including P. odontotermitis S. Handel, T. Wang, A.M. Yurkov & H. Koenig (Handel et al. 2016), P. leoncinii D.M. Pagani, L.R. Brandão, A.R.O. Santos, C.R. Félix, J.P. Ramos, L. Broetto, G. Scorzetti, J.W. Fell, C.A. Rosa, P. Valente & M.F. Landell and P. miconiae J.W. Fell, G. Scorzetii & M.F. Landell (Pagani et al. 2016), P. plantarum P. Into, A. Pontes, N. Jacques, S. Casarég, S. Limtong & J.P. Sampaio (Into et al. 2018), P. phichitensis P. Khunnamwong, J. Surussawadee, N. Srisuk, C. Boonmak & S. Limtong (Khunnamwong et al. 2018), P. zeae A.M. Yurkov & C.P. Kurtzman (Yurkov and Kurtzman 2019), P. horticola A.V. Kachalkin, A.M. Glushakova & M.A. Tomashevskaya (Crous et al. 2021), P. tapputiae Y.P. Tan, Bishop-Hurley & R.G. Shivas (Tan et al. 2023), P. castaneae Q.M. Wang and P. catalpae Q.M. Wang (Jiang et al. 2024) and P. fudaokuniae Y.P. Tan, Bishop-Hurley & Marney (Tan et al. 2024). To date, the genus Papiliotrema comprises 33 species. Amongst them, only three – P. bandonii, P. fusca and P. plantarum – have been reported with known sexual morphs (Sampaio et al. 2002; Sampaio et al. 2004; Into et al. 2018). These sexual states are characterised by clavate basidia with transverse septa, which clearly differentiate them from those of Tremella species (Liu et al. 2015; Into et al. 2018). The asexual morphs produce pale to brownish-yellow colonies and reproduce by polar budding (Liu et al. 2015). Physiologically, all members of Papiliotrema are non-fermentative, contain ubiquinone Q-10 as the major respiratory quinone and are capable of assimilating a variety of carbon sources, but not myo-inositol or nitrate (Liu et al. 2015). Papiliotrema species are widely distributed in diverse environments. The sexual morph of P. bandonii has been observed only on the inflorescences of Cortaderia selloana (Sampaio et al. 2002; Saluja and Prasad 2007), while the sexual states of P. fusca and P. plantarum have been recorded exclusively in culture, similar to the sexual forms of the sister genus Rhynchogastrema (Metzler et al. 1989; Sampaio et al. 2004; Into et al. 2018). The yeast morphs of Papiliotrema species are predominantly associated with plants, especially leaves (Surussawadee et al. 2014; Sylvester et al. 2015; Yurkov et al. 2015; Pagani et al. 2016; Into et al. 2018; Khunnamwong et al. 2018; Jiang et al. 2024; Tan et al. 2024). Additionally, strains of Papiliotrema species have also been isolated from soil (Crestani et al. 2009), glacier ice (de Garcia et al. 2012) and even aquatic environments (Fell et al. 2011; Pagani et al. 2016). Members of the genus Papiliotrema have attracted increasing attention due to their diverse biotechnological potential. Amongst them, P. laurentii (Kuff.) X.Z. Liu, F.Y. Bai, M. Groenew. & Boekhout was isolated from a wide range of natural, agricultural and anthropogenic environments (de Almeida et al. 2022). P. laurentii is known to produce biosurfactants, various enzymes and high levels of intracellular lipids, which are considered valuable feedstocks for fatty acid-derived industrial products (Vieira et al. 2020; Yalçın et al. 2021). Additionally, it can secrete mycocins with different inhibitory ranges, which has been utilised in the biocontrol of phytopathogenic fungi, thereby contributing to the preservation of fruit quality during both preand post-harvest stages (Castoria et al. 1997; Yurkov and Golubev 2013). The species also enhances mycorrhizal colonisation, improves nitrogen uptake and promotes plant growth (Moller et al. 2016). Furthermore, P. laurentii has demonstrated the ability to degrade poly- 173 MycoKeys 126: 171–186 (2025), DOI: 10.3897/mycokeys.126.163715 Wan-Li Gao et al.: Two new Papiliotrema species ester and diesel derivatives and to participate in the bioremediation of heavy metals (Hung et al. 2019). Other Papiliotrema species also exhibit functional versatility; for instance, P. terrestris (Crestani, Landell, Faganello, Vainstein, Vishniac & P. Valente) X. Z. Liu, F.Y. Bai, M. Groenew. & Boekhout produces exopolysaccharides with potential applications in biocompatible materials and wound healing (Hamidi et al. 2023), while P. flavescens (Saito) X.Z. Liu, F.Y. Bai, M. Groenew. & Boekhout has been reported to promote plant growth by modulating root architecture and inducing systemic resistance via volatile organic compounds in Arabidopsis (Liu et al. 2024). Furthermore, some species, as P. laurentii and P. flavescens, have also been isolated from clinical specimens and are considered opportunistic pathogens in humans (Fonseca et al. 2011; Zhang et al. 2022). During our recent surveys of epiphytic yeasts on leaf surfaces in different regions of China, five yeast strains belonging to the genus Papiliotrema were obtained. The objective of this study was to determine the taxonomic identity of these strains and to elucidate their phylogenetic relationships within Papiliotrema, based on a polyphasic approach including phenotypic characterisation, molecular phylogenetic analysis and ecological data. Materials and methods Sample collection and yeast isolation Plant leaf samples were collected from two locations in China: the Guiyang Medicinal Botanical Garden, Guizhou Province (26°34'51"N, 106°42'36"E) and Wuzhi Mountain, Hainan Province (18°17'21"N, 109°40'55"E). Yeast strains were isolated from the phylloplane using the washing and dilution method described by Jiang et al. (2024). Fresh leaves were aseptically cut into small pieces and placed into 10 ml sterile centrifuge tubes containing sterile water supplemented with 0.05% (v/v) Tween 80. The tubes were shaken for approximately 10 minutes and the resulting suspension was serially diluted to 10−2. Aliquots of 200 μl from each dilution were spread on to yeast extract–malt extract (YM) agar plates (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose and 2% agar) supplemented with 200 μg/ml chloramphenicol to suppress bacterial growth. The plates were incubated at 25 °C for 5–7 days. Distinct yeast colonies were selected and subcultured on fresh YM agar to obtain pure isolates. Purified strains were preserved in 20% (v/v) glycerol at −80 °C for longterm storage. Phenotypic characteriation Morphological, physiological, and biochemical characteristics were examined, following the standardised protocols outlined by Kurtzman et al. (2011). Colony morphology was recorded after 7 days of incubation at 20 °C on YM agar. Cellular morphology was observed after 3 days of cultivation in YM broth at 20 °C using a LEICA DM2500 microscope (LEICA, Wetzlar, Germany), equipped with LAS V4.13 software. Ballistoconidium formation was assessed using the inverted-plate method (do Carmo-Sousa and Phaff 1962) on corn meal agar (CMA: 2.5% corn meal infusion and 2% agar) at 17 °C. Discharged spores were 174 MycoKeys 126: 171–186 (2025), DOI: 10.3897/mycokeys.126.163715 Wan-Li Gao et al.: Two new Papiliotrema species collected on glass slides after 3 to 14 days and examined microscopically. The potential for sexual reproduction was evaluated on CMA, potato dextrose agar (PDA: 20% potato infusion, 2% glucose and 2% agar) and V8 agar (10% V8 juice and 2% agar). A loopful of cells from each test strain was streaked alone or mixed on the plates and incubated at 17 °C for up to two months, during which time, observations were made biweekly (Jiang et al. 2024). Glucose fermentation was tested in liquid medium using Durham fermentation tubes. Carbon and nitrogen assimilation tests were conducted in liquid media, with starved inocula used for nitrogen assimilation assessments (Kurtzman et al. 2011). Growth at various temperatures (15, 20, 25, 30, 35 and 37 °C) was assessed on YM agar plates. All novel species descriptions and proposed names were registered in the MycoBank database (http://www.mycobank.org; accessed 26 June 2025). DNA extraction, PCR amplification and sequencing Genomic DNA was extracted from actively growing yeast cultures on YM agar using the Ezup Column Yeast Genomic DNA Purification Kit (Sangon Biotech Co., Shanghai, China), following the manufacturer’s instructions. Two nuclear loci were targeted for amplification: the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit (LSU) rRNA gene. Amplifications were performed using the primer pairs ITS1/ITS4 (White et al. 1990) and NL1/ NL4 (Kurtzman and Robnett 1998), respectively. PCR reactions were carried out in a final volume of 25 μl, containing 9.5 μl of double-distilled water, 12.5 μl of 2× Taq PCR Master Mix with blue dye (Sangon Biotech), 1 μl of genomic DNA and 1 μl of each primer. The thermal cycling conditions were: initial denaturation at 98 °C for 2 min; 35 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 10 s and extension at 72 °C for 15 s; followed by a final extension at 72 °C for 5 min. PCR products were visualised on 1% agarose gels. Amplicons with single, clear bands were purified using the DNA Gel Extraction Kit (Sangon Biotech) and subjected to bidirectional Sanger sequencing at Sangon Biotech Co., Ltd. (Shanghai, China). Consensus sequences were assembled and edited using BioEdit v.7.1.3.0 (Hall 1999). Sequence identities were confirmed via BLASTn searches against the GenBank database. All newly-obtained sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Phylogenetic analyses For the phylogenetic analyses, sequences generated in this study along with additional related sequences derived from the GenBank database, were used (Table 1). Individual loci were aligned separately using MAFFT v.7.110 (Katoh and Standley 2013) with the G-INS-i algorithm, followed by manual refinement to remove ambiguous regions in BioEdit v.7.1.3.0 (Hall 1999). The resulting ITS and LSU alignments were concatenated into a single dataset using PhyloSuite v.1.2.3 (Zhang et al. 2020). Maximum Likelihood (ML) analysis was conducted using RAxML v.8.2.3 (Stamatakis 2014) under the GTRGAMMA model with 1, 000 rapid bootstrap replicates to assess nodal support. Bayesian Inference (BI) analysis was performed using MrBayes v.3.2.7a (Ronquist et al. 2012). Six Markov Chain Monte Carlo (MCMC) chains were run for 50 million generations, 175 MycoKeys 126: 171–186 (2025), DOI: 10.3897/mycokeys.126.163715 Wan-Li Gao et al.: Two new Papiliotrema species Table 1. Information of yeast species and strains used in phylogenetic analyses and their GenBank accession numbers. Sequences newly generated in this study are indicated in bold. Species Strain no. Country GenBank accession no. ITS LSU D1/D2 Papiliotrema anemochoreius CBS 10258 South Africa KY104455 KY108727 Papiliotrema aspenensis CBS 13867 USA NR_158801 NG_060109 Papiliotrema aurea CBS 318 Japan NR_130650 NG_148937 Papiliotrema baii PYCC 6523 China LK023827 LK023766 Papiliotrema bandonii CBS 9107 Portugal NR_121465 NG_042386 Papiliotrema castaneae YN83-2 China OP470272 OP470176 Papiliotrema catalpae YN109M3 China OP470271 OP470175 Papiliotrema flavescens CBS 942 Japan NR_130696 AB035042 Papiliotrema fonsecae CBS 12692 Svalbard NR_119972 JN193447 Papiliotrema frias CBS 12693 Patagonia GU997162 LK023834 Papiliotrema fudaokuniae BRIP 76370a Australia NR_199259 NG_244360 Papiliotrema fusca PYCC 5690 Portugal AF444668 AF444762 Papiliotrema hoabinhensis JCM 10835 Vietnam AB110695 AB193347 Papiliotrema horticola KBP MSU Y-6685 Russia NR_182874 MW579431 Papiliotrema japonica CBS 2013 Portugal NR_155613 NG_057690 Papiliotrema laurentii ATCC 18803 Congo NR_130670 NG_056281 Papiliotrema leoncinii CBS 13918 Brazil KP203864 KJ608554 Papiliotrema mangalensis CBS 10870 USA NR_144816 NG_057803 Papiliotrema miconiae CBS 8358 Brazil AF444387 AF444698 Papiliotrema millettiae NYNU 243102 China PP837692 PP837690 Papiliotrema millettiae NYNU 24472 China PV823290 PV823291 Papiliotrema mussaendae NYNU 23248 China OQ851892 OQ851890 Papiliotrema mussaendae NYNU 232142 China PV823294 PV823293 Papiliotrema mussaendae NYNU 23229 China PV823295 PV823292 Papiliotrema nemorosa CBS 9606 Russia NR_137534 NG_058365 Papiliotrema odontotermitis CBS 14181 Germany NR_156605 KU883278 Papiliotrema perniciosa CBS 9605 Russia NR_137653 NG_060063 Papiliotrema phichitensis DMKU-SP105 Thailand AB915388 AB826437 Papiliotrema plantarum CBS 15220 Thailand NR_164566 LC370335 Papiliotrema pseudoalba CBS 7227 Japan NR_073231 NG_058276 Papiliotrema rajasthanensis CBS 10406 India NR_155678 NG_058366 Papiliotrema ruineniae PYCC 6170 Indonesia LK023826 LK023764 Papiliotrema siamensis CBS 13330 Thailand NR_155608 NG_060062 Papiliotrema taeanensis CBS 9742 Korea NR_155679 AY422719 Papiliotrema tapputiae BRIP 75038a Australian NR_187102 NG_229127 Papiliotrema terrestris CBS 10810 USA NR_073350 NR_073350 Papiliotrema wisconsinensis CBS 13895 USA NR_160324 NG_060134 Papiliotrema zeae DSM 104035 Minnesota NR_168772 MH718306 ‘Cryptococcus’ sp. BI311 Brazil KR063666 KT318492 Rhynchogastrema aquatica CBS 12527 Brazil NR_120001 NG_042603 Rhynchogastrema complexa CBS 11570 Brazil NR_111476 NG_042525 Rhynchogastrema coronatum DSM 28188 Germany LN870267 LN870267 Rhynchogastrema fermentans CBS 12399 Taiwan, China NR_155732 NG_058388 Rhynchogastrema glucofermentans CBS 10381 Panama NR_119978 NG_042404 176 MycoKeys 126: 171–186 (2025), DOI: 10.3897/mycokeys.126.163715 Wan-Li Gao et al.: Two new Papiliotrema species with sampling every 1,000 generations. The optimal substitution model was selected using MrModelTest v.2.3 (Posada and Crandall 1998). The first 25% of sampled trees were discarded as burn-in and the remaining trees were used to estimate Bayesian posterior probabilities (BPPs) for the clades. Phylogenetic trees were visualised using FigTree v.1.4.3 (Andrew 2016) and final graphical editing was conducted in Adobe Illustrator CS v.5. Branches with bootstrap support (BS) values ≥ 50% and Bayesian posterior probabilities (BPPs) ≥ 0.95 were regarded as statistically significant. Results Molecular phylogeny In this study, a total of 76 yeast strains were isolated from 25 leaf samples collected. Based on the identification of the ITS and LSU sequences, the majority of these yeast strains were identified as 18 known species, including Bannoa ogasawarensis, Bullera mrakii, Bulleribasidium pseudovariabile, Cystobasidium pallidum, Derxomyces komagatae, Dioszegia hungarica, Erythrobasidium hasegawianum, Filobasidium magnum, Hannaella sinensis, Hannaella taiwanensis, Rosettozyma cystopteridis, Sporobolomyces carnicolor, Sporidiobolus pararoseus, Sporobolomyces roseus, Symmetrospora gracilis, Symmetrospora oryzicola, Tilletiopsis washingtonensis and Vishniacozyma carnescens. The remaining five strains, NYNU 243102, NYNU 24472, NYNU 23248, NYNU 232142 and NYNU 23229, which could not be identified as any known species, were selected for further taxonomic study. To determine the phylogenetic placements of these potential novel strains, phylogenetic analysis was conducted using the concatenated ITS and LSU sequences. The concatenated ITS and LSU dataset included 50 ITS and 50 LSU sequences from 50 strains, representing 46 ingroup taxa plus one outgroup (Table 1). The aligned dataset length was 1,096 nucleotides, comprising 491 from ITS and 605 from LSU region. MrModelTest identified GTR+I+G as the best-fit nucleotide substitution model for BI analysis. BI analysis produced tree topologies nearly identical to those from the ML analysis. Only the ML tree is shown in Fig. 1, with BS (≥ 50%) and BPPs (≥ 0.95) indicated on branches. Phylogenetic analyses revealed that five isolates from China clustered into two genetically distinct lineages, each representing a putative novel species within the genus Papiliotrema. Two strains, NYNU 243102 and NYNU 24472, were isolated from leaves of Millettia pachycarpa Benth. and Musa nana Lour., respectively, but shared identical D1/D2 and ITS sequences, indicating conspecificity. These strains formed Species Strain no. Country GenBank accession no. ITS LSU D1/D2 Rhynchogastrema nanyangensis CBS 12474 China NR_166792 JN564592 Rhynchogastrema noutii CBS 8364 Brazil NR_111072 NG_042389 Rhynchogastrema tunnelae CBS 8024 Finland NR_111074 NG_042390 Naematelia aurantia CBS 6965 China AF444315 AF189842 Naematelia encephala CBS 8207 Germany AF042402 AF042220 Kwoniella mangrovensis CBS 8507 Bahamas AF444646 AF444742 177 MycoKeys 126: 171–186 (2025), DOI: 10.3897/mycokeys.126.163715 Wan-Li Gao et al.: Two new Papiliotrema species Figure 1. Phylogram of Papiliotrema, based on the concatenated ITS-LSU dataset, with Kwoniella mangrovensis CBS 8507 as the outgroup. Bootstrap values (BS) ≥ 50% and Bayesian posterior probabilities (BPPs) ≥ 0.90 are indicated at the nodes. Newly-generated sequences are highlighted in bold. “T” denotes sequences from type strains. 178 MycoKeys 126: 171–186 (2025), DOI: 10.3897/mycokeys.126.163715 Wan-Li Gao et al.: Two new Papiliotrema species a distinct clade clustering with P. mangalensis, P. pseudoalba, P. hoabinhensis and P. anemochoreius in the concatenated ITS and LSU phylogeny (Fig. 1). They differed from these species by 13–17 substitutions (~ 2–2.8%) in the D1/D2 domain and 12–16 mismatches (~ 2.4–3%) in the ITS region, which are higher than the basidiomycetous yeasts thresholds of 0.49% for D1/D2 and 1.39% for ITS, recommended by Vu et al. (2016). These results support that strains NYNU 243102 and NYNU 24472 represent a novel Papiliotrema species, for which the name Papiliotrema millettiae sp. nov. is proposed. Three strains, NYNU 23248, NYNU 232142 and NYNU 23229, were isolated from three different leaf samples of Mussaenda pubescens Ait.f. and shared identical D1/D2 and ITS sequences, indicating conspecificity. These strains were closely related to P. wisconsinensis (Fig. 1) and differed from it by seven substitutions (~ 1.3%) in the D1/D2 domain and 10 mismatches (~ 2.2%) in the ITS region, respectively. Additionally, they also differed from other closely-related species, P. miconiae and P. plantarum, by 7–8 nucleotide substitutions (~ 1.3–1.4%) in the D1/D2 domain and 10–16 substitutions (~ 2.2– 3.6%) in the ITS region. These results support that the three strains represent a novel Papiliotrema species, for which the name Papiliotrema mussaendae sp. nov. is proposed. Taxonomy Papiliotrema millettiae C.Y. Chai & F.L. Hui, sp. nov. MycoBank No: 859778 Fig. 2 Etymology. The specific epithet millettiae refers to Millettia, the plant genus from which the type strain was isolated. Typus. China • Guizhou, Pingtang County, Sifangjing Village, 25°7'N, 107°2'E, on the phylloplane of Millettia pachycarpa, Feb 2024, D. Lu, NYNU 243102 (holotype CICC 33641 preserved in a metabolically inactive state, metabolically inactive ex-type culture PYCC 10057), GenBank Accession No.: PP837692 (ITS), PP837690 (LSU). Description. On YM agar after 7 days at 20 °C, the streak culture is whitecream, butyrous and smooth, with an entire margin. After 3 days in YM broth at 20 °C, cells are ovoid and ellipsoidal, 3.5–6.4 × 4.3–8.9 μm and single, budding is polar. After 1 month at 20 °C, a ring and a 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 not produced. Ballistoconidia are not produced. Glucose fermentation is absent. Glucose, inulin (weak and delayed), sucrose, raffinose, galactose, lactose, trehalose, maltose, melezitose, methyl-α-D-glucoside, cellobiose, salicin, L-rhamnose, D-xylose, L-arabinose, D-arabinose, 5-keto-D-gluconate, D-ribose, ethanol, glycerol, ribitol, galactitol, D-mannitol, D-glucitol, myo-inositol, DL-lactate, succinate, citrate (delayed), D-gluconate, D-glucosamine (weak), N-acetyl-D-glucosamine (weak), 2-keto-D-gluconate, D-glucuronate and glucono-1,5-lactone are assimilated as carbon sources. Melibiose, L-sorbose, methanol and erythritol are not assimilated. Ethylamine and L-lysine and cadaverine (delayed) are assimilated as nitrogen sources. Nitrate and nitrite are not assimilated. Growth is observed 179 MycoKeys 126: 171–186 (2025), DOI: 10.3897/mycokeys.126.163715 Wan-Li Gao et al.: Two new Papiliotrema species Figure 2. Morphology of Papiliotrema millettiae (NYNU 243102). A. Colony on YM agar after 7 days at 20 °C; B. Budding cells in YM broth after 3 days at 20 °C. at 20 °C and 25 °C, but not at 30 °C. Growth on 50% (w/w) glucose-yeast extract agar is positive. Vitamins are not necessary for growth. Starch-like compounds are produced. Diazonium blue B colour and urease reaction are positive. Additional strain examined. China, Hainan, Sanya City, Wuzhi Mountain, 32°45'N, 113°30'E, on the phylloplane of Musa nana, Mar 2024, S.L. Lv, NYNU 24472, GenBank Accession No.: PV823290 (ITS), PV823291 (LSU). Note. P. millettiae is phylogenetically closely related to P. mangalensis, P. pseudoalba, P. hoabinhensis and P. anemochoreius. Physiologically, P. millettiae differs from P. mangalensis in its ability to assimilate inulin, methyl-α-D-glucoside and DL-lactate. Compared to P. pseudoalba, it cannot assimilate melibiose or grow at 30 °C. It differs from P. hoabinhensis in its ability to assimilate inulin and DL-lactate and to grow on 50% glucose medium. Compared to P. anemochoreius, it cannot assimilate melibiose or L-sorbose and cannot grow at 30 °C (Table 2). Papiliotrema mussaendae C.Y. Chai & F.L. Hui, sp. nov. MycoBank No: 859779 Fig. 3 Etymology. The specific epithet mussaendae refers to Mussaenda, the plant genus from which the type strain was isolated. Typus. China • Guizhou, Pingtang County, Sifangjing Village, 25°7'N, 107°2'E, on the phylloplane of Mussaenda pubescens, Feb 2023, D. Lu, NYNU 23248 (holotype CICC 33595, preserved in a metabolically inactive state, metabolically inactive ex-type culture PYCC 9975), GenBank Accession No.: OQ851892 (ITS), OQ851890 (LSU). Description. On YM agar after 7 days at 20 °C, the streak culture is whitecream, mucoid, smooth and shiny, with an entire margin. 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