New species and records of ascomycetes on cypress in Beijing, China

271 New species and records of ascomycetes on cypress in Beijing, China Zixian Bi1, Yingying Wu1, Shuji Li1, Chengming Tian1 1 The Key Laboratory for Silviculture and Conservation of the Ministry of Education, Beijing Forestry University, Beijing 100083, China Corresponding author: Chengming Tian ([email protected]) Copyright: © Zixian Bi 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 Cypress, a collective term for plants belonging to the Cupressaceae family, is widely utilized in Chinese landscaping and holds considerable economic and medicinal importance. In recent years, dieback of branches and foliage in cypress has been widespread in the Ming Tombs area of Beijing, yet the associated ascomycetous fungi remain unidentified. During an investigation of the species of ascomycetes associated with three cypress species (Juniperus chinensis, J. procumbens, and Platycladus orientalis) in the Ming Tombs area of Beijing, 22 fungal strains were isolated from withered branches and diseased leaves to healthy strobili and mature cones. Based on integrated morphological and multi-gene phylogenetic analyses, these strains were identified as 13 fungal species belonging to 8 genera. Among these, two novel species—Nigrospora platycladiensis sp. nov. and Spegazzinia juniperi sp. nov.—and two new host records, Aplosporella hesperidica and Nigrospora philosophiae-doctoris, are reported herein. This study contributes to our understanding of the richness of ascomycetes on cypress. Key words: Ascomycota, cypress, morphology, multi-gene phylogenetic, taxonomy Introduction Nigrospora was introduced by Zimmerman (1902) with N. panici designated as the type species (Wang et al. 2017). As an important genus of ascomycetous fungi, Nigrospora commonly exists as plant pathogens, endophytes, or saprophytes and is widely distributed across various plants, as well as in soil and air (Wang et al. 2017; Raza et al. 2019). The defining characteristics of Nigrospora include its dark-pigmented conidia and conidiophores (Zimmerman 1902; Wang et al. 2024a; Zou et al. 2024). Initially, species identification within Nigrospora relied on morphological comparisons of conidial size and structure (Mason 1927; Zou et al. 2024). However, interspecific morphological distinctions in this genus are often subtle—thus taxonomic identification necessitates an integrative approach combining morphological examination with phylogenetic evidence (Hao et al. 2020; Wang et al. 2017). Since then, numerous novel Nigrospora species have been discovered (Raza et al. 2019; Tian et al. 2020; Liu et al. 2024; Wang et al. 2024a). Spegazzinia was established by Saccardo (1880) with S. ornata (now treated as a synonym of S. tessarthra) designated as its type species. Spegazzinia exhibits a remarkably broad geographic distribution, predominantly existing as Academic editor: Huzefa Raja Received: 21 July 2025 Accepted: 2 September 2025 Published: 16 October 2025 Citation: Bi Z, Wu Y, Li S, Tian C (2025) New species and records of ascomycetes on cypress in Beijing, China. MycoKeys 123: 271–317. https://doi.org/10.3897/ mycokeys.123.165848 MycoKeys 123: 271–317 (2025) DOI: 10.3897/mycokeys.123.165848 272 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China endophytes within host organisms or as saprophytes colonizing decaying plant debris (Hashemlou et al. 2023; Dai et al. 2025). Hyde et al. (1998) initially classified this genus within Sordariomycetes (Apiosporaceae) based on its morphological characteristic of basauxic conidiogenesis. Subsequently, Tanaka et al. (2015) reclassified the genus into Dothideomycetes (Didymosphaeriaceae) based on phylogenetic evidence derived from three multi-gene loci. In recent years, many new species of Spegazzinia have been described (Jayasiri et al. 2019; Samarakoon et al. 2020; Hashemlou et al. 2023; Zhang et al. 2024a; Dai et al. 2025). The genus is characterized by basauxic conidiogenesis, producing brown to dark brown conidia that may exhibit spine-like appendages. Notably, it develops two distinct conidial morphotypes: stellate α-conidia and cloverleaf-shaped β-conidia (Suwannarach et al. 2021; Hashemlou et al. 2023; Zhang et al. 2024a). Cypress belongs to the family Cupressaceae, a general term encompassing various cypress species, including Juniperus chinensis, Juniperus procumbens, and Platycladus orientalis (Su and Zou 2023). Cupressaceae plants are widely distributed worldwide, comprising approximately 22 genera and nearly 150 species (Shang et al. 2013). In China, there are about 8 genera and over 30 species of cypress, with 1 additional genus introduced through cultivation (Shang et al. 2013; Lin et al. 2021). Cypresses are extensively planted across all districts of Beijing, primarily featuring P. orientalis and J. chinensis, with P. orientalis designated as Beijing’s official city tree (Duan et al. 2024). Renowned for their elegant forms, exceptional environmental adaptability, and unique chemical composition, cypress trees play crucial roles in China’s urban landscaping, economic development, ecological conservation, and medical applications (Gong et al. 2020; Duan et al. 2024). The phylum Ascomycota, comprising the largest number of species within the fungal kingdom, is widely distributed across various host plants (Hyde et al. 2019). With the rise of molecular systematics, ascomycete groups related to cypresses have been further explored, and many new species have been successively reported (Pan et al. 2021; Peng et al. 2023; Jiao et al. 2024; Lin et al. 2024; Jiao et al. 2025b; Zhou et al. 2025). Zhu (1922) first discovered leaf blight disease of P. orientalis caused by Alternaria pruni in China. Later, Liu et al. (1997) observed a large-scale outbreak of shoot blight disease on J. chinensis in the Dalian region. They identified both the pathogen and its infection patterns, confirming that the disease was caused by a fungal species belonging to Coniothyrium sp. Li et al. (2010) reported for the first time in China that Neofusicoccum parvum caused cypress dieback disease. Subsequently, Mohammadi et al. (2014) isolated N. parvum from discolored, necrotic, cankered, and wilted wood tissues of cypress trees during their investigation of cypress decline disease in southeastern Iran. Cypress canker disease is a severe fungal disease worldwide (Boesewinkel 1983; Graniti 1986, 1998; Danti and Della Rocca 2017). This disease was first reported as an epidemic on Monterey cypress in California (Wagener 1928; Danti and Della Rocca 2017). Initially prevalent across North America, South America, Africa, Australia, and New Zealand, it subsequently spread eastward to other regions (Wagener 1928; Urbasch 1993). According to international literature, Seiridium cardinale, S. cupressi, and S. unicorne are among the most destructive pathogenic fungi affecting Cupressaceae plants and are recognized as the primary causative agents of cypress canker epidemics (Boesewinkel 1983; Graniti 1986, 1998; Danti and Della Rocca 2017; Bonthond et al. 2018). Guo (2023) made the first documented discovery of cypress canker dis- 273 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China ease caused by S. unicorne in China. In recent years, studies have demonstrated that Alternaria spp., Nothophoma spp., Bipolaris setariae, and B. sorokiniana are pathogenic fungi capable of causing leaf blight in ancient P. orientalis trees in the Beijing region (Jiao et al. 2024, 2025a, 2025b). While studies have documented ascomycete communities associated with cypress trees, research efforts have primarily concentrated on pathogenic groups, with cypress pathogens receiving significant attention from researchers both in China and internationally. In addition to pathogenic species, cypress-associated ascomycetes include diverse endophytic fungi whose secondary metabolites exhibit insecticidal, antimicrobial, and antitumor bioactivities (Rawat et al. 2010; Ismail et al. 2013). For instance, Zhang et al. (2014) isolated Chaetomium globosum from P. orientalis foliage, with subsequent bioassays demonstrating its metabolites’ potent inhibitory effects against pathogenic bacteria. However, current research on endophytic fungi of cypresses in China remains limited, with most studies primarily focusing on their antibacterial and antifungal activities (Wang et al. 2010; Zhang et al. 2014). In recent years, the phenomenon of branch and leaf withering in cypress has become increasingly common in the Ming Tombs area of Beijing. However, the diversity of Ascomycota fungi associated with these trees remains unclear. Most existing studies on cypress-related fungi focus primarily on pathogenic species, while research on endophytic and saprophytic fungi is relatively scarce. In this study, we collected 22 strains of Ascomycota fungi from various parts of three cypress species (J. chinensis, J. procumbens, and P. orientalis), including withered branches, diseased leaves, healthy strobili, and mature cones, in the Ming Tombs area of Beijing. Through morphological and molecular phylogenetic analyses, detailed identification of known species and potential new species was conducted. This research not only provides fundamental data for future studies on the diversity of cypress-associated Ascomycota but also expands the known diversity of these fungi. Materials and methods Sample collection and fungal isolation During a diversity survey of ascomycetous fungi associated with cypress in the Ming Tombs area of Beijing, specimens were collected from 3 cypress species (Juniperus chinensis, J. procumbens, and Platycladus orientalis) at multiple sites, including Dingling Tomb, Changling Tomb, Longshan Sub-Farm of the Ming Tombs, Beijing Mangshan National Forest Park, Beijing Dayu Mountain Scenic Area, and the Ming Tombs Reservoir. A total of 18 specimens were collected: P. orientalis (15 specimens), J. chinensis (2 specimens), and J. procumbens (1 specimen), including diseased branches, infected leaves, as well as healthy strobili and mature cones (Fig. 1). The fungal species isolated from various tissues of different host plants in this study are detailed in Suppl. material 1. All specimens were transported to the laboratory in sealed plastic bags for further analysis. Before separation, the surface of each specimen was rinsed with tap water to remove dust and then placed in a laminar flow cabinet. Using a sterilized blade, tissue blocks (approximately 0.5 × 0.5 cm) were cut from the disease–health junction, infection sites, and surfaces of healthy strobili and mature cones. The samples were sequentially treated by soaking 274 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China Figure 1. Collection type of Ascomycota specimens related to cypress in the Ming Tombs area, Beijing. A–H. Diseased leaves of Platycladus orientalis; I, J. Dieback twigs of Juniperus procumbens; K, L. Dead branches on Platycladus orientalis; M. Healthy strobili of Platycladus orientalis; N. Healthy strobili of Juniperus chinensis; O, P. Healthy cones of Juniperus chinensis. 275 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China in 75% ethanol for 30 seconds, followed by 5% NaClO for 1 minute, and then rinsed three times with sterile water. Surface moisture was absorbed with sterilized filter paper, and the samples were transferred to potato dextrose agar plates (PDA: prepared with 200 g potatoes, 20 g glucose, and 20 g agar; 1 liter of sterile water) and incubated at 25 °C for 2–5 days. After colonies appeared, a sterilized inoculation needle was used to pick hyphae from the edge of the colony and transfer them to PDA medium to obtain pure cultures (Yuan et al. 2024). For specimens with clearly visible fruiting bodies, spore masses were aseptically transferred to the surface of PDA medium and cultured under dark conditions at 25 °C (Wang et al. 2024b). After colony formation, they were transferred to fresh PDA plates for further cultivation. The specimens used in this study are deposited in the Museum of Beijing Forestry University (BJFC), and the fungal strains are preserved at the China Forestry Culture Collection Center (CFCC). Morphological observation The cultures were inoculated onto PDA medium and incubated at 25 °C under dark conditions for 14–30 days, with regular documentation of colony morphological characteristics (Yuan et al. 2024). After sporulation structures developed, the morphological features of fruiting bodies were examined using a stereomicroscope (OLYMPUS SZX2-FOF, Tokyo, Japan). Microscopic characteristics, including asci, ascospores, conidia, and conidiophores, were observed under a Nikon Eclipse 80i compound microscope equipped with differential interference contrast (DIC) illumination. Photographic documentation was performed using Nikon Nis-Elements F4.30.01 software. For naturally developed fruiting bodies on host branches, transverse and longitudinal sections were prepared using sterile double-edged razor blades for microscopic examination and documentation. Morphometric analyses were performed on 15–30 pycnidia, asci, and conidiogenous cells, along with 30–50 spores. DNA extraction, PCR amplification, and sequencing After approximately 10 days of culture, fungal DNA was extracted using a modified CTAB method (Doyle 1990). Based on genus-specific diagnostic characteristics documented in the literature, corresponding genetic markers were selected for PCR amplification. For Aplosporella, the internal transcribed spacer region rDNA (ITS) (primers; ITS1/ITS4) and the translation elongation factor 1-alpha (tef1) (primers; EF1-728F/EF1-986R) gene were amplified (Lin et al. 2023a). For Achaetomium, Arcopilus, and Chaetomium, the internal transcribed spacer region rDNA (ITS) (primers; ITS1/ITS4), large subunit ribosomal RNA (LSU) (primers; LR5/ LROR), the partial beta-tubulin (tub2) (primers; T1/TUB4Rd) gene, and the RNA polymerase II second largest subunit (rpb2) (primers; fRPB2-5F/fRPB2-7cR) loci were amplified (Wang et al. 2022). For Seiridium, the internal transcribed spacer region rDNA (ITS) (primers; ITS1/ITS4), large subunit ribosomal RNA (LSU) (primers; LR5/LROR), the translation elongation factor 1-alpha (tef1) (primers; EF1728F/EF2) gene, the partial beta-tubulin (tub2) (primers; T1/BT2b) gene, and the RNA polymerase II second largest subunit (rpb2) (primers; fRPB2-5F/fRPB2-7cR) loci were amplified (Dissanayake et al. 2021). For Nigrospora, the internal transcribed spacer region rDNA (ITS) (primers; ITS1/ITS4), the translation elongation 276 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China factor 1-alpha (tef1) (primers; EF1-728F/EF2 or EF1-728F/EF1-986R) gene, and the partial beta-tubulin (tub2) (primers; BT2a/BT2b) gene were amplified (Wang et al. 2017; Liu et al. 2024). For Neofusicoccum, the internal transcribed spacer region rDNA (ITS) (primers; ITS1/ITS4), the translation elongation factor 1-alpha (tef1) (primers; EF1-728F/EF1-986R) gene, the partial beta-tubulin (tub2) (primers; BT2a/BT2b) gene, and the RNA polymerase II second largest subunit (rpb2) (primers; fRPB2-5F/fRPB2-7cR) loci were amplified (Si et al. 2023). For Spegazzinia, the internal transcribed spacer region rDNA (ITS) (primers; ITS1/ITS4), the translation elongation factor 1-alpha (tef1) (primers; EF1-983F/EF1-2218R) gene, the large subunit ribosomal RNA (LSU) (primers; LR5/LROR), and the small subunit rDNA (SSU) (primers; NS1/NS4) were amplified (Zhang et al. 2024a). The primer sequences (forward and reverse) and PCR conditions for each genus are provided in Suppl. material 2. PCR amplification was performed in a 20 μL reaction system containing 10 μL 2×ES Taq Mastermix (Dye), 7 μL double deionized water, 1 μL template DNA, and 1 μL of each primer. Amplification products were electrophoresed on 2% agarose gels (Wu et al. 2024) and subsequently sequenced by Tsingke Biotechnology Co., Ltd. (Beijing). The sequences newly obtained in this study have been submitted to GenBank, and the accession numbers have been obtained. The sequences obtained in this study are in Suppl. material 2. Phylogenetic analyses The obtained sequences were first assembled using SeqMan v. 7.1.0 software. Subsequently, BLAST analysis was performed in the NCBI database (https:// www.ncbi.nlm.nih.gov/) to retrieve reference sequences for relevant genera from previously published articles (Samarakoon et al. 2020; Dissanayake et al. 2021; Li et al. 2022; Xu et al. 2022; Condé et al. 2023; Lin et al. 2023a; Si et al. 2023; Qian et al. 2024; Wu et al. 2024; Zhang et al. 2024a, 2024b, 2024c; Zou et al. 2024; Dai et al. 2025). The acquired sequences are listed in Suppl. material 2. Sequence alignment was conducted using MAFFT v. 7 (https://mafft.cbrc.jp/ alignment/server/) (Katoh and Standley 2013), and the sequences were aligned and edited using MEGA v. 6 software (Tamura et al. 2013). Phylogenetic trees based on multiple genes were constructed using Maximum Likelihood (ML) and Bayesian inference (BI) methods, implemented with PhyML v. 3.0 and MrBayes v. 3.1.2 software, respectively (Huelsenbeck and Ronquist 2001; Swofford 2003; Silvestro and Michalak 2012). The phylograms were visualized using FigTree v. 1.4.0 software and then edited using Adobe Illustrator CS v. 5. Finally, nodes with bootstrap support values ≥ 50% in the ML analyses, and posterior probabilities (PP) ≥ 0.90 in the BI analysis, were clearly labeled on each phylogram. Results Phylogenetic analyses Phylogenetic analyses of Nigrospora In this study, eight Nigrospora strains were isolated from Juniperus chinensis, Juniperus procumbens, and Platycladus orientalis. A multigene phylogenetic tree of the genus Nigrospora was constructed using concatenated sequences of the ITS, tef1-α, and tub2 genes, with Apiospora malaysiana (CBS 102053) and 277 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China Apiospora pseudoparenchymatica (LC 7234) designated as outgroups (Zou et al. 2024). The phylogenetic tree revealed that the eight strains formed four distinct clades: the strains CFCC 72628 and CFCC 72637 clustered with Nigrospora philosophiae-doctoris (CGMCC 3.20540) with strong support values of 100/1 (ML/BI); the strains CFCC 72638 and CFCC 72644 clustered with Nigrospora oryzae with strong support values of 100/0.99 (ML/BI); the strains CFCC 72646 and CFCC 72649 clustered with Nigrospora osmanthi (CGMCC 3.18126) with strong support values of 100/0.99 (ML/BI); and the strains CFCC 72630 and CFCC 72632 formed an independent branch, with strong support values of 100/1 (ML/BI), representing a novel species (Fig. 2). The phylogenetic tree contained a total of 1615 characters, comprising 935 constant characters, 129 variable characters, and 551 parsimony-informative characters. In the ML analysis based on the combined gene dataset, the matrix possessed 866 distinct alignment patterns. Estimated base frequencies are as follows: A = 0.214835, C = 0.305355, G = 0.239540, T = 0.240270, AC = 0.948986, AG = 2.819556, AT = 0.889264, CG = 0.897776, CT = 4.263318, GT = 1.000000, and gamma distribution shape parameter: α = 0.233074. The phylogenetic trees constructed using ML and BI exhibited identical topology. Phylogenetic analyses of Spegazzinia In this study, two Spegazzinia strains were isolated from healthy cones of Juniperus chinensis. A multigene phylogenetic tree of the Spegazzinia and its closely related genera was constructed using concatenated sequences of the ITS, LSU, SSU, and tef1-α genes, with reference sequences derived from previously published studies (Samarakoon et al. 2020; Hashemlou et al. 2023; Zhang et al. 2024a; Dai et al. 2025). The complete phylogenetic tree of Didymosphaeriaceae is provided in Suppl. material 3. Pleospora herbarum (CBS 191.86) and Stemphylium botryosum (CBS 714.68) were selected as the outgroup (Samarakoon et al. 2020). The phylogenetic tree revealed that the two isolated strains, CFCC 72641 and CFCC 72647, formed a distinct clade with strong support values of 99/1 (ML/BI) (Fig. 3). This clade did not cluster with any known species, indicating their status as a novel species. The phylogenetic tree contained a total of 3455 characters, comprising 2733 constant sites, 182 variable sites, and 540 parsimony-informative characters. In the ML analysis based on the combined gene dataset, the matrix possessed 829 distinct alignment patterns. Estimated base frequencies are as follows: A = 0.234832, C = 0.261337, G = 0.275448, T = 0.228383, AC = 1.194130, AG = 2.107716, AT = 1.188755, CG = 1.166085, CT = 5.918954, GT = 1.000000, and gamma distribution shape parameter: α = 0.161473. The phylogenetic trees constructed using ML and BI exhibited identical topology. Phylogenetic analyses of Aplosporella In this study, five Aplosporella strains were isolated from Platycladus orientalis in the Ming Tombs area of Beijing. A multi-gene phylogenetic tree of the genus Aplosporella was constructed based on the combined ITS and tef1-α gene regions, with Alanomyces indica (CBS 134264) selected as the outgroup (Lin et al. 2023a; Wu et al. 2024). The phylogenetic tree revealed that these five strains clustered into three distinct clades: strain CFCC 72635 clustered with Aplosporella hesperidica (CBS 732.79) with support values of 98/0.94 (ML/BI); 278 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China Figure 2. ML phylogenetic tree of Nigrospora based on combined ITS, tef1-α, and tub2 sequence data. The tree is rooted with Apiospora malaysiana (CBS 102053) and Apiospora pseudoparenchymatica (LC 7234). Bootstrap support values from ML analysis (ML ≥ 50%) and Bayesian posterior probabilities (BI ≥ 0.90) are shown at the nodes. Strains obtained in this study are marked in blue; ex-type strains are indicated in bold black type. 0.08 Nigrospora rubi CGMCC 3.18326 Nigrospora platycladiensis CFCC 72630 Nigrospora oryzae LC 2699 Nigrospora zimmermanii CBS 167.26 Nigrospora lacticolonia CGMCC 3.18123 Nigrospora sacchari-officinarum CGMCC 3.19335 Nigrospora guilinensis LC 7301 Nigrospora philosophiae-doctoris CFCC 72637 Nigrospora chinensis CGMCC3.18127 Nigrospora chinensis LC 4593 Nigrospora pyriformis LC 2688 Nigrospora osmanthi CFCC 72646 Nigrospora lacticolonia LC 7009 Nigrospora endophytica ARM 973 Nigrospora manihoticola ARM 645 Nigrospora yunnanensis GUCC 24-0010 Nigrospora philosophiae-doctoris LC 15838 Nigrospora osmanthi CGMCC 3.18126 Nigrospora osmanthi CFCC 72649 Nigrospora tomentosae ZHKUCC 22-0340 Nigrospora oryzae CFCC 72644 Nigrospora falsivesicularis CGMCC 3.1967 Nigrospora aurantiaca LC 7034 Nigrospora humicola CFCC 56885 Nigrospora bambusae LC 7244 Nigrospora macarangae MFLUCC 19-0141 Apiospora malaysiana CBS 102053 Nigrospora stoneae BRIP 75022a Nigrospora globosa CGMCC 3.19633 Nigrospora platycladiensis CFCC 72632 Nigrospora oryzae LC 6759 Nigrospora philosophiae-doctoris CFCC 72628 Nigrospora oryzae LC 6760 Nigrospora marylouisemclawsiae BRIP 74865 Nigrospora yunnanensis GUCC 24-0008 Nigrospora gorlenkoana CBS 480.73 Nigrospora guangdongensis Tly068 Nigrospora yunnanensis GUCC 24-0009 Nigrospora musae CBS 319.34 Nigrospora camelliae-sinensis CGMCC 3.18125 Nigrospora oryzae LC 2695 Nigrospora pernambucoensis ARM 974 Nigrospora saccharicola CGMCC 3.19362 Nigrospora humicola CFCC 56884 Nigrospora globospora CGMCC 3.20539 Nigrospora singularis CGMCC 3.19334 Nigrospora oryzae CFCC 72638 Nigrospora aurantiaca CGMCC 3.18130 Nigrospora vesicularis CGMCC 3.18128 Nigrospora guangdongensis CFCC 53917 Nigrospora camelliae-sinensis LC 4460 Apiospora pseudoparenchymatica LC 7234 Nigrospora tomentosae ZHKUCC 22-0339 Nigrospora sphaerica LC 7294 Nigrospora hainanensis LC 6979 Nigrospora coryli W 18 Nigrospora oryzae LC 6761 Nigrospora oryzae LC 2693 Nigrospora covidalis CGMCC 3.20538 Nigrospora musae LC 6385 Nigrospora guilinensis CGMCC 318124 Nigrospora pyriformis CGMCC 3.18122 Nigrospora sp. LC 4566 Nigrospora brasiliensis CMM 1214 Nigrospora zimmermanii CBS 290.62 Nigrospora globospora LC 15839 Nigrospora anhuiensis QY-2 Nigrospora sphaerica LC 7295 Nigrospora pernambucoensis ARM 651 Nigrospora bambusae CGMCC 3.18327 Nigrospora vesicularifera CGMCC 3.19333 Nigrospora magnoliae MFLUCC 19-0112 Nigrospora hainanensis CGMCC 3.18129 Nigrospora mercuriadeae BRIP 75764a Nigrospora sp. LC 2725 Nigrospora osmanthi LC 4487 Nigrospora vesicularis LC 0322 Nigrospora cooperae BRIP 72531c Nigrospora endophytica ARM 687 Nigrospora pyriformis LC 3099 Nigrospora magnoliae LC 6704 Nigrospora philosophiae-doctoris CGMCC 3.20540 100/0.99 100/1 97/0.99 100/0.99 96/1 100/1 70/- 74/- 98/0.95 63/- 99/1 90/0.99 100/1 84/0.98 100/1 51/- 90/0.98 100/0.99 100/1 95/0.92 100/0.99 87/0.92 100/0.99 100/0.90 69/- 92/0.92 75/0.95 100/0.99 100/1 99/0.99 100/0.99 100/0.99 99/0.91 100/1 100/1 76/0.91 91/0.94 100/1 63/- 100/0.99 100/0.97 94/0.99 100/1 100/0.99 96/0.92 100/0.97 100/0.99 98/1 100/0.99 100/1 100/0.99 100/1 100/0.99 57/- 100/1 100/0.99 72/- 82/0.95 68/0.99 Outgroup 279 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China strains CFCC 72633 and CFCC 72643 grouped with Aplosporella javeedii with support values of 99/1 (ML/BI); and strains CFCC 72634 and CFCC 72640 grouped with Aplosporella prunicola (CBS 121167) with support values of 84/1 (ML/BI) (Fig. 4). The phylogenetic tree contained a total of 831 characters, comprising 668 constant characters, 73 variable characters, and 90 parsimony-informative characters. In the ML analysis based on the combined gene dataset, the matrix possessed 207 distinct alignment patterns. Estimated base frequencies are as follows: A = 0.211875, C = 0.267409, G = 0.257202, T = 0.263513, AC = 2.898147, AG = 3.745261, AT = 1.882649, CG = 2.351749, CT = 6.230615, GT = 1.000000, and gamma distribution shape parameter: α = 0.214122. The phylogenetic trees constructed using ML and BI exhibited identical topology. Phylogenetic analyses of Neofusicoccum In this study, two Neofusicoccum strains were isolated from Platycladus orientalis in the Ming Tombs area of Beijing. Based on a multi-gene phylogenetic analysis using the concatenated sequences of the ITS, tef1-α, tub2, and rpb2 genes, with Botryosphaeria dothidea (CBS 115476) selected as the outgroup (Xu et al. 2022; Si et al. 2023), a multilocus phylogenetic tree of Neofusicoccum isolates was reconstructed (Fig. 5), with the complete phylogeny of the genus provided in Suppl. Figure 3. ML phylogenetic tree of Spegazzinia and its closely related genera based on combined ITS, LSU, SSU, and tef1-α sequence data. The tree is rooted with Pleospora herbarum (CBS 191.86) and Stemphylium botryosum (CBS 714.68). Bootstrap support values from ML analysis (ML ≥ 50%) and Bayesian posterior probabilities (BI ≥ 0.90) are shown at the nodes. Strains obtained in this study are marked in blue; ex-type strains are indicated in bold black type. 0.04 Spegazzinia musae MFLUCC 20-0001 Spegazzinia deightonii MFLUCC20-00027 Spegazzinia jinghaensis KUMCC 21-0495 Spegazzinia zhujiangyuanensis GMBCC 1002 Spegazzinia cryptica yone 279 Spegazzinia neosundara MFLUCC 15-0456 Sp egazzinia juniperi CFCC 72641 Spegazzinia camelliae WNA 03 Dictyoarthrinium musae MFLUCC 20-0105 Spegazzinia bromeliacearum URM 8084 Pleospora herbarum CBS 191.86 Spegazzinia tessarthra MFLUCC 18-1624 Dictyoarthrinium musae MFLUCC 20-0106 Spegazzinia intermedia CBS 249.89 Dictyoarthrinium hydei SQUCC 13296 Spegazzinia camelliae CMU 328 Spegazzinia menglaensis YMF 1.04800 Dictyoarthrinium sacchari CBS 529.73 Spegazzinia radermacherae MFLUCC 17-2285 Spegazzinia tessarthra SH 287 Dictyoarthrinium sacchari MFLUCC 20-0107 Stemphylium botryosum CBS 714.68 Spegazzinia juniperi CFCC 72647 Spegazzinia tessarthra IRAN 4932C Spegazzinia zhujiangyuanensis ZHKUCC 23-1020 Spegazzinia jinghaensis KUMCC 21-0496 Spegazzinia lobulata CBS 361.58 Dictyoarthrinium thailandicum KUMCC 21-0665 Dictyoarthrinium thailandicum KUMCC 21-0664 100/1 63/- 95/0.99 100/0.99 100/1 98/0.99 65/- 100/1 76/1 99/1 100/1 97/1 100/1 90/1 100/1 95/1 99/0.92 100/1 81/- 100/1 72/0.99 95/- Spegazzinia Dictyoarthrinium Outgroup x2 x2 286 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China known strains and formed a distinct clade with strong support values of 99/1 (ML/BI) (Fig. 3). This clade is clearly separated as a sister group to Spegazzinia tessarthra (support values ML/BI = 95/0.99) and shows close phylogenetic affinity to S. radermacherae (Fig. 3). Morphologically, S. juniperi differs from S. tessarthra and S. radermacherae in having granular, slightly moist sporodochia. The α-conidia of S. juniperi are larger than those of S. tessarthra (18.2– 28.3 × 15.8–24.3 µm vs. 15–20 × 14–18 µm), and its β-conidia are broader (13.4–17.8 µm vs. 8–12 µm) (Tennakoon et al. 2022). Compared to S. radermacherae, S. juniperi exhibits larger α-conidia (18.2–28.3 × 15.8–24.3 µm vs. 18–22 × 17.5–20 µm), broader β-conidia (13.4–17.8 µm vs. 8–10 µm), and longer spines (1.8–7.6 µm vs. 2–3 µm) (Jayasiri et al. 2019). Furthermore, this species could be differentiated from S. tessarthra (SH 287) at the ITS, LSU, SSU, and tef1-α loci with nucleotide differences of 2/355 bp in ITS, 7/890 bp in LSU, 12/925 bp in tef1, and 1/1008 bp in SSU. It was distinguishable from S. radermacherae (MFLUCC 17-2285) at the ITS and tef1-α loci, showing 4/355 bp differences in ITS and 73/925 bp in tef1-α. Therefore, based on phylogenetic and morphological data, S. juniperi collected from Juniperus chinensis is formally described as a new species. Sordariomycetes O.E. Erikss. & Winka Xylariales Nannf Apiosporaceae K.D. Hyde, J. Fröhl., Joanne E. Taylor & M.E. Barr Nigrospora Zimm Nigrospora platycladiensis Z.X. Bi & C.M. Tian, sp. nov. MycoBank No: 859531 Fig. 9 Etymology. Named after the host genus, Platycladus. Specimens examined. China • Beijing City, Changping District, Ming Tombs Reservoir, 40°14'57"N, 116°15'54"E, on the discolored scale leaves of Platycladus orientalis, 23 February 2025, Z.X. Bi, holotype BJFC-S2578, ex-type strain CFCC 72632. Description. Sexual morph: Not observed. Asexual morph: Hyphae Intertwined, hyaline to pale brownish, slightly thick-walled, smooth-surfaced, septate, branched, 1.6–5.1 µm in diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells Initially hyaline, becoming pale brown with age, solitary or aggregated in clusters, ampulliform to subcylindrical, 5.5–8.9 × 3.9– 6.9 µm (av. ± S.D. = 7.3 ± 0.9 × 5.3 ± 0.8; n = 30). Conidia mostly solitary and sparse, but capable of forming clusters under pine needle induction, initially hyaline, turning black to brown at maturity, smooth-walled, aseptate, subglobose, 10.4–17.5 × 9.7–17.3 µm (av. ± S.D.= 14.68 ± 1.75 × 13.6 ± 2.0; n = 50). Culture characteristics. When cultured on PDA at 25 °C under dark conditions for 7 days, the colony diameter reaches 60 mm. The colony appears fluffy with well-developed aerial hyphae. These hyphae later intertwine to form small aggregates. Initially white, the colony begins to produce light yellow hyphae after 10 days. The reverse side of the colony is pale brownish. After 20 days, brownish block-like spots start to develop on the reverse. By 30 days, deep black, irregular patches form near the bottom of the medium. 287 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China Figure 9. Nigrospora platycladiensis (CFCC 72632). A. Diseased scale leaves habit of Platycladus orientalis; B, C. Colony surface and reverse on PDA; D. Conidia; E–I. Conidiogenous cells; J, K. Hyphae growing at the bottom of PDA. Scale bars: 10 µm (D–K). 288 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China Notes. Phylogenetic analysis based on ITS, tub2, and tef1-α loci revealed that strains CFCC 72630 and CFCC 72632 (ex-type strain) formed a distinct clade with strong statistical support of 100/1 (ML/BI) and clustered as a sister clade to Nigrospora guangdongensis (ex-type strain CFCC 53917) with bootstrap support values of 98/1 (ML/BI) (Fig. 2). However, this species can be distinguished from N. guangdongensis by nucleotide differences at the ITS, tef1, and tub2 loci (1/534 bp with 4 gaps in ITS, 7/408 bp with 7 gaps in tub2, 36/495 bp with 10 gaps in tef1). Morphologically, the newly discovered species N. platycladiensis from Platycladus orientalis showed partial overlap in conidial size with its closely related species N. guangdongensis (10.4–17.5 μm vs. 13.6–20.9 μm). However, the average conidial length of N. platycladiensis was significantly smaller than that of N. guangdongensis (av. ± S.D. = 14.6 ± 1.7 μm vs. av. ± S.D. = 16.8 ± 1.9 μm). Additionally, the conidiogenous cells of N. platycladiensis were markedly narrower than those of N. guangdongensis (3.9–6.9 μm vs. 7.1– 9.9 μm) (Tian et al. 2020). Based on integrated phylogenetic and morphological data, Nigrospora platycladiensis is proposed as a novel species. Dothideomycetes O.E. Erikss. & Winka Botryosphaeriales C.L. Schoch, Crous & Shoemaker Aplosporellaceae Slippers, Boissin & Crous Aplosporella Speg Aplosporella was introduced by Spegazzini (1880), with Aplosporella chlorostroma designated as its type species. The type species defines the genus as characterized by multilocular conidiomata that produce brown, aseptate, verrucose conidia with filiform paraphyses (Damm et al. 2007). In this study, five fungal strains were isolated from dead twigs and healthy strobili of Platycladus orientalis, belonging to three species: A. hesperidica, A. javeedii, and A. prunicola. Aplosporella hesperidica Speg., Anal. Soc. cient. argent. 13(1): 18 (1882). Fig. 10 Description. Sexual form: Not observed. Asexual form: Fruiting bodies distributed on dead twigs of Platycladus orientalis, mostly breaking through the host epidermis, appearing brown-black or gray-black. Conidiomata pycnidial, immersed or semi-immersed, light brown, solitary, multiloculate, 205–588 µm diam., the outer wall composed of light brown textura angularis, gradually becoming lighter inward, with the inner region hyaline. Ostiole central, black or dark brown, 41.7–57.1 µm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells smooth, hyaline, nearly cylindrical, thin-walled, 5.8–11.9 × 1.8–3.5 µm (av. ± S.D. = 8.4 ± 2.1 × 2.4 ± 0.5). Paraphyses long-cylindrical, 31.4–87.1 × 1.6–4.9 µm, hyaline, thin-walled, smooth, occasionally branched at the base. Conidia initially hyaline with a truncate base, turning brown or black at maturity, aseptate, subellipsoid or broadly ellipsoid, 14.1–22.2 × 8.1–15.6 µm (av. ± S.D. = 16.7 ± 1.8 × 10.8 ± 1.4). Culture characteristics. On PDA at 25 °C under dark conditions for approximately 7 days, colonies reach a diameter of 60 mm. Initially white, the colonies exhibit radial growth patterns. The aerial mycelium appears appressed to floccose, ranging in color from white to smoke-grey. Mycelial density shows 289 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China regional variation—being relatively sparse near the central region while becoming more densely distributed towards the marginal zone. Specimens examined. China • Beijing City, Changping District, Ming Tombs Reservoir, 40°14'52"N, 116°15'30"E, on the dead branches of Platycladus orientalis, 2 October 2024, Z.X. Bi, BJFC-S2566, living culture CFCC 72635. Notes. Aplosporella hesperidica was first discovered on Citrus × aurantium in Argentina (Spegazzini 1882). Subsequently, Dissanayake et al. (2021) reported its first occurrence in China, followed by Lin et al. (2023b) detecting this fungal species on Euonymus japonicus. Additionally, A. hesperidica has been found to cause stem rot in cowpea in India (Deepika et al. 2020). Comprehensive phylogenetic and morphological analyses identified the fungal strain CFCC 72635 as A. hesperidica. This is the first report of A. hesperidica on Platycladus orientalis. Figure 10. Aplosporella hesperidica (CFCC 72635). A. Conidiomata on a dead twig of Platycladus orientalis; B. Cransverse section of conidioma; C. Longitudinal section of conidioma; D, E. Colony morphology on PDA front and reverse views; F. Conidia; G. Pycnidia; H–K. Conidiogenous cells and paraphyses. Scale bars: 200 µm (B, C); 100 µm (G); 20 µm (I–K); 10 µm (H). 290 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China Aplosporella javeedii Jami, Gryzenh., Slippers & M.J. Wingf., Fungal Biology 118(2): 174 (2013) Fig. 11 Description. Sexual form: Not observed. Asexual form: Sporulation began after 2 weeks of cultivation on PDA medium. Conidiomata pycnidial, immersed to semi-immersed, grey-olivaceous, solitary, subglobose, 529–883 µm diam., pycnidial wall consists of dark brown textura angularis in the outer layers, gradually becoming paler in coloration towards the interior, with the innermost layers thinning and becoming hyaline and transparent. Conidiophores reduced to conidiogenous cells. Conidiogenous cells smooth, hyaline, elongate-ellipsoidal, thinwalled, gradually tapering toward the apex, 9.3–18.0 × 2.0–6.8 μm (av. ± S.D. = 12.2 ± 3.0 × 3.6 ± 1.1). Paraphyses long-cylindrical, 22.9–37.2 × 2.0–5.0 µm, hyaline, thin-walled, smooth, occasionally branched. Conidia initially hyaline, gradually turning pale brown to yellowish-brown, and finally dark brown at maturity, aseptate, ellipsoidal, 18.3–22.2 × 6.8–9.0 µm (av. ± S.D. = 19.9 ± 1.2 × 7.9 ± 0.5). Cultural characteristics. On PDA at 25 °C under dark conditions, the colony reached approximately 60 mm in diameter after 7 days of incubation. The aerial mycelium was well-developed, appearing floccose and whitish-gray with sparse Figure 11. Aplosporella javeedii (CFCC 72643). A. Healthy strobili of Platycladus orientalis; B, C. Colony morphology on PDA front and reverse views; D. The conidiomata on PDA; E, F. Conidiogenous cells and paraphyses; G, H. Conidiogenous cells and conidia. Scale bars: 200 µm (D); 10 µm (E–H). 291 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China growth in the central region and denser growth at the periphery. After 20 days, the colony developed an olivaceous coloration, with abundant grayish-green aerial mycelium particularly concentrated near the marginal zone. Specimens examined. China • Beijing City, Changping District, Dingling, Ming Tombs Scenic Area, 40°17'23"N, 116°14'8"E, on the dead branches of Platycladus orientalis, 21 September 2024, Z.X. Bi, BJFC-S2567, living culture CFCC 72633; China • Beijing City, Changping District, Dingling, Ming Tombs Scenic Area, 40°17'28"N, 116°14'31"E, on the healthy strobili of P. orientalis, 31 March 2025, Z.X. Bi, BJFC-S2568, living culture CFCC 72643. Notes. Aplosporella javeedii was first described by Jami et al. (2013) and isolated from healthy branches of Celtis africana and Searsia lancea. Fan et al. (2015) subsequently reported its first occurrence in China, where it was isolated from five host plants, including Juniperus chinensis, exhibiting stem canker symptoms. Additionally, A. javeedii has been identified as the causal agent of mulberry (Morus alba) branch blight disease (Jia et al. 2019). According to literature records, this fungal species has now been documented across more than 10 plant families (Fan et al. 2015; Zhu et al. 2018; Pan et al. 2019; Lin et al. 2023b; Wu et al. 2024). Based on comprehensive phylogenetic and morphological analyses, strains CFCC 72633 and CFCC 72643 were identified as A. javeedii. Aplosporella prunicola Damm & Crous, Fungal Diversity 27: 39 (2007). Fig. 12 Description. Sexual form: Not observed. Asexual form: Fruiting bodies densely distributed on dead twigs of Platycladus orientalis, mostly immersed in the host epidermis. Conidiomata pycnidial, immersed, multilocular, solitary, 406– 651 μm diam., pycnidial wall composed of dark brown textura angularis in the outer layers, gradually becoming paler toward the interior, with the innermost region thin-walled and hyaline. Ostiole central, 67–122 μm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells cylindrical, smooth, hyaline, 6.7–14.6 × 2.1–5.9 μm (av. ± S.D. = 10.5 ± 2.5 × 3.7 ± 1.1). Paraphyses long-cylindrical, occasionally swollen at the apex, 30.7–92.5 × 1.1–7.0 μm, septate, hyaline, smooth-walled, and branched. Conidia 16.1–23.0 × 8.3– 14.2 μm (av. ± S.D. = 19.8 ± 1.6 × 11.1 ± 1.3), initially hyaline, gradually turning yellowish-brown, and finally dark brown at maturity, aseptate, smooth-walled. Cultural characteristics. On PDA at 25 °C under dark conditions, the colony reached approximately 60 mm in diameter after 4 days of incubation, exhibiting abundant floccose aerial mycelium. After 10 days, the colony developed pale grayish-green pigmentation, later transitioning to whitish-gray and ultimately olivaceous. Sporulation commenced after 2 weeks, forming subglobose pycnidia. Specimens examined. China • Beijing City, Changping District, Longshan SubFarm, Ming Tombs, 40°14'25"N, 116°13'17"E, on the dead branches of Platycladus orientalis, 18 July 2024, Z.X. Bi & C.M. Tian, BJFC-S2569, living culture CFCC 72634; China • Beijing City, Changping District, Dayu Mountain Scenic Area, Ming Tombs, 40°18'32"N, 116°11'47"E, on the dead branches of P. orientalis, 23 October 2024, Z.X. Bi & M.H. Wang, BJFC-S2570, living culture CFCC 72640. Notes. Aplosporella prunicola was first isolated by Damm et al. (2007) from Prunus persica var. nucipersica in South Africa. In China, A. prunicola has been 292 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China recorded on Castanea mollissima, Euonymus japonicus, and Zanthoxylum bungeanum (Jiang 2021; Li et al. 2023; Lin et al. 2023b). Based on comprehensive phylogenetic and morphological analyses, strains CFCC 72634 and CFCC 72640 were identified as A. prunicola. Dothideomycetes O.E. Erikss. & Winka Botryosphaeriales C.L. Schoch, Crous & Shoemaker Botryosphaeriaceae Theiss. & Syd Neofusicoccum Crous, Slippers & A.J.L. Phillips Neofusicoccum was established by Crous et al. (2006), with Neofusicoccum parvum designated as its type species. Members of this genus commonly exist as endophytes, saprobes, or latent pathogens across diverse host plants, being particularly notorious for causing dieback and canker diseases in woody hosts (Crous et al. 2006; Brewer et al. 2021; Guo 2023). These fungi predominantly reproduce asexually, with their sexual morph being relatively uncommon (Pennycook and Samuels 1985; Lopes et al. 2017). The genus exhibits a cosmopolitan distribution with a broad host range (Crous et al. 2006; Brewer et al. 2021). In the present study, two fungal strains isolated from withered branches of Platycladus orientalis were identified as N. occulatum. According to previous reports, this species of fungi is the pathogen causing canker and branch blight of cypress (Liu et al. 2022; Guo 2023). Figure 12. Aplosporella prunicola (CFCC 72634). A. Conidiomata on a dead twig of Platycladus orientalis; B. Transverse section of conidioma; C. Longitudinal section of conidioma; D, E. Colony morphology on PDA front and reverse views; G. Pycnidia; H. Conidia; I, J. Conidiogenous cells and paraphyses. Scale bars: 200 µm (B, C); 100 µm (G); 10 µm (H–J). 293 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China Neofusicoccum occulatum Sakalidis & T.Burgess, Molecular Phylogenetics and Evolution 60(3): 340(2011) Fig. 13 Description. Sexual morph: Not observed. Asexual morph: Fruiting bodies densely distributed on dead twigs of Platycladus orientalis. Conidiomata pycnidial immersed in bark surface, aggregated, unilocular or multilocular, subglobose, black, 58–194 µm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells thin-walled, hyaline, ovoid to cylindrical, 6.1–19.5 × 1.0–4.3 µm (av. ± S.D. = 13.3 ± 3.7 × 2.8 ± 0.9). Conidia unicellular, hyaline, fusiform to subellipsoid, containing granular inclusions, occasionally with 1–2 oil droplets, 14.0–22.9 × 4.3–8.1 µm (av. ± S.D. = 19.8 ± 1.9 × 6.1 ± 0.7). Cultural characteristics. On PDA at 25 °C under dark conditions, colonies reached approximately 60 mm in diameter after 7 days of incubation, exhibiting dense, floccose mycelium. After 10 days, the aerial hyphae developed a smokegray coloration, while the reverse side of colonies turned grayish-brown. With Figure 13. Neofusicoccum occulatum (CFCC 72629). A. Conidiomata on withered twigs of Platycladus orientalis; B. Colony morphology on PDA front views; C. The conidiomata on PDA; D, E. Pycnidia; F–H. Conidiogenous cells; I. Conidia. Scale bars: 100 µm (C); 10 µm (D–I). 294 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China prolonged cultivation, the mycelium darkened to blackish-brown, accompanied by black pigmentation on the colony reverse. At approximately 20 days, grayish-white to smoke-black pycnidia formed on the medium, often embedded in mycelial mats and appearing as irregular masses or subglobose structures. At maturity, these pycnidia produced pale yellow conidial masses. Specimens examined. China • Beijing City, Changping District, Mangshan National Forest Park, Ming Tombs, 40°16'5"N, 116°16'51"E, on the diseased branches of Platycladus orientalis, 23 November 2024, Z.X. Bi & W.K. Gao, BJFC-S2579, living culture CFCC 72629; China • Beijing City, Changping District, Mangshan National Forest Park, Ming Tombs, 40°16'5"N, 116°16'57"E, on the dead branches of P. orientalis, 23 November 2024, Z.X. Bi & W.K. Gao, BJFC-S2580, living culture CFCC 72636. Notes. Neofusicoccum occulatum was introduced by Sakalidis et al. (2011) and was isolated from Eucalyptus spp. and Wollemia nobilis in Australia. Previous studies have demonstrated that this fungus is a pathogen causing canker and shoot blight in Platycladus orientalis (Liu et al. 2022; Guo 2023). It has also been recorded on host plants such as Prunus persica and Dendrobium chrysanthum (Ma et al. 2021; Zhou et al. 2024). Based on comprehensive phylogenetic and morphological analyses, strains CFCC 72629 and CFCC 72636 were identified as Neofusicoccum occulatum. Sordariomycetes O.E. Erikss. & Winka Xylariales Nannf Apiosporaceae K.D. Hyde, J. Fröhl., Joanne E. Taylor & M.E. Barr Nigrospora Zimm Nigrospora oryzae (Berk. & Broome) Petch, J. Indian Bot. Soc. 4: 24 (1924) Fig. 14 Description. Sexual morph: Not observed. Asexual morph: Hyphae interwoven, initially hyaline, becoming brownish with age, septate, frequently branched, 2.4–6.7 µm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells predominantly clustered but occasionally solitary on hyphae, hyaline, ampulliform to subglobose, 3.1–13.8 × 3.4–7.4 µm (av. ± S.D. = 7.3 ± 2.2 × 5.5 ± 1.0). Conidia typically aggregated in slimy masses, initially white and hyaline, gradually turning pale brown, and finally black at maturity, smooth-walled, aseptate, globose to subellipsoid, 11.7–14.8 × 10.2–13.9 µm (av. ± S.D. = 12.8 ± 0.6 × 11.9 ± 0.9). Cultural characteristics. On PDA medium, colonies initially appeared white and cottony. After 7 days of incubation, the mycelium developed a smoke-gray coloration, with denser growth and darker pigmentation in the central region compared to the margins. By day 20, the colonies turned grayish-black throughout. Specimens examined. China • Beijing City, Changping District, Dayu Mountain Scenic Area, Ming Tombs, 40°18'20"N, 116°12'4"E, on the diseased scale leaves with lesions of Platycladus orientalis, 23 October 2024, Z.X. Bi & M.H. W, BJFC-S2574, living culture CFCC 72644. China • Beijing City, Changping District, Changling Scenic Area, Ming Tombs, 40°17'41"N, 116°14'24"E, on the withered leaf tips of Juniperus procumbens, 23 October 2024, Z.X. Bi, BJFC-S2575, living culture CFCC 72638. 295 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China Notes. Nigrospora oryzae is recognized as both an endophyte and a pathogen causing leaf spot disease on rice (Oryza sativa); it commonly colonizes diverse plants and plant debris in dual roles as a pathogen and endophyte (Wang et al. 2017; Liu et al. 2021; Liu et al. 2024). In this study, two fungal strains, CFCC 72638 and CFCC 72644, were isolated from Platycladus orientalis and Juniperus procumbens. Based on comprehensive phylogenetic and morphological analyses, strains CFCC 72644 and CFCC 72638 were identified as N. oryzae. Nigrospora osmanthi Mei Wang & L. Cai, Persoonia 39: 135 (2017) Fig. 15 Description. Sexual morph: Not observed. Asexual morph: Hyphae interwoven, initially hyaline, becoming pale brown to yellowish-brown with age, thickwalled, septate, frequently branched, 2.0–5.1 µm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells solitary on hyphae, smooth-walled, Figure 14. Nigrospora oryzae (CFCC 72638, CFCC 72644). A, B. Diseased scale leaves of Platycladus orientalis; C. Withered leaf tips of Juniperus procumbens; D, E. Colony surface and reverse on PDA; F, G. Conidia; H, I. Conidiogenous cells. Scale bars: 10 µm (F–I). 302 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China 2016). A. aureus is an endophyte widely associated with various plants (Zimowska and Nicoletti 2023) and also acts as a pathogenic fungus. Reported infections caused by A. aureus include leaf black spot disease in Pseudostellaria heterophylla (Yuan et al. 2021), leaf spot disease in Cucumis melo (Wei et al. 2024), and gray spot disease in tobacco (Yang et al. 2024). Comprehensive phylogenetic and morphological analyses identified the fungal strain CFCC 72639 as A. aureus. Chaetomium globosum Kunze, Mykol. Hefte 1: 16 (1817). Fig. 19 Description. Sexual morph: Ascomata densely distributed on the surface of PDA medium, initially pale yellow, maturing to yellowish-black after 2 weeks, superficial on the medium, globose to ovate, with an apical ostiole, 158–269 × 136–186 µm, surrounded by ascomatal hairs, the ascomatal wall is brownish and composed of textura intricate. Terminal hairs initially pale yellow, turning brownish-yellow with age, base dark brown, apex pale yellowish-brown, sinuous, septate, unbranched, 146–468 µm long, 1.7–3.9 µm wide at the base. Asci fasciculate, clavate, stipitate, hyaline, 8-spored, evanescent, 25.6–47.2 × 10.3–17.9 µm (av. ± S.D. = 37.5 ± 5.4 × 14.1 ± 2.1). Ascospores ovoid, hyaline when immature, becoming brown at maturity, 8.5–10.7 × 6.4–8.5 µm (av. ± S.D. = 9.6 ± 0.5 × 7.5 ± 0.5). Asexual morph: Not observed. Cultural characteristics. Initially, colonies on PDA medium appeared white. After approximately 7 days, they turned pale yellow and began producing golden-brown ascomata from the center. Within 10 days, the ascomata densely covered the entire medium surface. By 14 days, pale orange-yellow exudates were observed. Upon maturation, ascospores were released through the apical ostioles. Specimens examined. China • Beijing City, Changping District, Ming Tombs Reservoir, 40°14'47"N, 116°15'54"E”, on the diseased scale leaves of Platycladus orientalis, 23 February 2025, Z.X. Bi, BJFC-S2572, living cultures CFCC 72642; China • Beijing City, Changping District, Mangshan National Forest Park, Ming Tombs, 40°15'36"N, 116°16'40"E, on the diseased scale leaves of P. orientalis, 23 November 2024, Z.X. Bi &W.K. Gao, BJFC-S2573, living culture CFCC 72645. Notes. Chaetomium was introduced by Kunze, with Chaetomium globosum designated as the type species (Kunze and Schmidt 1817). C. globosum is a widely distributed endophytic fungus, recorded on numerous plants including Actinidia chinensis, Artemisia argyi, Descurainia sophia, Glycine max, Juncus sp., Oryza sativa, Platycladus orientalis, and Solanum lycopersicum (Guo 2012; Wang et al. 2016). As a significant resource fungus, it exhibits critical biological functions such as antimicrobial activity, biocontrol potential, and plant growth promotion (Ye et al. 2013; Zhao et al. 2017; Tian et al. 2022). In this study, two fungal strains isolated from diseased scale leaves of P. orientalis were analyzed. Based on comprehensive phylogenetic and morphological analyses, strains CFCC 72642 and CFCC 72645 were identified as C. globosum. 303 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China Sordariomycetes O.E. Erikss. & Winka Xylariales Nannf Sporocadaceae Corda Seiridium Nees The genus Seiridium was introduced by Nees (1817), with Seiridium marginatum designated as the type species (Bonthond et al. 2018; Li et al. 2022). Seiridium species primarily exist as phytopathogens, widely distributed globally Figure 19. Chaetomium globosum (CFCC 72642). A. Diseased scale leaves habit of Platycladus orientalis; B, C. Colony surface and reverse on PDA; D–F. Ascomata on PDA medium; G–I. Ascomata; J, K. Asci; L. Ascospores. Scale bars: 200 µm (D–F); 10 µm (G–L). 304 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China and causing significant economic losses, particularly through infections of Cupressaceae plants. Seiridium cardinale, S. cupressi, and S. unicorne are recognized as the most dangerous parasitic fungi for Cupressaceae, identified as the primary pathogens responsible for cypress canker pandemics (Boesewinkel 1983; Graniti 1986, 1998; Bonthond et al. 2018). In this study, one fungal strain isolated from cankered twigs of Platycladus orientalis in the Ming Tombs area was identified as S. unicorne. Seiridium unicorne (Cooke & Ellis) B. Sutton, Mycol. Pap. 138: 74 (1975) Fig. 20 Description. Sexual morph: Not observed. Asexual morph: Fruiting bodies scattered on the surface of Platycladus orientalis branches, carbon-black to jet-black; Conidiomata acervular, immersed to erumpent through bark tissue, black, subglobose, scattered, unilocular; wall brownish, 65–255 µm diam. Conidiophores long-cylindrical, hyaline, thin-walled, septate, occasionally branched, 16.3–51.4 × 1.0–2.6 µm; Conidiogenous cells hyaline, thin-walled, smooth, cylindrical, solitary, 5.9–17.5 × 1.1–4.2 µm (av. ± S.D. = 10.8 ± 3.2 × 2.0 ± 0.6). Conidia falcate to lunate, hyaline when immature, becoming pale brown to yellowish-brown at maturity, 5-septate, curved, with one hyaline apical appendage and one basal appendage, total conidial dimensions 19.4–29.8 × 6.2–11.9 µm (av. ± S.D. = 24.5 ± 0.4 × 9.6 ± 1.2), basal cell obconical, hyaline to pale brown, truncate, 2.5–7.1 µm long, the first cell from the basal cell upwards is 3.5–7.1 µm long, the second cell is 3.2–6.7 µm long, the third cell 3.1–6.0 µm long, the fourth cell 3.6–6.9 µm long, the apical cell conical, smooth, and hyaline, with a length of 1.6–5.7 µm. Appendages cylindrical, the apical appendages are mostly centric, 4.0–8.9 µm long, the basal appendages are mostly eccentric, 4.0–8.6 µm long. Cultural characteristics. On PDA medium, colonies exhibited appressed growth with a sparse, felt-like texture and slow expansion rates, reaching approximately 30 mm in diameter after 7 days of incubation. Aerial mycelium was poorly developed and diffuse. After 2 weeks, a pale yellow pigmentation became visible on the colony reverse. Specimens examined. China • Beijing City, Changping District, Ming Tombs Longshan Sub-farm, 40°14'21"N, 116°13'15"E, on the dead branches of Platycladus orientalis, 18 July 2024, Z.X. Bi & C.M. Tian. BJFC-S2581, living culture CFCC 72631. Notes. The genus Seiridium can be distinguished from other genera by its conidia with five septa (Li et al. 2022). Seiridium unicorne has been documented to infect hosts across diverse plant families, including Anacardiaceae, Caprifoliaceae, Cornaceae, Cupressaceae, Hamamelidaceae, Rosaceae, and Vitaceae (Guba 1961; Boesewinkel 1983; Cho and Shin 2004; Bonthond et al. 2018). Phylogenetic analysis revealed that the studied strains cluster within the same clade as reference strains of S. unicorne with a high support value of 100/1 (ML/BI) (Fig. 7). In terms of morphology, the maximum lengths of the basal cells (2.5–7.1 µm vs. 3–5.5 μm) and the first cell counted upwards from the basal cell (3.5–7.1 µm vs. 3.5–5.5 μm) in the conidia of the strains in this study are slightly larger than those of the reference species S. unicorne (Bonthond et al. 2018). However, the differences are not significant, and the remaining 305 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China morphological characteristics are basically consistent with the previous descriptions of this species. Therefore, based on the above evidence, we identified this strain as S. unicorne. Discussion This study isolated 22 fungal strains from diseased leaves and twigs, as well as healthy strobili and mature cones of cypress (Juniperus chinensis, J. procumbens, and Platycladus orientalis) in the Ming Tombs area of Beijing. Identification revealed that these isolates belong to 13 species across 8 fungal genera, including Achaetomium globosum, Aplosporella hesperidica, A. javeedii, A. prunicola, Arcopilus aureus, Chaetomium globosum, Neofusicoccum occulatum, Nigrospora oryzae, N. osmanthi, N. philosophiae-doctoris, N. platycladiensis, Seiridium unicorne, and Spegazzinia juniperi. Among these, N. platycladiensis and S. juniperi are described as novel species. A. hesperidica was recorded for the first time on P. orientalis. N. philosophiae-doctoris represents the first record on both J. chinensis and P. orientalis. In this study, a total of 12 fungal species were isolated from P. orientalis, Figure 20. Seiridium unicorne (CFCC 72631). A. Conidiomata on a diseased branch habit of Platycladus orientalis; B, C. Colony surface and reverse on PDA; D. Conidiomata and conidia; E, F. Conidiogenous cells; G. Conidia. Scale bars: 10 µm (D–G). 306 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China 3 species were obtained from J. chinensis, and 1 was isolated from J. procumbens. Furthermore, 12 ascomycete species were isolated from diseased cypress leaves and branches, whereas 4 fungal species were obtained from healthy tissues. Aplosporella is primarily characterized by the formation of multilocular pycnidia (multi-chambered fruiting bodies), producing brown, aseptate, verruculose conidia, and the presence of filiform paraphyses (Sutton 1980; Damm et al. 2007). In this study, 3 Aplosporella species were isolated from P. orientalis, namely A. hesperidica, A. javeedii, and A. prunicola. A. javeedii was found not only in withered branches but also within healthy strobili tissues of the host. According to the literature, A. javeedii exhibits the broadest host range within the genus, having been reported on hosts spanning more than 10 plant families (Fan et al. 2015; Zhu et al. 2018; Pan et al. 2019; Lin et al. 2023b; Wu et al. 2024). Significantly, although specimens from 3 different cypress species were collected, Aplosporella isolates were obtained exclusively from P. orientalis. This phenomenon warrants further investigation and provides new research directions for exploring the distribution patterns of this fungal genus within cypress hosts. This study isolated 3 species belonging to the family Chaetomiaceae from diseased scale-like leaves of P. orientalis: Arcopilus aureus, Achaetomium globosum, and Chaetomium globosum. Chaetomiaceae species exhibit remarkable phenotypic and ecological diversity and hold significant value in medical and economic contexts, representing important resource fungi (Wang et al. 2022). The family is distributed globally, existing primarily as saprophytes, endophytes, and pathogens in natural environments (Wang et al. 2022; Al-Rifaie and Ameen 2023; Condé et al. 2023). Studies indicate that A. aureus has a wide geographical distribution. It frequently exists as an endophyte in symbiotic relationships with host plants, demonstrating high adaptability to its ecological niche (Zimowska and Nicoletti 2023). Reports also suggest that this fungus possesses potential pathogenicity, capable of causing black spot, leaf spot, and grey spot diseases in plants (Yuan et al. 2021; Wei et al. 2024; Yang et al. 2024). In this study, A. aureus, A. globosum, and C. globosum were isolated from diseased scale-like leaves of P. orientalis. Whether these fungi can induce disease necessitates experimental confirmation through subsequent pathogenicity assays. Nigrospora is not only an endophyte widely present in various host plants but also a potential pathogen on many plants in different regions (Wang et al. 2016; Liu et al. 2024). In this study, 3 previously reported species of this genus (N. oryzae, N. osmanthi, and N. philosophiae-doctoris), as well as one new species, Nigrospora platycladiensis, were isolated from cypress. Among these, N. osmanthi and N. philosophiae-doctoris were isolated from both diseased leaves and healthy strobili of cypress. N. oryzae and N. platycladiensis were isolated from diseased leaves. Notably, N. oryzae was isolated from diseased parts of both P. orientalis and J. procumbens, indicating its potential broad host range. Early taxonomic studies of Nigrospora primarily relied on morphological characteristics for species delimitation (Mason 1927, 1933; Wang et al. 2017). However, research by Wang et al. (2017) demonstrated that although some species within Nigrospora exhibit extremely similar morphology, they belong to distinct phylogenetic clades, often showing overlapping conidial size ranges. Although the newly described species N. platycladiensis sp. nov. exhibits partially overlapping morphological characteristics with its closely related species N. guangdongensis, the two species show clear distinctions in their 307 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China geographical distribution and host origins: N. platycladiensis was isolated from P. orientalis in Beijing, China, whereas N. guangdongensis was collected from Cunninghamia lanceolata in Hebei, China (Tian et al. 2020). Therefore, the identification of Nigrospora cannot rely solely on morphological features and requires an integrated approach combining both morphological and phylogenetic analyses for proper species classification and delimitation (Wang et al. 2017). Neofusicoccum occulatum and Seiridium unicorne, identified as pathogens causing twig blight and canker diseases in cypress trees, have been confirmed to be closely associated with cypress diseases (Liu et al. 2022; Guo 2023). In this study, both of these fungal species were isolated from withered branches of P. orientalis, providing new corroborating evidence for previous research findings. N. occulatum is known to infect various Cupressaceae plants and has been reported on species such as Chamaecyparis lawsoniana, Cupressus funebris, Juniperus communis, and Thujopsis dolabrata (Zlatkovic 2016; Li et al. 2022). S. cardinale, S. cupressi, and S. unicorne are considered the most dangerous parasitic fungi for Cupressaceae plants (Bonthond et al. 2018). However, only S. unicorne was isolated in this study. This discrepancy might be attributed to factors such as host plant species, geographical location, and the scale of sampling. According to Guo (2023), reports on Seiridium fungi in China are relatively scarce. To date, no studies have been identified that investigate the presence of Seiridium on the branches and leaves of Cupressaceae plants in the Ming Tombs area of Beijing. Species of Spegazzinia exhibit an extremely wide geographical distribution across various ecosystems (Dai et al. 2025). They primarily exist as endophytes within host organisms or as saprobes on decaying plant debris (Hashemlou et al. 2023; Dai et al. 2025). Spegazzinia has been recorded on multiple host plants, including Brachypodium sp., Musa sp., Radermachera sinica, and Saccharum officinarum, and has also been reported in air samples (Mena-Portales et al. 2017; Jayasiri et al. 2019; Samarakoon et al. 2020; Hashemlou et al. 2023; Dai et al. 2025). In this study, a new species of this genus, Spegazzinia juniperi, was isolated from healthy cones of J. chinensis. Phylogenetically, S. juniperi formed a distinct branch (Fig. 3). Morphologically, it can be distinguished from other species by its granular, slightly moist sporodochia and characteristic conidial dimensions. A preliminary investigation into the diversity of ascomycetes on cypress trees in the Ming Tombs area of Beijing revealed that the ascomycete species in this region possess a certain degree of richness. However, the number of specimens collected in this study is limited. In the future, it will be necessary to expand the scale of specimen collection to more fully verify the existing research results and explore additional ascomycete groups on cypress. 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. 308 MycoKeys 123: 271–317 (2025), DOI: 10.3897/mycokeys.123.165848 Zixian Bi et al.: New species and records of ascomycetes on cypress in Beijing, China Funding This study was financed by the National Natural Science Foundation of China (Project No.: 32371887). Author contributions Conceptualization, Zixian Bi and Chengming Tian; data curation, Zixian Bi; funding acquisition, Chengming Tian; investigation, Zixian Bi and Chengming Tian; project administration, Chengming Tian; resources, Zixian Bi; supervision, Chengming Tian; writing—original draft, Zixian Bi; writing—review and editing, Zixian Bi, Yingying Wu, Shuji Li, and Chengming Tian. All authors have read and agreed to the published version of the manuscript. Author ORCIDs Zixian Bi https://orcid.org/0009-0008-1186-8995 Yingying Wu https://orcid.org/0009-0007-5095-2738 Chengming Tian https://orcid.org/0000-0002-3352-7664 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. References AL-Rifaie AA, Ameen MKM (2023) New endophytic fungal species of Chaetomiaceae (Ascomycota) in Iraq. 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