The addition of six novel species and a new record of Amphisphaeria from northern Thailand

1 The addition of six novel species and a new record of Amphisphaeria from northern Thailand Zaw Lin Tun1,2,3,4 , Digvijayini Bundhun2,4 , Chitrabhanu S. Bhunjun2,4 , Sajeewa S. N. Maharachchikumbura5,6 , Vinodhini Thiyagaraja1, Fatimah Al-Otibi7, Kevin D. Hyde1,4,7,8 1 CAS Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Science, Kunming, Yunnan 650201, China 2 School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand 3 Mushroom Research Foundation, 128 M.3 Ban Pa Deng T. Pa Pae, A. Mae Taeng, Chiang Mai 50150, Thailand 4 Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand 5 School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 611731, China 6 Department of Biosystems Technological Studies, Faculty of Technological Studies, Uva Wellassa University, Badulla, Sri Lanka 7 Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 22452, Riyadh 11495, Saudi Arabia 8 Department of Plant Pathology, College of Agriculture, Guizhou University, Guiyang Guizhou 550025, China Corresponding author: Kevin D. Hyde ([email protected]) Copyright: © Zaw Lin Tun 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 This study introduces six new Amphisphaeria species, discovered on dead twigs belonging to Calophyllaceae, Fabaceae, Sapotaceae, and Theaceae in northern Thailand, based on a morpho-phylogenetic approach. Our newly introduced Amphisphaeria species share morphological traits with other members of the genus, featuring ascomata with a two-layered peridium and unitunicate asci with either J+ or Japical ring. The newly identified species Amphisphaeria mesuae, A. mimusopis, A. paraserianthis, A. pseudomicheliae, A. pterocarpi, and A. schimae differ from previously known Amphisphaeria species in Amphisphaeriaceae. This distinction is supported by combined analyses using maximum likelihood and Bayesian inference of nuclear ribosomal large subunit rDNA (LSU) and the internal transcribed spacer (ITS) sequence matrix. Amphisphaeria mesuae can be distinguished from A. ailaoshanensis by the presence of larger ascomata, smaller asci, ellipsoidal ascospores, and the absence of a sheath surrounding the ascospores. Amphisphaeria mimusopis differs from its phylogenetically related sister taxon by possessing smaller asci, larger ascospores, and narrower paraphyses. Amphisphaeria paraserianthis differs from closely related taxa in its ostiolate ascomata and 3-septate ascospores. Amphisphaeria pterocarpi can be distinguished from the taxon with which it clusters by having smaller ascomata and ascospores. Amphisphaeria pseudomicheliae has larger ascomata and asci, but smaller ascospores, compared to its sister taxon, A. micheliae. Amphisphaeria schimae differs from closely related taxa in its larger ascomata, asci, and ascospores. Amphisphaeria micheliae was also collected here and is reported as a new record on Senna siamea. This study contributes to the expansion of the taxonomic framework of Amphisphaeria. Key words: 6 novel species, inconspicuous taxa, Sordariomycetes, woody twigs Academic editor: Samantha C. Karunarathna Received: 28 June 2025 Accepted: 15 August 2025 Published: 14 November 2025 Citation: Tun ZL, Bundhun D, Bhunjun CS, Maharachchikumbura SSN, Thiyagaraja V, Al-Otibi F, Hyde KD (2025) The addition of six novel species and a new record of Amphisphaeria from northern Thailand. MycoKeys 125: 1–31. https://doi.org/10.3897/ mycokeys.125.163523 MycoKeys 125: 1–31 (2025) DOI: 10.3897/mycokeys.125.163523 This article is part of: Exploring the Hidden Fungal Diversity: Biodiversity, Taxonomy, and Phylogeny of Saprobic Fungi Edited by Edited by Samantha C. Karunarathna, Danushka Sandaruwan Tennakoon, Ajay Kumar Gautam 2 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand Introduction Amphisphaeriales was described by Eriksson and Hawksworth in 1986 and is phylogenetically closely related to Xylariales within the Xylariomycetidae group (Senanayake et al. 2015; Samarakoon et al. 2022). The type genus Amphisphaeria was established by Cesati et al. in 1863 for the family Amphisphaeriaceae, and two additional genera, Griphosphaerioma and Lepteutypa, were later added to the family (Wijayawardene et al. 2018). However, Lepteutypa was ultimately synonymized with Amphisphaeria based on holomorphic morphology and multigene phylogeny by Samarakoon et al. (2020), while Griphosphaerioma was regarded as a synonym of Labridella by Rossman et al. (2016). Currently, Amphisphaeriaceae includes Amphisphaeria and Labridella (Hyde et al. 2024a). Amphisphaeria, typified by A. umbrina, generally comprises immersed, clypeate, and ostiolate ascomata that appear as elevated, blackened, round spots on the host surface (Samarakoon et al. 2020). The peridium layers comprise a hyaline inner layer and a brown outer layer of cells. Paraphyses are filamentous, septate, and flexuous (Wang et al. 2004). The cylindrical asci are 8-spored, with J+ or J-, discoid, tubular, or wedge-shaped apical rings, and the ascospores are 1–3 septate, ellipsoidal, and brown (Cesati and De 1863; Wang et al. 2004; Samarakoon et al. 2019, 2020, Samarakoon 2023). Both coelomycetous and hyphomycetous asexual morphs are reported in Amphisphaeria (Samarakoon et al. 2020). Saprobic Amphisphaeria species have been reported from woody branches and various monocotyledon hosts, including grasses from different geographical regions (Samarakoon et al. 2019). Members of Amphisphaeria are predominantly found on dead plant materials in both terrestrial and marine habitats (Senanayake et al. 2015, 2019; Samarakoon et al. 2019; Sun et al. 2025). Given the widespread nature and diverse lifestyles of Amphisphaeria species, further taxonomic studies are essential. In this study, we examine the taxonomy, lifestyle, ecological roles, and distribution of Amphisphaeria species in northern Thailand. We introduce six species of Amphisphaeria, along with a new host record. These taxa were isolated from dead twigs of Mesua sp., Mimusops elengi, Paraserianthes lophantha, Pterocarpus sp., Schima wallichii, Senna siamea, and an undetermined host, from two provinces in northern Thailand. Morphological illustrations of the taxa are provided. Phylogenetic studies incorporating combined nuclear ribosomal large subunit rDNA (LSU) and the internal transcribed spacer (ITS) region confirm the taxonomic placements of these species as novel within Amphisphaeria and also support the finding of a new host record of an existing Amphisphaeria taxon. Materials and methods Sample collection, isolation, and morphology Dead twigs were collected from the premises of the Mushroom Research Center (MRC) and Mae Fah Luang University during the cold (October, November) and wet (July) seasons in northern Thailand. After the collection details were recorded (Rathnayaka et al. 2025), specimens were brought to the laboratory in plastic bags and stored in a paper envelope. A Motic SMZ 168 Series stereo microscope (Leica Microsystems Company, Germany) was used to observe the fungi colonizing the host, and water-mounted slides were prepared to exam- 3 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand ine their micro-morphological characters. Melzer’s reagent and Indian ink were utilized for further morphological investigations. Digital images of the micro morphological features were captured using a Canon 750D camera (Canon, Tokyo, Japan) mounted on a Nikon ECLIPSE E600 compound microscope (Nikon, Tokyo, Japan), with objective lenses providing magnifications of 10×, 20×, 40×, 60×, and 100×. Photo plates were produced using Adobe Photoshop CS6 software (Adobe Systems, USA). Measurements of the fungal characteristics were conducted using the Tarosoft® Image Framework software (version 0.9.7). Single spore isolations were performed as outlined by Senanayake et al. (2020) to obtain pure cultures. Germinated spores were identified after 24 hours of growth on malt extract agar (MEA), then transferred to fresh MEA media and incubated at 25 °C. Pure cultures were maintained on malt extract agar (MEA) at 25 °C, and their cultural characteristics were observed after one month of growth. Herbarium materials and cultures were deposited in the Mae Fah Luang University Herbarium (MFLU) and Mae Fah Luang University Culture Collection (MFLUCC), respectively. Index Fungorum and faces of fungi numbers were obtained (Jayasiri et al. 2015; Index Fungorum 2025). The species descriptions were added to the Greater Mekong Subregion database (https://gmsmicrofungi.org/) (Chaiwan et al. 2021) and the Fungalpedia webpage (Hyde et al. 2023). DNA extraction, PCR amplification, and sequencing Genomic DNA was extracted from fresh mycelia grown on MEA for 15 days, or DNA was extracted directly from the fruiting bodies using a DNA Extraction Kit (Omega Biotek) following the manufacturer’s protocol. The polymerase chain reaction (PCR) was conducted in a total volume of 25 μL, comprising 12.5 μL of 2× Power TaqPCR Master Mix, 1 μL of each primer (20 M), 2 μL of genomic DNA, and 8.5 μL of distilled water. PCR was performed using an Eppendorf thermal cycler (Mastercycler X50s) to amplify the LSU and ITS loci under the conditions outlined in Table 1. Agarose gel electrophoresis was performed to assess the quality of PCR products prior to sequencing at SolGent Co., South Korea. The PCR thermal cycling protocol consists of an initial step at 95 °C for 5 minutes, followed by a final elongation step at 72 °C for 10 minutes, and concluding with a hold at 4 °C, as well as annealing at 55 °C. Table 1. Primers and adapted PCR conditions applied for individual locus. Gene region Primer pairs PCR conditions References LSU LR0R/LR5 95 °C/30 s, 55 °C/50 s, 72 °C/60 s Vilgalys and Hester (1999) ITS ITS5/ITS4 95 °C/30 s, 55 °C/50 s, 72 °C/60 s White et al. (1990) Phylogenetic analyses SeqMan (DNAStar, Inc., Madison, WI, USA) was used to generate consensus sequences from the forward and reverse chromatograms obtained. The sequences were subsequently subjected to a BLASTn search in NCBI (https://blast.ncbi.nlm. nih.gov/). The LSU and ITS sequences for Amphisphaeria species were retrieved from the GenBank database (Table 2). Each gene locus was aligned using the default settings in MAFFT v. 7 (https://mafft.cbrc.jp/alignment/server/) (Katoh et 4 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand Table 2. Taxa used in the phylogenetic analyses and their corresponding GenBank accession numbers. Sequences obtained in this study are in bold. Taxon Strain LSU ITS Reference Amphisphaeria acericola MFLU 16-2479* MK640424 MK640423 Senanayake et al. (2019) Amphisphaeria acericola MFLUCC 14-0842* MF614131 MF614128 Senanayake et al. (2019) Amphisphaeria ailaoshanensis KUNCC 23-15521 PP584771 PP584674 Dissanayake et al. (2024) Amphisphaeria ailaoshanensis KUNCC 23-15520* PP584770 PP584673 Dissanayake et al. (2024) Amphisphaeria camelliae HKAS 107021* MT756615 MT756621 Samarakoon et al. (2020) Amphisphaeria camelliae MFLU 20-0181* MT756616 MT756622 Samarakoon et al. (2020) Amphisphaeria chiangmaiensis CMUB 40017* OR507152 OR507139 Samarakoon (2023) Amphisphaeria chiangmaiensis MFLU 23-0411* OR507153 OR507140 Samarakoon (2023) Amphisphaeria curvaticonidia MFLU 18-0789* MT756617 MT756623 Samarakoon et al. (2020) Amphisphaeria curvaticonidia HKAS 102288* MT756618 MT756624 Samarakoon et al. (2020) Amphisphaeria falcata CGMCC3.23740* OQ645284 OQ645270 Chang et al. (2025) Amphisphaeria flava MFLU 18-0102* MH971234 MH971224 Samarakoon et al. (2019) Amphisphaeria fuckelii WU 33555 N/A KT949903 Jaklitsch et al. (2016) Amphisphaeria fuckelii CBS 140409* N/A KT949902 Jaklitsch et al. (2016); Voglmayr et al. (2019) Amphisphaeria guttulata MFLUCC 22-0052 N/A NR_190966 Liu et al. (2024b) Amphisphaeria guttulata MFLUCC 22-0078 OQ101583 OQ101582 Liu et al. (2024b) Amphisphaeria hibiscicola HKAS 136910 PQ570865 PQ570847 Sun et al. (2025) Amphisphaeria hongheensis MHZU 24-0515 PQ166524 PQ165968 Liu et al. (2024a) Amphisphaeria hydei CMUB 40016* OR507154 OR507141 Samarakoon (2023) Amphisphaeria hydei MFLU 23-0412* OR507155 OR507142 Samarakoon (2023) Amphisphaeria karsti GZAAS 20-0147* OR209622 OR224991 Zhang et al. (2023) Amphisphaeria karsti GZAAS 20-148 OR209623 OR224992 Zhang et al. (2023) Amphisphaeria kunmingensis KUNCC 23-15522* PP584772 PP584675 Dissanayake et al. (2024) Amphisphaeria kunmingensis KUNCC 23-15523 PP584773 PP584676 Dissanayake et al. (2024) Amphisphaeria magna HKAS 130271 PP584775 PP584678 Dissanayake et al. (2024) Amphisphaeria magna HKAS 130270* PP584774 PP584677 Dissanayake et al. (2024) Amphisphaeria mangrovi PUFD37 MG844275 MG844283 Dissanayake et al. (2024) Amphisphaeria mesuae MFLUCC 25-0197* PV299568 PV393830 (This study) Amphisphaeria micheliae UESTCC 23.0125 OR253277 OR253118 Li et al. (2024) Amphisphaeria micheliae UESTCC 23.0123 OR253249 OR253097 Li et al. (2024) Amphisphaeria micheliae UESTCC 23.0124 OR253280 OR253121 Li et al. (2024) Amphisphaeria micheliae HKAS 107012* MT756619 MT756625 Samarakoon et al. (2020) Amphisphaeria micheliae MFLUCC 24-0324 PQ340163 PQ340156 Pathirana et al. (2025) al. 2019) and trimmed with trimAl v. 1.2 (Capella-Gutiérrez et al. 2009). Single locus alignments were concatenated using BioEdit v. 7.0.5.2 (Hall 1999). ALTER (http://www.sing-group.org/ALTER/) was used to convert FASTA files into PHYLIP format. Single-locus and multi-locus aligned datasets were analyzed separately using maximum likelihood (ML) and Bayesian inference (BI). Maximum likelihood analysis was performed using IQ webserver (http://iqtree.cibiv.univie.ac.at/) with bootstrap support for 1000 replicates (Nguyen et al. 2015). 5 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand Taxon Strain LSU ITS Reference Amphisphaeria micheliae MFLU 20-0172* MT756620 MT756626 Samarakoon et al. (2020) Amphisphaeria micheliae MFLU 21-0207 OK179729 OK284457 De Silva et al. (2022) Amphisphaeria micheliae MFLU 25-077 PV299571 PV290310 (This study) Amphisphaeria mimusopis MFLUC 25-0076* PV299569 PV366837 (This study) Amphisphaeria mimusopis MFLU 25-0159 N/A PV522822 (This study) Amphisphaeria neoaquatica MFLUCC 14-0045* MK835805 MK828607 Luo et al. (2019) Amphisphaeria oleae UESTCC: 23.0120 OR253314 OR253157 Li et al. (2024) Amphisphaeria oleae CGMCC: 3.24959* OR253313 OR253156 Li et al. (2024) Amphisphaeria orixae GZCC 22-2031* OQ064543 OQ064541 Wang et al. (2023) Amphisphaeria orixae GZCC 22-2032* OQ064544 OQ064542 Wang et al. (2023) Amphisphaeria paraserianthis MFLU 25-0075* PV393833 PV393832 (This study) Amphisphaeria parvispora MFLU 18-0767* MW240574 MW240644 Samarakoon et al. (2022) Amphisphaeria pseudomicheliae MFLU 25-0074* PV299570 PV393834 (This study) Amphisphaeria pterocarpi MFLU 25-0073 PV299564 PV366837 (This study) Amphisphaeria pterocarpi MFLUCC 25-0195* PV299566 PV366836 (This study) Amphisphaeria qujingensis KUMCC 19-0187* MN556316 MN477033 Dissanayake et al. (2020) Amphisphaeria qujingensis KUMCC 19-0186* MN707566 MN707568 Dissanayake et al. (2020) Amphisphaeria sambuci WU 33557 N/A KT949905 Jaklitsch et al. (2016) Amphisphaeria sambuci CBS 131707* NG_066215 KT949904 Jaklitsch et al. (2016), Liu et al. (2019) Amphisphaeria schimae MFLUCC 25-0196 * PV299567 PX488288 (This study) Amphisphaeria schimae MFLU 25-0071 PV299565 PX488290 (This study) Amphisphaeria shangrilaensis HKAS 130273 PP584776 PP584679 Dissanayake et al. (2024) Amphisphaeria shangrilaensis HKAS 130272* PP584777 PP584680 Dissanayake et al. (2024) Amphisphaeria sorbi MFLUCC 13-0721* KP744475 KR092797 Liu et al. (2015) Amphisphaeria sp. KoLRI 053241 N/A MZ855365 Yang et al. (2022) Amphisphaeria thailandica MFLU 18-0794* MH971235 MH971225 Samarakoon et al. (2019) Amphisphaeria umbrina HKUCC 994 AF452029 AF009805 Jeewon and Hyde (2003), Jaklitsch et al. (2016) Amphisphaeria umbrina PRA-JV24328 N/A OL396664 Vondrák et al. (2022) Amphisphaeria uniseptata CBS 114967* MH554197 MH553979 Liu et al. (2019) Amphisphaeria verniciae UESTCC: 23.0122 OR253270 OR253155 Samarakoon (2023) Amphisphaeria verniciae CGMCC: 3.24960* OR253269 OR253154 Samarakoon (2023) Amphisphaeria xishuangbannaense KUNCC 23-15525 PP584779 PP584682 Dissanayake et al. (2024) Amphisphaeria xishuangbannaense KUNCC 23-15524* PP584778 PP584681 Dissanayake et al. (2024) Amphisphaeria yunnanensis KUMCC 19-0189* MN550992 MN550997 Dissanayake et al. (2020) Amphisphaeria yunnanensis KUMCC 19-0188* MN556306 MN477177 Dissanayake et al. (2020) Beltrania rhombica CBS 123.58* MH869260 MH857718 Liu et al. (2019); Vu et al. (2019) Beltraniella endiandrae CBS 137976* KJ869185 KJ869128 Crous et al. (2014) Beltraniopsis longiconidiophora MFLUCC 17-2139* MF580256 MF580249 Lin et al. (2017) Neoarthrinium moseri CBS 164.80* LN851049 LN850995 Sandoval-Denis et al. (2016) Neoarthrinium trachycarpi CFCC 53039* N/A MK301099 Yan et al. (2019) Pidoplitchkoviella terricola CBS 180.77* AF096197 MH861046 Suh and Blackwell (1999), Vu et al. (2019) 6 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand MrModeltest v. 2.2 was used to estimate the evolution model using the Akaike information criterion (AIC), implemented in PAUP v. 4.0b10 (Nylander 2004). MrBayes v. 3.1.2 was used to conduct Bayesian inference (BI) analyses for estimating posterior probabilities (PP) through Markov chain Monte Carlo sampling (MCMC) under the GTR+I+G model (Huelsenbeck et al. 2001; Ronquist and Huelsenbeck 2003). Markov chains were executed for 1,000,000 generations, with trees sampled every 100th generation. The initial 25% of trees were discarded during the burn-in phase, while the remaining trees were used to compute the posterior probability (PP) in the majority rule consensus tree. FigTree v.1.4 was employed to visualize the phylograms (Rambaut and Drummond 2012), which were subsequently edited using Microsoft PowerPoint. Abbreviations: CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; CFCC: China Forestry Culture Collection Center, Research Institute of Forest Ecology, Environment and Protection, Beijing, China; CGMCC: China General Microbiological Culture Collection Center, Beijing, China; CMUB: Chiang Mai University, Chiang Mai, Thailand; GZAAS and GZCC: Guizhou Academy of Agricultural Sciences, Guizhou, China; HKAS: Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica, China; HKUCC: University of Hong Kong Culture Collection, Department of Ecology and Biodiversity, Hong Kong, China; KUMCC: Kunming Institute of Botany Culture Collection, China; KoLRI: Kholodny Institute of Botany, Tereshchenkivska, Kiev, Ukraine; MFLU, MFLUCC: Mae Fah Luang University, Chiang Rai, Thailand; PRA: The Herbarium of the Institute of Botany, Czech Academy of Sciences, Průhonice, Czech Republic; UESTCC: University of Electronic Science and Technology culture Collection, Xiyuan, Chengdu, China; WU: The Herbarium of the University of Vienna, Austria. Type species are denoted in ‘*’; “N/A” indicates the sequences are not available. Results Sequence alignment and phylogenetic analyses The combined LSU and ITS sequence matrix comprised 74 Amphisphaeriaceae taxa, including our ten new strains. The tree is rooted with Beltrania rhombica (CBS 123.58), Beltraniella endiandrae (CBS 137976), and Beltraniopsis longiconidiophora (MFLUCC 17-2139). The combined alignment comprised 1976 characters, including gaps (LSU: 1–1,332 and ITS: 1,333–1,970). Both ML and BI analyses yielded trees with similar topologies. The ML phylogram was used as the backbone tree (Fig. 1). The best-scoring RAxML tree had an optimization likelihood value of -14759.462. The matrix contained 1,000 distinct patterns, with 29.25% of the characters being undetermined or gaps. Estimated base frequencies were: A = 0.250, C = 0.250, G = 0.250, T = 0.250; substitution rates AC = 0.72949, AG = 2.67549, AT = 1.00000, CG = 0.72949, CT = 3.60004, GT = 1.0; the gamma distribution shape parameter was 0.467, Tree Length: 2.512. For the Bayesian analysis, the best-fit models generated from MrModeltest under the Akaike information criterion (AIC) are as follows: LSU: TI M3+I+G and ITS: TVM+I+G. Amphisphaeria mesuae (MFLUCC 25-0197) clustered with Amphisphaeria hibiscicola (HKAS 136910) and Amphisphaeria umbrina (PRA JV24328). Amphisphaeria micheliae (MFLU 25-0077) clustered in a clade shared by 7 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand Figure 1. Phylogram generated from ML analysis based on combined LSU, ITS sequence data. Bootstrap support values for ML ≥75% and PP ≥0.90 are indicated near the corresponding nodes. The tree is rooted with Beltrania rhombica (CBS123.58), Beltraniella endiandrae (CBS:137976), and Beltraniopsis longiconidiophora (MFLUCC17-2139). Type and reference strains are in bold, while the novel strains are in bold red. 0.03 Amphisphaeria falcata CGMCC3.23740 Amphisphaeria verniciae UESTCC 23.0122 Beltrania rhombica CBS 123.58 Amphisphaeria schimae MFLUCC 25-0196 Amphisphaeria micheliae MFLU 25-0077 Amphisphaeria chiangmaiensis MFLU 23-0411 Amphisphaeria magna HKAS 130270 Amphisphaeria fuckelii WU 33555 Amphisphaeria ailaoshanensis KUNCC 23-15521 Amphisphaeria neoaquatica MFLUCC 14-0045 Amphisphaeria magna HKAS 130271 Amphisphaeria pterocarpi MFLU 25-0073 Amphisphaeria oleae UESTCC 23.0120 Amphisphaeria sp. KoLRI 053241 Amphisphaeria parvispora MFLU 18-0767 Amphisphaeria camelliae HKAS 107021 Amphisphaeria sambuci WU 33557 Amphisphaeria micheliae MFLUCC 21-0207 Amphisphaeria shangrilaensis HKAS 130272 Amphisphaeria hydei MFLU 23-0412 Amphisphaeria shangrilaensis HKAS 130273 Amphisphaeria hibiscicola HKAS 136910 Amphisphaeria acericola MFLU 16-2479 Amphisphaeria micheliae MFLU 20-0172 Amphisphaeria uniseptata CBS 114967 Amphisphaeria ailaoshanensis KUNCC 23-15520 Amphisphaeria orixae GZCC 22.2031 Amphisphaeria micheliae MFLUCC 24-0324 Amphisphaeria umbrina HKUCC 994 Amphisphaeria guttulata MFLU 22-0078 Amphisphaeria yunnanensis KUMCC 19-0188 Amphisphaeria pterocarpi MFLUCC 25-0195 Amphisphaeria chiangmaiensis CMUB 40017 Amphisphaeria micheliae UESTCC 23-0124 Beltraniella endiandrae CBS 137976 Amphisphaeria mangrovei PUFD37 Amphisphaeria kunmingensis KUNCC 23-15522 Beltraniopsis longiconidiophora MFLUCC 17-2139 Amphisphaeria schimae MFLU 25-0071 Amphisphaeria thailandica MFLU 18-0794 Amphisphaeria verniciae CGMCC 3.24960 Amphisphaeria qujingensis KUMCC 19-0186 Amphisphaeria paraserianthis MFLU 25-0075 Amphisphaeria umbrina PRA JV24328 Neoarthrinium trachycarpi CFCC 53039 Amphisphaeria camelliae MFLU 20-0181 Amphisphaeria yunnanensis KUMCC 19-0189 Amphisphaeria mimusopis MFLU 25-0159 Amphisphaeria karsti GZAAS 20-0147 Amphisphaeria hydei CMUB 40016 Amphisphaeria curvaticonidia MFLU 18-0789 Amphisphaeria qujingensis KUMCC 19-0187 Amphisphaeria pseudomicheliae MFLU 25-0074 Amphisphaeria mimusopis MFLU 25-0076 Amphisphaeria micheliae UESTCC 23-0125 Amphisphaeria sorbi MFLUCC 13-0721 Amphisphaeria kunmingensis KUNCC 23-15523 Amphisphaeria karsti GZAAS 20-148 Amphisphaeria curvaticonidia HKAS 102288 Amphisphaeria xishuangbannaense KUNCC 23.15525 Amphisphaeria micheliae HKAS 107012 Amphisphaeria guttulata MFLU 22-0052 Amphisphaeria oleae CGMCC 3.24959 Pidoplitchkoviella terricola CBS 180.77 Amphisphaeria flava MFLU 18-0102 Amphisphaeria sambuci CBS 131707 Amphisphaeria mesuae MFLUCC 25-0197 Amphisphaeria xishuangbannaense KUNCC 23.15524 Amphisphaeria hongheensis MHZU 24-0515 Amphisphaeria acericola MFLUCC 14-0842 Amphisphaeria micheliae UESTCC 23-0123 Neoarthrinium moseri CBS 164.80 Amphisphaeria fuckelii CBS 140409 Amphisphaeria orixae GZCC 22.2032 98/1 98/1 100/1 100/1 100/1 100/1 100/1 100/1 87/- 100/1 100/1 100/1 100/1 85/- 100/1 94/1 100/1 100/1 100/1 100/1 98/1 100/1 82/- 100/1 93/1 100/1 100/1 97/1 81/- 100/1 99/1 100/1 100/1 95/0.97 100/1 100/1 99/1 98/1 100/1 100/1 100/1 100/1 100/1 77/- 100/- 100/1 100/1 100/1 87/- 89/- 100/1 Genera incertae sedis Outgroup 8 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand other A. micheliae strains (HKAS 107012, MFLU 20-0172, MFLUCC 21-0207, MFLUCC 24-0324, UESTCC 23-0123, UESTCC 23-0124, UESTCC 23-0125). Amphisphaeria mimusopis (MFLU 25-0076, MFLU 25-0159) was sister to A. flava (MFLU 18-0102). Amphisphaeria paraserianthis (MFLU 25-0075) formed a separate lineage, basal to A. neoaquatica (MFLUCC 14-0045) and A. hongheensis (GMB1135). Amphisphaeria pseudomicheliae was a sister to the A. micheliae group. Amphisphaeria pterocarpi (MFLU 25-0073, MFLUCC 25-0195) formed a separate lineage, sister to A. curvaticonidia (MFLUCC 180620, HKAS 102288). Amphisphaeria schimae (MFLU 25-0071, MFLUCC 250196) is sister to A. ailaoshanensis (KUNCC 23-15520, KUNCC 23-15521). Taxonomy Amphisphaeria mesuae Z.L. Tun & K.D. Hyde, sp. nov. Index Fungorum: IF903423 Facesoffungi Number: FoF17659 Fig. 2 Etymology. The epithet refers to the host genus, Mesua, from which the fungus was isolated. Holotype. MFLU 25-0069. Description. Saprobic on decaying twigs of Mesua sp. Sexual morph: Ascomata 520–580 μm high, 260–290 µm wide (xˉ = 549 × 272 µm, n = 5), immersed, visible as black spots with tiny pores, flat, scattered or aggregated, globose to subglobose, ostiolate. Peridium 14–18 µm (xˉ = 15 µm, n = 5), two-layered; outer layer wide, comprising thick-walled, dark brown cells of textura angularis, inner layer thin, composed of hyaline textura angularis cells. Paraphyses 3–5 µm wide, hyaline, septate, longer than asci, narrow towards the apex. Asci 86–134× 7–13µm (xˉ = 115 × 9 µm, n = 20), 8-spored, unitunicate, cylindrical, with short pedicel, apically rounded, with a J+, apical ring. Ascospores 14–18 × 5–6.8 µm (xˉ = 15 × 6 µm, n = 20), uniseriate, ellipsoidal, hyaline when immature, turning sub-hyaline to pale brown at maturity, 1-septate, slightly constricted at the septum, guttulate, smooth-walled, lacking a sheath in Indian ink. Asexual morph: Not observed. Culture characteristics. Colonies on MEA, reaching 4 cm diam. after 15 days at 27 °C, from above white to pale yellow radiating outwards, dense, circular to slightly irregular, flattened with smooth surface, with smooth margin; reverse pale brown in the middle, yellowish brown at the margin. Material examined. Thailand • Chiang Rai Province, Mae Fah Luang University (20°02′42″N, 99°53′41″E), on decaying dead twigs of Mesua sp. (Calophyllaceae), 02 October 2023, Zaw Lin Tun P11 (holotype MFLU 25-0069); ex-type culture MFLUCC 25-0197. Notes. Phylogenetic analyses revealed that Amphisphaeria mesuae (MFLUCC 25-0197) clustered with Amphisphaeria hibiscicola (HKAS 136910) and Amphisphaeria umbrina (PRA JV24328) in (Fig. 1). The interspecies genetic distances between A. mesuae (MFLU250069) and A. hibiscicola (HKAS 136910) showed the following base pair differences (without gaps): 5.7% for LSU (49/935 bp) and 11.18% for ITS (66/559 bp). Amphisphaeria mesuae (MFLUCC 25-0197) and Amphisphaeria umbrina (PRA JV24328) showed the following base pair differences (without gaps): 3.3% for LSU (28/844 bp) and 9.45% for ITS 9 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand Figure 2. Amphisphaeria mesuae (MFLU 25-0069, holotype). A. Decaying woody twig; B. Appearance of an ascoma on the host; C, D. Vertical section through an ascoma; E. Vertical section of peridium; F. Paraphyses; G. J+ Apical ring; H, I. Asci; J–M. Ascospores; N. Upper view of culture; O. Reverse view of culture. Scale bars: 200 μm (B, C); 100 μm (D); 20 μm (E); 5 μm (F, G); 40 μm (H, I); 10 μm (J–M). 16 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand Amphisphaeria pseudomicheliae Z. L. Tun & K.D. Hyde, sp. nov. Index Fungorum: IF903743 Facesoffungi Number: FoF17662 Fig. 6 Etymology. Refers to the morphological similarity with Amphisphaeria micheliae. Holotype. MFLU 25-0074. Description. Saprobic on decaying twigs. Sexual morph: Ascomata 202– 273μm high, 349–400 µm diam (xˉ = 246 × 337 µm, n = 5), immersed, visible as tiny pores, flat, solitary to aggregated, scattered, globose to subglobose, brown, ostiolate. Peridium 17–23 µm wide (xˉ = 20.6 µm, n = 5), two-layered; outer layer wide, dark brown, comprising thick-walled cells of textura angularis, inner layer comprising hyaline cells of textura angularis, thin-walled. Paraphyses 4–4.4 µm (xˉ = 4.2 µm, n = 5) wide, hyaline, longer than asci, filiform, guttulate, septate, embedded in a gelatinous matrix. Asci 84–97× 7–9 µm (xˉ = 89.8 × 8.45 µm, n = 20), 8-spored, unitunicate, cylindrical, with short pedicel, apically rounded and narrowed, with a J+ inconspicuous apical ring. Ascospores 11–17 × 4–6µm (xˉ= 15 × 5 µm, n = 20), uniseriate, fusiform, hyaline when immature, turning yellow to yellowish brown on maturity, 1-septate, guttulate, smooth-walled, slightly constricted at septum, straight to slightly curved, surrounded by a thin mucilaginous sheath. Asexual morph: Not observed. Material examined. Thailand • Chiang Rai Province, Mae Fah Luang University premises (20°02′42″N, 99°53′41″E), on decaying dead twigs of an undetermined host, 06 July 2023, Zaw Lin Tun E15 (holotype MFLU 25-0074). Notes. Amphisphaeria pseudomicheliae is closely related to A. micheliae, receiving robust support with 97% ML and 1 PP bootstrap support (Fig. 1). However, it can be distinguished from A. micheliae by its larger ascomata (202–273× 349–400 µm vs. 180–210 × 225–370 µm) and smaller asci (84–97× 7–9 µm vs. 92–135 × 7–10.5 μm) (Samarakoon et al. 2020). Additionally, the ascospores of A. pseudomicheliae are smaller (11–17 × 4–6 µm) than those of A. micheliae (15.5–21 × 6–7.5 μm) (Samarakoon et al. 2020). Amphisphaeria pseudomicheliae has a thin mucilaginous sheath, whereas A. micheliae lacks (Samarakoon et al. 2020). The genetic analysis reveals interspecies distances of 4.3% base pair differences (without gaps) in the ITS (23/524 bp) and 0.43% in the LSU (4/873 bp) between A. pseudomicheliae (MFLU25-0074) and A. micheliae (HKAS 107012). Amphisphaeria micheliae is uncertain since our new species lack protein coding genes. Thus, numerous collections with the protein coding genes can provide better resolution between the species. Amphisphaeria pseudomicheliae is described as a new species based on its distinct morphology and phylogenetic data. Amphisphaeria pterocarpi Z.L. Tun & K.D. Hyde, sp. nov. Index Fungorum: IF903750 Facesoffungi Number: FoF17663 Fig. 7 Etymology. The epithet refers to the host genus Pterocarpus, from which the fungus was isolated. Holotype. MFLU 25-0073. 17 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand Figure 6. Amphisphaeria pseudomicheliae (MFLU 25-0077, holotype). A. Host; B. Appearance of ascomata on host (arrows indicate ascomata as tiny pores on host surface); C. Vertical section through an ascoma; D. Vertical section of peridium; E. Paraphyses; F. An inconspicuous J+ apical ring; G–I. Asci; J–Q. Ascospores; Q. An ascospore with a thin gelatinous sheath (in Indian ink). Scale bars: 100 μm (C); 10 μm (D–F); 20 μm (G–I); 10 μm (J–Q). Description. Saprobic on recently dead branches of Pterocarpus sp. Sexual morph. Ascomata 225–237 μm high, 355–373 µm wide, (xˉ = 231 × 360 µm, n = 5), immersed, visible as black spots, host tissue becoming reddish around the apical pores, solitary to aggregated, scattered, globose to sub-globose, brown. Ostiole central, 70–60 wide, comprising a short papilla. Peridium 12–16 µm wide (xˉ = 14 µm, n = 5), two-layered; outer layer wide, dark brown, thick-walled cells of 18 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand Figure 7. Amphisphaeria pterocarpi (MFLU 25-0073, holotype). A. Dead branches; B. Appearance of ascomata on host (arrows indicate ascomata as spots on host surface); C, D. Vertical sections through ascomata; E. Vertical section of peridium; F. Paraphyses; G, H. Asci; I. J+ apical ring; J–L. Ascospores; M. Germinated ascospore; N. Upper view of culture; O. Reverse view of culture. Scale bars: 100 μm (C, D); 10 μm (E); 5 μm (F, G); 50 μm (H, I); 20 μm (J–M). 19 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand textura angularis, inner layer comprising one layer of hyaline cells of textura angularis, thin-walled. Paraphyses 4–6 µm wide (xˉ = 4.8 µm, n = 5), hyaline, septate, guttulate, embedded in a gelatinous matrix. Asci 95–116 × 9–14 µm (xˉ= 106 × 11 µm, n = 20), 8-spored, unitunicate, cylindrical, deliquescing, with short pedicel, apically rounded, with a J+, wedge-shaped, apical ring. Ascospores 19–21 × 5–7 µm (xˉ = 20 × 6 µm, n = 20), ellipsoidal, hyaline when immature, turning yellow to yellowish-brown when mature, 3-septate, guttulate, smooth-walled, surrounded by a mucilaginous sheath. Asexual morph: Not observed. Culture characteristics. Colonies on MEA reaching 4 cm diam. after 15 days at 27 °C, from above white, dense, irregular, flattened with smooth surface, with lobate margin; reverse yellow red in the middle, yellow at the margin. Material examined. Thailand • Chiang Rai Province, Mae Fah Luang University premises (20°02′42″N, 99°53′41″E), on recently dead branches of Pterocarpus rotundifolius (Fabaceae), 06 November 2023, Zaw Lin Tun H62 (holotype MFLU 25-0073), ex-type culture MFLUCC 25-0195. Additional specimens examined. Thailand • Chiang Rai Province, Mae Fah Luang University premises (20°02′42″N, 99°53′41″E), on dead branches of Pterocarpus rotundifolius (Fabaceae), 06 November 2023, Zaw Lin Tun 2H62 (MFLU 25-0072). Notes. Based on our phylogenetic analyses, A. pterocarpi formed a separate lineage, sister to A. curvaticonidia (MFLUCC 18-0620, HKAS 102288), with 100% ML and 1.00 PP bootstrap support (Fig.1). Amphisphaeria pterocarpi can be distinguished from A. curvaticonidia by its smaller ascomata (225–237 × 355– 373 µm vs. 320–390 × 360–410 µm), asci (95–116 × 9–14 µm vs. 121–162 × 10.5–17.5 µm), and ascospores (19–21 × 5–7 µm vs. 17–23 × 6–9 µm) (Samarakoon et al. 2020). Additionally, the ascospores of A. pterocarpi are ellipsoidal, whereas A. curvaticonidia have oblong or narrowly fusiform ascospores. The asexual morph of A. curvaticonidia has been documented as coelomycetous in culture, while the asexual morph of A. pterocarpi has yet to be observed in culture (Samarakoon et al. 2020). When considering the base pair differences (without gaps) between A. pterocarpi and A. curvaticonidia (MFLUCC 18-0620, HKAS 102288), 2.5% base pair differences (without gaps) were revealed in LSU (27/1040 bp) and 3.30% base pair differences (without gaps) in ITS (18/545 bp). Due to the distinct morphology and phylogenetic evidence, along with the species delineation guidelines provided by Chethana et al. (2021), we introduce A. pterocarpi as a new species. Amphisphaeria schimae Z.L. Tun & K.D. Hyde, sp. nov. Index Fungorum: IF903771 Facesoffungi Number: FoF17664 Fig. 8 Etymology. The epithet refers to the host genus, Schima, from which the fungus was isolated. Holotype. MFLU 25-0070. Description. Saprobic on decaying twigs of Schima wallichii. Sexual morph: Ascomata 480–620 μm high × 520–683 µm wide, (xˉ = 531 × 583 µm, n = 5), immersed with ostiole breaking through host surface, visible as black spot, solitary 20 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand Figure 8. Amphisphaeria schimae (MFLU 25-0070, holotype). A. Host; B. Close-up of ascoma on host (Arrow indicates ascoma visible as a black spot, with ostiole breaking through the host); C. Vertical section of an ascoma; D. Ostiole; E.Vertical section of peridium; F. Paraphyses; G–I. Asci (I in Melzer’s reagent); J–N. Ascospores with sheaths; O. Germinated ascospore; P. Upper view of culture; Q. Reverse view of culture. Scale bars: 100 μm (C); 20 μm (D); 10 μm (E, F); 50μm (G–I); 20 μm (J–O). to aggregated, scattered, globose to subglobose, brown, ostiolate. Ostiole central, comprising a short papilla, with an ostiolar canal. Peridium 13–16 µm wide (xˉ = 14.2 µm, n = 5), two-layered; outer layer thick, dense, reddish-brown cells of 21 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand textura angularis, inner layer thin, hyaline cells of textura angularis. Paraphyses 3–5 µm wide, hyaline, filiform, septate, embedded in a gelatinous matrix. Asci 196–275 × 21–30 µm (xˉ = 236.2 × 22.9 µm, n = 20), 8-spored, unitunicate, cylindrical, with short pedicel, apically rounded, with J-, apical ring. Ascospores 36.9– 40 × 13.5–15 µm (xˉ = 38.6 × 14 µm, n = 20), uniseriate, broadly fusiform, hyaline, turning olivaceous brown to brown at maturity, one median and constricted euseptum, with two distosepta, guttulate (especially at immaturity), broad to conically rounded at both ends, smooth, surrounded by a thick mucilaginous sheath. Asexual morph: Not observed. Culture characteristics. Colonies on MEA slow-growing, reaching 3 cm in diam. after 15 days at 27 °C, from above white to pale yellow radiating outwards, dense, circular, irregular, flattened with smooth surface, with lobate margin; reverse yellow brown in the middle, white at the margin. Materials examined. Thailand • Chiang Mai Province, in forests around the Mushroom Research Center (19°07.200'N, 98°44.044'E), on fallen dead twigs of Schima wallichii (Theaceae), 14 November 2022, Zaw Lin Tun C1 (holotype MFLU 25-0070), ex-type culture MFLUCC 25-0196. Additional specimens examined. Thailand • Chiang Mai Province, in forests around the Mushroom Research Center (19°07.200'N, 98°44.044'E), on fallen dead twigs of Schima wallichii (Theaceae), 14 November 2022, Zaw Lin Tun C2 (MFLU 25-0071). Notes. Based on our phylogenetic analyses, Amphisphaeria schimae (MFLU 25-0071, MFLUCC 25-0196) is sister to A. ailaoshanensis (KUNCC 23-15520, KUNCC 23-15521) (Fig.1). However, A. schimae can be distinguished from A. ailaoshanensis by having larger ascomata (480–620 μm high × 520–683 μm vs. 100–140 μm high × 250–350 μm), asci (196–275 × 21–30 μm vs. 70–100 × 7–10 μm), and ascospores (36.9–40 × 13.5–15 μm vs. 14–20 × 5–8 μm) (Dissanayake et al. 2024). Additionally, the ascospores of A. schimae are broadly fusiform, hyaline, turning olivaceous brown to brown at maturity, with one median, conspicuously constricted euseptum, and surrounded by a thick mucilaginous sheath, whereas those of A. ailaoshanensis are fusiform, hyaline, guttulate, turning brown, 1–3-septate, and lack a mucilaginous sheath (Dissanayake et al. 2024). When comparing base pair differences (excluding gaps) between A. schimae (MFLUCC 25-0196) and A. ailaoshanensis (KUNCC 23-15520, KUNCC 23-15521), there is a 4.9% divergence in the LSU region (34/690 bp) and 8.2% divergence in the ITS region (43/525 bp). Based on the distinct morphological and phylogenetic evidence, along with the species delineation guidelines proposed by Chethana et al. (2021), we describe A. schimae as a new species. Discussion In this study, we introduce six new Amphisphaeria species, along with a new host and geographical record, based on morphological and multigene phylogenetic analyses of combined LSU and ITS alignments. This work significantly advances the taxonomy of Amphisphaeria by providing important insights into species delimitation, phylogenetic relationships, and morphological diversity. Moreover, the discovery of a new host and geographic record broadens our understanding of the ecological distribution and host specificity of these species, offering a more comprehensive framework for future taxonomic and ecological research in this group. 22 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand Most strains of Amphisphaeria lack protein gene sequences, with the exception of A. camelliae, A. flava, A. fuckelii, A. hongheensis, A. hydei, A. micheliae, A. parvispora, A. sambuci, A. thailandica, and A. uniseptata (Tusi et al. 2001; Jaklitsch et al. 2016; Voglmayr et al. 2019; Liu et al. 2019, 2024a; Samarakoon et al. 2019; Samarakoon 2023; Li et al. 2024). In this study, we encountered challenges in sequencing protein markers across our Amphisphaeria isolates. Specifically, all isolates lacked the β-tub and the rpb2 gene. These difficulties align with findings from previous studies, which also reported challenges in sequencing these protein markers for Amphisphaeria (Senanayake et al. 2019; Samarakoon et al. 2020; Zhang et al. 2023; Dissanayake et al. 2024; Sun et al. 2025). To address these limitations, we constructed two phylogenetic trees: one using a combination of LSU, ITS, rpb2, and β-tub (not shown), and another using only LSU and ITS (Fig. 1). Comparison of the topologies of the two-locus (LSU and ITS) and four-locus trees showed mostly similar phylogenetic placements of the taxa. Since most of our species lacked β-tub and rpb2, we predominantly relied on LSU and ITS for our phylogenetic analyses. This approach allowed us to achieve robust phylogenetic resolution despite the absence of key protein markers in most isolates. Currently, 312 names are listed under Amphisphaeria in Index Fungorum (July 2025). Wang et al. (2004) examined 170 type specimens and accepted only 12 species in Amphisphaeria, highlighting taxonomic confusion within this genus. The lack of molecular data for many Amphisphaeria species makes it difficult to confirm their taxonomic positions (Thiyagaraja et al. 2025). Additionally, the absence of protein gene sequences for Amphisphaeria species often leads to inaccurate identifications (Samarakoon 2023; Dissanayake et al. 2024). The high morphological similarity and lack of molecular data among Amphisphaeria species can cause misidentifications (Thiyagaraja et al. 2025). Therefore, incorporating new collections and multi-gene molecular data is essential for accurate species delimitation and a clearer understanding of species boundaries within Amphisphaeria. The saprobes were collected from forest areas during both the wet and cold seasons of 2022–2024. The distribution of Amphisphaeria species is shown in Table 3. Amphisphaeria species are found worldwide, with reports from 15 countries. They occur on 24 host genera across 17 different plant families, with Sapindaceae being the most diverse (Table 3). Additionally, Amphisphaeria has been reported in other families, including two species each in Theaceae, Leguminosae, and Malvaceae, three in Fabaceae, and one each in Proteaceae, Gramineae, Calophyllaceae, Magnoliaceae, Actinidiaceae, Apocynaceae, Rutaceae, Oleaceae, Asparagaceae, Agavaceae, Rosaceae, and Euphorbiaceae (Table 3). Therefore, Malvaceae and Fabaceae are the second most diverse families for Amphisphaeria species (Table 3). Amphisphaeria chiangmaiensis, A. curvaticonidia, A. flava, A. hydei, A. parvispora, A. micheliae, and A. thailandica have been previously documented in northern Thailand (Samarakoon et al. 2019–2023; Pathirana et al. 2025), highlighting the region as a hotspot for fungal diversity. In our study, we further contribute to the understanding of Amphisphaeria species in this region by describing six new species, each associated with different host families, as shown in Table 3. This discovery not only broadens the known taxonomic range of Amphisphaeria but also underscores the ecological adaptability of this genus to various plant hosts. Additionally, we report A. micheliae on Senna siamea for the first time in Thailand, 23 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand Table 3. Accepted species in Amphisphaeria, including their host and geographical location (NA = Lack of information). Species Host Country References Amphisphaeria acericola On a branch of Acer campestre L. (Sapindaceae) Italy Samarakoon et al. (2019) A. ailaoshanensis NA China Dissanayake et al. (2024) A. aquatica NA China Luo et al. (2019) A. bertiana In cavities at the end of a rotting log USA Wang et al. (2004) A. camelliae Camellia japonica (Theaceae) China Samarakoon et al. (2020) A. chiangmaiensis NA Thailand Samarakoon (2023) A. curvaticonidia NA Thailand Samarakoon et al. (2020) A. depressa Cassia bicapsularis (Leguminosae) USA, Petrak 1953; Wang et al. (2004) A. falcata The medullary tissue of the lichen Usneadiffracta China Chang et al. (2025) A. fallax Quercus robur (Leguminosae) Austria, Germany De not 1865; Wang et al. (2004) A. flava NA Thailand Samarakoon et al. (2019) A. fuckelii On attached branches of Tilia cordata, Acer campestre (Malvaceae, Sapindaceae) Germany, Austria, Belgium Jaklitsch et al. (2016); Liu et al. (2019); Voglmayr et al. (2019) A. gaubae Dead leaves of Lambertia formosa (Proteaceae) Australia Wang et al. (2004) A. guttulata NA Thailand Liu et al. (2024b) A. hibiscicola Hibiscus mutabilis (Malvaceae) China Sun et al. (2025) A. hongheensis NA China Liu et al. (2024a) A. hydei NA Thailand Samarakoon (2023) A. karsti NA China Zhang et al. (2023) A. kunmingensis NA China Dissanayake et al. (2024) A. lusitanica Arundo donax (Gramineae) Portugal Wang et al. (2004) A. magna NA China Dissanayake et al. (2024) A. mesuae Mesua sp. (Calophyllaceae) Thailand This study A. micheliae Alstonia scholaris, Acer truncatum, on a dead branch of Michelia alba, Senna siamea, Micromelum integerrimum (Apocynaceae, fabaceae, Magnoliaceae, Sapindaceae, Rutaceae) China, Thailand Samarakoon et al. (2020); Li et al. (2024); Pathirana et al. (2025), This study A. mimusopis Mimusops Elengi (Sapotaceae) Thailand This study A. multipunctate Ctinidia deliciosa (Actinidiaceae) New Zealand Petrak (1923); Wang et al. (2004) A. oleae On branches of Olea europaea (Oleaceae) China Li et al. (2024) A. orixae The healthy roots of Orixa japonica (Asparagaceae) China Wang et al. (2023) A. paedida NA Germany Saccardo (1882); Wang et al. (2004) A. paraserianthis Paraserianthes lophantha (Fabaceae) Thailand This study A. parvispora NA China Samarakoon et al. (2022) A. pseudomicheliae NA Thailand This study A. pseudoumbrina On bark of Acer campestre (Sapindaceae) Italy Wang et al. (2004) A. pterocarpi Pterocarpus rotundifolius (Fabaceae) Thailand This study A. qujingensis NA China Dissanayake et al. (2020) A. sambuci Sambucus nigra (Adoxaceae) England, France, Germany Liu et al. (2019); Jaklitsch et al. (2016) A. schimae Schima wallichii (Theaceae) Thailand This study A. seriata On leaf of Nolina sp. (Agavaceae) USA Barr and Ramaley (1996); Wang et al. (2004) A. shangrilaensis NA China Dissanayake et al. (2024) A. sorbi On branch of Sorbus aucuparia L. (Rosaceae) Italy Liu et al. (2015) A. thailandica NA Thailand Samarakoon et al. (2019) A. umbrina Tilia sp. (Malvaceae) Switzerland Jeewon and Hyde (2003) A. uniseptata NA China Tusi et al. (2001) A. verniciae Branches of Vernicia fordii (Euphorbiaceae) China Li et al. (2024) A. vibratilis On the stem of Prunus sp. (Rosaceae) Canada, British, Columbia Müller and Arx (1962); Wang et al. (2004) A. xishuangbannaense NA China Dissanayake et al. (2024) A. yunnanensis NA China Dissanayake et al. (2020) 24 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand marking an important addition to the fungal flora of the region. These findings collectively highlight the impressive diversity of Amphisphaeria species in northern Thailand and their associations with a wide array of host families, indicating a complex and dynamic ecological interaction that warrants further study. This genus also needs to be studied in other tropical regions to determine if it is equally diverse there (Hyde et al. 2024b). Historically, there have been no records of Amphisphaeria species being identified as pathogens (Wang et al. 2004). However, recent studies have revealed new insights into the ecological roles of these fungi (Sun et al. 2025). For instance, A. hibiscicola was reported on diseased leaves of Hibiscus mutabilis (Sun et al. 2025). Additionally, A. orixae has been identified as an endophyte, highlighting its ability to colonize plant tissues without causing disease (Wang et al. 2023). These findings indicate that Amphisphaeria species may exhibit diverse ecological roles, ranging from pathogenicity to endophytism, and may have evolved from endophytic species (Bhunjun et al. 2024). Furthermore, while most Amphisphaeria species are typically found in terrestrial environments (Dissanayake et al. 2024), the discovery of Amphisphaeria aquatica in freshwater habitats on submerged, decaying wood in China and Thailand (Tsui et al. 2001; Luo et al. 2019) expands our understanding of their ecological adaptability. This contrast between terrestrial and aquatic habitats highlights the ecological versatility of the genus and raises questions about how environmental factors influence their behavior and role in ecosystems. Therefore, future research should concentrate on clarifying the complex ecological interactions between these fungi and their hosts, as well as examining the impact of environmental factors on their distribution and behavior. Furthermore, it is essential to enhance the understanding of the ecological and evolutionary importance of Amphisphaeria species. This acknowledges the ongoing significance of northern Thailand as a vital center for continued research, as emphasized by Hyde et al. (2018). Acknowledgements Zaw Lin Tun is grateful to the National Research Council of Thailand (NRCT) grant “Total fungal diversity in a given forest area with implications towards species numbers, chemical diversity and biotechnology” (grant no. N42A650547) and Chinese Research Fund, grant number E1644111K1, titled “Flexible introduction of high-level expert program, Kunming Institute of Botany, Chinese Academy of Sciences” for its financial support. Zaw Lin Tun would like to thank Mae Fah Luang University for providing a scholarship and the Mushroom Research Foundation (MRF) for financial support. Zaw Lin Tun would like to thank Mae Fah Luang University for allocating the thesis writing grant (No. 0510). Zaw Lin Tun would like to thank Shaun Pennycook for his valuable suggestion on nomenclature for the new taxa and Achala Rathnayaka for her suggestion. Kevin D. Hyde and Fatimah Al-Otibi thank the Distinguished Scientist Fellowship Program (DSFP), King Saud University, Kingdom of Saudi Arabia. Additional information Conflict of interest The authors have declared that no competing interests exist. 25 MycoKeys 125: 1–31 (2025), DOI: 10.3897/mycokeys.125.163523 Zaw Lin Tun et al.: Addition of Amphisphaeria taxa from northern Thailand Ethical statement No ethical statement was reported. Use of AI No use of AI was reported. Funding This study was supported by the National Research Council of Thailand (NRCT) under the grant “Total fungal diversity in a given forest area with implications towards species numbers, chemical diversity and biotechnology” (Grant No. N42A650547); MFU thesis writing grant (No.0510). Additional support was provided by the Chinese Research Fund (Grant No. E1644111K1) for the project “Flexible introduction of high-level expert program, Kunming Institute of Botany, Chinese Academy of Sciences.” Author contributions Conceptualization: ZLT, CSB, KDH. Methodology: ZLT. Formal analysis: ZLT, DB, VT. Investigation: ZLT. Resources: KDH. Data curation: ZLT. Writing – original draft preparation: ZLT. Writing – review and editing: DB, CSB, SSNM, VT, FA-O, KDH. Supervision: CSB, KDH. Project administration: CSB, KDH. Funding acquisition: VT, KDH. Author ORCIDs Zaw Lin Tun https://orcid.org/0009-0001-3108-4425 Digvijayini Bundhun https://orcid.org/0000-0002-0790-215X Chitrabhanu S. Bhunjun https://orcid.org/0000-0001-8098-3390 Sajeewa S. N. Maharachchikumbura https://orcid.org/0000-0001-9127-0783 Vinodhini Thiyagaraja https://orcid.org/0000-0002-8091-4579 Fatimah Al-Otibi https://orcid.org/0000-0003-3629-5755 Kevin D. Hyde https://orcid.org/0000-0002-2191-0762 Data availability The DNA sequences produced in this study are available on NCBI GenBank (https:// www.ncbi.nlm.nih.gov). The vouchers are deposited in the the Mae Fah Luang University Herbarium and culture collection (MFLU, MFLUCC). References Barr ME, Ramaley AW (1996) Two more species of Amphisphaeria (Xylariales). Mycotaxon 58: 349–351. Bhunjun CS, Phukhamsakda C, Hyde KD, McKenzie EHC, Saxena RK, Li Q (2024) Do all fungi have ancestors with endophytic lifestyles? Fungal Diversity 125: 73–98. https:// doi.org/10.1007/s13225-023-00516-5 Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T (2009) TrimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics (Oxford, England) 25(15): 1972–1973. https://doi.org/10.1093/bioinformatics/btp348 Cesati V, De NG (1863) Classification scheme of the Aschigerian Italic spherical sphericae: More or less belonging to the genus Sphaeria in the ancient meaning attributed to it by Persoon. Italian Cryptogamic Herbarium 1: 177–240. Chaiwan N, Gomdola D, Wang S, Monkai J, Tibpromma S, Doilom M, Wanasinghe D, Mortimer P, Lumyong S, Hyde KD (2021) An online database providing updated informa-