Two New Species of Pleosporales from Pteridophytes in China

Two New Species of Pleosporales from Pteridophytes in China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Two New Species of Pleosporales from Pteridophytes in China Hua Li, Dhanushka N. Wanasinghe, Chitrabhanu Sharma Bhunjun, Chuanhao Zhou, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9126008/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Pleosporales is one of the largest and most diverse orders in Dothideomycetes. This order encompasses numerous families and genera characterized by considerable morphological diversity and a wide range of ecological lifestyles. During an investigation of fungal diversity associated with pteridophytes in South China, healthy samples of Diplopterygium chinense and Lepidogrammitis drymoglossoides were collected from Yunnan and Guangdong provinces. Endophytic fungi were isolated using the tissue isolation method. Among the isolates, four strains were identified as members of Pleosporales based on multi-locus phylogenetic analyses using the internal transcribed spacer (ITS), the large subunit (LSU), the β-tubulin ( tub 2), and the RNA polymerase II subunit ( rpb 2) sequence data, together with morphological characteristics of conidiation. These strains were assigned to two species each in Epicoccum and Paraconiothyrium and are herein described as two new species, Epicoccum diplopterygii and Paraconiothyrium drymoglossoidis . Detailed morphological illustrations, taxonomic descriptions, and molecular data are provided. This study expands the current knowledge of Pleosporales diversity and host associations in pteridophytic plants, providing a foundation for future taxonomic and ecological studies of fungi associated with pteridophytes in China. 2 new species Didymellaceae Didymosphaeriaceae Endophytic fungi Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Pleosporales was formally established by Luttrell ( 1955 ) and is currently recognized as the largest order in Dothideomycetes, with 91 families and 653 genera (Hyde et al. 2024 , Thiyagaraja et al. 2025 ). Pleosporales encompasses a wide range of morphological and ecological diversity (Kirk et al. 2008 , Zhang et al. 2012, Hyde et al. 2013 , Wijayawardene et al. 2014 , Bhunjun et al. 2021 , Thiyagaraja et al. 2025 ). Pleosporales species are characterized by perithecial ascomata, which are flask-shaped and typically possess papillae, and ostioles. The asexual morphs are coelomycetous and hyphomycetous (Hyde et al. 2011 , Pem et al. 2024 , He et al. 2025 ). The species exhibit remarkable ecological and morphological diversity, including saprobic, plant pathogenic, endophytic, and lichenicolous fungi, as well as species from freshwater and marine habitats (Shearer et al. 2009 , Hongsanan et al. 2020 , Phukhamsakda et al. 2020 , Yang et al. 2025 ; Thiyagaraja et al. 2025 ). Didymellaceae is a large family within the Pleosporales, which includes major genera of phoma-like fungi including Ascochyta , Didymella , and Stagonosporopsis , and represents an underestimated component of fungal diversity (Chen et al.2017, 2023, Gomzhina et al. 2025). Didymellaceae species are predominantly plant pathogens, such as Didymella pinodes , which causes Ascochyta blight of field pea, and Parastagonospora nodorum , which causes Stagonospora nodorum blotch of wheat (Bretag et al. 1995 , Solomon et al. 2006 , Quaedvlieg et al. 2013 ); however, several species have also been reported as opportunistic pathogens of humans and animals worldwide (Boerema et al. 2004, de Hoog et al. 2011). Moreover, numerous taxa occur as plant endophytes or saprobes, often in association with both host and non-host organisms, highlighting the ecological versatility of Didymellaceae (Schulz and Boyle 2005 , Tahtamouni et al. 2016). Therefore, investigating Didymellaceae species diversity is essential for understanding the taxonomy, evolution, and functional roles of plant-associated fungi (de Gruyter et al. 2009, Chen et al. 2015 , Hou et al. 2020 ). Didymosphaeriaceae represents a morphologically distinct but phylogenetically underexplored family within Pleosporales. Studies of its species diversity are important for resolving family boundaries and understanding evolutionary relationships among pleosporalean fungi (Hyde et al. 2013 , Ariyawansa et al. 2014 , Hyde et al. 2024 ). Didymosphaeriaceae is characterized by 1-septate ascospores and trabeculate pseudoparaphyses, which anastomose mostly above the asci (Liew et al. 2000 , Hyde et al. 2013 , Ariyawansa et al. 2014 , Wanasinghe et al. 2016 ). Wanasinghe et al. ( 2016 ) introduced two new genera, Laburnicola and Paramassariosphaeria , based on combined phylogenetic analyses of LSU, SSU, and ITS sequence data. The family comprises 39 genera, with species occurring worldwide as endophytes, pathogens, and saprobes on a wide range of plant substrates (Liu et al. 2015 , Wanasinghe et al. 2016 , Hyde et al. 2024 ). Pteridophytes are seedless vascular plants that reproduce by spores and typically inhabit moist environments (PPG 2016). In addition, they are an important part of the ground vegetation in many forest communities and, with about one-third of the species growing on the trunks and branches of trees, they are also an important component of many epiphytic plant communities (Yatskievych et al. 2003, PPG 2016). Recent studies have revealed that pteridophytes host a wide range of fungal taxa; however, knowledge of fungal diversity associated with pteridophytes remains limited (Bao et al. 2025, Zhang et al. 2025 ). This study is a part of an ongoing investigation of fungal diversity associated with pteridophytes in South China, in which we collected fresh healthy samples to understand the endophytic diversity in pteridophytes. The objectives of this study are to determine species composition, assess phylogenetic relationships, and enhance current knowledge of fungal diversity in pteridophyte hosts. Here we described four new taxa from Didymellaceae and Didymosphaeriaceae with updated taxonomic positions through morphology combined with phylogenetic analyses. These data will not only contribute to a better understanding of host associations and geographic distribution of Pleosporales in China but also highlight their potential as valuable fungal resources for future studies on ecosystem functions. 2. Materials and methods 2.1 Sample collection, isolation, and morphological examination Samples of fungi were obtained from Baiyun Mountain (23°09'35"N 113°17'40"E) in Guangzhou city, Guangdong Province, and Ailao Mountain (24°31'23"N 101°03'12"E) in Yunnan Province, China. Healthy fresh leaves of Diplopterygium chinense and Lepidogrammitis drymoglossoides were collected, and the specimens were brought to the lab in a Ziploc bag. The fresh leaves were washed with water to remove dirt and dust. Then, leaves were cut into small pieces (5×5 mm); these small pieces were soaked for 30 s in a 75% ethanol solution, soaked for 30 s in a 2.5% sodium hypochlorite solution, and washed three times with sterilized distilled water for 30 s each. The sterilized tissue fragments were dried on sterile filter paper and then transferred to Potato Dextrose Agar (PDA) (Senanayake et al. 2020 ). All PDA plates were cultured at a constant temperature of 25 ℃ for 2–4 days, and single hyphae were picked from the periphery of the grown colonies and inoculated on new PDA plates. After 2–3 weeks of purification, pure cultures were obtained. For morphological characterization, potato dextrose agar medium (PDA), malt extract agar medium (MEA), synthetic nutrient-poor agar medium (SNA), water agar medium (WA), as well as on PDA, SNA, and WA supplemented with sterile pine needle segments were used. Morphological characterization was done by observing the mucus clumps produced on these media, including hyphae, ascomata, and ascospores, were observed and photographed using a Nikon Eclipse 80i microscope (Nikon, Japan). The colony morphology was recorded with a Nikon D300S digital camera (Nikon, Japan). All microscopic structures were measured using Tarosoft® Image Frame Works Version 0.9.7 and images were processed in Adobe Photoshop 2019 (Adobe Systems, San Jose, CA, USA) (Senanayake et al. 2020 , Xiong et al. 2021, Manawasinghe et al. 2025 ). The morphological characteristics were examined using a stereomicroscope (Cnoptec sz650, China). The micro-morphological characteristics were observed and photographed using the Nikon Eclipse 80i microscope (Nikon, Japan). Colony colour (upper and reverse) was described using to the colour charts of Rayner ( 1970 ). All pure cultures were deposited in the culture collection of Zhongkai University of Agriculture and Engineering (ZHKUCC), China. Herbarium materials were deposited in the herbaria of Zhongkai University of Agriculture and Engineering (MHKU), China. Data on the new species were deposited in the Greater Mekong Subregion database (Chaiwan et al. 2021 ). 2.2 DNA extraction, PCR amplification, and sequencing Genomic DNA was extracted directly from one-week-old pure cultures using a Biospin Fungal Genomic DNA Extraction Kit (BioFlux, China) following the manufacturer’s instructions. The DNA was subjected to polymerase chain reaction (PCR) to amplify the internal transcribed spacer (ITS) gene using the primers ITS1 and ITS4 (White et al 1990 ); the large subunit (LSU) gene using the primers LROR and LR5 (Vilgalys and Hester 1990 ); the β-tubulin (tub 2) gene using the primers TUB-2Fd and TUB-4R (Glass and Donaldson 1995 ); and the RNA polymerase II subunit ( rpb 2) gene using the primers fRPB2-5F and fRPB2-7cr (Liu et al. 1999 ). The final Polymerase chain reaction (PCR) consisted of 12.5 µL of 2×Taq Master Mix (Dye Plus) (mixture of FastTaq TM DNA Polymerase, buffer, dNTP Mixture, and stabilizer) (Beijing Qingke Biological Technology Co., Ltd., Beijing, PR China), 9 µL of ddH 2 O, 1 µL of primers, and 1 µL of DNA template. PCR reactions were performed using the program for ITS, LSU, tub 2, and rpb 2, as described by Conforto et al. ( 2019 ) and Tennakoon et al. ( 2020 ). PCR products were analysed on 1% agarose gels and sent to Tianyi (Guangzhou, China) Co., Ltd for sequencing. All sequence data generated in this study were deposited in NCBI GenBank. 2.3 Phylogenetic analyses The chromatograms were examined using BioEdit version 7.0.5.2 (Hall 1999), and low–quality regions were trimmed before alignment. All sequences were subjected to BLASTn searches in the National Center for Biotechnology Information (NCBI). Based on the BLASTn search, we identified our isolates belonging to Epicoccum and Paraconiothyrium . All reference sequences used in this study were downloaded from GenBank following Cao et al. ( 2025 ), Gomzhina et al. (2025), and Manawasinghe et al. ( 2025 ) and are listed in Table 1. Alignments for each locus were generated using MAFFT 7 webservers ( http://mafft.cbrc.jp/alignment/server (accessed 20 January 2026) and were manually improved using BioEdit 7.0.5.2 (Hall 1999). Multigene phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI) analysis. Maximum likelihood analysis was performed using the IQ-TREE webserver (Trifinopoulos et al. 2016). The branch support of the phylogenetic tree was tested with 1000 replicates of Ultrafast bootstrap (Minh et al. 2013) and SH-aLRTtest (Guindon et al. 2010). Bayesian inference analysis was performed using MrBayes 3.2.7a on XSEDE on the CIPRES portal (Huelsenbeck et al. 2001, Miller et al. 2010, Stamatakis et al. 2014). Posterior probabilities (PP) were defined by the Markov chain Monte Carlo (MCMC) sampling method (Rannala & Yang 1996, Huelsenbeck & Ronquist 2001). Two parallel runs were conducted using the default settings. Six simultaneous Markov chains were run for 2 million generations, and trees were sampled every 1000th generation. The first 25% of trees were discarded as the burn-in phase. The remaining trees were used for calculating posterior probabilities in the majority rule consensus tree (Doll and Jacquemin 2018). Phylogenetic trees were visualized with FigTree 1.4.3 ( http://tree.bio.ed.ac.uk/software/figtree/ ), and layouts were carried out with Adobe Illustrator 2018 (Adobe, USA). 3. Result Four isolates were obtained and identified as two novel species, Epicoccum diplopterygii and Paraconiothyrium drymoglossoidis , belonging to Didymellaceae and Didymosphaeriaceae , respectively. For the updated taxonomic treatments of this study, Hyde et al. ( 2024 ) is followed. Identified taxa are listed alphabetically under the taxonomy and phylogeny section. 3.1 Taxonomy and Phylogeny Pleosporales Luttr. ex M.E. Barr (1987) Didymellaceae Gruyter, Aveskamp & Verkley, Mycol. Res.113(4): 516 (2009) Epicoccum Link, Mag. Gesell. naturf. Freunde, Berlin 7: 32 (1816) [1815] Epicoccum was introduced by Link (1816) and presently includes 186 records in Index Fungorum (2026). Xu et al. ( 2023 ) and Gomzhina et al. (2025) are followed for the taxonomic treatment of Epicoccum . Two isolates obtained in this study showed morphological resemblance to species of Epicoccum . The multi-gene phylogenetic tree consisted of LSU, ITS, rpb 2, and tub 2 gene regions. Macroventuria anomochaeta (CBS 525.71) and Neodidymelliopsis cannabis (CBS 121.75) were used as outgroup taxa. Ninety-eight strains were included in the combined gene analyses, comprising 3084 characters after alignment (580 for ITS, 969 for LSU, 988 for rpb 2, and 546 for tub 2). The best RAxML tree with a final likelihood value of -15602.333541 is presented (Fig. 1 ). The GTR + I+G evolutionary model was selected as the best-fit for the ITS, LSU, rpb 2, and tub 2 gene regions in the Bayesian analysis. The phylogenetic tree resulting had a topology closely resembling the ML tree presented. In both analyses (ML and BIPP). The sequences of our collected strains nested within the clades correspond to their respective genera, confirming their affinities. Certain nodes within the clades lacked robust support (they consistently appeared stable in repeated phylogenetic analyses). In the phylogenetic analysis, the two new isolats formed a distinct, well-supported clade, supporting their recognition as a novel species: Epicoccum diplopterygii . Epicoccum diplopterygii H. Li, K.D. Hyde & Manawas., sp. nov. Figure 2 Index Fungorum: IF 905123; Facesoffungi number: FoF 19502. Etymology The name refers to the host genus Diplopterygium chinense , from which it was isolated. Holotype MHKU 24–0837 Description : Endophytic in fresh leaves of Diplopterygium chinense . Sexual morph : Not observed. Asexual morph : Vegetative hyphae 3–6 µm gathering, branched, white to pale yellow. Conidiomata sporodochial 130–220 µm diam (n = 10) abundant, aggregated, superficial, pale brown initially, turning dark with age. Conidiophores 13–21 × 3–5 µm (x̅ = 18.2 × 4.6 µm, n = 10) µm, macronematous or semi-macronematous, branched, yellow to pale brown. Conidiophores cell 8–15 × 6–14 µm (x̅ = 11.6 × 10.1 µm, n = 20), verrucose, pale brown to dark brown. Conidia 14–20 × 11–15 µm (x̅ = 16.1 × 13.6 µm, n = 30), solitary, acrogenous, 1–3 septate, irregular in shape, sometimes subglobose, brown to dark brown, rough-walled. Culture characteristics Colony on PDA reaching 40 mm diam after 4 days at 25°C in the dark. Colonies fluffy with dense mycelia, on the surface, apricot in the centre with white margin, in reverse dark coral in the centre with orange irregular margin. Material examined China, Yunnan Province, Puer City, Ailao Mountain, from fresh leaves of Diplopterygium chinense , 16 June 2023, H. Li, ZHKUCC 24-1467 (MHKU 24–0837, holotype); ex-type living culture, ZHKUCC 24-1468. GenBank numbers : ZHKUCC 24-1467: ITS: PZ094857, LSU: PZ094861, rpb 2: PZ094688, tub 2: PZ094684; ZHKUCC 24-1468: ITS: PZ094858, LSU: PZ094862, rpb 2: PZ094689, tub 2: PZ094685. Notes In the phylogenetic analyses, our new isolates (ZHKUCC 24-1467 and ZHKUCC 24-1468) grouped with Epicoccum loliicola with 79% ML, 0.93 BIPP statistical support (Fig. 1 ). The asexual morph of E. loliicola was introduced by Xu et al. (2014) from Lolium multiflorum in China. Our isolates differ from Epicoccum loliicola (HMCE5) in having smaller conidia (14.8 × 11.4 µm vs. 16.1 × 13.6 µm), which are irregular in shape and 1–3-septate, whereas E. loliicola possesses globose to subglobose or pyriform, aseptate conidia that are brown to dark brown and verrucose (Xu et al. 2014). Pairwise sequence comparisons between the new isolate (ZHKUCC 24-1467) and E. loliicola (HMCE5) revealed nucleotide differences of 3/823 bp (0.4%) in LSU, 1/459 bp (0.2%) in ITS, 13/849 bp (1.5%) in rpb 2, and 13/269 bp (5%) in tub 2. Phylogeny also indicates that the clade containing our isolates and E. loliicola provides a sister lineage to E. yunnanense . The sexual morph of E. yunnanense differs from our isolate in having larger conidia (16.1 × 13.6 µm vs.14 × 7 µm), and the conidia surface of E. yunnanense are smooth, whereas those of our new isolates are irregular (Tian et al. 2024 ). Hence, based on significant differences, we present our isolate as a novel species, E. diplopterygii from Diplopterygium chinense . Didymosphaeriaceae Munk, Dansk bot. Ark. 15(no. 2): 128 (1953) Paraconiothyrium Verkley, Stud. Mycol. 50(2): 327 (2004) Paraconiothyrium was introduced by Verkley et al. ( 2004 ). Members of this genus play a crucial function as saprobes on dead plants, particularly dead wood, and occasionally on dead leaves (Verkley et al. 2004 , Hyde et al. 2013 , Ariyawansa et al. 2014 , Hongsanan et al. 2020 , Boonmee et al. 2021). Currently, there are 39 records of Paraconiothyrium in Index Fungorum (2026). In this study, the taxonomic treatment of Paraconiothyrium follows Tennakoon et al. ( 2022 ) and Xiong et al. ( 2024 ). Two isolates obtained in this study were morphologically similar to species of Paraconiothyrium . The multi-gene phylogenetic tree consists of LSU, ITS, and tub 2 gene regions. Tremateia arundicola (MFLU 16-1275) and T. guiyangensis (GZAAS01) were used as outgroup taxa (Tennakoon et al. 2022 ). Fourty-six strains were included in the combined gene analyses, comprising 2579 characters after the alignment (729 characters for ITS, 929 characters for LSU, and 685 characters for tub 2). The best RAxML tree with a final likelihood value of -9334.497796 is presented (Fig. 2 ). The GTR + I+G evolutionary model was selected as the best-fit for the LSU, ITS, and tub 2 gene regions in the Bayesian analysis. The phylogenetic tree resulting had a topology closely resembling the ML tree presented. In both analyses (ML and BIPP). The sequences of our collected strains nested within the clades correspond to their respective genera, confirming their affinities. Certain nodes within the clades lacked robust support (they consistently appeared stable in repeated phylogenetic analyses). In the phylogenetic analysis, the two new isolates formed a distinct, well-supported clade, supporting their recognition as a novel species: Paraconiothyrium drymoglossoidis . Paraconiothyrium drymoglossoidis H. Li, K.D. Hyde & Manawas., sp. nov. Figure 4 Index Fungorum number: IF 905124; Facesoffungi number: FoF 19503. Etymology The name refers to the host genus Lepidogrammitis drymoglossoides , from which it was isolated. Holotype MHKU 24–0614 Description : Endophytic in fresh leaves of Lepidogrammitis drymoglossoides . Sexual morph : Not observed. Asexual morph : Mycelium 3–5 µm wide, surface and endophytic hyphae consist of septate, branched hyphae that are white, smooth-walled and thin-walled. The hyphae are occasionally swollen in the septal regions. Chlamydospores 10–20 × 10–20 µm (x̅ = 16.8 × 15.6 µm, n = 30), globose, hyaline, integrated or terminal, mostly formed by the differentiation of swollen hyphae, aseptate, thick and smooth walled. Culture characteristics Colony on PDA reaching 20 mm diam after 6 days at 25°C in the dark. Colonies fluffy with dense mycelia, creamy white overall, slightly raised in the middle, reverse yellow spreads from dark to light from the center. Material examined China, Guangdong Province, Guangzhou City, Baiyun Mountain, from fresh leaves of Lepidogrammitis drymoglossoides , 16 August 2021, H. Li, ZHKUCC 24-1136 (MHKU 24–0614, holotype); ex-type living culture, ZHKUCC 24-1137. GenBank numbers : ZHKUCC 24-1136: ITS: PZ094859, LSU: PZ094863, tub 2: PZ094686; ZHKUCC 24-1137: ITS: PZ094860, LSU: PZ094864, tub 2: PZ094687. Notes : In the phylogenetic analyses, our new isolates (ZHKUCC 24-1136 and ZHKUCC 24-1137) grouped with Paraconiothyrium archidendri with 84% ML, 0.92 BIPP statistical support (Fig. 3 ). The asexual morph of P. archidendri was introduced by Verkley et al. ( 2014 ) from Pithecellobium bigeminum in Burma. Despite incubation for 2 months on various media (PDA, MEA, SNA, WA, and WA amended with pine needles), the isolate failed to sporulate. Only chlamydospores were produced on WA amended with pine needles, precluding morphological comparison with the asexual morph of P. archidendri . The compare the comparisons of our new isolate (ZHKUCC 24-1136) in LSU, ITS, and tub2 gene regions separately, showing P. archidendri (CBS 168.77, LSU: 5/905, 1%; ITS:3/605, 0.5%; tub2: 64/416, 15.4%). Thus, we present our isolate as a new species, P. drymoglossoidis from Lepidogrammitis drymoglossoides . 4. Discussion In this study, the asexual morphs of two novel Pleosporales species, Epicoccum diplopterygii and Paraconiothyrium drymoglossoidis , were identified using an integrative taxonomic approach combining multigene phylogenetic analyses with morphological characterization. Both taxa were isolated as endophytes from healthy pteridophytic hosts, highlighting the ecological breadth of Pleosporales and their ability to persist in asymptomatic associations with plants (Zhang et al. 2012). Notably, increasing evidence suggests that closely related taxa within Pleosporales may occupy distinct ecological niches, with frequent transitions among endophytic, saprobic, and pathogenic lifestyles (Zanne et al. 2020 , Hill et al. 2022 , Bhunjun et al. 2024 ). The discovery of these two endophytic species from ferns further supports the view that lifestyle plasticity is a common and evolutionary significant feature within the order (Hu et al. 2024 , Bhunjun et al. 2024 ). Species of Epicoccum are important plant-associated fungi, with numerous taxa reported as pathogens causing leaf spots, blights, and roots on a wide range of hosts, while others occur as endophytes or saprobes (Chen et al. 2020 , Chethana et al. 2019 , Lin et al. 2018 , Xu et al. 2022 ). The isolation of E. diplopterygii as an endophyte from Diplopterygium chinense is consistent with this ecological diversity. However, phylogenetic analyses revealed that E. diplopterygii is closely related to species with documented pathogenic or saprobic lifestyles ( E. loliicola and E. yunnanense ), indicating that endophytic isolation does not preclude pathogenic potential in phylogenetically related taxa. Such findings align with the concept of an endophyte–pathogen continuum, where asymptomatic colonization may represent a latent or conditional ecological state (Schulz and Boyle 2005 , Porras-Alfaro and Bayman 2011 , Hardoim et al. 2015 ). Consequently, the ecological role of E. diplopterygii warrants further investigation through pathogenicity assays and functional studies. Paraconiothyrium is a morphologically and ecologically diverse genus within Didymosphaeriaceae , typically characterized by pycnidial or eustromatic conidiomata and phialidic or annellidic conidiogenesis (Verkley et al. 2004 , Phukhamsakda et al. 2022, Yang et al. 2025 ). In the present study, Paraconiothyrium drymoglossoides was observed to form chlamydospores in culture, a feature that has not previously been reported for the genus. As we used molecular data, we were able to link the taxon to Paraconiothyrium (Shenoy et al. 2006). This first record of chlamydospore formation in Paraconiothyrium expands the known morphological concept of the genus and suggests that its structural diversity may have been underestimated. The presence of chlamydospores may reflect an adaptive strategy for survival under unfavorable environmental conditions (Watanabe 2002 , Agrios 2005 ), further emphasizing the ecological flexibility of the genus. Despite recent advances in multigene phylogenetic analyses, taxonomic instability persists within Didymellaceae and Didymosphaeriaceae , partly due to overlapping morphological characters and incomplete molecular sampling (Aveskamp et al. 2010 , Ariyawansa et al. 2014 , Tennakoon et al. 2022 ). The placement of newly described taxa, such as those reported here, underscores the necessity of integrative taxonomic approaches that combine detailed morphology with robust phylogenetic frameworks. Continued efforts to collect, culture, and sequence Pleosporales species from diverse hosts and habitats will be essential to improving phylogenetic resolution and achieving a more stable, natural classification within these families. Declarations Ethics approval: Not applicable. Consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors have no competing interests to declare that are relevant to the content of this article. Author Contribution All authors were involved in this research. H-L, IS-M and CH-Z sample collection, morphological observation, isolation, and preparation of related materials. The first draft of the manuscript was written by H-L. Research supervision, conceptualisation, and study design were provided by I-S-M and K-D-H. The manuscript was critically reviewed, revised, and edited by K-D-H, I-S-M, D-N-W, and C-S-B to improve clarity and meet the requirements for submission. All authors read and approved the final version of the manuscript Acknowledgement Hua Li would like to express gratitude to Mae Fah Luang University for awarding the tuition fee waiver scholarship for the PhD program. Data Availability The datasets generated for this study can be found in GenBank, NCBI and the accession numbers are given in Table 1. Newly introduced fungal names were registered at the Index Fungorum and the identification numbers are shown in their respective entries. References Adikaram NKB, Yakandawala DMD (2020) A checklist of plant pathogenic fungi and Oomycota in Sri Lanka. Ceylon J Sci 49:93–123. ttps://doi.org/10.4038/cjs.v49i1.7709 Agrios GN (2005) Plant Pathology. 5th ed. 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Mycosphere 16(1):536–1411. ttps://doi.org/10.5943/mycosphere/16/1/8 Tian Q, Liu JK, Hyde KD, Wanasinghe DN, Boonmee S, Jayasiri SC, Luo ZL, Taylor JE (2015) Phylogenetic relationships and morphological reappraisal of Melanommataceae (Pleosporales). Fungal Divers 74(1):267–324. ttps://doi.org/10.1007/s13225-015-0330-1 Tian XG, Bao DF, Karunarathna SC, Luo ZL, Hyde KD, Wanasinghe DN, Jayasiri SC, Tibpromma S, Li JF, Liu NG (2024) Taxonomy and phylogeny of ascomycetes associated with selected economically important monocotyledons in China and Thailand. Mycosphere 15:1–274. ttps://doi.org/10.5943/mycosphere/15/1/1 Verkley GJM, da Silva M, Wicklow DT, Crous PW (2004) Paraconiothyrium , a new genus to accommodate the mycoparasite Coniothyrium minitans , anamorphs of Paraphaeosphaeria , and four new species. Stud Mycol 50:323–336 Verkley GJM, Dukik K, Renfurm R, Göker M, Stielow JB (2014) Novel genera and species of coniothyrium-like fungi in Montagnulaceae (Ascomycota). Persoonia 32:25–51. ttps://doi.org/10.3767/003158514X679191 Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172:4238–4246. ttps://doi.org/10.1128/jb.172.8.4238-4246.1990 Wanasinghe DN, Phukhamsakda C, Hyde KD, Jeewon R, Lee HB, Jones EBG, Tibpromma S, Tennakoon DS, Dissanayake AJ, Jayasiri SC, Gafforov Y, Camporesi E, Bulgakov TS, Ekanayake AH, Perera RH, Samarakoon MC, Goonasekara ID, Mapook A, Li WJ, Senanayake IC, Li JF, Norphanphoun C, Doilom M, Bahkali AH, Xu JC, Mortimer PE, Tibell L, Tibell S, Karunarathna SC (2018) Fungal diversity notes 709–839: taxonomic and phylogenetic contributions to fungal taxa with an emphasis on fungi on Rosaceae. Fungal Divers 89:1–236. ttps://doi.org/10.1007/s13225-018-0395-7 Wanasinghe DN, Jones EBG, Camporesi E, Dissanayake AJ, Kamolhan S, Mortimer PE, Xu JC, Hyde KD (2016) Taxonomy and phylogeny of Laburnicola gen. nov. and Paramassariosphaeria gen. nov. ( Didymosphaeriaceae , Massarineae, Pleosporales). Fungal Biology 120:1354–1373. ttps://doi.org/10.1016/j.funbio.2016.07.004 Watanabe T (2002) Pictorial atlas of soil and seed fungi: morphologies of cultured fungi and key to species. CRC, Boca Raton White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR Protocols: A Guide to Methods and Applications. Academic, San Diego, pp 315–322. ttps://doi.org/10.1016/B978-0-12-372180-8.50042-1 Wijayawardene NN, Crous PW, Kirk PM, Hawksworth DL, Boonmee S, Braun U, Dai DQ, D’souza MJ, Diederich P, Dissanayake AJ, Doilom M, Hongsanan S, Jones EBG, Groenewald JZ, Jayawardena RS, Lawrey JD, Liu JK, Lücking R, Madrid H, Manawasinghe IS, Muggia L, Nelsen MP, Phookamsak R, Suetrong S, Tanaka K, Thambugala KM, Wanasinghe DN, Wikee S, Zhang Y, Aptroot A, Hyde KD (2014) Naming and outline of Dothideomycetes – 2014. Fungal Divers 69:1–55. ttps://doi.org/10.1007/s13225-014-0309-2 Xiong Y, Manawasinghe IS, Wanasinghe DN, Hyde KD, Jayawardena RS, Liu NG, Tian Q, Li JF (2024) Two new species and a new host record of Pleosporales (Dothideomycetes) from palm ( Arecaceae ) in Guangdong Province, China. N Z J Bot 62:165–191. ttps://doi.org/10.1080/0028825X.2023.2267429 Xu X, Li J, Yang X, Zhang H, Liu T, Zhang Y, Wang Y, Li Y (2022) Epicoccum spp. causing maize leaf spot in Heilongjiang Province, China. Plant Dis 106:3050–3060. ttps://doi.org/10.1094/PDIS-02-22-0352-RE Xu Z, Xu L, Liu J, Zhang Y, Hyde KD, Jayawardena RS, Wang Y (2023) High diversity of Epicoccum associated with leaf spot on Italian ryegrass in southwestern China: three new species and six new records. Plant Dis 107:2556–2570. ttps://doi.org/10.1094/PDIS-10-22-2432-RE Yang CL, Li XY, Xiang SS, Hyde KD, Jayawardena RS, Liu NG, Bao DF, Tian Q (2025) Microfungi associated with plant diseases on horticultural vegetation in southwestern China. Mycosphere 16:1861–2001. ttps://doi.org/10.5943/mycosphere/16/1/18 Yatskievych G, Ltd (2003) ttps://doi.org/10.1038/npg.els.0003679 Zanne AE, Abarenkov K, Afkhami ME, Aguilar-Trigueros CA, Bates S, Bhatnagar JM, Busby PE, Christian N, Cornwell WK, Crowther TW, Flores-Moreno H, Floudas D, Gazis R, Hibbett D, Kennedy P, Lindner DL, Maynard DS, Milo AM, Nilsson RH, Powell JR, Schildhauer M, Schilling JS, Treseder KK, Peay KG (2020) Fungal functional ecology: bringing a trait-based approach to plant-associated fungi. Biol Rev 95:409–433. ttps://doi.org/10.1111/brv.12570 Zhang JY, Hyde KD, Bao DF, Liu NG, Jayawardena RS, Tian Q, Li JF, Wang Y (2025) A worldwide checklist and morpho-molecular systematics of fungi associated with pteridophytes. Fungal Divers 131:1–273. ttps://doi.org/10.1007/s13225-024-00558-3 Zhang Y, Crous PW, Schoch CL, Bahkali AH, Guo LD, Hyde KD (2011) A molecular, morphological and ecological re-appraisal of Venturiales – a new order of Dothideomycetes. Fungal Divers 51:249–277. ttps://doi.org/10.1007/s13225-011-0147-8 Table 1 Table 1 is not available with this version. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 12 May, 2026 Reviewers invited by journal 29 Apr, 2026 Editor assigned by journal 18 Mar, 2026 Submission checks completed at journal 16 Mar, 2026 First submitted to journal 14 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9126008","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":632805470,"identity":"b0c0c09f-f152-4f2a-855f-2aa7f66781ee","order_by":0,"name":"Hua Li","email":"","orcid":"","institution":"Mae Fah Luang University","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Li","suffix":""},{"id":632805472,"identity":"5e1646f8-0b0c-463e-89a6-0913608d6e98","order_by":1,"name":"Dhanushka N. Wanasinghe","email":"","orcid":"","institution":"Kunming, Institute of Botany","correspondingAuthor":false,"prefix":"","firstName":"Dhanushka","middleName":"N.","lastName":"Wanasinghe","suffix":""},{"id":632805473,"identity":"931fd7be-45af-4a0c-b7f9-a006f546ca0c","order_by":2,"name":"Chitrabhanu Sharma Bhunjun","email":"","orcid":"","institution":"Mae Fah Luang University","correspondingAuthor":false,"prefix":"","firstName":"Chitrabhanu","middleName":"Sharma","lastName":"Bhunjun","suffix":""},{"id":632805474,"identity":"ff25a067-4612-407f-9e6a-2d7c30afb081","order_by":3,"name":"Chuanhao Zhou","email":"","orcid":"","institution":"Guangzhou Huali College","correspondingAuthor":false,"prefix":"","firstName":"Chuanhao","middleName":"","lastName":"Zhou","suffix":""},{"id":632805475,"identity":"d9d8525d-2658-4ade-aaf5-f512a4ff9b16","order_by":4,"name":"Kevin David Hyde","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBACCYYcBgYehho5EOfAAxK0HDMGa0kgQQtzYgOIR5QWyfbcYxJvKtjS54cdfgi0xU5Ot4GAFmmed2mSc87I5G68nWYA1JJsbHaAgBY5iRwzad42ttyNsxNAWg4kbiNOyz/mdMPZ6R+I0yIN1tLAnCAvnUOkLZI9b4wt5xw7ZrhBOqfgQIIBEX6ROJ5jeONNTY28/Oz0zR8+VNjJEdQCBCwSINIArNKAsHIQYP4AIuUbiFM9CkbBKBgFIxAAANTjQ9Bop9lxAAAAAElFTkSuQmCC","orcid":"","institution":"Mae Fah Luang University","correspondingAuthor":true,"prefix":"","firstName":"Kevin","middleName":"David","lastName":"Hyde","suffix":""},{"id":632805478,"identity":"f9e8d577-2c5a-4625-aead-42ee1468f6bd","order_by":5,"name":"Ishara Sandeepani Manawasinghe","email":"","orcid":"","institution":"Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ishara","middleName":"Sandeepani","lastName":"Manawasinghe","suffix":""}],"badges":[],"createdAt":"2026-03-15 03:38:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9126008/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9126008/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108808169,"identity":"812ea4a3-03db-4e28-89e6-ba4850c856da","added_by":"auto","created_at":"2026-05-08 15:40:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3024102,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree generated by maximum likelihood analyses based on the combined ITS,\u003cem\u003e \u003c/em\u003eLSU, \u003cem\u003erpb\u003c/em\u003e2, and \u003cem\u003etub\u003c/em\u003e2 sequence alignments of \u003cem\u003eEpicoccum\u003c/em\u003e. \u003cem\u003eMacroventuria anomochaeta \u003c/em\u003e(CBS 525.71)\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Neodidymelliopsis cannabis \u003c/em\u003e(CBS 121.75) are used as the outgroup taxa. Bootstrap support values for maximum likelihood (ML) equal to or greater than 70% and Bayesian posterior probabilities equal to or greater than 0.90 are indicated above branches as ML/BIPP. Ex-type strains are in bold, and the newly obtained strains are in red.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9126008/v1/cc39f1df5b9d9e43ce3d18ae.jpg"},{"id":108808143,"identity":"4f07b157-f714-41be-bcd1-af1a8994b081","added_by":"auto","created_at":"2026-05-08 15:40:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1177781,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEpicoccum diplopterygii\u003c/em\u003e (MHKU 24-0837, holotype). a, b Colony on PDA (front and reverse). c Sporodochia. d–e Conidiogenous cells with attached conidium. f–l Conidia. Scale bars: c = 0.1 mm, d–l = 10 μm.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9126008/v1/04f3c8f4f5433c50cb6660ba.jpg"},{"id":108808001,"identity":"2a347fb5-4b99-4852-8eef-70a0209c5c5a","added_by":"auto","created_at":"2026-05-08 15:38:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2651391,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree generated by maximum likelihood analyses based on the combined LSU, ITS, and \u003cem\u003etub\u003c/em\u003e2 sequence alignments of \u003cem\u003eParaconiothyrium\u003c/em\u003e. \u003cem\u003eTremateia arundicola \u003c/em\u003e(MFLU 16-1275) and \u003cem\u003eT\u003c/em\u003e.\u003cem\u003e guiyangensis \u003c/em\u003e(GZAAS01) are used as the outgroup taxa. Bootstrap support values for maximum likelihood (ML) equal to or greater than 70% and Bayesian posterior probabilities equal to or greater than 0.90 are indicated above branches as ML/BIPP. Ex-type strains are in bold, and the newly obtained strains are in red.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9126008/v1/b71db545b1db3dbcff1b891c.jpg"},{"id":108808097,"identity":"b4cab87d-fb0b-4ef6-b986-038575d4c4a6","added_by":"auto","created_at":"2026-05-08 15:39:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1701889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eParaconiothyrium drymoglossoidis \u003c/em\u003e(MHKU 24-0614, holotype). a, b Cultures on PDA from obverse and reverse. c Colonies on WA pine needle medium. d–g Mycelium and Chlamydospores. Scale bars: c = 1 mm, d–f = 10 μm.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9126008/v1/95f02f7b259434cfb1d17515.jpg"},{"id":108812261,"identity":"3f18a080-b45b-4fe6-a874-284d062025ba","added_by":"auto","created_at":"2026-05-08 16:10:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8918002,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9126008/v1/8eb1fbea-0b06-4d9c-82b7-c132f47806c6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Two New Species of Pleosporales from Pteridophytes in China","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePleosporales was formally established by Luttrell (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1955\u003c/span\u003e) and is currently recognized as the largest order in Dothideomycetes, with 91 families and 653 genera (Hyde et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Thiyagaraja et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Pleosporales encompasses a wide range of morphological and ecological diversity (Kirk et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Zhang et al. 2012, Hyde et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Wijayawardene et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Bhunjun et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Thiyagaraja et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Pleosporales species are characterized by perithecial ascomata, which are flask-shaped and typically possess papillae, and ostioles. The asexual morphs are coelomycetous and hyphomycetous (Hyde et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Pem et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, He et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The species exhibit remarkable ecological and morphological diversity, including saprobic, plant pathogenic, endophytic, and lichenicolous fungi, as well as species from freshwater and marine habitats (Shearer et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Hongsanan et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Phukhamsakda et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Yang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Thiyagaraja et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eDidymellaceae\u003c/em\u003e is a large family within the Pleosporales, which includes major genera of phoma-like fungi including \u003cem\u003eAscochyta\u003c/em\u003e, \u003cem\u003eDidymella\u003c/em\u003e, and \u003cem\u003eStagonosporopsis\u003c/em\u003e, and represents an underestimated component of fungal diversity (Chen et al.2017, 2023, Gomzhina et al. 2025). \u003cem\u003eDidymellaceae\u003c/em\u003e species are predominantly plant pathogens, such as \u003cem\u003eDidymella pinodes\u003c/em\u003e, which causes Ascochyta blight of field pea, and \u003cem\u003eParastagonospora nodorum\u003c/em\u003e, which causes Stagonospora nodorum blotch of wheat (Bretag et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1995\u003c/span\u003e, Solomon et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Quaedvlieg et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e); however, several species have also been reported as opportunistic pathogens of humans and animals worldwide (Boerema et al. 2004, de Hoog et al. 2011). Moreover, numerous taxa occur as plant endophytes or saprobes, often in association with both host and non-host organisms, highlighting the ecological versatility of \u003cem\u003eDidymellaceae\u003c/em\u003e (Schulz and Boyle \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Tahtamouni et al. 2016). Therefore, investigating \u003cem\u003eDidymellaceae\u003c/em\u003e species diversity is essential for understanding the taxonomy, evolution, and functional roles of plant-associated fungi (de Gruyter et al. 2009, Chen et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Hou et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eDidymosphaeriaceae\u003c/em\u003e represents a morphologically distinct but phylogenetically underexplored family within Pleosporales. Studies of its species diversity are important for resolving family boundaries and understanding evolutionary relationships among pleosporalean fungi (Hyde et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Ariyawansa et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Hyde et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eDidymosphaeriaceae\u003c/em\u003e is characterized by 1-septate ascospores and trabeculate pseudoparaphyses, which anastomose mostly above the asci (Liew et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Hyde et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Ariyawansa et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Wanasinghe et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Wanasinghe et al. (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) introduced two new genera, \u003cem\u003eLaburnicola\u003c/em\u003e and \u003cem\u003eParamassariosphaeria\u003c/em\u003e, based on combined phylogenetic analyses of LSU, SSU, and ITS sequence data. The family comprises 39 genera, with species occurring worldwide as endophytes, pathogens, and saprobes on a wide range of plant substrates (Liu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Wanasinghe et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Hyde et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePteridophytes are seedless vascular plants that reproduce by spores and typically inhabit moist environments (PPG 2016). In addition, they are an important part of the ground vegetation in many forest communities and, with about one-third of the species growing on the trunks and branches of trees, they are also an important component of many epiphytic plant communities (Yatskievych et al. 2003, PPG 2016). Recent studies have revealed that pteridophytes host a wide range of fungal taxa; however, knowledge of fungal diversity associated with pteridophytes remains limited (Bao et al. 2025, Zhang et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This study is a part of an ongoing investigation of fungal diversity associated with pteridophytes in South China, in which we collected fresh healthy samples to understand the endophytic diversity in pteridophytes. The objectives of this study are to determine species composition, assess phylogenetic relationships, and enhance current knowledge of fungal diversity in pteridophyte hosts. Here we described four new taxa from \u003cem\u003eDidymellaceae\u003c/em\u003e and \u003cem\u003eDidymosphaeriaceae\u003c/em\u003e with updated taxonomic positions through morphology combined with phylogenetic analyses. These data will not only contribute to a better understanding of host associations and geographic distribution of Pleosporales in China but also highlight their potential as valuable fungal resources for future studies on ecosystem functions.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sample collection, isolation, and morphological examination\u003c/h2\u003e \u003cp\u003eSamples of fungi were obtained from Baiyun Mountain (23\u0026deg;09'35\"N 113\u0026deg;17'40\"E) in Guangzhou city, Guangdong Province, and Ailao Mountain (24\u0026deg;31'23\"N 101\u0026deg;03'12\"E) in Yunnan Province, China. Healthy fresh leaves of \u003cem\u003eDiplopterygium chinense\u003c/em\u003e and \u003cem\u003eLepidogrammitis drymoglossoides\u003c/em\u003e were collected, and the specimens were brought to the lab in a Ziploc bag. The fresh leaves were washed with water to remove dirt and dust. Then, leaves were cut into small pieces (5\u0026times;5 mm); these small pieces were soaked for 30 s in a 75% ethanol solution, soaked for 30 s in a 2.5% sodium hypochlorite solution, and washed three times with sterilized distilled water for 30 s each. The sterilized tissue fragments were dried on sterile filter paper and then transferred to Potato Dextrose Agar (PDA) (Senanayake et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). All PDA plates were cultured at a constant temperature of 25 ℃ for 2\u0026ndash;4 days, and single hyphae were picked from the periphery of the grown colonies and inoculated on new PDA plates. After 2\u0026ndash;3 weeks of purification, pure cultures were obtained.\u003c/p\u003e \u003cp\u003eFor morphological characterization, potato dextrose agar medium (PDA), malt extract agar medium (MEA), synthetic nutrient-poor agar medium (SNA), water agar medium (WA), as well as on PDA, SNA, and WA supplemented with sterile pine needle segments were used. Morphological characterization was done by observing the mucus clumps produced on these media, including hyphae, ascomata, and ascospores, were observed and photographed using a Nikon Eclipse 80i microscope (Nikon, Japan). The colony morphology was recorded with a Nikon D300S digital camera (Nikon, Japan). All microscopic structures were measured using Tarosoft\u0026reg; Image Frame Works Version 0.9.7 and images were processed in Adobe Photoshop 2019 (Adobe Systems, San Jose, CA, USA) (Senanayake et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Xiong et al. 2021, Manawasinghe et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The morphological characteristics were examined using a stereomicroscope (Cnoptec sz650, China). The micro-morphological characteristics were observed and photographed using the Nikon Eclipse 80i microscope (Nikon, Japan). Colony colour (upper and reverse) was described using to the colour charts of Rayner (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). All pure cultures were deposited in the culture collection of Zhongkai University of Agriculture and Engineering (ZHKUCC), China. Herbarium materials were deposited in the herbaria of Zhongkai University of Agriculture and Engineering (MHKU), China. Data on the new species were deposited in the Greater Mekong Subregion database (Chaiwan et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 DNA extraction, PCR amplification, and sequencing\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted directly from one-week-old pure cultures using a Biospin Fungal Genomic DNA Extraction Kit (BioFlux, China) following the manufacturer\u0026rsquo;s instructions. The DNA was subjected to polymerase chain reaction (PCR) to amplify the internal transcribed spacer (ITS) gene using the primers ITS1 and ITS4 (White et al \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1990\u003c/span\u003e); the large subunit (LSU) gene using the primers LROR and LR5 (Vilgalys and Hester \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1990\u003c/span\u003e); the \u003cem\u003eβ-tubulin (tub\u003c/em\u003e2) gene using the primers TUB-2Fd and TUB-4R (Glass and Donaldson \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1995\u003c/span\u003e); and the RNA polymerase II subunit (\u003cem\u003erpb\u003c/em\u003e2) gene using the primers fRPB2-5F and fRPB2-7cr (Liu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The final Polymerase chain reaction (PCR) consisted of 12.5 \u0026micro;L of 2\u0026times;Taq Master Mix (Dye Plus) (mixture of FastTaq TM DNA Polymerase, buffer, dNTP Mixture, and stabilizer) (Beijing Qingke Biological Technology Co., Ltd., Beijing, PR China), 9 \u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO, 1 \u0026micro;L of primers, and 1 \u0026micro;L of DNA template. PCR reactions were performed using the program for ITS, LSU, \u003cem\u003etub\u003c/em\u003e2, and \u003cem\u003erpb\u003c/em\u003e2, as described by Conforto et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and Tennakoon et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). PCR products were analysed on 1% agarose gels and sent to Tianyi (Guangzhou, China) Co., Ltd for sequencing. All sequence data generated in this study were deposited in NCBI GenBank.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Phylogenetic analyses\u003c/h2\u003e \u003cp\u003eThe chromatograms were examined using BioEdit version 7.0.5.2 (Hall 1999), and low\u0026ndash;quality regions were trimmed before alignment. All sequences were subjected to BLASTn searches in the National Center for Biotechnology Information (NCBI). Based on the BLASTn search, we identified our isolates belonging to \u003cem\u003eEpicoccum\u003c/em\u003e and \u003cem\u003eParaconiothyrium\u003c/em\u003e. All reference sequences used in this study were downloaded from GenBank following Cao et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), Gomzhina et al. (2025), and Manawasinghe et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and are listed in Table\u0026nbsp;1. Alignments for each locus were generated using MAFFT 7 webservers (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mafft.cbrc.jp/alignment/server\u003c/span\u003e\u003cspan address=\"http://mafft.cbrc.jp/alignment/server\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed 20 January 2026) and were manually improved using BioEdit 7.0.5.2 (Hall 1999). Multigene phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI) analysis.\u003c/p\u003e \u003cp\u003eMaximum likelihood analysis was performed using the IQ-TREE webserver (Trifinopoulos et al. 2016). The branch support of the phylogenetic tree was tested with 1000 replicates of Ultrafast bootstrap (Minh et al. 2013) and SH-aLRTtest (Guindon et al. 2010). Bayesian inference analysis was performed using MrBayes 3.2.7a on XSEDE on the CIPRES portal (Huelsenbeck et al. 2001, Miller et al. 2010, Stamatakis et al. 2014). Posterior probabilities (PP) were defined by the Markov chain Monte Carlo (MCMC) sampling method (Rannala \u0026amp; Yang 1996, Huelsenbeck \u0026amp; Ronquist 2001). Two parallel runs were conducted using the default settings. Six simultaneous Markov chains were run for 2\u0026nbsp;million generations, and trees were sampled every 1000th generation. The first 25% of trees were discarded as the burn-in phase. The remaining trees were used for calculating posterior probabilities in the majority rule consensus tree (Doll and Jacquemin 2018). Phylogenetic trees were visualized with FigTree 1.4.3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tree.bio.ed.ac.uk/software/figtree/\u003c/span\u003e\u003cspan address=\"http://tree.bio.ed.ac.uk/software/figtree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and layouts were carried out with Adobe Illustrator 2018 (Adobe, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result","content":"\u003cp\u003eFour isolates were obtained and identified as two novel species, \u003cem\u003eEpicoccum diplopterygii\u003c/em\u003e and \u003cem\u003eParaconiothyrium drymoglossoidis\u003c/em\u003e, belonging to \u003cem\u003eDidymellaceae\u003c/em\u003e and \u003cem\u003eDidymosphaeriaceae\u003c/em\u003e, respectively. For the updated taxonomic treatments of this study, Hyde et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) is followed. Identified taxa are listed alphabetically under the taxonomy and phylogeny section.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Taxonomy and Phylogeny\u003c/h2\u003e \u003cp\u003e \u003cb\u003ePleosporales\u003c/b\u003e Luttr. ex M.E. Barr (1987)\u003c/p\u003e \u003cp\u003e \u003cb\u003eDidymellaceae\u003c/b\u003e Gruyter, Aveskamp \u0026amp; Verkley, Mycol. Res.113(4): 516 (2009)\u003c/p\u003e \u003cp\u003e \u003cb\u003eEpicoccum\u003c/b\u003e Link, Mag. Gesell. naturf. Freunde, Berlin 7: 32 (1816) [1815]\u003c/p\u003e \u003cp\u003e \u003cem\u003eEpicoccum\u003c/em\u003e was introduced by Link (1816) and presently includes 186 records in Index Fungorum (2026). Xu et al. (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Gomzhina et al. (2025) are followed for the taxonomic treatment of \u003cem\u003eEpicoccum\u003c/em\u003e. Two isolates obtained in this study showed morphological resemblance to species of \u003cem\u003eEpicoccum\u003c/em\u003e. The multi-gene phylogenetic tree consisted of LSU, ITS, \u003cem\u003erpb\u003c/em\u003e2, and \u003cem\u003etub\u003c/em\u003e2 gene regions. \u003cem\u003eMacroventuria anomochaeta\u003c/em\u003e (CBS 525.71) and \u003cem\u003eNeodidymelliopsis cannabis\u003c/em\u003e (CBS 121.75) were used as outgroup taxa. Ninety-eight strains were included in the combined gene analyses, comprising 3084 characters after alignment (580 for ITS, 969 for LSU, 988 for \u003cem\u003erpb\u003c/em\u003e2, and 546 for \u003cem\u003etub\u003c/em\u003e2). The best RAxML tree with a final likelihood value of -15602.333541 is presented (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The GTR\u0026thinsp;+\u0026thinsp;I+G evolutionary model was selected as the best-fit for the ITS, LSU, \u003cem\u003erpb\u003c/em\u003e2, and \u003cem\u003etub\u003c/em\u003e2 gene regions in the Bayesian analysis. The phylogenetic tree resulting had a topology closely resembling the ML tree presented. In both analyses (ML and BIPP). The sequences of our collected strains nested within the clades correspond to their respective genera, confirming their affinities. Certain nodes within the clades lacked robust support (they consistently appeared stable in repeated phylogenetic analyses). In the phylogenetic analysis, the two new isolats formed a distinct, well-supported clade, supporting their recognition as a novel species: \u003cem\u003eEpicoccum diplopterygii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEpicoccum diplopterygii\u003c/b\u003e H. Li, K.D. Hyde \u0026amp; Manawas., \u003cb\u003esp. nov.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e \u003cp\u003eIndex Fungorum: IF 905123; Facesoffungi number: FoF 19502.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEtymology\u003c/strong\u003e \u003cp\u003eThe name refers to the host genus \u003cem\u003eDiplopterygium chinense\u003c/em\u003e, from which it was isolated.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHolotype\u003c/strong\u003e \u003cp\u003eMHKU 24\u0026ndash;0837\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDescription\u003c/b\u003e: \u003cem\u003eEndophytic\u003c/em\u003e in fresh leaves of \u003cem\u003eDiplopterygium chinense\u003c/em\u003e. \u003cem\u003eSexual morph\u003c/em\u003e: Not observed. \u003cb\u003eAsexual morph\u003c/b\u003e: Vegetative hyphae 3\u0026ndash;6 \u0026micro;m gathering, branched, white to pale yellow. \u003cem\u003eConidiomata\u003c/em\u003e sporodochial 130\u0026ndash;220 \u0026micro;m diam (n\u0026thinsp;=\u0026thinsp;10) abundant, aggregated, superficial, pale brown initially, turning dark with age. \u003cem\u003eConidiophores\u003c/em\u003e 13\u0026ndash;21 \u0026times; 3\u0026ndash;5 \u0026micro;m (x̅ = 18.2 \u0026times; 4.6 \u0026micro;m, n\u0026thinsp;=\u0026thinsp;10) \u0026micro;m, macronematous or semi-macronematous, branched, yellow to pale brown. \u003cem\u003eConidiophores cell\u003c/em\u003e 8\u0026ndash;15 \u0026times; 6\u0026ndash;14 \u0026micro;m (x̅ = 11.6 \u0026times; 10.1 \u0026micro;m, n\u0026thinsp;=\u0026thinsp;20), verrucose, pale brown to dark brown. \u003cem\u003eConidia\u003c/em\u003e 14\u0026ndash;20 \u0026times; 11\u0026ndash;15 \u0026micro;m (x̅ = 16.1 \u0026times; 13.6 \u0026micro;m, n\u0026thinsp;=\u0026thinsp;30), solitary, acrogenous, 1\u0026ndash;3 septate, irregular in shape, sometimes subglobose, brown to dark brown, rough-walled.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCulture characteristics\u003c/strong\u003e \u003cp\u003eColony on PDA reaching 40 mm diam after 4 days at 25\u0026deg;C in the dark. Colonies fluffy with dense mycelia, on the surface, apricot in the centre with white margin, in reverse dark coral in the centre with orange irregular margin.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMaterial examined\u003c/strong\u003e \u003cp\u003eChina, Yunnan Province, Puer City, Ailao Mountain, from fresh leaves of \u003cem\u003eDiplopterygium chinense\u003c/em\u003e, 16 June 2023, H. Li, ZHKUCC 24-1467 (MHKU 24\u0026ndash;0837, holotype); ex-type living culture, ZHKUCC 24-1468.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGenBank numbers\u003c/b\u003e: ZHKUCC 24-1467: ITS: PZ094857, LSU: PZ094861, \u003cem\u003erpb\u003c/em\u003e2: PZ094688, \u003cem\u003etub\u003c/em\u003e2: PZ094684; ZHKUCC 24-1468: ITS: PZ094858, LSU: PZ094862, \u003cem\u003erpb\u003c/em\u003e2: PZ094689, \u003cem\u003etub\u003c/em\u003e2: PZ094685.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNotes\u003c/strong\u003e \u003cp\u003eIn the phylogenetic analyses, our new isolates (ZHKUCC 24-1467 and ZHKUCC 24-1468) grouped with \u003cem\u003eEpicoccum loliicola\u003c/em\u003e with 79% ML, 0.93 BIPP statistical support (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The asexual morph of \u003cem\u003eE. loliicola\u003c/em\u003e was introduced by Xu et al. (2014) from \u003cem\u003eLolium multiflorum\u003c/em\u003e in China. Our isolates differ from \u003cem\u003eEpicoccum loliicola\u003c/em\u003e (HMCE5) in having smaller conidia (14.8 \u0026times; 11.4 \u0026micro;m vs. 16.1 \u0026times; 13.6 \u0026micro;m), which are irregular in shape and 1\u0026ndash;3-septate, whereas \u003cem\u003eE. loliicola\u003c/em\u003e possesses globose to subglobose or pyriform, aseptate conidia that are brown to dark brown and verrucose (Xu et al. 2014). Pairwise sequence comparisons between the new isolate (ZHKUCC 24-1467) and \u003cem\u003eE. loliicola\u003c/em\u003e (HMCE5) revealed nucleotide differences of 3/823 bp (0.4%) in LSU, 1/459 bp (0.2%) in ITS, 13/849 bp (1.5%) in \u003cem\u003erpb\u003c/em\u003e2, and 13/269 bp (5%) in \u003cem\u003etub\u003c/em\u003e2. Phylogeny also indicates that the clade containing our isolates and \u003cem\u003eE. loliicola\u003c/em\u003e provides a sister lineage to \u003cem\u003eE. yunnanense\u003c/em\u003e. The sexual morph of \u003cem\u003eE. yunnanense\u003c/em\u003e differs from our isolate in having larger conidia (16.1 \u0026times; 13.6 \u0026micro;m vs.14 \u0026times; 7 \u0026micro;m), and the conidia surface of \u003cem\u003eE. yunnanense\u003c/em\u003e are smooth, whereas those of our new isolates are irregular (Tian et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Hence, based on significant differences, we present our isolate as a novel species, \u003cem\u003eE. diplopterygii\u003c/em\u003e from \u003cem\u003eDiplopterygium chinense\u003c/em\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDidymosphaeriaceae\u003c/b\u003e Munk, Dansk bot. Ark. 15(no. 2): 128 (1953)\u003c/p\u003e \u003cp\u003e \u003cb\u003eParaconiothyrium\u003c/b\u003e Verkley, Stud. Mycol. 50(2): 327 (2004)\u003c/p\u003e \u003cp\u003e \u003cem\u003eParaconiothyrium\u003c/em\u003e was introduced by Verkley et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Members of this genus play a crucial function as saprobes on dead plants, particularly dead wood, and occasionally on dead leaves (Verkley et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Hyde et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Ariyawansa et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Hongsanan et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Boonmee et al. 2021). Currently, there are 39 records of \u003cem\u003eParaconiothyrium\u003c/em\u003e in Index Fungorum (2026). In this study, the taxonomic treatment of \u003cem\u003eParaconiothyrium\u003c/em\u003e follows Tennakoon et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and Xiong et al. (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Two isolates obtained in this study were morphologically similar to species of \u003cem\u003eParaconiothyrium\u003c/em\u003e. The multi-gene phylogenetic tree consists of LSU, ITS, and \u003cem\u003etub\u003c/em\u003e2 gene regions. \u003cem\u003eTremateia arundicola\u003c/em\u003e (MFLU 16-1275) and \u003cem\u003eT. guiyangensis\u003c/em\u003e (GZAAS01) were used as outgroup taxa (Tennakoon et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Fourty-six strains were included in the combined gene analyses, comprising 2579 characters after the alignment (729 characters for ITS, 929 characters for LSU, and 685 characters for \u003cem\u003etub\u003c/em\u003e2). The best RAxML tree with a final likelihood value of -9334.497796 is presented (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The GTR\u0026thinsp;+\u0026thinsp;I+G evolutionary model was selected as the best-fit for the LSU, ITS, and \u003cem\u003etub\u003c/em\u003e2 gene regions in the Bayesian analysis. The phylogenetic tree resulting had a topology closely resembling the ML tree presented. In both analyses (ML and BIPP). The sequences of our collected strains nested within the clades correspond to their respective genera, confirming their affinities. Certain nodes within the clades lacked robust support (they consistently appeared stable in repeated phylogenetic analyses). In the phylogenetic analysis, the two new isolates formed a distinct, well-supported clade, supporting their recognition as a novel species: \u003cem\u003eParaconiothyrium drymoglossoidis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eParaconiothyrium drymoglossoidis\u003c/b\u003e H. Li, K.D. Hyde \u0026amp; Manawas., sp. nov. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e \u003cp\u003eIndex Fungorum number: IF 905124; Facesoffungi number: FoF 19503.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEtymology\u003c/strong\u003e \u003cp\u003eThe name refers to the host genus \u003cem\u003eLepidogrammitis drymoglossoides\u003c/em\u003e, from which it was isolated.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHolotype\u003c/strong\u003e \u003cp\u003eMHKU 24\u0026ndash;0614\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDescription\u003c/b\u003e: \u003cem\u003eEndophytic\u003c/em\u003e in fresh leaves of \u003cem\u003eLepidogrammitis drymoglossoides\u003c/em\u003e. \u003cb\u003eSexual morph\u003c/b\u003e: Not observed. \u003cb\u003eAsexual morph\u003c/b\u003e: \u003cem\u003eMycelium\u003c/em\u003e 3\u0026ndash;5 \u0026micro;m wide, surface and endophytic hyphae consist of septate, branched hyphae that are white, smooth-walled and thin-walled. The hyphae are occasionally swollen in the septal regions. \u003cem\u003eChlamydospores\u003c/em\u003e 10\u0026ndash;20 \u0026times; 10\u0026ndash;20 \u0026micro;m (x̅ = 16.8 \u0026times; 15.6 \u0026micro;m, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;30), globose, hyaline, integrated or terminal, mostly formed by the differentiation of swollen hyphae, aseptate, thick and smooth walled.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCulture characteristics\u003c/strong\u003e \u003cp\u003eColony on PDA reaching 20 mm diam after 6 days at 25\u0026deg;C in the dark. Colonies fluffy with dense mycelia, creamy white overall, slightly raised in the middle, reverse yellow spreads from dark to light from the center.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMaterial examined\u003c/strong\u003e \u003cp\u003eChina, Guangdong Province, Guangzhou City, Baiyun Mountain, from fresh leaves of \u003cem\u003eLepidogrammitis drymoglossoides\u003c/em\u003e, 16 August 2021, H. Li, ZHKUCC 24-1136 (MHKU 24\u0026ndash;0614, holotype); ex-type living culture, ZHKUCC 24-1137.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGenBank numbers\u003c/b\u003e: ZHKUCC 24-1136: ITS: PZ094859, LSU: PZ094863, \u003cem\u003etub\u003c/em\u003e2: PZ094686; ZHKUCC 24-1137: ITS: PZ094860, LSU: PZ094864, \u003cem\u003etub\u003c/em\u003e2: PZ094687.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNotes\u003c/b\u003e: In the phylogenetic analyses, our new isolates (ZHKUCC 24-1136 and ZHKUCC 24-1137) grouped with \u003cem\u003eParaconiothyrium archidendri\u003c/em\u003e with 84% ML, 0.92 BIPP statistical support (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The asexual morph of \u003cem\u003eP. archidendri\u003c/em\u003e was introduced by Verkley et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) from \u003cem\u003ePithecellobium bigeminum\u003c/em\u003e in Burma. Despite incubation for 2 months on various media (PDA, MEA, SNA, WA, and WA amended with pine needles), the isolate failed to sporulate. Only chlamydospores were produced on WA amended with pine needles, precluding morphological comparison with the asexual morph of \u003cem\u003eP. archidendri\u003c/em\u003e. The compare the comparisons of our new isolate (ZHKUCC 24-1136) in LSU, ITS, and tub2 gene regions separately, showing \u003cem\u003eP. archidendri\u003c/em\u003e (CBS 168.77, LSU: 5/905, 1%; ITS:3/605, 0.5%; tub2: 64/416, 15.4%). Thus, we present our isolate as a new species, \u003cem\u003eP. drymoglossoidis\u003c/em\u003e from \u003cem\u003eLepidogrammitis drymoglossoides\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, the asexual morphs of two novel Pleosporales species, \u003cem\u003eEpicoccum diplopterygii\u003c/em\u003e and \u003cem\u003eParaconiothyrium drymoglossoidis\u003c/em\u003e, were identified using an integrative taxonomic approach combining multigene phylogenetic analyses with morphological characterization. Both taxa were isolated as endophytes from healthy pteridophytic hosts, highlighting the ecological breadth of Pleosporales and their ability to persist in asymptomatic associations with plants (Zhang et al. 2012). Notably, increasing evidence suggests that closely related taxa within Pleosporales may occupy distinct ecological niches, with frequent transitions among endophytic, saprobic, and pathogenic lifestyles (Zanne et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Hill et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Bhunjun et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The discovery of these two endophytic species from ferns further supports the view that lifestyle plasticity is a common and evolutionary significant feature within the order (Hu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Bhunjun et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSpecies of \u003cem\u003eEpicoccum\u003c/em\u003e are important plant-associated fungi, with numerous taxa reported as pathogens causing leaf spots, blights, and roots on a wide range of hosts, while others occur as endophytes or saprobes (Chen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Chethana et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Lin et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Xu et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The isolation of \u003cem\u003eE. diplopterygii\u003c/em\u003e as an endophyte from \u003cem\u003eDiplopterygium chinense\u003c/em\u003e is consistent with this ecological diversity. However, phylogenetic analyses revealed that \u003cem\u003eE. diplopterygii\u003c/em\u003e is closely related to species with documented pathogenic or saprobic lifestyles (\u003cem\u003eE. loliicola\u003c/em\u003e and \u003cem\u003eE. yunnanense\u003c/em\u003e), indicating that endophytic isolation does not preclude pathogenic potential in phylogenetically related taxa. Such findings align with the concept of an endophyte\u0026ndash;pathogen continuum, where asymptomatic colonization may represent a latent or conditional ecological state (Schulz and Boyle \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Porras-Alfaro and Bayman \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Hardoim et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Consequently, the ecological role of \u003cem\u003eE. diplopterygii\u003c/em\u003e warrants further investigation through pathogenicity assays and functional studies.\u003c/p\u003e \u003cp\u003e \u003cem\u003eParaconiothyrium\u003c/em\u003e is a morphologically and ecologically diverse genus within \u003cem\u003eDidymosphaeriaceae\u003c/em\u003e, typically characterized by pycnidial or eustromatic conidiomata and phialidic or annellidic conidiogenesis (Verkley et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Phukhamsakda et al. 2022, Yang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In the present study, \u003cem\u003eParaconiothyrium drymoglossoides\u003c/em\u003e was observed to form chlamydospores in culture, a feature that has not previously been reported for the genus. As we used molecular data, we were able to link the taxon to \u003cem\u003eParaconiothyrium\u003c/em\u003e (Shenoy et al. 2006). This first record of chlamydospore formation in \u003cem\u003eParaconiothyrium\u003c/em\u003e expands the known morphological concept of the genus and suggests that its structural diversity may have been underestimated. The presence of chlamydospores may reflect an adaptive strategy for survival under unfavorable environmental conditions (Watanabe \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Agrios \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), further emphasizing the ecological flexibility of the genus.\u003c/p\u003e \u003cp\u003eDespite recent advances in multigene phylogenetic analyses, taxonomic instability persists within \u003cem\u003eDidymellaceae\u003c/em\u003e and \u003cem\u003eDidymosphaeriaceae\u003c/em\u003e, partly due to overlapping morphological characters and incomplete molecular sampling (Aveskamp et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Ariyawansa et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Tennakoon et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The placement of newly described taxa, such as those reported here, underscores the necessity of integrative taxonomic approaches that combine detailed morphology with robust phylogenetic frameworks. Continued efforts to collect, culture, and sequence Pleosporales species from diverse hosts and habitats will be essential to improving phylogenetic resolution and achieving a more stable, natural classification within these families.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval:\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to participate:\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication:\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests:\u003c/strong\u003e \u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors were involved in this research. H-L, IS-M and CH-Z sample collection, morphological observation, isolation, and preparation of related materials. The first draft of the manuscript was written by H-L. Research supervision, conceptualisation, and study design were provided by I-S-M and K-D-H. The manuscript was critically reviewed, revised, and edited by K-D-H, I-S-M, D-N-W, and C-S-B to improve clarity and meet the requirements for submission. All authors read and approved the final version of the manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eHua Li would like to express gratitude to Mae Fah Luang University for awarding the tuition fee waiver scholarship for the PhD program.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated for this study can be found in GenBank, NCBI and the accession numbers are given in Table 1. Newly introduced fungal names were registered at the Index Fungorum and the identification numbers are shown in their respective entries.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdikaram NKB, Yakandawala DMD (2020) A checklist of plant pathogenic fungi and Oomycota in Sri Lanka. Ceylon J Sci 49:93\u0026ndash;123. ttps://doi.org/10.4038/cjs.v49i1.7709\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgrios GN (2005) Plant Pathology. 5th ed. Elsevier Academic Press. ttps://doi.org/10.1016/C2009-0-02037-6\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAriyawansa HA, Camporesi E, Thambugala KM, Mapook A, Kang JC, Alias SA, Chukeatirote E, Thines M, McKenzie EHC, Hyde KD (2014) Confusion surrounding \u003cem\u003eDidymosphaeria\u003c/em\u003e\u0026mdash;phylogenetic and morphological evidence suggest \u003cem\u003eDidymosphaeriaceae\u003c/em\u003e is not a distinct family. 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Fungal Divers 51:249\u0026ndash;277. ttps://doi.org/10.1007/s13225-011-0147-8\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is not available with this version.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"2 new species, Didymellaceae, Didymosphaeriaceae, Endophytic fungi","lastPublishedDoi":"10.21203/rs.3.rs-9126008/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9126008/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePleosporales is one of the largest and most diverse orders in Dothideomycetes. This order encompasses numerous families and genera characterized by considerable morphological diversity and a wide range of ecological lifestyles. During an investigation of fungal diversity associated with pteridophytes in South China, healthy samples of \u003cem\u003eDiplopterygium chinense\u003c/em\u003e and \u003cem\u003eLepidogrammitis drymoglossoides\u003c/em\u003e were collected from Yunnan and Guangdong provinces. Endophytic fungi were isolated using the tissue isolation method. Among the isolates, four strains were identified as members of Pleosporales based on multi-locus phylogenetic analyses using the internal transcribed spacer (ITS), the large subunit (LSU), the \u003cem\u003eβ-tubulin\u003c/em\u003e (\u003cem\u003etub\u003c/em\u003e2), and the RNA polymerase II subunit (\u003cem\u003erpb\u003c/em\u003e2) sequence data, together with morphological characteristics of conidiation. These strains were assigned to two species each in \u003cem\u003eEpicoccum\u003c/em\u003e and \u003cem\u003eParaconiothyrium\u003c/em\u003e and are herein described as two new species, \u003cem\u003eEpicoccum diplopterygii\u003c/em\u003e and \u003cem\u003eParaconiothyrium drymoglossoidis\u003c/em\u003e. Detailed morphological illustrations, taxonomic descriptions, and molecular data are provided. This study expands the current knowledge of Pleosporales diversity and host associations in pteridophytic plants, providing a foundation for future taxonomic and ecological studies of fungi associated with pteridophytes in China.\u003c/p\u003e","manuscriptTitle":"Two New Species of Pleosporales from Pteridophytes in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 12:27:41","doi":"10.21203/rs.3.rs-9126008/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"268902809356768815514129924835437504711","date":"2026-05-12T08:47:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-29T08:10:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-18T18:33:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-16T19:22:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Current Microbiology","date":"2026-03-15T03:32:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"141b519f-3b6f-4934-886a-9c954e2faee7","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"268902809356768815514129924835437504711","date":"2026-05-12T08:47:14+00:00","index":15,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T12:27:41+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 12:27:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9126008","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9126008","identity":"rs-9126008","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}