683 MycoKeys MycoKeys 113: 101-121 (2025) DOI: 10.3897/mycokeys.113.137678 Research Article Two novel hyphomycetes associated with ferns from China Jing-Yi Zhang'2*®, Kevin D. Hyde?3*®, Li-Juan Zhang'2*®, Song Bai®®, Dan-Feng Bao™, Fatimah Al-Otibi*®, Yong-Zhong Lu'® an oF WO NY Province 550025, China School of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang 550003, China Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 22452, Riyadh 11495, Saudi Arabia Guizhou Industry Polytechnic College, Guiyang 550008, China Engineering and Research Center for Southwest Bio-Pharmaceutical Resources of National Education Ministry of China, Guizhou University, Guiyang, Guizhou Corresponding authors: Song Bai (basonmail@163.com); Yong-Zhong Lu (yzlu@git.edu.cn) This article is part of: Exploring the Hidden Fungal Diversity: Biodiversity, Taxonomy, and Phylogeny of Saprobic Fungi Edited by Samantha C. Karunarathna, Danushka Sandaruwan Tennakoon, Ajay Kumar Gautam OPEN Qaceess Academic editor: Danushka Sandaruwan Tennakoon Received: 23 September 2024 Accepted: 7 December 2024 Published: 31 January 2025 Citation: Zhang J-Y, Hyde KD, Zhang L-J, Bai S, Bao D-F, Al-Otibi F, Lu Y-Z (2025) Two novel hyphomycetes associated with ferns from China. MycoKkeys 113: 101-121. https://doi. org/10.3897/mycokeys.113.137678 Copyright: © Jing-Yi Zhang 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). Abstract During an ongoing investigation of fungi associated with ferns in southwestern Chi- na, three hyphomycetes were discovered on the dead rachises of Angiopteris fok- iensis and an unidentified fern. Based on morphology and multi-gene phylogenetic analyses, Arthrobotrys angiopteridis and Corynespora septata are introduced as new species. Arthrobotrys angiopteridis is a nematode-trapping fungus characterized by macronematous, mononematous, hyaline conidiophores, conidiogenous cells with polyblastic denticles at each node, and 0-1-septate, clavate to elongate pyriform, hyaline conidia. Corynespora septata features macronematous, mononematous, pale brown to dark brown conidiophores, integrated, monotretic conidiogenous cells and up to 7-distoseptate with one true septum, subcylindrical to obclavate, hyaline to pale brown conidia. Detailed descriptions and illustrations of these two new species are provided, along with morphological comparisons of the new taxa with closely related species. Key words: Asexual morph, new species, phylogeny, pteridophytes, taxonomy Introduction Fungi associated with ferns have historically been overlooked and have re- ceived insufficient research attention, despite being an immensely promising and diverse group (Kirschner and Liu 2014; Kirschner et al. 2019; Medel-Ortiz et al. 2020). Recent studies have provided evidence supporting this perspective, with many new species of fern-associated fungi being discovered (Guatimosim et al. 2016a; Guatimosim et al. 201 6b; Li et al. 2023a; Wu and Diao 2023; Zhang et al. 2023b). China has the highest fern species, with about 2,130 species, ac- counting for 19% of the total global fern and fern-allies species (Lin et al. 2013; Zhou et al. 2016). Notably, the Yunnan region houses approximately 1,500 fern species, while Guizhou has about 800, ranking them as the first and third most diverse regions for fern species in China (Li et al. 2015; Zhou et al. 2016). These 101 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China regions boast abundant and diverse fern resources, offering great potential for the discovery of even more interesting fungi (Zhang et al. 2023b; Hyde et al. 2024a; Phookamsak et al. 2024). Arthrobotrys was introduced by Corda (1839), with A. superba Corda as the type species and belongs in Orbiliaceae, Orbiliales, Orbiliomycetes (Wi- jayawardene et al. 2022; Hyde et al. 2024b). Arthrobotrys is characterized by simple or branched conidiophores and obovoid, elliptic, pyriform, 0-3-sep- tate conidia growing asynchronously on the nodes or short denticles of co- nidiophores (Yu et al. 2014; Zhang et al. 2022a, b, 2023a, 2024; Yang et al. 2023a; Jin et al. 2024). Arthrobotrys is the most complex and largest genus among Orbiliaceae nematode-trapping fungi, comprising 78 accepted spe- cies characterized by producing adhesive networks to capture nematodes (Li et al. 2005; Yang et al. 2012; Yu et al. 2014; Zhang and Hyde 2014; Jiang et al. 2017; Zhang et al. 2023a, 2024; Thiyagaraja et al. 2024). These fun- gal species mainly occur in soil or sediment in various ecosystems such as farmland, forests, mangroves, and freshwater. They have also been recorded in hot springs, animal waste, and tree trunks worldwide (Duddington 1954; Swe et al. 2008; Kim et al. 2001; Kumar et al. 2011; Yu et al. 2014; Jiang et al. 2017; Zhang et al. 2022a, b). Corynespora was established by Glssow (1905) with C. mazei Giissow as the type species. Corynespora was placed in Corynesporascaceae as the asex- ual morph associated with Corynesporasca, based on cultural studies (Sivane- san 1996), although the latter is still accepted as a distinct genus (Wijayawar- dene et al. 2022; Hyde et al. 2024b; Pem et al. 2024). Phylogenetic analyses demonstrated that Corynespora belongs to Corynesporascaceae, Pleosporales (Voglmayr and Jaklitsch 2017; Hyde et al. 2024b; Thiyagaraja et al. 2024). The genus is characterized by distinct conidiophores; integrated, terminal, mono- tretic, determinate, or percurrently extending conidiogenous cells; and acroge- nous, solitary or catenate, distoseptate conidia (Sharma and Chaudhary 2002; Pal et al. 2007; Voglmayr and Jaklitsch 2017; Capital and Lao 2020; Xu et al. 2020; Pem et al. 2024). Synopses of Corynespora species have been provided by Siboe et al. (1999), Kumar et al. (2021) and Xu et al. (2020). Subsequently, Liu et al. (2023) provided the latest list of identified and accepted species of Corynespora with major morphological features, host information, and locality data. Corynespora species have a wide distribution and can be found as sap- robes, pathogens, and endophytes on living leaves, or from decaying woody material of various plants, as well as on other fungi, nematodes, and human skin (Furukawa et al. 2008; Dixon et al. 2009; Kumar and Singh 2016; Capital and Lao 2020; Xu et al. 2020; Liu et al. 2022; Liu et al. 2023). Li et al. (2023b) then introduced a new species, on branches of /desia polycarpa from Sichuan Province, China. A total of 213 epithets were listed under Corynespora (http:// www.indexfungorum.org, accessed 20, September 2024), with 129 species be- ing accepted (Liu et al. 2023). In this study, collections representing two new species (Arthrobotrys angiop- teridis and Corynespora septata) associated with ferns were made in Yunnan and Guizhou provinces in southwestern China. The identification and establish- ment of these taxa were based on morphological characteristics and phyloge- netic evidence, a polyphasic approach, following the guidelines of Maharach- chikumbura et al. (2021). MycoKeys 113: 101-121 (2025), DOI: 10.3897/mycokeys.113.137678 102 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China Material and methods Collections, isolation and conservation Samples of dead fern tissues were collected from Yunnan and Guizhou Provinc- es, China. The samples were packed in plastic bags for transportation to the lab- oratory, and subsequently examined using the methods described in Senanay- ake et al. (2020). A stereomicroscope (Leica EZ4 Microsystems (Schweiz) AG, Singapore) was used to examine and observe fungal colonies on the host sur- face. Morphological characteristics were documented using a Nikon DS-Ri2 dig- ital camera fitted to a Nikon ECLIPSE Ni compound microscope (Nikon, Japan). Measurements of fungal structure were made using the Tarosoft (R) Image Frame Work, and the images used for figures were processed and combined in Adobe Illustrator CS6 (Adobe Systems, San Jose, CA, USA). Single spores were isolated following the method described by Chomnunti et al. (2014) to obtain pure cultures. Dried specimens were deposited in the Herbarium of Cryptog- ams, Kunming Institute of Botany, Academia Sinica (HKAS), Kunming, China, and the Herbarium of Guizhou Academy of Agricultural Sciences (GZAAS), Gui- yang, China. Pure cultures were deposited in Kunming Institute of Botany Cul- ture Collection (KUNCC), Kunming, China, and Guizhou Culture Collection, China (GZCC). Index Fungorum numbers (https://www.indexfungorum.org/Names/ Names.asp) and Facesoffungi numbers (Jayasiri et al. 2015) are provided. DNA Extraction, PCR amplification and sequencing Fresh fungal mycelia were scraped from the surface of colonies grown on PDA, which had been incubated at 25 °C-28 °C for one month. Fungal genomic DNA was then extracted using the Biospin Fungus Genomic DNA Extraction Kit (Bio- Flux®, Shanghai, China). Four partial gene regions, the nuclear ribosomal inter- nal transcribed spacer region (ITS: ITS1-5.8S-ITS2), the partial nuclear ribosom- al large subunit rRNA gene (LSU) and the partial second-largest subunit of the RNA polymerase II gene (rpb2), were amplified using polymerase chain reac- tion (PCR). The primers used were ITS5/ITS4 for ITS (White et al. 1990), LROR/ LR5 for LSU (Vilgalys and Hester 1990) and fRPB2-5F/fRPB2-7cR for rpb2 (Liu et al. 1999). The quality of the PCR products was checked on 1% agarose elec- trophoresis gels stained with ethidium bromide. Purification and sequencing of PCR products were performed by Beijing Qingke Biotechnology Co., Ltd. Phylogenetic analyses Original sequences were checked using BioEdit v. 7.1.3.0 (Hall 1999) and assem- bled using SeqMan v. 7.0.0 (DNASTAR, Madison, WI, USA). The newly assembled sequences were subjected to BLAST searches in NCBI-GenBank to preliminarily determine related taxa. The sequence data obtained from the BLAST search re- sults and the latest publications (Li et al. 2023b; Liu et al. 2023; Jin et al. 2024; Zhang et al. 2024) were used for phylogenetic analyses. Alignments for sequenc- es of each locus were performed with the online multiple alignment program MAFFT version 7 (https://mafft.cbrc.jp/alignment/server/, accessed on Septem- ber 2024; Katoh et al. 2019), and the alignment files were further trimmed in tri- mAI version 1.2 (Capella-Gutiérrez et al. 2009) with the option “-gt 0.6”. Multi-gene MycoKeys 113: 101-121 (2025), DOI: 10.3897/mycokeys.113.137678 103 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China alignments were combined using Sequence Matrix 1.7.8 (Vaidya et al. 2011). Se- quences generated in this study were deposited in GenBank (Table 1 and Table 2). Table 1. Taxa used in the phylogenetic analyses for Arthrobotrys genus, and their Gen- Bank accession numbers. Taxa robe CBS 268.83 NR_159625 N/A N/A Arthrobotrys amerospora Arthrobotrys anomala YNWS02-5-1 AY773451 | AY773393 | AY773422 AOAC MF926580 N/A Arthrobotrys arthrobotryoides Arthrobotrys blastospora 0Q341651 | 0Q341649 Arthrobotrys botryospora N/A Arthrobotrys cibiensis OR882797 Arthrobotrys cibiensis OR882802 Arthrobotrys cladodes MH179893 Arthrobotrys clavispora Arthrobotrys conoides Arthrobotrys cookedickinson Arthrobotrys cystosporia Arthrobotrys dendroides Arthrobotrys dianchiensis Arthrobotrys elegans Arthrobotrys eryuanensis Arthrobotrys eudermata Arthrobotrys flagrans Arthrobotrys gampsospora Arthrobotrys globospora Arthrobotrys gongshanensis Arthrobotrys guizhouensis Arthrobotrys heihuiensis DLUCC 108-1 OR880378 | OR882791 | OR882796 Arthrobotrys heihuiensis Y710 OR902194 | OR882786 | OR882801 Arthrobotrys hengjiangensis CGMCC 3.24983 | 0Q946587 | 0Q989312 | 0Q989302 Arthrobotrys hyrcanus IRAN 3650C OP351540 N/A YMF 1.01845 N/A N/A Arthrobotrys indica Arthrobotrys iridis Arthrobotrys janus Arthrobotrys javanica Arthrobotrys jindingensis OP236810 OP272515 Arthrobotrys jinpingensis OM850305 Arthrobotrys jinshaensis OR882799 Arthrobotrys jinshaensis OR902197 OR882804 Arthrobotrys koreensis N/A Arthrobotrys lanpingensis OM850302 Arthrobotrys latispora N/A Arthrobotrys longiphora MH179815 Arthrobotrys lunzhangensis OM621810 Arthrobotrys luquanensis OM850303 Arthrobotrys mangrovispora N/A Arthrobotrys megalospora N/A MycoKeys 113: 101-121 (2025), DOI: 10.3897/mycokeys.113.137678 104 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China Taxa Arthrobotrys microscaphoides Arthrobotrys multiformis Arthrobotrys musiformis Arthrobotrys musiformis Arthrobotrys nonseptata Arthrobotrys obovata Arthrobotrys oligospora Arthrobotrys paucispora Arthrobotrys polycephala Arthrobotrys pseudoclavata Arthrobotrys psychrophila Arthrobotrys pyriformis Arthrobotrys reticulata Arthrobotrys robusta Arthrobotrys salina Arthrobotrys scaphoides Arthrobotrys shizishanna Arthrobotrys shuifuensis Arthrobotrys sinensis Arthrobotrys sphaeroides Arthrobotrys superba Arthrobotrys thaumasia Arthrobotrys tongdianensis Arthrobotrys vermicola Arthrobotrys weixiensis Arthrobotrys xiangyunensis Arthrobotrys yangbiensis Arthrobotrys yangbiensis Arthrobotrys yangjiangensis Arthrobotrys yangjiangensis Arthrobotrys yunnanensis Arthrobotrys zhaoyangensis Dactylellina cangshanensis Dactylellina copepodii Strain Number YMEF 1.00028 CBS 773.84 SQ77-1 1.03481 YMF 1.01852 YMF 1.00011 920 ATCC 96704 1.01888 1130 1.01412 YNWS02-3-1 CBS 550.63 nefuA4 SF 0459 1.01442 YMF 1.00022 CGMCC 3.19716 105-1 1.0141 127 917 CGMCC 3.20942 629 CGMCC 3.24984 YXY10-1 DLUCC 36-1 Y678 DLUCC 124 ¥iB12 YMF 1.00593 CGMCC 3.20944 CGMCC 3.19714 CBS 487.90 ITS MF948395 MH861834 AY773469 MH179783 FJ185261 MF948389 AY773462 EF445991 MH179760 AY773446 MH179727 AY773450 MH858355 MZ326655 KP036623 MH179732 MF948392 MT612334 AY773445 MH179726 EU977558 AY773461 OP236809 AY773454 0Q946585 MK537299 OR880382 OR902198 OR880380 OR902196 AY50993 OM855568 MK372062 U51964 tef1-a MF948552 N/A AY773411 MH179607 N/A MF948546 AY773404 N/A MH179592 AY773388 MH179578 AY773392 N/A N/A N/A MH179580 MF948549 OM850306 AY773387 MH179577 N/A AY773403 OP272509 AY773396 0Q989310 N/A OR882795 OR882790 OR882793 OR882788 N/A OM850310 MN915115 DQ999835 rpb2 MF948476 N/A AY773440 MH179883 N/A MF948470 AY773433 N/A MH179862 AY773417 MH179832 AY773421 N/A N/A N/A MH179836 MF948473 OM850300 AY773416 MH179831 N/A AY773432 OP272513 AY773425 0Q989300 N/A OR882800 OR882805 OR882798 OR882803 N/A OM850304 MN915114 DQ999816 Note: “N/A” indicates no data are available in GenBank. The newly generated sequences are indicated in blue. The fasta files were converted to the formats required for the AliView pro- gram (Larsson 2014), PHYLIP for maximum likelihood analysis (ML), and NEX- US for Bayesian analysis (BYPP). Maximum likelihood (ML) analyses were performed using RAXML-HPC Blackbox (8.2.10) tool on the XSEDE Teragrid at the CIPRES Science Gateway (https://www.phylo.org; accessed on 10 Sep- tember 2024), with rapid bootstrap analysis followed by 1,000 bootstrap repli- cates (Miller et al. 2010; Stamatakis 2014). The final tree was selected from the suboptimal trees of each run by comparing likelihood scores under the GTR- GAMMA substitution model. Bayesian analyses were performed by MrBayes 3.2.7a on XSED via CIPRES (Miller et al. 2010). MrModeltest v.2.3 was used to determine the best nucleotide substitution model for each data partition (Nylander 2004). Posterior probabilities (PP) (Rannala and Yang 1996) were MycoKeys 113: 101-121 (2025), DOI: 10.3897/mycokeys.113.137678 105 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China calculated using the Bayesian Markov Chain Monte Carlo (BMCMC) sampling method (Huelsenbeck 2001; Zhaxybayeva and Gogarten 2002). Four simulta- neous Markov chains were run for 1 million generations, with trees sampled every 100° generations, yielding 10,000 trees. Phylogenetic trees were visual- ized using FigTree v. 1.4.4 (Rambaut 2014), and the layouts were created using Adobe Illustrator CS5 software (Adobe Systems, San Jose, CA, USA). The newly obtained sequences in this study were deposited in GenBank. Table 2. Taxa used in the phylogenetic analyses for Corynespora genus, and their Gen- Bank accession numbers. Taxa Strain Number ITS LSU Corynespora cassiicola CBS 100822 N/A GU301808 Corynespora citricola CBS 169.77 FJ852594 N/A Corynespora doipuiensis MFLUCC 14-0022 MN648322 | MN648326 Corynespora encephalarti CBS 145555 MK876383 MK876424 Corynespora lignicola MFLUCC 16-1301 MN860549 MN860554 Corynespora mengsongensis HJAUP C2000T 0Q060574 0Q060578 Corynespora nabanheensis HJAUP C2048T 0Q060577 0Q060580 Corynespora pseudocassiicola CPC 31708 MH327794 : D/ i 208 KY984297 Corynespora smithii L120 Corynespora smithii | L130 KY984298 KY984298 Corynespora smithii | CABI 5649b FJ852597 GU323201 Corynespora smithii CBS 139925 KY984299 KY984299 Corynespora submersa MFLUCC 16-1101 MN860548 MN860553 Corynespora torulosa CBS 136419 MH866095 MH877634 Corynespora thailandica CBS 145089 MK047455 MK047505 Corynespora yunnanensis HJAUP C2132T 0Q060579 0Q060583 Periconia byssoides H 4600 LC014581 AB807570 Periconia digitata CBS 510.77 LC014584 AB807561 Periconia pseudodigitata KT 1395 NR_153490 NG_059396 Periconia pseudodigitata UESTCC 23.0022 OR253146 OR253305 Periconia pseudodigitata UESTCC 23.0023 OR253147 OR253306 Note: “N/A” indicates no data are available in GenBank. The newly generated sequences are indicated in blue. Taxonomy Arthrobotrys angiopteridis J.Y. Zhang, Y.Z. Lu & K.D. Hyde, sp. nov. MycoBank No: 902682 Facesoffungi number: FoF16618 Fig. 2 Etymology. Named after the fungal host genus Angiopteris. Holotype. HKAS 129855. Description. Saprobic on dead rachis of Angiopteris fokiensis in terres- trial habitats. Sexual morph Undetermined. Asexual morph Colonies on nat- ural substrate superficial, effuse, hyaline, with white and glistening masses MycoKeys 113: 101-121 (2025), DOI: 10.3897/mycokeys.113.137678 106 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China of conidia on the apex of conidiophores. Mycelium partly superficial, partly immersed, composed of septate, branched, smooth hyphae. Conidiophores 345-502 um long, 6-8.5 um wide at the base (x = 418 x 6.9 um, n = 20), mac- ronematous, mononematous, solitary, erect, straight or slightly flexuous, un- branched, cylindrical, septate, smooth-walled, hyaline. Conidiogenous cells 95-176 x 2-4.5 um (x = 129 x 3.5 um, n = 20), polyblastic, producing 1-5 sep- arate nodes by the repeated elongation, with multi polyblastic denticles at each node, hyaline. Conidia 25-35 x 8-11 um (x = 28.8 x 9 um, n = 25), aseptate, or 1-septate at the median to submedian, not constricted or slightly constricted at the septum, clavate to elongate pyriform, broadly rounded at apex, pointed or sometimes truncate at the base, sometimes with a bud-like projection at base, straight or slightly curved, smooth-walled or rough walled, guttulate, hyaline. Culture characteristics. Conidia germinating on WA within 15 h and germ tube produced from conidia. Colonies growing on PDA, reaching 60 mm diameter in 10 days at 26 °C, circular, cottony, white, and not producing pigmentation in culture. Material examined. CHINA * Guizhou Province, Zunyi City, Xishui County (28°22'19"N, 106°0'35"E), on dead rachis of Angiopteris fokiensis (Marattiaceae) in a disturbed forest nearby the roadside, 13 April 2023, J.Y. Zhang, ZY06 (HKAS 129855, holotype; GZAAS 23-0758, isotype), ex-type living culture, KUNCC 23- 14121; : ibid., ZY02 (HKAS 129854, paratype), ex-paratype living culture, KUNCC 23-14119. Additional sequence: KUNCC 23-14121: PQ346313 (SSU) and PQ346310 (LSU); KUNCC 23-14119: PQ346312 (SSU) and PQ346309 (LSU). Notes. Phylogenetically, the new isolates KUNCC 23-14121 and KUNCC 23- 14119 of Arthrobotrys angiopteridis clustered together formed a separate clade with 100% ML/1.00 PP bootstrap support and are sister to A. pyriformis (Fig. 1). A comparison of nucleotide base pairs between them reveals differences of 30/459 (6.5%, including 15 gaps) and 82/730 bp (11%, no gap) in the ITS and rpb2 sequences, respectively. This indicates that they are distinct species. Mor- phologically, A. angiopteridis aligns well with the generic concept and resem- bles A. oligospora in having hyaline conidiophores with the successive produc- tion of additional denticle nodes (Yu et al. 2014). However, A. angiopteridis can be easily distinguished from A. oligospora by its longer conidiophores (345- 502 um vs. 110-440 um) and clavate to elongate pyriform conidia, with 0-1 septate near the middle, whereas A. oligospora has pyriform or obovoid conid- ia with 1-septate near the base. Therefore, we introduce A. angiopteridis as a novel species based on its distinct morphological and phylogenetic evidence following the guidelines of Maharachchikumbura et al. (2021). Corynespora septata J.Y. Zhang, Y.Z. Lu & K.D. Hyde, sp. nov. MycoBank No: 902683 Facesoffungi number: FoF16619 Fig. 4 Etymology. Named after the presence of eu-septate conidia. Holotype. HKAS 129839. Description. Saprobic on dead rachis of an unidentified fern in terrestri- al habitats. Sexual morph undetermined. Asexual morph Colonies on natu- ral substrate superficial, effuse, gregarious, hairy, brown to black. Mycelium MycoKeys 113: 101-121 (2025), DOI: 10.3897/mycokeys.113.137678 107 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China 1400/1; Arthrobotrys robusta nefuA4 87/0.99| | Arthrobotrys cladodes 1.03514 99/1 Arthrobotrys latispora H.B. 8952 95/1 Al Arthrobotrys botryospora CBS 321.83" 67/0.98| “| arthrobotrys jindingensis CGMCC 3.2 100/1 100/1]\ Arthrobotrys tongdianensis CGMCC 3. Arthrobotrys koreensis C45 Arthrobotrys scaphoides 1.01442 38/0.98 Arthrobotrys obovata YMF 1.00011" B6/1L__ arthrobotrys lunzhangensis CGMCC 3.20941 Arthrobotrys clavispora CBS 545.63! 100/0.98 ;Arthrobotrys yangbiensis Y678 ; 95/1] 'Arthrobotrys yangbiensis DLUCC 36-1 67/1 70K Arthrobotrys gampsospora CBS 12 100/41 Arthrobotrys salina SF 0459 ae 6 Arthrobotrys psychrophila 1.01412 lL Arthrobotrys cystosporia CBS 439.54 Arthrobotrys paucispora ATCC 96704 Arthrobotrys heihuiensis Y710 p0/1| 1909/1! arthrobotrys heihuiensis DLUCC 108-1" 400/47 Arthrobotrys lanpingensis CGMCC 3.20 97/1 s T 64/1 Arthrobotrys gongshanensis CGMC\ 93/17 Arthrobotrys elegans 1.00027 74/1 Arthrobotrys cookedickinson YMF 1.00024 63/-| '-Arthrobotrys sinensis 105-1 Arthrobotrys indica YMF 1.01845 mi Arthrobotrys microscaphoides YMF 99/1) j4,-Arthrobotrys megalospora TWF800 — Arthrobotrys sphaeroides 1.0141 ' 87/1] Arthrobotrys weixiensis CGMCC 3.24984" 98/1 Arthrobotrys globospora 1.00537 1400/1 |A’throbotrys shuifuensis CGM 90/1 Arthrobotrys arthrobotryoide: Arthrobotrys musiformis 1.03481 400/17—Arthrobotrys shizishanna YMF 1. Figure 1. Phylogram generated from maximum likelihood analysis based on combined ITS, tef7-a and rpb2 sequence data. Seventy-eight taxa were included in the combined analyses, which comprised 1920 characters (ITS = 583 bp, tef7-a = 512 bp and rpb2 = 825 bp) after alignment. The best scoring RAxML tree with a final likelihood value of -22800.405782 is presented. The matrix had 983 distinct alignment patterns, with 25.05% of undetermined characters or gaps. Estimat- ed base frequencies were as follows: A = 0.260557, C = 0.263561, G = 0.230377, T = 0.245504; substitution rates: AC = 1.414531, AG = 3.978691, AT = 1.319991, CG = 0.945884, CT = 6.618473, GT = 1.000000; gamma distribution shape parameter a = 0.262034. Bootstrap support values for ML equal to or greater than 60% and prior probabilities (PPs) equal to or greater than 0.95 are given above the nodes as ML/PP. The tree was rooted to Dactylellina copepodii (CBS 487.90) and D. cangshanensis (CGMCC 3.19714). The strain numbers are noted after the species names with ex-type strains indicated by '. The newly generated sequences are indicated in blue. MycoKeys 113: 101-121 (2025), DOI: 10.3897/mycokeys.113.137678 108 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China 81/- (Athrobotrys superba 127 100/1} Arthrobotrys blastospora CGMCC 3.20940! 94/1 Arthrobotrys oligospora 920 Arthrobotrys conoides 670 10.96 Arthrobotrys flagrans 1.01471 82/1 Arthrobotrys pseudoclavata 1130 100/17 A'throbotrys hyrcanus IRAN 3650C' Arthrobotrys dianchiensis 1.00571 Arthrobotrys eryuanensis CGMCC 3.1971 5! 100/1|A/throbotrys angiopteridis KUNCC 23-14121! 99/1 Arthrobotrys angiopteridis KUNCC 23-14119 Arthrobotrys pyriformis YNWS 02-3-1 Arthrobotrys javanica 105 Arthrobotrys zhaoyangensis CGMCC 3.20944! 400/17 -—Arthrobotrys jinpingensis CGMCC 3.20896" 87/1 Arthrobotrys hengjiangensis CGMCC 3.24983! 99/0.98 |A/throbotrys yunnanensis YMF 1.00593" 100/1 |' Arthrobotrys iridis 521 100/1 Arthrobotrys multiformis CBS 773.84! Arthrobotrys lugquanensis CGMCC 3.20894! 400/1|Arthrobotrys jinshaensis DLUCC 133" Arthrobotrys jinshaensis MA142 76/0.95 rahe Talos Arthrobotrys mangrovispora MGDW 17 400/1|A/throbotrys janus 85-1 Arthrobotrys 100/1|' Arthrobotrys eudermata SDT24 89/1 Arthrobotrys thaumasia 917 400/1|A/throbotrys yangjiangensis YB19 67/1 Arthrobotrys yangjiangensis DLUCC 124! 100/17 Arthrobotrys anomala YNWS 02-5-1 Arthrobotrys dendroides YMF 1.00010 74). - Arthrobotrys xiangyunensis YXY10-1' UAL Arthrobotrys reticulata CBS 550.63 100/1 Arthrobotrys longiphora 1.00538 00 Arthrobotrys cibiensis EY10 100/1! arthrobotrys cibiensis DLUCC 109! Arthrobotrys polycephala 1.01888 Arthrobotrys nonseptata YMF 1.01 52! 400/1 Dactylellina copepodii CBS 487.90" Dactylellina cangshanensis CGMCC 3.1 9714" Outgroup 0.03 Figure 1. Continued. MycoKeys 113: 101-121 (2025), DOI: 10.3897/mycokeys.113.137678 109 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China Figure 2. Arthrobotrys angiopteridis (HKAS 129855, holotype) a the host b colonies on the host c, d conidiophores with conidiogenous cells e-h conidiogenous cells i-k conidia | trapping mycelia: adhesive networks m pure culture from front and reverse. Scale bar: 100 um (ce, d); 20 um (e-I). MycoKeys 113: 101-121 (2025), DOI: 10.3897/mycokeys.113.137678 110 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China partly superficial, partly immersed, composed of branched, septate, pale brown to brown, smooth-walled hyphae. Conidiophores 490-671 um long, 3.5- 6.5 um wide at the base (x = 600 x 5 um, n = 15), macronematous, mononem- atous, erect, straight or flexible, unbranched, or occasionally branched, septate, smooth, dark brown at the base, pale towards the apex. Conidiogenous cells 21-60 x 3-5.5 um (x = 36.3 x 3.8 um, n = 15), integrated, terminal, mono- tretic, cylindrical, smooth, pale brown to brown. Conidia 42-74 x 4.5-7.5 um (x = 54 x 5.7 um, n = 25), acrogenous, solitary, up to 7-distoseptate with one true septum, straight or slightly curved, subcylindrical to obclavate, rounded at the apex, base short obconically truncate, somewhat thickened and darkened, sometimes with percurrent proliferation which forms another conidium from the conidial apex, hyaline to pale brown. Culture characteristics. Conidia germinating on WA within 15 h and germ tube produced from conidia. Colonies growing on PDA, reaching 55 mm diame- ter in 10 days at 26 °C, circular, flat with entire margin, velvety, fluffy, white from above, reverse dark brown at center, paler to light yellow towards margin, and not producing pigmentation in culture. Material examined. CHINA * Yunnan Province, Xishuangbanna Dai Autono- mous Prefecture, Mengla County, Menglun Town, Xishuangbanna Tropical Botan- ical Garden, Chinese Academy of Sciences (21°55'39"N, 101°15'15"E), on dead ra- chis of an unidentified fern, 16 November 2019, J.Y. Zhang, Y159 (HKAS 129839, holotype; GZAAS 23-0769, isotype), ex-type living culture, GZCC 23-0741. Notes. A BLASTn search in NCBI-GenBank revealed that the LSU and ITS sequences of our newly collected strain of Corynespora septata exhibited 99% similarity to C. encephalarti (NG_067878) and 95.62% similarity to C. cassiicola (MN648322), respectively. Phylogenetic analysis confirmed that C. septata formed a distinct clade within Corynespora and shared a sister relationship with C. pseudocassiicola Crous & M.J. Wingf. (Fig. 3). There are 10 bp (10/841 bp with 0 gap, 1%) and 38 bp (38/527 bp with 13 gaps, 7%) differences between the C. pseudocassiicola and C. septata in the LSU and ITS gene regions, respectively. Morphologically, C. septata has longer conidiophores (490-671 um vs. 200-400 um), and smaller conidia (42-74 x 4.5-7.5 um vs. 95-160 x 9-10 um) compared to C. pseudocassiicola (Crous et al. 2018). Similarly, C. septata is most similar to C. lignicola Z.L. Luo, H.Y. Su & K.D. Hyde in the shapes of conidiophores, conid- iogenous cells, and conidia (Capital and Lao 2020). However, C. septata differs from C. lignicola in having narrower conidiophores (3.5-6.5 um vs. 9-13 um) and notably smaller conidia (42-74 x 4.5—7.5 um vs. 110-156 x 7-9 um). Discussion During a survey of bracken (Pteridiurn aquilinum (L.) Kuhn) petiole decomposi- tion in the United Kingdom, Arthrobotrys megalosporus (Drechsler) M. Scholler, Hagedorn &A. Rubner (Synonym: Dactylella megalospora Drechsler) was found to be a member of the common fungi (Frankland 1976). This is also the only record of Arthrobotrys species being associated with ferns. Corynespora cassii- cola (Berk. & M.A. Curtis) C.T. Wei and an unidentified Corynespora species have been discovered on ferns in United States (Alfieri et al. 1984; Smith 2008; Farr et al. 2021). Specifically, C. cassiicola has been found on six fern species includ- ing Arachniodes aristata (Davalliaceae), Athyrium niponicum (Dryopteridaceae), MycoKeys 113: 101-121 (2025), DOI: 10.3897/mycokeys.113.137678 111 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China a Corynespora nabanheensis HJAUP C2048" 78/0.99 Corynespora mengsongensis HJAUP C2000' 75/- Corynespora yunnanensis HJAUP C2132! 68/0.99|' Corynespora submersa MFLUCC 16-1101' Corynespora doipuiensis MFLUCC 14-0022' Corynespora thailandica CBS 145089' gg] Corynespora idesiae UESTCC 23.0022" 7710.99 | Corynespora idesiae UESTCC 23.0023 Corynesporascaceae Corynespora lignicola MFLUCC 16-1301" cree Corynespora septata GZCC 23-0741" Corynespora pseudocassiicola CPC 31708' Corynespora cassiicola CBS 100822 saont Corynespora torulosa CBS 136419' 1/2 Corynespora smithii CAB| 5649b Corynespora smithii CBS 139925 100 Corynespora smithii L130 KY984419 Corynespora smithii L120 Si Corynespora citricola CBS 169.77' 96/1 Corynespora encephalarti CBS 145555" 4100/1 Periconia digitata CBS 510.77 100/1 Periconia pseudodigitata KT 1395' a Outgroup Periconia byssoides H 4600 0.02 Figure 3. Phylogram generated from maximum likelihood analysis based on combined LSU and ITS sequence data. Twen- ty-two taxa were included in the combined analyses, which comprised 1393 characters (LSU = 844 bp and ITS = 549 bp) after alignment. The best scoring RAXML tree with a final likelihood value of -4677.993509 is presented. The matrix had 336 distinct alignment patterns, with 9.65% of undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.243499, C = 0.247400, G = 0.289966, T = 0.219136; substitution rates: AC = 3.067252, AG = 2.397685, AT = 1.551875, CG = 1.069828, CT = 6.624253, GT = 1.000000; gamma distribution shape parameter a = 0.240919. Bootstrap support values for ML equal to or greater than 60% and prior probabilities (PPs) equal to or greater than 0.95 are given above the nodes as ML/PP. The tree was rooted to Periconia byssoides (H 4600), P digitata (CBS 510.77) and P pseudodigitata (KT 1395). The strain numbers are noted after the species names with ex-type strains indicated by ". The newly generated sequences are indicated in blue. Adiantum cuneatum (Adiantaceae), Adiantum tenerum (Adiantaceae), Davallia repens (Davalliaceae), and Platycerium spp. (Pteridaceae) (Alfieri et al. 1984; Smith 2008). Additionally, an unidentified Corynespora species was collected from Nephrolepis exaltata (Davalliaceae) (Alfieri et al. 1984; Farr et al. 2021). Based on phylogenetic and morphological evidence, Arthrobotrys angiopteridis and Corynespora septata, isolated from ferns are reported as new species in this study from Yunnan and Guizhou provinces. These findings contribute to MycoKeys 113: 101-121 (2025), DOI: 10.3897/mycokeys.113.137678 2 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China Figure 4. Corynespora septata (HKAS 1298339, holotype) a, b colonies on the host c-e conidiophores with conidiogenous cells f-k conidia I pure culture from front and reverse. Scale bar: 200 um (b); 100 um (ce); 20 um (f-k). MycoKeys 113: 101-121 (2025), DOI: 10.3897/mycokeys.113.137678 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China a better understanding of fern-related fungi and aim to enhance attention and awareness of fungal communities associated with ferns. Most Corynespora species were introduced based on morphology (Sivane- san 1996; Siboe et al. 1999; Sharma and Chaudhary 2002; Kumar and Singh 2016; Liu et al. 2023). However, distinguishing between Corynespora and some similar genera, especially Helminthosporium, based solely on morphology has proven to be challenging (Castafeda Ruiz et al. 2004; Voglmayr and Jaklitsch 2017). The application of molecular data has confirmed this difficulty. For ex- ample, Corynespora caespitosa, C. endiandrae, C. leucadendri and C. olivacea were transferred to Helminthosporium based on phylogenetic evidence (Vogl- mayr and Jaklitsch 2017). Currently, sequence data is available in GenBank for only 15 species. Therefore, it is essential to obtain more collections with sequence data for verification of Corynespora species. Arthrobotrys angiopteridis sp. nov., isolated from Angiopteris fokiensis, is a member of nematode-trapping fungi with trapping device of adhesive networks (Fig. 2l). Nematode-trapping fungi are crucial for preserving ecological balance and possess the potential for biologically controlling harmful nematodes (Jiang et al. 2017; Zhang et al. 2024). Arthrobotrys angiopteridis is a valuable fungus that is expected to contribute to the exploration of ecological protection in the future. Yunnan and Guizhou provinces are not only the most abundant areas for fern plants in China (Li et al. 2015; Zhou et al. 2016), but also hotspots for the dis- covery of new fungal species (Wang et al. 2021; Yang et al. 2023b; Zhang et al. 2023b; Dissanayake et al. 2024). The introduction of A. angiopteridis and C. sep- tata adds to the growing evidence of high fungal diversity in Guizhou and Yun- nan province, China (Dissanayake et al. 2024; Dong et al. 2024; Li et al. 2024). Acknowledgements We would like to thank Shaun Pennycook (Manaaki Whenua Landcare Re- search, New Zealand) for advising on the fungal names and Yu Yang for helping in experiment. Jing-Yi Zhang thanks the Mae Fah Luang University for granting her the tuition scholarship and the dissertation writing grant (Grant number: 7702(6)/842 (no.0320). Dan-Feng Bao would like to thank the Postdoctoral Fel- lowship Program of CPSF under Grant Number GZC20240346. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding This work was funded by the National Natural Science Foundation of China (NSFC 32060013) and the Youth Science and Technology Talent Development Project from Guizhou Provincial Department of Education (QJHKYZ[2022]345). The authors also ex- tend their appreciation to the Researchers Supporting Project number (RSP2024R114), King Saud University, Riyadh, Saudi Arabia. MycoKeys 113: 101-121 (2025), DOI: 10.3897/mycokeys.113.137678 114 Jing-Yi Zhang et al.: Two novel hyphomycetes associated with ferns from China Author contributions Conceptualization: KDH, JYZ. Data curation: JYZ. Formal analysis: DFB, JYZ, LUZ. Fund- ing acquisition: YZL, LUZ. Investigation: JYZ. Methodology: JYZ, FAO. Project adminis- tration: YZL. Supervision: KDH. Writing - original draft: JYZ. Writing - review and editing: SB, DFB, FAO, KDH. 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