<8) MycoKeys MycoKeys 116: 327-344 (2025) DOI: 10.3897/mycokeys.116.146683 Research Article Biconidium sinense gen. et sp. nov. (Hypocreales, Bionectriaceae) and Didymocyrtis shanxiensis sp. nov. (Phaeosphaeriaceae, Didymocyrtis) isolated from urban soil in China Hai-Yan Wang'™®, Chunbo Dong’®, Yan-Wei Zhang2®, Wan-Hao Chen?®, Yan-Feng Han'® 1 Institute of Fungus Resources, Department of Ecology, College of Life Science, Guizhou University, Guiyang 550025, Guizhou, China 2 Key Laboratory of Development and Utilization of Biological Resources in Colleges and Universities of Guizhou Province/Key Laboratory of Ecology and Management on Forest Fire in Higher Education Institutions of Guizhou Province, Guizhou Education University, Guiyang 550018, Guizhou, China 3 Center for Mycomedicine Research, Basic Medical School, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, Guizhou, China Corresponding authors: Yan-Wei Zhang (zyw_email@163.com); Yan-Feng Han (swallow1128@126.com) OPEN Qaceess Academic editor: Ajay Kumar Gautam Received: 14 January 2025 Accepted: 20 March 2025 Published: 29 April 2025 Citation: Wang H-Y, Dong C, Zhang Y-W, Chen W-H, Han Y-F (2025) Biconidium sinense gen. et sp. nov. (Hypocreales, Bionectriaceae) and Didymocyrtis shanxiensis sp. nov. (Phaeosphaeriaceae, Didymocyrtis) isolated from urban soil in China. Mycokeys 116: 327-344. https://doi. org/10.3897/mycokeys.116.146683 Copyright: © Hai-Yan Wang 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 a fungal diversity survey in various urban habitats across China, 5 fungal isolates were discovered from soil samples. Detailed morphological observations and multi- gene phylogenetic analyses confirmed the identification of two novel taxa: Biconidium sinense gen. et sp. nov. and Didymocyrtis shanxiensis sp. nov. These species were for- mally described, illustrated, and discussed, highlighting their distinct characteristics and taxonomic placement. The study expands our understanding of fungal diversity in urban environments, emphasizing the importance of combining morphological and molecular approaches for accurate species delineation and discovery. Key words: Fungal taxonomy, mycodiversity, new taxa, phylogeny Introduction Soil fungi play an important role in mediating the processes of geochemical cycling, ecosystem material cycling and energy flow. For instance, they influ- ence soil fertility, mineral breakdown, and organic matter cycling, as well as plant health and nutrition (Guo et al. 2017; Lu 2018). Moreover, some soil fun- gi can produce a lot of metabolites that are essential for human life and pro- duction (Zhang et al. 2023; Wang et al. 2024). For example, among the fungal species in the soil, the strains of the genera Aspergillus, Penicillium, Paecilo- myces and Trichoderma produce flavins, ankaflavin, quinones, and anthraqui- none (Akilandeswari and Pradeep 2016). Penicillium griseofulvum can produce a range of secondary metabolites including chanoclavine |, elymoclavine, fulvic acid, and griseofulvin, all of which can be used for antimicrobial activity (Yo- gabaanu et al. 2017). Acrophialophora levis QHDZ1-2 isolated from a zoo soil can produce some compounds, such as amino acid, amines, fatty acid, and vitamins (Wang 2024). Due to a multitude of factors, it is suspected that spe- cies are disappearing before they are discovered in many habitats (Wang et al. 2018; L6bl et al. 2023; Wang et al. 2024). This implies that we still need to make 327 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed more efforts to delve deeper into soil fungi resources, contribute to the study of our Earth’s fungal diversity, and provide fungal resources for social industrial production. Currently, numerous studies have investigated fungal diversity in various soil habitats across China, including caves, forests, farmland, deserts and grasslands (Guo et al. 2017; Ma et al. 2021; Ma 2023; Guo 2024; Song et al. 2024). However, the composition and diversity of soil fungi in various urban various environments appear to have been neglected. Urbanization has been the most impactful human activity in altering landscape patterns over the past century and is widely regarded as a significant threat to global biodiversity (Grimm et al. 2008; Nugent and Allison 2022). Developing countries are experiencing the swiftest rates of urbanization, with projections indicating that approximately 68% of the global population will reside in urban areas by 2050 (Desa 2019). The process of urbanization will reshape the land landscape, impacting elements such as surface vegetation, hydrology and soil, which in turn affects biodiversity and can lead to species homogenization or even extinction of species (Buczkowski and Richmond 2012; Yan et al. 2022). Urbanization has had a profound impact on soil fungi. It fragments the original habitats, resulting in a decline in fungal diversity and the potential disappear- ance of some native fungal species (Zhao et al. 2012; Hou et al. 2014; Rai et al. 2018). Consequently, in the context of urbanization, the composition and distri- bution of soil fungi across various urban habitats should be paid more attention. In recent years, the composition and diversity of green soil fungi in different urban habitats were explored (Zhang et al. 2021, 2023, 2024; Li et al. 2022a, 2022b; Ren et al. 2022; Wang et al. 2023, 2024). Fortunately, many new species and genera have been discovered and documented in these urban settings. Bionectriaceae Samuels & Rossman was proposed by Rossman et al. (1999) based on the sexual morph-typified genus Bionectria Speg. (Spegazzini 1919). It is including 26 genera. Its diagnostic characteristics are the presence of white, pale tan orange or brown, uniloculate, perithecial, rarely cleistothecial ascomata and generally not changing color in KOH. Barr (1979) proposed the family Phaeosphaeriaceae using Phaeosphaeria with Ph. oryzae as the type species. The new genus Diederichomyces was de- scribed by Trakunyingcharoen et al. (2014) to include most of the lichenicolous Phoma species that were assigned to the Phaeosphaeriaceae by Lawrey et al. (2012). Vainio (1921) established Didymocyrtis Vain., based on the type species Didymocyrtis consimilis Vain. With the development of phylogeny, the lichenico- lous species of genus Didymocyrtis had been assigned to Diederichia D. Hawk- sw., Diederichomyces Crous & Trakun., Leptosphaeria Pass. and Phoma Sacc. (Trakunyingcharoen et al. 2014). Recently, the genus Didymocyrtis was resurrect- ed for these species, and the new combinations, Didymocyrtis bryonthae (Arnold) Hafellner, Didymocyrtis cladoniicola (Diederich, Kocourk. & Etayo) Ertz & Died- erich, Didymocyrtis foliaceiphila (Diederich, Kocourk. & Etayo) Ertz & Diederich, Didymocyrtis infestans (Speg.) Hafellner, Didymocyrtis kaernefeltii (S.Y. Kondr.) Hafellner, Didymocyrtis melanelixiae (Brackel) Diederich, R.C. Harris & Etayo, Didymocyrtis pseudeverniae (Etayo & Diederich) Ertz & Diederich, Didymocyrtis ramalinae (Roberge ex Desm.) Ertz, Diederich & Hafellner, Didymocyrtis slapto- niensis (D. Hawksw.) Hafellner & Ertz, and Didymocyrtis xanthomendozae (Died- erich & Freebury) Diederich & Freebury were created (Ertz et al. 2015). Presently, the genus Didymocyrtis includes twenty-nine species in the Index Fungorum. Mycokeys 116: 327-344 (2025), DOI: 10.3897/mycokeys.116.146683 328 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed During a continuous survey of fungal diversity exploration from different urban green soils in China, five strains from green soils of sewage treatment plant were isolated and purified. Based on the multi-gene phylogeny and mor- phological characteristics, these isolated strains were identified as two new taxa, Biconidium sinense gen. et sp. nov. and Didymocyrtis shanxiensis sp. nov., which are described and illustrated. Materials and methods Sample collection and fungal isolation Soil samples, from 3-10 cm below the soil surface, were collected from green soil of sewage treatment plant in some cities in China. Samples were placed in sterile Ziploc plastic bags, and brought back to the laboratory. Then, the 2 g of each soil samples for fungal isolation, were placed into a sterile conical flask containing 20 mL sterile water in a 50 mL sterile conical flask, and thorough- ly shaken using a Vortex vibration meter. Subsequently, the soil suspension was diluted to a concentration of 10°. Then, 1 mL of the diluted sample was transferred to a sterile Petri dish with Sabouraud’s dextrose agar (SDA; peptone 10 g/L, dextrose 40 g/L, agar 20 g/L, 3.3 mL of 1% Bengal red aqueous solution) medium containing 50 mg/L penicillin and 50 mg/L streptomycin. The plates were incubated at 25 °C for 1 week, then every single colony was selected from the plates and transferred to new potato dextrose agar (PDA, potato 200 g/L, dextrose 20 g/L, agar 20 g/L) plates. Morphological study Strains of potentially new species were transferred to plates of malt extract agar (MEA), oatmeal agar (OA) and potato dextrose agar (PDA), and were incubated at 25 °C for examining their colony morphology and microscopic morphology. After 7 days, the colony colors according to national standard color card and diameters on the surface and reverse of inoculated Petri dishes were observed and recorded. Meanwhile, fungal hyphae and conidiogenous structures were ex- amined, and images were captured by making direct wet mounts with 25% lactic acid on PDA, with an optical microscope (DM4 B, Leica). Strains of two novel species were deposited in the Institute of Fungus Resources, Guizhou University (GZUIFR = GZAC). Taxonomic descriptions and nomenclature of one new genus and two new species were uploaded in MycoBank (https://www.mycobank.org/). DNA extraction, PCR amplification and sequencing Using the BioTeke Fungus Genomic DNA Extraction kit (DP2032, BioTeke), to- tal genomic DNA was extracted following the manufacturer's instruction. The extracted DNA was stored at -20 °C. Primer combinations: ITS1/ITS4 (White et al. 1990), LROR/LR5 (Wang et al. 2022) and T1/TUB4Rd (O’Donnell and Cigelnik 1997; Woudenberg et al. 2009) were used for amplification of the internal tran- scribed spacers (ITS), the 28S nrRNA locus (LSU) and beta-tubulin gene (tub2), respectively. The PCR amplification conditions: ITS, 94 °C: 5 min, (94 °C: 30 s, 51 °C: 50s, 72°C: 45s) x 35 cycles, 72 °C: 10 min (White et al. 1990); LSU, 94 °C: Mycokeys 116: 327-344 (2025), DOI: 10.3897/mycokeys.116.146683 329 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed 5 min, (94 °C: 30 s, 51 °C: 1 min, 72 °C: 2 min) x 35 cycles, 72 °C: 10 min (Zhang et al. 2023); tub2, 94 °C: 5 min, (94 °C: 30 s, 52 °C: 30s, 72 °C: 30s) x 35 cycles 72 °C: 10 min (Woudenberg et al. 2009). In this study, the PCR products were sent to Quintarabio (Wuhan, China) for purification and sequencing. Strains se- quences of two new species were submitted to GenBank (https://www.ncbi. nlm.nih.gov/) (Table 1 and Table 2). Phylogenetic analysis The relevant strains sequences were downloaded from GenBank in this paper (Table1 and Table2). Flammocladiella decora (Wallr.) Lechat & J. Fourn. and Flammocladiella aceris Crous, L. Lombard & R.K. Schumach. were used as the outgroup in phylogenetic tree 1 (Fig. 1). Parathyridaria philadelphi Crous & R.K. Schumach. was used as the outgroup in phylogenetic tree 2 (Fig. 3). The mul- tiple datasets of ITS, LSU and tub2 were aligned and trimmed in MEGA v.6.06 (Tamura et al. 2013). Using the “Concatenate Sequence” function, the concat- enation of loci was conducted in PhyloSuite v.1.16 (Zhang et al. 2020). Then, the phylogenetic construction of each loci dataset was processed by both Max- imum Likelihood (ML) and the Bayesian Inference (BI) methods. In ModelFind- er, the Akaike Information Criterion correction (AlCc) was used for the best-fit substitution model (Kalyaanamoorthy et al. 2017). With 1000 bootstrap tests using the ultrafast algorithm (Minh et al. 2013), the ML analysis was conduct- ed in IQ-TREE v.1.6.11 (Nguyen et al. 2015). The BI analysis was performed in MrBayes v.3.2 (Ronquist et al. 2012) and Markov chain Monte Carlo (MCMC) simulations were used for 2x10° generations. Using FigTree version 1.4.3, the phylogenetic trees were visualized and edited in Microsoft PowerPoint. Results Phylogenetic analysis In this study, using ITS sequences, our five isolates were identified and assigned to potential genera and species based on a BLASTn in NCBI. Five strains be- longing to Bionectriaceae or Didymocyrtis were screened and tested for further identification through morphological characterization and phylogenetic anal- yses. Using ML and BI analyses, the two phylogenetic trees were consistent and supported strongly in branches. The ML analysis for the combined dataset provided the best scoring tree. The concatenated sequences of Fig. 1 and Fig. 3 included 90 and 16 taxa, respectively. The dataset in Fig. 1 was composed of ITS (1-382 bp) and LSU (383-782 bp) sequence data. The dataset in Fig. 3 was composed of ITS (1-402 bp) and tub2 (403-731 bp) sequence data. The phylogeny shows that each genus clusters into a monophyletic clade, and three strains of the genus Biconidium clustered in a well-separated clade, with a high support value (ML/BI 100/1) (Fig. 1). Two strains of the genus Didymocyrtis also clustered together, with a high support value (ML/BI 98/1) (Fig. 3). Therefore, a new genus, Biconidium H.Y. Wang & Y.F. Han, is intro- duced, and Biconidium sinense H.Y. Wang & Y.F. Han and Didymocyrtis shanx- iensis H.Y. Wang & Y.F. Han as new species are proposed according to the phylogenetic analysis. Mycokeys 116: 327-344 (2025), DOI: 10.3897/mycokeys.116.146683 330 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed Table 1. Strains of Bionectriaceae and corresponding GenBank numbers included in phylogenetic analyses. Species Gliomastix murorum Gliomastix murorum Gliomastix roseogrisea Gliomastix tumulicola Paracylindrocarpon aloicola Paracylindrocarpon aloicola Paracylindrocarpon aurantiacum Paracylindrocarpon multiseptatum Fusariella curvata Fusariella atrovirens Fusariella arenula Fusariella arenula Selinia pulchra Roumegueriella rufula Verrucostoma martinicense Verrucostoma freycinetiae Synnemellisia aurantia Musananaesporium tectonae Gossypinidium sporodochiale Caespitomonium squamicola Caespitomonium squamicola Monohydropisphaera fusigera Hydropisphaera fungicola Hydropisphaera suffulta Paragliomastix rosea Paragliomastix chiangraiensis Septofusidium berolinense Pseudoacremonium sacchari Lasionectria olida Lasionectria olida Lasionectria castaneicola Lasionectria atrorubra Verruciconidia persicina Verruciconidia persicina Verruciconidia erythroxyli Verruciconidia infuscata Verruciconidia quercina Verruciconidia quercina Lasionectriopsis dentifera Lasionectriopsis dentifera Ochronectria thailandica Lasionectriopsis germanica Ochronectria calami Lasionectriella arenuloides Lasionectriella marigotensis Lasionectriella rubioi Ramosiphorum polyporicola Ramosiphorum polyporicola Ramosiphorum thailandicum MycoKeys 116: 327-344 (2025), DOI: Strains CBS 154.25T CBS 253.79 CBS 134.56T CBS 127532T CBS 141300T CBS 135907 CBS 135909T CBS 337.77T MFLUCC 15-0844T CBS 311.73 CBS: 330-77 CBS 329.77 A.R. 2812 CBS 346.85 CBS 138731T MAFF 240100T COAD 2070 T CBS 725.87T CBS 101694T CBS 701.73 CBS 392.73 CBS 124147T CBS 122304T CBS 122.87 CBS 277.80AT MFLUCC 14-0397T CBS 731.70 CBS 137990T CBS 799.69T CBS 798.69 CBS 122792T CBS 123502T CBS 310.59T GBS: 113716 CBS 728.87T CBS 100888T CBS 469.67T CBS:355-77 CBS 650.75 CBS 574.76T MFLUCC 15-0140T CBS 143538T CBS 134535 CBS 576.76T CBS 131606T CBS 140157T CBS 123779T CBS 109.87 CBS 101914T 10.3897/mycokeys.116.146683 ITS 0Q429613 0Q429614 0Q429639 0Q429641 KX228277 0Q429762 0Q429763 0Q429768 KX025152 0Q429594 0Q429592 0Q429593 HM484859 0Q429827 0Q429934 HM484866 KX866395 0Q429714 0Q429643 0Q429515 0Q429514 0Q429713 0Q429666 0Q429672 0Q429775 MN648324 0Q429859 KJ869144 0Q429693 0Q429692 0Q429680 0Q429674 0Q429921 0Q429922 0Q429910 0Q429911 0Q429925 0Q429927 0Q429700 KY607540 KU564071 0Q429701 0Q429755 0Q429696 0Q429698 0Q429699 0Q429823 0Q429822 0Q429825 LSU HQ232063 0Q055521 0Q055545 0Q055547 KX228328 0Q055661 0Q055662 0Q055666 KX025154 ORO52105 0Q055503 0Q055504 GQ505992 0Q430088 ORO52121 GQ506013 KX866396 0Q055615 0Q055549 0Q055426 0Q055425 0Q055614 ORO52107 0Q055577 0Q055673 MN648329 0Q430110 KJ869201 0Q055598 0Q055597 0Q055585 0Q055579 0Q430172 0Q430173 0Q430161 0Q430162 0Q430176 0Q430178 0Q055602 KY607555 KU564069 MK276528 0Q055654 0Q055601 KR105613 KU593581 0Q430084 0Q430083 0Q430086 Reference Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) 331 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed Species Protocreopsis rutila Protocreopsis rutila Protocreopsis finnmarkica Protocreopsis phormiicola Protocreopsis freycinetiae Nectriopsis lindauiana Nectriopsis fuliginicola Nectriopsis violacea Nectriopsis violacea Nectriopsis sporangiicola Clonostachys spinulosispora Clonostachys phyllophila Stephanonectria keithii Stephanonectria keithii Mycocitrus odorus Mycocitrus odorus Mycocitrus zonatus Mycocitrus phyllostachydis Emericellopsis fuci Emericellopsis fuci Emericellopsis maritima Emericellopsis pallida Emericellopsis brunneiguttula Stanjemonium grisellum Stanjemonium ochroroseum Proliferophialis apiculata Proliferophialis apiculata Acremonium subulatum Acremonium subulatum Acremonium aerium Acremonium longiphialidicum Acremonium purpurascens Acremonium ellipsoideum Acremonium ellipsoideum Acremonium brunneisporum Acremonium brunneisporum Acremonium multiramosum Waltergamsia pilosa Waltergamsia pilosa Waltergamsia alkalina Waltergamsia dimorphospora Geosmithia microcorthyli Geosmithia pallidum Bulbithecium spinosum Bulbithecium spinosum Bulbithecium arxii Bulbithecium ellipsoideum Ovicillium oosporum Ovicillium asperulatum Ovicillium asperulatum Strains CBS 396.66T CBS 229.70 CBS 147428T CBS 567.76T CBS 573.76T CBS 897.70T CBS 400.82T CBS 914.70T CBS 849.70 CBS 166.74T CBS 133762T CBS 921.97T CBS 943.72 CBS 100007 CBS 100104T CBS 120610 CBS 400.70 CBS 330.69 CBS 116467 CBS 485.92 CBS 491.71T CBS 490.71T CBS 111360T CBS 655.79T CBS 656.79T CBS 303.64T CBS 365.64 CBS 588.73AT CBS 115996 CBS 189.70T CBS 451.70T CBS 149.62T CBS 147433T CBS 147434 CBS 413.76T CBS 142823 CBS 147436T CBS 124.70T GBS:511.82 CBS 741.94T CBS 139050T CCF 3861T CBS 260;337 CBS 136.33T CBS 915.85 CBS 737.84T CBS 993.69T CBS 110151T CBS 130362T CBS 426.95 MycoKeys 116: 327-344 (2025), DOI: 10.3897/mycokeys.116.146683 ITS 0Q429814 0Q429813 0Q429803 0Q429806 0Q429804 0Q429729 KU382175 0Q429733 ORO50510 AF210661 MH634702 AF210664 0Q429872 0Q429871 0Q429717 0Q429715 0Q429719 0Q429718 0Q429564 0Q429565 0Q429566 0Q429574 0Q429545 0Q429868 0Q429869 0Q429796 0Q429797 0Q429491 0Q429490 0Q429441 0Q429475 0Q429485 0Q429468 0Q429467 0Q429444 0Q429445 0Q429476 0Q429949 0Q429948 0Q429935 LN810515 NR_137566 0Q429599 0Q429512 0Q429510 0Q429505 0Q429507 0Q429758 0Q429756 KU382192 LSU 0Q430077 0Q430076 0Q055699 0Q430069 ORO52113 0Q055629 0Q055628 0Q055632 MH871773 AF210662 KY006568 0Q055445 0Q430121 0Q430120 0Q055618 0Q055616 0Q055620 0Q055619 0Q055477 0Q055478 0Q055480 0Q055487 0Q055457 0Q430117 0Q430118 0Q055692 0Q055693 0Q055402 0Q055401 0Q055352 0Q055386 0Q055396 0Q055379 0Q055378 0Q055355 0Q055356 0Q055387 0Q430199 0Q430198 0Q430185 LN810506 NG_067560 0Q055509 0Q055423 0Q055421 0Q055416 0Q055418 0Q055657 0Q055655 KU382233 Reference Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) 332 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed Species Strains Proxiovicillium blochii CBS 427.93T Proxiovicillium blochii CBS 324.33 Proxiovicillium lepidopterorum CBS: 1012391 Hapsidospora flava CBS 596.70T Hapsidospora flava CBS 316.72 Hapsidospora variabilis CBS 100549T Hapsidospora stercoraria CBS 516.70T Alloacremonium humicola CBS 613.82T Alloacremonium ferrugineum CBS 102877T Stilbocrea walteri CBS 144627T Stilbocrea macrostoma CBS 114375 Flammocladiella decora CBS 142776 Flammocladiella aceris CBS 138906T GZUIFR 24.013T GZUIFR 24.014 GZUIFR 24.015 Biconidium sinense Biconidium sinense Biconidium sinense “ n Note: T = Ex-type; New isolates in this study are in bold; The line “—” represents the absence of GenBank record. ITS: the internal tran- scribed spacer region and intervening 5.8S nrRNA; LSU: 28S large subunit. ITS 0Q429816 0Q429815 0Q429817 0Q429649 0Q429648 0Q429663 0Q429662 0Q429496 0Q429495 ORO50519 0Q429873 MF611693 0Q429591 PQ595985 PQ595986 PQ595987 LSU 0Q430079 0Q430078 0Q430080 0Q055555 0Q055554 0Q055569 0Q055568 0Q055407 0Q055406 0Q430124 0Q430122 MF614949 KR611901 PQ595988 PQ595989 PQ595990 Reference Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) Hou et al. (2023) This study This study This study Table 2. Strains of Didymocyrtis and corresponding GenBank numbers included in phylogenetic analyses. Species Strains Didymocyrtis banksiae CSN1049 Didymocyrtis banksiae CSN1065 Didymocyrtis brachylaenae CPC 32651 Didymocyrtis cladoniicola CBS 131731 Didymocyrtis cladoniicola CBS 131732 Didymocyrtis consimilis CBS 129140 Didymocyrtis consimilis CBS 129338 Didymocyrtis epiphyscia Freebury 1411 Didymocyrtis foliaceiphila CBS 131729 Didymocyrtis foliaceiphila €BS'131730 Harris 57476 (NY) Harris 57475 (NY) CAA 1002 T CAA 1003 Diederich 17327b Diederich 17327a Didymocyrtis melanelixiae Didymocyrtis melanelixiae Didymocyrtis pini Didymocyrtis pini Didymocyrtis pseudeverniae Didymocyrtis pseudeverniae Didymocyrtis ramalinae Paul 10113 Didymocyrtis ramalinae Paul 27113 Didymocyrtis septata KNU-JJ-1827 Didymocyrtis slaptonensis MoraA (BR) Didymocyrtis trassii AB298 Didymocyrtis trassii AB297 Didymocyrtis xanthomendozae CBS 129666 Parathyridaria philadelphi CBS 143432 GZUIFR 24.004T GZUIFR 24.005 Didymocyrtis shanxiensis Didymocyrtis shanxiensis a“ an” ITS MT813909 MT813919 MH327821 KP170644 KP170645 MH865190 MH865230 KT383824.1 KP170649 KP170650 KT383831 KT383828 MW732246 MW732247 KT383833 KT383832 KT383839 KT383836 LC552949 KT383841 MG519614 MG519613 KP170651 MH107905 PQ065635 PQ065636 tub2 MH327896 KP170694 KP170695 KP170699 KP170700 MW759031 MW759030 KP170701 PQ119783 PQ119784 Reference Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) Monteiro et al. (2022) This study This study Note: T = Ex-type; New isolates in this study are in bold; The line “—” represents the absence of GenBank record. ITS: the internal tran- scribed spacer region and intervening 5.8S nrRNA; tub2: B-tubulin. MycoKeys 116: 327-344 (2025), DOI: 10.3897/mycokeys.116.146683 333 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed 100/1.00, Caespitomonium squamicola CBS 392.73 i i Cacdpliomonium SauaMicolA CBS 701.73 Caespitomonium 58/0.68 _ |100/1.00 ; Fusariella arenula CBS 329.77 66/0.53 Fusariella arenula CBS 330.77 F t : —- Fusariella atrovirens CBS 311.73 usariella 86/0.75. 100/1.06 Sata ht MFLUCC 15-0844T = -/0,52 -———— Gossypinidium sporodochiale CBS 101694T Gossypinidium 100/1.00 ————- peat emelsie aurantia COAD 2070T Synnemellisia 100/1.00 ee eae wea ran ee ae ‘aracylindrocarpon aloicola : DE) Paracylindrocarpon aurantiacum CBS 135909T Paracylindrocarpon 80/0.88 __ Paracylindrocarpon multiseptatum CBS 337.7717 66/0.63. 100/0.98 eee murorum cB 154.25T 100/1.00 fiomastix murorum CBS 253 79 Gliomastix 93/0.96 30.66 — Gliomastix roseogrisea CBS 134.56T 88/0.93 94/0.79\_ Gliomastix tumulicola CBS 127532T . $8/0.9: MS ba ., EE Te Sas fusigera CBS 124147T Monohydropisphaera 100/1.00 , Hydropisphaera fungicola CBS 122304T ; sso / me op opi fecha ngico la CBS 132-81 Hydropisphaera oumegueriella rufula b i 69/0.51 7 Selinia pulchra Ate 83 40100 ROU rE ene i 100/1.00 — Verrucostoma freycineti srl C Verrucostoma Indrdiicense CBS 187510 Verrucostoma : Musananaesporium tectonae CBS 725.87T Musananaesporium Paragliomastix chiangraiensis MFLUCC 14-0397T i A 56/0.57 100/1.00 Pacaghormasty rate CRS 277.80AT Par agliomastix A -————_——_Septofusidium berolinense CBS 731.70 Septofusidium 100/1.00 Verruciconidia erythroxyli CBS 728.87T 98/1.00 Verruciconidia infuscata CBS 100888T Verruciconidia persicina CBS 113716 Verruciconidia Verruciconidia persicina CBS 310,59T 99/1100, Verruciconidia quercina CBS 355.77 98/1.00 ~ Verruciconidia quercina CBS 469.67T 99/1.00 Lasionectria atrorubra CBS 123502T Tood.00) L4sionectria castaneicola CBS 122792T Lasionectria 5 ae \ Lasionectria olida CBS 798.69 100/1.00! Lasionectria olida CBS 799.69T . apt Fae sacchari BS PRG IIESE Pseudoacremonium 52/1.00. 100/1.00 chronectria calami ; 86/0.62.~s ae sesinstess cs ea 15.0140T Ochronectria 100/0,82 ~*+ Lasionectriella arenuloides : ? : area 1010.52 Filomena marigotensis CBS 131606T Lasionectriella 66/1.00 r Lasionectriella rubiot CBS oot asionectriopsis dentifera E TINE pss Lastonectridpsis denttf era, cB 650.75 Lasionectriopsis 96/1.00 100/1.00- Lasionectriopsis germanica CBS 143538T 95/0.60| 98/0.81, Ramosiphorum polyporicola CBS 109.87 i - Ramosiphorum polyporicola CBS 123779T Ramosiphorum eM 0s7|\. 100/1.00 Ramosiphorum Tiatlandicum CBS 101914T “ Ls Protocreopsis freycinetiae CBS 573.76T 100/1.00 Protocreopsis finnmarkica CBS 147428T i : Ly Protocreopsis rutila CBS 229.70 Protocreopsis 100/1.00| 100/1.00' Protocreopsis rutila CBS 396,66) 00 Protocreopsis phormiicola CBS 567.76T [|___ amu sdi drees RH SB iconidium sinense fl iconidi Biconidium sinense GLUIFR 24.015 USE: 88/0.77— Nectriopsis fuliginicola CBS 400.82T 10/1200 90/1.00)" Nectriopsis lindauiana CBS 897,70T Werrione 2 100/1.00 Los Nectriopsis violacea CBS 849.70 ectriopsis 100/1.00' Nectriopsis violacea CBS 914,70T |__________ Nectriopsis sporangiicola CBS 166.74T 83/1.00 ———— Clonostachys phyllophila CBS 921.97T '— Clonostachys spinulosispora CBS 133762T Clonostachys paOaeae My Coe peones cs tidal ds . ycocitrus odorus i 83/0.99| 100/100! |_— Wycocitrus zonatus CBS 400.70 Mycocitrus 91/0.92| 100/1.00- Steph eae Ms Dy Hee h ares hae i : ‘ephanonectria keithii i, 100/18 Stephanonectria keithit CBS 943.72 Stephanonectria 100/1.00. Acremonium aerium 189. 99/1.00 Acremonium purpurascens CBS 149.62T ied ibedi longiphialidicum Mae ae cremonium brunnei 835/064 Lean SraineCponianC Ee alsteU Acremonium Acremonium subulatum CBS 115996 Acremonium subulatum CBS 588 7. TNT ay tama eee feum PS gz gRaT cremonium ellipsoide 95/0.98 L____ 4cremonium multiramosum CBS 147436T 100/1.00 100/1.00 Emericellopsis brunneiguttula CBS 111360T Emericellopsis fuci CBS 116467 92/0.99| 98/1.00 ALO Emericellopsis fuci CBS 485.92 Emericellopsis 98/0.99. Emericellopsis maritima CBS 491,71T 68/062) \, Emericellopsis pallida CBS 490.71T. Ly Stanjemonium grisellum CBS 655.79T Stanjemonium 100/0.99! Stanjemonium ochroroseum CBS 656 79T 96/0.78 i ial: i i * aay eee Lroorro Proiiferophialis dpicudara CBS 308-04 Pron ferophiahs ; 100/1.00;— Alloacremonium ferrugineum CBS 102877T i Sigs Alloacremonium humicola CBS 613.827 Alloacremonium Waltergamsia alkalina CBS 741.94T Waltergamsia pilosa CBS 124.70T ‘ 100/1.00 PETC BE BOSE CBS 511.82 Waltergamsia ~ Waltergamsia GAIL URES CBS 139050T 100/1.007 _ Bulbithecium arxti CBS 737.8: = EE GLa che eA) S Bulbithecium o6—___ Bu ecium spinosum i —— L| 99/1.00 gee 100/1.00 Buinecin spinosum CBS 915.85 100/1.00 Ovicillium asperulatum CBS 130362T — 2 Ovicillium asperulatum CBS 426.95 Ovicillium L— Oveilli 110151T 100/1.00 100/0.99 Proxiovicilium blochit CBS" 5S BS; iter a : 97/1.00| 100/1.00 1 Proxiovicillium blochii CBS 427.937 Proxiovicillium L Proxiovicillium epidopterorum CBS 101239T 100/1.00 , Hapsidospora flava CBS 316.72 , 710,97] Hapsidospora flava CBS 596.70T Hapsidospora __ Hapsidospora variabilis CBS_100549T. 99/1.00|___ Hapsidospora stercoraria CBS 516.70T 100/1.00— Geosmithia microcorthyli CCF 3861T Geosmithia '— Geosmithia pallidum CBS 260.33T 99/1.00 — Stilbocrea macrostoma CBS_114375 100/1.00 — Stilbocrea walteri CBS 144627T Stilbocrea <"| Flammocladiella decora CBS 142776 2 Flammocladiella aceris CBS 138906T Flammocladiellaceae (Outgroup) 0.06 Figure 1. Phylogenetic tree of Bionectriaceae constructed from the dataset of ITS and LSU. Notes: Statistical support values (ML/BI) were shown at nodes. ML bootstrap values >50% and posterior probabilities => 0.50 are shown above the internal branches. ‘—’ indicates the absence of statistical support (< 50% for bootstrap proportions from ML analysis; < 0.50 for posterior probabilities from Bayesian analysis). Three new strains are shown in blue font. Sordariomycetes O.E. Erikss. & Winka Hypocreales Lindau Bionectriaceae Samuels & Rossman Biconidium H.Y. Wang & Y.F. Han, gen. nov. MycoBank No: MB857281 Etymology. Referring to the bicellular conidia. MycoKeys 116: 327-344 (2025), DOI: 10.3897/mycokeys.116.146683 334 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed Description. Mycelium hyaline, septate, smooth, thin-walled. Conidiophores hyaline, septate, smooth-walled, solitary, straight, (sub-)erect, arising directly from hyphae, unbranched or branched, bearing 1-5 levels with 1-6 phialides per node. Conidiogenous cells enteroblastic, monophialidic, lateral or terminal, awl-shaped, hyaline, smooth, with globose to cylindriform thickening at con- idiogenous loci. Conidia bicellular, podiform, unsymmetrically at both ends, hyaline, thick-walled, smooth, arranged in slimy heads. Chlamydospores and sexual morph absent. Type species. Biconidium sinense H.Y. Wang & Y.F. Han Notes. Three isolates from green soil of sewage treatment plant clearly form an independent clade on the ITS and LSU tree (Fig. 1), and are phylogenetically segregated from other genera, representing the new species with conidioge- nous cells with globose to cylindriform thickening at conidiogenous loci and podiform conidia arranged in slimy heads. Therefore, we introduce Biconidium as anew genus to accommodate this species. Biconidium sinense H.Y. Wang & Y.F. Han, sp. nov. MycoBank No: MB857282 Fig.s2 Etymology. Referring to China where the species was isolated. Type. CHINA * Zhejiang Province, Hangzhou City, sewage treatment plant (30°10'53'"N, 120°10'2"E), soil, August 2021, Yulian Ren, ex-type culture GZUIFR 24.013, dried holotype GZAC 24.013. ITS sequences, GenBank PQ595985; LSU sequences, GenBank PQ595988. Description. Culture characteristics (7 days of incubation at 25 °C): Colony on PDA, 20-30 mm diam., fleshy, plicated, beige (RAL1001) at the center, villiform, traffic white (RAL 9016) at the edge, reverse, light Ivory (RAL1015) at the cen- ter, cream (RAL9001) at the edge, nearly round, margin partial; Colony on MEA, 25-30 mm diam., flocculence, traffic white (RAL 9016), reverse, broom yellow (RAL1032), margin entire, round. Colony on OA, 30-35 mm diam., thin, short villous, signal white (RAL9003), reverse, cream (RAL9001), margin entire, round. On PDA, Mycelium hyaline, septate, smooth, thin-walled 1.2-2.7 um wide. Conidiophores hyaline, septate, smooth, solitary, straight, (sub-)erect, arising directly from hyphae, branched or unbranched, bearing 1-5 levels with 1-6 phialides, 1-3 septate at base or middle, 20-52 um long, 1.5-2.7 um wide at base. Phialides lateral or terminal, from the conidiophores or directly from the mycelia, awl-shaped, hyaline, smooth-walled, 9.5-35 um long, 1-2.3 um wide at base, with globose to cylindriform thickening at conidiogenous loci. polyphi- alides not observed. Conidia podiform, 1-septate, 2.5-6.0 x 1.0-3.0 um (mean + SD =3.5+1.0 x 2.0 + 0.5 um, n = 30), center-empty, unsymmetrically at both ends, apex angular, base subobtuse, hyaline, thick-, smooth-walled, arranged in slimy heads. Chlamydospores and sexual morph not observed. Additional specimens examined. CHINA * Zhejiang Province, Hangzhou City, sewage treatment plant (30°10'53"N, 120°10'2"E), soil, August 2021, living cul- tures GZUIFR 24.014 (ITS sequences, GenBank PQ595986; LSU sequences, GenBank PQ595989), GZUIFR 24.015 (ITS sequences, GenBank PQ595987; LSU sequences, GenBank PQ595990). Mycokeys 116: 327-344 (2025), DOI: 10.3897/mycokeys.116.146683 335 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed £ h t Figure 2. Morphological characteristics of Biconidium sinense sp. nov. a-c front and reverse of colony on PDA, OA and MEA after 7 days at 25 °C d, e conidiophores and conidial heads f-h conidiophores and conidia i conidia. Scale bars: 10 mm (a-c); 50 um (d); 20 um (e); 10 um (f-i). Notes. Phylogenetically, our three strains (GZUIFR 24.013, GZUIFR 24.014 and GZUIFR 24.015) can apparently separate with other species in Bionectri- aceae, and clustered in a single clade with a high support value (BI pp = pos- terior probability 1, ML BS 100) (Fig. 1). Biconidium sinense is distinguished from other species of Bionectriaceae by conidiogenous cells with globose to cylindriform thickening at conidiogenous loci, and podiform conidia arranged in slimy heads in the morphological characteristics. Dothideomycetes O.E. Erikss. & Winka Pleosporales Luttr. ex M.E. Barr Phaeosphaeriaceae M.E. Barr Didymocyrtis Vain. Didymocyrtis shanxiensis H.Y. Wang & Y.F. Han, sp. nov. MycoBank No: MB857280 Fig. 4 Etymology. shanxiensis, referring to Shanxi province where the type locality was isolated. MycoKkeys 116: 327-344 (2025), DOI: 10.3897/mycokeys.116.146683 336 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed 51/0.7 Didymocyrtis brachylaenae CPC 32651 T 98/1.00 Ht, Didymocyrtis shanxiensis GZUIFR 24.004T Didymocyrtis shanxiensis GZUIFR 24.005 100/1.00 49/1.00| Didymocyrtis pini CAA 1002 T Didymocyrtis pini CAA 1003 62/0.69 67/0.67 Didymocyrtis septate KNU-JJ-1827 93/0.97 Didymocyrtis ramalinae Paul 10i13 65/0.73 >| | Didymocyrtis ramalinae Paul 27i13 | Didymocyrtis consimilis CBS 129140 Pua Didymocyrtis consimilis CBS 129338 Didymocyrtis melanelixiae Harris 57475 (NY) 93/0.98 Didymocyrtis melanelixiae Harris 57476 (NY) Didymocyrtis pseudeverniae Diederich 17327a 81/0.85 Didymocyrtis pseudeverniae Diederich 173276 _ Phaeosphaeriaceae 95/0.94 68/0.79 NY Didymocyrtis trassii AB297 i. Didymocyrtis trassii AB298 98/1.00 | Diederichomyces foliaceiphila CBS 131729 Diederichomyces foliaceiphila CBS 131730 96/0.99. Didymocyrtis epiphyscia Freebury 1411 62/0.53|\) Diederichomyces cladoniicola CBS 131731 Diederichomyces cladoniicola CBS 131732 Didymocyrtis slaptoniensis MoraA (BR) 99/1.00 Didymocyrtis xanthomendozae CBS 129666 T Didymocyrtis banksiae CSN1049 88/0.88 Didymocyrtis banksiae CSN1065 Parathyridaria philadelphi CBS 143432 0.08 Thyridariaceae (Outgroup) Figure 3. Phylogenetic tree of the genus Didymocyrtis constructed from the dataset of ITS and tub2. Notes: Statistical support values (ML/BI) were shown at nodes. ML bootstrap values >50% and posterior probabilities => 0.50 are shown above the internal branches. Two new strains are shown in blue font. Type. CHINA * Shanxi Province, Datong City, sewage treatment plant (40°2'42"N, 113°20'48"E), soil, August 2021, Yulian Ren, ex-type culture GZUIFR 24.004, dried holotype GZAC 24.004. ITS sequences, GenBank PQ065635; tub2 sequences, GenBank PQ119783. Description. Culture characteristics (7 days of incubation at 25 °C): Colo- ny on PDA, 30-35 mm diam., thin, villiform, cream (RAL9001), reverse cream (RAL9001), regular in the margin; Colony on MEA, 20-25 mm diam., thick, villi- form, light lvory (RAL1015), reverse dahlia yellow (RAL1033), regular in the mar- gin; Colony on OA, 30-35 mm diam.., texture velvety, olive yellow (RAL1020), re- verse stone gray (RAL7030), regular in the margin. Black spots produced after incubating 15 days on PDA. On PDA medium after 30 days of incubation at 25 °C, Hyphae septate, hya- line, smooth, thick-walled, 1.0-2.5 um wide. Conidiomata submersed, brown to black, globose, 150-250 um diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells globose to subglobose, also ampulliform, aseptate, hyaline, smooth, thick-walled, 4.5-10.0 x 2.0-6.0 um (mean + SD = 7.0 + 1.9 x 3.5 + 1.0 um, n = 15). Conidia abundant, cymbiform mostly, brown, smooth, apex subobtuse, base truncate, 1-septate, 5.0-11.0 x 1.5-3.0 um (mean + SD = 7.5+1.6 x 2.0 + 0.4 um, n = 30). MycoKeys 116: 327-344 (2025), DOI: 10.3897/mycokeys.116.146683 337 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed Figure 4. Morphological characteristics of Didymocyrtis shanxiensis sp. nov. a-c front and reverse of colony on PDA, OA and MEA after 7 days at 25 °C d, e conidiomata on culture f hyphae g, h conidiomata and ruptured conidiomata with conidia mass i conidia j, k conidiogenous cells. Scale bars: 10 mm (a-c); 20 um (g, h); 10 um (i); 20 um (j, k). Additional specimens examined. CHINA * Shanxi Province, Datong City, sewage treatment plant (40°2'42'N, 113°20'48’E), soil, August 2021, living cultures GZUIFR 24.005. ITS sequences, GenBank PQ065636; tub2 sequences, GenBank PQ119784. Notes. Twenty-nine species of the genus Didymocyrtis are recorded in the Index Fungorum. However, the DNA sequence data of fifteen species have no MycoKeys 116: 327-344 (2025), DOI: 10.3897/mycokeys.116.146683 338 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed records in NCBI database. Phylogenetically, our two strains (GZUIFR 24.004 and GZUIFR 24.005) clustered in a single clade with a high support value (ML/ BI 98/1) (Fig. 3). In the phylogenetic tree, although our new species D. shanxien- sis and Didymocyrtis brachylaenae Crous are closely related species, they were obviously different in morphological characteristics. Didymocyrtis shanxiensis, having conidiophores reduced to conidiogenous cells, globose to subglobose and ampulliform conidiogenous cells, and cymbiform conidia, can be distin- guished from D. brachylaenae with subcylindrical and branched conidiophores, lining the inner cavity and ampulliform to doliiform conidiogenous cells, and fusoidellipsoid to subcylindrical conidia (Crous et al. 2018). Discussion Hou et al. (2023) revaluated acremonium-like fungi in Hypocreales, and found most species of Acremonium s. lat. grouped in genera of Bionectriaceae. There- fore, the phylogenetic tree of Bionectriaceae is provided based on multi-locus (ITS, LSU, rpb2, tef-1a) DNA sequencing analyses to accommodate 183 spe- cies and 39 genera including 10 new genera. In this study, employing ITS and LSU sequences can well distinguish the species of Bionectriaceae. From the phylogenetic tree (Fig. 1), three strains of our new species Biconidium sinense cluster in a well-separated clade with a high support value (ML/BI 100/1). Meanwhile, B. sinense having conidiogenous cells with globose to cylindriform thickening at conidiogenous loci, and podiform conidia arranged in slimy heads differs from all other species of Bionectriaceae. Therefore, Biconidium is intro- duced to accommodate a new species B. sinense combined with phylogenetic and morphological analyses. Bionectriaceae are including both sexual morphs and asexual taxa (Hou et al. 2023). Species of the Bionectriaceae are mostly found in terrestrial or freshwater environments, with fewer commonly found in marine habitats, and they are common coprophilous, corticolous, fungicolous, lichenicolous or herbicolous (Zhao et al. 2023). In this study, our three strains of B. sinense were isolated from green soils of sewage treatment plant. In this study, although D. shanxiensis, D. brachylaenae, D. pini and D. septata clustered as the sister subclades, they were obviously different in morphologi- cal characteristics. Morphologically, the main characteristics of D. shanxiensis are having globose conidiomata, conidiophores reduced to conidiogenous cells, globose to subglobose and ampulliform conidiogenous cells, and the smaller size of cymbiform conidia (mean size = 7.5 x 2.0 um). While, D. brachy- laenae can be distinguished from D. shanxiensis by having subcylindrical and branched conidiophores, and fusoidellipsoid to subcylindrical conidia (Crous et al. 2018); Didymocyrtis pini can be distinguished from D. shanxiensis by hav- ing fusiform conidia (mean size = 8.5 x 2.4 um) (Monteiro et al. 2022); Didymo- cyrtis septate differed from D. shanxiensis by having irregular conidiomata, and fusiform, clavate to subcylindrical conidia (mean size = 8.2 x 2.3 ym) (Das et al. 2021). At the same time, D. shanxiensis has a clear morphological difference from fifteen species without DNA sequence data (Joshi et al. 2024), so it is proposed as a new species in the genus Didymocyrtis. Up to now, the most spe- cies of Didymocyrtis are lichenicolous fungi living parasitic life-styleare (Ertz et al. 2015; Suija et al. 2021). Some Didymocyrtis spp. are pathogenic fungi and saprophytic fungi. For example, D. brachylaenae and D. pini as pathogeny Mycokeys 116: 327-344 (2025), DOI: 10.3897/mycokeys.116.146683 339 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed live on plant leaves (Crous et al. 2018; Monteiro et al. 2022), and D. septata is saprophytic in containing plant soil (Das et al. 2021). Our two strains of new species were also isolated from green land soil in this study and possible to be saprophytic. Presently, this genus includes twenty-nine species in the Index Fungorum (http://www.indexfungorum.org/Names/Names.asp, retrieval on 10 January 2025). Here, together with D. shanxiensis, the genus Didymocyrtis has a total of thirty species. Though two new species were reported in this study, we believed that more new taxa will be found and reported from the various soil habitats, which are deserving to be explored in the future. Acknowledgements We appreciate Tianpeng Wei for the photomicrograph. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding The work was supported by the National Natural Science Foundation of China (no. 32160007, 32260003) and “Hundred” Talent Projects of Guizhou Province (Qian Ke He [2020] 6005). Author contributions Hai-Yan Wang: Writing — review & editing, Formal analysis, Project administration. Chun- bo Dong, Yan-Wei Zhang and Wan-Hao Chen: Data acquisitio, Data analysis, Investiga- tion, Data curation. Yan-Wei Zhang and Yan-Feng Han: Supervision, Project administra- tion, Funding acquisition. Author ORCIDs Hai-Yan Wang ® https://orcid.org/0000-0001-9190-0490 Chunbo Dong ® hittps://orcid.org/0000-0001-7074-5700 Yan-Wei Zhang ® https://orcid.org/0000-0003-1251-5821 Wan-Hao Chen ® hittps://orcid.org/0000-0001-7240-6841 Yan-Feng Han ® https://orcid.org/0000-0002-8646-3975 Data availability All of the data that support the findings of this study are available in the main text. References Akilandeswari P, Pradeep B (2016) Exploration of industrially important pigments from soil fungi. Applied Microbiology and Biotechnology 100(4): 1631-1643. https://doi. org/10.1007/s00253-015-7231-8 MycoKeys 116: 327-344 (2025), DOI: 10.3897/mycokeys.116.146683 340 Hai-Yan Wang et al.: One new genus and two new species we identified and proposed Barr ME (1979) A classification of Loculoascomycetes. 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