<8) MycoKeys MycoKeys 119: 95-121 (2025) DOI: 10.3897/mycokeys.119.145174 Research Article Overview of hirsutella-like anamorphs in Ophiocordyceps (Sordariomycetes, Ophiocordycipitaceae): introducing two new species and one new record from China Shi-Wen Xie'23®, De-Ping Wei22®, Jun-Zhi Qiu’, Xing-Can Peng2*5*®, Ji-Chuan Kang'22®, Zhang-Jiang He'22®, Zeng-Zhi Li’, Chun-Ru Li’, Shi-Ke Huang2®, Xian Zhang2*5*®, Zhong-Liang Liu’23®, Jing Bu'?%®, Nalin N. Wijayawardene®®, Ting-Chi Wen'23© 1 School of Pharmacy, Guizhou University, Guiyang 550025, Guizhou, China 2 State Key Laboratory of Green Pesticide; Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, China aN Do Fe WwW Engineering Research Center of Southwest Bio-Pharmaceutical Resources, Ministry of Education, Guizhou University, Guiyang 550025, China State Key Laboratory of Agricultural and Forestry Biosecurity, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, 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 Zhejiang BioAsia Life Science Institute, Pinghu 314200, China Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biology and Food Engineering, Qujing Normal University, Qujing 655011, China Corresponding author: Ting-Chi Wen (tingchiwen@yahoo.com) OPEN Qrceess This article is part of: Diversity, taxonomy, and systematics of macrofungi from tropical Asia Edited by Olivier Raspé, Rui-Lin Zhao, Jennifer Luangsa-ard Academic editor: Jennifer Luangsa-ard Received: 24 December 2024 Accepted: 15 May 2025 Published: 1 July 2025 Citation: Xie S-W, Wei D-P Qiu J-Z, Peng X-C, Kang J-C, He Z-J, Li Z-Z, Li C-R, Huang S-K, Zhang X, Liu Z-L, Bu J, Wijayawardene NN, Wen T-C (2025) Overview of hirsutella-like anamorphs in Ophiocordyceps (Sordariomycetes, Ophiocordycipitaceae): introducing two new species and one new record from China. MycoKeys 119: 95-121. https://dol.org/10.3897/ mycokeys.119.145174 Copyright: © Shi-Wen Xie 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 Ophiocordyceps, a species-rich genus in Ophiocordycipitaceae, is a holomorphic genus in which most of the species are reported with hirsutella-like anamorphs. In this study, we introduce two new species of hirsutella-like anamorphs from lepidopteran larvae (viz., Ophiocordyceps tielingensis sp. nov. and Ophiocordyceps keginii sp. nov.). Ophiocordy- ceps radiata (syn. Hirsutella radiata), a new combination, exhibits a pathogenic associa- tion with a fly, and it is reported as a new geographic record from China, based on inte- grated morphological and molecular analyses. We provide a checklist of Ophiocordyceps species with hirsutella-like anamorphs and comprehensively review their characteristics of anamorphs and teleomorphs. These definitive findings establish a foundation for the classification and diversity of Ophiocordyceps species with hirsutella-like anamorphs. Key words: Entomopathogenic fungi, hirsutella-like, Ophiocordyceps Introduction The clavicipitoid fungi are an ecologically important group that are classified into Clavicipitaceae, Cordycipitaceae, Ophiocordycipitaceae, and Polycephalomyceta- ceae (Xiao et al. 2023). Members of these families establish close associations with insects (up to 13 orders of Insecta) and other arthropods (Wei et al. 2022). Ophio- cordycipitaceae is a diverse family encompassing fungi with significant ecologi- cal, economic, medicinal, and cultural importance. Sung et al. (2007a) established Ophiocordycipitaceae based on molecular data, and this family currently compris- es more than 500 species and eight genera, including Drechmeria, Harposporium, Hantamomyces, Ophiocordyceps, Paraisaria, Purpureocillium, Tolypocladium, and 95 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps Torrubiellomyces (Quandt et al. 2014; Spatafora et al. 2015; Mongkolsamrit et al. 2019; Crous et al. 2020; Araujo et al. 2022). The type genus of Ophiocordycipita- ceae, Ophiocordyceps, was erected by Petch (1931) to accommodate O. blattae, O. unilateralis, O. peltata, and O. rhizoidea. These four mentioned species share similarities in producing fibrous, tough, pliant to wiry, dark to brightly colored stro- mata; superficial to immersed perithecia; clavate asci with thickened apex; and whole, hyaline, fusiform, multiseptate ascospores. Ophiocordyceps is the most species-rich genus within Ophiocordycipitaceae, with a wide distribution ranging from tropical forests to temperate ecosystems. Anamorphs belonging to Hirsute- lla, Hymenostilbe, Syngliocladium, Paraisaria, and Tilachlidiopsis have been linked to species of Ophiocordyceps (Quandt et al. 2014; Mongkolsamrit et al. 2019). Hirsutella is a widely distributed entomopathogenic genus with a broad host range, primarily infecting arthropods and nematodes (Liang 1990b). Hirsutella was originally classified as a clavarioid basidiomycete (Patouillard 1892). Speare (1920) re-evaluated the type species and clarified the taxonomic placement of this genus. Gams and Zare (2003) summed up that Hirsutella is distinguished by its basally subulate phialides, which taper into one (typically) or occasional- ly several very slender needle-like necks, either on synnemata or mononema- tous mycelium. Quandt et al. (2014) proposed that species with hirsutella-like anamorphs are phylogenetically spread throughout Ophiocordyceps, for which Hirsutella was suppressed in favor of Ophiocordyceps. However, there are still a few new species being introduced to this genus since then, viz., H. tortricicola (Zou et al. 2016a), H. shennongjiaensis (Zou et al. 2016b), Hirsutella changbei- sanensis (Qu et al. 2017), H. hongheensis (Yuan et al. 2020), H. flava (Qu et al. 2021), and H. kuankuoshuiensis (Qu et al. 2021). On the contrary, most research- ers accepted the suggestion of Quandt et al. (2014) and added new species with hirsutella-like anamorph to Ophiocordyceps, such as O. myrmicarum (Simmons et al. 2015a), O. nooreniae (Crous et al. 2016), O. retorta (Qu et al. 2018), O. uni- tuberculata (Wang et al. 2018), O. sporangifera (Xiao et al. 2019), O. delicatula (Clifton et al. 2021), O. pingbianensis (Chen et al. 2021), O. flavida (Mongkolsam- rit et al. 2021), O. nujiangensis (Sun et al. 2022), O. lilacina (Mongkolsamrit et al. 2023), O. maybankeae (Tan et al. 2023), and O. albastroma (Sun et al. 2024). During our field surveys of entomopathogenic fungi in southwestern China, we collected several samples of dead insects. In morphology, three fungal species were identified as hirsutella-like anamorphs. DNA sequence-based phylogenetic analyses confirmed two species (from lepidopteran larvae) are new to Ophiocordy- ceps S. Str. (viz., O. tielingensis sp. nov. and O. keginii sp. nov.). H. radiata has been reclassified as O. radiata based on a newly collected specimen (associated with a fly), and itis the first time to report this species from China. Furthermore, a checklist of Ophiocordyceps species with hirsutella-like anamorphs and a comprehensive re- view of their teleomorphic and anamorphic characteristics are also provided. Methods and materials Sample collection and morphological study A survey of entomopathogenic fungi was conducted in mixed forests in Yunnan and Liaoning Provinces of China. Two species were found infecting lepidopteran larvae, with their synnemata protruding from the host on the ground, while one MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 96 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps species was found infecting flies attached to fresh fern leaves. High-resolution images and morphological data were collected in the field for subsequent tax- onomic validation. The fresh samples were collected into sterilized self-sealing bags or centrifuge tubes and labeled appropriately. For a more detailed exam- ination of the morphology of the specimens, freehand sections were made. Fol- lowing sectioning, the tissue slices were carefully transferred onto slides using sterile water or Congo red solution for mounting. Subsequently, the prepared specimens were examined under a compound microscope (Nikon ECLIPSE Ni) to discern the intricate microstructures, including synnemata, phialides, and conidia. The dried specimens were deposited in the Herbarium of Cryptogams, Kunming Institute of Botany, Academia Sinica (KUN-HKAS). Index Fungorum identifiers were obtained following the protocols described in Index Fungorum (http://www.indexfungorum.org/, retrieved on 23 May 2025). DNA extraction, PCR amplification, and sequencing Genomic DNA was extracted from fungal tissues using a DNA extraction kit (Omega Bio-Tek, Norcross, GA, USA) in accordance with the manufacturer’s protocol. The obtained total genomic DNA was stored at -20 °C. PCR amplifica- tion was performed for five loci, including the partial small subunit rRNA gene (SSU), the partial large subunit rRNA gene (LSU), the internal transcribed spac- er encompassing the 5.8S rDNA gene (ITS), the translation elongation factor 1-alpha gene (tef7-a), and the partial RNA polymerase II largest subunit (rpb7). The corresponding primers that were used for the amplification and sequenc- ing of these loci were NS1/NS4 for SSU (White et al. 1990), LROR/LR5 for LSU (Vilgalys and Hester 1990), ITS5/ITS4 for ITS (White et al. 1990), EF1-983F/EF1- 2218R for tef1-a (Rehner and Buckley 2005), and CRPB1A/RPB1Cr for RPB1 (Castlebury et al. 2004). The polymerase chain reaction (PCR) was performed ina 25 uL volume, including 12.5 uL of PCR mixture (2x Rapid Taq Master Mix, Vazyme Biotech), 7.5 uL of double-distilled water, 1 uL of each primer (10 uM), and 3 uL of 30 ng/L DNA template. Amplifications of ITS, SSU, and LSU genes were carried out using a BioRAD T100 Thermal Cycler (Singapore) with the PCR program as follows: initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 50 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 10 min. The PCR conditions of tef1-a and rpb7 were as follows: 95 °C for 5 min, followed by 10 cycles of 95 °C for 30 s, 56 °C for 50 s, 72 °C for 50 s, 30 cycles of 95 °C for 30 s, 52 °C for 50 s, and 72 °C for 50 s, and end with 72 °C for 10 min. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. in Chongqing, China, for sequencing using the aforementioned primers. The generated sequences were manually edited using BioEdit v.7.0.5.3 (Hall 1999) and submitted to GenBank. The accession numbers of newly generated sequences are listed in Table 1. Phylogenetic analyses The taxa included in the phylogenetic analyses were selected based on BLAST search results in NCBI and relevant literature (Quandt et al. 2014; Simmons et al. 2015b; Qu et al. 2021; Peng et al. 2024). Each locus was independent- ly aligned using MAFFT version v.7 (Kuraku et al. 2013; Katoh et al. 2019). MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 97 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps Table 1. GenBank accession numbers of the taxa used in the phylogenetic analyses; the newly generated sequences are in bold. Ex-type strains are indicated by ‘T.’ Species Hirsutella cf. haptospora H. changbeisanensis citriformis cryptosclerotium eleutheratorum fusiformis gigantea guyana haptospora hongheensis illustris XietpPiLiTiyT T\IirI = kuankuoshuiensis leizhouensis liboensis necatrix x)rtj)r\}x nodulosa x radiata’ rhossiliensis satumaensis x)r}x shennongjiaensis sinensis strigosa subulata thompsonii thompsonii uncinata Xj rprlpri ri rit versicolor Ophiocordyceps acicularis O. acroasca O. ansiformis O. arborescens O. australis O. basiasca O. bifertilis O. blattae O. blattae O. borealis O. campes O. camponoti-balzani 0. camponoti-bispinosi OBIS4 Hymenoptera O. camponoti-femorati O. camponoti- hippocrepidis O. clavata O. communis O. communis O. communis O. contiispora O. delicatula O. elongata Voucher | Host, =| TS Diptera KM652166 ARSEF 2228 ARSEF 30 Hymenoptera =e KM651995 | KM652036 kY4652172 | KME5T956| KY415582 | KY415590 ARSEF 9603 Lepidoptera | KM652163 | KM652072 | KM652115; si ARSEF 5549 KM652164 | KM652073 | KM652116 | KM651999 ARSEF 2226 KM652159 HKAS 1024517 MNO17176 ARSEF 5539 KM652160 GZUIFR Lepidoptera KY415575 2012KKS3-1 GZUIFR hir130707 | Lepidoptera KY415573 ass" MNO17177 KM652069 KM652118 | KM652001 | KM652041 “GZUFRbiNG0527 Homoptera | Kvaiss7e | || VAISS TARSEFS975 | Coleopters || MOS7794 |_| MHOS7799 | MHOS7799. ARSEF 5474 | Coleoters | ———_—_~| KM52067 | KMES2I10 | KMGSI999 | KMS2039_ | se506077 | oxs66900 | KMssz00 | KM652035 KM651996 | KM652037 KY945360 KY945358 KM652039 TARSEFTa69 |_Diptera |_| KM52076 | KMES2119 | KMGS2002 | KMGS2042 OF el Le Snj121022' —ARSEF253 | Aeari_—_|KMBS2179 | KMES2088 | KMG52I59|KMGS2016 | MTCC 10896 KJ524691 | ss, 24712) = s«| Ss sSsefert and Boulay (2004) KY945364 ARSEF 1037 Hemiptera KM652102 | KM652150 | KM652029 | KM652063 OSC 128580 JN049820 | DQ522543 | DQ518757 | DQ522326 | DQ522371 Sung et al. (2007a) —yroosois' | Formicinee |__| ONS5B47 | ONSSSGZ2 | ONSS7761 | ONSEa6aT ~YaH2z10007 | Formicinae |_| oRo4sza0 |__| OR098495 | ORGSI952 NoRC 1056517 | Lepidortera | ABSEEGBD | ApS6aGE6 | ABDGOATA | AB560572 | —y20101_|Formicine |__| 0N55828 | ONSSEBTO | ONS67748 | ONS69672 HUA 186097 Hymenoptera = YFCC 9012" | Formicinae | — —_—_| ON555843 | ON555923 | ON567763 ON568143 Reference Simmons et al. (2015b) Qu et al. (2017) Simmons et al. (2015b) Simmons et al. (2015b) Wraight et al. (2018) Simmons et al. (2015b Simmons et al. (2015b Simmons et al. (2015b Simmons et al. (2015b Yuan et al. (2020) Simmons et al. (2015b) Qu et al. (2017) ) ) ) ) Qu et al. (2017) Simmons et al. (2015b Simmons et al. (2015b Simmons et al. (2015b Simmons et al. (2015b Simmons et al. (2015b Simmons et al. (2015b Qu et al. (2021) we | mo FV |Home Simmons et al. (2015b) Simmons et al. (2015b) Simmons et al. (2015b) Simmons et al. (2015b) Simmons et al. (2015b) Simmons et al. (2015b) Tang et al. (2023c) Tang et al. (2023b) Ban et al. (2015) Sanjuan et al. (2015) Tang et al. (2023c) Tang et al. (2023c) ace se241 | Blatodea =) ~SS*UMS T2087 | TSGEABS | MITSEGATD ~MFLU1@0163 | Coleoptera MKAS251 | MKBEGO44 | MKESDS1 | MKBE0I09 | Bcc 36558" Lepidoptera MT7an955 |_| MTITBI75 | mTiven67 | MTTT@109 G143 Hymenoptera = FEMO2 — KX713637 | KX713615 | KX713692 | KX713720 Formicidae | KX713663 | KX713590 | KX713678 | KX713702 HIPPOC a KX713655 | KX713597 | KX713673 | KX713707 O. camponoti-nidulantis NIDUL2 | Formicidae | == ——'|- KX713640 | KX713611 | KX713669 | KX713717 O. camponoti-rufipedis G108 Hymenoptera | | KX713659 | KX713594 | KX713679 | KX713704 NBRC 106961 Coleoptera JN943327 | JN941727 | JN941414 | AB968586 ~scc1e42—Termiidse ~aco1s4 | Termmigee “yroc 9027" Formicnge ARSE MAT Hemiptes osc 110989 Lepidoptera MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 ON555832 MZ198251 JN992461 MK284266 | MK214110 MK284267 | MK214109 MH753681 | M K284268 | MK214111 ON555913 | ON567752 | ON568142 MZ246828 | MZ246829 EF468808 | EF468748 EF468856 Sung et al. (2007a) Zha et al. (2021) Tasanathai et al. (2020) Aratijo et al. (2018) Evans et al. (2011 Araujo et al. (2015 Araujo et al. (2018 Araujo et al. (2018 ~ eS wma a Araujo et al. (2018) Evans et al. (2011) Sung et al. (2007a) Tasanathai et al. (2019) Tasanathai et al. (2019) Sung et al. (2007a) Tang et al. (2023c) Clifton et al. (2021) Sung et al. (2007a) 98 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps Species O. entomorrhiza O. flabellata O. flavida O. formosana O. fusiformis O. geometridicola O. globiceps O. globiperitheciata O. globosa O. hydrangea O. isopterae O. issidarum O. kegqinii O. khokpasiensis O. khonkaenensis O. kimflemingiae O. kobayasii O. krachonicola O. lanpingensis O. laojunshanensis O. laotii O. liangshanensis O. lloydii O. longissima O. longistipes O. longistromata O. macroacicularis O. maybankeae O. mosingtoensis O. multiperitheciata O. myrmecophila O. nooreniae O. nuozhaduensis O. ootakii O. ovatospora O. pingbianensis O. pseudoacicularis O. pseudorhizoidea O. pseudorhizoidea O. pseudorhizoidea O. purpureostromata O. radiata O. radiciformis O. ravenelii O. salganeicola O. salganeicola O. satoi O. sinensis O. sobolifera O. spataforae O. spataforae O. spataforae O. sphecocephala O. spicatus O. stylophora O. subtiliphialida O. termiticola Voucher | Host —=|=SOS=|~SSUCS*Y~=CLSUS=«d;SCe@® | ~Kewsa4ed | Insecta | —=~SC*d:«F 68954 | EFaco@09 | EFaGQ749 | ~yroce79s" | Formicinae |_| otst0721 | oLaioza | oLzzz6e8 | 01322607 | “TNMF13899_Tenebronoidea | ——=*|«KUSTBO0G | =| ~KJ78956 | KJ87H900 | Bcc 93025" | Termitidae | MZ676740 |__| MZ675422 | MZ7O7849 | WZ707955 | 0079023 | Lepidoptera || =S*‘MF514648 | MFG14632 | MF614669 | “MFLUCC 18-0495" Diptera WH725815 | MH725011 | NeosEa74 | MH727G07| cc 93023 | Termitdae | Mze76740 |__| MZ675419 | MZ707846 | WZ707861 “rocesca" | Hemiptera |__| OMB04695 | oMa04699 | OMBa1z76 | OMEIT279 | wv 129760 | Termitidae | MZ676741 |__| MZ675420 | MZ7O7847 | WZ707959 | MFLU 17-0751" HKAS 1356147 BCC 48071 BCC 81463 SCO9B BCC 75694" BCC 79667 Hemiptera Lepidoptera Termitidae Hemiptera Formicinae Gryllidae nereoasr | (| Neoeaasa || ‘wn7se72e | | MH759602 | wk2e4269 | woerraest | wr13620 | 1713668 | MK632047 Ls MK632081 | MK632055 MK214112 MK632169 KX713724 MK632112 | MK632082 | MK632056 | MK632172 MK632162 ~YHOSO7O7" | Lepidoptera|———_—_~(|-KOAT7ASB | KoaT7461 | K04I7469 | KOAI7465 HKAS 126087 Lepidoptera 00935368 | | 0P962579 | oquan732| “Yoo 15099244 | Hepiaidae || ———=*|- 9608804 | 00622100 | 00622106 | ~0sc1s1913_| Formicidae | _——_—|~KU79904 | KJB78001 | KJ878970 | KJ8790OA | NeRC 108589 | Odonata 8968407 | _—=«|—ABOGA21 | ABNEESES | HKAS 126186 ORO15960 | ORO82947 | ORO15966 | ORO30S31 BCC 4449’T NBRC 105888 BRIP 72909b' BCC 30904 BCC 69008 — BRP Ss36T | Temiidse | ——_—_—|-KXG7GBIi | Kxe7aeT0 | KKe7aBIZ | YH 20168 | Formicinae |__| ONS65e49 | ON5S5927 | ONSG7769 | ONS6O6ES_ —s3_) Formicinae | KX19652_| XTI9600 | KXTI86B1 | KXTIB7OB yoo 807 | Coleoptera MT276118 |__| Ma70099 | MT270097 | MT270058_ pc 59843 | Lepldopters ||| MF514546 | MF614690 | MF614661_ MK284262 | MK751469 BCC 864317 NHJ 12522 NHJ 12529 TNS F18430 Termitidae Termitidae Termitidae Coleoptera Diptera MH754721 ‘pposeeso | pposes22 | Peel a) =e — | See MT118178 | MT118170 rr178170 | ORO62225 MH753686 | MK284273 | MK214115 MF614657 | MF614641 MH028156 | MH028183 EF 468764 MH028168 EF468873 EF468872 KJ879011 BCC 93036 Mz676746 | ~=~—*| Mz675425 | MZ707852 | MZ707857 DQ522550 | DQ518764 | DQ522334 | DQ522379 MT741719 | MT759575 | MT759578 OSC 110995 NHJ 12525 YFCC 8815° ~coleopters ec Moro tataa | MAT T¥B | IT7S0570 | WT759579 | | Temtsae || K7T9659 | Kxria500 | exria6es | KXTTSTI | W205 | Leidoptea_| =| MKB04560 | MK04500 | MK9E4572 | WKORASOT | Kew 7e0e2 | Hemiptera | ———~—~=*d;sirasa972 | Fasaaze |_| EFASO075 | MY11765 Hemiptera/ MG831747 | MG831746 | MG831748 | Luangsa-Ard et al. (2018) Coleoptera eee sl EF469125 | EF469078 | EF469063 | EF469092 Sung et al. (2007a) OSC 128575 Hemiptera/ | JNO49845 | EF469126 | EF469079 | EF469064 | EF469093 Coleoptera OSC 110998 | Hymenoptera | S| DQ522551 | DQ518765 | DQ522336 | DQ522381 MFLU 18-0164 MK863254 | MK863047 | MK863054 | MK860192 ia) OSC 111000 JNO49828 | DQ522552 | DQ518766 | DQ522337 | DQ522382 Hemiptera/ Coleoptera ON555833 | ON555914 | ON567753 | ON568673 BCC 1770 _ Temitidae | | MH753677 | MK284264 | MK214107 MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 Reference Sung et al. (2007a) Tang et al. (2023a) Wang et al. (2015) Tasanathai et al. (2022) Luangsa-ard et al. (2018) Xiao et al. (2019) Fan et al. (2024) Tasanathai et al. (2022) Zou et al. (2022) Tasanathai et al. (2022) Hyde et al. (2017) This study Tasanathai et al. (2019) Crous et al. (2019) Araujo et al. (2018) Thanakitpipattana et al. (2020) Thanakitpipattana et al. (2020) Chen et al. (2013) Chen et al. (2011) Wang et al. (2021b) Sung et al. (2007a) Sung et al. (2007a) Fan et al. (2024) Tasanathai et al. (2020) Ban et al. (2015) Tan et al. (2023) Tasanathai et al. (2019) Luangsa-ard et al. (2018) Sung et al. (2007a) Crous et al. (2016) Tang et al. (2023c) Araujo et al. (2018) Tang et al. (2022) Luangsa-ard et al. (2018) Chen et al. (2021) Luangsa-ard et al. (2018) Tasanathai et al. (2019) Tasanathai et al. (2019) Tasanathai et al. (2019) Quandt et al. (2014) This study Tasanathai et al. (2022) Sung et al. (2007a) Araujo et al. (2021) Aratijo et al. (2021) Araujo et al. (2018) Wang et al. (2022) Sung et al. (2007a) Sung et al. (2007b) Sung et al. (2007a) Zha et al. (2021) Sung et al. (2007a) Tang et al. (2023c) Tasanathai et al. (2019) oh) Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps Species O. termiticola O. tricentri O. tielingensis O. unilateralis O. unituberculata O. variabilis O. xuefengensis Tolypocladium cylindrosporum T. pseudoalbum T. reniformisporum T. subparadoxum T. yunnanense Voucher Host ITS SSU LSU tef1-a rpb1 Reference BCC 1920 Termitidae MH753678 | MK284265 | MK214108 Tasanathai et al. (2019) NBRC 106968 Hemiptera AB968410 | AB968393 | AB968423 | AB968593 Sung et al. (2007a) HKAS 135612" Lepidoptera PP951446 | PP958848 | PP956621 | PP966945 | PP955355 This study OSC 128574 Hymenoptera DQ522554 | DQ518768 | DQ522339 | DQ522385 Sung et al. (2007a) YHH HU 13017 Lepidoptera KY923211 | KY923213 KY923215 | KY923217 Wang et al. (2018) ARSEF 5365 Dipteran DQ522555 | DQ518769 | DQ522340 | DQ522386 Sung et al. (2007a) GZUH2012HN14" Coleoptera KC631802 | KC631789 KC631793 | KC631798 Wen et al. (2013) YFCC 1805001 Soil MK984581 | MK984565 | MK984577 | MK984569 | MK984584 Wang et al. (2022) Soil OP207725 | OP207717 | OP207737 | OP223151 | OP223129 Dong et al. (2022) YFCC 1805002 T Lepidoptera MK984582 | MK984566 | MK984578 | MK984570 | MK984585 Wang et al. (2022) Soil OP207727 | OP207715 | OP207735 | OP223149 | O0P223127 Dong et al. (2022) Soil OP207730 | OP207720 | OP207740 | OP223154 | O0P223132 Dong et al. (2022) Abbreviations: ARSEF: The Agricultural Research Service Collection of Entomopathogenic Fungi, USDA, USA; BCC: BIOTEC Culture Collection, Klong Luang, Thailand; BRIP: Queensland Plant Pathology Herbarium, Australia; GZUH/GACP: Herbarium of Guizhou University, China; GZUIFR: Institute of Fungal Resources of Guizhou University, China; HKAS: Kunming Institute of Botany, Academia Sinica, China; HUA: Herbarium Antioquia University, Medellin, COL; KEW: Mycology collection of Royal Botanical Garden, Surrey, UK; MFLU: Mae Fah Luang University Herbarium, Thailand; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; MTCC: Microbial Type Culture Collection and Gene Bank, India; MY: Mycology Laboratory in BIOTEC, Thailand; NBRC: Biological Resource Center, the National Institute of Technology and Evaluation, Japan; OSC: Oregon State University Herbarium, Corvallis, Oregon, USA; TNS F: The mycological herbarium of the National Museum of Nature and Science, Tsukuba, Ibaraki, Japan; YFCC: Yunnan Fungal Culture Collection of Yunnan University, China; YHH: Yunnan Herbal Herbarium, China; YHOL: Yunnan Herbal Laboratory, Institute of Herb Biotic Resources, China; Holotype of specimens ATRI3/G143/OBIS4/FEMO2/HIPPOC/NIDUL2/G108/SCO9B and J13 were deposited in INPA herbarium (Instituto Nacional de Pesquisas da Amazonia, Brazil). Uninformative gaps and ambiguous regions were removed using Trimal v.1.2 (Capella-Gutiérrez et al. 2009) with the -gt value set to 0.6. SequenceMatrix 1.7.8 (Vaidya et al. 2011) was used to combine the five trimmed alignments. AliView v. 1.26 (Larsson 2014) was used to convert the format to a FASTA file for maximum likelihood (ML) analysis and a NEXUS file for Bayesian inference (BI) analysis. The final combined alignment was used for ML and BI analysis. ML analysis was performed using RAXxML-HPC2 on ACCESS (8.2.12) (Stamat- akis 2014) available in the CIPRES Science Gateway platform with the GTRCAT model and bootstrap iterations setting to 1000. The best-fit models for each gene were independently determined by MrModeltest version 2.3 (Nylander 2004) with Akaike Information Criterion (AIC), resulting in the selection of GTR+I+G for SSU, LSU, ITS, tef1-a, and rpb7. BI analysis was performed with MrBayes on XSEDE version 3.2.7a on the CIPRES Science Gateway portal, employing the suggested best-fit models and launching two parallel runs with four parallel Markov Chain Monte Carlo chains sampled every 1000 steps for 100,000,000 generations until the average standard deviation reached 0.01. The first 20% of trees represented burn-in fractions were discarded, and the remaining trees were used to calculate the posterior probabilities (PP) of each clade (Alfaro and Holder 2006). Phylo- grams generated from ML and BI analyses were viewed with the FigTree v.1.4.0 program (Rambaut and Drummond 2012) and edited with Adobe Illustrator. Results Phylogenetic analyses The combined dataset of 122 taxa consisted of 3959 characters (SSU: 1028 bp, LSU: 839 bp, ITS: 547 bp, tef1-a: 903 bp, and rpb1: 642 bp), of which 2238 char- acters were constant, 356 variable characters were parsimony-uninformative, and 1365 characters were parsimony-informative. Four strains of Tolypocladium were selected as the outgroup taxon. Both maximum likelihood (ML) and Bayes- ian inference (BI) analyses produced congruent tree topologies. The optimal ML tree with a likelihood score of -52,290.517614 (Fig. 1) resolved nine strongly MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 100 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps supported clades, namely O. sinensis, O. issidarum, O. acicularis, O. blattae, O. uni- lateralis, O. elongata, O. ravenelii, O. sohecocephala, and O. sobolifera. Ophiocordy- ceps tielingensis and Hirsutella kuankuoshuiensis formed a monophyletic group sister to O. elongata and H. gigantea (100% ML/1 PP; Fig. 1), nested within the O. elongata clade. Ophiocordyceps radiata (HKAS 135613) clustered with H. ra- diata and H. fusiformis, forming a clade sister to H. shennongjiaensis (100% ML; Fig. 1), also within the O. elongata clade. Ophiocordyceps keqinii was resolved as sister to a clade containing O. macroacicularis and H. changbeisanensis with moderate support (89% ML/0.99 PP; Fig. 1). The alignments used in this study are available on Figshare (https://doi.org/10.6084/m9.figshare.29075552). Taxonomy Ophiocordyceps tielingensis S. W. Xie, T. C. Wen & D. P Wei, sp. nov. Index Fungorum: IF903218 Fig. 2 Etymology. Named after the location where the type specimen was found, ‘Tiel- ing’ County, Liaoning Province, China. Description. Anamorph: Stromata extending from the body of a lepidopteran larva, simple, up to 70 mm long and 1 mm wide, with irregularly branches 0.8-17.0 x 0.1-1.0 mm, brown, becoming pale white toward the apex due to the formation of hymenium, fibrous, gradually attenuating toward the apex. Phialides emerging from the middle to upper regions of stromata, lageniform, broadly cylindrical, or swollen at base, hyaline, slightly guttulate,6-11 x 3-8 (x = 7 x 5,n = 20) um, abrupt- ly narrowing into a thin neck with slightly guttulate, 16-28 x 1-3 (x = 22 x 2,n = 20) um. Conidia 8-17 x 2-5 (x = 13 x 3, n = 35) um, narrowly cymbiform, clavate, and elongated fusiform, one-celled, hyaline, enveloped in a mucous sheath forming a globose head 3-8 (x = 5,n = 15) um in diameter. Teleomorph: Undetermined. Material examined. CHINA : Liaoning Province, Tieling City, on a dead larva of Lepidoptera, Ting-Chi Wen, TLO3 (HKAS 135612, holotype). Notes. Multigene phylogenetic analysis showed that O. tielingensis forms a sister clade to Hirsutella kuankuoshuiensis with lower statistical values (76% ML / 0.95 PP) and grouped with O. elongata (anamorph: Hirsutella gigantea) (Sung et al. 2007a; Simmons et al. 2015b) (Fig. 1). All species share similarity in forming parasitic associations with larvae of Lepidoptera (Qu et al. 2018). Ophiocordyceps tielingensis and H. kuankuoshuiensis were known only from their anamorphs. However, notable differences can be observed between O. tielingensis and H. kuankuoshuiensis in the morphologies of stromata, phial- ides, and conidia (Table 2). Hence, based on the biphasic approach, we confirm that our collection is qualified as a novel species of Ophiocordyceps s. str. Ophiocordyceps keginii S. W. Xie, T. C. Wen & D. P Wei, sp. nov. Index Fungorum: IF903219 Fig. 3 Etymology. Named after an eminent Chinese mycologist, Prof. Ke-Qin Zhang, who has made a significant contribution to the studies of fungi in China. MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 101 Shi-Wen Xie et al.: Figure 1. Phylogram generated from maximum likelihood analysis based on combined SSU, LSU, ITS, tef7-a, and rpb1 sequence data. ML bootstrap values equal to or greater than 50% and PP values equal to or greater than 0.90 are given Two new Species, one new record and an overview of Ophiocordyceps 99) Ophiocordyceps macroacicularis NBRC 10 068 890.99 Hirsutella changbeisanensis GZUIFR hirl ¢ co Ophiocordyceps keqinii HKAS. 135614" oon Hirsutella illustris ARSEF 5539 ; Ophiocordyceps xuefengensis GZUH20 7/08 svo.or Hirsutella sinensis ARSEF 6282 soi!" Ophiocordyceps sinensis YHH 1805 Sa Ophiocordyceps laojunshanensis | $3. por Ophiocordyceps lanpingensis YHOS0707" Ophiocordyceps liangshanensis YFCC ‘sbi Tor Ophiocordyceps multi z 10.96 Hirsutella strigosa Al Hirsutella uncinata MTCC 10896 __ Ophiocordyceps stylophora OSC 111000 _ io.) Hirsutella cf haptospora ARSEF 2228 vot Hirsutella rhossiliensis ARSEF 2931 Ophiocordyceps arborescens NBRC 1058917 “Hirsutella ‘hongheensis Hi ton Ophiocordyceps flav B nn Hirsutella vers Hirsutella hapt Hirsutella guyana Ophiocordyceps issidarum MFLU 17 075 soioosf Ophiocordyceps spataforae NH. 125 Lo Ophiocordyceps spataforae Osc I } ou Ge 56/-- 961 Sieh Ophiocordyceps campes COs Ophiocordyceps pseudoacicularis BCC Ophiocordyceps acicularis OS' 28580 (Hirsutella nodulosa ARSEF 5473, i” Hirsutella satumaensis ARSEF 996 Hirsutella subulata ARSEF 2227 Ophiocordyceps geometridico. Ophiocordyceps vo Ophiocordyceps unituberculata YHI Hirsutella leizhouensis GZUIFR hir13( too) Hirsutella thompsonii ARSEF 253 to} ' Hirsutella thompsonii ARSEF 3323 Ton Phiocordyceps termiticola BCC 1770 Ophiocordyceps termiticola BCC 1920 Hirsutella necatrix ARSEF 5549 ‘ T- sy. Ophiocordyceps communis BCC 1842 a Ophiocordyceps communis BCC 2754 6510.90 Ophiocordyceps communis BCC 1874 Hirsutella cryptosclerotium ARSEF 4: ir aa Ophiocordyceps glol toa1~— Ophiocordyceps khokpasiensis BCC 48071 00/1 Ophiocordyceps ovatospora YHH 22060017 } ny Ophiocordyceps globosa MY 12356 Ophiocordyceps mosingtoensis BCC 30904 _ Ophiocordyceps fusiformis BCC 93025" Ophiocordyceps longistipes HKAS 126186 4100/1 730.99 9010.99 om 95/1 54/— owt sr Ophiocordyceps pseudorhizoidea NHJ 12522 svoesl! Ophiocordyceps pseudorhizoidea NHJ 12529 wi}! Ophiocordyceps pseudorhizoidea BCC 86431" 09/1 Ophiocordyceps isopterae MY 12376 Ophiocordyceps radiciformis MY 12367 01 _ tou Ophiocordyceps. fabellata ‘ eS 4100/1 5510.92 Ophiocordyceps Ophiocordyceps unila Ophiocordyceps satoi 37 — Ophiocordyceps bifertilis an Ophiocordyceps Ophiocordyceps ansiformi Ophiocordyceps = yfdphiocordyceps basiasca YHH Ophiocordyceps subtiliphial Ophiocordyceps campono Ophiocordyceps Ophiocordyceps kimflemin, Ophiocordyceps camponoti balzani Ophiocordyceps camponoti bi 86/0,99 Ophiocordyceps campon J00/1 Ophiocordyceps camp not Ophiocordyceps cai Ophiocordyceps maybankeae BRIP 72909b™ Hirsutella citriformis ARSEF 1035 wor f— Hirsutella eleutheratorum ARSEF 13375 Ophiocordyceps delicatula ARSEF 14442™ ssosof Hirsutella gigantea ARSEF 30 101 | Ophiocordyceps elongata OSC 110989 Feo@2Phiocordyceps tielingensis HKAS 135612" ‘sik "Hirsutella kuankuoshuiensis GZUIFR 2012KKS3-1 7o-~ ‘Hirsutella radiata! ARSEF 1369 su Ophiocordyceps radiata HKAS 135613 —_ Hirsutella fusiformis ARSEF 5474 Hirsutella shennongjiaensis GZUIFR Snj 121022" Ophiocordyceps entomorrhiza KEW 53484 _ — Ophiocordyceps formosana TNM F 13893 5810.93 Ophiocordyceps spicatus MFLU 18 0164 pace Ophiocordyceps ra Loo oe Ophiocordyceps clavata NBE Tones Ophiocordyceps variabilis ARSEF 5365 25/1 Ophiocordyceps krachonicola BCC79667 100/1 Ophiocordyceps purpureostromata TNS F 1843 Pu Ophiocordyceps borealis MFLU 18 0163 _ 10/1 4100/1 100/1 96/1 6610.99) 50/0.97 ont 10/1 7210.94 ar Ophiocordyceps longissima NBRC 108989 L0OrT Ophiocordyceps hydrangea YFCC 8834" ib Ophiocordyceps sobolifera KEW 78842 Ophiocordyceps khonkaenensis BCC81463 10/1 Tolypocladium cylindrosporum YFCC 1805001 530g Tolypocladium reniformisporum YFCC 1805002" Tolypocladium yunnanense YFCC 878" Tolypocladium pseudoalbum YFCC 875 Tolypocladium subparadoxum NBRC 106958 10/1 100/41 0.05 above each node. The newly generated sequences are indicated in red. MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps L Figure 2. Ophiocordyceps tielingensis (HKAS 135612, holotype) a stromata growing from the lepidopteran larva b, ¢ close- up of branching stromata d close-up of host e enlargement of stromata f phialides with conidial mass g-k phialides I, m co- nidia limited in mucus sheath n-p conidia. Scale bars: 2 mm (a, b); 200 um (ec); 2 mm (d); 10 um (e, h-p); 20 um (f-g). MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 103 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps Table 2. Morphological differences between O. tielingensis and H. kuankuoshuiensis. Species O. tielingensis H. kuankuoshuiensis Stromata (mm) 70, branched 86, unbranched Phialides (um) Lageniform, broadly cylindrical, or swollen verrucose base, with a thin Subulate or slender columnar base, with a long and narrow neck, and verrucose neck, 16-28 x 1-3 30-45 x 1-3 Conidia (um) 8-17 x 2-5, narrowly cymbiform, clavate, and elongated fusiform, 9.9-12.6 x 2.7-4.5, clavate, narrow fusiform, or botuliform, with a mucus with a mucus References This study Qu et al. 2018 oS = — LL == ——— —_—_—— —- —- = = — a (oO) == ee —— oe —— =a —— __ —_ oO = a a — — aa a ~] : = —_, ar) — — —— — — —J (8 9 ees —— __——_! —! aa —! = Figure 3. Ophiocordyceps keginii (HKAS 135614, holotype) a, b stromata growing from the insect larva c, d close-up of stromata e stromata covered with hymenium f-h phialides i-k conidia. Scale bars: 200 um (c-e); 15 um (fh); 5 um (i-k). MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 104 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps Table 3. Differences in morphological characteristics of Hirsutella changbeisanensis, Ophiocordyceps keginii, and O. macroacicularis. Species H. changbeisanensis O. keginii 0. macroacicularis Stromata (mm) None 15-90 x 0.3-1.1, branched 97-166 x 1.3-2.4, branched Phialides (um) Cylindrical base, 6.5-20.0 x 1.8-5.4, with a Cylindrical base 4-12 x 2-4, with a neck, Awl-shaped, 21-63 long, 3-3.8 wide at base, slender and verruculose neck 8.1-18.0 6-16 x 0.7-2 1.8-2.0 wide at neck Conidia (um) Ellipsoid or orange-segment, 4.0-7.0 x 2.5- Semielliptical, ovoid with a round apex, 3-12 | Orange-segment or oval, 8.1-10.8 x 2.7—-5.4, 3.5, with a mucus x 2-5 with a mucus References Liang 1991; Qu et al. 2017 This study Ban et al. 2015; Zhou et al. 2015 Description. Anamorph: Stromata extending from the head of the lepidopter- an larva, 15-90 x 0.3-1.1 mm, irregularly branched at upper part, cylindrical, fi- brous, dark brown at base, becoming white toward the apex due to the formation of hymenium. Phialides exclusively formed at the apical region of stromata, hya- line, smooth-walled, cylindrical at the base 4-12 x 2-4 (X = 7 x 3, n = 20) um, nar- rowing rapidly to a long neck 6-16 x 0.7—2 (X = 11 x 1,n = 20) um. Conidia 3-12 x 2-5 (X = 9 x 4,n = 20) um, hyaline, semielliptical, ovoid with a round apex and obvious scars at base, one-celled, smooth-walled. Teleomorph: Undetermined. Material examined. CHINA * Yunnan Province, Honghe Prefecture, Amushan natural reserve, on a dead larva of Lepidoptera on the ground, Shi-Wen Xie, YO8 (HKAS 135614, holotype). Notes. Phylogenetic analyses revealed that O. keginii is sister to a clade comprising O. macroacicularis and Hirsutella changbeisanensis, with strong statistical support (89% ML / 0.99 PP Fig. 1). Ophiocordyceps macroacicularis was found infecting lepidopteran larvae in Japan (Ban et al. 2015). Accord- ing to the studies by Ban et al. (2015) and Zhou et al. (2015), they identified polyphialidic phialides in their strains of O. macroacicularis, which were absent in our collection. The comparison of nucleotide sequences showed that there are 17 bp differences (5 bp in ITS, 12 bp in tef1-a) between O. keqinii and O. macroacicularis, suggesting they are separate species. Hirsutella changbeisanensis was initially discovered on leafhoppers (Hemip- tera) by Liang (1991) and restudied by Qu et al. (2017) based on a new col- lection occurring on Cicadellidae (Homoptera). Hirsutella changbeisanensis is distinct from O. keginii in having a verruculose neck, which is smooth-walled in our collection (Qu et al. 2017). Additionally, there are 23 bp differences in nucleotides (6 bp in ITS, 17 bp in tef1-a) between O. keqginii HKAS 135612 and H. changbeisanensis GZUIFR-hir160527, suggesting they are not conspecific. Hence, based on the differences in morphological characteristics (Table 3), multi-locus phylogenetic analyses, and base pair differences, we introduce O. keginii as a new species of Ophiocordyceps. Ophiocordyceps radiata (Petch) S. W. Xie, D. P Wei & T. C. Wen, comb. nov. Index Fungorum: IF903448 Fig. 4 Basionym. Hirsutella radiata Petch, Trans. Br. Mycol. Soc. 19(3): 184 (1935) [1934]. Description. Anamorph: Synnemata up to 5.4 mm long, 0.04 mm wide, emerging from neck and leg joints of the host, multiple, unbranched, brown, filiform, slender, wiry, gradually attenuating toward the apex. Subiculum form- MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 105 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps Figure 4. Ophiocordyceps radiata a, b synnemata growing from the fly host ¢ synnema-bearing conidiophores d, i sporo- dochia emerging from leg joints of host e, f synnema g, j-I conidia mass on tip of phialides h phialides m conidia mass n-q conidia. Scale bars: 2 mm (a, b); 200 um (c-e); 50 um (f); 15 um (g-l); 5 um (mq). MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 106 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps ing from leg joints of the host, white, composed of interlaced hyphae. Phialides laterally formed along synnemata or produced from subiculum, hyaline, asep- tate, smooth-walled, cylindrical, 4-16 x 3-7 (x = 10 x 4, n = 30) um at the base, nrowing rapidly into a long neck 10-40 x 0.8-2 (X = 19 x 1,n = 30) um. Conidia 6-10 x 2-5 (X = 9 x 3, n = 20) um, hyaline, cymbiform, one-celled, smooth- walled, enveloped in a mucous sheath, forming a globose head 8-13 (x = 10, n = 10) um in diameter. Teleomorph: Undetermined. Material examined. CHINA * Yunnan Province, Honghe Prefecture, Amushan natural reserve, on fly (Diptera) attached to lower side of a living fern leaf, Shi- Wen Xie, TSQ13 (HKAS 135613). Notes. In the phylogenetic analyses, our new collection clustered with Hirsutella radiata and H. fusiformis, forming a monophyletic clade with high statistical support (97% ML / 1 PP Fig. 1). Hirsutella radiata was initial- ly found infecting a small fly attached to a leaf from Great Britain. It was characterized by filiform, brown, branched synnemata; phialides with con- ical to cylindrical bases and stout necks; cymbiform to oval conidia; and oval conidial masses (Petch 1935). Hirsutella fusiformis was introduced by Speare (1920) from a cricket in Hawaii. It has erect, straight, unbranched, nearly black synnemata; simple phialides with inflated basal portions ta- pering to a neck; and fusoid-cylindrical conidia. For the first time, Simmons et al. (2015b) used the DNA sequences of LSU, SSU, tef7-a, and rpb7 gene regions of ‘H. radiata’ (from a specimen occurring on Diptera in Poland) and H. fusiformis (from a specimen occurring on Brachyderes incanus in the Netherlands) in their phylogenetic analyses. However, these sequences have not been linked to any morphological description, and epitypes were not designated. The close phylogenetic relationship between H. radiata and H. fusiformis was observed in this study and that of Simmons et al. (201 5b), while it is undetermined whether they are conspecific. Morphologi- cally, our specimen shares similarities with H. radiata in the association with a dipteran host, the filiform brown synnemata, and the cymbiform conidia; thus, we concluded our collection was H. radiata. According to our knowl- edge, this is the first geographical record of H. radiata in China. Besides, for the first time, we created the linkage between molecular data and the mor- phological characteristics of this species, thereby formally synonymizing H. radiata as Ophiocordyceps radiata. Discussion Systematics of Ophiocordyceps subclades with hirsutella-like anamorphs Species with hirsutella-like anamorphs are distributed in most clades of Ophio- cordyceps, with the exception of the O. sphecocephala clade (Fig. 1). The mor- phological characteristics of hirsutella-like phialides (including shape, size, branching patterns, and surface texture), along with their teleomorphs, exhib- it significant variation across Ophiocordyceps clades (Table 4). Historically, Simmons et al. (2015b) established a foundational classification system for hirsutella-like anamorphs, delineating six subclades: H. citriformis, H. guyana, H. nodulosa, H. sinensis, H. thompsonii, and a distinct ‘ant pathogen’ sub- clade. Qu et al. (2018) subsequently provided comprehensive morphological MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 107 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps Table 4. The synopsis of the phylogenetic lineage of hirsutella-like anamorphs in Ophiocordyceps s. str. Hirsutella-like subclade O. sinensis clade O. issidarum clade O. acicularis clade O. blattae clade O. elongata clade O. unilateralis clade O. sobolifera clade O. ravenelii clade MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 Description This clade comprises taxa characterized by phialides with cylindrical, slender, or subulate bases that gradually taper into a warted neck (Simmons et al. 2015b; Qu et al. 2018). The teleomorphs of this clade produce superficial perithecia and filiform, multiseptate, whole ascospores (Dai et al. 2024). This clade shares large phialides with a cylindrical basal portion (Qu et al. 2018). The teleomorphs of this clade have been known from O. issidarum (Hyde et al. 2017) and O. spataforae (Luangsa-ard et al. 2018). Both of them produce superficial perithecia and filiform, multiseptate, whole ascospores (Hyde et al. 2017; Luangsa-ard et al. 2018). This clade is composed of many cryptic species occurring on lepidopteran larvae, except for H. leigongshanensis, which infects coleopteran larvae (Tasanathai et al. 2020). The anamorphs of this clade are characterized by the helical neck of the phialides (Mains 1950; Liang 1990a, 1990b). The teleomorphs of this clade produce superficial perithecia and needle-like or filiform, whole ascospores (Tasanathai et al. 2020). The members of this clade are specialized parasites on Blattodea (cockroaches and termites) and produce superficial or immersed perithecia and filiform, multiseptate, whole ascospores (Araujo et al. 2021). The anamorphs in this clade are produced at the terminal part of the stromata (Tasanathai et al. 2019). Its phialides are inflated at the base, and the conidia are globose or fusiform with a warty surface or mucous sheath (Qu et al. 2018, Tasanathai et al. 2019, 2022). The teleomorphs of this clade have been known from O. alboperitheciata, O. elongata, O. capilliformis, and O. xifengensis (Fei et al. 2024; Mongkolsamrit et al. 2024). This clade contains species pathogenic to a variety of insect taxa and produces terminal or intercalary fertile parts, immersed or superficial perithecia, and narrowly fusiform, whole ascospores (Fei et al. 2024; Mongkolsamrit et al. 2024). The anamorphs of this clade are unique in producing many branches along the stromata, and the conidia usually are encompassed by a mucous sheath (Tan et al. 2023; Mongkolsamrit et al. 2024). This clade consists of the O. unilaterali core clade and O. kniphofioides subclade. These two groups are different in the ascomata morphologies (Araujo et al. 2018). Species in the O. unilateralis core clade produce brown to black ascomata laterally attached to stromata, while species in the O. kniphofioides subclade produce orange ascomata covering 360° of the stalk (Araujo et al. 2018). Phialides in this clade generally are monophialidic and produce limoniform conidia at the tip. Some species of this clade produce polymorphic phialides, which are defined as Hirsutella A-type, Hirsutella B-type, and Hirsutella C-type (Evans and Samson 1982; Evans and Samson 1984; Evans et al. 2011; Araujo et al. 2018). This clade encompasses fungi pathogenic to cicada and coleopteran larvae (Zou 2022; Mongkolsamrit et al. 2024), sharing morphological traits in producing cylindrical ascomata at the subterminal part of stromata ended with fertile tips, immersed perithecia, multiseptate, disarticulating ascospores. The anamorphs in this clade are nomo- to polyphialidic phialides with a nearly globose base abruptly narrowing into a hair-like, long neck terminated in subglobose conidia without an evident mucous sheath (Lao et al. 2021; Zou 2022; Mongkolsamrit et al. 2024). This clade comprises the taxa that prefer larvae of Coleoptera and share morphology in producing yellow, orange, or brown stromata, forming immersed perithecia on terminal or lateral fertile parts, and filiform, multiseptate ascospores fragmenting into cylindrical secondary ascospores at maturity (Wang et al. 2015; Mongkolsamrit et al. 2024). The mucous sheaths are commonly absent in species of this clade (Wang et al. 2015). descriptions for the first five subclades. Notably, Araujo et al. (2018) rede- fined the “ant pathogen” subclade as the O. unilateralis clade. Building on these frameworks, recent studies have expanded the phylogenetic scope by proposing additional hirsutella-like clades, such as the O. sobolifera and O. ravenelii clades (Wang et al. 2018; Fei et al. 2024; Sun et al. 2024). However, taxonomic inconsistencies persist: Dai et al. (2024) merged four subclades (H. guyana, H. nodulosa, H. sinensis, and H. thompsonii) into a broader O. sin- ensis clade. Mongkolsamrit et al. (2024) incorporated four hirsutella-linked clades (0. blattae, O. elongata, O. ravenelii, and O. sobolifera) in their analysis, revealing partial overlap between groups (e.g., O. blattae with H. citriform- is; O. elongata with H. thompsonii). These conflicting nomenclature systems across studies highlight the taxonomic complexity of Ophiocordyceps sub- clades. Critically, the morphological diversity of hirsutella-like anamorphs re- mains systematically unclear, obscuring potential correlations between ana- morphs, teleomorphs, and host-specific adaptations. This study reassessed the subclades of Ophiocordyceps with hirsutelloid anamorphs and proposed two novel clades (0. issidarum and O. acicularis), which have not been recog- nized in prior taxonomic classifications. Furthermore, we have prepared a checklist of Ophiocordyceps species with hirsutella-like anamorphs (see the Suppl. material 1). There are 95 species of Ophiocordyceps that have been reported to produce hirsutella-like anamorphs that are various in shape, branching, ornamentation, and arrangement of phial- ides, as shown in Fig. 5. 108 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps 00000 y “ ‘ . 0° 1C-type 1A-type 1B-type on 1D-type Vv Ae ANA 1F-type 1G-type 1H-type 1E-type O oO? {e) 26 1I-type 1L-type 2A-type 2B-type 2C-type 2D-type 2E-type 4B-type 4C-type Figure 5. The anamprphs and teleomorphs characteristics of Ophiocordyceps species with hirsutella-like anamorph 1A-1L type hirsutella-like types 2A—2F type stromatal types 3A-3C perithecial arrangements 4A—4C type ascospore shapes 2A-type, terminal 2B, 2C type subterminal 2D, 2E type intercalary 2F-type lateral 3A-type immersed 3B-type obliquely immersed 3C-type superficial 4A-type filiform, multiseptate, disarticulating 4B-type needle-like or filiform, whole 4C-type vermiform. MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 109 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps 1A-type phialide The 1A-type phialide corresponds to Hirsutella Type A as described by Araujo et al. (2018). It is characterized by monophialidic, cylindrical at the base abruptly narrowing into a thin neck, and it is commonly found in O. unilateralis clade. Teleomorphs of this clade are featured with lateral fertile cushions, immersed perithecia, and whole ascospores (Araujo et al. 2018; Tang et al. 2023b, 2023c). The 1A-type phialides usually co-occur with the teleomorph of O. unilateralis clade, and they are associated with apical region of stromata (Wei et al. 2022). 1B-type phialide The 1B-type phialide corresponds to Hirsutella Type B as described by Evans and Samson (1982). It is cylindrical, finely echinulate, and accumulated at ter- minal regions and only found from O. camponoti-novogranadensis (Evans and Samson 1982; Evans et al. 2011). Phialides develop acrogenously at the synne- matal apex, with their supporting synnemata arising from joint or foot of all legs. Synnemata upright, black, cylindrical at the base, tapering towards apex and broadening into a globose head. 1C-type phialide The 1C-type phialide is unique by its intergraded, septate conidiophores termi- nating in flask-shaped phialides, which can be seen in the Hirsutella C-type of O. unilateralis complex (Evans and Samson 1984; Kobmoo et al. 2015; Wei et al. 2022). Hirsutella C-type phialides are produced from brown cushions (sporodo- chia) on the leg and antennal joints of ants. 1D-type phialide The 1D-type phialide is unique with an undulate neck, which is only found in H. dendritica, a species without molecular data (Samson and Evans 1991). 1E-type phialide The 1E-type phialide is curved and gradually attenuated toward the apex from the middle part, and the conidia are cylindrical. This type of hirsutella has been linked to O. formosana, which has a terminal fertile part, obliquely immersed perithecia, and filiform ascospores fragmenting into cylindrical and truncated part-spores (Li et al. 2005; Wang et al. 2015). 1F-type phialide The 1F-type phialide is branching and becoming thread-like at the subterminal region. This type of phialide has been found from culture of O. kobayasii, with its anamorphs being defined as hymenostilbe-like on the natural specimen and as acremonium-like in artificial culture (Thanakitpipattana et al. 2020). How- ever, we recognize both of the mentioned anamorphs as hirsutella-like follow- ing the line drawing provided by Mongkolsamrit et al. (2023). Additionally, the MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 110 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps significant morphological difference of anamorphs on natural specimens and in culture suggests that the substrate can shape anamorphic traits. Thus, phialide morphology from different substrates is incomparable for species delimitation. 1G-type phialide The 1G-type phialide is characterized by the globose base and short neck ter- minating in a single globose conidium. This was observed from cultures of Hir- sutella minnesotensis, a species pathogenic to nematodes (Chen et al. 2000). 1H-type phialide The 1H-type phialide presents a flask shape with the base tapering towards a short, thread-like neck. This type of phialide has been reported from O. spa- taforae (Luangsa-ard et al. 2018), O. geometridicola (Luangsa-ard et al. 2018), O. flavida (Mongkolsamrit et al. 2021), and O. ovatospora (Tang et al. 2022). 11-type phialide The 1I-type phialide is mono- to polyphialidic and can be recognized by its inflated base and filiform, long neck-producing globose conidia. Mongkolsamrit et al. (2024) have described O. ratchaburiensis, O. brunnea, and O. kohchangensis, with the 1l-type phialide being observed from cultures. The three mentioned species are featured with intercalary fertile parts ending with sterile tips, immersed perith- ecia, filiform, disarticulating ascospores, and occurrences on coleopteran larvae. 1J-type phialide The 1J-type phialide is polyphialidic with cylindrical, multiseptate base and short, thread-like necks and often intergraded into a hymenial layer. This type of phialide was only found on natural specimens of O. ratchaburiensis and O. nao- mipierceae (Araujo et al. 2018; Mongkolsamrit et al. 2024). 1K-type phialide The distinctiveness of 1K-type phialides is the inflated base narrowing into one to several thin necks apically twisted in a characteristic helix. It is worth men- tioning that the co-occurrence of twisted neck and smooth neck can be ob- served in one species such as O. pseudoacicularis, O. longistromata, and O. re- torta (Luangsa-ard et al. 2018; Qu et al. 2018; Tasanathai et al. 2020), indicating that the ornamentation of the neck is not significant for interspecific demar- cation. The 1K-type phialides commonly are observed from cultures isolated from Lepidoptera-pathogenic species with intercalary fertile parts, superficial perithecia, and needle-like, filiform, whole ascospores. 1L-type phialide 1L-type phialides are slenderer than 1A-type phialides, narrowing gradually into a neck with warty protrusions and often coming with conidia enveloped in a MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 111 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps Stromata Perithecia Ascospore Hirsutella Numbers of types arrangements shapes types species Terminal Fertile region is formed the apex of the stromata Immersed Subterminal Fertile region is formed near the apex of the stromata Morphological diversity Intercalary Fertile region is formed in the central part of the stromata Lateral Fertile region of lateral cushions partially enclosed by stromata Figure 6. The relationship of anamorphs and teleomorphs’ characteristic state of the Suppl. material 1. mucous sheath. This type of phialide often is found on the culture of Lepidop- tera-pathogenic species with intercalary fertile parts, immersed or superficial perithecia, and whole ascospores (Ban et al. 2015; Luangsa-ard et al. 2018). Morphological diversity of teleomorphs with hirsutella-like anamorphs The morphological diversity of teleomorphs linked with hirsutella-like ana- morph is based on the Suppl. material 1 and presented in Fig. 6. It is shown that terminal, subterminal, intercalary, and lateral stromata types are linked with hirsutella-like anamorphs. Teleomorphs with lateral stromata type, immersed perithecia, and whole ascospores often come with 1A, 1B, and 1C-type phial- ides. This combination of teleomorphs and anamorphs has been found in up to 20 species. Significantly, 1L-type phialides are often found from cultures of the O. unilateralis complex. Up to 19 species have been described to have an intercalary stromatal type, superficial perithecia, needle-like or filiform whole ascospores, and 1A, 1F, 1H, 1K, and 1L type phialides. We categorized these teleomorph-anamorph combinations into several different groups, which pro- vides the guideline for species identification for future work. 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 supported by the National Natural Science Foundation of China (No. 31760014, 32270029) and the Science and Technology Foundation of Guizhou Province (No. [2019]2451-3). Shaun Pennycook is thanked for checking the Latin diagnosis of the new genus and species. MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 2 Shi-Wen Xie et al.: Two new species, one new record and an overview of Ophiocordyceps Author contributions Specimens were collected by Shi-Wen Xie, De-Ping Wei, and Ting-Chi Wen; morphological data, photo-plates, and phylogenetic analyses were completed by Shi-Wen Xie, De-Ping Wei, Zhong-Liang Liu, and Jing Bu. The original draft was by Shi-Wen Xie and De-Ping Wei. De- Ping Wei, Nalin N. Wijayawardene, Xing-Can Peng, Shi-Ke Huang, Xian Zhang, Ji-Chuan Kang, Zhang-Jiang He, Ting-Chi Wen, Chun-Ru Li, Zeng-Zhi Li, and Jun-Zhi Qiu revised the paper. Author ORCIDs Shi-Wen Xie © https://orcid.org/0009-0005-2803-0302 De-Ping Wei © https://orcid.org/0000-0002-6576-2239 Xing-Can Peng © https://orcid.org/0000-0002-7271-7639 Ji-Chuan Kang ® https://orcid.org/0000-0002-6294-5793 Zhang-Jiang He © https://orcid.org/0000-0002-7120-1227 Shi-Ke Huang ® https://orcid.org/0000-0002-2936-396X Xian Zhang © https://orcid.org/0009-0008-0919-4303 Zhong-Liang Liu © https://orcid.org/0009-0007-9519-1418 Jing Bu © https://orcid.org/0009-0006-6861-7770 Nalin N. Wijayawardene © https://orcid.org/0000-0003-0522-5498 Ting-Chi Wen ® https://orcid.org/0000-0003-1744-5869 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. References Alfaro ME, Holder MT (2006) The Posterior and the Prior in Bayesian Phylogenetics. 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Mycosystema 35(7). https:// doi.org/10.13346/j.mycosystema.150189 Zou X, Zhou JX, Liang ZQ, Han YF (2016b) Hirsutella shennongjiaensis, a new entomo- pathogenic species infecting Earwing (Dermaptera). Mycosystema 35(9): 1070- 1079. https://doi.org/10.13346/j.mycosystema.160077 Zou WQ, Tang DX, Xu ZH, Huang O, Wang YB, Tran NL, Yu H (2022) Multigene phyloge- ny and morphology reveal Ophiocordyceps hydrangea sp. nov. and Ophiocordyceps bidoupensis sp. nov. (Ophiocordycipitaceae). MycoKeys 92: 109-130. https://doi. org/10.3897/mycokeys.92.86160 Supplementary material 1 The relationship of anamorph and teleomorph characteristics state. Authors: Shi-Wen Xie, De-Ping Wei, Jun-Zhi Qiu, Xing-Can Peng, Ji-Chuan Kang, Zhang-Ji- ang He, Zeng-Zhi Li, Chun-Ru Li, Shi-Ke Huang, Xian Zhang, Zhong-Liang Liu, Jing Bu, Nalin N. Wijayawardene, Ting-Chi Wen Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.119.145174.suppl1 MycoKeys 119: 95-121 (2025), DOI: 10.3897/mycokeys.119.145174 121