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Research Article
Overview of hirsutella-like anamorphs in Ophiocordyceps (Sordariomycetes, Ophiocordycipitaceae): introducing two new species and one new record from China
expand article infoShi-Wen Xie‡, De-Ping Wei‡, Jun-Zhi Qiu§, Xing-Can Peng|‡, Ji-Chuan Kang‡, Zhang-Jiang He‡, Zeng-Zhi Li, Chun-Ru Li, Shi-Ke Huang‡, Xian Zhang‡|, Zhong-Liang Liu‡, Jing Bu‡, Nalin N. Wijayawardene#, Ting-Chi Wen‡
‡ Guizhou University, Guizhou, China
§ Fujian Agriculture and Forestry University, Fuzhou, China
| Mae Fah Luang University, Chiang Rai, Thailand
¶ Zhejiang BioAsia Life Science Institute, Pinghu, China
# Qujing Normal University, Qujing, China

Corresponding author: Ting-Chi Wen ( tingchiwen@yahoo.com )

Academic editor: Jennifer Luangsa-ard
© 2025 Shi-Wen Xie, De-Ping Wei, Jun-Zhi Qiu, Xing-Can Peng, Ji-Chuan Kang, Zhang-Jiang He, Zeng-Zhi Li, Chun-Ru Li, Shi-Ke Huang, Xian Zhang, Zhong-Liang Liu, Jing Bu, Nalin N. Wijayawardene, Ting-Chi Wen.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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://doi.org/10.3897/mycokeys.119.145174
Open Access

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 keqinii sp. nov.). Ophiocordyceps radiata (syn. Hirsutella radiata), a new combination, exhibits a pathogenic association with a fly, and it is reported as a new geographic record from China, based on integrated 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 Polycephalomycetaceae (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). Ophiocordycipitaceae is a diverse family encompassing fungi with significant ecological, economic, medicinal, and cultural importance. Sung et al. (2007a) established Ophiocordycipitaceae based on molecular data, and this family currently comprises more than 500 species and eight genera, including Drechmeria, Harposporium, Hantamomyces, Ophiocordyceps, Paraisaria, Purpureocillium, Tolypocladium, and Torrubiellomyces (Quandt et al. 2014; Spatafora et al. 2015; Mongkolsamrit et al. 2019; Crous et al. 2020; Araújo et al. 2022). The type genus of Ophiocordycipitaceae, 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 stromata; 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 Hirsutella, 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 occasionally several very slender needle-like necks, either on synnemata or mononematous 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 changbeisanensis (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 researchers 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. unituberculata (Wang et al. 2018), O. sporangifera (Xiao et al. 2019), O. delicatula (Clifton et al. 2021), O. pingbianensis (Chen et al. 2021), O. flavida (Mongkolsamrit 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 Ophiocordyceps s. str. (viz., O. tielingensis sp. nov. and O. keqinii sp. nov.). H. radiata has been reclassified as O. radiata based on a newly collected specimen (associated with a fly), and it is the first time to report this species from China. Furthermore, a checklist of Ophiocordyceps species with hirsutella-like anamorphs and a comprehensive review 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 species was found infecting flies attached to fresh fern leaves. High-resolution images and morphological data were collected in the field for subsequent taxonomic validation. The fresh samples were collected into sterilized self-sealing bags or centrifuge tubes and labeled appropriately. For a more detailed examination of the morphology of the specimens, freehand sections were made. Following 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 amplification was performed for five loci, including the partial small subunit rRNA gene (SSU), the partial large subunit rRNA gene (LSU), the internal transcribed spacer encompassing the 5.8S rDNA gene (ITS), the translation elongation factor 1-alpha gene (tef1-a), and the partial RNA polymerase II largest subunit (rpb1). The corresponding primers that were used for the amplification and sequencing 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 in a 25 µL volume, including 12.5 µL of PCR mixture (2× Rapid Taq Master Mix, Vazyme Biotech), 7.5 µL of double-distilled water, 1 µL of each primer (10 µM), and 3 µL of 30 ng/µL 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 rpb1 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.

Table 1.
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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 Voucher Host ITS SSU LSU tef1-a rpb1 Reference
Hirsutella cf. haptospora ARSEF 2228 Diptera KM652166 KM652075 KM652118 KM652001 KM652041 Simmons et al. (2015b)
H. changbeisanensis GZUIFR hir160527 Homoptera KY415578 KY415592 Qu et al. (2017)
H. citriformis ARSEF 1035 Hemiptera KM652153 KM652064 KM652105 KM651989 KM652030 Simmons et al. (2015b)
H. cryptosclerotium ARSEF 4517 Hemiptera KM652157 KM652066 KM652109 KM651992 KM652032 Simmons et al. (2015b)
H. eleutheratorum ARSEF 13375 Coleoptera MH057734 MH057732 MH057733 Wraight et al. (2018)
H. fusiformis ARSEF 5474 Coleoptera KM652067 KM652110 KM651993 KM652033 Simmons et al. (2015b)
H. gigantea ARSEF 30 Hymenoptera JX566977 JX566980 KM652034 Simmons et al. (2015b)
H. guyana ARSEF 878 Hemiptera KM652158 KM652068 KM652111 KM651994 KM652035 Simmons et al. (2015b)
H. haptospora ARSEF 2226 Acari KM652159 KM651995 KM652036 Simmons et al. (2015b)
H. hongheensis HKAS 102451T Insecta MN017176 MN017177 MN017175 MN733824 Yuan et al. (2020)
H. illustris ARSEF 5539 Hemiptera KM652160 KM652069 KM652112 KM651996 KM652037 Simmons et al. (2015b)
H. kuankuoshuiensis GZUIFR 2012KKS3-1 Lepidoptera KY415575 KY415582 KY415590 KY945360 Qu et al. (2017)
H. leizhouensis GZUIFR hir130707 Lepidoptera KY415573 KY415587 KY945358 Qu et al. (2017)
H. liboensis ARSEF 9603 Lepidoptera KM652163 KM652072 KM652115 Simmons et al. (2015b)
H. necatrix ARSEF 5549 Acari KM652164 KM652073 KM652116 KM651999 KM652039 Simmons et al. (2015b)
H. nodulosa ARSEF 5473 Lepidoptera KM652165 KM652074 KM652117 KM652000 KM652040 Simmons et al. (2015b)
‘H. radiata’ ARSEF 1369 Diptera KM652076 KM652119 KM652002 KM652042 Simmons et al. (2015b)
H. rhossiliensis ARSEF 2931 Nematodes KM652168 KM652078 KM652121 KM652004 KM652043 Simmons et al. (2015b)
H. satumaensis ARSEF 996 Lepidoptera KM652172 KM652082 KM652125 KM652008 KM652047 Simmons et al. (2015b)
H. shennongjiaensis GZUIFR Snj121022T Dermaptera KY945357 KY945364 Qu et al. (2021)
H. sinensis ARSEF 6282 Lepidoptera KM652173 KM652083 KM652126 KM652009 KM652048 Simmons et al. (2015b)
H. strigosa ARSEF 2197 Hemiptera KM652175 KM652085 KM652129 KM652012 KM652050 Simmons et al. (2015b)
H. subulata ARSEF 2227 Lepidoptera KM652176 KM652086 KM652130 KM652013 KM652051 Simmons et al. (2015b)
H. thompsonii ARSEF 3323 Acari KM652188 KM652096 KM652143 KM652024 KM652059 Simmons et al. (2015b)
H. thompsonii ARSEF 253 Acari KM652179 KM652088 KM652133 KM652016 Simmons et al. (2015b)
H. uncinata MTCC 10896 Myrtales KJ524691 KJ524712 Seifert and Boulay (2004)
H. versicolor ARSEF 1037 Hemiptera KM652102 KM652150 KM652029 KM652063 Simmons et al. (2015b)
Ophiocordyceps acicularis OSC 128580 Coleoptera JN049820 DQ522543 DQ518757 DQ522326 DQ522371 Sung et al. (2007a)
O. acroasca YFCC 9016T Formicinae ON555841 ON555922 ON567761 ON568681 Tang et al. (2023c)
O. ansiformis YHH 2210007 Formicinae OR345230 OR098435 OR351952 Tang et al. (2023b)
O. arborescens NBRC 105891T Lepidoptera AB968398 AB968386 AB968414 AB968572 Ban et al. (2015)
O. australis HUA 186097 Hymenoptera KC610786 KC610765 KC610735 KF658662 Sanjuan et al. (2015)
O. basiasca YHH 20191 Formicinae ON555828 ON555910 ON567748 ON568672 Tang et al. (2023c)
O. bifertilis YFCC 9012T Formicinae ON555843 ON555923 ON567763 ON568143 Tang et al. (2023c)
O. blattae BCC 34765 Blattodea MT533484 MT533478 Mongkolsamrit et al. (2020)
O. blattae BCC 38241 Blattodea MT512657 MT533485 MT533479 Sung et al. (2007a)
O. borealis MFLU 18-0163 Coleoptera MK863251 MK863044 MK863051 MK860189 Zha et al. (2021)
O. campes BCC 36938T Lepidoptera MT783955 MT118175 MT118167 MT118183 Tasanathai et al. (2020)
O. camponoti-atricipis ATRI3 Hymenoptera KX713666 KX520652 KX713677 Araújo et al. (2018)
O. camponoti-balzani G143 Hymenoptera KX713658 KX713595 KX713690 KX713705 Evans et al. (2011)
O. camponoti-bispinosi OBIS4 Hymenoptera KX713637 KX713615 KX713692 KX713720 Araújo et al. (2015)
O. camponoti-femorati FEMO2 Formicidae KX713663 KX713590 KX713678 KX713702 Araújo et al. (2018)
O. camponoti-hippocrepidis HIPPOC Formicidae KX713655 KX713597 KX713673 KX713707 Araújo et al. (2018)
O. camponoti-nidulantis NIDUL2 Formicidae KX713640 KX713611 KX713669 KX713717 Araújo et al. (2018)
O. camponoti-rufipedis G108 Hymenoptera KX713659 KX713594 KX713679 KX713704 Evans et al. (2011)
O. clavata NBRC 106961 Coleoptera JN943327 JN941727 JN941414 AB968586 JN992461 Sung et al. (2007a)
O. communis BCC 1842 Termitidae MH753680 MK284266 MK214110 Tasanathai et al. (2019)
O. communis BCC 1874 Termitidae MH753679 MK284267 MK214109 Tasanathai et al. (2019)
O. communis BCC 2754 Termitidae MH754727 MH753681 MK284268 MK214111 Sung et al. (2007a)
O. contiispora YFCC 9027T Formicinae ON555832 ON555913 ON567752 ON568142 Tang et al. (2023c)
O. delicatula ARSEF 14442T Hemiptera MZ198251 MZ246828 MZ246829 Clifton et al. (2021)
O. elongata OSC 110989 Lepidoptera EF468808 EF468748 EF468856 Sung et al. (2007a)
O. entomorrhiza KEW 53484 Insecta EF468954 EF468809 EF468749 Sung et al. (2007a)
O. flabellata YFCC 8795T Formicinae OL310721 OL310724 OL322688 OL322687 Tang et al. (2023a)
O. flavida BCC 84256T Hemiptera MT512655 MT533482 MT533476 Mongkolsamrit et al. (2021)
O. formosana TNM F13893 Tenebrionoidea KJ878908 KJ878956 KJ878988 Wang et al. (2015)
O. fusiformis BCC 93025 T Termitidae MZ676743 MZ675422 MZ707849 MZ707855 Tasanathai et al. (2022)
O. geometridicola BCC 79823 Lepidoptera MF614648 MF614632 MF614663 Luangsa-ard et al. (2018)
O. globiceps MFLUCC 18-0495T Diptera MH725815 MH725811 NG068274 MH727387 Xiao et al. (2019)
O. globiperitheciata HKAS 126130T Termitidae OR015963 OR082950 OR015968 OR030532 OR119834 Fan et al. (2024)
O. globosa BCC 93023 Termitidae MZ676740 MZ675419 MZ707846 MZ707861 Tasanathai et al. (2022)
O. hydrangea YFCC 8834T Hemiptera OM304635 OM304639 OM831276 OM831279 Zou et al. (2022)
O. isopterae MY 12376.01 Termitidae MZ676741 MZ675420 MZ707847 MZ707859 Tasanathai et al. (2022)
O. issidarum MFLU 17-0751T Hemiptera NR160481 NG064454 Hyde et al. (2017)
O. keqinii HKAS 135614 T Lepidoptera PP951447 PP958849 PP956623 PP966946 This study
O. khokpasiensis BCC 48071 Termitidae MH754728 MH753682 MK284269 MK214112 Tasanathai et al. (2019)
O. khonkaenensis BCC 81463 Hemiptera MK632044 MK632127 MK632102 MK632076 MK632169 Crous et al. (2019)
O. kimflemingiae SC09B Formicinae KX713631 KX713620 KX713698 KX713724 Araújo et al. (2018)
O. kobayasii BCC 75694T Gryllidae MK632030 MK632112 MK632082 MK632056 MK632172 Thanakitpipattana et al. (2020)
O. krachonicola BCC 79667 Gryllidae MK632047 MK632081 MK632055 MK632162 Thanakitpipattana et al. (2020)
O. lanpingensis YHOS0707T Lepidoptera KC417459 KC417461 KC417463 KC417465 Chen et al. (2013)
O. laojunshanensis HKAS 126041 Lepidoptera OQ935368 OP962379 OQ440732 Chen et al. (2011)
O. laotii BCC 76495T Formicidae ON763786 ON764219 ON759347 ON759354 Mongkolsamrit et al. (2023)
O. liangshanensis YFCC 15099244 Hepialidae OQ608804 OQ622100 OQ622106 Wang et al. (2021b)
O. lloydii OSC 151913 Formicidae KJ878924 KJ878891 KJ878970 KJ879004 Sung et al. (2007a)
O. longissima NBRC 108989 Odonata AB968407 AB968421 AB968585 Sung et al. (2007a)
O. longistipes HKAS 126186 Termitidae OR015960 OR082947 OR015966 OR030531 OR062225 Fan et al. (2024)
O. longistromata BCC 44497T Lepidoptera MT783956 MT118178 MT118170 Tasanathai et al. (2020)
O. macroacicularis NBRC 105888 Lepidoptera AB968401 AB968389 AB968417 AB968575 Ban et al. (2015)
O. maybankeae BRIP 72909bT Coccinellidae OR750694 OR731501 OR737805 Tan et al. (2023)
O. mosingtoensis BCC 30904 Termitidae MH754732 MH753686 MK284273 MK214115 Tasanathai et al. (2019)
O. multiperitheciata BCC 69008 Lepidoptera MF614657 MF614641 Luangsa-ard et al. (2018)
O. myrmecophila BCC 82255 Hymenoptera MH028143 MH028156 MH028183 MH028168 Sung et al. (2007a)
O. nooreniae BRIP 55363T Termitidae KX673811 KX673810 KX673812 Crous et al. (2016)
O. nuozhaduensis YHH 20168 Formicinae ON555849 ON555927 ON567769 ON568683 Tang et al. (2023c)
O. ootakii J13 Formicinae KX713652 KX713600 KX713681 KX713708 Araújo et al. (2018)
O. ovatospora YHH 2206001T Termitidae OP295105 OP295110 OP295113 OP313801 OP313803 Tang et al. (2022)
O. pauciovoperitheciata BCC 45562 Lepidoptera MF614651 MF614634 MF614666 Luangsa-ard et al. (2018)
O. pingbianensis YFCC 8075T Coleoptera MT273118 MT270099 MT270097 MT270098 Chen et al. (2021)
O. pseudoacicularis BCC 53843 Lepidoptera MF614646 MF614630 MF614661 Luangsa-ard et al. (2018)
O. pseudorhizoidea BCC 86431T Termitidae MH754721 MH753674 MK284262 MK751469 Tasanathai et al. (2019)
O. pseudorhizoidea NHJ 12522 Termitidae JN049857 EF468970 EF468825 EF468764 EF468873 Tasanathai et al. (2019)
O. pseudorhizoidea NHJ 12529 Termitidae EF468969 EF468824 EF468765 EF468872 Tasanathai et al. (2019)
O. purpureostromata TNS F18430 Coleoptera KJ878931 KJ878897 KJ878977 KJ879011 Quandt et al. (2014)
O. radiata HKAS 135613 Diptera PP958850 PP956622 This study
O. radiciformis BCC 93036 Termitidae MZ676746 MZ675425 MZ707852 MZ707857 Tasanathai et al. (2022)
O. ravenelii OSC 110995 Coleoptera DQ522550 DQ518764 DQ522334 DQ522379 Sung et al. (2007a)
O. salganeicola Mori01 Blattaria MT741719 MT759575 MT759578 Araújo et al. (2021)
O. salganeicola Mori02 Blattaria MT741718 MT759572 MT759579 Araújo et al. (2021)
O. satoi J7 Termitidae KX713653 KX713599 KX713683 KX713711 Araújo et al. (2018)
O. sinensis YHH 1805 Lepidoptera MK984568 MK984580 MK984572 MK984587 Wang et al. (2022)
O. sobolifera KEW 78842 Hemiptera EF468972 EF468828 EF468875 Sung et al. (2007a)
O. spataforae MY11765 Hemiptera/Coleoptera MG831747 MG831746 MG831748 Luangsa-Ard et al. (2018)
O. spataforae NHJ 12525 Hemiptera/Coleoptera EF469125 EF469078 EF469063 EF469092 Sung et al. (2007a)
O. spataforae OSC 128575 Hemiptera/Coleoptera JN049845 EF469126 EF469079 EF469064 EF469093 Sung et al. (2007b)
O. sphecocephala OSC 110998 Hymenoptera DQ522551 DQ518765 DQ522336 DQ522381 Sung et al. (2007a)
O. spicatus MFLU 18-0164 Coleoptera MK863254 MK863047 MK863054 MK860192 Zha et al. (2021)
O. stylophora OSC 111000 Coleoptera JN049828 DQ522552 DQ518766 DQ522337 DQ522382 Sung et al. (2007a)
O. subtiliphialida YFCC 8815T Formicinae ON555833 ON555914 ON567753 ON568673 Tang et al. (2023c)
O. termiticola BCC 1770 Termitidae MH753677 MK284264 MK214107 Tasanathai et al. (2019)
O. termiticola BCC 1920 Termitidae MH753678 MK284265 MK214108 Tasanathai et al. (2019)
O. tricentri NBRC 106968 Hemiptera AB968410 AB968393 AB968423 AB968593 Sung et al. (2007a)
O. tielingensis HKAS 135612T Lepidoptera PP951446 PP958848 PP956621 PP966945 PP955355 This study
O. unilateralis OSC 128574 Hymenoptera DQ522554 DQ518768 DQ522339 DQ522385 Sung et al. (2007a)
O. unituberculata YHH HU 1301T Lepidoptera KY923211 KY923213 KY923215 KY923217 Wang et al. (2018)
O. variabilis ARSEF 5365 Dipteran DQ522555 DQ518769 DQ522340 DQ522386 Sung et al. (2007a)
O. xuefengensis GZUH2012HN14 T Coleoptera KC631802 KC631789 KC631793 KC631798 Wen et al. (2013)
Tolypocladium cylindrosporum YFCC 1805001 Soil MK984581 MK984565 MK984577 MK984569 MK984584 Wang et al. (2022)
T. pseudoalbum YFCC 875T Soil OP207725 OP207717 OP207737 OP223151 OP223129 Dong et al. (2022)
T. reniformisporum YFCC 1805002 T Lepidoptera MK984582 MK984566 MK984578 MK984570 MK984585 Wang et al. (2022)
T. subparadoxum NBRC 106958 Soil OP207727 OP207715 OP207735 OP223149 OP223127 Dong et al. (2022)
T. yunnanense YFCC 878 Soil OP207730 OP207720 OP207740 OP223154 OP223132 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/SC09B and J13 were deposited in INPA herbarium (Instituto Nacional de Pesquisas da Amazônia, Brazil).

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 independently aligned using MAFFT version v.7 (Kuraku et al. 2013; Katoh et al. 2019). 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 RAxML-HPC2 on ACCESS (8.2.12) (Stamatakis 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 rpb1. 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). Phylograms 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-α: 903 bp, and rpb1: 642 bp), of which 2238 characters 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 Bayesian inference (BI) analyses produced congruent tree topologies. The optimal ML tree with a likelihood score of -52,290.517614 (Fig. 1) resolved nine strongly supported clades, namely O. sinensis, O. issidarum, O. acicularis, O. blattae, O. unilateralis, O. elongata, O. ravenelii, O. sphecocephala, and O. sobolifera. Ophiocordyceps 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. radiata 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).

Figure 1. 

Phylogram generated from maximum likelihood analysis based on combined SSU, LSU, ITS, tef1-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 above each node. The newly generated sequences are indicated in red.

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, ‘Tieling’ 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 × 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 × 3–8 (x̄ = 7 × 5, n = 20) μm, abruptly narrowing into a thin neck with slightly guttulate, 16–28 × 1–3 (x̄ = 22 × 2, n = 20) μm. Conidia 8–17 × 2–5 (x̄ = 13 × 3, n = 35) μm, narrowly cymbiform, clavate, and elongated fusiform, one-celled, hyaline, enveloped in a mucous sheath forming a globose head 3–8 (x̄ = 5, n = 15) μm in diameter. Teleomorph: Undetermined.

Figure 2. 

Ophiocordyceps tielingensis (HKAS 135612, holotype) a stromata growing from the lepidopteran larva b, c close-up of branching stromata d close-up of host e enlargement of stromata f phialides with conidial mass g–k phialides l, m conidia limited in mucus sheath n–p conidia. Scale bars: 2 mm (a, b); 200 μm (c); 2 mm (d); 10 μm (e, h–p); 20 μm (f–g).

Material examined.

China • Liaoning Province, Tieling City, on a dead larva of Lepidoptera, Ting-Chi Wen, TL03 (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, phialides, 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.

Table 2.
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Morphological differences between O. tielingensis and H. kuankuoshuiensis.

Species O. tielingensis H. kuankuoshuiensis
Stromata (mm) 70, branched 86, unbranched
Phialides (μm) Lageniform, broadly cylindrical, or swollen verrucose base, with a thin and verrucose neck, 16–28 × 1–3 Subulate or slender columnar base, with a long and narrow neck, 30–45 × 1–3
Conidia (μm) 8–17 × 2–5, narrowly cymbiform, clavate, and elongated fusiform, with a mucus 9.9–12.6 × 2.7–4.5, clavate, narrow fusiform, or botuliform, with a mucus
References This study Qu et al. 2018

Ophiocordyceps keqinii 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.

Figure 3. 

Ophiocordyceps keqinii (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 μm (c–e); 15 μm (f–h); 5 μm (i–k).

Description.

Anamorph: Stromata extending from the head of the lepidopteran larva, 15–90 × 0.3–1.1 mm, irregularly branched at upper part, cylindrical, fibrous, dark brown at base, becoming white toward the apex due to the formation of hymenium. Phialides exclusively formed at the apical region of stromata, hyaline, smooth-walled, cylindrical at the base 4–12 × 2–4 (x̄ = 7 × 3, n = 20) μm, narrowing rapidly to a long neck 6–16 × 0.7–2 (x̄ = 11 × 1, n = 20) μm. Conidia 3–12 × 2–5 (x̄ = 9 × 4, n = 20) μm, 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, Y08 (HKAS 135614, holotype).

Notes.

Phylogenetic analyses revealed that O. keqinii 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). According 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 (Hemiptera) by Liang (1991) and restudied by Qu et al. (2017) based on a new collection occurring on Cicadellidae (Homoptera). Hirsutella changbeisanensis is distinct from O. keqinii 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. keqinii 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. keqinii as a new species of Ophiocordyceps.

Table 3.
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Differences in morphological characteristics of Hirsutella changbeisanensis, Ophiocordyceps keqinii, and O. macroacicularis.

Species H. changbeisanensis O. keqinii O. macroacicularis
Stromata (mm) None 15–90 × 0.3–1.1, branched 97–166 × 1.3–2.4, branched
Phialides (μm) Cylindrical base, 6.5–20.0 × 1.8–5.4, with a slender and verruculose neck 8.1–18.0 Cylindrical base 4–12 × 2–4, with a neck, 6–16 × 0.7–2 Awl-shaped, 21–63 long, 3–3.8 wide at base, 1.8–2.0 wide at neck
Conidia (μm) Ellipsoid or orange-segment, 4.0–7.0 × 2.5–3.5, with a mucus Semielliptical, ovoid with a round apex, 3–12 × 2–5 Orange-segment or oval, 8.1–10.8 × 2.7–5.4, with a mucus
References Liang 1991; Qu et al. 2017 This study Ban et al. 2015; Zhou et al. 2015

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].

Figure 4. 

Ophiocordyceps radiata a, b synnemata growing from the fly host c synnema-bearing conidiophores d, i sporodochia emerging from leg joints of host e, f synnema g, j–l conidia mass on tip of phialides h phialides m conidia mass n–q conidia. Scale bars: 2 mm (a, b); 200 μm (c–e); 50 μm (f); 15 μm (g–l); 5 μm (m–q).

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 forming from leg joints of the host, white, composed of interlaced hyphae. Phialides laterally formed along synnemata or produced from subiculum, hyaline, aseptate, smooth-walled, cylindrical, 4–16 × 3–7 (x̄ = 10 × 4, n = 30) μm at the base, nrowing rapidly into a long neck 10–40 × 0.8–2 (x̄ = 19 × 1, n = 30) μm. Conidia 6–10 × 2–5 (x̄ = 9 × 3, n = 20) μm, hyaline, cymbiform, one-celled, smooth-walled, enveloped in a mucous sheath, forming a globose head 8–13 (x̄ = 10, n = 10) μm 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 initially found infecting a small fly attached to a leaf from Great Britain. It was characterized by filiform, brown, branched synnemata; phialides with conical 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 tapering to a neck; and fusoid-cylindrical conidia. For the first time, Simmons et al. (2015b) used the DNA sequences of LSU, SSU, tef1-a, and rpb1 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. (2015b), while it is undetermined whether they are conspecific. Morphologically, 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 knowledge, 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 morphological 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 Ophiocordyceps, with the exception of the O. sphecocephala clade (Fig. 1). The morphological characteristics of hirsutella-like phialides (including shape, size, branching patterns, and surface texture), along with their teleomorphs, exhibit 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’ subclade. Qu et al. (2018) subsequently provided comprehensive morphological descriptions for the first five subclades. Notably, Araújo et al. (2018) redefined 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. sinensis clade. Mongkolsamrit et al. (2024) incorporated four hirsutella-linked clades (O. blattae, O. elongata, O. ravenelii, and O. sobolifera) in their analysis, revealing partial overlap between groups (e.g., O. blattae with H. citriformis; O. elongata with H. thompsonii). These conflicting nomenclature systems across studies highlight the taxonomic complexity of Ophiocordyceps subclades. Critically, the morphological diversity of hirsutella-like anamorphs remains systematically unclear, obscuring potential correlations between anamorphs, teleomorphs, and host-specific adaptations. This study reassessed the subclades of Ophiocordyceps with hirsutelloid anamorphs and proposed two novel clades (O. issidarum and O. acicularis), which have not been recognized in prior taxonomic classifications.

Table 4.
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The synopsis of the phylogenetic lineage of hirsutella-like anamorphs in Ophiocordyceps s. str.

Hirsutella-like subclade Description
O. sinensis clade 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).
O. issidarum clade 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).
O. acicularis clade 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).
O. blattae clade The members of this clade are specialized parasites on Blattodea (cockroaches and termites) and produce superficial or immersed perithecia and filiform, multiseptate, whole ascospores (Araújo 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).
O. elongata clade 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).
O. unilateralis clade This clade consists of the O. unilateralis core clade and O. kniphofioides subclade. These two groups are different in the ascomata morphologies (Araújo 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 (Araújo 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; Araújo et al. 2018).
O. sobolifera clade 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).
O. ravenelii clade 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).

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 phialides, as shown in Fig. 5.

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.

1A-type phialide

The 1A-type phialide corresponds to Hirsutella Type A as described by Araújo 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 (Araújo 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 terminal regions and only found from O. camponoti-novogranadensis (Evans and Samson 1982; Evans et al. 2011). Phialides develop acrogenously at the synnematal 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 terminating 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 (sporodochia) 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). However, we recognize both of the mentioned anamorphs as hirsutella-like following the line drawing provided by Mongkolsamrit et al. (2023). Additionally, the 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 terminating in a single globose conidium. This was observed from cultures of Hirsutella 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. spataforae (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).

1I-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 1I-type phialide being observed from cultures. The three mentioned species are featured with intercalary fertile parts ending with sterile tips, immersed perithecia, 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. naomipierceae (Araújo 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 mentioning that the co-occurrence of twisted neck and smooth neck can be observed in one species such as O. pseudoacicularis, O. longistromata, and O. retorta (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 demarcation. 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 mucous sheath. This type of phialide often is found on the culture of Lepidoptera-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 anamorph 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 phialides. 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 provides the guideline for species identification for future work.

Figure 6. 

The relationship of anamorphs and teleomorphs’ characteristic state of the Suppl. material 1.

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.

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.

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Supplementary material

Supplementary material 1 

The relationship of anamorph and teleomorph characteristics state.

Shi-Wen Xie, De-Ping Wei, Jun-Zhi Qiu, Xing-Can Peng, Ji-Chuan Kang, Zhang-Jiang 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

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.
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