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Research Article
Three new species of Ophiocordyceps (Hypocreales, Ophiocordycipitaceae) and a new host record for Hirsutella vermicola from China
expand article infoYu Yang‡§|, Yuan-Pin Xiao‡, Ruvishika S. Jayawardena§|, Kevin D. Hyde§, Somrudee Nilthong|, Ausana Mapook§, Yong-Zhong Lu‡, Shu-Qiong Xie‡, Fatimah Al-Otibi, Xiao Wang#, Kang Luo#, Li-Ping Luo‡
‡ School of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang, China
§ Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand
| School of Science, Mae Fah Luang University, Chiang Rai, Thailand
¶ King Saud University, Riyadh, Saudi Arabia
# Guizhou Xishui National Nature Reserve Management Bureau, Xishui, China

Corresponding author: Yuan-Pin Xiao ( emmaypx@gmail.com )

Academic editor: Jennifer Luangsa-ard
© 2025 Yu Yang, Yuan-Pin Xiao, Ruvishika S. Jayawardena, Kevin D. Hyde, Somrudee Nilthong, Ausana Mapook, Yong-Zhong Lu, Shu-Qiong Xie, Fatimah Al-Otibi, Xiao Wang, Kang Luo, Li-Ping Luo.
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: Yang Y, Xiao Y-P, S. Jayawardena R, Hyde KD, Nilthong S, Mapook A, Lu Y-Z, Xie S-Q, Al-Otibi F, Wang X, Luo K, Luo L-P (2025) Three new species of Ophiocordyceps (Hypocreales, Ophiocordycipitaceae) and a new host record for Hirsutella vermicola from China. MycoKeys 117: 289-313. https://doi.org/10.3897/mycokeys.117.144875
Open Access

Abstract

Ophiocordyceps, a genus of invertebrate-pathogenic fungi in the family Ophiocordycipitaceae (order Hypocreales), is globally distributed. Over 300 species have been described and accepted as members of the genus. This study introduces three new Ophiocordyceps species from China: O. liaoningensis, O. muscidarum, and O. neocommunis. Remarkably, O. muscidarum, hosted on flies (Muscidae, Diptera), is characterized by its larger perithecia and longer secondary ascospores. Ophiocordyceps neocommunis is also introduced based on morphological distinctions from closely related species. Ophiocordyceps liaoningensis produces dark brown superficial perithecia with an asexual morph at the apex and does not break into part-spores. Phylogenetic analyses using six loci (LSU, ITS, SSU, tef-1α, rpb1, and rpb2) robustly support the placement of these new species within the Ophiocordyceps. Additionally, we report a new host record for Hirsutella vermicola. Detailed descriptions, illustrations, color photo plates, and a phylogenetic tree are provided.

Key words:

Morphology, novel taxa, Ophiocordycipitaceae, phylogeny, taxonomy

Introduction

Fungi are essential to ecosystems as decomposers, mutualists, and pathogens, contributing to nutrient cycling and decomposition (Hyde et al. 2020, 2024; Niego et al. 2023). Invertebrate-associated fungi, ranging from mutualistic to parasitic, play crucial roles in ecological dynamics across diverse environments (Niego et al. 2023). Cordyceps sensu lato refers to a group of insect-infecting fungi that are globally distributed, particularly in forest ecosystems (Evans 1982; Sanjuan et al. 2015). This group encompasses four families: Clavicipitaceae, Cordycipitaceae, Ophiocordycipitaceae, and Polycephalomycetaceae (Sung et al. 2007a; Xiao et al. 2023).

Ophiocordyceps, initially considered a subgenus of Cordyceps, was reclassified as a distinct genus within Ophiocordycipitaceae based on DNA-based phylogenetic analyses (Sung et al. 2007b). With over 300 accepted species, Ophiocordyceps is the largest genus within this family and is especially prevalent in tropical and subtropical regions, with Asia being a hotspot of species diversity (Petch 1931, 1933; Sung et al. 2007a; Sanjuan et al. 2015; Spatafora et al. 2015; Hyde et al. 2016, 2017, 2018; Araújo et al. 2018; Luangsa-ard et al. 2018; Khonsanit et al. 2019; Yang et al. 2024). Most species in this genus produce fibrous, tough stromata with cylindrical asci and multi-septate ascospores, exhibiting a wide range of morphologies (Sung et al. 2007a; Quandt et al. 2014; Ban et al. 2015; Xiao et al. 2019). The asexual morphs of Ophiocordyceps are primarily characterized by Hirsutella, Hymenostilbe, Paraisaria, and Syngliocladium (Sung et al. 2007a; Maharachchikumbura et al. 2015; Mongkolsamrit et al. 2019; Tehan et al. 2023). Historically, Ophiocordyceps and Hirsutella were considered closely related, with Hirsutella initially regarded as a synonym of Ophiocordyceps (Evans and Samson 1982; Quandt et al. 2014). However, it was later recognized as valid due to its morphological diversity within Hypocreales (Quandt et al. 2014; Spatafora et al. 2015), and several Hirsutella sexual morphs were linked to Ophiocordyceps, reinforcing their teleomorphic-anamorphic connection (Simmons et al. 2015).

Species in the genus Ophiocordyceps parasitize a wide variety of insect hosts across different orders, demonstrating broad host range and adaptability (Araújo and Hughes 2016; Yang et al. 2021). They infect a diverse range of insect orders, including Blattaria, Coleoptera, Dermaptera, Diptera, Hemiptera, Hymenoptera, Isoptera, Lepidoptera, Megaloptera, Mantodea, Odonata, and Orthoptera, as well as other organisms like protozoans, rotifers, nematodes, and fungi (Ban et al. 2015; Xiao et al. 2019). Ophiocordyceps species are known to parasitize insects at various life stages, including larvae, pupae, nymphs, and adults (Kobayasi 1941; Sung et al. 2007a).

During surveys of the taxonomy and diversity of entomopathogenic fungi in various regions of Guizhou and Liaoning provinces, China, eight fungal specimens were collected and preliminarily identified as belonging to Ophiocordyceps based on morphological characteristics. Phylogenetic analyses using a multi-locus dataset (LSU, ITS, SSU, tef-1α, rpb1, and rpb2) confirmed their placement within Ophiocordyceps. Combined molecular and morphological analyses revealed three previously undescribed species and a new host record of Hirsutella, expanding the known fungal diversity in China.

Materials and methods

Sample collection, macro- and micro-morphological examination

Eight samples were carefully collected from natural habitats in Guizhou and Liaoning provinces, China, from decaying leaf litter or soil, ensuring minimal disturbance to the environment. During collection, relevant metadata, including location, longitude, and latitude, were recorded for each sample (Rathnayaka et al. 2024). The samples were then transported to the laboratory in plastic containers for further examination. In the laboratory, fruiting bodies were sectioned by hand and examined using a stereomicroscope (SMZ 745 and SMZ 800N, Nikon, Tokyo, Japan) to observe macroscopic features. Micromorphological characteristics, including perithecia, asci, ascospores, secondary ascospores, synnemata, conidiophores, phialides, and conidia, were documented using a Nikon DS-Ri2 digital camera connected to a Nikon ECLIPSE microscope.

Isolation and material deposition

Pure cultures were obtained by germinating mature ascospores (sexual morphs) or conidia (asexual morphs) or by isolating fungal tissues from surface-sterilized substrates or infected insect hosts, followed by purification (Senanayake et al. 2020). Cultures were grown on potato dextrose agar (PDA, Oxoid, UK) at 25 °C. Living strains were deposited in the Guizhou Culture Collection, China (GZCC), and dried specimens were preserved in the Herbarium of Cryptogams at the Kunming Institute of Botany, Academia Sinica (HKAS). Morphological data were analyzed using Tarosoft (R) v.0.9.7 Image Framework. Photographic plates were prepared and edited using Adobe Photoshop CC 2022 (Adobe Systems, USA). The Faces of Fungi and Index Fungorum numbers were assigned to the new species in accordance with the guidelines of Jayasiri et al. (2015) and https://www.indexfungorum.org/.

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from fresh mycelia grown on PDA or directly from dried fungal specimens using the Fungal DNA MiniKit (Biotech, USA), following the directions of the manufacturer. For amplification, six nuclear loci —LSU, ITS, SSU, tef-1α, rpb1, and rpb2—were amplified using their respective primers: LR0R/LR5, ITS5/ITS4, NS1/NS4, EF1-983F/EF1-2218R, CRPB1A/RPB1Cr, and fRPB2-5F/fRPB2-7Cr (Vilgalys and Hester 1990; White et al. 1990; Hopple 1994; Castlebury et al. 2004; Sung et al. 2007b). PCR amplifications were conducted with an initial denaturation at 98 °C for 2 minutes, followed by 40 cycles of 98 °C for 10 seconds, 55 °C for 1 minute, and 72 °C for 30 seconds, with a final extension step at 72 °C for 2 minutes. The PCR products were verified by electrophoresis on a 1% agarose gel stained with ethidium bromide in a TBE buffer. The PCR products were purified and sequenced using the Sanger method at Shenggong Biological Engineering Co. (Shanghai, China). All newly generated sequences were uploaded to GenBank, and the corresponding accession numbers are provided in Table 1.

Table 1.
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Names, voucher numbers, references, and corresponding GenBank accession numbers of the taxa used in the phylogenetic analysis of this study.

Taxa names Specimen/ Strain number GenBank accession numbers References
LSU ITS SSU tef-1α rpb1 rpb2
Hirsutella gigantea ARSEF 30 JX566977 JX566980 KM652034 — Simmons et al. 2015
Hirsutella guyana ARSEF 878 KM652111 KM652158 KM652068 KM651994 KM652035 — Simmons et al. 2015
Hirsutella lecaniicola ARSEF 8888 KM652114 KM652162 KM652071 KM651998 KM652038 — Simmons et al. 2015
Hirsutella minnesotensis SB3612 — EF194145 — — — — Balazy et al. 2008
Hirsutella minnesotensis CBS.115627 — DQ078757 — — — — Xiang et al. 2010
Hirsutella nodulosa ARSEF 5473 KM652117 KM652165 KM652074 KM652000 KM652040 — Simmons et al. 2015
Hirsutella radiata ARSEF 1369 KM652119 — KM652076 KM652002 KM652042 — Simmons et al. 2015
Hirsutella rhossiliensis ARSEF 2931 KM652121 KM652168 KM652078 KM652004 KM652043 — Simmons et al. 2015
Hirsutella strigosa ARSEF 2197 KM652129 KM652175 KM652085 KM652012 KM652050 — Simmons et al. 2015
Hirsutella thompsonii ARSEF 257 KM652136 KM652182 — KM652019 KM652054 — Simmons et al. 2015
Hirsutella thompsonii ARSEF 414 KM652139 KM652184 — KM652021 KM652056 — Simmons et al. 2015
Hirsutella vermicola AS3.7879 — DQ345581 — — — — Xiang et al. 2006
Hirsutella vermicola CGMCC 3.7877T — NR_137547 — — — — Xiang et al. 2006
Hirsutella vermicola AS3.7878 — DQ345592 — — — — Xiang et al. 2006
Hirsutella vermicola HKAS 132167 PQ423697 PQ423678 PQ424974 PQ569874 PQ569888 PQ569904 This study
Hirsutella vermicola HKAS 132168 PQ423698 PQ423679 PQ424975 PQ569875 PQ569889 PQ569905 This study
Hirsutella versicolor ARSEF 1037 KM652150 — KM652102 KM652029 KM652063 — Simmons et al. 2015
Hymenostilbe dipterigena NHJ12170 — GU723771 — GU797127 — — Luangsa-ard et al. 2011
Ophiocordyceps longistipes HKAS126185T OR015966 OR015960 OR082947 OR030531 OR062225 — Fan et al. 2024
Ophiocordyceps acicularis OSC 110988 EF468804 — EF468951 EF468745 EF468853 — Sung et al. 2007b
Ophiocordyceps acicularis OSC 110987 EF468805 — EF468950 EF468744 EF468852 — Sung et al. 2007b
Ophiocordyceps agriotidis ARSEF 5692 DQ518754 JN049819 DQ522540 DQ522322 DQ522368 DQ522418 Spatafora et al. 2007
Ophiocordyceps annulata CEM 303 — — KJ878915 KJ878962 KJ878995 — Quandt et al. 2014
Ophiocordyceps aphodii ARSEF 5498 DQ518755 — DQ522541 DQ522323 — DQ522419 Spatafora et al. 2007
Ophiocordyceps appendiculata NBRC 106959 JN941412 JN943325 JN941729 AB968578 JN992463 AB968540 Ban et al. 2015
Ophiocordyceps asiatica BCC 30516T MH753675 MH754722 — MK284263 MK214105 MK214091 Tasanathai et al. 2019
Ophiocordyceps bidoupensis YHH 20036T — — OK571396 OK556893 OK556897 OK556899 Zou et al. 2022
Ophiocordyceps bispora KVL 606 AF009654 — KX713641 — KX713716 — Araújo et al. 2018
Ophiocordyceps borealis MFLU 18-0163T MK863052 MK863252 MK863045 MK860190 — — Zha et al. 2021
Ophiocordyceps brunneiperitheciata BCC 49312 MF614660 — — MF614642 — MF614686 Luangsa-ard et al. 2018
Ophiocordyceps brunneipunctata OSC 128576 DQ518756 —— DQ522542 DQ522324 DQ522369 DQ522420 Spatafora et al. 2007
Ophiocordyceps brunneirubra BCC 14478T MH753688 MH754734 — GU797122 MK751466 MK214102 Tasanathai et al. 2019
Ophiocordyceps brunneirubra BCC 14384 MH753690 MH754736 — GU797121 MK751465 MK751468 Tasanathai et al. 2019
Ophiocordyceps brunneirubra BCC 14477 MH753689 MH754735 — GU797123 MK751467 MK214103 Tasanathai et al. 2019
Ophiocordyceps camponoti-hippocrepidis HIPPOCT KX713597 — KX713655 KX713673 KX713707 — Araújo et al. 2018
Ophiocordyceps camponoti-nidulantis NIDUL2T KX713611 — KX713640 KX713669 KX713717 — Araújo et al. 2018
Ophiocordyceps camponoti-rufipedis G108 KX713594 — KX713659 KX713679 KX713704 — Araújo et al. 2018
Ophiocordyceps clavata NBRC 106961 JN941414 JN943327 JN941727 AB968586 JN992461 AB968547 Ban et al. 2015
Ophiocordyceps communis BCC 1842 MH753680 MH754726 — MK284266 MK214110 MK214096 Tasanathai et al. 2019
Ophiocordyceps communis NHJ 1131 MH753679 MH754725 — MK284267 MK214109 MK214095 Tasanathai et al. 2019
Ophiocordyceps communis NHJ 10673T MH753681 MH754727 — MK284268 MK214111 MK214097 Tasanathai et al. 2019
Ophiocordyceps curculionum OSC 151910 KJ878885 — KJ878918 — KJ878999 — Quandt et al. 2014
Ophiocordyceps dipterigena HUA 186102 KJ917568 — KC610787 — KF658664 KC610715 Quandt et al. 2014
Ophiocordyceps dipterigena OSC 151911 KJ878886 — KJ878919 KJ878966 KJ879000 — Quandt et al. 2014
Ophiocordyceps dipterigena OSC 151912 KJ878887 — KJ878920 KJ878967 KJ879001 — Quandt et al. 2014
Ophiocordyceps dipterigena MRCIF71 — EU573346 — — — — Freire 2015
Ophiocordyceps dipterigena MY621 — GU723764 — GU797126 — — Luangsa-ard et al. 2011
Ophiocordyceps ferruginosa NBRC 101743 — AB968405 — — — — Sung et al. 2007b
Ophiocordyceps formosana TNM F13893 — — KJ878908 KJ878956 KJ878988 KJ878943 Quandt et al. 2014
Ophiocordyceps forquignonii OSC 151908 KJ878889 KJ878922 — — KJ879003 KJ878947 Quandt et al. 2014
Ophiocordyceps forquignonii OSC 151902 KJ878876 KJ878912 — — KJ878991 KJ878945 Quandt et al. 2014
Ophiocordyceps furcatosubulata YFCC 904T MT774222 — MT774215 MT774243 MT774229 MT774236 Wang et al. 2021
Ophiocordyceps fusiformis BCC 93025T MZ675422 MZ676743 — MZ707849 MZ707855 MZ707805 Tasanathai et al. 2022
Ophiocordyceps globiceps MFLUCC 18-0495 MH725829 MH725815 MH725811 MH727387 — — Xiao et al. 2019
Ophiocordyceps globiceps MFLU 18-0661T MH725830 MH725816 MH725812 MH727388 — — Xiao et al. 2019
Ophiocordyceps globiperitheciata HKAS126130T OR015968 OR015963 OR082950 OR030532 OR119834 — Fan et al. 2024
Ophiocordyceps globosa BCC 93023T MZ675419 MZ676740 — MZ707846 MZ707861 — Tasanathai et al. 2022
Ophiocordyceps gracillima HUA 186132 KC610768 KF937353 — KC610744 KF658666 — Sanjuan et al. 2015
Ophiocordyceps hemisphaerica FLOR 59525T — KX197233 — — — — Hyde et al. 2016
Ophiocordyceps hemisphaerica FLOR 59542 — KX197234 — — — — Hyde et al. 2016
Ophiocordyceps hemisphaerica FLOR 59553 — KX197235 — — — — Hyde et al. 2016
Ophiocordyceps isopterorum MY 12376.01 MZ675420 MZ676741 — MZ707847 MZ707859 MZ707803 Tasanathai et al. 2022
Ophiocordyceps isopterorum BCC 93042T MZ675421 MZ676742 — MZ707848 — — Tasanathai et al. 2022
Ophiocordyceps khokpasiensis BCC 1764 MH753684 MH754730 — MK284271 MK214114 MK214098 Tasanathai et al. 2019
Ophiocordyceps khokpasiensis BCC 48072 MH753682 MH754728 — MK284269 MK214112 — Tasanathai et al. 2019
Ophiocordyceps khokpasiensis BCC 48071T MH753683 MH754729 — MK284270 MK214113 — Tasanathai et al. 2019
Ophiocordyceps kimflemingiae SJS4Oph MH536516 — MH536517 MN785130 MN785132 — Araújo et al. 2018
Ophiocordyceps konnoana EFCC 7315 — — EF468959 EF468753 EF468861 EF468916 Sung et al. 2007b
Ophiocordyceps kuchinaraiensis BCC 95830T OQ627397 OQ627396 — OQ625474 — OQ625475 Tasanathai et al. 2019
Ophiocordyceps lacrimoidis FLOR 59552T — KX197231 — — — — Freire 2015
Ophiocordyceps liaoningensis HKAS 132185 PQ423690 PQ423671 — PQ569869 PQ569883 PQ569897 This study
Ophiocordyceps liaoningensis HKAS 132189 PQ423691 PQ423672 PQ424968 PQ569870 PQ569884 PQ569898 This study
Ophiocordyceps liaoningensis HKAS 132276 T PQ423692 PQ423673 PQ424969 PQ569871 PQ569885 PQ569899 This study
Ophiocordyceps macroacicularis NBRC 100685T AB968416 AB968400 AB968388 AB968574 — AB968536 Ban et al. 2015
Ophiocordyceps mosingtoensis BCC 30904 MH753686 MH754732 — MK284273 MK214115 MK214100 Tasanathai et al. 2019
Ophiocordyceps mosingtoensis BCC 36921T MH753685 MH754731 — MK284272 MK214116 MK214099 Tasanathai et al. 2019
Ophiocordyceps muscidarum HKAS 132178 T PQ423695 PQ423676 PQ424972 PQ675604 — PQ569900 This study
Ophiocordyceps muscidarum HKAS 132275 PQ423696 PQ423677 PQ424973 PQ675605 — PQ569901 This study
Ophiocordyceps neocommunis HKAS 132236 T PQ423693 PQ423674 PQ424970 PQ569872 PQ569886 PQ569902 This study
Ophiocordyceps neocommunis GZCC 24-0158 PQ423694 PQ423675 PQ424971 PQ569873 PQ569887 PQ569903 This study
Ophiocordyceps neovolkiana OSC 151903 KJ878896 — KJ878930 KJ878976 KJ879010 — Quandt et al. 2014
Ophiocordyceps ovatospora YHH 2206001T OP295113 OP295105 OP295110 OP313801 OP313803 OP313805 Tang et al. 2022
Ophiocordyceps pseudocommunis BCC 16757T MH753687 MH754733 — MK284274 MK214117 MK214101 Tasanathai et al. 2019
Ophiocordyceps pseudocommunis NHJ 12581 EF468831 — — EF468775 — EF468930 Sung et al. 2007b
Ophiocordyceps pseudocommunis NHJ 12582 EF468830 — — EF468771 — EF468926 Sung et al. 2007b
Ophiocordyceps pseudorhizoidea BCC 86431T MH753674 MH754721 — MK284262 MK751469 MK214090 Tasanathai et al. 2019
Ophiocordyceps pseudorhizoidea BCC 48879 MH753673 MH754720 — MK284261 MK214104 MK214089 Tasanathai et al. 2019
Ophiocordyceps radiciformis BCC 93036T MZ675425 MZ676746 — MZ707852 MZ707857 MZ707808 Tasanathai et al. 2022
Ophiocordyceps robertsii YHORZT007 KC561978 — KC561978 KC561979 KC561980 — Sung et al. 2007b
Ophiocordyceps rubiginosiperitheciata NBRC 100946 JN941436 JN943341 JN941705 AB968581 JN992439 AB968543 Ban et al. 2015
Ophiocordyceps ryogamiensis NBRC 101751 KF049633 — KF049614 KF049688 KF049650 — Sanjuan et al. 2015
Ophiocordyceps satoi J19 KX713601 — KX713650 KX713684 KX713710 — Araújo et al. 2018
Ophiocordyceps sinensis EFCC7287 EF468827 JN049854 EF468971 EF468767 EF468874 EF468924 Sung et al. 2007b
Ophicordyceps sp. FMF147 — KX197238 — — — — Freire 2015
Ophiocordyceps spataforae OSC 128575 EF469079 JN049845 EF469126 EF469064 EF469093 EF469110 Sung et al. 2007b
Ophiocordyceps spicatus MFLU 18-0164T MK863054 MK863254 MK863047 MK860192 — — Tasanathai et al. 2019
Ophiocordyceps stylophora OSC 111000 DQ518766 JN049828 DQ522552 DQ522337 DQ522382 DQ522433 Spatafora et al. 2007
Ophiocordyceps termiticola BCC 1920T MH753678 MH754724 — MK284265 MK214108 MK214094 Tasanathai et al. 2019
Ophiocordyceps termiticola BCC 1770 MH753677 GU723780 — MK284264 MK214107 MK214093 Tasanathai et al. 2019
Ophiocordyceps unilateralis SERI1 KX713626 — KX713628 KX713675 KX713730 — Araújo et al. 2018
Ophiocordyceps unituberculata YHH HU1301T — KY923211 KY923213 KY923215 KY923217 — Wang et al. 2018
Ophiocordyceps unituberculata YFCC HU1301 — KY923212 KY923214 KY923216 KY923218 KY923220 Wang et al. 2018
Ophiocordyceps variabilis ARSEF 5365 DQ518769 — DQ522555 DQ522340 DQ522386 DQ522437 Spatafora et al. 2007
Ophiocordyceps variabilis OSC 111003 EF468839 — EF468985 EF468779 EF468885 EF468933 Sung et al. 2007b
Ophiocordyceps xuefengensis GZUH2012HN14T — KC631800 KC631786 KC631791 KC631796 — Wen et al. 2013
Ophiocordyceps melolonthae OSC 110993 DQ518762 — DQ522548 DQ522331 DQ522376 — Spatafora et al. 2007
Tolypocladium inflatum OSC 71235 EF469077 JN049844 EF469124 EF469061 EF469090 EF469108 Sung et al. 2007b
Tolypocladium ophioglossoides NBRC 106332 JN941409 JN943322 JN941732 — JN992466 — Schoch et al. 2012

Note: The symbol “—” means the sequence unavailability, and sequences newly generated for this study are in bold. “T” represents the type strain.

Phylogenetic analyses

All newly generated sequences were assembled using SeqMan (Clewley 1995). Reference taxa for phylogenetic analyses were identified via BLAST searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and referenced from previous studies (Table 1). Individual sequences were aligned with MAFFT v.7 (https://mafft.cbrc.jp/alignment/server/) and refined using TrimAl (Capella-Gutiérrez et al. 2009; Katoh and Standley 2013). Alignments were manually inspected and adjusted in BioEdit (Hall et al. 2011). Phylogenetic relationships were conducted using maximum likelihood (ML) and Bayesian inference (BI). Tolypocladium inflatum (OSC 71235) and T. ophioglossoides (NBRC 106332) were selected as outgroup taxa (Xiao et al. 2019; Yang et al. 2021, 2024).

Maximum likelihood analyses were conducted using IQ-TREE 2 under partitioned models and 1,000 bootstrap replicates to assess branch support (Minh et al. 2020). ModelFinder was used to select the optimal substitution model for each locus (Kalyaanamoorthy et al. 2017). Bayesian inference was performed using MrBayes v.3.1.2 (Ronquist et al. 2012), employing Markov Chain Monte Carlo (MCMC) sampling with six parallel chains, run for 1,000,000 to 5,000,000 generations. Convergence was determined by achieving a standard deviation of split frequencies below 0.01, with trees sampled every 1,000 generations. The first 25% of sampled trees (25,000) were discarded as burn-in, and the remaining trees were used to calculate posterior probabilities (PP). The resulting maximum likelihood tree was visualized using FigTree v.1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/).

Phylogenetic analysis results

Reference sequences (Table 1) were downloaded from NCBI GenBank based on previous studies (Sung et al. 2007a; Ban et al. 2015; Sanjuan et al. 2015; Xiao et al. 2019; Tang et al. 2022). In this study, we collected eight specimens and one strain, from which 51 new sequences were generated, including 9 LSU, 9 ITS, 8 SSU, 9 tef-1α, 7 rpb1, and 9 rpb2. Six loci—LSU, ITS, SSU, tef-1α, rpb1, and rpb2—were used to determine the phylogenetic placement of the new collections. The concatenated matrix consisted of 108 taxa with a total of 4,653 base pairs (bp) across the loci (LSU: 1–832 bp; ITS: 833–1,319 bp; SSU: 1,319–2,258 bp; tef-1α: 2,259–3,152 bp; rpb1: 3,153–3,806 bp; rpb2: 3,807–4,653 bp). This matrix was deposited in TreeBASE (accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S31902). Single-locus analyses were performed to compare the topologies and clade stabilities. The optional models selected by ModelFinder were as follows: LSU: TNe+I, ITS: TIM3e+G4, SSU: K2P, tef-1α: TN+F+G4, rpb1: TNe+G4, rpb2: TNe+I. The ML and BI analyses produced nearly congruent topologies; therefore, the best-scoring ML tree is presented in Fig. 1. Ophiocordyceps muscidarum (HKAS 132178 and HKAS 132275) was sister to O. globiceps (MFLU 18-0661 and MFLUCC 18-0495) and formed a strongly supported monophyletic lineage (100% MLBP, 1.00 PP). Ophiocordyceps neocommunis was sister to O. communis (BCC 1842, NHJ 1131, and NHJ 10676) and Hirsutella minnesotensis (SB3612 and CBS.115627) in the phylogenetic tree, with strong support (100% MLBP, 1.00 PP). Ophiocordyceps liaoningensis (HKAS 132276, HKAS132189, HKAS 132185) was sister to O. acicularis (OSC 110988 and OSC 110987) and also formed a strongly supported monophyletic lineage (100% MLBS/1.00 BIPP). Two collections (HKAS 132167 and HKAS 132168) were nested with Hirsutella vermicola (AS3.7878, AS3.7879, and CGMCC 3.7877) without branch length support. Based on these phylogenetic analyses, the collections were identified as three new species, namely Ophiocordyceps liaoningensis, O. muscidarum, and O. neocommunis, as well as a new host record for Hirsutella vermicola.

Figure 1. 

A phylogram constructed using a maximum likelihood (ML) analysis with RAxML based on concatenated LSU, ITS, SSU, tef-1α, rpb1, and rpb2 sequence data. Tolypocladium inflatum and T. ophioglossoides were employed as outgroup taxa. Nodes with maximum likelihood bootstrap values ≥ 75% and Bayesian posterior probabilities ≥ 0.90 are indicated on the phylogram. Type strains are in bold and marked with T, and the new species are in red.

Taxonomy

Ophiocordyceps liaoningensis Y. P. Xiao, K.D. Hyde & Y. Yang, sp. nov.

Index Fungorum: IF902881
Facesoffungi Number: FoF16768
Fig. 2

Etymology.

The epithet “liaoningensis” refers to the type location “Liaoning Province, China”.

Holotype.

China • Liaoning Province, Tieling City, Xifeng County, at 377 masl, 42.653°N, 124.452°E, parasitic on the larva of Coleoptera, buried in soil, 20 July 2023, Yuanpin Xiao (HKAS 132276).

Description

(HKAS 132276). Parasitic on the larva of Coleoptera (Elateridae), buried in the soil. Sexual morph: Host 2.0 cm long, 2–4 mm wide, without hyphae on the surface. Stromata 5–6 cm long, 1–3 mm wide, single, stipitate, cylindrical, pale brown, arising from the host head. Fertile head 1–2 cm long, 2–3 mm wide, with superficial perithecia along the surface of the stipe, cylindrical, dark brown, with asexual morph at the apex. Perithecia 310–415 × 170–290 μm (x– = 362.5 × 230 µm, n = 20), superficial, dark brown, ovoid to flask-shaped, thick-walled. Peridium 30–50 µm (x– = 40 µm, n = 30) wide, two layers, textura angularis outer layer to textura porrecta inner layer, outer layer brownish, inner layer hyaline. Asci 205–255 × 7–11 μm (x– = 230 × 9 µm, n = 30), 8-spored, cylindrical, hyaline, with a thin apex. Apical cap 5.5–6.5 × 3.5–4.7 μm (x– = 6 × 4.1 µm, n = 40), hyaline. Ascospores 150–200 × 2–4 μm (x– = 175 × 3 µm, n = 60), multiseptate, slender filiform, not breaking into secondary ascospores. Asexual morph: Hymenostilbe-like. Synnemata 0.5 cm long, 0.5–1 mm wide, single, cylindrical, light brown, tapering upwards. Conidiophores 10–28 µm wide (x– = 19 µm, n = 40), usually simple, branched or unbranched, septate, hyaline, bearing conidia. Phialide 15–33 × 3.5–6.5 µm (x– = 24 × 5 µm, n = 40), polyblastic, hyaline, clavate or bottle-shaped, forming a dense palisade layer covering the synnemata. Conidia 5–9 × 4.2–6.4 µm (x– = 7 × 5.3 µm, n = 60), 1-celled, hyaline, ovoid or subglobose, developing along the tip of the phialide.

Additional material.

China • Liaoning Province, Tieling City, Xifeng County, at 394 masl, 42.656°N, 124.449°E, 20 July 2023, Yuanpin Xiao, TL2356 (HKAS 132189, paratype); China • Liaoning Province, Tieling City, Xifeng County, at 368 masl, 42.654°N, 124.454°E, 20 July 2023, Yuanpin Xiao, TL01 (HKAS 132185).

Figure 2. 

Ophiocordyceps liaoningensis (HKAS 132276, holotype) a habitat b overview of the host and stroma c host (larva of Elateridae) d fertile part on stroma showing superficial perithecia e–f cross section of perithecia g–i asci j–n, p ascospores o apical cap q synnema r–t conidiophores u–t phialide w conidia. Scale bars: 1 cm (b); 0.5 cm (c, q); 0.1 cm (d); 500 μm (e); 100 μm (f–n); 30 μm (r–t, p); 10 μm (u, v); 5 μm (w).

Notes.

Ophiocordyceps liaoningensis clustered with O. acicularis in the phylogenetic tree with support (100% MLBP, 1.00 PP) (Fig. 1). Nucleotide differences of LSU, ITS, tef-1α, rpb1, and rpb2 sequences of O. acicularis are 2.09% (17/810), 6.74% (34/504), 6.77% (63/931), 5.61% (39/695), and 9.44% (105/1112), respectively. Morphologically, Ophiocordyceps acicularis produces shorter perithecia (280 × 250 μm vs. 310–415 μm), longer asci (260–290 × 7–10 μm vs. 205–255 × 7–11 μm), and longer ascospores (150–240 × 3–4 μm vs. 150–200 × 2–4 μm), compared to O. liaoningensis (Petch 1933). Ophiocordyceps agriotidis is similar to O. liaoningensis in having superficial perithecia and multiseptate ascospores. However, O. agriotidis differs by producing larger perithecia (380–550 × 280–350 μm vs. 310–415 × 170–290 μm) and longer asci (260–280 × 8.5–9.0 μm vs. 205–255 × 7–11 μm) (Kobayasi and Shimizu 1980). Furthermore, the phylogenetic tree clearly distinguishes O. agriotidis from O. liaoningensis (Fig. 1). Zha et al. (2021) provided the first comprehensive review of wireworm-infecting Cordyceps sensu lato species, documenting 27 species within Ophiocordyceps. These fungi are phylogenetically distinct from O. liaoningensis. However, eight species were described solely based on morphological characteristics, highlighting the need for further molecular studies to clarify their taxonomic status (Zha et al. 2021). In contrast, Ophiocordyceps liaoningensis is characterized by superficial perithecia, multiseptate ascospores that do not break into part-spores, bottle-shaped phialides, and ovoid or subglobose conidia (Table 2). Both morphological observation and phylogenetic analyses of combined LSU, ITS, SSU, tef-1α, rpb1, and rpb2 sequence data support that this fungus is a distinctive species in Ophiocordyceps.

Table 2.
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Synopsis of a comparison of O. liaoningensis sp. nov. and its closely related Ophiocordyceps species.

Species Distribution Stromata (mm) Perthecia (μm) Asci (μm) Ascospores (μm) Secondary ascospores (μm) References
O. asyuensis Japan 7–15 × 0.5 Superficial, 600–650 × 450 400–500 × 7–8 — 7–8 × 1.5 Kobayasi and Shimizu 1982
O. elateridicola Japan 70 × 3 Immersed, 400–450 × 120–150 4–3 in diam — 3–4 × 1 Kobayasi and Shimizu 1982
O. falcatoides Japan 17–24 × 0.8–1 Superficialia, 350–400 × 200–250 150–175 × 7–8 125–150 × 2.5–3 9–11 × 3 Kobayasi and Shimizu 1980
O. larvicola France 5–6 Ovoid, small, purplish brown. 5–8, formed by three globules Quélet 1879
O. liaoningensis China 50–60 × 1–3 Superficial, 310–415 × 170–290 205–255 × 7–11 150–200 × 2–4 — This study
O. nigripoda Japan 25–30 × l Immersed, 320–350 × 150–180 μ 115–140 × 10 57–90 × 3.3–5 septatae — Kobayasi and Shimizu 1982
O. rubripunctata Ghana 60 × 0.5–1.5 Embedded, 360–600 × 145–325 Cylindrical with a 2–3 thick Filiform 6–9 × 1.0–2.5 Samson et al. 1982
O. salebrosa Panama Canal Zone 35–40 Immersed, 840–1200 × 240–300 600–660 × 64 Filiformibus 6–10 × 1–1.5 Mains 1947
O. subflavida Venezuela 2–4 500–600 × 150–200 225–300 × 3–4 Filiform — Mains 1959

The symbol “—” means that the data is unavailable.

Ophiocordyceps muscidarum Y. P. Xiao, K.D. Hyde & Y. Yang, sp. nov.

Index Fungorum: IF902879
Facesoffungi Number: FoF16766
Fig. 3

Etymology.

The epithet “muscidarum” refers to its host belonging to the family Muscidae (Diptera).

Holotype.

China • Liaoning Province, Tieling City, Xifeng County, at 356 masl, 42.663°N, 124.482°E, parasitic on the fly (Muscidae, Diptera), collected on a tree stem, 20 July 2023, Yuanpin Xiao (HKAS 132178).

Description

(HKAS 132178). Parasitic on flies (Muscidae, Diptera), collected on a tree stem. Host 6–8 mm long, 3–5 mm wide, without hyphae on the surface. Sexual morph: Stromata 5–7 mm long, 1–4 mm diam., one or several growing from the host prothorax, stipitate, capitate, unbranched, cinnamon to pale yellow. Stipe 3–5 mm long, 1–2 mm diam, cinnamon to pale brown, cylindrical, with a fertile apex. Fertile head hemispherical to globose, 1.5–4 mm, cinnamon to pale yellow, single. Perithecia 570–760 × 190–310 μm (x–= 665 × 250 µm, n = 30), immersed, ovoid to flask-shaped, thick-walled. Peridium 30–50 µm (x–= 40 µm, n = 60) wide, hyaline, three layers: outer layer to textura porrecta, middle layer textura prismatica, inner layer textura angularis. Asci 280–430 × 5.4–7.5 μm (x–= 355 × 6.5 µm, n = 60), 8-spored, hyaline, cylindrical, with a thick apex. Apical cap 5.2–7.6 × 4.4–5.2 μm (x–= 6.4 × 4.8 µm, n = 60), thick, hyaline. Ascospores as long as asci, filiform, hyaline, easily breaking into part-spores. Secondary ascospores 7–10.5 × 1.6–2.5 μm (x–= 8.8 × 2.1 µm, n = 60) fusiform, 1-celled, mostly straight, hyaline, smooth-walled. Asexual morph Not observed in natural substrates.

Additional material.

China • Liaoning Province, Tieling City, Xifeng County, at 374 masl, 42.665°N, 124.487°E, parasitic on the fly (Muscidae, Diptera), collected on a tree stem, 20 July 2023, Yuanpin Xiao TL2378 (HKAS 132275, paratype).

Figure 3. 

Ophiocordyceps muscidarum (HKAS 132178, holotype) a habitat b, c stromata d host (Muscidae, Diptera) e perithecia f peridium g-j asci k ascus apical cap l–n secondary ascospores. Scale bars: 0.1 cm (c, d); 200 μm (e); 50 μm (f, k); 100 μm (g–j); 5 μm (l–o).

Notes.

Phylogenetically, Ophiocordyceps muscidarum is closely related to O. globiceps, with support (100% MLBP, 1.00 PP) (Fig. 1). Base pair differences of LSU, ITS, and tef-1α sequences of O. globiceps are 4.14% (34/820), 7.43% (35/471), and 2.17% (20/920), respectively. O. muscidarum is similar to O. globiceps in having a fly (Muscidae, Diptera) as the host, whereas O. muscidarum differs from O. globiceps in having larger perithecia and longer secondary ascospores (Xiao et al. 2019; Table 3). Additionally, several Ophiocordyceps species exhibit a specific affinity for parasitizing dipteran flies. Notable examples include Ophiocordyceps dipterigena, O. globiceps, O. hemisphaerica, and O. lacrimoidis, all of which can be distinguished from O. muscidarum through phylogenetic analyses (Berkeley and Broome 1873; Freire 2015; Hyde et al. 2016; Xiao et al. 2019). Another species, Ophiocordyceps discoideicapitata, has also been reported to infect flies but lacks molecular data (Kobayasi and Shimizu 1982). Ophiocordyceps discoideicapitata differs from O. muscidarum in having smaller perithecia and shorter, cylindrical, truncated secondary ascospores (Kobayasi and Shimizu 1982, Table 3). Based on morphology and phylogeny, Ophiocordyceps muscidarum is introduced as a new species in Ophiocordyceps.

Table 3.
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Synopsis of a comparison of O. muscidarum sp. nov. and its closely related Ophiocordyceps species.

Species Distribution Stromata (mm) Perthecia (μm) Asci (μm) Ascospores (μm) Secondary ascospores (μm) Reference
O. dipterigena
(First record)		 Sri Lanka 5–10 × 1, pale — — — 10 × 1.5 Berkeley and Broome 1873, Freire 2015
O. dipterigena Japan 5–8 × 1–2 700–900 × 240–400 480–600 Filiform, multiseptate 6–12 × 1–1.5, cylindric or fusoid fragments Kobayasi 1941
O. dipterigena Thailand 4–10 800–1000 × 200–300 450–600 × 4–6 Filiform, breaking up
into 64 part-spore		 6–12 × 1–1.5, cylindrical to fusiform Luangsa-ard et al. 2008
O. dipterigena Brazil 9–13 × 0.5–1 850–920 × 230–300 550–700 × 5, filiform 650–700 × 2, 64 part-spores 8–10 × 1–2, cylindrical, fusoids
O. discoideicapitata Japan 2.5–3.5 × 0.7–1.2 620–700 × 200–250 5–6 diam. — 6–9 × 1, cylindrical, truncated Kobayasi and Shimizu 1982
O. forquignonii — — — — — Oval, 8 Saccardo 1891
O. globiceps Thailand 4–8 × 0.5–1 538–663 × 182–247 373–454 × 5.7–8 240–303 × 1.8–2.3, filiform 4–5.4 × 1.2–1.9, cylindrical to fusoid Xiao et al. 2019
O. hemisphaerica Brazil 12–20 × 0.8–1 780–860 × 220–290 500–640 × 5–6 Filiform, more than 52 septa 7–10 × 1–1.5, cylindrical to unusually fusoid Hyde et al. 2016
O. lacrimoidis Brazil 4–5 × 1 650–700 × 200–250 350–450 × 5 Filiform, more than 56 septa 8–14 × 2, cylindrical Hyde et al. 2016
O. muscidarum China 5–7 × 1–4 570–760 × 190–310 280–430 × 5.4–7.5 as long as asci, filiform 7–10.5 × 1.6–2.5 This study

The symbol “—” means that the data is unavailable.

Ophiocordyceps neocommunis Y. Yang, K.D. Hyde & Y. P. Xiao, sp. nov.

Index Fungorum: IF902880
Facesoffungi Number: FoF16767
Fig. 4

Etymology.

The epithet “neocommunis” refers to the new species’ similarity to its close relative, O. communis.

Holotype.

China • Guizhou Province, Qiandongnan Miao and Dong Autonomous Prefecture, Rongjiang County, at 382 masl, 25.934°N, 108.479°E, parasitic on termites in soil, 10 June 2023, Yu Yang (HKAS 132236).

Description

(HKAS 132236). Parasitic on termite (Blattodea: superfamily Blattoidea), buried in the soil, the synnemata erect in the air. Sexual morph not observed in natural substrates and in culture on PDA. Asexual morph: Hirsutella-like, the host covered with white mycelium. Synnemata 3–6 cm long, white to yellow bottom to top. Conidiophores absent. Phialides single, borne laterally on synnemata, smooth, hyaline 6.5–12.5 × 3–4.5 µm (x–= 9.5 × 3.8 µm, n = 50), basal part strongly swollen, globose, subglobose, or ellipsoid 4.5–8.5 × 3.5–4.8 µm (x–= 6.5 × 4.2 µm, n = 50), usually tapering abruptly to a slender neck 0.5–1.2 µm diam. Conidia 3.0–5.5 × 2.2–4.2 µm (x– = 4.2 × 3.2 µm, n = 50), 1-cell, hyaline, oval to teardrop-shaped.

Cultural characteristics.

Colonies on PDA grow slowly, reaching 2 cm in diameter after 25 days at 25 °C, ivory yellow, flat, and closely appressed to the agar surface. Synnemata are produced after 40 days, with the reverse side showing a warm orange. No phialides or conidia found.

Figure 4. 

Ophiocordyceps neocommunis (HKAS 132236, holotype) a, b habitat c overview of the fungus d host (an unidentified termite species) e synnemata f–h phialides i, j conidia k–l culture on PDA. Scale bars: 10 μm (f–h); 5 μm (i–j).

Additional material.

CHINA, Guizhou Province, Qiandongnan Miao and Dong Autonomous Prefecture, Rongjiang County, at 382 masl, 25.934°N, 108.479°E, parasitic on termites in soil, 10 June 2023, Yu Yang, RJ2363J (GZCC 24-0158; ex-type living culture).

Notes.

Ophiocordyceps neocommunis clustered with O. communis and Hirsutella minnesotensis in the phylogenetic tree, supported by 100% MLBP and 1.00 PP (Fig. 1). Notably, the differences between O. communis and H. minnesotensis are minimal, as indicated by the short branch length in the phylogenetic tree. However, further evidence is needed to determine whether they represent the same species. Ophiocordyceps communis shares its host, termites (Blattodea, superfamily Blattoidea), with O. neocommunis but differs in the morphology of its phialides. Specifically, O. communis produces longer and narrower phialides, as well as longer conidia (Sung et al. 2007b; Table 4). Hirsutella minnesotensis is distinct from O. neocommunis, as it is isolated from second-stage juveniles of the soybean cyst nematode and has longer phialides (9–15 µm vs. 6.5–12.5 µm) (Chen et al. 2000). Comparing the ITS, tef-1α, rpb1, and rpb2 sequences of Ophiocordyceps neocommunis and O. communis revealed 98.83% (6 bp differences), 97.57% (22 bp differences), 98.59% (10 bp differences), and 98.76% (13 bp differences) sequence similarities, respectively. Comparing the ITS sequences of Ophiocordyceps neocommunis and Hirsutella minnesotensis revealed 98.83% (6 bp differences). Thus, we would like to introduce Ophiocordyceps neocommunis as a new species based on phylogenetic and morphological analyses.

Table 4.
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Synopsis of a comparison of O. neocommunis sp. nov. and its closely related Ophiocordyceps species.

Species Distribution Asexual morph Phialides (µm) Conidia (µm) Reference
O. asiatica Thailand Hirsutella-like 15–20 × 2–3 Fusiform, 7–9 × 2–3 Tasanathai et al. 2019
O. brunneirubra Thailand Hirsutella-like 32–50 × 2–3 Fusiform, 12–17 × 2–4 Tasanathai et al. 2019
O. communis Thailand Hymenostilbe/Hirsutella-like 10–14 × 2.7–3.3 Almond-shaped, 7–9 × 2.5–3 Sung et al. 2007a
O. fusiformis Thailand Hymenostilbe-like 9–24 × 2–4 Fusiform, 6–18 × 2–4 Tasanathai et al. 2022
O. globosa Thailand Hirsutella-like 9–15 × 3–5 Globose, 2–4 Tasanathai et al. 2022
O. isopterae Thailand Hirsutella-like 14–28 × 2–4 Fusiform, 6–11 × 1.5–3 Tasanathai et al. 2022
O. khokpasiensis Thailand Hirsutella-like 15–28 × 3–5 Globose to oval, 4–6 × 2.5–4 Khonsanit et al. 2019
O. longistipes China Hirsutella-like 29–60 × 4–4.5 Citriform or oval, 7–10 × 4.5–7 Fan et al. 2024
O. mosingtoensis Thailand Hirsutella-like 10–17 × 2–3 Oval, 3–5 × 2–3 Tasanathai et al. 2019
O. neocommunis China Hirsutella-like 6.5–12.5 × 3–4.5 Oval to teardrop-shaped 3.0–5.5 × 2.2–4.2 This study
O. ovatospora China Hirsutella-like 15–35 × 3–6 Oval, 3–5 × 3–4 Tang et al. 2022
O. pseudocommunis Thailand Hymenostilbe-like 17–22 × 2–8 Fusiform, (6–)6.5–7.5(–8) × 2–3 Tasanathai et al. 2019
O. pseudorhizoidea Thailand Hirsutella-like 9–21 × 2–4 Fusiform, 5–10 × 1–2 Tasanathai et al. 2019
O. puluongensis Vietnam Hirsutella-like 7.9–21.2 × 1.7–5.0 Fusiform or citriform, 2.8–6.1 × 1.9–3.4 Xu et al. 2022
O. radiciformis Thailand Hirsutella-like 6–15 × 2–5 Fusiform, 5–7 × 2–3 Tasanathai et al. 2022
O. termiticola Thailand Hirsutella-like 7–11 × 2.5–4 Globose, 2.5–3.5 Tasanathai et al. 2019

Hirsutella vermicola M.C. Xiang & Xing Z. Liu, in Xiang, Yang, Xiao, Liu & Chen, Fungal Diversity 22: 258 (2006)

Index Fungorum: IF500924
Fig. 5

Description

(HKAS 132167). Parasitic pupa of Lepidoptera, buried in soil. Sexual morph not observed in natural substrates. Asexual morph: Synnemata 2–6 cm long, 1–3 mm wide, cylindrical with tapering tip, stipitate, gradually become white or green yellowish to the upper portion, scattered on the insect head, without fertile head. Phialide solitary along synnema, 15.2–25.5 × 1.3–4.8 µm (x–= 20.3 × 3.1 µm, n = 60), solitary, upright, typically cylindrical, with a wider base that gradually narrows towards the top 7.2–15.3 × 2.4–4.8 µm (x–= 11.2 × 3.6 µm, n = 60), tapering into a long neck 7.5–11.5 µm long, has a rough surface, featuring verrucose projections. Conidia 4.6–6.8 × 2.2–3.2 µm (x– = 5.7 × 2.7 µm, n = 60), fusiform or oval, 1-celled, hyaline.

Material examined.

China • Guizhou Province, Zunyi City, Xishui National Nature Reserve, at 1069 masl, 28.494°N, 106.388°E, parasitic on pupa of Lepidoptera, buried in soil, 10 April 2023, Yu Yang, XS2310, XS2311 (HKAS 132167, HKAS 132168).

Figure 5. 

Hirsutella vermicola (New host, HKAS 132167) a habitat b synnemata on host c synnema d host (pupa of Lepidoptera) e–h phialides i phialides with conidia j–l conidia. Scale bars: 50 µm (e–f); 20 µm (g–i); 5 µm (j–l).

Notes.

Hirsutella vermicola, introduced by Xiang et al. (2006), was found in bacteria-feeding nematodes in the United States. Phylogenetic analysis showed that our new isolates, HKAS 132167 and HKAS 132168, are nested with Hirsutella vermicola (CGMCC 3.7877, AS3.7878, and AS3.7879) (Fig. 1). Morphologically, our new isolate is almost identical to Hirsutella vermicola except for the conidia (Xiang et al. 2006). Our new isolate has smaller conidia than the holotype of H. vermicola (4.6–6.8 µm vs. 6–8 µm) (Xiang et al. 2006). The molecular data of our new isolate (HKAS 132168) are not significantly different from those of Hirsutella vermicola (CGMCC 3.7877, AS3.7878, and AS3.7879). Thus, we identified our new isolate as H. vermicola. This is the first report of H. vermicola parasitic on the pupa of Lepidoptera, which broadens the range of hosts. Furthermore, this is the first report of H. vermicola in China.

Discussion

The taxonomy of entomopathogenic fungi has undergone significant revisions in the molecular era, moving toward a monophyletic classification (Tasanathai et al. 2016; Dong et al. 2022). Initially, ITS sequences were inadequate for differentiating closely related species due to their limited variability (Chen et al. 2002; Liu et al. 2002; Stensrud et al. 2005). To enhance phylogenetic resolution, additional genes were incorporated into analyses, including the ribosomal small subunit (SSU), large subunit (LSU), and protein-coding genes such as the elongation factor 1-alpha (tef-1α), RNA polymerase II largest subunit (rpb1), second largest subunit (rpb2), and β-tubulin (TUB). These combined analyses provided more comprehensive phylogenetic insights (Sung et al. 2007b; Khonsanit et al. 2020; Fan et al. 2024). The adoption of Genealogical Concordance Phylogenetic Species Recognition (GCPSR) has since improved species identification and classification within the Ophiocordyceps, leading to more accurate taxonomic delineations (Sung et al. 2007b; Khonsanit et al. 2020; Mongkolsamrit et al. 2021). As genomic sequencing of Ophiocordyceps species has expanded, our understanding of their phylogenetic relationships and taxonomic framework has deepened, deepening our understanding of their phylogenetic relationships and taxonomic framework (Wang et al. 2016; Shu et al. 2020; de Menezes et al. 2023).

Ophiocordyceps exhibit remarkable parasitic adaptability, infecting a wide range of hosts. Our research has identified new species that parasitize insects from the orders Blattodea, Coleoptera, Diptera, and Lepidoptera. Termites, particularly those from the superfamily Blattoidea, are also known hosts for some Ophiocordyceps species (Tasanathai et al. 2019; Tasanathai et al. 2022; Fan et al. 2024). Despite the global presence of over 300 Ophiocordyceps species, fewer than 19 are reported to parasitize termites, with occurrences documented in diverse regions including China, Indonesia, Japan, Kenya, Mexico, Tanzania, and Thailand (Stifler 1941; Blackwell and Gilbertson 1981; Ochiel et al. 1997; Tasanathai et al. 2019; Tasanathai et al. 2022; Fan et al. 2024). Termite-infecting Ophiocordyceps typically reside 5 to 15 cm underground, mirroring the subterranean habits of their termite hosts (Martelossi et al. 2023). However, identifying these fungi poses challenges due to the potential loss of fragile stromata during excavation (Tasanathai et al. 2022). In China, four termite-associated species have been identified: Ophiocordyceps longistipes, O. globiperitheciata, O. ovatospora, and O. puluongensis (Tang et al. 2022; Xu et al. 2022; Fan et al. 2024). Comparatively, Ophiocordyceps neocommunis features white to yellow synnemata, with strongly swollen basal part phialides, and single, oval to teardrop-shaped conidia.

The taxonomic distinction between Ophiocordyceps and Hirsutella remains unclear, as evidenced by their recurrent clustering in phylogenetic trees (Evans and Samson 1982; Quandt et al. 2014; Spatafora et al. 2015). This study, which introduces three new Ophiocordyceps species and a new host record for H. vermicola, underscores the need for further phylogenetic clarification, as our phylogenetic tree also shows that both genera are clustered together. Extensive research has demonstrated the widespread parasitism of both genera across a variety of insect hosts (Spatafora et al. 2015; Araújo et al. 2018; Xiao et al. 2019; Mongkolsamrit et al. 2021; Tasanathai et al. 2022; Yang et al. 2024). The three new species and the new host record identified in our study further reinforce this pattern, with hosts spanning multiple insect orders, families, and genera, including flies, lepidopteran pupae, termites, and wireworms (Tasanathai et al. 2019; Zha et al. 2021). Importantly, the adaptation of Ophiocordyceps and Hirsutella species to specific hosts underscores the influence of host diversity on the genetic diversity and geographic distribution of both genera.

Ophiocordyceps species in the unilateralis complex exhibit remarkable specificity, targeting exclusively the ant genus Camponotus, which significantly alters the ant’s behavior to facilitate its reproduction (de Bekker et al. 2014; Araújo et al. 2018). Similarly, Ophiocordyceps sinensis has a specialized association with ghost moth larvae (Thitarodes spp.), parasitizing the larvae to produce the medicinally valuable fungus known as “yarsagumba” (Baral 2017). Furthermore, Somavilla et al. (2019) investigated how Ophiocordyceps humbertii influences the behavior of social wasps in Neotropical forests, highlighting the fungus’s adaptability and its impact on its host. These instances underscore how Ophiocordyceps species adapt to specific hosts, influencing their genetic diversity and distribution across ecosystems.

Advancements in phylogenetic research and taxonomy, particularly through DNA sequencing, are crucial for accurately identifying and classifying Ophiocordyceps and Hirsutella species (Tasanathai et al. 2016; Xiao et al. 2019; Dong et al. 2022; Yang et al. 2024). Moreover, the potential of Ophiocordyceps in pest management is increasingly recognized. For example, Ophiocordyceps unilateralis regulates ant populations by infecting and controlling their behavior, which helps protect crops from ant damage (Tasanathai et al. 2019; Moon et al. 2023; Fan et al. 2024), and O. nutans is being developed as a natural medicine and biopesticide to protect crops and forest trees (Paul et al. 2020). Utilizing Ophiocordyceps in biological control highlights its significant potential for promoting sustainable agriculture and protecting ecological environments. In this study, three new species, Ophiocordyceps muscidarum, O. neocommunis, and O. liaoningensis, and one new host record for Hirsutella vermicola, associated with insects from China, were introduced based on phylogenetic inferences of a combined LSU, ITS, SSU, tef-1α, rpb1, and rpb2 DNA sequence dataset and morphological evidence.

Acknowledgements

We thank the Mushroom Research Foundation, Chiang Rai, Thailand, for supporting this research. The authors also thank Shaun Pennycook (Manaaki Whenua Landcare Research, New Zealand) for advising us on fungal nomenclature.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was funded by the National Natural Science Foundation of China (NSFC32060013), Guizhou Provincial Basic Research Program (MS [2025] No. 193), and the High-level Talent Research Initiation Fund Project in Guizhou Instituteof Technology (2023GCC063). The authors extend their appreciation to the ResearchersSupporting Project number (RSP2025R114), King Saud University, Riyadh, Saudi Arabia.The work was also funded by Guizhou Institute of Technology 2024 Academic New Bud Cultivation and Innovation Exploration Project (No. 2024XSXM008) and the survey and assessment of macrofungal biodiversity inXishui National Nature Reserve (TYJZB-20214013).

Author contributions

Morphological data, photo-plates and phylogenetic analyzes were completed by Yu Yang. The original draft was written by Yu Yang, and Yuan-Pin Xiao, Ruvishika S. Jayawardena, Kevin D. Hyde, Somrudee Nilthong, Ausana Mapook, Yong-Zhong Lu, Su-Qiong Xie, Fatimah Al-Otibi, Xiao Wang, Kang Luo revised the paper.

Author ORCIDs

Yu Yang https://orcid.org/0000-0001-8268-487X

Ruvishika S. Jayawardena https://orcid.org/0000-0001-7702-4885

Kevin D. Hyde https://orcid.org/0000-0002-2191-0762

Yong-Zhong Lu https://orcid.org/0000-0002-1033-5782

Fatimah Al-Otibi https://orcid.org/0000-0003-3629-5755

Data availability

All of the data that support the findings of this study are available in the main text.

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