Nine new species of cordycipitoid fungi (Hypocreales, Sordariomycetes) from China

Abstract

Cordycipitoid fungi, encompassing the four families Clavicipitaceae, Cordycipitaceae, Ophiocordycipitaceae, and Polycephalomycetaceae, have been the focus of intensive molecular phylogenetic and taxonomic research over the past twenty years. Utilizing a six-locus dataset (LSU, ITS, SSU, tef1-α, rpb1, and rpb2) with expanded taxon sampling, this study establishes a robust phylogenetic framework for this group, with particular emphasis on the Ophiocordycipitaceae. Our work significantly expands the documented diversity of cordycipitoid fungi in China through the description of nine new species: Cordyceps multisynnematosa, Metarhizium acrididarum, Ophiocordyceps sungii, O. brunneascospora, O. ramosiphialidica, O. rubella, Paraisaria clavata, Par. cocoonihabita, Purpureomyces fenggangensis and reports three new records: Jenniferia thomisidarum, O. brunneipunctata, and Pleurocordyceps heilongtanensis. Macro- and micro-morphological descriptions are provided to support the taxonomic placement of these new taxa. Beyond taxonomic additions, we have accessed to the morphological diversity of the asexual morphs of Cordyceps, Metarhizium, Ophiocordyceps, Paraisaria, and Purpureomyces, elucidating conserved and divergent features in phialides and conidia. Beyond enhancing knowledge of cordycipitoid fungal phylogeny and diversity in China, this study highlights the pressing need for sustained exploration of entomopathogenic fungi and their ecological roles.

Citation: Peng XC, Wei DP, Zhang X, Tangtrakulwanich K, Wang Y, Hyde KD, Kang JC, Jayawardena RS, Liu ZL, Xie SW, Bu J, Wen TC (2026). Nine new species of cordycipitoid fungi (Hypocreales, Sordariomycetes) from China. Persoonia 56: 381–419. doi: 10.3114/persoonia.2026.56.06

INTRODUCTION

Hypocreales constitutes the most species-rich order of entomopathogenic fungi, which are taxonomically mainly distributed across four families, viz. Clavicipitaceae, Cordycipitaceae, Ophiocordycipitaceae, and Polycephalomycetaceae (Araújo & Hughes 2016, Wei et al. 2022, Xiao et al. 2023). Members of these families exhibit broad ecological plasticity, colonising diverse hosts including arthropods, plant, fungi and soil substrates (Mongkolsamrit et al. 2020a, Xiao et al. 2023, Pu et al. 2025). This group of entomopathogenic fungi plays a crucial role in maintaining ecosystem balance, as source for pharmaceutical industry and acting as biocontrol agents (Erler & Ates 2015, Das et al. 2021, Apirajkamol et al. 2023, Shweta et al. 2023, Sharma et al. 2024). Taxonomy of hypocrealean entomopathogenic fungi is essential for the downstream industries, drawing extensive attention from mycologists around the world (Simmons et al. 2015, Kepler et al. 2017, Araújo et al. 2018, Wang et al. 2020, Tanaka et al. 2023, Chuang et al. 2024).

The taxonomic systematics of hypocrealean entomopathogenic fungi have undergone iterative revisions in response to advancing molecular methodologies. Sung et al. (2007) subdivided the paraphyletic Clavicipitaceae sensu lato into three monophyletic families viz. Clavicipitaceae sensu stricto, Cordycipitaceae, and Ophiocordycipitaceae through combined morphological assessments and multi-gene phylogenetic analyses (SSU, LSU, tef1-α, rpb1, rpb2, tub, and atp6). Quandt et al. (2014) and Kepler et al. (2017) resolved competing genera within Ophiocordycipitaceae and Cordycipitaceae respectively, utilizing a molecular dataset (SSU, LSU, tef1-α, rpb1, and rpb2). Xiao et al. (2023) segregated a novel family Polycephalomycetaceae from Ophiocordycipitaceae to accommodate genera Polycephalomyces, Perennicordyceps, and Pleurocordyceps based on multi-gene phylogenetic analysis. Current systematic inventories respectively recognise 56, 37, 8, and 5 genera within Clavicipitaceae, Cordycipitaceae, Ophiocordycipitaceae, and Polycephalomycetaceae respectively (Hyde et al. 2024). Despite recent advancements, persistent challenges hinder the taxonomic resolution of certain historic genera (e.g., Cordyceps, Metarhizium, and Ophiocordyceps), which contain numerous morphospecies with unresolved phylogenetic affinities. Additionally, modern molecular phylogenetic analyses further reveal extensive cryptic speciation within these lineages, underscoring the necessity for revisions of misclassified taxa with freshly collected specimens complemented by museum collections, and for exploration of biodiversity by targeting both cryptic species complexes and understudied ecological niches.

Southwest China has emerged as an important region for the diversity and documentation of entomopathogenic fungi belonging to Hypocreales. The past decade has witnessed the sharply increasing description of entomopathogenic species from these areas (Zhang et al. 2017, Wang et al. 2020, 2024a, Chen et al. 2021, Wei et al. 2021, 2022, Tang et al. 2023, Chuang et al. 2024, Peng et al. 2024). Through integrative taxonomic approaches combining morphological phenomics with multilocus phylogenetics (LSU, ITS, SSU, tef1-α, rpb1, and rpb2), this study aims to: (i) describe previously unknown entomopathogenic species within Hypocreales, (ii) to report new hosts or new geographic species, and (iii) to reveal their phylogenetic relationship within Hypocreales. These findings not only contribute to the understanding of biodiversity in these regions but also provide critical baseline data for biocontrol applications targeting agricultural pests in subtropical ecosystems.

MATERIALS AND METHODS

Fungal materials and isolation

Entomopathogenic fungal specimens were systematically surveyed across two distinct microhabitats in subtropical forest ecosystems of southern China: (1) floor substrates including soil, leaf litter and decaying wood; and (2) phyllosphere surfaces encompassing both upper and underside of leaves. Field documentation employed a Canon 6D digital camera for morphological characterization, with georeferencing data concurrently recorded using Biotracks v. 3.6 software. A total of 17 fresh specimens associated with host insects were collected from three Chinese provinces: Guizhou (n = 7), Yunnan (n = 9), and Guangdong (n = 1).

Axenic cultures were established through aseptic transfer of either mycelial mass from insect cadavers or ascospores/conidia onto potato dextrose agar (PDA) plates using sterile inoculation needles, followed by incubation at 25 °C under controlled conditions. The resultant pure cultures have been accessioned in the Guizhou University Herbarium (GACP) and the China General Microbiological Culture Collection Center (CGMCC), while voucher specimens were processed through desiccation with colour-indicating silica gel and permanently preserved in the Herbarium of Cryptogams (HKAS) at the Kunming Institute of Botany, Chinese Academy of Sciences.

Morphological observations

The macro-morphological characters and relevant data of the fungus, such as the host and the colour and shape of the stromata were examined under a dissecting microscope (Leica S9E). For micro-morphological characterization including perithecia, peridium, asci, ascospores, secondary ascospores, mycelium, metulae, phialides and conidia, structures were mounted in ultrapure water or lactophenol cotton blue solution, and photographed using a compound microscope (Nikon ECLIPSE Ni). Measurements of the microstructures were taken using Adobe Photoshop CC 2019, with a minimum of twenty replicates obtained for each structure. The minimum, maximum, and mean values were calculated for each feature. Asexual morphs were observed from cultures grown on PDA at 25 °C in the dark for 2 wk. Slow-growing species were incubated for 4–10 wk until sporulation was observed.

DNA extraction and PCR amplification

A small piece of tissues from fungal specimens or mycelial mass from pure culture was transferred to a 2.0 mL tube and homogenized with glass beads using a high throughput tissue mill (POWTEQ® GT50, China). The genomic DNA was extracted using a DNA extraction kit (E.Z.N.A.^® SP Fungal DNA Kit, Omega, USA), following the protocol of the manufacturer. The obtained total genomic DNA was stored at −20 °C. Six genetic makers including the nuclear ribosomal large subunit (LSU), the internal transcribed spacer region of the nuclear ribosomal DNA (ITS), the nuclear ribosomal small subunits (SSU), the translation elongation factor-1 alpha (tef1-α), and the genes for RNA polymerase II largest (rpb1) and second largest (rpb2) subunits were amplified and sequenced. The primer pairs used were: LROR/LR5 for LSU (Vilgalys & Hester 1990, Rehner & Samuels 1994), ITS5/ITS4 for ITS (White et al. 1990), NS1/NS4 for SSU (White et al. 1990), EF1-983f/EF1-2218r for tef1-α (Rehner & Buckley 2005), CRPB1A/RPB1Cr for rpb1 (Castlebury et al. 2004), and fRPB2-5F/fRPB2-7cR for rpb2 (Liu et al. 1999). The PCR amplification system and thermal cycling conditions followed the protocol described by Peng et al. (2024). The resultant amplicons were purified and subsequently sequenced by Tsingke Biological Technology (Chongqing, China) using the aforementioned primers.

Phylogenetic analyses

The forward and reverse reads generated in this study were assembled with BioEdit v. 7.0.5.3 (Hall et al. 2011). A comprehensive analysis based on a large-scaled dataset was constructed following the OFPT framework (Zeng et al. 2023) with default parameters to preliminarily determine the phylogenetic placement of our specimens. A refined dataset (Table 1) was subsequently generated by reducing phylogenetically distant taxa, followed by multiple sequence alignment using MAFFT v. 7 (Katoh et al. 2019). The alignment was automatically adjusted with trimAL v. 1.2 (Capella-Gutiérrez et al. 2009). The concatenation of six genetic loci (LSU, ITS, SSU, tef1-α, rpb1, rpb2) was performed using SequenceMatrix v. 1.8 (Vaidya et al. 2011). The resulting alignment was converted to FASTA files for maximum likelihood (ML) analyses and to NEXUS files for Bayesian inference (BI) analyses using AliView v. 1.19 (Larsson 2014). The data matrix comprised 203 taxa from Hypocreales with extensive sampling from four families as follow: 37 taxa from Clavicipitaceae, 54 taxa from Cordycipitaceae, 87 taxa from Ophiocordycipitaceae, 20 taxa from Polycephalomycetaceae. Three taxa from Bionectriaceae and two taxa from Calcarisporiaceae were treated as outgroup. The ML analysis was performed using IQ-TREE v. 1.6.12 with the “Auto” model option, and branch support was estimated from 1000 ultrafast bootstraps replicates (Minh et al. 2020). For BI analysis, MrModeltest v. 2.3 as implemented in MrMTgui (Nuin 2007) was used to select the best-fit evolution model for each locus under the Akaike Information Criterion (AIC). MrBayes on ACCESS v. 3.2.7a in the CIPRES Science Gateway was used to evaluate posterior probabilities (PP) with the Markov Chain Monte Carlo (MCMC) sampling method. Two independent runs with four Markov chains each were conducted for up to 1000 M generations, sampling every 5000 generations. The first 20 % of sampled trees were discarded as burn-in, and the remaining trees were used to calculate PP in a majority rule consensus tree. Convergence of BI analyses was considered achieved when the average standard deviation of split frequencies reached 0.01. The GTR+I+G model was deemed as the most suitable for Bayesian inference analysis. Calcarisporium cordycipiticola (MFLUCC 15-0686 and MFLUCC 15-0685) in Calcarisporiaceae, and Amphichorda excrementa (YFCC AECCS848), A. felina (CBS 250.34), and A. kunmingensis (YFCC AKYYH8414) in Bionectriaceae, Hypocreales were used as outgroups. Phylogenetic trees (Fig. 1) were visualized in FigTree v. 1.4.3 (Rambaut 2016) and edited in Adobe Illustrator CC 2019. The ML bootstrap values equal or greater than 80 % are shown above each node, and the PP values equal to or greater than 0.90 are indicated in Fig. 1.

Table 1.

List of taxa included in the phylogenetic analyses and their GenBank accession numbers. The accession numbers marked in bold font refer to new sequences generated in this study.

Current name1	Voucher2,3	Location4	Host / substratum	GenBank accession numbers4,5						References
				LSU	ITS	SSU	tef1-α	rpb1	rpb2
Akanthomyces fusiformis	BCC 40756T	Thailand	Lepidoptera, Pyralidae	ON008549	—	—	ON013552	ON013567	ON013576	Khonsanit et al. (2024)
Akanthomyces niveus	BCC 79887T	Thailand	Lepidoptera	ON008551	—	—	ON013554	—	ON013578	Khonsanit et al. (2024)
Amphichorda excrementa	YFCC AECCS848T	China	animal faeces	OR913439	—	OR913433	OR917446	OR917451	OR917443	Wang et al. (2024b)
Amp. felina	CBS 250.34	United Kingdom	pressed yeast	OR913440	—	OR913436	OR917447	OR917450	OR917444	Wang et al. (2024b)
Amp. kunmingensis	YFCC AKYYH8414T	China	animal faeces	OR913438	—	OR913435	OR917448	OR917452	—	Wang et al. (2024b)
Arachnidicola waltergamsii	BBH 32434T	Thailand	Araneae	MF140714	MF140748	—	MF140834	MF140782	MF140806	Mongkolsamrit et al. (2018)
Ascopolyporus albus	BCC 48975T	Thailand	Hemiptera, Coccidae	OL322048	OL331502	—	OL322035	OL322056	OL322065	Thanakitpipattana et al. (2022)
Beauveria blattidicola	MCA 1727T	Guyana	Blattodea, Blattidae	MF416539	—	MF416593	MF416483	MF416640	—	Kepler et al. (2017)
B. caledonica	ARSEF 2567	Scotland	soil	AF339520	HQ880817	AF339570	EF469057	EF469086	HQ880961	Chiriví-Salomón et al. (2015)
B. scarabaeidicola	ARSEF 5689	—	Coleoptera, Scarabaeidae	AF339524	AY245639	AF339574	DQ522335	DQ522380	DQ522431	Spatafora et al. (2007)
Blackwellomyces aurantiacus	BCC 85060T	Thailand	Lepidoptera	MT003028	MT000692	—	MK411598	MK411600	MT017819	Mongkolsamrit et al. (2020b)
Calcarisporium cordycipiticola	MFLUCC 15-0686T	China	Hypocreales, Cordycipitaceae	KX442599	KT944999	KT944998	KX442593	—	KX442594	Sun et al. (2017)
	MFLUCC 15-0685	China	Hypocreales, Cordycipitaceae	KX442604	KT945001	KT945000	KX442605	—	KX442607	Sun et al. (2017)
Claviceps quebecensis	DAOM 867491T	Canada	Poales, Poaceae	MH477795	MH477795	—	MH397459	—	MH349067	Tanaka et al. (2023)
Claviceps setariicola	PRM 915379T	Brazil	Poales, Poaceae	AJ557074	AJ557074	—	LT216533	—	LT216606	Píchová et al. (2018)
Cordyceps amoene-rosea	CBS 107.73T	Thailand	Coleoptera	MF416550	AY624168	AY526464	MF416494	MF416651	MF416445	Luangsa-ard et al. (2005), Kepler et al. (2017)
C. araneae	BCC 85066T	Thailand	Araneae	MT003038	MT000703	—	MT017851	MT017811	MT017829	Mongkolsamrit et al. (2020b)
C. bifusispora	EFCC 8260	—	Lepidoptera	EF468807	—	EF468953	EF468747	EF468855	EF468910	Sung et al. (2007)
C. cf. takaomontana	NHJ 12623	—	Lepidoptera	EF468838	—	EF468984	EF468778	EF468884	EF468932	Sung et al. (2007)
C. chaetoclavata	YHH 15101T	China	Lepidoptera	MN576778	—	MN576722	MN576948	MN576838	MN576894	Wang et al. (2020)
C. javanica	TBRC 7261	Thailand	Lepidoptera	MF140709	MF140743	—	MF140829	MF140778	MF140802	Mongkolsamrit et al. (2018)
	TBRC 7260	Thailand	Lepidoptera	MF140710	MF140744	—	MF140830	MF140779	MF140803	Mongkolsamrit et al. (2018)
C. kyusyuensis	EFCC 5886	—	Lepidoptera	EF468813	—	EF468960	EF468754	EF468863	EF468917	Sung et al. (2007)
C. lepidopterorum	BBH 40735T	Thailand	Lepidoptera	MF140699	MF140765	—	MF140819	MF140768	MF140792	Mongkolsamrit et al. (2018)
C. multisynnematosa	HKAS 149957 T	China	Lepidoptera	PX411458	—	PX414344	PX409302	PX423491	PX423508	This study
	GACP JK22052023	China	Lepidoptera	PX411459	PX411440	PX414345	PX409303	PX423492	PX423509	This study
C. parvistroma	BCC 79829T	Thailand	Lepidoptera	MT003046	MT000710	—	MT017857	MT017813	MT017837	Mongkolsamrit et al. (2020b)
C. pleuricapitata	NBRC 100745	Japan	Hemiptera	JN941391	JN943304	JN941750	KF049679	JN992484	KF049667	Schoch et al. (2012), Kepler et al. (2013)
	NBRC 100746	Japan	Hemiptera	JN941392	JN943306	JN941749	KF049680	JN992483	KF049668	Schoch et al. (2012), Kepler et al. (2013)
C. takaomontana	BCC 12688	Thailand	Lepidoptera	MF416545	EU807996	MF416599	MF416489	MF416646	—	Luangsa-ard et al. (2009), Kepler et al. (2017)
C. tenuipes	OSC 111007	—	Lepidoptera	DQ518774	—	DQ522559	DQ522349	DQ522395	DQ522449	Spatafora et al. (2007)
	ARSEF 5135	—	Lepidoptera	JF415980	AY624196	AY526492	JF416020	JN049896	JF416000	Luangsa-ard et al. (2005), Kepler et al. (2012)
Corniculantispora psalliotae	CBS 532.81T	—	soil	AF339560	JN049846	AF339609	EF469067	EF469096	EF469112	Sung et al. (2007), Kepler et al. (2012)
Dingleyomyces lloydii	PDD 121254	New Zealand	Hypocreales, Ophiocordycipitaceae	OR602640	OR602634	OR647563	OR588853	OR588860	OR588858	Johnston and Park (2023)
Drechmeria gunnii	OSC 76404	—	Lepidoptera	AF339522	JN049822	AF339572	AY489616	AY489650	DQ522426	Kepler et al. (2012)
Drechmeria sinensis	CBS 567.95	—	Nematoda	AF339545	MH862540	AF339594	DQ522343	DQ522389	DQ522443	Spatafora et al. (2007), Vu et al. (2019)
Engyodontium aranearum	CBS 309.85	—	Araneae	AF339526	—	AF339576	DQ522341	DQ522387	DQ522439	Spatafora et al. (2007)
Flavocillium acerosum	CBS 418.81T	Brazil	Agaricales, Marasmiaceae	KM283786	EF641893	KM283762	KM283810	KM283832	KM283852	Kaifuchi et al. (2013), Park et al. (2015)
F. bifurcatum	YFCC 6101T	China	Lepidoptera, Noctuidae	MN576781	MN576833	MN576725	MN576951	MN576841	MN576897	Wang et al. (2020)
Gamszarea coprophilum	CGMCC 3.18987	China	animal faeces	MH177618	MH177615	MH177626	MH184586	MH177621	MH177623	Su et al. (2019)
	CGMCC 3.18986T	China	animal faeces	MH177619	MH177616	MH177627	MH184587	MH177622	MH177624	Su et al. (2019)
Gamszarella antillana	CBS 350.85T	—	Hymenomycetes	AF339536	AJ292392	AF339585	DQ522350	DQ522396	DQ522450	Spatafora et al. (2007)
Gibellula fusiformispora	BCC 56802T	Thailand	Araneida	MT477063	MT477070	—	MT503329	MT503322	MT503337	Kuephadungphan et al. (2020)
Gibellula scorpioides	BCC 47976T	Thailand	Araneae, Salticidae	MT477066	MT477078	—	MT503335	MT503325	MT503339	Kuephadungphan et al. (2020)
Hevansia minuta	BCC 47519T	Thailand	Araneae, Theridiidae	MZ684002	MZ684087	—	MZ707811	MZ707826	MZ707833	Mongkolsamrit et al. (2022)
Hevansia novoguineensis	NHJ 11923	—	Araneae	EU369032	—	EU369095	EU369013	EU369052	EU369072	Johnson et al. (2009)
Hirsutella cryptosclerotium	ARSEF 4517	Togo	Hemiptera, Pseudococcidae	KM652109	KM652157	KM652066	KM651992	KM652032	—	Simmons et al. (2015)
H. minnesotensis	3608	China	Nematoda	JPUM01000376	JPUM01000376	JPUM01000376	JPUM01000211	JPUM01000139	JPUM01000138	Lai et al. (2014)
	SB3612	Germany	Trombidiformes	—	EF194145	—	—	—	—	Bałazy et al. (2008)
	SB3730/e	Poland	Trombidiformes	—	EF194144	—	—	—	—	Bałazy et al. (2008)
H. necatrix	ARSEF 5549	United Kingdom	Acari	KM652116	KM652164	KM652073	KM651999	KM652039	—	Simmons et al. (2015)
Jenniferia cinerea	NHJ 3510T	—	Araneae, Amyciaea	GQ249970	GQ249999	EU369091	EU369009	EU369048	EU369070	Johnson et al. (2009)
J. griseocinerea	BCC 42062T	Thailand	Araneae, Thomisidae	MZ684006	MZ684091	—	MZ707815	MZ707828	MZ707837	Mongkolsamrit et al. (2022)
J. thomisidarum	BCC 48932	Thailand	Araneae, Thomisidae	MZ684012	MZ684095	—	MZ707819	—	MZ707841	Mongkolsamrit et al. (2022)
	BCC 37882	Thailand	Araneae, Thomisidae	MZ684011	MZ684100	—	MZ707824	MZ707831	MZ707844	Mongkolsamrit et al. (2022)
	BCC 37881T	Thailand	Araneae, Thomisidae	MZ684010	MZ684099		MZ707823	MZ707830	MZ707843	Mongkolsamrit et al. (2022)
	HKAS 149963	China	Araneae	PX411470	PX411449	PX414355	PX409311	—	—	This study
Kanoksria zaquensis	HMAS 246917	China	Hypocreales, Ophiocordycipitaceae	MT789696	MT789698	MT789700	MT797811	MT797809	—	Wang et al. (2023b)
	HMAS 246915T	China	Hypocreales, Ophiocordycipitaceae	MT789697	MT789699	MT789701	MT797812	MT797810	—	Wang et al. (2023b)
Keithomyces carneus	CBS 239.32T	France	Sand dune	EF468843	AY624171	EF468988	EF468789	EF468894	EF468938	Luangsa-ard et al. (2005), Sung et al. (2007)
Keithomyces echinosporus	CGMCC 3.25517	China	soil	OR680609	OR680542	OR680904	OR858936	—	OR842957	Zhang et al. (2024)
Liangia sinensis	YFCC 3103T	China	Hypocreales, Cordycepitaceae	MN576782	MN576831	MN576726	MN576952	MN576842	MN576898	Wang et al. (2020)
	YFCC 3104	China	Hypocreales, Cordycepitaceae	MN576783	MN576832	MN576727	MN576953	MN576843	MN576899	Wang et al. (2020)
Marquandomyces marquandii	CBS 182.27	USA	soil	EF468845	AY624193	EF468990	EF468793	EF468899	EF468942	Luangsa-ard et al. (2005), Sung et al. (2007)
Metapochonia bulbillosa	CBS 145.70T	Denmark	Pinales, Pinaceae	AF339542	AJ292410	AF339591	EF468796	EF468902	EF468943	Sung et al. (2007), Kepler et al. (2012)
Metarhizium biotecense	BCC 51812T	Thailand	Hemiptera, Delphacidae	MN781838	MN781878	MN781937	MN781693	MN781745	MN781792	Mongkolsamrit et al. (2020a)
Metar. culicidarum	BCC 7600T	Thailand	Diptera, Culicidae	MN781852	MN781889	MN781951	MN781707	MN781754	MN781803	Mongkolsamrit et al. (2020a)
Metar. acrididarum	HKAS 149984 T	China	Orthoptera, Acrididae	PX411471	PX411451	PX414357	PX789407	PX789406	—	This study
Metar. phuwiangense	BCC 85068	Thailand	Coleoptera	MN781864	MN781912	—	MN781720	MN781766	MN781813	Mongkolsamrit et al. (2020a)
	BCC 85069T	Thailand	Coleoptera	MN781865	MN781913	—	MN781721	MN781767	MN781814	Mongkolsamrit et al. (2020a)
Metar. putuoense	HMAS 285457T	China	Coleoptera, Elateridae	OQ981970	OQ981963	OQ981977	OQ980403	OQ980411	—	Li et al. (2023)
	HMAS 285458	China	Coleoptera	OQ981971	OQ981964	OQ981978	OQ980404	OQ980412	—	Li et al. (2023)
Metar. reniforme	ARSEF 429	Philippines	Orthoptera, Tettigoniidae	HQ165733	—	HQ165671	HQ165690	—	HQ165650	Luangsa-ard et al. (2017)
	ARSEF 577	Indonesia	Orthoptera, Tettigoniidae	HQ165734	—	HQ165672	HQ165691	—	HQ165651	Luangsa-ard et al. (2017)
Metar. taii	ARSEF 5714	China	Lepidoptera	AF543787	JN049829	AF543763	AF543775	EU248908	EU248936	Bischoff et al. (2009), Kepler et al. (2012)
Metar. takense	BCC 30934	Thailand	Hemiptera	HQ165720	HQ165698	HQ165658	HQ165679	HQ165740	HQ165639	Luangsa-ard et al. (2017)
Moelleriella raciborskii	Afr 28	Ghana	—	DQ070113	—	—	EU392675	EU392727	—	Chaverri et al. (2008)
Nigelia aurantiaca	BCC 37621T	Thailand	Lepidoptera	GU979946	KY348783	GU979937	GU979955	GU979964	GU979970	Luangsa-ard et al. (2017)
N. martiale	HMAS 197472	China	Coleoptera, Cerambycidae	JF415975	JN049881	JF415956	JF416015	JN049892	JF415994	Nonaka et al. (2013)
Niveomyces hirsutellae	BCC 36631T	Thailand	Hypocreales, Ophiocordycipitaceae	ON103164	ON103039	—	ON125022	ON286882	ON125033	Kobmoo et al. (2023)
Ophiocordyceps acroasca	YFCC 9049	China	Hymenoptera, Formicidae	ON555918	—	ON555837	ON567757	ON568677	ON568130	Tang et al. (2023)
O. albostroma	BBH 39783T	Thailand	Coleoptera	OR805251	—	—	OR855785	OR855805	OR855829	Mongkolsamrit et al. (2024)
O. aphodii	ARSEF 5498	—	Coleoptera, Scarabaeidae	DQ518755	—	DQ522541	DQ522323	—	DQ522419	Spatafora et al. (2007)
O. araracuarensis	HUA 186135	Colombia	Hemiptera	KC610769	KP200891	KC610788	KC610738	KF658665	KC610716	Sanjuan et al. (2015)
O. bidoupensis	YHH 20036T	Vietnam	Coleoptera, Elateridae	—	—	OK571396	OK556893	OK556897	OK556899	Zou et al. (2022)
O. bifertilis	YFCC 9012T	China	Hymenoptera, Formicidae	ON555923	—	ON555843	ON567763	ON568143	ON568135	Tang et al. (2023)
O. bispora	KVL 606	Kenya	Blattodea, Termitidae	AF009654	—	AH006986	—	—	—	Suh et al. (1998)
O. brunnea	BCC 93057T	Thailand	Coleoptera	—	—	—	OR855787	OR855807	OR855831	Mongkolsamrit et al. (2024)
	BBH 49819	Thailand	Coleoptera	—	—	—	OR855788	OR855808	OR855832	Mongkolsamrit et al. (2024)
O. brunneipunctata	OSC 128576T	—	Coleoptera, Elateridae	DQ518756	—	DQ522542	DQ522324	DQ522369	DQ522420	Spatafora et al. (2007)
	BCC 2218	Thailand	Coleoptera	—	—	—	OR855789	OR855809	OR855833	Mongkolsamrit et al. (2024)
	HKAS 149958	China	Lepidoptera	—	PX411450	PX414356	—	PX423503	—	This study
O. camponoti-bispinosi	OBIS	Brazil	Hymenoptera, Formicidae	KX713612	—	KX713639	KX713694	KX713718	—	Araújo et al. (2018)
O. clavata	BCC 95653	Thailand	Coleoptera, Tenebrionidae	—	—	—	OR855796	OR855816	OR855838	Mongkolsamrit et al. (2024)
O. communis	BCC 1874	Thailand	Blattodea, Termitidae	MH753679	MH754725	—	MK284267	MK214109	MK214095	Tasanathai et al. (2019)
	BCC 1842	Thailand	Blattodea, Termitidae	MH753680	MH754726	—	MK284266	MK214110	MK214096	Tasanathai et al. (2019)
	BCC 2754	Thailand	Blattodea, Termitidae	MH753681	MH754727	—	MK284268	MK214111	MK214097	Tasanathai et al. (2019)
O. desmidiospora	SJS3Des	USA	Hymenoptera, Formicidae	MH536514	—	MH536515	MN785129	MN785131	—	Saltamachia & Araújo (2020)
O. entomorrhiza	KEW 53484	—	Lepidoptera	EF468809	JN049850	EF468954	EF468749	EF468857	EF468911	Sung et al. (2007), Kepler et al. (2012)
O. furcatosubulata	YFCC 904T	China	Coleoptera, Elateridae	MT774223	—	MT774216	MT774244	MT774230	MT774237	Wang et al. (2021a)
	YHH 17005T	China	Coleoptera, Elateridae	MT774224	—	MT774217	MT774245	MT774231	MT774238	Wang et al. (2021a)
O. gracillima	HUA 186132	Colombia	Coleoptera, Scarabeidae	KC610768	KF937353	—	KC610744	KF658666	—	Sanjuan et al. (2015)
O. houaynhangensis	TBRC 8428T	Thailand	Coleoptera	MH092902	MH092891	—	MH092894	—	—	Crous et al. (2018)
O. hydrangea	YFCC 8834T	China	Hemiptera	OM304639	—	OM304635	OM831276	OM831279	OM831282	Zou et al. (2022)
O. khonkaenensis	BCC 81462T	Thailand	Hemiptera	—	—	MK632126	MK632075	MK632168	MK632157	Crous et al. (2019)
	BCC 81464	Thailand	Hemiptera	MK632103	MK632043	MK632128	MK632077	MK632170	MK632159	Crous et al. (2019)
O. kohchangensis	BCC 90811	Thailand	Coleoptera, Elateridae	OR805254	—	—	OR855797	OR855818	OR855839	Mongkolsamrit et al. (2024)
O. krachonicola	BCC 79666T	Thailand	Orthoptera, Gryllotalpidae	MK632080	—	—	MK632054	MK632161	MK632132	Thanakitpipattana et al. (2020)
O. langbianensis	DL0017T	Vietnam	Coleoptera	MT928306	—	MT928355	—	—	—	Lao et al. (2021)
O. liangii	HKAS 125845T	China	Lepidoptara	OR527543	OR527536	OR527539	OR526347	—	—	Peng et al. (2024)
O. megacuculla	OSC 110993	—	Coleoptera, Scarabaeidae	DQ518762	—	DQ522548	DQ522331	DQ522376	—	Spatafora et al. (2007)
O. mosingtoensis	BCC 36921	Thailand	Blattodea, Termitidae	MH753685	MH754731	—	MK284272	MK214116	MK214099	Tasanathai et al. (2019)
O. nigrella	EFCC 9247	—	Lepidoptera	EF468818	JN049853	EF468963	EF468758	EF468866	EF468920	Sung et al. (2007), Kepler et al. (2012)
O. ovatospora	YFCC 22069184T	China	Blattodea, Termitidae	OP295114	OP295106	OP295111	OP313802	OP313804	—	Tang et al. (2022)
O. phitsanulokensis	BCC 85328T	Thailand	Coleoptera	OR805257	—	—	OR855798	OR855822	OR855843	Mongkolsamrit et al. (2024)
O. pseudovariabilis	BCC 88308T	Thailand	Coleoptera, Lycidae	—	—	—	OR855799	—	—	Mongkolsamrit et al. (2024)
	BCC 88311	Thailand	Coleoptera, Lycidae	—	—	—	OR855800	OR855824	OR855844	Mongkolsamrit et al. (2024)
O. ramosiphialidica	HKAS 149959	China	Diptera	PX411460	—	PX414346	—	PX423493	PX423510	This study
	HKAS 149960 T	China	Diptera	PX411461	—	PX414347	—	PX423494	PX423511	This study
O. ratchaburiensis	BCC 48033T	Thailand	Coleoptera	OR805259	—	—	OR855802	OR855826	OR855846	Mongkolsamrit et al. (2024)
	BCC 48035	Thailand	Coleoptera	OR805258	—	—	OR855803	OR855827	OR855847	Mongkolsamrit et al. (2024)
O. rubella	HKAS 149979 T	China	Hemiptera, Cicadidae	PX411456	PX411438	PX414342	PX409300	PX423489	—	This study
	GACP FS24061001	China	Hemiptera, Cicadidae	PX411457	PX411439	PX414343	PX409301	PX423490	—	This study
O. sinensis	EFCC 7287	—	Lepidoptera	EF468827	JN049854	EF468971	EF468767	EF468874	EF468924	Kepler et al. (2012)
O. sobolifera	NBRC 106967	Japan	Hemiptera	AB968422	AB968409	AB968395	AB968590	—	AB968551	Ban et al. (2015)
	TNS F18521	Japan	Hemiptera	KJ878898	—	KJ878933	KJ878979	KJ879013	—	Quandt et al. (2014)
O. subtiliphialida	YFCC 8815T	China	Hymenoptera, Formicidae	ON555914	—	ON555833	ON567753	ON568673	ON568126	Tang et al. (2023)
O. sungii	HKAS 149952 T	China	Coleoptera, Elateridae	PX411453	—	PX414338	—	PX423486	PX423505	This study
	HKAS 149953	China	Coleoptera, Elateridae	PX411452	—	PX414339	—	PX423485	PX423504	This study
O. brunneascospora	HKAS 149962	China	insect	PX411462	PX411441	—	PX409304	PX423495	—	This study
	HKAS 149964 T	China	insect	PX411463	PX411442	PX414353	PX409305	PX423496	—	This study
O. taiwanensis	TNM F0037796T	China	Blattodea, Termitidae	PP926235	PP926231	PP926233	—	—	—	Samarakoon and Chiu (2024)
O. termiticola	BCC 1770	Thailand	Blattodea, Termitidae	MH753677	GU723780	—	MK284264	MK214107	MK214093	Tasanathai et al. (2019)
O. variabilis	ARSEF 5365	—	Diptera	DQ518769	—	DQ522555	DQ522340	DQ522386	DQ522437	Spatafora et al. (2007)
	OSC 111003	—	Diptera	EF468839	—	EF468985	EF468779	EF468885	EF468933	Sung et al. (2007)
O. yakusimensis	HMAS 199604	—	Hemiptera	KJ878902	—	KJ878938	—	KJ879018	KJ878953	Quandt et al. (2014)
Papiliomyces longiclavatus	HKAS 115914	China	Lepidoptera	MZ702103	MZ702082	MZ702114	MZ955882	MZ955878	OM419143	Zhang et al. (2023a)
Papiliomyces shibinensis	GZUH SB13050311	China	Lepidoptera	—	—	KR153588	KR153589	KR153590	—	Wen et al. (2015)
Paradingleyomyces lepidopterorum	HKAS 131927	China	Hypocreales, Polycephalomycetaceae	OR828239	OR878364	—	OR880679	OR829675	—	Wang et al. (2024a)
	HKAS 131926T	China	Hypocreales, Polycephalomycetaceae	OR828238	OR878363	—	—	OR829674	OR880683	Wang et al. (2024a)
Paraisaria alba	HKAS 102484T	Thailand	Orthoptera	MN943839	MN947219	MN943843	MN929085	MN929078	MN929082	Wei et al. (2021)
Par. arcta	HKAS 102552	China	Lepidoptera	MN943840	MN947220	MN943844	MN929086	MN929079	MN929083	Wei et al. (2021)
Par. cascadensis	OSC-M-052017T	USA	Orthoptera, Prophalangopsidae	OQ708934	OQ709240	OQ800921	OR199817	OR199831	—	Tehan et al. (2023)
	OSC-M-052010	USA	Orthoptera, Prophalangopsidae	OQ708931	OQ709237	OQ800918	OR199814	OR199828	OR199838	Tehan et al. (2023)
Par. clavata	HKAS 149976 T	China	Coleoptera, Cerambycidae	PX411464	PX411443	PX414348	PX409306	PX423497	—	This study
	HKAS 149977	China	Coleoptera, Cerambycidae	PX411465	PX411444	PX414349	—	PX423498	—	This study
	GACP SB22051601	China	Coleoptera, Cerambycidae	PX411466	PX411445	PX414350	PX409307	PX423499	PX423512	This study
Par. cocoonihabita	HKAS 149975 T	China	Lepidoptera	PX411467	PX411446	PX414351	PX409308	PX423500	—	This study
	GACP SB22050202	China	Lepidoptera	PX411468	PX411447	PX414352	PX409309	PX423501	—	This study
Par. coenomyiae	NBRC 108993T	Japan	Diptera, Xylophagidae	AB968412	AB968396	AB968384	AB968570	—	AB968532	Ban et al. (2015)
Par. gracilioides	HUA 186095	Colombia	Coleoptera, Elateridae	—	—	KJ917556	KM411994	KP212914	—	Sanjuan et al. (2015)
Par. gracilis	OSC 151906	—	Lepidoptera	KJ878890	—	KJ878923	KJ878969	—	—	Quandt et al. (2014)
Par. heteropoda	BCC 18235	Japan	Hemiptera	JN941421	—	JN941720	AB968594	JN992454	AB968555	Ban et al. (2015)
	NBRC 100643	Japan	Hemiptera	JN941422	—	JN941719	AB968595	JN992453	AB968556	Ban et al. (2015)
Par. insignis	OSC-M-052013T	USA	Coleoptera	OQ708938	OQ709244	OQ800924	OR199820	OR199834	—	Tehan et al. (2023)
Par. pseudoarcta	GMBC 3064T	China	Lepidoptera	PQ785777	PQ787759	PQ785774	PQ789220	PQ789223	PQ789226	Chen et al. (2025)
	GMBC 3065	China	Lepidoptera	PQ785778	PQ787760	PQ785775	PQ789221	PQ789224	PQ789227	Chen et al. (2025)
Par. pseudoheteropoda	OSC-M-052022T	USA	Hemiptera	OQ708939	OQ709245	OQ800925	OR199821	OR199835	OR199841	Tehan et al. (2023)
Par. rosea	HKAS 102546T	China	Coleoptera	MN943842	MN947222	MN943846	MN929088	MN929081	MN929084	Wei et al. (2021)
Paraisaria sp.	OSC-M-052011	USA	Insecta	OQ708932	OQ709238	OQ800919	OR199815	OR199829	OR199839	Tehan et al. (2023)
Parametarhizium changbaiense	CGMCC 19143T	China	forestlitters	MN589994	MN589741	MN590231	MN908589	MN917168	MT921829	Gao et al. (2021)
Parametarhizium hingganense	CGMCC 19144T	China	forestlitters	MN061635	MN055703	MN055706	MN065770	MN917170	MT939494	Gao et al. (2021)
Perennicordyceps paracuboidea	NBRC 100942	Japan	Coleoptera	JN941430	JN943337	JN941711	AB972954	JN992445	AB972958	Schoch et al. (2012), Ban et al. (2015)
Perennicordyceps prolifica	NBRC 101750	Japan	Hemiptera	JN941433	JN943340	JN941708	AB972953	JN992442	AB972957	Ban et al. (2015)
Pleurocordyceps fusiformispora	YFCC 07239279T	China	Hypocreales, Ophiocordycipitaceae	PP410610	PP002030	—	PP254877	PP581807	PP581824	Liu et al. (2024)
Ple. heilongtanensis	KUMCC 3008T	China	Hypocreales, Ophiocordycipitaceae	OQ172063	OQ172091	OQ172111	OQ459731	OQ459759	OQ459805	Xiao et al. (2023)
	HKAS 148574	China	Hypocreales, Ophiocordycipitaceae	—	PV737571	PV739068	PV740522	—	PV740523	Unpublished
	HKAS 149961	China	Hymenoptera, Formicidae	PX411469	PX411448	PX414354	PX409310	PX423502	PX423513	This study
Ple. lanceolata	GACP 17-2004T	China	Lepidoptera	OQ172046	OQ172076	OQ172110	OQ459726	OQ459754	OQ459800	Xiao et al. (2023)
Ple. neoagarica	GZCC 22-2043T	China	Hypocreales, Ophiocordycipitaceae	OQ968794	OQ968791	OQ968804	OQ982007	OQ981996	OQ981999	Xiao et al. (2024)
Ple. nutansis	MFLU 21-0275T	China	Hypocreales, Ophiocordycipitaceae	OQ172048	OQ172073	OQ172119	OQ459739	OQ459765	OQ459811	Xiao et al. (2023)
Ple. parvicapitata	MFLU 21-0271	China	Hypocreales, Ophiocordycipitaceae	OQ172055	OQ172083	OQ172106	OQ459723	OQ459752	OQ459797	Xiao et al. (2023)
Ple. sanduensis	GZCC 22-2044T	China	Hypocreales, Ophiocordycipitaceae	OQ968799	OQ968787	OQ968806	OQ982006	OQ981995	OQ982001	Xiao et al. (2024)
Ple. yunnanensis	YHH PY1006T	China	Hypocreales, Ophiocordycipitaceae	KF977849	KF977849	KF977849	KF977851	KF977853	KF977855	Wang et al. (2015)
Pleurodesmospora acaricola	R. Kirschner 4968T	China	mite	MZ435415	MZ435417	—	LC629776	—	—	Yeh et al. (2021)
Pleurodesmospora entomophila	BRIP 72652a	Australia	insect	OR527526	OR527518	—	OR514842	—	OR514850	Tan and Shivas (2023)
Pochonia sinensis	ZY 22.009T	China	soil	OQ709263	OQ709257	OQ709251	OQ719629	—	OQ719624	Zhang et al. (2023b)
Polycephalomyces albiramus	GACP 21-XS08T	China	Lepidoptera	OQ172037	OQ172092	OQ172115	OQ459735	OQ459761	OQ459807	Xiao et al. (2023)
Pol. formosus	CGMCC 5.2208	—	Coleoptera	MN586844	MN586835	MN586826	MN598059	MN598050	MN598066	Wang et al. (2021b)
Pol. tengchongensis	HKAS 131923T	China	Hypocreales, Polycephalomycetaceae	OR828240	OR878365	PP129612	—	OR829676	OR880685	Wang et al. (2024a)
Polystromomyces araneae	BCC 93301T	Thailand	Arachnida	MZ684016	MZ684101	—	MZ707825	MZ707832	MZ707845	Mongkolsamrit et al. (2022)
Pseudomeria mucosa	CBS 487.83	Canada	—	—	MH861630	—	—	—	—	Vu et al. (2019)
Pseudoniveomyces arachnovorum	BCC 95818T	Thailand	Arachnida	—	OR098526	—	OR133172	OR133173	OR133174	Kobmoo et al. (2023)
Pseudoniveomyces blattae	BCC 53567T	Thailand	Blattodea	ON103167	ON103042	—	—	ON286885	ON125036	Kobmoo et al. (2023)
Purpureocillium lilacinum	CBS 431.87	Philippines	Tylenchida, Heteroderidae	EF468844	AY624188	—	EF468791	EF468897	EF468940	Sung et al. (2007)
	CBS 284.36	USA	soil	FR775484	AY624189	AY526475	EF468792	EF468898	EF468941	Sung et al. (2007)
Purpureomyces fenggangensis	HKAS 149956 T	China	Lepidoptera	PX411455	PX411437	PX414341	PX409299	PX423488	PX423507	This study
	GACP FG21042639	China	Lepidoptera	PX411454	PX411436	PX414340	PX409298	PX423487	PX423506	This study
Pur. khaoyaiensis	BCC 1376	Thailand	Lepidoptera	KX983462	—	KX983468	KX983457	—	KX983465	Luangsa-ard et al. (2017)
	BCC 14290	Thailand	Lepidoptera	JF415970	JN049869	—	JF416012	JN049888	—	Kepler et al. (2012)
Pur. maesotensis	BCC 88441	Thailand	Lepidoptera	MN781877	MN781916	—	MN781734	MN781779	MN781824	Mongkolsamrit et al. (2020a)
	BCC 89300T	Thailand	Lepidoptera	MN781876	MN781917	—	MN781733	MN781778	—	Mongkolsamrit et al. (2020a)
Pur. pyriformis	BCC 85348	Thailand	Lepidoptera	MN781871	MN781927	—	MN781728	MN781773	MN781820	Mongkolsamrit et al. (2020a)
	BCC 85074T	Thailand	Lepidoptera	MN781873	MN781929	—	MN781730	MN781775	MN781821	Mongkolsamrit et al. (2020a)
Samsoniella farinospora	YFCC 8774T	Vietnam	Araneae	ON621672	—	ON563168	ON676516	ON676504	ON568687	Wang et al. (2022)
S. sinensis	YFCC 8766T	China	Lepidoptera	ON621679	—	ON563175	ON676523	ON676511	ON568694	Wang et al. (2022)
Shimizuomyces paradoxus	EFCC 6564	Korea	Liliales, Smilacaceae	EF469083	—	EF469130	EF469072	EF469101	EF469118	Sung et al. (2007)
Sungia yongmunensis	EFCC 2131	Korea	Lepidoptera	EF468833	JN049856	EF468977	EF468770	EF468876	KJ398690	Sung et al. (2007), Kepler et al. (2014)
Tolypocladium inflatum	OSC 71235	—	Coleoptera	EF469077	JN049844	EF469124	EF469061	EF469090	EF469108	Sung et al. (2007)
T. paradoxum	NBRC 100945	Japan	Hemiptera	JN941410	JN943323	JN941731	AB968599	JN992465	AB968560	Schoch et al. (2012), Ban et al. (2015)
Yosiokobayasia kusanagiensis	TNS F18494	Japan	Coleoptera	JF415972	JN049873	JF415954	JF416014	JN049890	—	Kepler et al. (2012)
Zarea flavidum	CBS 300.70D	Austria	Hymenochaetales, Hymenochaetaceae	KM283789	MH859668	KM283765	KM283813	—	KM283855	Park et al. (2015)
Zouia cauligalbarum	GZUIFRZHJ01T	China	Lepidoptera	MH730667	MH730663	MH730665	MH801920	MH801922	MH801924	Zhou et al. (2018)

¹Amp.: Amphichorda; Metar.: Metarhizium; Par.: Paraisaria; Ple.: Pleurocordyceps; Pol.: Polycephalomyces; Pur.: Purpureomyces.

²Superscript T (^T) after the voucher number represents type specimens, type strain, or neotype.

³ARSEF, Afr 28: Agricultural Research Service Entomopathogenic Fungus Collection, USDA, Ithaca, USA; BBH: BIOTEC Bangkok Herbarium, Thailand; BCC: BIOTEC Culture Collection, Thailand; BRIP: Queensland Plant Pathology Herbarium, Brisbane, Australia; CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands; CGMCC: China General Microbiological Culture Collection Center, China; DAOMC: Canadian Collection of Fungal Cultures, Ottawa, Canada; EFCC: Entomopathogenic Fungal Culture Collection, Chuncheon, Korea; GZUIFR, GZUH, GACP, and ZY 22.009: Herbarium of Guizhou University, Guizhou, China; GZCC: Guizhou Culture Collection, China; HKAS: Kunming Institute of Botany, Academia Sinica, China; HUA: Herbarium Antioquia University, Medellin, COL; KEW: mycology collection of Royal Botanical Garden, Surrey, UK; KUMCC: the Kunming Culture Collection, China; KVL: Entomopathogenic Fungus Culture Collection, Section for Organismal Biology, Department of Plant and Environmental Sciences, University of Copenhagen, Denmark; MCA: Marie Catherine Aime personal collection; MFLU: Mae Fah Luang University herbarium, Thailand; MFLUCC: Mae Fah Luang University Culture Collection, Thailand; MTCC: Microbial Type Culture Collection & Gene Bank, India; NBRC: Biological Resource Center, the National Institute of Technology and Evaluation, Japan; NHJ: Nigel Hywel-Jones personal collection, Thailand; OSC: Oregon State University Herbarium, Oregon, USA; PDD: the New Zealand Fungarium, New Zealand; PRM: National Museum, Prague, Czech Republic; RCEF: Research Center for Entomogenous Fungi, Anhui Agricultural University, Anhui, China; TBRC: Thailand Bioresources Research Center, Thailand; TNM: National Museum of Natural Science, Taichung, Taiwan, China; TNS: National Museum of Nature and Science, Tsukuba, Ibaraki, Japan; YFCC: Yunnan Fungal Culture Collection of Yunnan University, China; YHH: Yunnan Herbal Laboratory, Institute of Herbal Biotic Resources, Yunnan University, Yunnan, China; DL0017 was deposited in Dalat University, Dalat, Lam Dong, Vietnam; OBIS was deposited in D.P. Hughes personal collection, Penn State University, USA; R. Kirschner 4968 was deposited in the National Museum of Natural Science, Taichung, Taiwan, China; SJS3Des was deposited University of Louisiana at Lafayette, Lafayette, Louisiana, USA.

⁴“—” indicates unavailability of information.

⁵LSU: the nuclear ribosomal DNA large subunit; ITS: the internal transcribed spacer regions of the nuclear ribosomal DNA; SSU: the nuclear ribosomal DNA small subunits; tef1-α: the partial translation elongation factor-1 alpha gene; rpb1: the partial gene for RNA polymerase II largest subunit; rpb2: the partial gene for RNA polymerase II second largest subunit.

Fig. 1.

Phylogram generated from maximum likelihood analysis based on combined LSU-ITS-SSU-tef1-α-rpb1-rpb2 sequence data of 203 taxa. Maximum-likelihood bootstrap (MLB) values ≥ 80 % and Bayesian posterior probabilities (PP) ≥ 0.90 are presented near to the nodes. Genera and families are indicated at the right side of the tree. Newly generated sequences are shown in bold blue. ^T indicates a holotype or ex-type strains.

RESULTS

Molecular phylogeny

A total of 101 new sequences were generated (20 LSU, 16 ITS, 20 SSU, 16 tef1-α, 21 rpb1, and 10 rpb2) in this study (Table 1). The combined dataset (Supplementary Data S1) comprised 203 taxa, with multi-loci sequences totalling an alignment length of 4920 characters (bp), including gaps (LSU: 836 bp, ITS: 566 bp, SSU: 1016 bp, tef1-α: 904 bp, rpb1: 680 bp and rpb2: 928 bp). The matrix had 3096 distinct patterns, 1955 parsimony-informative, 381 singleton sites, and 2594 constant sites. The respective partition best-fit models were TIM3+F+I+G4 for LSU, tef1-α and rpb2, TIM2+F+I+G4 for ITS and rpb1, K2P+I+G4 for SSU. The likelihood of the best-scoring IQ tree was −107724.723. The phylogenetic tree from maximum likelihood phylogenetic analysis with maximum likelihood bootstrap (MLB) values is shown in Fig. 1. The nodes were also evaluated with Bayesian posterior probabilities (PP).

Similar topologies were recovered from both BI and ML phylogenetic analyses, with families resolved as monophyletic and supported by moderate to strong statistical values (Fig. 1). Within Ophiocordycipitaceae, the new collections O. rubella and O. sungii nested within the O. sobolifera clade as a distinct group. Specimen HKAS 149958 together with O. brunneipunctata were placed in a distinct clade (100 % MLB/1 PP). Ophiocordyceps ramosiphialidica and O. brunneascospora were respectively nested within the O. ravenelii clade and the O. blattae clade, both strongly supported (100 % MLB/1 PP). Paraisaria clavata and Par. cocoonihabita respectively formed sister relationships with Par. heteropoda and Par. cascadensis (100 % MLB/1 PP). Specimen HKAS 149961 grouped with Ple. heilongtanensis with a fully supported clade (100 % MLB/1 PP) in Polycephalomycetaceae. In Clavicipitaceae, Pur. fenggangensis formed a distinct clade with the highest support (100 % MLB/1 PP) in Purpureomyces. The collection Metar. acrididarum branches off from a clade of Metar. reniforme and Metar. phuwiangense with maximum support (100 % MLB/1 PP). In Cordycipitaceae, C. multisynnematosa was resolved as sister to C. cf. takaomontana NHJ 12623 with strong support (100 % MLB/1 PP). In addition, specimen HKAS 149963 clustered with J. thomisidarum in a distinct clade (100 % MLB/1 PP).

TAXONOMY

Clavicipitaceae Rogerson, Mycologia 62(5): 900. 1970.

Metarhizium Sorokīn, Veg. Parasitenk. Mensch Tieren 2: 268. 1879, emend. Kepler et al. (2014).

Notes: Metarhizium is known as the “green muscardine fungus” due to the production of green conidia covering its hosts (Wei et al. 2024). The genus was established by Sorokīn (1883) based on the type species Metar. anisopliae (syn. Entomophthora anisopliae), which parasitises Anisoplia austriaca in Ukraine (Mechnikov 1879). Metarhizium has been linked to the sexual morph genus Metacordyceps through culturing and molecular methods (Sung et al. 2007). However, based on the “one fungus one name” standard, morphological observation and phylogenetic tree analysis of combined tub, rpb1, rpb2 and tef1-α sequences, Kepler et al. (2014) revised the taxonomic history of Metarhizium and provided a list of species within the genus. Mongkolsamrit et al. (2020a) established six new genera including Keithomyces, Marquandomyces, Papiliomyces, Purpureomyces, Sungia, and Yosiokobayasia to accommodate phylogenetically distinct lineages of Metarhizium sensu lato. Mongkolsamrit et al. (2020a) categorised the phialide morphology of Metarhizium sensu stricto into three types: metarhizium-like, nomuraea-like and paecilomyces-like. Here a new species exhibiting paecilomyces-like phialides characterized by a globose base with a distinct neck was introduced.

Metarhizium acrididarum X.C. Peng & T.C. Wen, sp. nov. MB 860846. Fig. 2.

Fig. 2.

Metarhizium acrididarum (HKAS 149984). A, B. Fungus on stick insect hosts. C–G. Conidiophores, phialides and conidia. H. Conidia. Scale bars: B = 5 mm; C–H = 5 µm.

Etymology: Named after the host family, Acrididae.

Typus: China, Guizhou Province, Qiannan Buyei and Miao Autonomous Prefecture, Sandu Shui Autonomous County, Yaorenshan national forest park (25°55′19″N, 107°56′54″E), on dead adult of Acrididae, Orthoptera, on the leaf litter, 9 Jul. 2023, J. Bu, YRS23070910 (holotype HKAS 149984; GenBank LSU: PX411471, ITS: PX411451, SSU: PX414357, tef1-α: PX789407, rpb1: PX789406).

Parasitic on adult locust (Acrididae, Orthoptera), ca 11 × 4 mm. The host body is covered with green and yellow mycelium and conidia, which are more densely distributed on the head, thorax, and abdominal segments. Sexual morph: Undetermined. Asexual morph: Paecilomyces-like. Conidiophores micronematous, smooth-walled, irregularly branched. Phialides globose to ellipsoidal at basal portion, tapering into a distinct neck, smooth, borne single or in clusters on conidiophores, 4.0–10.0 × 2.0–3.9 µm ($\stackrel{\_}{\text{x}}=6.1\times 3.0\mu \text{m}$, n = 100). Conidia long ovoid to cylindrical, smooth-walled, aseptate, light green, 5.2–7.3 × 2.4–3.7 μm ($\stackrel{\_}{\text{x}}=6.4\times 3.0\mu \text{m}$, n = 80), L/W ratio 1.7–2.7 ($\stackrel{\_}{\text{x}}=2.1$, n = 80).

Notes: Multi-gene phylogenetic analyses resolve Metar. acrididarum within a clade comprising Metar. reniforme and Metar. phuwiangense. While these species share globose to broadly ellipsoidal phialides with pronounced necks, they exhibit divergence in phialide arrangement and conidial morphology. Metarhizium acrididarum produces phialides that arise singly or in groups from conidiophores without metulae, forming elongate-ovoid to cylindrical conidia. In contrast, Metar. phuwiangense develops phialides singly along hyphae in sympodial succession, bearing ellipsoidal-cylindrical conidia (Mongkolsamrit et al. 2020a). Metarhizium reniforme, on the other hand, produces phialides either directly on hyphae in septal whorls or on metulae (2–3 per node), and reniform conidia (Samson 1974). Host specificity further distinguishes these species: Metar. acrididarum parasitises Orthoptera (Acrididae), Metar. phuwiangense infects Coleoptera, and Metar. reniforme targets Orthoptera (Tettigoniidae) (Samson 1974, Mongkolsamrit et al. 2020a).

Purpureomyces Luangsa-ard et al., Stud. Mycol. 95: 241. 2020.

Notes: Purpureomyces, a sister genus to Marquandomyces, was segregated from Metarhizium by Mongkolsamrit et al. (2020a) to accommodate three species (Pur. khaoyaiensis, Pur. maesotensis, and Pur. pyriformis) based on morphological characteristics and phylogenetic analyses of SSU, LSU, tef1-α, rpb1, rpb2, and ITS sequences. Purpureomyces is characterized by its solitary purple stroma, obliquely immersed perithecia, cylindrical asci, septate ascospores that do not disarticulate into part-spores, and a lecanicillium-like asexual morph (Mongkolsamrit et al. 2020a).

Purpureomyces fenggangensis X.C. Peng & T.C. Wen, sp. nov. MB 860855. Fig. 3.

Fig. 3.

Purpureomyces fenggangensis (A–F. HKAS 149956, G–N. Culture GACP FG21042640). A, B. Synnemata emerging from infected insect. C–E, J–M. Conidiophores, phialides and conidia. F, N. Conidia. G–I. Culture on PDA, black arrows show pale purplish red droplets in panel I. Scale bars: B = 10 mm; C = 20 µm; D–F, J–N = 10 µm; G–I = 20 mm.

Etymology: Named after the locality where the type was collected, Fenggang County, Guizhou Province, China.

Typus: China, Guizhou Province, Fenggang County, Tianqiao Town (27°37′31″N, 107°51′07″E), on dead larvae of Lepidoptera immersed into soil, 26 Apr. 2021, X.C. Peng, FG21042640 (holotype HKAS 149956; culture ex-type GACP FG21042640 = CGMCC 3.29472; GenBank LSU: PX411455, ITS: PX411437, SSU: PX414341, tef1-α: PX409299, rpb1: PX423488, rpb2: PX423507).

Parasitic on caterpillar (Lepidoptera larvae), ca 10 × 2 mm. Several off-white synnemata arising from the insect host. Sexual morph: Undetermined. Asexual morph: Isaria-like. Two to five synnemata arising from the insect host, cylindrical, unbranched, white to light-purple, 5–10 mm long and 2 mm wide. Mycelium septate, branched, smooth, hyaline, 1.5–3.0 μm ($\stackrel{\_}{\text{x}}=2.3\mu \text{m}$, n = 20) in diam. Conidiophores dense, branches bearing 1–4 phialides per branch. Metulae cylindrical, smooth, hyaline, 5.0–10.0 × 1.5–3.7 µm ($\stackrel{\_}{\text{x}}=7.2\times 2.8\mu \text{m}$, n = 30). Phialides ampulliform, sparsely scattered, usually solitary or in pairs on metulae, occasionally in groups of three to four, smooth, hyaline, 5.2–13.7 × 2.0–3.5 µm ($\stackrel{\_}{\text{x}}=9.7\times 2.6\mu \text{m}$, n = 30). Conidia globose, subglobose to lemon-shaped, smooth, hyaline, 2.1–3.6 × 2.0–3.1 μm ($\stackrel{\_}{\text{x}}=3.0\times 2.5\mu \text{m}$, n = 60), L/W ratio 1.0–1.4 ($\stackrel{\_}{\text{x}}=1.2$, n = 60).

Culture characteristics: Colonies on PDA, attaining 31–42 mm diam. within 16 d at 25 °C, leathery, off-white, circular, flat, curled margin, pale purplish red droplets, reverse dark grey gradually lightened, radial striation. At later stages, off-white synnemata emerge from the centre of the colony. Mycelium septate, branched, smooth, hyaline, 1.0–4.3 μm ($\stackrel{\_}{\text{x}}=2.3\mu \text{m}$, n = 70) in diam. Conidiophores dense, branches bearing 1–3 phialides per branch. Metulae cylindrical, smooth, hyaline, 2.2–11.2 × 1.9–6.1 µm ($\stackrel{\_}{\text{x}}=6.4\times 3.0\mu \text{m}$, n = 20). Phialides ampulliform, sparsely scattered, usually solitary or in pairs on metulae, occasionally in groups of three, smooth, hyaline, 6.3–19.1 × 1.2–5.0 µm ($\stackrel{\_}{\text{x}}=11.9\times 2.7\mu \text{m}$, n = 80). Conidia globose, subglobose to lemon-shaped, smooth, hyaline, 2.1–3.6 × 2.0–3.6 μm ($\stackrel{\_}{\text{x}}=3.0\times 2.7\mu \text{m}$, n = 80), L/W ratio 1.0–1.4 ($\stackrel{\_}{\text{x}}=1.1$, n = 80).

Additional material examined: China, Guizhou Province, Fenggang County, Tianqiao Town (27°37′31″N, 107°51′07″E) on dead larvae of Lepidoptera, in the soil, 26 Apr. 2021, X.C. Peng, FG21042639 (paratype HKAS 149955; culture ex-paratype GACP FG21042639 = CGMCC 3.29471).

Notes: Phylogenetically, this new species forms an independent lineage sister to a clade comprising Pur. pyriformis, Pur. maesotensis, and Pur. khaoyaiensis, with strong statistical support (100 % MLB/1 PP; Fig. 1). The sexual morphs of Pur. pyriformis, Pur. maesotensis, and Pur. khaoyaiensis were observed in nature (Mongkolsamrit et al. 2020a), while their asexual morphs were described from culture. These three species share similarities in producing lecanicillium-like conidiophores and ovoid conidia arranged in chains. In contrast, Pur. fenggangensis (asexual morph observed in nature) produces ampulliform phialides with swollen bases and a neck, along with subglobose to lemon-shaped conidia that distinguish it from other members of the genus.

Cordycipitaceae Kreisel ex G.H. Sung et al., Stud. Mycol. 57: 48. 2007.

Cordyceps Fr., Observ. mycol., Cancellans Edn (Havniae) 2: 316. 1818.

Notes: Cordyceps is a diverse genus in terms of species number and host range (Sung et al. 2007). The genus was first introduced by Fries (1818) to accommodate species that were previously classified under Clavaria in the Hypocreales and Sphaeria in the Sphaeriales (Kobayasi 1941). However, multi-gene phylogenetic analyses revealed that Cordyceps is not monophyletic within the Clavicipitaceae (Sung et al. 2007); thus, the authors proposed the Cordycipitaceae to include most species of Cordyceps with brightly coloured, fleshy stromata. More recently, Dong et al. (2022) recognised five major clades within Cordyceps based on phylogenetic and morphological analyses, and our species belongs to clade IV.

Cordyceps multisynnematosa X.C. Peng & T.C. Wen, sp. nov. MB 860845. Fig. 4.

Fig. 4.

Cordyceps multisynnematosa (B–G. HKAS 149957, H–O. Culture GACP JK22052023). A. Habitat. B, C. Synnemata emerging from infected insect, black arrows indicate long synnemata, and yellow arrows indicate short synnemata. D. Close-up of short synnema. E, F, J–L. Conidiophores and phialides. G, M–O. Conidia H, I. Reverse and front view of the culture. Scale bars: B, C = 5 mm; D = 500 µm; E, F, J–L = 10 µm; G, M–O = 5 µm; H, I = 10 mm.

Etymology: The name reflects the production of multiple synnemata.

Typus: China, Guizhou Province, Jiangkou County, Taiping Town (27°46′03″N, 108°44′33″E), on dead larvae of Lepidoptera sp., on the leaf litter, 20 May 2022, X.C. Peng, JK22052023 (holotype HKAS 149957; culture ex-type GACP JK22052023; GenBank LSU: PX411458, SSU: PX414344, tef1-α: PX409302, rpb1: PX423491, rpb2: PX423508).

Parasitic on caterpillar (Lepidoptera larvae), ca 14 × 5 mm. Most white synnemata arise from the insect body. Sexual morph: Undetermined. Asexual morph: Isaria-like. Two types of synnemata were produced on insect hosts. Long synnemata fasciculata, linear or lanceolate, tapering gradually toward the apex, unbranched, white to cream, 5–15 mm long, ca 1 mm wide, no conidial state was observed. Short synnemata cylindrical with subglobose or oblong end, unbranched, white to cream, 1–3 mm long. Mycelium septate, branched, smooth, hyaline, 1.0–3.5 μm ($\stackrel{\_}{\text{x}}=2.2\mu \text{m}$, n = 40) in diam. Conidiophores dense, branched, bearing 1–3 phialides per branch. Metulae cylindrical, smooth, hyaline, 5.0–12.5 × 2.0–3.5 µm ($\stackrel{\_}{\text{x}}=8.0\times 2.9\mu \text{m}$, n = 30). Phialides ampulliform, smooth, hyaline, 6.0–14.5 × 1.5–4.0 µm ($\stackrel{\_}{\text{x}}=10.3\times 2.2\mu \text{m}$, n = 50), 1–3 phialides on one metulae. Conidia subglobose to ellipsoidal, smooth, hyaline, 3.0–7.5 × 1.3–3.0 μm ($\stackrel{\_}{\text{x}}=4.5\times 1.8\mu \text{m}$, n = 80), L/W ratio 2.3–2.6 ($\stackrel{\_}{\text{x}}=2.5$, n = 80).

Culture characteristics: Colonies on PDA, attaining 16–20 mm diam. within 14 d at 25 °C, 26–32 mm within 22 d at 25 °C, dense, irregular, cottony, white, wrinkled, reverse light brown to grey. Mycelium septate, branched, smooth, hyaline, 1.5–4.5 μm ($\stackrel{\_}{\text{x}}=2.5\mu \text{m}$, n = 60) in diam. Conidiophores dense, branched, bearing 1–3 phialides per branch. Metulae cylindrical, smooth, hyaline, 7.0–13.5 × 2.0–4.5 µm ($\stackrel{\_}{\text{x}}=8.8\times 3.2\mu \text{m}$, n = 25). Phialides ampulliform, smooth, hyaline, 10.0–21.5 × 1.5–4.0 µm ($\stackrel{\_}{\text{x}}=14.0\times 3.0\mu \text{m}$, n = 40), 1–3 phialides per metulae. Conidia ellipsoidal to cylindrical, smooth, hyaline, 2.5–7.5 × 1.5–3 μm ($\stackrel{\_}{\text{x}}=5.0\times 2.0\mu \text{m}$, n = 35), L/W ratio 2.2–2.8 ($\stackrel{\_}{\text{x}}=2.4$, n = 35).

Notes: Phylogenetically, the specimen clustered with C. cf. takaomontana (NHJ 12623). Nucleotide sequence comparisons between NHJ 12623 and HKAS 149957 revealed no differences in the SSU and LSU regions, three base pair differences in tef1-α and rpb2, respectively, and one base pair difference in rpb1. The molecular analysis indicated that the specimen HKAS 149957 is conspecific with C. cf. takaomontana (NHJ 12623) that is not linked to morphological illustration. Cordyceps takaomontana was introduced by Kobayasi (1941) from a lepidopteran chrysalis in Japan based on its sexual morph. The asexual morph of Isaria japonica produces a single type of synnemata that is longer than that of C. multisynnematosa, with a distinct fertile part and mealy conidia measuring 4–5 × 1.5 µm (Yasuda 1921). On the other hand, the phylogenetic analysis (Fig. 1) showed that C. takaomontana (BCC 12688) and C. multisynnematosa are distantly related. Therefore, we describe this taxon as a new species of Cordyceps, and C. cf. takaomontana NHJ 12623 is here recognized as belonging to this newly described species.

Jenniferia thomisidarum Mongkols. et al., MycoKeys 91: 135. 2022. Fig. 5.

Fig. 5.

Jenniferia thomisidarum (HKAS 149963). A. Synnemata arising from spider. B–D. Phialides with conidia. E. Conidia. Scale bars: A = 5 mm; B–E = 10 µm.

Parasitic on a spider, ca 8 × 5 mm. Sexual morph: Not observed. Asexual morph: Synnemata arising from the host, numerous, cylindrical to clavate, dense, greyish, ca 1 mm long. Conidiophores numerous, branched, numerous, arising directly from the mycelium. Metula cylindrical, hyaline, with 1–2 phialides, 5.0–10.0 × 2.5–5.0 µm ($\stackrel{\_}{\text{x}}=7.2\times 3.5\mu \text{m}$, n = 50). Phialides cylindrical, tapering into a distinct neck, smooth, forming from metula, hyaline, 8–17.5 × 2.5–5.5 µm ($\stackrel{\_}{\text{x}}=11.5\times 3.8\mu \text{m}$, n = 90). Conidia cylindrical with round ends, smooth-walled, aseptate, 7.5–11.5 × 1.5–4.0 μm ($\stackrel{\_}{\text{x}}=9.6\times 2.5\mu \text{m}$, n = 90), L/W ratio 2.1–5.5 ($\stackrel{\_}{\text{x}}=3.9$, n = 90).

Material examined: China, Yunnan Province, Pu’er City, Simao District, Yixiang Town (22°36′56″N, 101°05′49″E), on dead a spider (Araneae), on the back of a living leaf, 6 Aug. 2024, X.C. Peng, SM240806118 (HKAS 149963).

Notes: The multi-gene phylogenetic analysis showed that our specimen HKAS 149963 clusters with J. thomisidarum, which was first reported from Thailand. Our specimen was collected from China and shares key morphological and ecological characteristics with J. thomisidarum in its parasitism of spiders attached to living leaves, cylindrical phialides with a distinct neck, and cylindrical conidia. Notably, the dimensions of the phialides (8–17.5 × 2.5–5.5 μm vs 7.0–16.0 × 2.0–5.0 μm) and conidia (7.5–11.5 × 1.5–4.0 μm vs 3.0–12.0 × 1.0–3.0 μm) overlap with those reported for J. thomisidarum (Mongkolsamrit et al. 2022), supporting their conspecificity. Comparative nucleotide sequence analyses between HKAS 149963 and J. thomisidarum BCC37881 revealed one bp difference in LSU, seven bp in ITS (including one bp gap), and two bp in tef1-α. Based on both molecular and morphological evidence, we report HKAS 149963 as a new geographic record from China.

Ophiocordycipitaceae G.H. Sung et al., Stud. Mycol. 57: 35. 2007

Ophiocordyceps Petch, Trans. Brit. Mycol. Soc. 16: 73. 1931, emend. Sung et al. (2007).

Notes: Ophiocordyceps was first introduced by Petch (1931) to accommodate four species (O. blattae, O. unilateralis, O. rhizoidea and O. peltata) of Cordyceps that produce non-disarticulating ascospores. However, subsequent mycologists did not accept this genus and instead reclassified these species as Cordyceps subg. Ophiocordyceps, or placed them in various subgenera within Cordyceps (Kobayasi 1941, Mains 1958). Later, Sung et al. (2007) proposed Ophiocordycipitaceae with Ophiocordyceps as the type genus, which is characterized by dark, fibrous stromata; perithecia superficial to immersed, arranged vertically or obliquely; asci typically cylindrical with a thickened apex; and ascospores usually cylindrical, multiseptate, and either disarticulating into part-spores or remaining intact.

Ophiocordyceps sungii X.C. Peng & T.C. Wen, sp. nov. MB 860852. Fig. 6.

Fig. 6.

Ophiocordyceps sungii (HKAS 149952). A, B. Stroma arising from Elateridae larvae. C. Perithecia. D. Peridium. E, F. Immature asci. G. Mature ascus. H–J. Ascospores. K. Apical cap of ascus. L. Secondary ascospores. Scale bars: B = 20 mm; C = 100 µm; D–J = 25 µm; K = 10 µm; L = 5 µm.

Etymology: Named in honour of Prof. Jae Mo Sung, who has made a significant contribution to the studies of entomopathogenic fungi.

Typus: China, Yunnan Province, Xishuangbanna Dai Autonomous Prefecture, Menghai County, Mengzhe Town (21°56′60″N, 100°23′03″E), on Elateridae sp. larvae (Coleoptera), buried in soil, 10 Aug. 2024, X.C. Peng, BN240810130-1 (holotype HKAS 149952; GenBank LSU: PX411453, SSU: PX414338, rpb1: PX423486, rpb2: PX423505).

Parasitic on Elateridae larvae (Coleoptera), buried in soil of broadleaf forests, ca 20 mm in length, 3 mm wide. Sexual morph: Stroma arising from the head of the insect, solitary, cylindrical, unbranched main axis with a branched sterile tip, light brown to reddish brown, fibrous, 77 × 1–2 mm. Stipe cylindrical, brown, 60 × 1 mm. Fertile part distinctly subterminal, erect, cylindrical, reddish brown, 15 × 2 mm. Sterile tip branched, grey, 2 mm long. Perithecia immersed, flask-shaped, light reddish brown, ostiolate, 315–420 × 100–220 μm ($\stackrel{\_}{\text{x}}=357\times 157\mu \text{m}$, n = 40). Peridium 15–45 µm ($\stackrel{\_}{\text{x}}=27.8\mu \text{m}$, n = 30) wide. Asci filiform, cylindrical, hyaline, 8-spored, 115–200 × 3.5–6.5 μm ($\stackrel{\_}{\text{x}}=155\times 5.3\mu \text{m}$, n = 50), with thickened ascus apices. Apical cap hemispherical, with a small channel in the centre, 2.0–3.0 × 3.5–5 μm ($\stackrel{\_}{\text{x}}=2.5\times 4.6\mu \text{m}$, n = 30). Ascospores filiform, smooth, hyaline, 95–165 × 1.3–2 µm ($\stackrel{\_}{\text{x}}=145\times 1.6\mu \text{m}$, n = 20), breaking into 32 small truncate part-spores. Secondary ascospores cylindrical, smooth, hyaline, 5.3–8.2 × 1.5–2.4 μm ($\stackrel{\_}{\text{x}}=6.5\times 1.9\mu \text{m}$, n = 50), L/W ratio 2.5–5.1 ($\stackrel{\_}{\text{x}}=3.4$, n = 50). Asexual morph: Undetermined.

Additional material examined: China, Yunnan Province, Xishuangbanna Dai Autonomous Prefecture, Menghai County, Mengzhe Town (21°56′60″N, 100°23′03″E), on dead larvae of Elateridae sp. (Coleoptera), immersed in soil, 10 Aug. 2024, X.C. Peng, BN240810130-2 (paratype HKAS 149953).

Notes: The perithecia of O. sungii are restricted to the subterminal region of the stroma, which terminates in a branched sterile apex. This species exhibits striking morphological affinities with O. langbianensis (Lao et al. 2021) and O. furcatosubulata (Wang et al. 2021a), specifically in host specificity, the presence of a bifurcated sterile apex, and comparable dimensions of both fertile structures and perithecia. However, the asci of O. langbianensis (200–250 × 5.0–6.0 μm) are longer than those of O. sungii (115–200 × 3.5–6.5 μm) and O. furcatosubulata (138.8–202.5 × 4.3–6.0 μm). The secondary ascospores of O. furcatosubulata (3.7–5.3 × 1.3–2.0 µm) are shorter than O. langbianensis (5.0–7.5 × 1.3–2.0 µm) and O. sungii (5.3–8.2 × 1.5–2.4 μm). It is noteworthy that the length of the sterile tip and the colour of the fertile part of O. sungii are significantly different from those of the other two species. The length of the sterile tip of O. langbianensis and O. furcatosubulata is more than 4 mm, and the fertile part is yellowish brown, while the length of the sterile tip of O. sungii is only 2 mm, and the fertile part is reddish brown. Based on the multi-gene phylogenetic analyses, O. sungii is more closely related to O. langbianensis than O. furcatosubulata (Fig. 1). The comparison of the nucleotide sequences between O. sungii and O. langbianensis showed three bp (including one gap) differences in the LSU and 15 bp in SSU (including six gaps) sequences. Therefore, we describe this taxon as a new species in Ophiocordyceps.

Ophiocordyceps brunneascospora X.C. Peng & T.C. Wen, sp. nov. MB 860849. Fig. 7.

Fig. 7.

Ophiocordyceps brunneascospora (HKAS 149964). A. Stroma protruding from soil. B. Stroma. C. Perithecia. D. Immature asci. E–G. Mature asci. H, I. Immature ascospores. J, K. Mature ascospores. Scale bars: B = 20 mm; C = 500 µm; D–G = 50 µm; H–K = 20 µm.

Etymology: brunneascospora, referring to the brown ascospores.

Typus: China, Yunnan Province, Pu’er City, Simao District, Yixiang Town (22°36′56″N, 101°05′49″E), the host has decayed and cannot be identified, probably on larvae of Lepidoptera buried in soil, 6 Aug. 2024, X.C. Peng, SM240806126 (holotype HKAS 149964; GenBank LSU: PX411463, ITS: PX411442, SSU: PX414353, tef1-α: PX409305, rpb1: PX423496).

Parasitic on insects, buried in soil of broadleaf forests. Sexual morph: Stroma arising from the insect, solitary, cylindrical, unbranched, brown, 95 × 1–2 mm. Stipe cylindrical, light brown, 55 × 1 mm. Fertile part subterminal, erect, cylindrical, tapering gradually toward the apex, brown, 27 × 2 mm. Sterile tip solitary, grey, 13 mm long. Perithecia pseudo-immersed, ovoid, light brown, 295–400 × 125–260 μm ($\stackrel{\_}{\text{x}}=346\times 198\mu \text{m}$, n = 35). Peridium 18.0–44.0 µm ($\stackrel{\_}{\text{x}}=29.0\mu \text{m}$, n = 20) wide. Asci filiform, cylindrical with acute base, hyaline to light brown, 8-spored, 122–193 × 5.5–12.0 μm ($\stackrel{\_}{\text{x}}=151\times 8.6\mu \text{m}$, n = 30), with thickened ascus apices. Apical cap hemispherical, with a small channel in the centre, 2.8–4.5 × 5–7.0 μm ($\stackrel{\_}{\text{x}}=3.6\times 5.9\mu \text{m}$, n = 30). Ascospores filiform, with pointed end, smooth, hyaline to light brown, 2–13-septate, 73–188 × 2.5–4.5 µm ($\stackrel{\_}{\text{x}}=117\times 3.3\mu \text{m}$, n = 60), not breaking into part-spores. Asexual morph: Undetermined.

Additional material examined: China, Yunnan Province, Pu’er City, Simao District, Yixiang Town (22°36′56″N, 101°05′49″E), the host has decayed and cannot be identified, probably on larvae of Lepidoptera immersed in soil, 6 Aug. 2024, X.C. Peng, SM001 (paratype HKAS 149962).

Notes: Molecular phylogenetic analyses (Fig. 1) resolve that O. brunneascospora is closely related to O. communis and Hirsutella minnesotensis. Although both O. brunneascospora and O. communis possess subterminal fertile regions, they exhibit striking morphological distinctions in perithecial arrangement (pseudo-immersed in O. brunneascospora vs superficial in O. communis), and dimensions of perithecia (295–400 × 125–260 μm vs 285–675 × 195–390 μm), asci (122–193 × 5.5–12.0 μm vs 215–250 × 15 μm), and ascospores (73–188 × 2.5–4.5 μm vs 100–180 × 5.0–6.0 μm) (Sung et al. 2007). Furthermore, morphological comparison with H. minnesotensis is currently untenable, as this taxon is known only from its asexual morph, whereas O. brunneascospora has been documented exclusively from the sexual morph. Nucleotide sequence comparisons between H. minnesotensis (3608) and O. brunneascospora (HKAS 149964) revealed two bp differences in LSU, one bp in ITS, 13 bp in tef1-α, and 10 bp in rpb1, respectively. Therefore, we describe this taxon as a new species within Ophiocordyceps.

Ophiocordyceps ramosiphialidica X.C. Peng & T.C. Wen, sp. nov. MB 860850. Fig. 8.

Fig. 8.

Ophiocordyceps ramosiphialidica (HKAS 149960). A–C. Fungus on insect larvae. D, E. Phialides. F, G. Conidia attached to the tip of phialides. H. Conidia. Scale bars: A–C = 2 mm; D, E = 25 µm; F–H = 10 µm.

Etymology: Name refers to its branched phialides.

Typus: China, Yunnan Province, Kunming City, Panlong District, Longchuanqiao Forest Park (25°07′49″N, 102°47′34″E), on dead larvae (Diptera) immersed in the rotten trunk, 24 Jul. 2024, X.C. Peng, LCQ240724107 (holotype HKAS 149960; GenBank LSU: PX411461, SSU: PX414347, rpb1: PX423494, rpb2: PX423511).

Parasitic on the larvae of Diptera, buried in rotten wood of broadleaf forests, 2–4 mm long, ca 0.5 mm wide. Sexual morph: Undetermined. Asexual morph: Hirsutella-like. Synnemata solitary, cylindrical, unbranched, light brown, yellow to off-white, 0.5–4 mm long, 100–200 µm wide. Phialides lanceolate, branched, smooth, crowed, hyaline, singly and directly borne on hyphae, 15–42 × 0.8–1.9 μm ($\stackrel{\_}{\text{x}}=26.8\times 1.4\mu \text{m}$, n = 40). Conidia fusiform, smooth-walled, hyaline, 1-celled, aseptate, 3.0–5.0 × 1.3–2.6 μm ($\stackrel{\_}{\text{x}}=4.0\times 2.0\mu \text{m}$, n = 30), L/W ratio 1.5–2.6 ($\stackrel{\_}{\text{x}}=2.0$, n = 30).

Additional material examined: China, Yunnan Province, Kunming City, Panlong District, Longchuan Bridge Forest Park (25°07′49″N, 102°47′34″E), on dead Diptera larvae, in the rotten trunk, 24 Jul. 2024, X.C. Peng, LCQ240724108 (paratype HKAS 149959).

Notes: Ophiocordyceps ramosiphialidica is closely related to O. pseudovariabilis and O. variabilis based on multilocus phylogenetic analyses (Fig. 1). Ophiocordyceps pseudovariabilis and O. variabilis exhibit pleomorphic life cycles, while O. ramosiphialidica has been found only as an asexual morph. A comparative analysis demonstrated that O. ramosiphialidica is distinguished from the two allied species by possessing lanceolate, branched, monophialidic, erect phialides measuring 15–42 × 0.8–1.9 μm and fusiform conidia measuring 3.0–5.0 × 1.3–2.6 μm. In contrast, O. pseudovariabilis produces phialides that are mono- or polyphialidic, hirsutella-like with a swollen base (4.0–15.0 × 2.0–5.0 µm), and fusoid conidia (4.0–8.0 × 0.5–1.0 µm) (Mongkolsamrit et al. 2024). Ophiocordyceps variabilis produces enteroblastic phialides arranged in monoverticillate whorls or pairs. These phialides have a subcylindrical base (10.0–19.2 × 2.5 μm) that abruptly narrows into a short, tapering neck strongly hooked or bending, and produce subcylindrical conidia (8.0–12.4 × 1.9–3.1 μm) (Petch 1937, Hodge et al. 1998).

Ophiocordyceps rubella X.C. Peng & T.C. Wen, sp. nov. MB 860851. Fig. 9.

Fig. 9.

Ophiocordyceps rubella (HKAS 149979). A, B. Stromata arising from a cicada nymph. C, D. Perithecia. E–G. Immature asci. H. Apical caps of asci. I. Part of ascospores. J, K. Secondary ascospores. L, M. Culture on PDA after 7 wk. N. Culture on PDA after 10 wk. O–Q. Conidiophores, phialides and conidia. R, S. Conidia. Scale bars: A, N = 10 mm; B = 20 mm; C, D = 100 µm; E–G = 50 µm; H, O–R = 20 µm; I = 10 µm; J, K, S = 5 µm; L, M = 5 mm

Etymology: The specific epithet “rubella” refers to the red colour of the stroma.

Typus: China, Guangdong Province, Fatshan City, on dead nymph of cicada (Cicadidae, Hemiptera), in the soil, 10 Jun. 2024, T.C. Wen, FS24061001 (holotype HKAS 149979; culture ex-type GACP FS24061001 = CGMCC 3.29473; GenBank LSU; PX411456, ITS: PX411438, SSU: PX414342, tef1-α: PX409300, rpb1: PX423489).

Parasitic on the nymph of cicada (Cicadidae, Hemiptera) living underground, 21 × 7 mm. Sexual morph: Stroma arising from the head, paired, cylindrical, unbranched, red, 44 × 3–5.5 mm. Stipe cylindrical, red, 29–33 × 3–4 mm. Fertile part terminal, cylindrical, red, 11–15 × 5.5 mm. Perithecia immersed, long ovoid or flask-shaped, 500–730 × 85–295 μm ($\stackrel{\_}{\text{x}}=628\times 167\mu \text{m}$, n = 70). Peridium 15–75 µm ($\stackrel{\_}{\text{x}}=35.7\mu \text{m}$, n = 35) wide. Asci filiform, cylindrical, 8-spored, hyaline, 155–285 × 3.5–8.0 μm ($\stackrel{\_}{\text{x}}=214.3\times 5.2\mu \text{m}$, n = 45), with thickened apices. Apical cap hemispherical, with a small channel in the centre, 3–6 × 3.5–7 μm ($\stackrel{\_}{\text{x}}=4\times 6.4\mu \text{m}$, n = 30). Ascospores filiform, smooth, hyaline, 1.5–2.5 µm wide ($\stackrel{\_}{\text{x}}=2\mu \text{m}$, n = 20), multiseptate, breaking into secondary ascospores. Secondary ascospores cylindrical, smooth, part septate, hyaline, 8–16.5 × 1.5–3.5 μm ($\stackrel{\_}{\text{x}}=11.9\times 2.3\mu \text{m}$, n = 120), L/W ratio 3.2–8.0 ($\stackrel{\_}{\text{x}}=5.2$, n = 120). Asexual morph: Only formed in culture.

Culture characteristics: Colonies on PDA attaining 19–24 mm diam. within 7 wk at 25 °C, circular, dense, surface rough, leathery, brown green in the middle, light yellow green at the edges, raised, entire, radially and concentrically sulcate, reverse brown. Mycelium cylindrical, smooth or rough, branched, septate, hyaline, 2.0–4.5 µm diam ($\stackrel{\_}{\text{x}}=3\mu \text{m}$, n = 35). Synnemata emerging from centre and margin of colonies, pink, cylindrical at base, branched at apex. Conidiophores micronematous, hyaline, smooth-walled. Phialides syngliocladium-like, form along conidiophores, phialidic, solitary, with a swollen base and tapering gradually toward the apex, 6–21 × 2.5–5.5 μm ($\stackrel{\_}{\text{x}}=12.2\times 3.9\mu \text{m}$, n = 150). Conidia ellipsoidal, smooth-walled, hyaline, 8.5–11 × 3–4.5 µm ($\stackrel{\_}{\text{x}}=10\times 3.6\mu \text{m}$, n = 70), L/W ratio 2.0–3.4 µm ($\stackrel{\_}{\text{x}}=2.8\mu \text{m}$, n = 70).

Notes: Based on the multi-gene phylogenetic analyses, O. rubella is closely related to O. khonkaenensis and is placed within the O. sobolifera clade (Fig. 1). Both species share several morphological traits, including parasitism on cicada nymphs, red terminal fertile parts, white rhizoids, and overlapping size ranges of perithecia, asci, and secondary ascospores. However, O. rubella differs by producing cylindrical fertile heads which present a similar colour to the stipe, whereas O. khonkaenensis has a globose fertile head with a darker colour than the stipe (Crous et al. 2019). Significant differences are also observed in their culture characteristics on PDA. Ophiocordyceps rubella develops larger phialides with a pronounced neck (6.0–21 × 2.5–5.5 μm) and cylindrical conidia (8.5–11 × 3.0–4.5 µm), while O. khonkaenensis produces hirsutella-like phialides (5.5–11.0 × 2.0–3.0 µm) and fusiform conidia (3.0–5.5 × 1.0–3.0 µm) (Crous et al. 2019). Additionally, O. khonkaenensis was described from Thailand, while O. rubella is recorded from China. Nucleotide sequence comparisons between O. rubella (HKAS 149979) and O. khonkaenensis (BCC81462) revealed one bp difference in SSU, 10 bp in tef1-α, and eight bp in rpb1. Comparisons with O. khonkaenensis BCC81464 showed nine bp differences (including five bp gaps) in ITS and 69 bp differences (including 17 bp gaps) in LSU. Therefore, based on both morphological characteristics and phylogenetic analyses, we introduce our new collection as a new species.

Ophiocordyceps brunneipunctata (Hywel-Jones) G.H. Sung et al., Stud. Mycol. 57: 40. 2007. Fig. 10.

Fig. 10.

Ophiocordyceps brunneipunctata (HKAS 149958). A. Habitat. B. Stroma arising from host. C. Fertile part and sterile tip. D, E. Perithecia. F–J. Immature asci. K. Part of ascospores. L, M. Secondary ascospores. Scale bars: B = 10 mm; C = 2 mm; D = 5 µm; E = 100 µm; F–J = 30 µm; K = 10 µm; L = 5 µm; M = 2 µm.

Basionym: Cordyceps brunneipunctata Hywel-Jones, Mycol. Res. 99: 1195. 1995. [as C. ‘brunneapunctata’].

Parasitic on insect pupa (Lepidoptera), 14 × 3 mm. Sexual morph: Stroma arising from the head of an insect, single, flexuous, cylindrical with acute ends, unbranched, reddish brown, 59 × 1–2 mm. Stipe cylindrical, reddish brown, 50 × 1 mm. Fertile part subterminal, cylindrical, reddish brown, 8 × 2 mm. Sterile tip conical, light yellow, 2 × 1 mm. Perithecia immersed, flask-shaped, reddish brown, 355–481 × 71–206 μm ($\stackrel{\_}{\text{x}}=421.5\times 134.9\mu \text{m}$, n = 40). Asci narrowly filiform, cylindrical, hyaline, 117–220 × 3.0–7.4 μm ($\stackrel{\_}{\text{x}}=171.2\times 5.2\mu \text{m}$, n = 50), with thickened apices. Apical cap hemispherical, 1.6–4.4 × 4.1–6.3 μm ($\stackrel{\_}{\text{x}}=2.7\times 5.3\mu \text{m}$, n = 70). Ascospores filiform, hyaline, multiseptate breaking into secondary ascospores. Secondary ascospores cylindrical, aseptate, hyaline, 4.6–10.2 × 0.8–2.6 µm ($\stackrel{\_}{\text{x}}=6.24\times 1.8\mu \text{m}$, n = 30), L/W ratio 2.7–5.0 ($\stackrel{\_}{\text{x}}=3.5$, n = 30). Asexual morph: Not observed.

Material examined: China, Yunnan Province, Tengchong County, Jietou Town (25°18′50″N, 98°37′08″E), on dead pupa of Lepidoptera sp. immersed in soil, 20 Aug. 2022, X.C. Peng, TC22082008 (HKAS 149958).

Notes: According to the multi-gene phylogenetic analysis, our specimen (HKAS 149958) clustered with O. brunneipunctata (OSC 128576 and BCC 2218) (Fig. 1). Our specimen shares key morphological and ecological characteristics with O. brunneipunctata in its parasitic association with a lepidopteran insect that is immersed in soil, a reddish brown subterminal stroma with an unbifurcated sterile apex, immersed perithecia, and multiseptate ascospores that disarticulate into secondary ascospores. However, some differences were observed in micro-morphological structures. Specimen HKAS 149958 exhibits a broader size range in micro-structures than previously described, perithecia (355–481 × 71–206 μm vs 270–335 × 110–160 μm), asci (117–220 × 3.0–7.4 μm vs 280–295 × 6–7 μm), and secondary ascospores (4.6–10.2 × 0.8–2.6 μm vs 4.0–6.0 × 1.0–1.5 μm) (Hywel-Jones 1995). Comparative nucleotide sequence analyses between HKAS 149958 and OSC 128576 revealed five bp differences in SSU and three bp in rpb1. Based on both morphological and molecular evidence, we report our study species as a new geographic and host record from China.

Paraisaria Samson & B.L. Brady, Trans. Br. Mycol. Soc. 81(2): 285. 1983.

Notes: Paraisaria was established by Samson & Brady (1983) to accommodate the Par. dubia, which was originally described as Isaria dubia by Delacroix (1893) based on specimens found on larvae of Hepialus lupulinus in France. Paraisaria gracilis was identified as the sexual morph of Isaria dubia (Samson & Brady 1983). Mongkolsamrit et al. (2019) subsequently designated Par. gracilis as the type species of the genus Paraisaria. Paraisaria forms a monophyletic clade nested within Ophiocordyceps, demonstrating polyphyly in the latter genus (Mongkolsamrit et al. 2019, Wei et al. 2021, Tehan et al. 2023). To date, 21 species are accepted in Paraisaria (https://www.indexfungorum.org/Names/Names.asp, 3 Jan. 2026) and all members have been verified with molecular phylogenetic analysis. The sexual morph of this genus is characterized by robust stroma, globose or ovoid fertile head, ovoid-ellipsoid or obclavate, immersed perithecia; cylindrical, eight-spored asci; filiform, multiseptate ascospores breaking into cylindrical secondary ascospores. The asexual morph is characterized by verticillate conidiophores, phialides with swollen bases and a thin tapering neck, and narrowly cylindrical to fusiform conidia (Mongkolsamrit et al. 2019).

Paraisaria clavata X.C. Peng & T.C. Wen, sp. nov. MB 860853. Fig. 11.

Fig. 11.

Paraisaria clavata (A–I. HKAS 149976, J–Q. Culture GACP SB22050201). A. Stroma arising from the host. B. Fertile part. C. Host. D. Cross-section of fertile part showing perithecia. E. Immersed perithecia. F, G. Immature asci. H. Apical cap of ascus. I. Secondary ascospores. J, K. Colony on PDA. L–O. Conidiophores and phialides. P, Q. Conidia. Scale bars: A, J, K = 20 mm; B = 5 mm; C = 10 mm; D = 2 mm; E = 500 µm; F–G = 50 µm; H, L, M = 20 µm; I, O = 10 µm; N, P, Q = 5 µm.

Etymology: Referring to the clavate fertile part of its sexual morph.

Typus: China, Guizhou Province, Shibing County, on dead larvae of Cerambycidae, Coleoptera, 2 May 2022, T.C. Wen, SB22050201 (holotype HKAS 149976; culture ex-type GACP SB22050201 = CGMCC 3.29468; GenBank LSU: PX411464, ITS: PX411443, SSU: PX414348, tef1-α: PX409306, rpb1: PX423497).

Parasitic on beetle larvae (Cerambycidae, Coleoptera), 35–43 × 5–10 mm. Sexual morph: Stroma arising from the insect head, single, flexuous, clavate, unbranched, yellow, 75–95 × 2.5–9 mm, composed of rhizoids, stipe, and fertile part. Rhizoids arising from the head of larvae and remaining buried in soil, flexuous, tangled, one or two, yellow brown, ca 20–35 mm long. Stipe emerging from the soil surface, flexuous, unbranched, cylindrical, yellow, 40–65 × 2.5–4 mm. Fertile part terminal, clavate to cylindrical, yellow to light brown, 11–15 × 4.5–9 mm. Perithecia immersed, ellipsoidal with a beak-like ostiole, yellow, 570–730 × 230–370 μm ($\stackrel{\_}{\text{x}}=659\times 294\mu \text{m}$, n = 25). Peridium 20–45 µm ($\stackrel{\_}{\text{x}}=27.8\mu \text{m}$, n = 25) in width. Asci narrowly cylindrical, hyaline, 178–557 × 2.2–7.2 μm ($\stackrel{\_}{\text{x}}=347\times 4.2\mu \text{m}$, n = 40), with thickened ascus apices. Apical cap hemispherical, hyaline, 2.7–4.4 × 4–7 μm ($\stackrel{\_}{\text{x}}=3.6\times 5.7\mu \text{m}$, n = 40). Ascospores filiform, smooth, hyaline, easily breaking into secondary ascospores. Secondary ascospores cylindrical, smooth, hyaline, 6.5–10.0 × 1.0–1.6 μm ($\stackrel{\_}{\text{x}}=8.3\times 1.3\mu \text{m}$, n = 30), L/W ratio 4.6–8.0 ($\stackrel{\_}{\text{x}}=6.3$, n = 30). Asexual morph: Only formed in culture.

Culture characteristics: Colonies on PDA, attaining 21–25 mm diam. within 28 d at 25 °C, dense, cottony, white to grey brown to yellowish, reverse cyan green to white, produces light cyan green to yellow translucent pigment. Mycelium septate, 1.8–5 µm ($\stackrel{\_}{\text{x}}=3.3\mu \text{m}$, n = 40) in diam. Conidiophores branched and septate. Phialides paraisaria-like, elongated lageniform, swollen at the base and tapering gradually toward the apex, solitary or in whorls of 2–4, hyaline, 5.5–12 × 2.0–3.4 µm ($\stackrel{\_}{\text{x}}=8.5\times 2.8\mu \text{m}$, n = 60). Conidia fusiform, with acute base and rounded apex, smooth-walled, 2.8–5.6 × 0.8–2.2 µm ($\stackrel{\_}{\text{x}}=4.7\times 1.2\mu \text{m}$, n = 65), L/W ratio 2.0–5.5 ($\stackrel{\_}{\text{x}}=3.9$, n = 65).

Additional material examined: China, Guizhou Province, Shibing County, on dead larvae of Cerambycidae, Coleoptera, 16 May 2022, T.C. Wen, SB22051601 (paratype HKAS 149977, culture ex-paratype GACP SB22051601 = CGMCC 3.29470).

Notes: Paraisaria clavata is phylogenetically closely related to Par. heteropoda, with strong statistical support (100 % MLB/1 PP, Fig. 1). However, Par. clavata can be distinguished from Par. heteropoda in having yellow to light brown, larger fertile part (11–15 mm vs 6–7 mm) and cylindrical conidia with one acute end. Paraisaria heteropoda has a brown fertile part and broadly ellipsoidal conidia (Kobayasi 1939, Mongkolsamrit et al. 2019). Comparative analyses of nucleotide sequences between Par. heteropoda BCC 18235 and Par. clavata HKAS 149976 revealed 14 bp differences in LSU (including two bp gaps), 13 bp differences in SSU, 13 bp in tef1-α, 10 bp in rpb1, and 19 bp in rpb2. Based on its distinct morphological features and molecular divergence, Par. clavata is introduced here as a new species in Paraisaria.

Paraisaria cocoonihabita X.C. Peng & T.C. Wen, sp. nov. MB 860854. Fig. 12.

Fig. 12.

Paraisaria cocoonihabita (A–J. HKAS 149975, K–R. Culture GACP SB22050202). A. Stroma arising from host. B. Host. C. Fertile part of stroma. D. Cross section of fertile part showing arrangement of perithecia. E. Immersed perithecia. F–I. Immature asci. J. Secondary ascospores. K, L. Colony on PDA. M–P. Phialides. Q, R. Conidia. Scale bars: A, K, L = 10 mm; B = 5 mm; C, D = 1 mm; E = 250 µm; F–I, M = 50 µm; J, N, O = 10 µm; P–R = 5 µm.

Etymology: Referring to the cocoon of the Lepidoptera host.

Typus: China, Guizhou Province, Shibing County, on dead cocoon of Lepidoptera, 2 May 2022, T.C. Wen, SB22050202 (holotype HKAS 149975; culture ex-type GACP SB22050202 = CGMCC 3.29469; GenBank LSU: PX411467, ITS: PX411446, SSU: PX414351, tef1-α: PX409308, rpb1: PX423500).

Parasitic on pupae (Lepidoptera), 20 × 7–8 mm. Sexual morph: Stroma arising in the middle of body, single, flexuous, unbranched, dark brown to yellow brown, 45 × 1–4 mm. Stipe flexuous, cylindrical, dark brown to yellow brown, 41 × 1–2 mm. Fertile part terminal, conical, yellow brown, 4 × 4 mm. Perithecia immersed, flask-shaped, yellow, 454–663 × 142–293 μm ($\stackrel{\_}{\text{x}}=535\times 194\mu \text{m}$, n = 20). Peridium 20–32 µm ($\stackrel{\_}{\text{x}}=24.8\mu \text{m}$, n = 20) wide. Asci narrowly cylindrical, hyaline, 156–388 × 2.3–5.2 μm ($\stackrel{\_}{\text{x}}=245.6\times 4\mu \text{m}$, n = 50), with thickened apices. Apical cap 1.7–4.6 × 4.4–7.6 μm ($\stackrel{\_}{\text{x}}=3.4\times 5.8\mu \text{m}$, n = 40). Ascospores filiform, smooth, hyaline, easily breaking into secondary ascospores. Secondary ascospores cylindrical, smooth, hyaline, 4.5–8.5 × 1.0–1.6 μm ($\stackrel{\_}{\text{x}}=5.8\times 1.4\mu \text{m}$, n = 30), L/W ratio 3.0–5.5 ($\stackrel{\_}{\text{x}}=4.0$, n = 30). Asexual morph: Only formed in culture.

Culture characteristics: Colonies on PDA attaining 20–28 mm diam. within 71 d at 25 °C, dense, leathery to cottony, thick, white, wrinkled bulge, reverse brown, produces light brown translucent pigment. Mycelium septate, 2–5.9 µm ($\stackrel{\_}{\text{x}}=3.3\mu \text{m}$, n = 40) in diam. Phialides akanthomyces-like, single, swollen at the base, apically narrowing into a thin neck, hyaline, smooth, 2.9–10.5 × 2–4.3 µm ($\stackrel{\_}{\text{x}}=5.6\times 3.1\mu \text{m}$, n = 45). Conidia cylindrical with rounded ends, hyaline, smooth-walled, 3.6–5.4 × 0.8–1.6 µm ($\stackrel{\_}{\text{x}}=4.6\times 1.2\mu \text{m}$, n = 50), L/W ratio 3.4–4.5 ($\stackrel{\_}{\text{x}}=3.9$, n = 50).

Notes: Paraisaria cocoonihabita is closely related to Par. cascadensis with significant bootstrap support (100 % MLB/1 PP, Fig. 1). Paraisaria cocoonihabita was found parasitizing an adult of Cyphoderris monstrosa (Prophalangopsidae, Orthoptera), characterized by conspicuous rhizomorphs and subglobose fertile parts (Tehan et al. 2023). However, Par. cocoonihabita is featured with the parasitic association with lepidopteran cocoon, absence of rhizomorphs, and production of conical, smaller fertile part (4 × 4 mm vs. 8–9 × 6–9 mm), smaller perithecia (454–663 × 142–293 μm vs. 800–970 × 105–150 μm), and smaller secondary ascospores (4.5–8.5 × 1.0–1.6 μm vs. 6.3–10.3 × 1.6–2.4 μm). Comparative nucleotide sequence analyses between Par. cascadensis (OSC-M-052017) and Par. cocoonihabita (HKAS 149975) revealed seven bp differences in LSU (including one bp gap), 20 bp in ITS (including seven bp gaps), three bp in SSU, 23 bp in tef1-α, and 17 bp in rpb1 (including a nine bp continuous gap). Based on distinct morphological features and clear molecular divergence, Par. cocoonihabita is introduced here as a new species within the genus Paraisaria.

Polycephalomycetaceae Y.P. Xiao et al., Fungal Diversity 120: 23. 2023.

Pleurocordyceps Y.J. Yao, et al., J. Syst. Evol. 59(5): 1074. 2021.

Notes: Wang et al. (2021b) introduced Pleurocordyceps within Ophiocordycipitaceae based on multi-gene phylogeny analyses and morphological characteristics. Later, Pleurocordyceps was moved to Polycephalomycetaceae by Xiao et al. (2023). Up to now, 23 species are accepted within Pleurocordyceps (https://www.indexfungorum.org/Names/Names.asp, 3 Jan. 2026), and sequence data are available for all members of this genus (Xiao et al. 2023). The sexual morph of this genus is characterized by reddish brown to yellow stroma, capitate or laterally fertile parts, pyriform to ovoid and immersed perithecia, cylindrical asci with an apical cap, filiform ascospores breaking into cylindrical secondary spores. The asexual morph is characterized by light-coloured, clustered synnemata apically bearing globose fertile parts and dimorphic phialides and conidia (Wang et al. 2021b, Xiao et al. 2023).

Pleurocordyceps heilongtanensis Y.B. Wang et al., Fungal Diversity 120: 61. 2023. Fig. 13.

Fig. 13.

Pleurocordyceps heilongtanensis (HKAS 149961). A. Fungus on insect hosts. B. Synnemata. C, D. α-phialides with conidia. E, F. β-phialides with conidia. G. α-conidia. H. β-conidia. Scale bars: B = 100 µm; C = 20 µm; D–H = 10 µm.

Parasitic on ant (Formicidae, Hymenoptera), superficially buried in leaf litter, ca 4 mm long. Sexual morph: Undetermined. Asexual morph: Synnemata numerous, cylindrical with fertile head, arising from multiple parts of the thoracic, abdominal, and leg structures of the host, unbranched, 1–4 mm long, ca 0.2–0.4 mm wide. Stipes cylindrical, white to light brow towards the base, 1–3.6 mm long, 0.5–1 mm wide. Fertile heads globose to subglobose, white, with conidia masses, 0.3–0.4 mm in diam. Conidiophores arranged in a parallel palisade-like layer on the surface of stipe or fertile head, hyaline, 25–70 μm ($\stackrel{\_}{\text{x}}=45.4$, n = 40) long, usually bearing 2–4 phialides. α-phialides caespitose, elongated lageniform, smooth, hyaline, gathered at the top of synnema, 5–27 × 0.5–1.5 μm ($\stackrel{\_}{\text{x}}=13.3\times 0.96\mu \text{m}$, n = 60). β-phialides solitary, lanceolate with ovate base, scattered along the stipe, 8.2–15.0 × 1.2–2.5 μm ($\stackrel{\_}{\text{x}}=11.3\times 1.9\mu \text{m}$, n = 30). α-conidia subglobose to ovoid, one-celled, smooth-walled, hyaline, aseptate, 2.2–3.4 × 1.0–1.6 μm ($\stackrel{\_}{\text{x}}=2.8\times 1.3\mu \text{m}$, n = 30), L/W ratio 1.4–2.4 ($\stackrel{\_}{\text{x}}=2.0$, n = 30). β-conidia fusiform, one-celled, smooth-walled, hyaline, aseptate, 2.3–3.5 × 1.0–1.6 μm ($\stackrel{\_}{\text{x}}=2.9\times 1.2\mu \text{m}$, n = 35), L/W ratio 1.7–3.0 ($\stackrel{\_}{\text{x}}=2.4$, n = 35).

Material examined: China, Yunnan Province, Pu’er City, Simao District, Nanping Town (22°44′19″N, 100°58′46″E), on Camponotus sp. (Formicidae, Hymenoptera), on the leaf litter, 5 Aug. 2024, X.C. Peng, SM240805109 (HKAS 149961).

Notes: According to the multi-gene phylogenetic analysis (Fig. 1), specimen HKAS 149961 clusters with Ple. heilongtanensis, a species previously reported by Xiao et al. (2023). Our specimen shared key morphological characteristics with Ple. heilongtanensis such as dimorphic phialides and conidia with overlapping measurements. However, it parasitises ants, contrasting with being hyperparasitic on Ophiocordyceps sp. as observed in the type specimens (Xiao et al. 2023). Comparative nucleotide sequence analyses between HKAS 149961 and KUMCC 3008 revealed four bp differences in LSU (including two bp gaps), two bp in ITS, zero bp in tef1-α and rpb2, and one bp in rpb1. Thus, we report ants (Formicidae, Hymenoptera) as a new host for Ple. heilongtanensis. However, given that Polycephalomyces species are often hyperparasitic, a secondary parasitism on an entomopathogenic fungus infecting the ant cannot be excluded.

DISCUSSION

Entomogenous fungi have long attracted scientific interest due to their specialized insect-associated lifestyles, ecological roles, and economic potential. The multigene-based phylogenetic analyses combined with morphological and ecological observations have resolved the newly collected specimens as nine new species and three new records in four families (Clavicipitaceae, Cordycipitaceae, Ophiocordycipitaceae, and Polycephalomycetaceae) and seven genera (Cordyceps, Jenniferia, Metarhizium, Ophiocordyceps, Pleurocordyceps, Paraisaria, and Purpureomyces). Interestingly, the asexual morphs of Metar. acrididarum, Pur. fenggangensis, C. multisynnematosa, O. ramosiphialidica, O. rubella, Par. clavata, and Par. cocoonihabita exhibit unusual morphological traits that provide new insights into the diversity and evolution of asexual morphs within these groups. To better understand the morphological diversity of asexual morphs of Metarhizium, Purpureomyces, Cordyceps, Paraisaria and Ophiocordyceps, we comprehensively review the literature and redraw illustrations of the different types of asexual morph of these genera. Metarhizium comprises eight types of asexual morphs, Cordyceps five types, Paraisaria three types and Purpureomyces two types (Fig. 14). We found that Ophiocordyceps comprises 15 main types of asexual morphs (Fig. 15), representing the richest of morphological diversity.

Fig. 14.

The anamorph characteristics of Metarhizium, Cordyceps, Purpureomyces, and Paraisaria. A–H. Metarhizium. (A–D. Metarhizium-like phialide with conidia. E, F. Nomuraea-like phialide with different conidia. G, H. Paecilomyces-like phialide with conidia). I–M. Cordyceps. (I–K. Isaria-like phialide with conidia. L, M. Lecanicillium-like). N, O. Purpureomyces (N. Isaria-like, Pur. fenggangensis. O. Lecanicillium-like). P–R. Paraisaria (P, Q. Paraisaria-like. R. Akanthomyces-like, Par. cocoonihabita). Scale bars = 10 µm.

Fig. 15.

The anamorphs characteristics of Ophiocordyceps. A–I. Hirsutella-like. J. H. stilbelliformis var. myrmicarum. K. O. buquetii / O. laotii. L. Syngliocladium-like. M. H. uncinate. N. Hymenostilbe-like. O. Tilachlidiopsis-like. Scale bars = 10 µm.

Diversity of asexual morphs of Metarhizium and Purpureomyces

Mongkolsamrit et al. (2020a) classified the phialides of Metarhizium into three distinct morphological types: i) metarhizium-like, characterized by a candelabrum-like arrangement of phialides on conidiophores, with conidia exhibiting ovoid (Fig. 14D), cylindrical (Fig. 14A, C), or globose (Fig. 14B) shapes; ii) nomuraea-like, featuring oval-cylindrical phialides with very short necks arranged in whorls, conidia scattered (Fig. 14E) or dimorphic in chains (Fig. 14F); iii) paecilomyces-like phialides, comprising a globose basal portion and a distinct slender neck, with conidia occurring in subglobose (Fig. 14G) or cylindrical (Fig. 14H) chains.

Most Metarhizium species produce metarhizium-like asexual morphs and are distributed across four phylogenetic clades labelled by Mongkolsamrit et al. (2020a) as: the “Metar. anisopliae complex”, the “Metar. flavoviride complex”, the “on Coleoptera larva, ovoid perithecia, ordinal clade”, and the “small hopper clade”. Species exhibiting nomuraea-like phialides are primarily distributed within the “cicada clade” and the “nomuraea-like clade”. Within the “cicada clade”, all species except Metar. megapomponiae and Metar. owariense produce dimorphic conidia. In contrast, species within the “nomuraea-like clade” typically bear subglobose conidia. Species with paecilomyces-like phialides produce subglobose (e.g., Metar. granulomatis, Metar. viride; Fig. 14G) or cylindrical or reniform conidia (e.g., Metar. acrididarum, Metar. phuwiangense, Metar. reniforme; Fig. 14H). It is worth noting that the phialides of Metar. dendrolimi were described by Chen et al. (2017), have a swollen base with a short neck, corresponding to the paecilomyces-like type. However, BLAST analyses of its tef1-α, rpb1, and tub loci indicate a closer phylogenetic affinity to C. cicada, suggesting its placement requires further verification. The newly introduced species Metae. acrididarum phylogenetically groups with paecilomyces-like Metarhizium species; however, it can be distinguished from its allied species in producing phialides with a globose base and a short neck with long ovoid to cylindrical conidia.

Purpureomyces is a genus phylogenetically close to Metarhizium. This genus is characterised by the production of white to purplish stromata and lecanicillium-like asexual morph (Fig. 14O) (Mongkolsamrit et al. 2020a). The new species Pur. fenggangensis produces an isaria-like asexual morph (Fig. 14N) that is new to this genus. Host records for this genus remain limited, mostly associated with lepidopteran larvae or pupae, suggesting potential host specificity; however, further ecological data are needed to confirm this pattern.

Morphological diversity of Cordyceps and the unusual synnemata in C. multisynnematosa

Cordyceps is a monophyletic genus within the Cordycipitaceae. The asexual morph of this genus exhibits two distinct phialide types: isaria-like and lecanicillium-like. Isaria-like phialides are flask-shaped, with a subglobose to broadly cylindrical base tapering into a neck; they are often densely clustered and bear globose (Fig. 14I) to cylindrical (Fig. 14K) conidia arranged in line chains to imbricate chains (Fig. 14J), or aggregated masses. In contrast, lecanicillium-like phialides are slender and cylindrical at the base, tapering toward the apex, typically arising directly from hyphae, and are usually solitary and sparse, with cylindrical conidia arranged in an imbricate chain (Fig. 14L, 14M). Based on phylogenetic analyses, Dong et al. (2022) divided Cordyceps into five clades and revealed a strong correlation between phialide type and clade placement. However, this correlation is not absolute, as isaria-like phialides are widely distributed across multiple clades, indicating that asexual morph alone cannot be used to delimit clades within Cordyceps. Species producing lecanicillium-like phialides are mainly concentrated in Clade I (Fig. 14L, M), although a subclade comprising C. brevistroma, C. kuiburiensis, C. araneae, and C. nabanheensis bears isaria-like phialides (Fig. 14I–K). In Clade II, the two phialide types are intermingled with C. shuifuensis, C. hehuanensis, C. pseudorosea, and C. inthanonensis which exhibit lecanicillium-like phialides (Fig. 14L, M), whereas C. polystromata, C. sapaensis, and C. mexicana display isaria-like phialides (Fig. 14J, K). Notably, C. militaris has both types of phialides (Wang et al. 2023a). In Clade III, all species possess isaria-like phialides except C. bifusispora (Fig. 14L), which has lecanicillium-like phialides. All members of Clade IV also exhibit isaria-like phialides. Clade V species similarly display isaria-like phialides. However, Zha et al. (2020) treated C. grylli as a synonym of B. grylli and transferred it to Beauveria.

The new species C. multisynnematosa is notable for its production of multiple synnemata, a feature uncommon among its relatives. Although its phialides conform to the isaria-like type, the formation of numerous synnemata represents a distinct morphological adaptation. Whether this characteristic reflects a specific ecological function or host interaction requires further study. Phylogenetically, the species falls within a clade where both phialide types occur, reinforcing the need to use integrative approaches for species delimitation within this morphologically variable genus.

Diversity and phylogenetic discordance of asexual morphs in Ophiocordyceps

Ophiocordyceps is the most species-rich and morphologically complex lineage within the Ophiocordycipitaceae. Its asexual morphs exhibit diverse phialidic structures, which can be classified into three major types including hirsutella-like type, hymenostilbe-like and syngliocladium-like. The hirsutella-like type is characterized by flask-shaped phialides with a swollen or subulate base that gradually tapers toward the apex, usually solitary, monophialidic (Fig. 15A, B, D–F, H) or polyphialidic (Fig. 15C, G, I), sometimes ornamented with warts (Fig. 15D–F), one (Fig. 15C, F, G) or several (Fig. 15B) conidia that are often enveloped in a hyaline mucus. The hymenostilbe-like type is defined by cylindrical, polyblastic phialides arranged in fascicles to form a palisade layer, with apices crowded ornamented with short denticles (Fig. 15N). The syngliocladium-like type is defined by solitary or clustered flask-shaped phialides with a swollen base and a straight or hooked neck, producing ovoid to cylindrical conidia (Fig. 15L). Xie et al. (2025) divided Ophiocordyceps species into nine phylogenetic clades, each showing distinct host associations and asexual morphologies. The hirsutella-like morph is the most common and occurs in all clades except the O. sphecocephala clade (= Hymenostilbe clade), exhibiting predominantly hymenostilbe-like morphology. Notably, some clades contain more than one asexual morph type. For example, the hymenostilbe-like clade also exhibits species producing stilbella-like asexual morphs.

The O. acicularis clade and O. issidarum clade are both characterized by polyphialidic hirsutella-like phialides (Fig. 15C, G) but differ markedly in host spectrum, with the former primarily infecting Lepidoptera and the latter parasitizing a wider array of insect orders. Members of the O. sinensis clade also produce hirsutella-like phialides and primarily infect Lepidoptera, except H. uncinata, which produces multiple whorls of uncinate phialides with subfusiform conidia (Fig. 15M) on follicles of Hakea sp. (Seifert & Boulay 2004). Within the O. blattae clade, most species are restricted to termites (Blattodea), while several taxa indicate that host associations in this lineage are diverse and extend across multiple arthropod orders. In this clade, hirsutella-like or hymenostilbe-like morphs are observed on natural substrate, whereas cultures typically produce a hirsutella-like morph. The O. unilateralis clade, including the O. unilateralis core clade and the O. kniphofioides sub-clade (Araújo et al. 2018), generally produces hirsutella-like asexual morphs, except that H. stilbelliformis var. myrmicarum in the O. kniphofioides sub-clade produces terminal, finely echinulate, subulate phialides (Fig. 15J). The O. sobolifera clade comprises two subclades: one parasitizing cicadas (Hemiptera) and the other wireworms (Coleoptera). While most species in this clade bear hirsutella-like phialides, O. rubella and O. sobolifera produce syngliocladium-like phialides. The O. elongata clade exhibits a wide host range across Lepidoptera, Coleoptera, Diptera, Hemiptera, and Dermaptera, most species produce hirsutella-like phialides, except O. yushensis, which develops hymenostilbe-like phialides. The O. ravenelii clade is primarily associated with Coleoptera and typically exhibits hirsutella-like phialides, although O. clavata, O. pseudovariabilis, and O. variabilis form syngliocladium-like structures in culture. The O. sphecocephala clade parasitises Hymenoptera, Hemiptera, Diptera, and Megaloptera, and is consistently characterized by hymenostilbe-like phialides, except O. buquetii and O. laotii which produce cylindrical phialides with papillate end with narrowly obclavate conidia (Fig. 15K) (Mongkolsamrit et al. 2023), and O. myrmecophila, which develops tilachlidiopsis-like phialides consisting of broadly cylindrical metulae usually bearing two phialides without collarettes, cylindrical conidia in chains (Fig. 15O) (Papierok & Charpentie 1982). These findings highlight the remarkable plasticity and evolutionary innovation in fungal reproductive modes of asexual morph. It further underscores the limitations of relying exclusively on morphological characteristics of reproductive structures for determining phylogenetic affiliations.

Morphological innovation in Paraisaria: Par. cocoonihabita

Paraisaria currently comprises 21 species with available molecular data, and its re-establishment has rendered Ophiocordyceps paraphyletic. Except for Par. myrmicarum, all species of Paraisaria have been known from their sexual morph. Among them, the asexual morphs of Par. myrmicarum, Par. gracilis, Par. tettigoniae, and Par. gracilioides are observed from nature specimens, whereas those of the remaining species were primarily characterized from culture. All asexual morphs of Paraisaria are characterized by paraisaria-like phialides, which have a swollen base tapering toward the apex and are commonly borne on a metula; conidia are predominantly cylindrical (Fig. 14P, 14Q). Notably, Par. yodhathaii, Par. phuwiangensis, Par. orthopterorum, and Par. myrmicarum produce two distinct types of conidia (Fig. 14Q). Interestingly, the phialides of Par. gracilis, Par. myrmicarum, and Par. orthopterorum are polyphialidic, bearing multiple necks (Fig.14P). Moreover, the newly described Par. cocoonihabita exhibits phialides that arise directly from mycelium, displaying akanthomyces-like phialides (Fig. 14R). This remarkable morphological consistency, coupled with intriguing variations in conidial production and phialidic complexity, underscores Paraisaria as a distinct and evolutionarily cohesive genus. The observed diversity in reproductive structures, particularly the development of polyphialidic and dimorphic conidia, likely reflects sophisticated adaptations to their specific hosts and ecological niches, offering a fascinating glimpse into the evolutionary trajectory of fungal pathogenicity within the cordycipitoid clade. In addition, the genus displays a broad host range, parasitizing insects from Lepidoptera, Orthoptera, Coleoptera, Hymenoptera, Hemiptera, Diptera, and Blattodea, and has also been recorded from soil.

SUPPLEMENTARY MATERIAL

Data S1

Multilocus alignment in FASTA format used to generate the phylogenetic trees.