Uncovering cryptic species diversity of Ophiocordyceps (Ophiocordycipitaceae) associated with Coleoptera from Thailand

S Mongkolsamrit; W Noisripoom; K Tasanathai; A Khonsanit; D Thanakitpipattana; S Lamlertthon; W Himaman; PW Crous; M Stadler; JJ Luangsa-ard

doi:10.3114/fuse.2024.14.15

. 2024 Jul 26;14:223–250. doi: 10.3114/fuse.2024.14.15

Uncovering cryptic species diversity of Ophiocordyceps (Ophiocordycipitaceae) associated with Coleoptera from Thailand

S Mongkolsamrit ¹, W Noisripoom ¹, K Tasanathai ¹, A Khonsanit ¹, D Thanakitpipattana ¹, S Lamlertthon ², W Himaman ³, PW Crous ^4,^5,⁶, M Stadler ⁷, JJ Luangsa-ard ^1,^*

PMCID: PMC11736259 PMID: 39830293

Abstract

This study advances our understanding of Ophiocordyceps, an extensively studied entomopathogenic fungus within the Ophiocordycipitaceae, particularly in Thailand. We introduce seven novel species associated with Coleoptera – O. albostroma, O. brunnea, O. capilliformis, O. kohchangensis, O. phitsanulokensis, O. pseudovariabilis, and O. ratchaburiensis. Remarkably, O. brunnea, O. kohchangensis, and O. ratchaburiensis exhibit ascomata on the subterminal region of the stromata, with the asexual form appearing at the apex of the stipe, reminiscent of O. brunneipunctata. In contrast, O. phitsanulokensis produces its ascomata in the upper region of the stipe. Shared traits include immersed perithecia and part-spores production. Ophiocordyceps albostroma and O. pseudovariabilis produce pseudo-immersed perithecia, with the former producing ascospores breaking into four part-spores, and the latter displaying 32 part-spores. Ophiocordyceps capilliformis is also introduced due to morphological distinctions from closely related species. Phylogenetic analyses based on multigene loci (LSU, TEF1, RPB1, RPB2) robustly confirm the placement of these new species within Ophiocordyceps. Additionally, we report a new record of O. clavata in Thailand.

Citation: Mongkolsamrit S, Noisripoom W, Tasanathai K, Khonsanit A, Thanakitpipattana D, Lamlertthon S, Himaman W, Crous PW, Stadler M, Luangsa-ard JJ (2024). Uncovering cryptic species diversity of Ophiocordyceps (Ophiocordycipitaceae) associated with Coleoptera from Thailand. Fungal Systematics and Evolution 14: 223–250. doi: 10.3114/fuse.2024.14.15

Keywords: Ascomycota, Coleoptera, new taxa, Ophiocordyceps, pathogenic fungi, phylogeny

INTRODUCTION

Ophiocordyceps (Ophiocordycipitaceae, Hypocreales) was established as a genus of entomopathogenic fungi by Petch (1931) with O. blattae as the type species occurring on forest cockroaches, collected in Sri Lanka. This genus is one of the most populous genera of entomopathogenic fungi with a worldwide distribution. Ophiocordyceps spp. infect a wide range of insect orders, affecting insects at various life stages, including larvae and adults, which display a diverse range of morphological characteristics, spanning from soft, tough, and dark stromata to vividly coloured ones (ranging from yellow to red). The perithecia associated with these species can either be completely embedded or superficial. Their ascospores are filiform, whole with multiple septa, or they may form part-spores. The predominant asexual morphs associated with Ophiocordyceps are hirsutella-like, followed by hymenostilbe-like and syngliocladium-like. Species in Ophiocordyceps are widely recognised as famous traditional Chinese medicine, and a rich herbal source of bioactive compounds e.g., O. formosana on Coleoptera larvae and O. sinensis on the larvae of hepialid moths (Lepidoptera) (Wang et al. 2015, 2023, Wei et al. 2021). In recent years, other species in Ophiocordyceps have attracted increasing attention as a source of biologically active compounds, for example, O. irangiensis on ants has shown promising activity against Mycobacterium tuberculosis, and polysaccharides derived from O. sobolifera on cicada nymphs showing promise for potential biomedical applications (Zhang et al. 2020, Saepua et al. 2021, Tran et al. 2021). In addition, Hirsutella citriformis is being used to control hemipteran Diaphorina citri for agricultural insect pests (Pérez-González et al. 2022, Cantú-Bernal et al. 2023).

Previous studies have revealed that many entomopathogenic fungi within Ophiocordyceps exist as diverse species complexes which makes it difficult to identify individual species using only morphological characteristics. The use of DNA sequences of conserved loci combined with morphological data has improved our knowledge of fungal phylogeny (Mongkolsamrit et al. 2020a, b, Kuephadungphan et al. 2022, Thanakitpipattana et al. 2022). Numerous studies have shown that phylogenetic analyses can help clarify the taxonomy and identification of Ophiocordyceps spp. New species of Ophiocordyceps are increasingly described based on molecular results in association with their insect hosts and ecology (Sanjuan et al. 2014, Araújo et al. 2020, Saltamachia & Araújo 2020, Tasanathai et al. 2020, Khao-ngam et al. 2021). Species in Ophiocordyceps discovered in the last decade occur on several insect orders such as Blattodea, Coleoptera, Diptera, Hemiptera, Hymenoptera, Isoptera and Lepidoptera (Luangsaard et al. 2018, Khonsanit et al. 2019, Tasanathai et al. 2019, 2022, Araújo et al. 2021, Mongkolsamrit et al. 2021, 2023).

Coleoptera, or beetles, are one of the most diverse and abundant groups of insects on Earth and are classified into 176 families, with an estimate of 350 000 to 400 000 described species of beetles worldwide (Bouchard et al. 2017). They are found in a wide range of terrestrial and freshwater ecosystems, with some species inhabiting marine environments (Perissinotto et al. 2016, Hodge et al. 2019). Many species of entomopathogenic fungi in Cordyceps sensu lato (Hypocreales, Sordariomycetes) have been found in forests occurring on Coleoptera (adults or larvae) including Blackwellomyces calendulinus, B. minutus, Cordyceps chiangdaoensis, C. nirtolii, Metarhizium clavatum, Nigelia martiale, Paraisaria phuwiangensis, Perennicordyceps ryogamiensis, among others (Negi et al. 2012, Matočec et al. 2014, Tasanathai et al. 2016, Luangsa-ard et al. 2018, Mongkolsamrit et al. 2019, 2020a, b). In two studies, Shrestha et al. (2016) and Zha et al. (2021) conducted a comprehensive review and taxonomic summary on the distribution of numerous Ophiocordyceps species associated with Coleoptera. Most of these species were originally reported from the Old World, such as O. formosana, O. kuchinaraiensis and O. sporangifera from Thailand (Xiao et al. 2019, Crous et al. 2023), O. bidoupensis from Vietnam (Zou et al. 2022) and O. rubripunctata, known from Congo and Ghana (Samson et al. 1982). However, they have also been found in the New World, including O. melolonthae and O. michiganensis from the USA. Currently, there are approximately 60 known species of Ophiocordyceps associated with Coleoptera, including both larvae and adults (www.indexfungorum.org, October 15, 2023).

In our study of the diversity of pathogenic fungi on adults and larvae of Coleoptera in Thailand, we examined fungal specimens provisionally identified as Ophiocordyceps spp. These specimens were sourced from the BIOTEC Bangkok Herbarium (BBH) and the BIOTEC Culture Collection (BCC). Applying an integrative approach, we conducted taxonomic investigations, leading to the discovery of seven new species and one new record, Ophiocordyceps clavata. All species introduced here are presented with both morphological and phylogenetic illustrations, along with the identification of their respective Coleoptera hosts.

MATERIALS AND METHODS

Collections and isolation

The fungal specimens were collected from different forests in Thailand, located in Chiang Mai, Nan, Nakhon Ratchasima, Phitsanulok, Ratchaburi and Trad Provinces following searches on the ground and in decayed wood. Specimens were placed singly in plastic boxes, returned to the laboratory, and examined under an Olympus SZ61 dissecting microscope. The protocol for isolating ascospores and conidia from fertile parts followed previous studies (Luangsa-ard et al. 2018, Mongkolsamrit et al. 2018) using potato dextrose agar (PDA) plates (PDA: freshly diced potato 200 g/L, dextrose 20 g/L, agar 15 g/L). For isolation of the sexual morph, the fertile head containing mature perithecia was placed over a fresh PDA plate to ensure it remained elevated and did not contact the agar surface. These setups were then placed in a plastic box with moist tissue paper and examined daily for discharged ascospores. In the isolation of the asexual morph, a flame-sterilised inoculation needle was used to collect conidia from sporulating structures. The conidia were transferred to PDA plates, which were then incubated in a plastic box at room temperature. Daily examinations using a dissecting microscope were conducted to observe germinated conidia. Any discharged ascospores and germinated conidia were carefully removed with a sterile needle from the agar and transferred to a fresh PDA plate without antibiotics. The cultures were deposited at the BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology, Thailand. The Coleoptera hosts were identified, based on comparison of morphological characters of the dried specimens preserved at the Insect Museum Thailand of the Department of Agriculture, Thailand. All fungal specimens were dried in an electric food dryer (50–55 °C) overnight and accessioned in the BIOTEC Bangkok Herbarium (BBH), National Biobank of Thailand.

Morphological study

Macroscopic characters were observed based on natural specimens. Important microscopic characters such as perithecia, asci, ascospores, phialides and conidia from the specimens were mounted on a microscope slide containing a drop of lactophenol cotton blue solution. The shapes, sizes, and colours of individual characters were determined and measured according to Mongkolsamrit et al. (2020b). Fungal strains were grown on malt extract yeast agar (MYA, Gibco: malt extract 20 g/L, Gibco: yeast 2 g/L, agar 15 g/L), oatmeal agar (OA, Difco: oatmeal 60 g/L, agar 12.5 g/L) and PDA agar plates at 25 °C under light/ dark conditions (L:D 14:10) for 30–35 d. Cultures were observed to compare morphological characters including synnematal characteristics, phialides, and colony pigmentation. The colours of fresh specimens and cultures incubated on MYA, OA and PDA were described and codified following the Royal Horticultural Society (RHS) Colour Chart (RHS Media 2015).

DNA extraction, amplification and sequencing

Genomic DNA was extracted either from actively growing mycelial mass of PDA or from small pieces of tissues of fungal specimens using the modified cetyltrimethylammonium bromide (CTAB) method (Doyle 1987) as previously described by Mongkolsamrit et al. (2020b). Nuclear loci, including the nuclear 28S rDNA (LSU), the translation elongation factor 1-α gene (TEF1) and the genes for RNA polymerase II largest (RPB1) and second largest (RPB2) subunits were amplified and sequenced. The primer pairs used were: LR0R/LR7 (Vilgalys & Hester 1990) for LSU, EF1-983f/ EF1-2218r for TEF1 (Rehner & Buckley 2005), CRPB1/RPB1Cr for RPB1 (Castlebury et al. 2004) and RPB2-5F2/RPB2-7Cr for RPB2 (Liu et al. 1999, O’Donnell et al. 2007). Amplification reactions were performed in 25 μL reaction volumes containing 1 × PCR buffer, 2.5 mM MgCl₂, 0.4 M betaine, 200 μM of each of the four dNTPs, 0.5 μM of each forward and reverse primer, 1 U Taq DNA polymerase (Thermo Scientific) and 50–100 ng DNA templates. Amplifications were conducted under the following conditions: 2 min at 95 °C, 34 cycles of 1 min at 95 °C, 1 min at 55 °C, 2 min at 72 °C, with 2 min 72 °C for the TEF1 region; 3 min at 94 °C, 34 cycles of 1 min at 94 °C, 1 min at 50 °C, 1.3 min at 72 °C, with 8 min 72 °C for the RPB1 and RPB2 regions. The purified PCR products were sequenced with PCR amplification primers for Sanger dideoxy sequencing (Macrogen Inc., Seoul, South Korea).

Phylogenetic analyses

The DNA sequences generated in this study were examined for ambiguous base calls using BioEdit v. 7.2.5; unambiguous sequences were then submitted to GenBank. Sequences of LSU, TEF1, RPB1 and RPB2 for alignment were collated to include sequences generated from previous studies, as shown in Table 1. Sequences were aligned using the ClustalW (Thompson et al. 1994) and manually edited employing BioEdit. The phylogenetic analyses for combined and single-locus alignments were performed using RAxML-HPC2 on XSEDE v. 8.2.12 (Stamatakis 2014) in the CIPRES Science Gateway portal, using the GTRGAMMA+I model with 1 000 bootstrap iterations. Bayesian inference (BI) of phylogenetic relationships was performed in MrBayes v. 3.2.7a (Ronquist et al. 2012), with best-fit models selected using MrModeltest v. 2.2 (Nylander 2004). The best model was GTR + G + I. Markov chain Monte Carlo (MCMC) simulations were run for 5 000 000 generations, sampling every 1 000 and discarding the first 10 % as burn-in after which the Bayesian posterior probabilities (PP) were calculated on the remaining trees. RAxML and BI output were imported into TreeView v. 1.6.6 to visualise the phylogenetic trees (Page 1996).

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 or have been generated by our group in Thailand.

Species	Strain	Host/Substratum	GenBank Accession no.^*

			LSU	*TEF1*	*RPB1*	*RPB2*
Drechmeria balanoides	CBS 250.82	Nematoda	AF339539¹	DQ522342²	DQ522388²	DQ522442²
Drechmeria gunnii	OSC 76404	Lepidoptera	AF339522 ¹	AY489616 ³	AY489650 ³	DQ522426 ²
Drechmeria sinensis	CBS 567.95	Nematoda	AF339545 ¹	DQ522343 ²	DQ522389 ²	DQ522443 ²
Drechmeria zeospora	CBS 335.80	Nematoda	AF339540 ¹	EF469062 ⁴	EF469091 ⁴	EF469109 ⁴
Hirsutella citriformis	ARSEF 1446	—	KM652106 ⁵	KM651990 ⁵	KM652031 ⁵	—
Hirsutella gigantea	ARSEF 30	—	JX566977 ⁶	JX566980 ⁶	KM652034 ⁵	—
Harposporium harposporiferum	ARSEF 5472	—	AF339519 ¹	DQ118747 ⁷	DQ127238 ⁷	—
*Ophiocordyceps albostroma*	BBH 39783	*Coleoptera*	—	OR855785	OR855805	OR855829
	BBH 39788	*Coleoptera*	OR805251	OR855786	OR855806	OR855830
Ophiocordyceps alboperitheciata	YHH 16755	Lepidoptera	MT222278 ⁸	MT222279 ⁸	MT222280 ⁸	MT222281 ⁸
Ophiocordyceps aphodii	ARSEF 5498	Coleoptera	DQ518755 ²	DQ522323 ²	—	DQ522419 ²
Ophiocordyceps araracuarensis	HUA 186135	Hemiptera	KC610769 ⁹	KC610738 ⁹	KF658665 ⁹	KC610716 ⁹
Ophiocordyceps asiana	BCC 84234	Coreidae	MW280201 ¹⁰	MW292438 ¹⁰	—	—
Ophiocordyceps barnesii	BCC 28560	Coleoptera	—	—	EU408773 ¹¹	EU418599 ¹¹
	BCC 28561	Coleoptera	—	—	EU408774 ¹¹	EU418572 ¹¹
Ophiocordyceps bidoupensis	YHH 20036	Coleoptera	OK556893 ¹²	OK556897 ¹²	OK556899 ¹²	—
	YFCC 8793	Coleoptera	OK556894 ¹²	OK556898 ¹²	OK556900 ¹²	—
Ophiocordyceps blattae	BCC 34765	Blattodea (Blattoidea)	—	MT533484 ¹³	MT533478 ¹³	—
	BCC 38241	Blattodea (Blattoidea)	MT512657 ¹³	MT533485 ¹³	MT533479 ¹³	—
Ophiocordyceps borealis	MFLU 18 0163	Coleoptera	MK863051 ¹⁴	MK860189 ¹⁴	—	—
	GACPR 16002	Coleoptera	MK863052 ¹⁴	MK860190 ¹⁴	—	—
*Ophiocordyceps brunnea*	BCC 93057	*Coleoptera*	—	OR855787	OR855807	OR855831
	BBH 49819	*Coleoptera*	—	OR855788	OR855808	OR855832
*Ophiocordyceps brunneipunctata*	BCC 48060	*Coleoptera*	—	OR855791	OR855811	—
	BCC 48686	*Coleoptera*	—	OR855792	OR855812	OR855835
	OSC 128576	Coleoptera	DQ518756 ²	DQ522420 ²	DQ522369 ²	DQ522542 ²
	BCC 2218	*Coleoptera*	—	OR855789	OR855809	OR855833
	BCC 43276	*Coleoptera*	—	OR855790	OR855810	OR855834
*Ophiocordyceps capilliformis*	BCC 82180	*Coleoptera*	—	OR855794	OR855814	OR855837
	BCC 76486	*Coleoptera*	OR805252	OR855793	OR855813	OR855836
*Ophiocordyceps clavata*	BCC 95652	*Coleoptera*	—	OR855795	OR855815	—
	BCC 95653	*Coleoptera*	—	OR855796	OR855816	OR855838
	BUO 545	Coleoptera	MH879601 ¹⁵	MH879672 ¹⁵	MH885450 ¹⁵	—
	NBRC 106961	Coleoptera	JN941414 ¹⁶	—	JN992461 ¹⁶	—
	NBRC 106962	Coleoptera	JN941415 ¹⁶	—	JN992460 ¹⁶	—
Ophiocordyceps communis	BCC 1874	Termitidae	MH753679 ¹⁷	MK284267 ¹⁷	MK214109 ¹⁷	MK214095 ¹⁷
	BCC 1842	Termitidae	MH753680 ¹⁷	MK284266 ¹⁷	MK214110 ¹⁷	MK214096 ¹⁷
Ophiocordyceps cossidarum	MFLU 17-0752	Lepidoptera	MF398187 ³³	MF928403 ³³	MF928404 ³³	—
Ophiocordyceps elongata	OSC 110989	Lepidoptera	EF468808 ⁴	EF468748 ⁴	EF468856 ⁴	—
Ophiocordyceps entomorrhiza	KEW 53484	Coleoptera	EF468809 ⁴	EF468749 ⁴	EF468857 ⁴	EF468911 ⁴
	TNS 16250	—	—	KJ878987 ²⁸	KJ879021 ²⁸	—
	TNS 16252	—	KJ878906 ²⁸	KJ878986 ²⁸	—	—
Ophiocordyceps formosana	NTU00035	Coleoptera	—	KT275192¹⁹	KT275190¹⁹	KT275191¹⁹
	MFLU 15-3889	Coleoptera	—	KU854950 ²⁰	KU854948 ²⁰	—
Ophiocordyceps furcatosubulata	YFCC 903	Coleoptera	MT774222 ²¹	MT774243 ²¹	MT774244 ²¹	MT774236 ²¹
	YFCC 904	Coleoptera	MT774223 ²¹	MT774244 ²¹	MT774230 ²¹	MT774237 ²¹
Ophiocordyceps houaynhangensis	MY11460	Coleoptera	MH092908 ²²	MH092899 ²²	—	—
	MY11461	Coleoptera	MH092909 ²²	MH092900 ²²	—	—
Ophiocordyceps humbertii	MF116A	Hymenoptera	MK875537 ²³	—	MK863828 ²³	—
	MF116B	Hymenoptera	MK875536 ²³	—	MK863829 ²³	—
Ophiocordyceps irangiensis	NBRC 101400	Hymenoptera	JN941426 ¹⁷	—	JN992449 ¹⁷	—
Ophiocordyceps khaoyaiensis	BCC 82796	Hymenoptera	MH028153 ²⁴	MH028187 ²⁴	MH028165 ²⁴	MH028175 ²⁴
*Ophiocordyceps kohchangensis*	BCC 88229	*Coleoptera*	OR805253	—	OR855817	—
	BCC 90828	*Coleoptera*	OR805255	—	OR855819	OR855840
	BCC 90811	*Coleoptera*	OR805254	OR855797	OR855818	OR855839
Ophiocordyceps konnoana	EFCC 7315	Coleoptera	—	EF468753 ⁴	EF468861 ⁴	EF468916 ⁴
	EFCC 7295	Coleoptera	—	—	EF468862 ⁴	EF468915 ⁴
Ophiocordyceps krachonicola	BCC 79667	Coleoptera	MK632081 ²⁵	MK632055 ²⁵	MK632162 ²⁵	MK632133 ²⁵
	BCC 79666	Coleoptera	MK632080 ²⁵	MK632054 ²⁵	MK632161 ²⁵	MK632132 ²⁵
Ophiocordyceps kuchinaraiensis	BCC 95830	Coleoptera	OQ627397 ²⁶	OQ625474 ²⁶	—	OQ625475 ²⁶
Ophiocordyceps langbianensis	DL 0017	Coleoptera	MT928306 ²⁷	—	—	—
Ophiocordyceps longissima	HMAS 199600	—	—	KJ878972 ²⁸	KJ879006 ²⁸	KJ878949 ²⁸
	EFCC 6814	Hemiptera	EF468817 ⁴	EF468757 ⁴	EF468865 ⁴	−
Ophiocordyceps megacuculla	BCC 82262	Hymenoptera	MH028161 ²⁴	MH028191 ²⁴	MH028172 ²⁴	MH028180 ²⁴
Ophiocordyceps myrmicarum	ARSEF 11864	Hemiptera	JX566965⁶	JX566973⁶	KJ680151⁶	−
Ophiocordyceps nigrella	EFCC 9247	Lepidoptera	EF468818 ⁴	EF468758 ⁴	EF468866 ⁴	EF468920 ⁴
	OSC 110994	Hemiptera	—	DQ522333 ²	DQ522378 ²	—
*Ophiocordyceps phitsanulokensis*	BCC 85323	*Coleoptera*	OR805256	—	OR855820	OR855841
	BCC 85324	*Coleoptera*	—	—	OR855821	OR855842
	BCC 85328	*Coleoptera*	OR805257	OR855798	OR855822	OR855843
Ophiocordyceps pseudorhizoidea	NHJ12522	Termite	EF468825 ⁴	EF468764 ⁴	EF468873 ⁴	EF468923 ⁴
	NHJ12529	Termite	EF468824 ⁴	EF468765 ⁴	EF468872 ⁴	EF468922 ⁴
*Ophiocordyceps pseudovariabilis*	BCC 88308	*Coleoptera*	—	OR855799	OR855823	—
	BCC 88311	*Coleoptera*	—	OR855800	OR855824	OR855844
Ophiocordyceps purpureostromata	TNSF18430	Coleoptera	KJ878897 ²⁸	KJ878977 ²⁸	KJ879011 ²⁸	—
Ophiocordyceps ravenelii	OSC 110995	Coleoptera	DQ518764 ²	DQ522334 ²	DQ522379 ²	DQ522430 ²
Ophiocordyceps salganeicola	Mori01	Blattodea	MT741719 ²³	MT759575 ²³	MT759578 ²³	MT759580 ²³
	Mori02	Blattodea	MT741718 ²³	MT759572 ²³	MT759579 ²³	MT759581 ²³
Ophiocordyceps sobolifera	TNSF 18521	Hemiptera	KJ878898 ²⁸	KJ878979 ²⁸	KJ879013 ²⁸	—
	KEW 78842	Hemiptera	EF468828 ⁴	—	EF468875 ⁴	EF468925 ⁴
Ophiocordyceps sphecocephala	NBRC 101416	—	JN941443 ¹⁶	—	JN992432 ¹⁶	—
Ophiocordyceps spicatus	MFLU 18-0164	Coleoptera	MK863054 ¹⁴	MK860192 ¹⁴	—	—
Ophiocordyceps sporangifera	MFLUCC 18-0492	Lepidoptera	MH725832¹⁸	MH727390¹⁸	MH727392¹⁸	—
MFLU 18-0658	Lepidoptera	MH725831¹⁸	MH727389¹⁸	MH727391¹⁸	—
Ophiocordyceps termiticola	BCC 1770	Termitidae	MH753677 ¹⁷	MK284264 ¹⁷	MK214107 ¹⁷	MK214093 ¹⁷
	BCC 1920	Termitidae	MH753678 ¹⁷	MK284265 ¹⁷	MK214108 ¹⁷	MK214094 ¹⁷
Ophiocordyceps tessaratomidarum	MY 10830	Tessaratomidae	MW280218 ¹⁰	MW292434 ¹⁰	—	—
*Ophiocordyceps ratchaburiensis*	BCC 48035	*Coleoptera*	OR805258	OR855803	OR855827	OR855847
	BCC 48033	*Coleoptera*	OR805259	OR855802	OR855826	OR855846
	BCC 48038	*Coleoptera*	—	OR855804	OR855828	OR855848
Ophiocordyceps variabilis	OSC 111003	Dipteran	EF468779 ⁴	EF468885 ⁴	—	EF468933 ⁴
	ARSEF 5365	Dipteran	DQ522340 ²	DQ522386 ²	—	DQ522437 ²
Ophiocordyceps yakusimensis	HMAS 199604	Hemiptera	KJ878902 ²⁸	—	KJ879018 ²⁸	KJ878953 ²⁸
Paraisaria amazonica	HUA 186143	Orthoptera	KJ917571 ⁹	KM411989 ⁹	KP212902 ⁹	KM411982 ⁹
	HUA 186136	Orthoptera	—	KM411991 ⁹	KP212905 ⁹	—
Paraisaria gracilis	EFCC 3101	Lepidoptera	EF468810 ⁴	EF468750 ⁴	EF468858 ⁴	EF468913 ⁴
	EFCC 8572	Lepidoptera	EF468811 ⁴	EF468751 ⁴	EF468859 ⁴	EF468912 ⁴
Paraisaria heteropoda	EFCC 10125	Hemiptera	EF468812 ⁴	EF468752 ⁴	EF468860 ⁴	EF468914 ⁴
	OSC 106404	Hemiptera	AY489722³	AY489617³	AY489651³	—
Paraisaria phuwiangensis	TBRC 9709	Coleoptera	MK192057 ²⁹	MK214082 ²⁹	MK214086 ²⁹	—
Polycephalomyces formosus	CGMCC 5-2203	Coleoptera	MN586844 ³⁰	MN598059 ³⁰	MN598050 ³⁰	MN598066 ³⁰
	CGMCC 5-2204	Coleoptera	MN586839 ³⁰	MN598054 ³⁰	MN598045 ³⁰	MN598061 ³⁰
Purpureocillium lilacinum	CBS 431.87	Nematoda	EF468844 ⁴	EF468791 ⁴	EF468897 ⁴	EF468940 ⁴
	CBS 284.36	Soil	AY624227 ³¹	EF468792 ⁴	EF468897 ⁴	EF468940 ⁴
	ARSEF 2181	—	AF339534 ¹	EF468790 ⁴	EF468896 ⁴	—
Tolypocladium inflatum	OSC 71235	Coleoptera	EF469077 ⁴	EF469061 ⁴	EF469090 ⁴	EF469108 ⁴
Tolypocladium ophioglossoides	NBRC 8992	Elaphomyces sp.	JN941405 ¹⁶	—	JN992470 ¹⁶	—
Tolypocladium paradoxum	NBRC 100945	Hemiptera	JN941410 ¹⁶	—	JN992465 ¹⁶	—
Torrubiellomyces zombiae	NY 04434801	O. camponoti-floridani	ON493602 ³²	ON513396 ³²	ON513398 ³²	ON513402 ³²

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^*:

¹Sung et al. (2001),

²Spatafora et al. (2007),

³Castlebury et al. (2004),

⁴Sung et al. (2007),

⁵Simmons et al. (2015a),

⁶Simmons et al. (2015b),

⁷Chaverri et al. (2005),

⁸Fan et al. (2021),

⁹Sanjuan et al. (2015),

¹⁰Khao-ngam et al. (2021),

¹¹Luangsa-Ard et al. (2010a),

¹²Zou et al. (2022),

¹³Mongkolsamrit et al. (2021),

¹⁴Zha et al. (2021),

¹⁵Chen et al. (2019),

¹⁶Schoch et al. (2012),

¹⁷Tasanathai et al. (2019),

¹⁸Xiao et al. (2019),

¹⁹Wang et al. (2015),

²⁰Li et al. (2016),

²¹Wang et al. (2021a),

²²Crous et al. (2018),

²³Araujo et al. (2020),

²⁴Khonsanit et al. (2019),

²⁵Thanakitpipattana et al. (2020),

²⁶Crous et al. (2023),

²⁷Lao et al. (2021),

²⁸Quandt et al. (2014),

²⁹Mongkolsamrit et al. (2019),

³⁰Wang et al. (2021b),

³¹Luangsa-ard et al. (2005),

³²Araújo et al. (2022),

³³Hyde et al. (2017).

RESULTS

Molecular phylogeny

A total of 70 new sequences were generated (9 LSU, 19 TEF1, 23 RPB1 and 19 RPB2) in this study (Table 1). The combined dataset comprised 109 taxa, with multi-loci sequences totalling an alignment length of 3 589 characters (bps), including gaps (LSU: 972 bps, TEF1: 979 bps, RPB1: 753 bps and RPB2: 885 bps). Polycephalomyces formosus (CGMCC5.2204 and CGMCC5.2203) in Polycephalomycetaceae (Xiao et al. 2023) were used as outgroups. 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 (BPP). In the generated phylogenetic tree (Fig. 1), the family Ophiocordycipitaceae is represented by seven genera and seven subclades of Ophiocordyceps including 1) “O. ravenelii” (< 50 % MLB/0.85 BPP), 2) “O. entomorrhiza” (100 % MLB/1 BPP), 3) “O. purpureostromata” (100 % MLB/1 BPP), 4) “O. blattae” (95 % MLB/1 BPP), 5) “O. elongata” (99 % MLB/1 BPP), 6) “O. sphecocephala” (100 % MLB/1 BPP) and 7) “O. sobolifera” (76 % MLB/0.92 BPP) clades. Individual gene trees were generated using the same analytical tools as above and are shown in Supplementary Figs S1–S4. The sequence alignments for all datasets employed in this study were uploaded to Figshare at https://10.6084/m9.figshare.24495169.

Fig. 1 — RAxML tree of *Ophiocordyceps* and related genera in the *Ophiocordycipitaceae* from a combined LSU, *TEF1*, *RPB1* and *RPB2* dataset. Numbers at the major nodes represent Maximum Likelihood Bootstrap (MLB) support values and Bayesian Posterior Probabilities (BPP).

The results from multi-locus phylogenetic analyses strongly support the placement of O. clavata and O. pseudovariabilis within the “O. ravenelii” clade, while O. albostroma and O. capilliformis are nested in the “O. purpureostromata” and “O. elongata” clades, respectively. The four newly identified taxa, namely O. brunnea, O. kohchangensis, O. phitsanulokensis, and O. ratchaburiensis, along with known species O. aphodii, O. bidoupensis, O. houaynhangensis, O. furcatosubulata, O. cossidarum, O. langbianensis, form distinct lineages within the “O. brunneipunctata” species complex subclade, which is part of the “O. sobolifera” clade. Based on these results, species descriptions are provided for seven novel species, including O. clavata, which is which is a newly recorded species.

Taxonomy

Ophiocordyceps albostroma Khonsanit, Mongkolsamrit, Noisripoom & Luangsa-ard, sp. nov. MycoBank MB 851828. Fig 2.

Etymology: Latin “albus” means white, referring to the colour of the stipes of fresh specimens.

Typus: Thailand, Chiang Mai Province, Chiang Dao Wildlife Sanctuary, Doi Chiang Dao Wildlife Research Station, on Coleoptera larva, buried in soil, 31 Oct. 2014, A. Khonsanit, D. Thanakitpipattana, K. Tasanathai, S. Mongkolsamrit & W. Noisripoom (holotype BBH 39783).

Sexual morph: Stromata solitary or several, cylindrical, gradually tapering toward the apex, yellowish white (155D), arising from head, thorax and abdomen of Coleoptera larvae, 30–60 mm long. Stipes cylindrical, smooth, white to white cream, 0.5–2 mm wide. Fertile parts developing at middle to upper region of stipes, occasionally produced on one side of surface of stipes, light yellow (17D). Perithecia pseudo-immersed, tightly packed, ovoid, (390–)406–456(–490) × (220–)235.5–267.5(–295) μm. Asci cylindrical, (123–)155–213(–250) × (4–)5–6.5(–8) μm with asci caps, 2–3 × 3.5–4 μm. Ascospores filiform, (118–)134–160(– 178) × 1–2 μm, breaking into 4 part-spores, (25–)31–44(–55) × 1–2 μm. Asexual morph: Unknown.

Distribution: Found in the northern region of Thailand.

Additional materials examined: Thailand, Chiang Mai Province, Chiang Dao Wildlife Sanctuary, Doi Chiang Dao Wildlife Research Station, on Coleoptera larvae, buried in soil, 31 Oct. 2014, A. Khonsanit, D. Thanakitpipattana, K. Tasanathai, S. Mongkolsamrit & W. Noisripoom (specimens BBH 39781; BBH 39782; BBH 39784; BBH 39785; BBH 39786; BBH 39787; BBH 39788; BBH 39789; BBH 39790).

Notes: This is a fastidious species, and no cultures could be obtained from any of the collected specimens, hence DNA extraction was performed on the stroma. Ophiocordyceps albostroma has exclusively been found in the Chiang Dao Wildlife Sanctuary in northern Thailand. The phylogenetic analyses presented in Fig. 1 clearly show a close genetic affinity between O. albostroma and its counterparts, O. purpureostromata and O. borealis. The latter two were originally found on Elateridae larvae in Japan and Russia, respectively (Kobayasi & Shimizu 1980, Zha et al. 2021). A distinctive characteristic among these species is the densely packed structures of perithecia on the stipes, occasionally found on one side of the stipes. However, they notably differ in stromata coloration: O. albostroma exhibits white to cream stromata, O. purpureostromata displays purple stromata, and O. borealis produces grey stromata. These three species share the presence of part-spores. Ophiocordyceps albostroma produces ascospores that break into four part-spores, similar to O. purpureostromata, while the number of part-spores for O. borealis is not indicated. However, the part-spores in O. albostroma are longer than in the other two species (Table 2).

Table 2.

Morphological comparisons between Ophiocordyceps species occurring on Coleoptera and other hosts.

Species	Host	Stromata/Synnemata	Perithecia	Asci	Ascospores	Conidiogenous cells	Conidia	Reference
Ophiocordyceps albostroma	Larva of Coleoptera	Solitary, several, cylindrical, yellowish white, 30–60 mm long, 0.5−2 mm wide	Pseudo-immersed, 390–490 × 220–295 μm	Cylindrical, 123–250 × 4–8 μm	Filiform, breaking into 4 part-spores 25–55 × 1–2 μm	—	—	This study
O. alboperitheciata	Larva of Noctuidae (Lepidoptera)	Two, brown to dark brown, unbranched, 69–71 × 0.6–1.2 mm	Superficial, 408–549 × 233–321 μm	Cylindrical, 144–246 × 3.5–4.7 μm	Cylindrical, multiseptate, 1.1–1.3 × 0.5–0.6 μm. Part-spores were not seen.	—	—	Fan et al. (2021)
O. bidoupensis	Larva of Elateridae (Coleoptera)	Solitary, solid, cylindrical, yellow, 11.8–22.5 cm long	Immersed, pyriform to lanceolate, brown-yellow, 213.4–405.9 × 74.8–192.4 μm	Slender, 116.1–192.7 × 4.8–7.5 μm	Filiform, multiseptate	Cone, hyaline, spetate, smooth-walled, forming on hyphae, with a hypertrophic base, tapering abruptly into a thin neck, smooth-walled, 13.8–46.4 × 0.42–5.13 μm	Oval or briolette, 2.24–3.61 × 1.49–2.70 μm	Zou et al. (2022)
O. borealis	Larva of Elateroidea (Coleoptera)	Single or paired, grey, 10–13 mm long, 0.25–0.6 mm wide	Immersed, obliquely or at right angles to the surface of stipe, pyriform, 220–290 × 120–150 μm	Cylindrical, 6–8 μm wide	Cylindrical, part-spore, 10–15 × 2 μm	—	—	Zha et al. (2021)
O. brunnea	Larva of Elateridae (Coleoptera)	Solitary, or pairs, cylindrical, gradually tapering toward the apex, brownish orange, 20–35 mm long, 0.7–1 mm wide	Completely immersed, obpyriform, 450–500 × 150–240 μm	Cylindrical, 125–200 × 5–8 μm	Cylindrical, 125–195 μm long, breaking into 32 part-spores, 4–6 × 1–2 μm	Monophialidic, polyphialidic, cylindrical basal portion, 5–22 × 3–5 μm with a distinct, filiform, long neck up to 21 μm long	Globose, 3–5 μm diam.	This study
O. brunneipunctata	Larva of Elateridae (Coleoptera)	Solitary, rarely up to 3, simple, 25–90 mm high	Immersed, ovate to pyriform, brown-walled, 270–335 × 110–160 μm	Cylindric, 280–295 × 6–7 μm	Filiform, multiseptate, 4–6 × 1–1.5 μm	Monophialidic, rarely polyphialidic, hyaline, 5.5–7.5 × 2.5–3 μm at the base, cylindrical base, up to 15 × 0.5 μm above	Spherical, 1.5–2.5 μm diam., enveloped by a mucous sheath	Hywel-Jones (1995)
O. capilliformis	Larva of Meracantha sp. (Tenebrionidae, Coleoptera), adult of Platydracus sp. (Staphylinidae, Coleoptera)	Solitary, greyish reddish brown to moderate brown 80–100 mm long, 0.8–1.2 mm wide. Synnemata indeterminate, multiple, produced along the stipe below the ascomata, cylindrical, up to 8 mm long	Completely immersed, pyriform, 395–420 × 190–250 μm	Cylindrical, 125–200 × 5–8 μm	Whole, filiform, 130–210 × 2–3 μm with 5- or 6-septa	Hirsutella-like, solitary, cylindrical to ovoid basal portion, 29–33 × 4–5 μm, tapering to a narrow neck up to 25–28 μm long	Fusiform, occasionally slightly curved, 9–15 × 3–8 μm with a mucous sheath	This study
O. clavata	Larva of Tenebrionidae (Coleoptera)	Multiple, clavate, pale yellow, 2–10 mm long, 0.5–1.5 mm wide	Immersed, broad ovoid, 400–500 × 250–350 μm	Cylindrical, 200–300 × 6–8 μm	Cylindrical, 200–250 μm long, breaking into 32 part-spores, 5–10 × 1.5–3 μm	—	—	This study
O. cossidarum	Larva of Cossidae (Lepidoptera)	Solitary, simple, 40–70 mm high	Immersed, red, ovate to phialide, red-walled, 355–454 × 136–171 μm	Cylindrical, 8-spores with a thickened apex, 174–221 × 5.7–7 μm	Filiform, multiseptated, 131–153 × 1.8–2.2 μm, breaking into 32 part-spores	—	—	Hyde et al. (2017)
O. elongata	Pupae and Larva of Apatela americana (Lepidoptera)	The stalk is flexuose, longitudinally sulcate and twisted, 110 mm long	Immersed, ovatoconoid, 0.5 × 0.3 mm	220 μm long, 8 μm wide	Cylindrical, 2 um wide, with septa, 4–12 μm apart. Part-spores were not seen.	—	—	Petch (1937)
O. formosana	Larva of Tenebrionoidea (Coleoptera)	Several, yellow to orange, 14 mm, 2-5 mm wide, simple or branched	Completely immersed, 453–546 × 265–298 μm	366–498 × 8–11 μm	Cylindrical, part-spores, 2–6 × 1–3 μm	—	—	Li et al. (2016)
O. furcatosubulata	Larva of Elateridae (Coleoptera)	Single, solid, yellow to brown, 40–80 mm long, 1.5–2.2 mm wide	Immersed, long ovoid or pyriform, 289.6–405.8 × 87–159.2 μm	Cylindrical, 138.8–202.5 × 4.3–6 μm	Filiform, multiseptated, finally breaking into secondary ascospores, 3.7–5.3 × 1.3–2 μm	Polyphialidic, forming on conidiophores or side branches, with a slender or subulate base, tapering gradually, smooth-walled or verruculose, 3.5 –15.8 × 0.9–1.7 μm	Solitary, aseptate, smooth-walled, broadly ellipsoid or ellipsoid, 1.5–2.5 × 1.2–1.9 μm	Wang et al. (2021a)
O. houaynhangensis	Larva of Coleoptera	Solitary, cylindrical, cream, up to 11 cm long, 1.5–2.5 mm wide	Completely immersed, obclavate, 300–450 × 80–170 μm	Cylindrical, 100–250 × 4–7.5 μm	Cylindrical, part-spores, 4–7 × 1–2 μm	Monophialidic, phialides flasked-shaped with long necks, up to 30 μm long and 2–4 μm in breadth; phialide necks up to 18 μm long and 0.5 μm in breadth	Spherical, 2–3 μm	Crous et al. (2018)
O. kohchangensis	Larva of Elateridae (Coleoptera)	Solitary, cylindrical, gradually tapering toward the apex, moderate yellow to light yellow, up to 75 mm long, 1–1.5 mm wide	Completely immersed, pyriform, 320–400 × 100–200 μm	Cylindrical, 90–200 × 4–6 μm	Cylindrical, breaking into 32 part-spores, 4–7 × 1.5–2 μm	Hirsutella-like, monophialidic or polyphialidic, 5–40 × 3–5 μm with a distinct, filiform, long neck up to 25 μm long	Globose, 2–4 μm	This study
O. krachonicola	Nymph of Gryllotalpa orientalis (Gryllidae)	Solitary, cylindrical, dark grey, 37–40 × 1–1.5 mm	Completely immersed, ovoid to obclavate, 460–580 × 180–300 μm	Cylindrical, 250–400 × 4–5 μm	Cylindrical, breaking into 64 part-spores, 4–10 × 1 μm	—	—	Thanakitpipattana et al. (2020)
O. kuchinaraiensis	Larva of Coleoptera	Single, or double, brown, cylindrical, dark brown to black, slightly curved, 5.5–10 cm long, 2–3 mm wide	Semi-immersed, obclavate, 630–820 × 210–300 μm	Cylindrical, 305–525 × 4.5–5.5 μm	filliform, multiseptate, 430–615 × 1–2 μm, breaking into 64 part-spores, 5–20 × 1–2 μm	—	—	Crous et al. (2023)

O. langbianensis	Larva of Coleoptera	Solitary, cylindrical, 40–100 mm long, pale yellow, 0.7–1 mm wide,	Immersed, ovate or pyriform, 260–400 × 100–190 μm	Cylindrical, 200–250 × 5–6 μm	Cylindrical, part-spores, 5–7.5 × 1.3–2 μm	—	—	Lao et al. (2021)
O. phitsanulokensis	Larva of Elateridae (Coleoptera)	Solitary, multiple, cylindrical, greyish red to reddish brown, up to 15 cm long, 1–2 mm wide	Completely immersed, narrowly ovoid, 480–550 × 110–170 μm	Cylindrical, up to 250 μm long, 5–7 μm wide	Cylindrical, slightly curved, breaking into 32 part-spores, 5–8 × 1–1.5 μm	Hirsutella-like, polyphilalidic, cylindrical to subcylindrical basal part, occasionally slightly curved, 5–15 × 2.5–5 μm with a short neck up to 2–5 μm long	Fusoid, 5–8 × 1–2 μm	This study
O. pseudovariabilis	Lycidae larva (Coleoptera)	Solitary, multiple, brownish orange to moderate orange, 4–18 mm long, 0.5–1 mm wide	Pseudo-immersed, pyriform, 380–480 × 220–320 μm	Cylindrical, up to 360 μm long, 5–8 μm wide	Cylindrical, breaking into 32 small truncate part-spores, 5–10 × 1–1.5 μm	Hirsutella-like, monophialidic, polyphialidic, cylindrical basal portion, 4–15 × 2–5 μm	Fusoid, 4–8 × 0.5–1 μm	This study
O. purpureostromata	Larva of Elateridae (Coleoptera)	Cylindrical, purple, 7–23 mm, 0.6–1 mm wide	Immersed, pyriform, 430–450 × 220–250 μm	150–160 × 10 μm	13–23 × 2.5–3 μm	—	—	Kobayasi & Shimizu (1980)
O. ratchaburiensis	Larva of Coleoptera	Solitary, or pairs, cylindrical, gradually tapering toward the apex, strong orange yellow to moderate yellow, up to 25 mm long, 0.5–1 mm wide	Completely immersed, pyriform, 220–530 × 110–280 μm	Cylindrical, 153–225 × 5–10 μm	Cylindrical, part-spores, 3–9 × 1.5–2 μm	Monophialidic, polyphialidic, cylindrical basal portion, 8–19 × 3–4 μm with a distinct, filiform, long neck up to 20 μm	Globose, subglobose, 2–3.5 × 2–3 μm, a mucous sheath observed in mature conidia	This study
O. spicatus	Tenebrionoidea larva (Coleoptera)	Single, yellow, 5 mm in length	Partially immersed, obliquely or at right angles to the surface of stipe, broadly pyriform, 200–250 × 170–200 μm	Cylindrical, 5–9 μm	Cylindrical, part-spores cylindrical, 3.5–6.5 × 1.7–2 μm	—	—	Zha et al. (2021)

O. sporangifera	Larva of Elateridae (Coleoptera)	Primary synnemata; 9–18 cm high, 1–2 mm wide, brown to deep brown	—	—	—	Hirsutella-like, solitary, 25–40 × 1.3–2.5 μm, with narrow	Subglobose, reniform, 6.7–9.8 × 2.5–3.8	Xiao et al. (2019)
O. variabilis	Xylophagidae (Diptera)	Solitary, several, chrome yellow, 2–24 mm long, 0.2–3 mm wide	Immersed, obpyriform, 350–590 × 200–370 μm	Cylindrical, 210–330 × 6 μm	Cylindrical, part-spores cylindrical, 5–10 × 1.5–3 μm	Strongly hooked or bent, 10–19.2 × 2.5 μm, with subcylindric base and abruptly narrowing to form a short, tapering neck	Subcylindrical, 8–12.4 × 1.9–3.1 μm	Hodge et al. (2016)

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Ophiocordyceps brunnea Mongkolsamrit, Himaman, Noisripoom & Luangsa-ard, sp. nov. MycoBank MB 851829. Fig. 3.

Etymology: Referring to the brown colour of the ascomata.

Typus: Thailand, Nan Province, Ban Sali deciduous dipterocarp forest trail, on Elateridae larva (Coleoptera), buried in soil, 6 Aug. 2020, B. Sakolrak & W. Himaman (holotype BBH 49817, culture ex-type BCC 93057).

Sexual morph: Stromata solitary or in pairs, cylindrical, gradually tapering toward the apex, brownish orange (166B–C), 20–35 mm long. Stipes cylindrical, smooth, pale yellow (161D), 0.7–1 mm wide. Rhizoids flexuous, 2–3 cm long buried under the ground, arising from head and thorax of Coleoptera larvae. Fertile parts distinctly subterminal with asexual morph at the apex. Ascomata subterminal, cylindrical, moderate reddish brown to brownish orange (166B–C), crowded perithecia, 2–4 mm long, 1.2–2 mm wide. Perithecia completely immersed, obpyriform, (450–)455–485(–500) × (150–)160–220(–240) μm. Asci cylindrical, (125–)135–185(–200) × 5–8 μm with asci caps 1.5–2 × 5 μm. Ascospores cylindrical, 125–195 μm long, breaking into 32 small truncate part-spores, 4–5(–6) × 1–1.5(–2) μm. Asexual morph: Conidiogenous cells hirsutella-like, mono- or polyphialidic, cylindrical basal portion, 5–22 × 3–5 μm with a distinct, filiform, long neck up to 21 μm long. Conidia hyaline, smooth-walled, globose, 3–5 μm diam.

Culture characteristics: Colonies on MYA attaining a diam of 6–8 mm in 30 d, light yellowish pink (36A), colonies reverse dark greyish yellow brown (N199B). Conidiogenous cells hirsutellalike, phialidic, arising from either the lateral or terminal regions of the hyphae, cylindrical or ellipsoidal basal portion, 10–22(–25) × 3–4.5(–5) μm with a distinct, filiform, long neck up to 15 μm long. Conidia hyaline, smooth-walled, globose, 2–2.5(–3) μm diam. Poor sporulation was observed. Colonies on OA attaining a diam of 10–16 mm in 30 d, yellowish white (156D) to greyish red (182B), colonies reverse dark greyish yellow brown (N199B), sparse synnemata producing on edge of colonies, white, 2–3 mm long, 200–300 μm wide. Conidiogenous cells hirsutella-like, phialidic, emerging along the synnemata, subglobose, cylindrical or ellipsoidal basal portion, (8–)10.5–21.5(–25) × 3–4.5(–5) μm with a distinct, filiform, long neck up to 20 μm long. Conidia hyaline, smooth-walled, globose, 2–3 μm diam. Colonies on PDA attaining a diam of 10–15 mm in 30 d, white, greyish red (182B) in the centre of colonies, colonies reverse white and dark greyish yellow brown (N199B) in the centre of colonies. Conidiogenous cells hirsutella-like, phialidic, arising from either the lateral or terminal regions of the hyphae, cylindrical or ellipsoidal basal portion, (9.5–)10–17.5(–20) × 2–4 μm with a distinct, filiform, long neck up to 18 μm long. Conidia hyaline, smooth-walled, globose, 2–3 μm diam. Poor sporulation was observed.

Distribution: Found in the northern region of Thailand.

Additional materials examined: Thailand, Nan Province, Ban Sali deciduous dipterocarp forest trail, on Coleoptera larva, buried in soil, 6 Aug. 2020, B. Sakolrak & W. Himaman (BBH 48915, paratype, culture ex-paratype BCC 93059); idem., (BBH 49818, culture BCC 93058); idem., (BBH 49819).

Notes: Based on macro-morphology in the field, O. brunnea appears similar to O. brunneipunctata and O. ratchaburiensis as it produces ascomata on the subterminal region of the stroma, with the asexual form appearing at the apex of the stipe. All three species have immersed perithecia, and their ascospores break into 32 part-spores. However, molecular phylogenetic analysis (Fig. 1) strongly supports the close relationship between O. brunnea and O. ratchaburiensis, but not forming a monophyletic clade with O. brunneipunctata. When comparing the colour of the stromata, those of O. ratchaburiensis are pale yellow, while those of O. brunnea range from brown to light brown. Furthermore, when examining the characteristics of colonies on three different media (MYA, OA, and PDA), it is observed that O. brunnea and O. ratchaburiensis generally produce reproductive structures on those media. However, an exception is noted for O. ratchaburiensis on MYA, where it exhibits flattened white mycelium without any accompanying reproductive structures (Supplementary Fig. S5). Ophiocordyceps brunnea produces sparse synnemata on OA, a characteristic not observed in O. ratchaburiensis. Conversely, synnemata were observed in PDA for O. ratchaburiensis, a feature not observed in O. brunnea.

Ophiocordyceps capilliformis Tasanathai, Mongkolsamrit, Noisripoom & Luangsa-ard, sp. nov. MycoBank MB 851830. Fig 4.

Etymology: Refers to synnemata of the fungus which exhibit an outer appearance resembling hair.

Typus: Thailand, Phitsanulok Province, Ban Phaothai community forest, on larva of Meracantha sp. (Tenebrionidae, Coleoptera), buried in soil, 3 Aug. 2016, K. Tasanathai, S. Mongkolsamrit, A. Khonsanit, W. Noisripoom, D. Thanakitpipattana & R. Somnuk (holotype BBH 41761, culture ex-type BCC 82180).

Sexual morph: Stromata solitary, greyish reddish brown (200B) to moderate brown (200C), 80–100 mm long. Stipe slender, slightly curved, 0.8–1.2 mm wide. Rhizoids flexuous, 3–4 cm long buried under the ground, arising from head of larva of Meracantha sp. Ascomata on upper part of the stipe, cylindrical, strong yellowish brown (N199D), densely packed perithecia, 20–25 mm long, 1.5–2 mm wide. Perithecia completely immersed, pyriform, (395–)400–410(–420) × 190–240(–250) μm. Asci cylindrical, (125–)148–193(–200) × 5–8 μm with asci caps, 3–5 × 5–7 μm. Ascospores whole, filiform, (130–)143–190(–210) × 2–3 with 5- or 6-septa. Asexual morph: Synnemata indeterminate, multiple, slender projections produced along the stipe below the ascomata, light brownish grey (200C), or light yellow (162C), up to 8 mm long, 80–120 mm wide. Conidiogenous cells hirsutellalike, solitary, arising individually from synnemata, cylindrical to ovoid basal portion, 29–33 × 4–5 μm, tapering to a narrow neck up to 25–28 μm long. Conidia hyaline, smooth-walled, fusiform, occasionally slightly curved, (9–)10–13(–15) × 3–5(–8) μm with a mucous sheath. Some strains are found only producing their asexually reproductive form by producing abundant synnemata along the stipes.

Culture characteristics: Colonies on MYA attaining a diam of 18–20 mm in 30 d, pale yellow (161D), strong orange yellow (163B) in the centre of colonies, colonies reverse strong orange yellow (163B). Conidiogenous cells monophialidic, arising from either the lateral or terminal regions of the hyphae, up to 50 μm long, 2–3 μm wide, narrow cylindrical basal portion, gradually tapering from the base to the apex. Conidia hyaline, smooth-walled, fusiform, (7–)9–12(–14) × 2–3.5(–5) μm. Colonies on OA attaining a diam of 15–20 mm in 30 d, white, moderate brown (200C) in the centre of colonies, colonies reverse moderate brown (200C). Synnemata multiple, slender, moderate brown, arising from the centre of colonies, up to 20 mm long, 100– 200 μm wide. Conidiogenous cells monophialidic, arising from terminal region of the hyphae. Conidia hyaline, smooth-walled, fusiform, 9–11(–12) × 2–3 μm, a mucous sheath observed in mature conidia. Colonies on PDA attaining a diam of 12–15 mm in 30 d, white, brownish orange (166C) in the centre of colonies, colonies reverse brownish orange (171B). Conidiogenous cells monophialidic, arising from terminal region of the hyphae. Conidia hyaline, smooth-walled, fusiform, (9–)10–12(–13) × 2–3(–4) μm, a mucous sheath observed in mature conidia.

Distribution: Found in the central and northern regions of Thailand Thailand.

Additional material examined: Thailand, Chiang Mai Province, Ban Hua Thung community forest, on adult beetle of Platydracus sp. (Staphylinidae, Coleoptera), in leaf litter, 30 Oct. 2014, K. Tasanathai, A. Khonsanit, W. Noisripoom, D. Thanakitpipattana, P. Srikitikulchai & S. Wongkanoun (paratype BBH 41871, culture ex-paratype BCC 76486).

Notes: In terms of asexual morphology, O. capilliformis closely resembles O. sporangifera, both characterised by the production of multiple slender synnemata along the stipe. Ophiocordyceps capilliformis distinguishes itself by the absence of sclerotia at the apex of the stipe. Additionally, while both species have conidia enveloped in a mucous sheath, O. capilliformis features fusiform conidia, while O. sporangifera exhibits subglobose to reniform conidia. Ophiocordyceps sporangifera is commonly found on Elateridae larvae, whereas O. capilliformis was typically associated with larva of Meracantha sp. (Tenebrionidae), which is buried in the soil, as well as on adult beetles of Platydracus sp. (Staphylinidae) in the leaf litter. It is noteworthy that sexual morph have been observed in O. capilliformis, whereas O. sporangifera has not exhibited any sexual morph. The sexual morph morphology of O. capilliformis resembles that of O. obtusa, characterised by the production of cylindrical ascomata, immersed perithecia, and filiform ascospores. Ophiocordyceps obtusa was discovered on Coleoptera larvae in Java, Indonesia. Upon comparison, it is noted that the perithecia and ascospores in O. capilliformis are larger and longer than those reported for O. obtusa (395–420 × 190–250 μm vs 360–370 × 110–150 μm; 130–210 × 2–3 μm vs 150 × 1.5 μm, respectively) (Penzig & Saccardo 1904).

Ophiocordyceps clavata (Kobayasi & Shimizu) G.H. Sung et al., Stud. Mycol. 57: 40. 2007. MycoBank MB 504237. Fig. 5.

The description and illustrations are based on O. clavata specimens collected in Thailand.

Sexual morph: Stromata multiple, clavate, pale yellow (161C–D), arising from head, thorax and abdomen of Tenebrionidae larvae (Coleoptera), 2–10 mm long. Stipes cylindrical, smooth, pale yellow (161D), 0.5–1.5 mm wide. Ascomata formed on upper part of the stipe, or subterminal, occasionally located on one side of the stipe, pale yellow (161C–D), densely packed perithecia, 2–4 mm long, 1.5–2 mm wide. Perithecia immersed, tightly packed, broad ovoid, 400–470(–500) × (250–)275–335(– 350) μm. Asci cylindrical, (200–)207–300 μm long, 6–8 μm wide with asci caps 3.5–7 × 3.5–8 μm. Ascospores cylindrical, 200–250 μm long, breaking into 32 small truncate part-spores, (5–)6.5–8.5(–10) × 1.5–2(–3). Asexual morph: Unknown.

Culture characteristics: Colonies on MYA attaining a diam of 10–15 mm in 30 d, white, colonies reverse moderate orange yellow (164B). Synnemata multiple, white. Conidiogenous cells syngliocladium-like, monophialidic, cylindrical at the base or slightly flask-shaped, slightly curved, 5.5–15.5(–20) × 2–3 μm. Conidia hyaline, smooth-walled, cylindrical, 5–8.5(–10) × 1.5–2.5(–3) μm, mostly aggregated in slimy heads at the apex phialides. Colonies on OA attaining a diam of 5–8 mm in 30 d, mycelium sparse, white to moderate orange yellow (165C), colonies reverse moderate orange yellow (164B). Conidiogenous cells monophialidic, cylindrical basal portion, slightly curved, (5–)7–14(–18) × 2–3 μm. Conidia hyaline, smooth-walled, cylindrical, (6–)7–14(–18) × 1.5–2.5(–3) μm, not aggregated in slimy heads at the apex phialides. Poor sporulation was observed. Colonies on PDA attaining a diam of 12–15 mm in 30 d, white, colonies reverse greyish brown (166A). Conidiogenous cells monophialidic, cylindrical basal portion with acute end, 5–9.5(–15) × (1–)2–3 μm. Conidia hyaline, smooth-walled, cylindrical, (5–)7–8.5(–10) × 1.5–2.5(–3) μm, not aggregated in slimy heads at the apex phialides. Poor sporulation was observed.

Distribution: Found in Japan, known from Daisen mountain in Tottori Prefecture, Ryōkami mountain in Saitama Prefecture (Kobayasi & Shimizu 1980); Thailand, known from Doi Inthanon National Park and Phu Suan Sai National Park; China (Chen et al. 2019).

Additional materials examined: Thailand, Chiang Mai Province, Doi Inthanon National Park, on larvae of Tenebrionidae (Coleoptera), buried in the bark of a living tree. 24 Aug. 2022, J. Luangsa-ard, S. Mongkolsamrit, W. Noisripoom, U. Pinruan, S. Sommai, P. Khamsuntorn & C. Suriyachadkun (BBH 49815, culture BCC 95652); idem., (BBH 49816, culture BCC 95653); Loei Province, Phu Suan Sai National Park, on Coleoptera larva, buried in soil, 16 Jul. 2008, K. Tasanathai, S. Mongkolsamrit, B. Thongnuch, P. Srikitikulchai, A. Khonsanit, N.T. Toan & N.T. Vui (BBH 24029).

Notes: According to the multi-gene phylogenetic analysis presented in Fig. 1, our study revealed that O. clavata strains (BCC 95652 and BCC 95653) cluster together with O. clavata previously reported from China (BUO545) and Japan (NBRC106961, NBRC 10962). Ophiocordyceps clavata was initially described and illustrated based on Japanese specimens infecting Coleoptera larvae by Kobayasi & Shimizu (1980). When comparing the of morphological features of the Thai and Japanese materials, it becomes evident that the size of both the perithecia and part-spores are in the same range (400–500 × 250–350 μm vs 420–550 × 230–330 μm; 5–10 × 1.5–3 μm vs 5–9 × 1.5 μm, respectively). We obtained specimens of this species from Chiang Mai and Loei Provinces. In this study, we present the morphological characters through a colour figure for this species for the first time.

Ophiocordyceps kohchangensis Mongkolsamrit, Noisripoom, Himaman & Luangsa-ard, sp. nov. MycoBank MB 851832. Fig. 6.

Etymology: Refers to the locality where the type specimen was found, Mu Koh Chang National Park.

Typus: Thailand, Trad Province, Mu Koh Chang National Park. on Elateridae larva (Coleoptera), buried in soil, 9 May 2018, J. Luangsaard, S. Mongkolsamrit, W. Noisripoom, B. Sakolrak & W. Himaman (holotype BBH 44483, culture ex-type BCC 88229).

Sexual morph: Stromata solitary, cylindrical, gradually tapering toward the apex, moderate yellow to light yellow (162B–C), up to 75 mm long. Stipes cylindrical, smooth, pale yellow (162C–D), 1–1.5 mm wide. Rhizoids flexuous, 5 cm long buried under the ground, arising from head of Elateridae larva (Coleoptera). Fertile parts distinctly subterminal with asexual morph at apex. Ascomata subterminal, cylindrical, moderate yellow to light yellow (162B–C), 10–20 mm long, 2–2.5 mm wide. Perithecia completely immersed, pyriform, (320–)340–380(–400) × (100– )105–160(–200) μm. Asci cylindrical, 90–150(–200) × 4–5.5(–6) μm with asci caps, 2–3 × 3–5 μm. Ascospores cylindrical, breaking into 32 small truncate part-spores, (3–)4–5.5(–7) × 1.5–2 μm. Asexual morph: Synnemata solitary, cylindrical, produced at upper region of the stipe, up to 60 mm long. Conidiogenous cells hirsutella-like, monophialidic or polyphialidic, cylindrical basal portion, (5–)6.5–23(–40) × 3–4.5(–5) μm with a distinct, filiform, long neck up to 25 μm long. Conidia hyaline, smooth, globose, (2–)2.5–4 μm.

Culture characteristics: Colonies on MYA attaining a diam of 10–14 mm in 30 d, white, colonies reverse pale yellow (161C). Conidiogenous cells polyphialidic, cylindrical to subcylindrical basal portion, (5–)7–23(–40) × 3–4.5(–5) μm with a distinct, filiform, long neck up to 25 μm. Conidia hyaline, smooth-walled, globose 3–4 μm. Colonies on OA attaining a diam of 14–17 mm in 30 d, white, colonies reverse pale yellow (164D). Mycelium hyaline, septate, smooth-walled. Conidiogenous cells polyphialidic, cylindrical to subcylindrical basal portion, (10– )13–37(–45) × 3–4(–5) μm with a distinct, filiform, long neck up to 38 μm long. Conidia hyaline, smooth-walled, globose, 2–3(–5) μm. Colonies on PDA attaining a diam of 15–18 mm in 30 d, white, colonies reverse brownish orange (165B). Conidiogenous cells polyphialidic, cylindrical to subcylindrical basal portion, 10– 29(–45) × 3–4(–5) μm with a distinct, filiform, long neck up to 35 μm long. Conidia hyaline, smooth-walled, globose, (2–)3–4 μm.

Distribution: Found in the eastern region of Thailand.

Additional materials examined: Thailand, Trad Province, Mu Koh Chang National Park, on Elateridae larvae (Coleoptera), buried in soil, 9 July 2019, K. Tasanathai, S. Mongkolsamrit, B. Sakolrak, W. Himaman & P. Jangsantear (paratype BBH 48326, culture exparatype BCC 90828); idem., (BCC 90811); idem., 9 May 2018, J. Luangsa-ard, S. Mongkolsamrit, W. Noisripoom, B. Sakolrak & W. Himaman (BBH 88234); idem., (BBH 88226, culture BCC 44479); idem., (BBH 88227, culture BCC 44480).

Notes: Ophiocordyceps kohchangensis produces ascomata on the subterminal region of the stroma, with the asexual form appearing at the apex. It closely resembles O. houaynhangensis, O. bidoupensis and O. langbianensis. Ophiocordyceps kohchangensis, O. houaynhangensis, O. bidoupensis and O. langbianensis share remarkable similarities, including their stromata being pale yellow-brown and the size of their perithecia, asci, and ascospores falling within the same range. Difficulties in using morphological discrimination has often been encountered in these fungi since there is an overlap in the measurements of important morphological characters such as phialides, conidia, and ascospores. Furthermore, these species are associated with the larval stages of Coleoptera. It is noteworthy that only O. bidoupensis and O. kohchangensis have been reported to occur on Elateridae larvae (Coleoptera). However, the results of a molecular phylogenetic analysis clearly indicate that O. kohchangensis forms a distinct clade separate from the previously mentioned species (Fig. 1).

Ophiocordyceps phitsanulokensis Mongkolsamrit, Noisripoom, Lamlertthon & Luangsa-ard, sp. nov. MycoBank MB 851833. Fig 7.

Etymology: Refers to the locality where the type specimen was found, Phitsanulok Provice.

Typus: Thailand, Phitsanulok Province, Ban Phaothai community forest, on Coleoptera larva, buried in soil, 28 Jun. 2017, S. Mongkolsamrit, K. Tasanathai, W. Noisripoom & U. Pinruan (holotype BBH 44437, culture ex-type BCC 85328).

Sexual morph: Stromata solitary or multiple, cylindrical, greyish red (178A–B) to reddish brown (175A), arising from head region of Elateridae larvae (Coleoptera), up to 15 cm long. Stipes cylindrical smooth, moderated orange yellow (165C), 1–2 mm wide. Rhizoids flexuous, 3–8 cm long buried under the ground. Ascomata terminal, cylindrical, 20–40 mm long, 1–3.5 mm wide. Perithecia completely immersed, narrowly ovoid, (480–)490– 540(–550) × (110–)120–155(–170) μm. Asci cylindrical, up to 250 μm long, 5–7 μm with asci caps, 2.5–3 × 5–6 μm. Ascospores cylindrical, slightly curved, breaking into 32 small truncate part-spores, 5–7(–8) × 1–1.5 μm. Asexual morph: Synnemata solitary or multiple, cylindrical, produced at upper region of the stipe, up to 12 cm long. Conidiogenous cells hirsutella-like, polyphialidic, cylindrical to subcylindrical basal part, occasionally slightly curved, 5–15 × 2.5–5 μm with a short neck up to 2–5 μm. Conidia hyaline, smooth-walled, fusoid, 5–7(–8) × 1–1.5 μm. Some strains are occasionally found producing the asexual morph at the subterminal part of stipe connected with the ascomata.

Culture characteristics: Colonies on MYA attaining a diam of 8 mm in 30 d, brownish orange (165B), low mycelial density, colonies brownish orange (165B). Conidia and reproductive structures not observed (Supplementary Fig. S5). Colonies on OA attaining a diam of 5–8 mm in 30 d, greyish red (182C), with low mycelial density, colonies reverse light reddish brown (177B). Conidiogenous cells hirsutella-like, monophialidic or polyphialidic, cylindrical, swollen basal portion, (5–)6–16(–25) × (2–)3–5 μm, forming a short neck up to 2 μm long. Conidia hyaline, smooth-walled, fusoid, 5–7 × 1–2.5 μm. Colonies on PDA attaining a diam of 10–15 mm in 30 d, white to pale greenish yellow (9D), colonies reverse greyish brown (166A). Conidiogenous cells hirsutella-like monophialidic or polyphialidic, awl-shaped, or swollen basal portion, (5–)6.5–18.5(–30) × 2–4 μm, forming a short neck up to 2.5 μm long. Conidia hyaline, smooth-walled, fusoid, 5–6.5(–8) × 1.5–2.5 μm.

Distribution: Found in the central region of Thailand.

Additional materials examined: Thailand, Phitsanulok Province, Ban Phaothai community forest, on Coleoptera larva, buried in soil, 28 Jun. 2017, S. Mongkolsamrit, K. Tasanathai, W. Noisripoom & U. Pinruan (paratype BBH 43368, culture exparatype BCC 85323); idem., (BBH 43336, culture BCC 85324); idem., (BBH 43369, culture BCC 85325); idem., (BBH 43337, culture BCC 85326); idem., (BBH 43338, culture BCC 85327); idem., (BBH 44438, culture BCC 85329); idem., (BBH 44439, culture BCC 85333); idem., (BBH 44440, culture BCC 85334); idem., (BBH 44441, culture BCC 85335).

Notes: Based on the molecular phylogenetic analyses (Fig. 1), O. phitsanulokensis is closely related to O. bidoupensis, O. houaynhangensis, O. kohchangensis, O. brunneipunctata, O. furcatosubulata, O. langbianensis, O. cossidarum, O. brunnea and O. ratchaburiensis. When examining these natural specimens, it becomes evident that O. phitsanulokensis shares morphological similarities with these species, characterised by immersed perithecia and ascospores disarticulating into part-spores. However, the fertile components of the ascomata in O. phitsanulokensis are located at the terminal end of the stipe, while the above-mentioned species produce fertile components of the ascomata at the subterminal region of the stipe. Moreover, O. phitsanulokensis resembles O. longissima, particularly in the production of the long solitary stroma. The fertile component of its ascomata exhibits cylindrical structures located at the terminal end of the stipe, displaying a reddish brown colour. However, they differ significantly in their hosts, with O. phitsanulokensis found on Coleoptera larvae, while O. longissima was found on cicada nymphs (Luangsa-ard et al. 2010b).

Ophiocordyceps pseudovariabilis Mongkolsamrit, Noisripoom, Himaman & Luangsa-ard, sp. nov. MycoBank MB 851834. Fig. 8.

Etymology: Named after the similarity to Ophiocordyceps variabilis.

Typus: Thailand, Nakhon Ratchasima Province, Khao Yai National Park, on Lycidae larva (Coleoptera), buried in decaying wood, 31 May 2018, J. Luangsa-ard, K. Tasanathai, S. Mongkolsamrit & W. Noisripoom (holotype BBH 47453, culture ex-type BCC 88308).

Sexual morph: Stromata solitary or multiple, cylindrical, gradually tapering toward the apex, brownish orange to moderate orange (172C–D), arising from head, thorax and abdomen of Lycidae larvae (Coleoptera), 4–18 mm long. Stipes cylindrical, smooth, brownish orange, 0.5–1 mm wide. Ascomata distinctly subterminal, developed lateral cushion on stipes, slightly pulvinate, densely packed perithecia, 1.5–2 mm diam, 1.5 mm thick. Perithecia pseudo-immersed, pyriform, (380–)388–460(– 480) × (220–)290–320 μm. Asci cylindrical, up to 360 μm long, 5–6(–8) μm wide with asci caps, 2–5 × 5–6 μm. Ascospores cylindrical, breaking into 32 small truncate part-spores, (5– )6–9(–10) × 1–1.5 μm. Asexual morph: Synnemata multiple, cylindrical, brownish orange to pale yellow, up to 8 mm long, 0.5–1 mm wide, occasionally located at the terminal of stipe connected with the ascomata. Conidiogenous cells hirsutellalike, monophialidic or polyphialidic, swollen basal portion, 4–15 × 2–5 μm, gradually tapering from the base to the apex up to 8 μm long. Conidia hyaline, smooth-walled, fusoid, (4–)5–7.5(–8) × 0.5–1 μm.

Culture characteristics: Colonies on MYA attaining a diam of 8–10 mm in 30 d, pale yellow (20C), colonies reverse moderate reddish brown (166B). Synnemata arising on edge of colonies, up to 8 mm long, 0.5–1 mm wide. Conidiogenous cells syngliocladiumlike, phialidic, cylindrical to subcylindrical basal portion, slightly curved, (5–)6.5–12.5(–15) × 1.5–2 μm. Conidia hyaline, smooth-walled, falcate, slightly truncate at the ends, (9–)9.5(–11) × 2–3 μm. Colonies on OA attaining a diam of 5–8 mm in 30 d, white, low mycelial density, colonies reverse uncoloured. Conidia and reproductive structures not observed (Supplementary Fig. S5). Colonies on PDA attaining a diam of 10 mm in 30 d, strong orange yellow (N163D), reverse deep orange yellow (163A). Conidiogenous cells syngliocladium-like, phialidic, cylindrical to subcylindrical basal portion, slightly curved, (5–)8–14.5(–15) × 1.5–2 μm, forming a short neck up to 3 μm long. Conidia hyaline, smooth-walled, falcate, slightly truncate at the ends, (8–)8.5(–10) × 2–3.5 μm.

Distribution: Found in the northeastern region of Thailand.

Additional materials examined: Thailand, Nakhon Ratchasima Province, Khao Yai National Park, on Lycidae larva (Coleoptera), buried in decaying wood, 31 May 2018, J. Luangsa-ard, K. Tasanathai, S. Mongkolsamrit & W. Noisripoom (paratype BBH 47458, culture ex- paratype BCC 88311); idem., (BBH 47454, culture BCC 88309); idem., (BBH 47455, culture BCC 88310); idem., (BBH 43791, culture BCC 88268).

Notes: In Thailand, O. pseudovariabilis was rarely documented in natural forests. Despite extensive surveys conducted in various forests throughout the country at different times of the year, our collections were limited to Khao Yai National Park in May. The small size of the natural specimens adds to the challenge of distinguishing O. pseudovariabilis from Pleurocordyceps phaothaiensis, based on macromorphology, as both produce stroma with subterminal perithecial plates, and their ascospores form part-spores (Crous et al. 2017, Wang et al. 2021b). However, the asexual morphologies observed on PDA culture differ. Ophiocordyceps pseudovariabilis forms syngliocladiumlike colonies, with falcate conidia. In contrast, P. phaothaiensis exhibits verticillate branches or singly arranged phialides, and its conidia come in two types: fusoid to globose and fusiform in chains.

Ophiocordyceps ratchaburiensis Mongkolsamrit, Khonsanit, Noisripoom & Luangsa-ard, sp. nov. MycoBank MB 851835. Fig. 9.

Etymology: Refers to the locality where the type specimen was found, Ratchaburi Province.

Typus: Thailand, Ratchaburi Province, Chaloem Phrakiat Thai Prachan National Park, on Coleoptera larva, buried in soil, 26 May 2011, K. Tasanathai, P. Srikitikulchai, A. Khonsanit & W. Noisripoom (holotype BBH 30544, culture ex-type BCC 48033).

Sexual morph: Stromata solitary or in pairs, cylindrical, gradually tapering toward the apex, strong orange yellow to moderate yellow (163B–C), up to 25 mm long. Stipes cylindrical, smooth, light orange yellow (22B), 0.5–1 mm wide. Rhizoids flexuous, 1–2 cm long buried under the ground, arising from head of Elateridae larvae (Coleoptera). Fertile parts distinctly subterminal with asexual morph at apex. Ascomata subterminal, strong orange yellow (163B), crowded perithecia, 2–6 mm long, 1.5–2 mm wide. Perithecia completely immersed, pyriform, (220–)316– 446(–530) × (110–)154–222(–280) μm. Asci cylindrical, (153– )162–198(–225) × (5–)5.5–8.5(–10) μm with asci caps, 1.5–2.5 × 3–4 μm. Ascospores cylindrical, breaking into truncate part-spores, (3–)4–6(–9) × 1.5–2 μm. Asexual morph: Conidiogenous cells monophialidic or polyphialidic, cylindrical basal portion, (8– )9.5–15(–19) × 3–4 μm with a distinct, filiform, long neck up to 20 μm. Conidia hyaline, smooth-walled, globose to subglobose, (2–)2.5–3(–3.5) × 2–3 μm diam, a mucous sheath observed in mature conidia.

Culture characteristics: Colonies on MYA attaining a diam of 8 mm in 30 d, white, flattened, colonies reverse moderate orange yellow (164B). Conidia and reproductive structures not observed (Supplementary Fig. S5). Colonies on OA attaining a diam of 10–15 mm in 30 d, white, greyish red (182B) in the middle of colonies, colonies reverse greyish reddish orange (177C). Conidiogenous cells monophialidic or polyphialidic, arising from either the lateral or terminal regions of the hyphae, cylindrical basal portion, (5–)8–29.5(–42) × (2–)3–4 μm with a distinct, filiform, long neck up to 25 μm. Conidia hyaline, smooth-walled, globose to subglobose, 2–3(–4) × (2–)2.5–3.5(–4) μm with a mucous sheath. Colonies on PDA attaining a diam of 15–20 mm in 30 d, white, colonies reverse moderate reddish brown (177A). Synnemata white to dark pink (182C), in the middle of colonies. Conidiogenous cells monophialidic or polyphialidic, arising from either the lateral or terminal regions of the hyphae, cylindrical basal portion, 10–28.5(–44) × 2–4 μm with a distinct, filiform, long neck up to 28 μm. Conidia hyaline, smooth-walled, globose to subglobose, (1.5–)2–3.5(–4) × 1.5–2(–3.5) μm with a mucous sheath.

Distribution: Found in the central region of Thailand.

Additional materials examined: Thailand, Ratchaburi Province, Chaloem Phrakiat Thai Prachan National Park, on Coleoptera larvae, buried in soil, 26 May 2011, K. Tasanathai, P. Srikitikulchai, A. Khonsanit & W. Noisripoom (paratype BBH 30553, culture exparatype BCC 48037); idem., (BBH 30545, culture BCC 48034); idem., (BBH 30546, culture BCC 48035); idem., (culture BCC 48873); idem., (BBH 30551, culture BCC 48036); idem., (BBH 30552, culture BCC 48682); idem., (BBH 30554, culture BCC 48039).

Notes: Ophiocordyceps ratchaburiensis exhibits the formation of ascomata on the subterminal portion of its stipe, with the asexual morph located at the apex, similar to O. brunnea, O. brunneipunctata and O. houaynhangensis. However, O. ratchaburiensis distinguishes itself significantly from O. brunnea and O. brunneipunctata, by featuring pale orange stromata, whereas O. brunnea and O. brunneipunctata exhibit stromata in shades of dark brown to cinnamon. Moreover, O. ratchaburiensis shares a stroma colour similar to O. houaynhangensis, characterised by shades of yellow to orange. Nevertheless, molecular phylogenetic analysis supports the clear distinction of O. ratchaburiensis from O. brunnea, O. brunneipunctata and O. houaynhangensis.

DISCUSSION

In the present study, we introduce seven new species and document one new record of Ophiocordyceps clavata associated with Coleoptera in Thailand. These findings are the result of comprehensive phylogenetic analyses that incorporate sequences from LSU, TEF1, RPB1, and RPB2, complemented by compelling morphological evidence. Notably, the type of Ophiocordyceps is O. blattae, known for parasitising a cockroach, identified as Blatta germanica (Araújo et al. 2021). Initially, only two specimens were collected in Sri Lanka (Petch 1924) with more recent records from Thailand (Luangsa-ard et al. 2018, Mongkolsamrit et al. 2021), placing them in the “O. blattae” clade. Ophiocordyceps blattae forms cylindrical ascoma producing elongated-fusoid ascospores that do not disarticulate into part-spores. The seven proposed taxa, distributed into four subclades in Ophiocordyceps, are discussed below.

Species discovered in the “O. ravenelii” clade

This clade was recognised based on Ophiocordyceps ravenelii (Sung et al. 2007). Our comprehensive multi-locus analyses clearly show species like O. clavata, O. formosana, O. spicatus, O. pseudovariabilis, O. variabilis, O. krachonicola, and O. kuchinaraiensis are nested within this distinct clade. Most species in this clade infect larvae of Coleoptera, except for O. krachonicola on Gryllotalpa orientalis nymphs and O. variabilis on Xylophagidae larvae, Diptera. The seven aforementioned species share common features, such as solitary or multiple yellowish, orange to brown stromata and have ascospores that break into cylindrical part-spores, with overlapping sizes within a similar range. The phylogenetic analyses (Fig. 1) reveal a close relationship between O. pseudovariabilis and O. variabilis forming a sister group, closely related to the Thai species O. kuchinaraiensis and O. krachonicola (Thanakitpipattana et al. 2020, Crous et al. 2023), as well as the Chinese O. spicatus and the Taiwanese O. formosana. Both O. pseudovariabilis and O. variabilis produce perithecial cushions on the upper end of the stipe, but O. pseudovariabilis has smaller perithecia compared to O. variabilis. Ophiocordyceps pseudovariabilis displays a hirsutella-like asexual morph on the synnemata in natural samples, while a syngliocladium-like morph is observed in cultures on MYA and PDA. On the other hand, O. variabilis displays a syngliocladium-like asexual form either on immature stromata or on the mycelium covering the host. Cultures on OA and mOA have revealed blunt orange synnemata with abundant conidiogenesis and slime production, as observed by Hodge et al. (1998). These two species, commonly found in decaying wood, differ in their preferred insect hosts, with O. variabilis associated with Xylophagidae larvae (Diptera) and O. pseudovariabilis found on Lycidae larvae (Coleoptera).

Genetic confirmation of O. clavata strains from China, Japan, and Thailand indicates a shared genetic identity (Fig. 1). The morphological characteristics of perithecia and part-spores observed in Japanese and Thai specimens fall within the same range, suggesting that these populations represent the same species (Kobayasi & Shimizu 1980). This finding suggests a broader distribution for O. clavata, transcending geographical boundaries. Interestingly, this shared genetic identity is not exclusive to O. clavata; it is a phenomenon observed in various Ophiocordyceps species. Examples include O. buquetii, present in Brazil, China, Ghana, Taiwan and Thailand, while O. spataforae occurs in Thailand and the USA (Luangsa-ard et al. 2018, Araújo et al. 2020, Mongkolsamrit et al. 2023). These instances underscore similar genetic signatures, emphasizing the potential for widespread distribution and gene flow among these entomopathogenic fungi. The genetic uniformity observed across diverse geographic regions offers valuable insights into the dispersal mechanisms and ecological adaptability of Ophiocordyceps species (Castillo et al. 2018, Dai et al. 2020).

In this study, careful examination revealed that the majority of collected specimens showcase both sexual and asexual morphs, except O. clavata, which exclusively displayed the sexual morph. For O. pseudovariabilis, the natural specimens exhibited a hirsutella-like asexual morph. We used three different media to study the asexual morphological traits in these collections. The results revealed that both O. clavata and O. pseudovariabilis produce syngliocladium-like asexual morphs, with both species displaying the syngliocladium-like morphology when cultured on MYA and PDA. The variations observed in morphologies between specimens in their natural habitat and those cultured may arise from distinctions in nutritional conditions, a phenomenon that has been observed in many invertebrate pathogenic fungi. For instance, cultures of Polystromomyces araneae and Metarhizium rileyi exhibit a yeast-like form when cultivated (Boucias et al. 2016, Mongkolsamrit et al. 2022, Iwanicki et al. 2023). Among the syngliocladium-like asexual morphs, reports have been rare, and O. variabilis was found on immature stromata or in tufts or patches on the surface of the host (Hodge et al. 1998). The presence of this asexual morph represents a distinctive feature of O. pseudovariabilis and O. clavata, as revealed through comprehensive morphological and ecological comparisons with closely related species. Understanding this distinctiveness is important for generic or species circumscription.

New species in the “O. purpureostromata” clade

The “O. purpureostromata” clade encompasses three distinct species: O. albostroma, O. borealis, and O. purpureostromata, all occurring on Coleoptera larvae. The relationship of these three species that morphologically look the same but are genetically distinct represents a significant contribution to the taxonomy and understanding of this clade that it comprises a species complex. A noteworthy shared unique characteristic within this clade is the production of ascospores that divide into four part-spores, featuring long part-spores, as observed in O. purpureostromata (13–23 μm) (Kobayasi & Shimizu 1980) and O. albostroma (25–55 μm). Although the specific number of part-spores was not specified for O. borealis, it was mentioned that these part-spores are notably long (10–15 μm) (Zha et al. 2021). In Thailand, the production of elongated part-spores in Ophiocordyceps species is a rare occurrence. The first formally recorded instance of this characteristic is attributed to O. barnesii, as reported by Luangsa-ard et al. (2010a). To enhance our understanding of the “O. purpureostromata” clade, it is required to collect more samples, including additional isolates and a broader spectrum of insect hosts, for a comprehensive analysis. This approach is essential for refining taxonomy, elucidating evolutionary history, and uncovering the ecological roles of these fungi with various insect hosts.

New species in the “O. elongata” clade

The distinctiveness of O. capilliformis is evident through morphological comparisons with seven closely related species exhibiting the hirsutella-like asexual morph. Ophiocordyceps capilliformis stands out conspicuously from the seven other species within the “O. elongata” clade (Fig. 1). Sexual morphs in the “O. elongata” clade species are rarely reported, with exceptions including O. elongata and O. humbertii. A recent study by Fan et al. (2021) further described O. alboperitheciata from China. It is notable, that O. capilliformis shares comparable perithecia and ascospore morphology with O. elongata by having immersed perithecia and whole, filiform, multiseptate ascospores (Petch 1937). Nevertheless, the perithecia in O. capilliformis are smaller than those reported in O. elongata. Conversely, the distance between each septum in O. capilliformis is longer than that in O. elongata (20–35 μm vs 4–12 μm). Examining the morphology of perithecia and ascospores of the sexual morph of this clade reveals diverse characteristics. In the case of O. alboperitheciata, which is closely related to O. elongata, the perithecia are superficial, and the distances between septa in the ascospores are short (1.1–1.3 μm). On the other hand, in O. humbertii, perithecia are pseudo-immersed, with whole ascospores that are filiform and multiseptate (Petch 1935, Luangsa-ard et al. 2012). Ophiocordyceps capilliformis and O. sporangifera share similar features, producing multiple synnemata along stipes. However, O. capilliformis distinguishes itself by the absence of sclerotia at the apex of its stipe. The observed similarities and distinctions in asexual morphology between these two species raise interesting questions about their ecological roles and host specificity. Sclerotia form from dense aggregations of fungal hyphae (Smith et al. 2015). Sclerotia production was not reported among the species within the “O. elongata” clade. However, instances of sclerotia production were noted in Hirsutella cryptosclerotium and H. subramaniiani var. myrmicarum, which are part of the “H. thompsonii” clade and “Hirsutella ant-pathogen” clade presented by Simmons et al. (2015). The “O. elongata” clade reveals a wide range of insect hosts spanning various orders. Ophiocordyceps alboperitheciata, O. elongata and H. gigantea cluster together, predominantly associated with Lepidoptera, while O. myrmicarum and O. humbertii are linked with Hymenoptera. Ophiocordyceps sporangifera and O. capilliformis are affiliated with Coleoptera. Additionally, H. citriformis exhibits an association with Hemiptera. According to multigene phylogenies presented by Sanjuan et al. (2015) and Araújo et al. (2018), it is evident that O. unilateralis sensu lato and O. sinensis belong to the “O. elongata” clade. Ophiocordyceps unilateralis sensu lato is well known for its role as a common pathogenic fungus that infects ants (Hymenoptera) (Araújo et al. 2015, 2018, Kobmoo et al. 2015, Tang et al. 2023), while O. sinensis is an important component of the famous traditional Chinese medicine associated with larvae of Hepialidae (Lepidoptera) (Dai et al. 2020, 2024).

Diversity of new cryptic species in the “O. brunneipunctata complex” subclade in the “O. sobolifera” clade

The “O. sobolifera” clade encompasses fungi that are pathogens associated with cicada nymphs such as O. sobolifera, O. yakusimensis, O. longissima, which also include an endosymbiotic lineage (Sanjuan et al. 2015, Matsuura et al. 2018). Phylogenetic analyses revealed that O. brunnea, O. kohchangensis, O. phitsanulokensis and O. ratchaburiensis are nested well within the “O. sobolifera” clade (Fig. 1), forming a cluster with O. aphodii, O. bidoupensis, O. houaynhangensis, O. brunneipunctata, O. langbianensis, O. cossidarum and O. furcatosubulata. Within this clade, we identified a distinct subclade (supported by 99 % MLB/1 BPP) known as the “O. brunneipunctata complex” subclade. All species within this subclade primarily infect Coleoptera larvae, with the exception of O. cossidarum, which targets the larvae of Cossidae (Lepidoptera) (Hyde et al. 2017). The stromata typically exhibit a colour range from reddish brown to pale yellow, showcasing shared morphological traits including cylindrical structures, immersed perithecia, and mostly an asexual morph situated at the end of the stroma. Additionally, all species feature ascospores with short part-spores (Table 2). Our fungal collections feature O. brunnea from Nan (Northern Thailand), O. brunneipunctata from Khao Yai National Park (Northeast Thailand), O. ratchaburiensis from Ratchaburi Province (West-central Thailand), O. phitsanulokensis from Phitsanulok Province (North-central Thailand), and O. kohchangensis from Mu Koh Chang National Park, an island in eastern Thailand. The observation of these five species within this subclade, each exhibiting apparent specificity to distinct locations and possibly hosts in Thailand, implies an ecological preference or adaptation to the specific conditions prevalent in each location. Based on natural specimens, we recognise O. brunnea as a newly discovered cryptic species that parasitises coleopterans. Despite their morphological similarities, such as red-brown stromata, these species were previously misclassified under O. brunneipunctata. Molecular phylogenetic analyses revealed that O. brunnea forms a sister lineage with O. ratchaburiensis. The asexual morph of both is closely associated, characterised by the production of mono- or polyphialidic, cylindrical basal portion, while the asexual morph in O. brunneipunctata produces a hirsutella-like asexual morph. However, all three species produce conidia in globose to subglobose forms. The sexual morph of O. phitsanulokensis exhibits morphological traits shared with several species within the “O. brunneipunctata complex” subclade. However, upon observing the asexual morph of O. phitsanulokensis, a distinct characteristic emerges. The phialides are produced at the subterminal part of the stipe, which is connected with the ascomata or formed independently of the sexual morph. Notably, the phialides in O. phitsanulokensis significantly differ from those observed in related species in Thailand, as they produce a hirsutella-like asexual morph with polyphialidic structure, and the conidia exhibit a fusoid form both in natural specimens and on culture media. The identification of cryptic species in this fungal study has become more attainable, owing to the widespread integration of molecular data and the consistent application of phylogenetic species concepts, aligning with previous research findings (Xiao et al. 2019, Lao et al. 2021, Zha et al. 2021, Zou et al. 2022).

Diversity of host life stages and habitats

Understanding the host range and habitat preferences of entomopathogenic fungi provides helpful information for their initial taxonomic classification, particularly at the familial or genus level (Araújo et al. 2020, Mongkolsamrit et al. 2020a, Kobmoo et al. 2023, Xiao et al. 2023). For example, species in Ophiocordyceps exhibit a diverse range of nutritional preferences, infecting diverse orders of insect hosts and host stages, as extensively studied (Luangsa-ard et al. 2018, Tasanatai et al. 2020, 2021, Khao-ngam et al. 2021, Fan et al. 2024 ). In this study, the majority of Ophiocordyceps species were found predominantly buried in the soil or lying on the leaf litter. However, exceptions were noted for O. pseudovariabilis and O. clavata, which were discovered on decaying wood and in the bark of living trees, respectively. Identifying insect hosts during their larval stages, especially those from the Coleoptera or Lepidoptera, has posed challenges due to the limited availability of taxonomic features for classification as well as the availability of expert entomologist in the area. However, certain species of entomopathogenic fungi have coleopteran larvae hosts that can be classified at the genus level, as seen with examples like Metarhizium clavatum and M. purpureum infecting larvae of Oxynopterus sp. (Mongkolsamrit et al. 2020a). Similarly, O. sinensis infects lepidopteran larvae belonging to the ghost moth Thitarodes sp. (Wang et al. 2020, Dai et al. 2024). Based on the review conducted by Zha et al. (2021) and this study, Ophiocordyceps species associated with Coleoptera were primarily identified as belonging to the subfamilies Elateridae and Tenebrionidae, we hypothesise that Ophiocordyceps species associated with Coleoptera might have a broader range of hosts and manifest their host-specificity at the genus or family level. This stands in contrast to the compelling evidence of high host-specificity observed in Ophiocordyceps unilateralis complex species on ant species in the tribe Camponotini (Evans et al. 2011, Araújo et al. 2015, 2018, Kobmoo et al. 2012, 2015). To enhance our understanding of the relationships between fungi associated with Coleoptera, it is crucial to collect molecular data directly from the host, particularly during its larval stages.

Regarding O. capilliformis, its occurrence across disparate beetle host families and different stages of host development is fascinating, including adult beetles of Platydracus sp. and larvae of Meracantha sp. A similar phenomenon, where the sexual morph is found on larval stages while the asexual morph occurs on the adult of Coleoptera, was reported in Beauveria thailandica (Kobmoo et al. 2021). Additionally, M. chaiyaphumense also exhibits a similar pattern, with the sexual morph found on cicada nymphs buried in the soil and the asexual morph found on adult cicadas attached to the underside of leaves (Luangsa-ard et al. 2017).

ACKNOWLEDGEMENTS

This research was supported by the National Science, Research and Innovation Fund, Thailand Science Research and innovation (TSRI) and CPMO (Grant No. P15-51452), National Science and Technology Development Agency (NSTDA) and European Union’s Horizon 2020 Research and Innovation Staff exchange program (RISE) under the Marie Skłodowska-Curie grant agreement No. 101008129, project acronym “MycoBiomics” (lead beneficiaries J.J.L., M.S and P.C.). We are indebted to the Department of National Parks, Wildlife and Plant Conservation (DNP) and Ms. Mananee Patayacheva from Thairakpa Foundation for their cooperation and support of our research project. We also would like to thank Mr. Itsarapong Voraphab from the DNP for his help in the identification of the insect hosts and Dr. Baramee Sakolrak and Miss Panrada Jangsantear from DNP for surveying and collecting fungi.

Conflict of interest: The authors declare that there is no conflict of interest.

Supplementary Material: http://fuse-journal.org/

Fig. S1.

RAxML tree of Ophiocordyceps species based on LSU sequences. Numbers at the major nodes represent maximum likelihood bootstrap (MLB) support values. The new species presented here are indicated in blue font.

fuse-2024-14-223-FigsS1-S4.pdf^{(226.2KB, pdf)}

Fig. S2.

RAxML tree of Ophiocordyceps species based on TEF1 sequences. Numbers at the major nodes represent maximum likelihood bootstrap (MLB) support values. The new species presented here are indicated in blue font.

fuse-2024-14-223-FigsS1-S4.pdf^{(226.2KB, pdf)}

Fig. S3.

RAxML tree of Ophiocordyceps species based on RPB1 sequences. Numbers at the major nodes represent maximum likelihood bootstrap (MLB) support values. The new species presented here are indicated in blue font.

fuse-2024-14-223-FigsS1-S4.pdf^{(226.2KB, pdf)}

Fig. S4.

RAxML tree of Ophiocordyceps species based on RPB2 sequences. Numbers at the major nodes represent maximum likelihood bootstrap (MLB) support values. The new species presented here are indicated in blue font.

fuse-2024-14-223-FigsS1-S4.pdf^{(226.2KB, pdf)}

Fig. S5.

Culture characteristics of Ophiocordyceps phitsanulokensis on MYA, O. pseudovariabilis on OA and O. ratchaburiensis on MYA at 30 d.

fuse-2024-14-223-FigS5.psd^{(10.5MB, psd)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1.

fuse-2024-14-223-FigsS1-S4.pdf^{(226.2KB, pdf)}

Fig. S2.

fuse-2024-14-223-FigsS1-S4.pdf^{(226.2KB, pdf)}

Fig. S3.

fuse-2024-14-223-FigsS1-S4.pdf^{(226.2KB, pdf)}

Fig. S4.

fuse-2024-14-223-FigsS1-S4.pdf^{(226.2KB, pdf)}

Fig. S5.

Culture characteristics of Ophiocordyceps phitsanulokensis on MYA, O. pseudovariabilis on OA and O. ratchaburiensis on MYA at 30 d.

fuse-2024-14-223-FigS5.psd^{(10.5MB, psd)}

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PERMALINK

Uncovering cryptic species diversity of Ophiocordyceps (Ophiocordycipitaceae) associated with Coleoptera from Thailand

S Mongkolsamrit

W Noisripoom

K Tasanathai

A Khonsanit

D Thanakitpipattana

S Lamlertthon

W Himaman

PW Crous

M Stadler

JJ Luangsa-ard

Abstract

INTRODUCTION

MATERIALS AND METHODS

Collections and isolation

Morphological study

DNA extraction, amplification and sequencing

Phylogenetic analyses

Table 1.

RESULTS

Molecular phylogeny

Fig. 1.

Taxonomy

Fig. 2.

Table 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Fig. 8.

Fig. 9.

DISCUSSION

Species discovered in the “O. ravenelii” clade

New species in the “O. purpureostromata” clade

New species in the “O. elongata” clade

Diversity of new cryptic species in the “O. brunneipunctata complex” subclade in the “O. sobolifera” clade

Diversity of host life stages and habitats

ACKNOWLEDGEMENTS

Supplementary Material: http://fuse-journal.org/

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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