Untapped Diversity of Termite-Associated Ophiocordyceps and a New Species from China

Abstract

Termite-associated Ophiocordyceps species remain understudied despite the high diversity of the genus. Here we describe Ophiocordyceps minuta (holotype CXAC 0026) from termites (Termitidae, Macrotermitinae) collected in Yunnan, China. Phylogenetic analyses based on nrLSU (nuclear large subunit ribosomal RNA), tef1-α (translation elongation factor 1-alpha), and rpb2 (RNA polymerase II second largest subunit) sequences resolve this fungus as a distinct lineage. Notably, deep genetic divergence (65 bp in tef1-α) between the two ex-type strains of the allied O. fusiformis (BCC 93025 and BCC 93026) reveals cryptic diversity within that nominal species. Beyond the new species, a morphological assessment of termite-associated Ophiocordyceps indicates that perithecial immersion status, ascospore morphology, and conidial features are the most taxonomically valuable characters for this ecological group. These findings expand the known diversity in southwest China and underscore the importance of integrating phylogenetic data with key morphological traits for species delimitation in under-explored habitats.

1. Introduction

The genus Ophiocordyceps Petch (Ophiocordycipitaceae, Hypocreales) ranks among the most species-rich lineages of entomopathogenic fungi, with over 370 accepted names distributed globally (www.indexfungorum.org, accessed on 15 March 2026). Unlike their close relatives in Cordyceps sensu stricto, which typically produce brightly coloured, fleshy stromata, Ophiocordyceps species are characterized by dark-pigmented, wiry to pliant stromata and are associated with anamorphic genera including Hirsutella, Hymenostilbe, Paraisaria, and Syngliocladium [1,2,3]. Members of this genus exploit an exceptionally broad range of insect hosts, attacking various life stages across Blattodea, Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, Megaloptera, Neuroptera and Odonata [3,4,5,6,7]. However, the association with Blattodea is not restricted to termites; several Ophiocordyceps species, including the type species O. blattae, as well as O. asiatica, O. cf. blattae, and O. salganeicola, have been confirmed from non-termite Blattodea [8,9]. This ecological diversity is matched by morphological plasticity: perithecia may be superficial, immersed, or partially embedded, while ascospores either remain intact as multiseptate units or dissociate into individual part-spores [2,10].

Despite this overall diversity, the distribution of Ophiocordyceps species across host groups is markedly uneven [11,12,13]. Certain host associations, particularly with ants and beetle larvae, have yielded numerous described species, while others remain poorly documented [14,15]. Termites (Blattodea: Termitidae) represent one such understudied host group [16]. Termites were historically treated as a separate order, Isoptera, but molecular phylogenetic studies have firmly placed them within Blattodea [17], a placement further corroborated by recent genomic-scale analyses [18]. Termitidae is the most speciose termite family, comprising approximately 2000 described species [19]. As eusocial insects inhabiting subterranean nests in tropical and subtropical ecosystems worldwide, termites play critical roles in decomposition and nutrient cycling [20]. Their constant exposure to soil-borne microorganisms creates opportunities for fungal pathogens, yet the diversity of termite-associated Ophiocordyceps has only recently begun to receive sustained attention [8,21,22,23].

Historical records of termite-pathogenic Ophiocordyceps were limited to three species described over a span of six decades: O. bispora from East Africa, O. koningsbergeri from Java, and O. octospora from Mexico [24,25,26]. Following these early reports, progress remained slow until intensive surveys in Thailand during the past decade uncovered 11 additional species from forests [8,27]. The remaining species have been documented from other East and Southeast Asian localities: O. globiperitheciata, O. longistipes, O. ovatospora and O. taiwanensis from China, and O. puluongensis from Vietnam [21,22,23,28]. Thus, a total of 20 Ophiocordyceps species have been reported from termite hosts worldwide to date, not including the new species described in the present study [2,8,21,23,24,25,26,29]. These discoveries demonstrated that termite-pathogenic lineages harbor substantially greater diversity than previously recognized and suggested that similar surveys in other under-explored regions would likely yield additional taxa.

Southwest China, particularly Yunnan Province, has emerged as a promising area for discovering novel Cordyceps sensu lato species [30,31]. The region’s complex topography, elevational gradients, and high forest cover support diverse insect communities and their associated fungal pathogens [32,33]. Recent investigations have documented multiple new Ophiocordyceps taxa from Yunnan across various insect hosts, including two termite-associated species described in the past two years [21,22]. These findings indicate that the termite-pathogenic Ophiocordyceps diversity in this region remains incompletely sampled, with additional species awaiting discovery through continued field efforts. In the present study, we describe the new termite-associated species O. minuta using morphological and multigene phylogenetic evidence, reassess the key morphological characters for delimiting termite-associated Ophiocordyceps, and discuss the group’s diversity in southwest China.

2. Materials and Methods

2.1. Specimen Collection

Fungal specimens were collected from ground-dwelling termites (Termitidae, Macrotermitinae) in Taiyanghe National Nature Reserve, Yunnan Province, China. Specimens were carefully removed to preserve host integrity, placed in sterile plastic containers, and transported to the laboratory. Upon arrival, they were examined under a stereomicroscope (OLYMPUS SZ61, Olympus Corporation, Tokyo, Japan), cleaned of surface debris, assigned accession numbers CXAC 0026–0027, and air-dried. Hosts were identified using morphological characters and with guidance from termite taxonomists; finer identification was precluded by the condition of the fungus-infected specimens. Voucher specimens are deposited at the College of Agronomy Herbarium (CXAC), Chuxiong Normal University, China, for taxonomic identification and future studies.

2.2. Morphological Characterization

Specimen data, including host association and geographic origin, were documented. Sexual structures (perithecia, asci, ascospores, part-spores) were mounted in lactophenol cotton blue and measured under an Olympus BX53 compound microscope (Olympus Corporation, Tokyo, Japan). For each structure, 20–50 measurements were recorded, and the range was calculated.

2.3. Extraction of DNA, Polymerase Chain Reaction (PCR), and Molecular Sequencing

Genomic DNA was extracted from fungal mycelia using a commercial plant DNA isolation kit (FOREGENE, Chengdu, China) and amplified via polymerase chain reaction (PCR) for three genetic regions: nrLSU, tef1-α, and rpb2, with primers as following: the LSU region was amplified with the primer pair LR0R/LR5 [34,35], and the tef1-α and rpb2 genes using the primer sets EF1α-EF/EF1α-ER [2,36] and RPB2-5′F/RPB2-5′R [2,36] respectively. Each 25 μL PCR contained 2.5 μL of 10× buffer (2 mM Mg²⁺; Transgen Biotech, Beijing, China), 0.25 μL of Taq polymerase, 2 μL of dNTPs (2.5 mM each), 1 μL of DNA template (~500 ng/μL), 1 μL of each primer (10 μM), and 17.25 μL of deionized water. Amplification was conducted in a BIO-RAD T100™ Thermal Cycler following the gene-specific thermal cycling protocols detailed in

Table 1. PCR cycling conditions for each gene.

The Programs	nrLSU/tef1-α	rpb2
Initial denaturation	95 °C 4 min	95 °C 4 min
Phase 1	16 cycles	17 cycles
	95 °C 50 s	94 °C 50 s
	60 °C 50 s −0.5 °C/cycle	55 °C 50 s
	72 °C 60 s	72 °C 90 s
Phase 2	22 cycles	19 cycles
	94 °C 50 s	94 °C 50 s
	51 °C 50 s	51 °C 50 s
	72 °C 60 s	72 °C 90 s
Final extension	72 °C 8 min	72 °C 10 min

. Successful amplification was verified by gel electrophoresis; the resulting amplicons were then purified prior to bidirectional Sanger sequencing at the Beijing Genomics Institute (Shenzhen, China) with the same PCR primers.

2.4. Phylogenetic Analyses

To establish the phylogenetic placement of our taxa, we analyzed a three-gene matrix (nrLSU, tef1-α, and rpb2). Newly obtained sequences were confirmed via BLAST searches and aligned with reference sequences from GenBank (

Table 2. Specimen information and GenBank accession numbers for sequences used in this study.

Current Name	Voucher	GenBank Accession Number			References
		nrLSU	tef1-α	rpb2
Hirsutella citriformis	ARSEF 1446	KM652106	KM651990	–	[44]
H. fusiformis	ARSEF 5474	KM652110	KM651993	–	[44]
H. gigantea	ARSEF 30	JX566977	JX566980	–	[44]
H. guyana	ARSEF 878	KM652111	KM651994	–	[44]
H. illustris	ARSEF 5539	KM652112	KM651996	–	[44]
H. kirchneri	ARSEF 5551	KM652113	KM651997	–	[44]
H. lecaniicola	ARSEF 8888	KM652114	KM651998	–	[44]
H. liboensis	ARSEF 9603	KM652115	–	–	[44]
H. necatrix	ARSEF 5549	KM652116	KM651999	–	[44]
H. nodulosa	ARSEF 5473	KM652117	KM652000	–	[44]
H. radiata	ARSEF 1369	KM652119	KM652002	–	[44]
H. rhossiliensis	ARSEF 3747	KM652123	KM652006	–	[44]
H. strigosa	ARSEF 2197	KM652129	KM652012	–	[44]
H. subulata	ARSEF 2227	KM652130	KM652013	–	[44]
H. thompsonii	ARSEF 414	KM652143	KM652021	–	[44]
H. thompsonii var. vina	ARSEF 254	KM652149	KM652028	–	[44]
H. versicolor	ARSEF 1037	KM652150	KM652029	–	[44]
Ophiocordyceps acicularis	OSC 110988	EF468804	EF468745	–	[2]
O. agriotidis	ARSEF 5692	DQ518754	DQ522322	DQ522418	[45]
O. appendiculata	NBRC 106960	JN941413	AB968577	AB968539	[46]
O. arborescens	NBRC 105891	AB968414	AB968572	AB968534	[47]
O. asiatica	BCC 30516	MH753675	MK284263	MK214091	[8]
O. bidoupensis	YFCC 8793	–	OK556894	OK556900	[13]
O. brunneinigra	BCC 69032	MF614654	MF614638	MF614681	[48]
O. brunneiperitheciata	BCC 66167	MF614659	MF614644	MF614684	[48]
O. brunneipunctata	OSC 128576	DQ518756	DQ522324	DQ522420	[45]
O. brunneirubra	BCC 14384	MH753690	GU797121	MK751468	[8]
O. campes	BCC 36938	MT118175	MT118167	MT118188	[49]
O. communis	BCC 1842	MH753680	MK284266	MK214096	[8]
O. cossidarum	MFLU 17-0752	MF398187	MF928403	–	[50]
O. fusiformis	BCC 93025	MZ675422	MZ707849	MZ707805	[27]
O. fusiformis	BCC 93026	MZ675423	MZ707850	MZ707806	[27]
O. geometridicola	BCC 35947	MF614647	MF614631	MF614678	[48]
O. globiperitheciata	HKAS 126130	OR015968	OR030532	–	[21]
O. globosa	BCC 93023	MZ675419	MZ707846	–	[27]
O. hydrangea	YFCC 8832	OM304640	OM831277	OM831283	[13]
O. irangiensis	BCC 82795	–	MH028186	MH028174	[8]
O. isopterae	MY12376	MZ675420	MZ707847	MZ707803	[27]
O. isopterae	BCC 93042	MZ675421	MZ707848	MZ707804	[27]
O. khokpasiensis	BCC 48071	MH753682	MK284269	–	[8]
O. kimflemingiae	SC09B	KX713620	KX713698	–	[3]
O. konnoana	EFCC 7315	–	EF468753	EF468916	[2]
O. kuchinaraiensis	BCC 95830	OQ627397	OQ625474	OQ625475	[8]
O. linyphiidarum	HKAS 132197	PV139245	PV156008	PV155994	[51]
O. linyphiidarum	HKAS 132196	PV139246	PV156009	PV155995	[51]
O. longistipes	KUNCC 5224	OR015967	OR030530	OR113082	[21]
O. longistromata	BCC 44497	MT118178	MT118170	MT118191	[49]
O. macroacicularis	NBRC 100685	AB968416	AB968574	AB968536	[47]
O. melolonthae	OSC 110993	DQ518762	DQ522331	–	[52]
O. mosingtoensis	BCC 30904	MH753686	MK284273	MK214100	[8]
O. minuta	CXAC 0026	PX973468	PX984915	PX984944	This study
O. minuta	CXAC 0027	PX973469	PX984916	PX984945	This study
O. multiperitheciata	BCC 22861	MF614656	MF614640	MF614683	[48]
O. myrmecophila	CEM 1710	KJ878894	KJ878974	–	[53]
O. neovolkiana	OSC 151903	KJ878896	KJ878976	–	[53]
O. nigrella	EFCC 9247	EF468818	EF468758	EF468920	[2]
O. nutans	OSC 110994	DQ518763	DQ522333	–	[45]
O. pauciovoperitheciata	TBRC 8096	MF614649	MF614636	MF614672	[48]
O. pruinosa	NHJ 12994	EU369041	EU369024	EU369084	[54]
O. pseudoacicularis	BCC 53843	MF614646	MF614630	MF614677	[48]
O. pseudocommunis	BCC 16757	MH753687	MK284274	MK214101	[8]
O. pseudorhizoidea	BCC 86431	MH753674	MK284262	MK214090	[8]
O. puluongensis	YFCC 6442	MT270528	MT270520	MT270526	[22]
O. radiciformis	BCC 93036	MZ675425	MZ707852	MZ707808	[27]
O. radiciformis	BCC 93035	MZ675426	MZ707853	MZ707809	[27]
O. ramosissimum	GZUHHN8	–	KJ028014	–	[55]
O. ravenelii	OSC 110995	DQ518764	DQ522334	DQ522430	[50]
O. ravenelii	OSC 151914	–	KJ878978	KJ878950	[53]
O. rhizoidea	NHJ 12522	EF468825	EF468764	EF468923	[8]
O. rhizoidea	NHJ 12529	EF468824	EF468765	EF468922	[8]
O. robertsii	KEW 27083	EF468826	EF468766	–	[2]
O. rubiginosiperitheciata	NBRC 106966	JN941437	AB968582	AB968544	[56]
O. salganeicola	Mori01	MT741719	MT759575	MT759580	[9]
O. satoi	J7	KX713599	KX713683	–	[3]
O. sinensis	ARSEF 6282	KM652126	KM652009	–	[44]
O. sinensis	EFCC 7287	EF468827	EF468767	EF468924	[56]
O. sobolifera	NBRC 106967	AB968422	AB968590	–	[47]
O. spataforae	NHJ 12525	EF469078	EF469063	EF469111	[2]
O. spataforae	OSC 128575	EF469079	EF469064	EF469110	[2]
O. sphecocephala	NBRC 101752	JN941445	AB968591	AB968552	[51]
O. spicatus	MFLU 18-0164	MK863054	MK860192	–	[57]
O. stylophora	OSC 111000	DQ518766	DQ522337	DQ522433	[55]
O. stylophora	OSC 110999	EF468837	–	EF468931	[2]
O. thanathonensis	MFLU 16-2909	MF850377	MF872613	–	[58]
O. tricentri	NBRC 106968	AB968423	AB968593	AB968554	[47]
O. trichospora	CBS 109876	AF543790	AF543779	DQ522457	[2]
O. unilateralis	OSC 128574	DQ518768	DQ522339	DQ522436	[50]
O. unituberculata	YFCC HU1301	–	KY923216	KY923220	[7]
Tolypocladium inflatum	OSC 71235	EF469077	EF469061	EF469108	[59]
T. ophioglossoides	CBS 100239	KJ878874	KJ878958	KJ878944	[53]

Boldface: data generated in this study, – means no data.

) using MAFFT v7.526 (L-INS-i strategy) (http://mafft.cbrc.jp/alignment/server/, accessed 15 March 2026) and refined manually in BioEdit v7.7.1. These alignments were concatenated into a supermatrix with FASconCAT-G v1.06 [37]. The partition homogeneity test in PAUP v5.0 (1000 reps, p > 0.01) revealed no significant conflict between gene partitions. Optimal partitioning schemes and models of evolution were selected under the BIC criterion using PartitionFinder2 v2.0.0 (greedy algorithm) [38]. Phylogenetic relationships were then inferred using two complementary methods. First, a Maximum Likelihood (ML) analysis was performed in IQ-TREE v3.0.1 under the optimal partition scheme (GTR + G + I model), with nodal support assessed via 1000 ultrafast bootstrap replicates [39]. Second, Bayesian Inference (BI) was conducted in MrBayes v3.2.7 using the GTR + G + I model (selected by MrModeltest v2.2) [40]. MCMC chains were run for 5 million generations, sampling every 1000 generations [41]. Convergence (ESS > 200) was verified in Tracer v1.7.2, after which the first 25% of samples were discarded as burn-in [42]. The resulting trees were visualized and annotated in FigTree v1.4.4 (https://tree.bio.ed.ac.uk/software/figtree/ (accessed on 18 may 2026), with the final artwork prepared in Adobe Illustrator CS6 according to the standards of Xie et al. [43].

3. Results

3.1. Phylogenetic Analyses

Phylogenetic analyses of Ophiocordyceps were based on a dataset of 92 sequences (Table 2). Tolypocladium inflatum OSC 71235 and T. ophioglossoides CBS 100239 were used as the outgroups. The final concatenated matrix comprised 2927 characters (including gaps), with three partitions: nrLSU 908 bp, tef1-α 957 bp, and rpb2 1062 bp. Both Bayesian inference (BI) and maximum likelihood (ML) analyses produced congruent trees, recovering Ophiocordyceps as distinct, well-supported clades (Figure 1). The new species Ophiocordyceps minuta formed a distinct lineage within a well-supported clade, clearly separated from its close relatives (Figure 1).

3.2. Taxonomy

Ophiocordyceps minuta Q.Y. Dong & C. D. Xu, sp. nov.

Figure 2

MycoBank no: 862337

Etymology: minuta = minute, referring to the distinctly small size of the perithecia, a key diagnostic feature that distinguishes this species from its closely related congeners.

Holotype: China, Yunnan Province, Pu’er City, Simao District, Taiyanghe National Nature Reserve, on termite (Termitidae, Macrotermitinae), 15 August 2025, Quanying Dong (holotype CXAC 0026).

Stromata morph: Stromata arising from the segment between the prothorax and mesothorax of termite hosts buried in soil, solitary, simple, cylindrical, flexible, leathery, 6–14 cm long, 0.5–1.0 mm wide, brown to yellowish brown. Fertile parts cylindrical, yellowish brown, 3.5–5.5 cm long, 4–5 mm wide, located on the upper part of the stromata, with a spinous surface and a sterile apical tip 13–28 mm long, 0.5–1.0 mm wide. Perithecia superficial, pale yellow when young, turning brown at maturity, ovoid to flask-shaped, 151.5–221 × 115–188 µm, densely and irregularly scattered over the mid portion of the stromata. Ascospores dimorphic: type I fusiform, 80–92.5 × 5.0–6.5 µm, whole, hyaline, multiseptate, septa 8–14.5 µm long; type II vermiform or lanceolate, 32.5–52.5 × 2.0–4.5 µm.

Asexual morph: Undetermined.

Host: Parasitic on termites buried in soil.

Known distribution: Yunnan Province, China.

Additional specimens examined: China, Yunnan Province, Pu’er City, Simao District, Taiyanghe National Nature Reserve, on termite (Termitidae, Macrotermitinae), 15 August 2025, Quanying Dong (paratype CXAC 0027).

Commentary: The diagnostic morphology of Ophiocordyceps minuta conforms to the generic concept of Ophiocordyceps. The species produces dimorphic ascospores comprising two types: cylindrical, with pointed ends, and vermiform. The species can be distinguished by the following combination of morphological characteristics: perithecia distinctly small (151.5–221 × 115–188 µm), which is the smallest among its closely related congeners (

Table 3. Morphological comparison between Ophiocordyceps species associated with termites.

Species	Distribution	Stromata (cm)	Perithecia (µm)	Asci (µm)	Ascospore (µm)	Asexual Morph	Conidiogenous Cells (µm)	Conidia (µm)	References
O. asiatica	Thailand	Solitary, simple, filiform, orange brown, up to 15 long	Superficial, globose to subglobose, 240–320 × 180–260	Filiform, 92.5–175 × 5–6.3	Filiform, septate, whole, 80–132.5 × 1–2	Hirsutella-like	Monophialidic or rarely polyphialidic, 15–20 × 2–3	Fusiform, 7–9 × 2–3	[8]
O. bispora	Tanzania, Kenya	Multiple (20–30), simple or branched, clavate	Immersed, globose, 300–375 × 375	Clavate, 162–163 × 58–61	Elliptical closely appressed, septate, 95–105 × 34–35.4	–	–	–	[24,29]
O. brunneirubra	Thailand	Solitary, simple or branched, narrowly clavate, orange brown to red brown, 9.5 long	Immersed, ovoid, 300–400 × 130–200	Cylindrical, 155–225 × 4.5–8	Filiform, septate, whole, 156.5–197.5 × 2–3	–	Monophialidic, 32–50 × 2–3	Fusiform, 12–17 × 2–4	[8]
O. communis	Thailand	Solitary, simple, filiform, base whitish-grey, upper part yellow-brown, 5–13 long	Superficial, 285–675 × 195–390	Filiform, 215–250 × 15	Filiform, septate, whole, 100–180 × 5–6	Hymenostilbe/Hirsutella-like	Monophialidic or rarely polyphialidic, 10–14 × 2.7–3.3	Almond-shaped, 7–9 × 2.5–3	[2]
O. fusiformis	Thailand	Solitary, simple, cylindrical, brown, up to 6 long	Superficial, ovoid, 300–360 × 180–270	Cylindrical, 141–227 × 7–15	Cylindrical, septate, whole, 36–78 × 5–6.5	Hymenostilbe	Monophialidic, 9–24 × 2–4	Fusiform, 6–18 × 2–4	[27]
O. globosa	Thailand	Solitary, simple, cylindrical, brown, up to 8 long	Pseudo-immersed, ovoid, 190–245 × 120–190	Filiform, 100–157 × 7–13	Filiform, septate, whole, 58–118 × 2–3	Hirsutella-like	Monophialidic or polyphialidic, 9–15 × 3–5	Globose, 2–4	[27]
O. globiperitheciata	China	Multiple (2–5), unbifurcated, clavate, base brown, tip gray, 8–15 long	Superficial, subglobose, 240–295 × 215–280	Filiform, 135–170 × 8.5–13.5	Filiform, septate, whole, 85–110 × 3.5–4.5	–	–	–	[21]
O. isopterae	Thailand	Solitary, simple, cylindrical, brown, up to 10 long	Superficial, ovoid, 270–320 × 140–180	Filiform, 81–137 × 5–9	Filiform, septate, whole, 55–78 × 2–2.5	Hirsutella-like	Monophialidic, 14–28 × 2–4	Fusiform, 6–11 × 1.5–3	[27]
O. khokpasiensis	Thailand	Solitary, simple, cylindrical, brown, 16 long	Pseudo-immersed, subglobose, 200–250 × 120–200	Filiform, 62.5–125 × 4–5	Filiform, whole, 46–90 × 2–3	Hirsutella-like	Monophialidic or polyphialidic, 15–28 × 3–5	Globose to oval, 4–6 × 2.5–4	[8]
O. koningsbergeri	Jawa	Solitary, filiform, gray-white, 8–10 long	Immersed, 450 × 90	Cylindrica, 180–200 × 4–5	Filiform, whole, 150 × 1	–	–	–	[25]
O. longistipes	China	Solitary, unbifurcated, cylindrical, grayish white to yellowish brown, 17–24 long	Superficial, pyramidal to oval, 390–420 × 295–350	Filiform, 160–195 × 4.5–6.5	Filiform, septate, whole, 70–85 × 3.5–4.5	Hirsutella-like	Monophialidic or rarely polyphialidic, 29–60 × 4–4.5	Citriform or oval, 7–10 × 4.5–7	[21]
O. minuta	China	Solitary, simple, cylindrical, brown to yellowish brown, 6–14 long,	Superficial, ovoid to flask-shaped, 151.5–221 × 115–188	–	Dimorphic: type I fusiform, septate, whole, 80–92.5 × 5.0–6.5; type II vermiform or lanceolate, 32.5–52.5 × 2.0–4.5	–	–	–	This study
O. mosingtoensis	Thailand	Solitary, simple, cylindrical, brown to grey, 11 long	Pseudo-immersed, ovoid, 400–500 × 200–300	Filiform, 187.5–287.5 × 4.5–7.5	Filiform, septate, whole, 230–315 × 1.5–3	Hirsutella-like	Monophialidic, 10–17 × 2–3	Oval, 3–5 × 2–3	[8]
O. octospora	Mexico	Multiple, clavate, white to pale tan, 0.2–0.3 long	Immersed, subglobose to ovoid, 180–220 × 200	Clavate, about 250 × 60	Cylindrical, septate, 40–70 × 15–30	–	–	–	[26]
O. ovatospora	China	Solitary, simple, cylindrical or clavate, light-yellow, up to 13 long	Pseudo-immersed, ovoid to pyriform, 110–140 × 80–110	Filiform, 110–125 × 5–7	Filiform, septate, whole, 110–130 × 1–2	Hirsutella-like	Monophialidic or rarely polyphialidic, 15–35 × 3–6	Oval, 3–5 × 3–4	[28]
O. pseudocommunis	Thailand	Solitary, simple, cylindrical, brown, 21 long	Superficial, Subglobose, 520–600 × 360–440	Filiform, 160–165 × 14–17	Filiform, septate, whole, 107.5–147.5 × 6–7.5	Hymenostilbe	–	Fusiform, septate (2–3), 13–27 × 3–5	[8]
O. pseudorhizoidea	Thailand	Solitary, simple, filiform, light brown, up to 21 long	Superficial, ovoid, 280–390 × 160–220	Cylindrical, 120–150 × 5–7	Filiform, whole, 65–82.5 × 2–3	Hirsutella-like	Monophialidic, 9–21 × 2–4	Fusiform, 5–10 × 1–2	[8]
O. puluongensis	Vietnam	Solitary, simple or branched, filiform, pale orange to red brown, 7.1–13.3 long	Superficial, subglobose, 181.8–251.0 × 123.7–205.4	Filiform, 74.3–138.5 × 4.6–6.5	Filiform, septate, whole, 67.0–124.5 × 1.5–2.5	Hirsutella-like	Monophialidic or rarely polyphialidic, 7.9–21.2 × 1.7–5.0	Fusiform or citriform, 2.8–6.1 × 1.9–3.4	[22]
O. radiciformis	Thailand	Solitary, simple, cylindrical, brown, up to 11 long	Superficial, ovoid, 330–460 × 200–320	Cylindrical, 140–296 × 6–10	Filiform septate, whole, 154–215 × 2–3	Hirsutella-like	6–15 × 2–5	Fusiform, 5–7 × 2–3	[27]
O. taiwanensis	China	Grayish light brown, clavate, simple, 0.4–2.6 × 0.5–0.7 mm	Immersed, subglobose to ovoid, 280–365 × 140–210	Cylindrical to clavate, 87–100 × 21–37	Ellipsoidal to ellipsoidal-fusiform, 78–95 × 20–27	–	–	–	[23]
O. termiticola	Thailand	Solitary, simple, filiform, yellow brown, up to 14 long	Pseudo-immersed, globose to subglobose, 200–280 × 150–250	Filiform 62.5–110 × 4–6	Filiform, whole, 85 × 2	Hymenostilbe-like	Monophialidic to polyphialidic, 7–11 × 2.5–4	Globose, 2.5–3.5	[8]

); host specificity to termites (Termitidae, Macrotermitinae); producing dimorphic ascospores, type I fusiform and multiseptate (80–92.5 × 5.0–6.5 µm), type II vermiform or lanceolate (32.5–52.5 × 2.0–4.5 µm); and asci with conspicuous apical caps (3.5–5.5 μm high, 6.5–8.0 μm wide).

Our multi-locus phylogenetic data (Figure 1) indicate that Ophiocordyceps minuta is related to O. fusiformis Tasan. et al. and O. longistipes Y.B. Wang et al. However, it forms a distinct clade separate from these taxa (Figure 1). Morphologically, O. minuta differs from related species in the following ways. Ophiocordyceps fusiformis, a species described from Thailand, also similar to O. minuta in appearance, has relatively larger perithecia 300–360 × 180–270 µm, longer asci 141–227 × 7–15 µm and shorter ascospores 6–78 × 5–6.5 µm, shorter septa 5–12 (19) µm long [27]. Moreover, in our phylogenetic tree, the two strains of O. fusiformis (BCC 93025 and BCC 93026) did not form a single clade but instead separated into two distinct branches. Comparison of the tef1-α gene sequences between the two strains revealed a 65 bp difference (out of a total length of 903 bp). Ophiocordyceps longistipes can be distinguished from O. minuta by its grayish white to yellowish brown with relatively longer stromata, larger perithecia measuring 390–420 × 295–350 µm; filiform asci, 160–195 × 4.5–6.5 µm; and filiform shorter ascospores 70–85 × 3.5–4.5 µm [21].

3.3. Morphological Characters and Their Taxonomic Significance in Termite-Associated Ophiocordyceps Species

Analysis of termite-associated Ophiocordyceps (Table 3) shows that morphological characters differ in taxonomic usefulness, a pattern seen in other Ophiocordycipitaceae. Host association is uniform (all on termites, Isoptera), so it does not help distinguish species but defines the group as a specialized ecological guild. Stromatal characters provide moderate to high resolution. Stromatal color (orange-brown, brown, yellowish-brown, grayish-white to yellowish-brown, or pale to red-brown) aids species recognition. Stromatal size shows clear extremes: diminutive in O. octospora and O. taiwanensis, elongate in O. asiatica, O. longistipes, O. pseudocommunis, and O. pseudorhizoidea. Multiple stromata (O. bispora: 20–30; O. globiperitheciata: 2–5) are also diagnostic. Perithecial immersion status (superficial, immersed, or pseudo-immersed) reliably separates species, as does perithecial size (large in O. communis and O. pseudocommunis; small in O. ovatospora and O. khokpasiensis). Ascus characters are less informative. Ascospores vary strikingly: most species have filiform, septate, whole ascospores; exceptions include elliptical to ellipsoidal–fusiform, non-disarticulating ascospores (O. bispora, O. octospora) and dimorphic ascospores (O. minuta), the latter being a unique autapomorphy. Asexual morphs (Hirsutella- or Hymenostilbe-like), conidiogenous cells (monophialidic or polyphialidic), and conidia (fusiform, globose, oval, or citriform) offer additional characters. In summary, perithecial immersion status, ascospore architecture, and conidial features are the most taxonomically valuable traits.

4. Discussion

The description of Ophiocordyceps minuta from termite hosts in Yunnan Province adds a new species to the entomopathogenic fungal diversity of Southeast Asia. The morphological and phylogenetic data presented here contribute to ongoing discussions on species delimitation, ascospore variation, and biogeography within the genus Ophiocordyceps.

Taxonomic implications of O. fusiformis strain divergence. In our phylogenetic analysis, the two ex-type strains of O. fusiformis (BCC 93025 and BCC 93026) occupied distinct positions (Figure 1), with a 65 bp divergence in the tef1-α region. Although these strains were originally described as conspecific from Thailand [27], this level of sequence divergence suggests that the current concept of O. fusiformis may encompass multiple species. Confirming species boundaries would require re-examination of type material and additional collections from the type locality. This finding reinforces the importance of including multiple representatives per putative species in phylogenetic studies, as single-strain typification may not fully capture intraspecific variation or detect cryptic diversity [48,60].

Ophiocordyceps minuta produces two morphologically distinct ascospore types. The vermiform Type II spores (32.5–52.5 × 2.0–4.5 µm) are similar in size and shape to microconidia produced by some Hirsutella species associated with termite-pathogenic Ophiocordyceps [8,21,22]. Whether the two ascospore types differ in biological function remains unknown; infection assays comparing their relative infectivity would help clarify their respective roles in the life cycle of this species.

Biogeographic patterns. The discovery of O. minuta in Yunnan adds to the growing number of termite-associated Ophiocordyceps species recorded from the Indochinese Peninsula, including recent reports from Thailand and Vietnam [21,22,27]. Multiple species from this ecological group have now been documented in the Mekong River basin and adjacent areas [6]. The 65 bp divergence observed between the two O. fusiformis strains, both collected in Thailand, further indicates that sequence variation exists within what is currently recognized as a single species. This observation highlights the need for more intensive sampling across the region, particularly collecting multiple specimens per morphospecies and from different host colonies [61].

Ophiocordyceps minuta is a morphologically and phylogenetically distinct species. The genetic divergence detected among O. fusiformis strains and the presence of dimorphic ascospores in O. minuta point to several directions for future research, including taxonomic revision of the O. fusiformis complex and functional studies of ascospore variation in O. minuta. These findings underscore the value of continued exploration of termite-associated Ophiocordyceps in Southeast Asia.