Ophiocordyceps duyunensis X. C. Peng & T. C. Wen sp. nov.

Index Fungorum number: IF 900184; Mycobank number: MB 900184; Facesoffungi number: FoF 13974; Fig. 1

Etymology – Referring to the collecting site “Duyun District”.

Holotype – HKAS 125850.

Parasitic on the cocoon or larva. Sexual morph: Not observed. Asexual morph: Primary synnemata 5–7.3 cm long, 0.7–1 mm wide, arising from the insect body, solitary, or several, stipitate, multiply branched, grey to brown, not smooth, cylindrical, or rhizoid, tapering gradually toward the apex. Secondary synnemata observed, without an enlarged globose fertile head on the top. Phialides hirsutella like, scattered, smooth or rough, tapering to a neck, lageniform to conical, lower portions amygdaliform to ovoid, tapering abruptly, forming a long neck, 9–44×2.8–7.7 µm (x̅=19.6×4.7 µm, n=60). Conidia fusiform to limoniform, rough, 8.7–15.5 × 2.7–6.5 μm (x̅= 11.2 × 5 µm, n = 20). Mycelium rough or smooth, branched, hyaline, septate, 1.4–3.7 μm (x̅=2.5 μm, n=20) diam.

Culture characteristics – colonies on PDA, attaining a diameter of27–34 mm within 22 d at 25 ℃, dense, leathery or villous, the center is brown, the edges are white, flat, reverse off-white to dark grey. Synnemate was produced in the later stage. Phialides scattered, hirsutella-like, hyaline, solitary, branched or unbranched, narrow slender, smooth, 11.5–41.5 ×1–6 µm (x̅=26×3 µm, n=50). Conidia fusiform to limoniform, rough, 6–15×4–8.5 μm (x̅= 11 × 5.5 µm, n = 80). Mycelium rough or smooth, branched, hyaline, hyaline, septate, 2.5–5.5 μm (x̅=3.8 μm, n=30) diam.

Habitat and distribution – The new species grow on the ground of coniferous and broad leaf forests and is currently only found in China.

Material examined – China, Guizhou, Duyun City, Lvyinhu street, 26° 23′ 47′′ N, 107° 30′ 42′′ E, 946 m alt., parasitise on cocoon of Lepidoptera, 20 May 2021, X. C. Peng, (HKAS 125850, holotype, ex-type DY21052060, HKAS 125849, paratype). China, Guizhou, Duyun City, Yangliu street, 26° 23′ 46′′ N, 107° 30′ 43′′ E, 929 m alt., parasitise on pupa of Lepidoptera, 20 May 2021, X. C. Peng, (HKAS 125843, paratype, HKAS 125844, paratype).

GenBank numbers – HKAS 125850: ITS= OQ110574; SSU=OQ110579, LSU=OQ110572; tef1-α=OQ116922, rpb1 = OQ116925. HKAS 125843: LSU = OQ110570, tef1-α = OQ116920, rpb1 = OQ116923. HKAS 125849: LSU=OQ110571, tef1-α=OQ116921, rpb1=OQ116924.

Notes – Three specimens were obtained from the same region in Guizhou, China. Ophiocordyceps duyunensis is phylogenetically distinct and is sister to a clade containing O. elongate (its anamorphic stage is Hirsutella gigantea; Simmons et al. 2015), H. kuankuoshuiensis, and O. alboperitheciata with strong support value (100% ML/1.00 PP). They are all parasitic on Lepidoptera, but at different metamorphosis stages. Ophiocordyceps elongate (≡Cordyceps elongata) is on larvae and pupa (Petch 1937), H. kuankuoshuiensis and O. alboperitheciata are on larvae (Qu et al. 2021; Fan et al. 2021), and O. duyunensis is on cocoon and pupa. The morphology of O. duyunensis is different from O. elongate and H. kuankuoshuiensis since the former have secondary synnemata and bigger conidia (8.7–15.5×2.7–6.5 vs. 9–10×3–4 vs. 9.9–12.6×2.7–4.5) (Qu et al. 2021; Petch 1937), O. alboperitheciata is only known from its sexual morph (Qu et al. 2021) (Fig. 2).

Figure 1 – Ophiocordyceps duyunensis (a–b HKAS 125850, holotype. c HKAS 125850, paratype. d HKAS 125843, paratype) a Habitat. b–d Synnemata emerging from the infected insect. e–g Overview of the host. h–i Philidies. j–k Philidie with conidia. l conidia. m–n Lower side and upper side of the culture, o Synnemata growing on PDA medium, p–q Phialides on culture, r Philidies with conidia. s Conidia on culture. Scale Bars: b, m, n=10 mm, h, j, l, p–r=10 µm, i, k, s=5 µm

Figure 2 – Phylogram generated from maximum likelihood analysis based on combined LSU, tef1-α, and rpb1 sequence data. The maximum likelihood (ML) analysis was performed in RAxML-HPC BlackBox (8.2.12), and Bayesian inference (BI) analysis was performed the MrBayes on XSEDE (3.2.7a) (Miller et al. 2010). Bootstrap support values for maximum likelihood, greater than 75% and Posterior Probabilities from Bayesian Inference≥0.80 are given above branches. Sixty-nine strains are included in the combined gene analyses, which comprise 2450 characters after alignment (836 characters for LSU, 918 characters for tef1-α, and 696 characters for rpb1). Two specimens of Cordyceps militaris (OSC 93623 and YFCC 6587) are used as the outgroup taxa. The tree topology derived from the Bayesian analysis was similar to the maximum likelihood analysis. The best RAxML tree with a final likelihood value of − 25,546.230423 is presented. The matrix had 1262 distinct alignment patterns, with 16.37% undetermined characters or gaps. Estimated base frequencies were as follows: A=0.226310, C=0.299137, G=0.286722, T=0.187831; substitution rates AC=1.097283, AG=3.441517, AT=1.035933, CG=1.353883, CT=7.600215, GT=1.000000; gamma distribution shape parameter α=0.588153. The newly generated sequences are indicated in blue bold