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Research Article
Morphology and molecular study of three new Cordycipitoid fungi and its related species collected from Jilin Province, northeast China
expand article infoJia-Jun Hu‡§, Gui-Ping Zhao§, Yong-Lan Tuo§, Dan Dai§, Di-Zhe Guo§|, Gu Rao§, Zheng-Xiang Qi§, Zhen-Hao Zhang§, Yu Li§, Bo Zhang§
‡ Northeast Normal University, Changchun, China
§ Jilin Agricultural University, Changchun, China
| Hebei Normal University of Science and Technology, Qinghuangdao, China

Corresponding author: Yu Li ( fungi966@126.com )

Corresponding author: Bo Zhang ( zhangbofungi@126.com )

Academic editor: Thorsten Lumbsch
© 2021 Jia-Jun Hu, Gui-Ping Zhao, Yong-Lan Tuo, Dan Dai, Di-Zhe Guo, Gu Rao, Zheng-Xiang Qi, Zhen-Hao Zhang, Yu Li, Bo Zhang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Hu J-J, Zhao G-P, Tuo Y-L, Dai D, Guo D-Z, Rao G, Qi Z-X, Zhang Z-H, Li Y, Zhang B (2021) Morphology and molecular study of three new Cordycipitoid fungi and its related species collected from Jilin Province, northeast China. MycoKeys 83: 161-180. https://doi.org/10.3897/mycokeys.83.72325
Open Access

Abstract

Cordyceps species are notable medicinal fungi in China, which are pathogenic on insects and exhibit high biodiversity in tropical and subtropical regions. Recently, three new Cordyceps species, Cordyceps changchunensis and Cordyceps jingyuetanensis growing on pupae of Lepidoptera and Cordyceps changbaiensis growing on larvae of Lepidoptera, were found in Jilin Province, China and are described, based on morphological and ecological characteristics. These three new species are similar to the Cordyceps militaris group, but are distinctly distinguishable from the known species. Cordyceps changchunensis, characterised by its small and light yellow to orange stromata which is occasionally forked, covered with white mycelium at the base of stipe, globose to ovoid perithecia, is macroscopically similar to Cordyceps militaris. Cordyceps changbaiensis is clearly discriminated from other Cordyceps species by its white to orange and branched stromata, clavate to cylindrical fertile apical portion, immersed and globose to ovoid perithecia. Moreover, unbranched, clavate and orange to light red stromata, almond-shaped to ovoid and immersed perithecia separate Cordyceps jingyuetanensis from other Cordyceps species. nrITS, nrLSU and EF-1α sequences were undertaken and phylogenetic trees, based on Maximum Likelihood and Bayesian Inference analysis showed that the three new species clustered with Cordyceps militaris, but formed individual clades, as well as confirmed the results of our morphological study.

Keywords

Cordyceps, host, new species, phylogenetic study, relationship

Introduction

The family Cordycipitaceae belongs to Hypocreales with plant-, animal- and fungus-based nutrition modes (Sung et al. 2007; Vega et al. 2009). The species of Cordycipitaceae are a wide variety which infect invertebrates and, in the tropics and subtropics, are known to have the highest species diversity (Kobayasi 1941, 1982). According to current data, over 900 species of Cordycipitoid fungi are reported worldwide (Yan and Bau 2015; Zha et al. 2018). In China, more than 146 species are recorded (Yan and Bau 2015).

Cordycipitoid fungi were first described in 1753 as Clavaria militaris L., later being recognised as Cordyceps militaris (L.) Fr. The genus Cordyceps Fr. was established by Fries in 1818, encompassing over 450 species (Kobayasi 1982; Luangsa-ard et al. 2007). Compared with a large number of species, subdivisions into infrageneric groups, for example, subgenera and sections, have been proposed in the Cordyceps classification, traditionally based on morphological and ecological characters (Stensrud et al. 2005). The classification of Cordyceps, based on the studies of Kobayasi (1941, 1983), three subgenera, C. subg. Cordyceps, C. subg. Ophiocordyceps and C. subg. Neocordyceps were recognised. Subg. Cordyceps was characterised by the production of either immersed or superficial perithecia, which are approximately at right angles to the surface of stroma and ascospores break into part-spores at maturity. Mains proposed a different viewpoint, two subgenera, C. subg. Cryptocordyceps and C. subg. Racemella, were added (Mains 1958). Based on nrITS, nrSSU, nrLSU, EF-1α, RPB1, RPB2, TUB and ATP6 sequences, the phylogenetic study implied that the Cordycipitoid fungi belong to six genera (Cordyceps Fr., Metacordyceps G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, Tyrannicordyceps Kepler & Spatafora, Elaphocordyceps G.H. Sung & Spatafora, Ophiocordyceps Petch and Polycephalomyces Kobayasi) across three families, Cordycipitaceae, Clavicipitaceae and Ophiocordycipitaceae (Sung et al. 2007; Yan and Bau 2015).

The host of Cordycipitoid fungi is varied and the fungi are always parasitic on larvae of swifts, pupae of Lepidoptera, spiders etc. Cordycipitoid fungi have a strong relationship with the environment and its host (Zha et al. 2019).

In this study, three new species of Cordyceps are reported, based on morphology and molecular studies. Furthermore, the relationship between the host and Cordyceps species is analysed.

Material and methods

Sampling and morphological studies

The specimens were photographed in situ. The size of the stromata was measured when fresh. After examination and description of the fresh macroscopic characters, the specimens were dried in an electric drier at 40–45 °C.

Descriptions of macroscopic characters were based on field notes and photographs. The colours correspond to the “Flora of British fungi: colour identification chart” (Royal Botanic Garden 1969). The dried specimens were rehydrated in 94% ethanol for microscopic examination and then mounted in 3% potassium hydroxide (KOH), 1% Congo Red, Cotton Blue and Melzer’s Reagent (Torres et al. 2005), along with a Zeiss Axio Lab. A1 microscope for observation. For each species, a minimum of 40 part-spores was measured from two different ascocarps, part-spores are given as length × width (l × w). The specimens examined are deposited in the Herbarium of Mycology of Jilin Agricultural University (HMJAU).

DNA extraction, PCR amplification and sequencing

Total DNA was extracted from dried specimens using the NuClean Plant Genomic DNA Kit (Kangwei Century Biotechnology Company Limited, Beijing, China). Sequences of the internal transcribed spacer region (ITS), nuclear large ribosomal subunits (LSU) and translation elongation factor 1-alpha (EF-1α) were used for phylogenetic analysis. The ITS sequence was amplified using the primer pair ITS4 and ITS5 (White et al. 1990), LSU sequence was amplified using the primer pair LROR and LR7 (Stensrud et al. 2005) and EF-1α sequence was amplified using the primer pair 983F and 2218R (Castlebury et al. 2004).

Reaction programmes followed Yan and Bau (2015), Castillo et al. (2018) and Ban et al. (2015), respectively. PCR products were visualised via UV light after electrophoresis on 1% agarose gels stained with ethidium bromide and purified using Genview High-Efficiency Agarose Gels DNA Purification Kit (Gen-View Scientific Inc., Galveston, TX, USA). The purified PCR products were sent to Sangon Biotech Limited Company (Shanghai, China) for sequencing using the Sanger method. The new sequences were deposited in GenBank.

Data analysis

Based on the results of BLAST and morphological similarities, the sequences obtained and related to these samples are listed in Table 1. A dataset comprising of sequences from this study, 31 representative sequences showing the highest similarity to Cordyceps spp. and the outgroup Metacordyceps taii (Z.Q. Liang & A.Y. Liu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, Metarhizium yongmunense (G.H. Sung, J.M. Sung & Spatafora) Kepler, S.A. Rehner & Humber, Nigelia martiale (Speg.) Luangsa-ard & Thanakitp., Ophiocordyceps spp. and Tolypocladium ophioglossoides (J.F. Gmel.) C.A. Quandt, Kepler & Spatafora, retrieved from GenBank, were aligned with using ClustalX (Thompson et al. 1997), MACSE V2.03 (Ranwez et al. 2018) and MAFFT (Katoh and Standley 2013), then manually adjusted in BioEdit (Hall 1999). The datasets were aligned first and then, nrITS, nrLSU and EF-1α sequences were combined with Mesquite. The tree construction procedure was performed in PAUP* version 4.0b10 (Swofford 2002) as described by Jiang et al. (Jiang et al. 2011). All characters were equally weighted and gaps were treated as missing data.

Table 1.
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Voucher information and GenBank accession numbers of ITS, LSU and EF-1α DNA sequences of Cordyceps changchunensis, Cordyceps changbaiensis, Cordyceps jingyuetanensis and related species used in this study.

Species name Specimen/Strain number Host/Substratum GenBank accession numbers References
ITS LSU EF-1α
Akanthomyces lecanii CBS101247 Homopteran JN049836 AF339555 DQ522359 (Kepler et al. 2012)
A. tuberculatus NBRC106949 Lepidoptera JN943318 JN941400 MF416490 (Kepler et al. 2017; Schoch et al. 2012)
Blackwellomyces cardinalis CBS113414 Lepidoptera MH862930 MH874497 EF469059 (Sung et al. 2007; Vu et al. 2019)
B. pseudomilitaris NBRC101411 Lepidoptera JN943308 JN941395 MT017849 (Mongkolsamrit et al. 2020; Schoch et al. 2012)
Cordyceps bassiana IFO4848 Lepidoptera AB027382 AB027382 MN401498 (Khonsanit et al. 2020; Nikoh and Fukatsu 2000)
C. bifusispora ARS5690/EFCC8260 Lepidoptera AY245627 EF468807 EF468747 (Kuo et al. 2005; Sung et al. 2007)
C. brongniartii NBRC101395 Lepidopteran pupae JN943298 JN941382 JF416009 (Kepler et al. 2012; Schoch et al. 2012)
C. cateniobliqua CBS153.83 Lepidoptera MH861560 MT017860 (Vu et al. 2019)
C. changbaiensis HMJAU48255 Lepidoptera MW893252 MW893277 MZ616772 This study
C. changbaiensis HMJAU48260 Lepidoptera MW893270 MW893272 MZ616774 This study
C. changchunensis HMJAU48251 Lepidoptera MW893249 MW893274 MZ616769 This study
C. changchunensis HMJAU48252 Lepidoptera MW893250 MW893275 MZ616775 This study
C. changchunensis HMJAU48259 Lepidoptera MW893251 MW893276 MZ616773 This study
C. chiangdaoensis BCC75734/TBRC7274 Coleopteran larvae KT261394 MF140732 KT261404 (Mongkolsamrit et al. 2018; Tasanathai et al. 2016)
C. coleopterorum CBS110.73 Coleoptera AY624177 JF415988 JF416028 (Kepler et al. 2012; Luangsa-Ard et al. 2005)
C. exasperata MCA2155 Lepidoptera MF416542 MF416486 (Kepler et al. 2017)
C. farinosa CBS111113 Lepidoptera AY624181 MF416554 MF416499 (Kepler et al. 2017; Luangsa-Ard et al. 2005)
C. fumosorosea CBS244.31 Coleoptera AY624182 MF416557 MF416503 (Kepler et al. 2017; Luangsa-Ard et al. 2005)
C. hepialidicola Lepidoptera AF315649 Unpublished
C. jingyuetanensis HMJAU48253 Lepidoptera MW893253 MW893278 MZ616770 This study
C. jingyuetanensis HMJAU48261 Lepidoptera MW893271 MW893273 This study
C. kyushuensis HMAS78115 Lepidoptera EF368021 EF468813 EF468754 (Sung et al. 2007; Wang et al. 2008)
C. militaris OSC93623 Lepidopteran pupae JN049825 AY184966 DQ522332 (Sung et al. 2007)
C. militaris HMJAU48256 Lepidopteran pupae MW888227 MW893279 This study
C. morakotii BCC55820/TBRC7276 Hymenoptera KT261389 MF140731 KT261399 (Mongkolsamrit et al. 2018; Tasanathai et al. 2016)
C. ninchukispora BCC30937 Lepidoptera FJ765274 FJ765242 MF416477 (Kepler et al. 2017)
C. ningxiaensis HMJAU25074 Diptera KF309668 KF309671 (Yan and Bau 2015)
C. polyarthra 6578 Lepidoptera AJ536548 Unpublished
C. pruinosa ARSEF5413 Lepidoptera JN049826 MK761215 DQ522351 (Kepler et al. 2012; Zha et al. 2019)
C. qingchengensis MFLU17-1022 Lepidoptera KY423506 MK761211 MK770630 (Zha et al. 2019)
C. rosea Spat09-053 Lepidoptera MF416536 MF416480 (Kepler et al. 2017)
C. roseostromata ARSEF4870 Larva, not specified AY245637 AF339523 (Kuo et al. 2005; Sung et al. 2001)
C. scarabaeicola ARSEF5689 Coleoptera JN049827 AF339524 DQ522335 (Kepler et al. 2012; Sung et al. 2007)
C. scarabaeicola Arsef5689 Coleoptera JN049827 AF339524 (Kepler et al. 2012; Sung et al. 2001)
Cordyceps sp. HMJAU48254 Lepidoptera MW888228 MW893280 MZ616771 This study
C. spegazzinii ARSEF7850 Diptera DQ196435 DQ196435 GU734752 (Torres et al. 2005)
C. taishanensis A-1 Lepidoptera FJ008927 Unpublished
C. tenuipes TBRC7266 Lepidoptera MF140742 MF140828 (Mongkolsamrit et al. 2018; Vu et al. 2019)
Isaria cicadae GACP07071701 Hemiptera KX017277 MK761212 MT268245 (Zhi et al. 2021)
I. japonica BCC2808 Lepidoptera AY624199 (Luangsa-Ard et al. 2005)
Metarhizium yongmunense EFCC2131 Lepidoptera JN049856 EF468833 EF468770 (Kepler et al. 2012; Sung et al. 2007)
Metacordyceps taii ARSEF5714 Lepidoptera JN049829 AF543787 AF543775 (Sung et al. 2007)
Nigelia martiale HMAS197472(S) Coleoptera JN049881 JF415975 JF416016 (Kepler et al. 2012)
Ophiocordyceps acicularis OSC12858/OSC110987 Coleoptera JN049820 DQ518757 DQ522326 (Kepler et al. 2012)
O. clavata NBRC106961 Coleoptera JN943327 JN941414 MH879672 (Schoch et al. 2012)
O. gracilis EFCC8572 Lepidoptera HM142942 EF468811 EF468751 (Sung et al. 2007; Zhong et al. 2010)
O. rubiginosoperitheciata NBRC106966 Coleoptera JN943344 JN941437 (Schoch et al. 2012)
O. sinensis ARSEF6282 Lepidopteran pupae HM595981 HM595885 EF468767 (Chan et al. 2011; Sung et al. 2007)
Tolypocladium ophioglossoides NBRC106331 Elaphomyces sp. JN943320 JN941408 (Schoch et al. 2012)

MrModeltest 2.3 was used to determine the best fitting substitution model for each dataset for Bayesian Inference, which was calculated with MrBayes 3.2.6 with a general time-reversible DNA substitution model and a gamma distribution rate variation across sites (Ronquist and Huelsenbeck 2003). Four Markov chains were run for two runs from random starting trees for four million generations until the split deviation frequency value was < 0.01 and trees were sampled every 100 generations. raxmlGUI 2.0 (Edler et al. 2020) was used for Maximum Likelihood (ML) analysis with 1,000 bootstrap replicates using the GTRGAMMA algorithm to perform a tree inference and search for optimal topology (Vizzini et al. 2015).

Results

Phylogenetic analysis

The phylogenetic tree, based on ITS from Bayesian analysis, included sequences from 46 fungal samples representing 43 taxa and the results are shown in Fig. 1. According to the phylogenetic tree, the three new species gather into one branch with C. militaris, C. roseostromata Kobayasi & Shimizu, C. taishanensis B. Liu, P.G. Yuan & J.Z. Cao, C. kyushuensis A. Kawam. and C. hepialidicola Kobayasi & Shimizu, but the species C. jingyuetanensis does not gather into one branch by itself. Meanwhile, the genus Cordyceps was divided into three independent clades. Furthermore, Cordyceps and Akanthomyces Lebert are a sister clade to Blackwellomyces Spatafora & Luangsa-ard.

Figure 1. 

Phylogenetic tree of Cordycepitiod fungi, based on ITS from Bayesian analysis; self-examined sequences are shown in bold.

For these reasons, the combined ITS, LSU and EF-1α dataset including 121 fungal samples representing 48 taxa was used for analysis and the results are shown in Fig. 2. In these data, the three new species are in three independent clades included in the C. militaris complex, C. jingyuetanensis was close to C. hepialidicola Kobayasi & Shimizu and is different from Fig. 1. From the phylogenetic tree (Fig. 2), the species of Cordyceps are mainly divided into three independent clades. Moreover, the family Cordycipitaceae clustered into three clades and the genus Akanthomyces formed a sister clade to the genus Cordyceps.

Figure 2. 

Phylogenetic tree of Cordycepitiod fungi, based on concatenated ITS, LSU and EF-1α from Bayesian analysis and Maximum Likelihood analysis; self-examined sequences are shown in bold.

Taxonomy

Cordyceps changchunensis J.J. Hu, Bo Zhang & Y. Li, sp. nov.

MycoBank No: 839249
Figs 3, 4

Holotype

China. Jilin Province: Changchun City, Jingyuetan National Forest Park, 43.77°N, 125.47°E, 27 August 2018, Jia-Jun Hu, Bo Zhang & Gui-Ping Zhao (HMJAU 48251, holotype, GenBank Acc. nos.: ITS = MW893249, LSU = MW893274, EF-1α = MZ616769).

Etymology

changchunensis: referring to Changchun, the location of the holotype.

Diagnosis

Cordyceps changchunensis can be easily differentiated from closely-related species C. militaris by its unique host, smaller stromata, immersed perithecia and larger part-spores (2.6–6 × 1.0–1.4 μm).

Description

Sexual Morph. Stromata 2.4–4.5 cm long, single or multiple, solitary to gregarious, arising from pupa; branched, sometimes single at base, then branched into two forks. Fertile apical portion, orange, clavate to globose, sometimes irregular, 2.0–3.5 cm long and 0.4–0.6 cm wide, distinctly distinguishable from the stipe. Sterile stipe fleshy, light yellow to orange, cylindrical, 1.3–3.3 cm long and ca. 0.4 cm wide, usually with white mycelium at the base. Perithecia immersed at right angles to the surface of the fruiting body, globose to ovoid, 180–600 × 180–520 μm, with a thick wall about 10–15 μm. Asci cylindrical, 80–300 × 2.5–5 μm, 8–spored, apex of ascus hemispherical, 3.0–4.0 × 2.0–3.0 μm. Part-spores oblong, 2.6–6 × 1.0–1.4 μm, smooth, hyaline in 3% KOH, thin-walled, inamyloid.

Figure 3. 

Morphological characters of Cordyceps changchunensis (HMJAU 48251, holotype) a, b, e stromata and host of Cordyceps changchunensis c surface of fertile apex of ascostroma d host of Cordyceps changchunensis f–h apex of ascus i–k ascus l–n part-spores. Scale bars: 1 cm (a, b); 2 mm (c, e); 1 mm (d); 10 μm (f–h); 50 μm (i–k); 5 μm (l–n).

Asexual Morph. Unknown.

Host

Growing on pupae of Lepidoptera.

Other specimens examined

China. Jilin Province: Changchun City, Jingyuetan National Forest Park, 20 August 2015, Bo Zhang (HMJAU 48259, GenBank Acc. nos.: ITS = MW893251, LSU = MW893276, EF-1α = MZ616773); Changchun City, Jingyuetan National Forest Park, 18 August 2018, Bo Zhang (HMJAU 48252, isotype, GenBank Acc. nos.: ITS = MW893250, LSU = MW893275, EF-1α = MZ616775).

Distribution

China (Jilin Province).

Note

C. changchunensis is easily confused with C. militaris due to highly similar morphology and sharing the same habitat. Morphologically, the stromata of C. militaris are larger than C. changchunensis, single or gregarious, larger perithecia (500–1089 × 132–264 μm) and smaller part-spores (2–4 × 1 μm) (Li et al. 2015). In the phylogenetic analysis, the three specimens of C. changchunensis were placed in separate monophyletic lineages (BPP = 0.91, MLBS = 78%).

Figure 4. 

Microscopical characters of Cordyceps changchunensis (HMJAU 48251, holotype) a perithecia b apex of ascus c part-spores. Scale bars: 100 μm (a); 5 μm (b, c).

Cordyceps changbaiensis J.J. Hu, Bo Zhang & Y. Li, sp. nov.

MycoBank No: 839250
Figs 5, 6

Holotype

China. Jilin Province, Yanbian Korean Autonomous Prefecture, Antu County, Changbai Mountain, 42.19°N, 128.18°E, 4 September 2019, Jia-Jun Hu & Bo Zhang (HMJAU 48255, holotype, GenBank Acc. nos.: ITS = MW893252, LSU = MW893277, EF-1α = MZ616772).

Etymology

changbaiensis: referring to Mt. Changbai, the location of the holotype.

Diagnosis

The species is characterised by orange to white and branched stromata, globose to ovoid perithecia and larger part-spores (3.0–7.0 × 1.0–1.4 μm).

Figure 5. 

Morphological characters of Cordyceps changbaiensis (HMJAU 48255, holotype) a stromata and host of Cordyceps changbaiensis b host of Cordyceps changbaiensis c, d surface of fertile apex of ascostroma e–h ascus i–k apex of ascus l–m part-spores. Scale bars: 1 cm (a); 5 mm (b–c); 200 μm (d); 20 μm (e–h); 10 μm (i–k); 5 μm (l–m).

Description

Sexual Morph. Stromata 2.4–5.2 cm long, single or multiple, solitary, arising from the head of the host insect covered with white mycelia. Fertile apical portion, orange, clavate to cylindrical, 0.6–1.5 cm long and 0.2–0.6 cm wide, obviously distinguishable from the stipe. Sterile stipe fleshy, white to light yellow, cylindrical, 1.8–3.7 cm long and 0.2–0.5 cm wide. Perithecia immersed to the surface of the fruiting body, globose to ovoid, 120–230 × 90–170 μm, with a thick wall about 15 μm. Asci cylindrical, 225–625 × 4–5 μm, 8–spored, apex of ascus hemispherical, 3.0–4.0 × 2.2–3.2 μm. Part-spores oblong, 3.0–7.0 × 1.0–1.4 μm, smooth, hyaline in 3% KOH, thin-walled, inamyloid.

Figure 6. 

Microscopical characters of Cordyceps changbaiensis (HMJAU 48255, holotype) a perithecia b apex of ascus c part-spores. Scale bars: 100 μm (a); 5 μm (b, c).

Asexual Morph. Unknown.

Host

Growing on larvae of Lepidoptera.

Distribution

China (Jilin Province).

Other specimen examined

China. Jilin Province: Baishan City, Fusong County, Quanyang Town, 42.30°N, 127.29°E, 22 August 2021, Jia-Jun Hu, Bo Zhang & Gui-Ping Zhao (HMJAU 482260, isotype, GenBank Acc. nos.: ITS = MW893270, LSU = MW893272, EF-1α = MZ616774)

Note

C. changbaiensis has orange to white and branched stromata. Morphologically, C. roseostromata Kobayasi & Shimizu is similar to C. changbaiensis due to the single or branched stromata. C. kyushuensis A. Kawam. is also close to C. changbaiensis because of the host and the stromata being similar in colour. However, both C. roseostromata and C. kyushuensis have a larger perithecia and smaller part-spores. Furthermore, the stromata of C. kyushuensis is gregarious or fascicled and grows from the head or abdomen of the host (Li et al. 2015); C. roseostromata has pyriform perithecia and host on larva of Coleoptera (Kobayasi 1983). In the phylogenetic analysis, C. changbaiensis was placed in separate monophyletic lineages (BPP = 0.95, MLBS = 97%) and formed a sister relationship with C. rosea.

Cordyceps jinyuetanensis J.J. Hu, Bo Zhang & Y. Li, sp. nov.

MycoBank No: 839251
Figs 7, 8

Holotype

China. Jilin Province: Changchun City, Jingyuetan National Forest Park, 43.80°N, 125.50°E, 27 August 2018, Jia-Jun Hu, Bo Zhang & Gui-Ping Zhao (HMJAU 48253, holotype, GenBank Acc. nos.: ITS = MW893253, LSU = MW893278, EF-1α = MZ616770).

Etymology

jinyuetanensis: referring to Jingyuetan National Forest Park, the location of the holotype.

Diagnosis

C. jingyuetanensis is different from other species by growing on pupae, orange to light red stromata, immersed and almond-shaped to ovoid perithecia.

Figure 7. 

Morphological characters of Cordyceps jingyuetanensis (HMJAU 48253, holotype) a stromata and host of Cordyceps jingyuetanensis b host of Cordyceps jingyuetanensis c, d surface of fertile apex of ascostroma e, f part-spores g–k ascus l–o apex of ascus. Scale bars: 1 cm (a); 2 mm (b, c); 500 μm (d); 5 μm (e, f); 20 μm (g–k); 10 μm (i–o).

Description

Sexual Morph. Stromata 4–4.5 cm long, multiple, solitary, arising from pupae of Lepidoptera. Fertile apical portion, orange to light red, clavate, 0.8–1.3 cm long and 0.1–0.2 cm wide, obviously distinguishable from the stipe. Sterile stipe fleshy, light yellow to orange, cylindrical, 2.7–3.7 cm long and 0.1–0.2 cm wide, usually with white mycelium at the base. Perithecia immersed to the surface of the fruiting body, almond-shaped to ovoid, 220–340 × 110–220 μm, with a thick wall about 15–20 μm. Asci cylindrical, 225–475 × 3–5 μm, 8-spored, apex of ascus hemispherical to irregular, 3.0–4.0 × 1.4–2.8 μm. Part-spores oblong, 2.8–5.0 × 1.0–1.4 μm, smooth, hyaline in 3% KOH, thin-walled, inamyloid.

Figure 8. 

Microscopical characters of Cordyceps jingyuetanensis (HMJAU 48253, holotype) a perithecia b apex of ascus c part-spores. Scale bars: 100 μm (a); 5 μm (b, c).

Asexual Morph. Unknown.

Host

Growing on pupae of Lepidoptera.

Distribution

China (Jilin Province).

Other specimen examined

China. Jilin Province: Baishan City, Fusong County, Quanyang Town, 42.30°N, 127.29°E, 22 August 2021, Jia-Jun Hu, Bo Zhang & Gui-Ping Zhao (HMJAU 482261, isotype, GenBank Acc. nos.: ITS = MW893271, LSU = MW893273)

Note

A review of literature revealed that there are about 20 species of Cordycipitiod fungi growing on pupae, like the unusual medicinal fungi O. sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, C. militaris, I. cicadae Miq. and also like the two new species, C. ningxiaensis T. Bau & J.Q. Yan and C. qingchengensis L.S. Zha & T.C. Wen, reported from China in 2015 and 2019. Nevertheless, C. jingyuetanensis is different from these Cordycipitiod species; C. ningxiaensis grows on the pupae of Diptera, I. cicadae grows on the pupae of Hemiptera and the stromata of C. qingchengensis are yellow, single or branched on the top. C. hepialidicola Kobayasi & Shimizu from Japan is similar to C. jingyuetanensis in its phylogenetic relationship, but there are distinct morphological differences. Morphologically, the stromata of C. hepialidicola are multiple, branched on the top sometimes, grow from the head of larva of Hepialida or Lepidoptera, have larger perithecia (300–350 × 500 μm) and smaller part-spores (3–4 × 1 μm) (Kobayasi 1983). In the phylogenetic analysis, C. changbaiensis was placed in separate monophyletic lineages (BPP = 0.92, MLBS = 79%).

Cordyceps militaris (L.) Fr., Observ. mycol. (Havniae) 2: 317 (cancellans) (1818)

Fig. 9

Specimens examined

China. Yunnan Province: Qujin City, Huize County, 26.24°N, 103.25°E, 30 July 2019, Jia-Jun Hu, Bo Zhang & Di-Zhe Guo (HMJAU 48256, GenBank Acc. nos.: ITS = MW888227, LSU = MW893279); Jilin Province: Changchun City, Jingyuetan National Forest Park, 43.80°N, 125.50°E, 25 August 2018, Jia-Jun Hu & Yong-Lan Tuo (HMJAU 48257); Changchun City, Jingyuetan National Forest Park, 43.80°N, 125.50°E, 25 August 2018, Jia-Jun Hu, Bo Zhang & Gui-Ping Zhao (HMJAU 48258); Tonghua City, Ji’an County, Wunvfeng National Forest Park, 41.28°N, 126.14°E, 25 August 2019, Yong-Lan Tuo (HMJAU 48262); Heilongjiang Province: Daxing’an Mountains, Shuanghe National Nature Reserve, 52.44°N, 125.40°E, 23 June 2019, Di-Zhe Guo (HMJAU 48263).

Figure 9. 

Macrocharacter of Cordyceps militaris a–e stromata and host of Cordyceps militaris (a collected from Daxing’an Mountains, Heilongjiang Province b collected from Ji’an County, Tonghua City, Jilin Province c, e collected from Changchun City, Jilin Province d collected from Qujin City, Huize County, Yunnan Province). Scale bars: 1 cm (a–e).

Note

C. militaris is a widely distributed species and also a well-known medicinal fungus in China. At this time, we collected samples from many different places. The morphological evidence shows no apparent differences between each other. However, the habitat is markedly different.

Table 2.
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Morphological comparisons of sexual states of Cordyceps changchunensis, Cordyceps changbaiensis and Cordyceps jingyuetanensis.

Species Host Stromata Fertile part Perithecia Asci Ascospores Reference
Beauveria bassiana Larvae of Lepidoptera Single or several, unbranched, slender and cylindrical, brownish- yellow to yellowish 18.7–33. 3 × 2.8–8.0 mm Elliptical, 610–720 × 230–320 µm, immersed to surface Cylindrical, 230–590 × 3.5–4.0 µm with ascus cap 3.6–4.0 µm in diameter Filamentous, 300–570 × 1.0 µm, not broken into part-spores (Li et al. 2001)
Blackwellomyces pseudomilitaris Larvae of Lepidoptera Single or cluster, simple or branched, cylindrical, white to white-orange 15–30 × 0.9–3 mm Elongate-ellipsoid or elongate-ovoid, 290–570 × 120–245 µm, superficial Filiform, 290–410 × 5–6 µm Filiform, 280–390 × 1 µm, not broken into part-spores (Hywel-Jones 1994)
Cordyceps bifusispora Larvae of Lepidoptera Simple, cylindrical clavate, whitish 6 × 1.3 mm Pyriform, with protruding apices, yellowish, 300 × 150–170 µm, immersed Cylindrical, 200–220 × 3–4.5 µm Bifusiform, 145–220 µm in length, central part filiform about 0.4 µm wide, terminal parts narrowly fusiform, about 30 × 1.6 µm and 3 septate (Eriksson 1982)
C. kyushuensis Larvae of Lepidoptera Cluster, cylindrical, Light yellow to orange red 20–30 × 5–8 mm Elliptical, 300–500 × 200–300 µm, half-buried Cylindrical, 3–4.5 µm wide Short cylindrical, part-spores 5–7 × 0.7–1 µm (Guo and Li 2000; Li et al. 2015)
C. militaris Lepidopteran pupae Single or several, clavate, orange 10–20 × 3–5 mm Conical, half-buried Clavate, 300–400 × 4–5 µm Filiform, part spores 2–3 × 1 µm (Li et al. 2015)
C. ningxiaensis Fly pupae (Diptera) One to two in a group, clavate, orange 1.2–3 × 1.2–2.8 mm Ellipsoid to ovoid, 288–400 × 103–240 μm, with a wall about 10 μm thick, loosely embedded at right angles to the surface Cylindrical, 168–205 × (3.7–)4.1–5.5(–6.6) μm, with oblate spheroid or hemispherical refractive cap 3.4–3.8 × 2.9–3.4 μm at apex Filiform, irregularly multiseptate, part-spores 3.6–7.8 × 1.0–1.4 μm (Yan and Bau 2015)
C. polyarthra Larvae of Lepidoptera Cespitose, narrowly clavate, light yellow to reddish-brown Ovoid, 250–450 × 125–250 μm, brown, with a definite wall 25 μm thick, embedded at right angles to the surface Cylindrical, 150–260 × 3–4 μm, with a 1.5–2 μm thick cap Filiform, part-spores 4–6 × 0.75–1 μm (Mains 1958)
C. pruinosa Larvae of Lepidoptera Solitary or several, clavate, orange to red 2–8 × 1–3 mm Ovoid to fusiform, 360–400 × 130–200 μm, crowded, red, ordinal in orientation, immersed Cylindrical, 100–200 × 2.5–4 μm Filiform, part-spores 4–6 × 1 μm (Li et al. 2015)
C. qingchengensis Lepidopteran pupae Branched, yellow 7–9 × 2.0–2.5 mm Ovoid but apex sharply pointed, 335–490 × 145–240 μm, partially immersed at right angle to the surface Cylindrical, 180–200 × 2.4–4.0 μm wide, caps hemispherical, 1.8–2.2 × 2.5–3.2 μm Filiform, 180–220 × 0.45–0.65 μm, not at all bifusiform and not broken into part-spores (Zha et al. 2019)
C. roseostromata Larva, not specified Single or branched 1.2–5 × 1.5–2.2 mm Pyriform, 280–300 × 140–160 μm, Superficial 3–3.5 × 2.5–3 μm 4–5 × 1 μm (Kobayasi 1983)
C. changchunensis Lepidopteran pupae Single or multiple, clavate, orange 2.0–3.5 × 0.4–0.6 mm Globose to ovoid, 180–600 × 180–520 μm, with a thick wall about 10–15 μm, partially immersed at right angles to the surface Cylindrical, 80–300 × 2.5–5 μm, caps hemispheric, 3.0–4.0 × 2.0–3.0 μm at apex Oblong, 2.6–6 × 1.0–1.4 μm This study
C. changbaiensis Larvae of Lepidoptera Single or multiple, clavate, white to orange 0.6–1.5 × 0.2–0.6 mm Globose to ovoid, 120–230 × 90–170 μm, with a thick wall about 15 μm, immersed to surface Cylindrical, 225–625 × 4–5 μm, caps hemispherical, 3.0–4.0 × 2.2–3.2 μm at apex Oblong, 3.0–7.0 × 1.0–1.4 μm This study
C. jingyuetanensis Lepidopteran pupae Single or multiple, clavate, orange to light red 0.8–1.3 × 0.1–0.2 mm Almond-shaped to ovoid, 220–340 × 110–220 μm, with a thick wall about 15–20 μm, immersed to surface Cylindrical, 225–475 × 3–5 μm, caps hemispherical to irregular, 3.0–4.0 × 1.4–2.8 μm at apex Oblong, 2.8–5.0 × 1.0–1.4 μm This study

Key to reported species in this study

1 Stromata arise from pupae 2
– Stromata arise from larvae Cordyceps changbaiensis
2 Stromata branched into two forks sometimes Cordyceps changchunensis
– Stromata not branched 3
3 Part-spores over 3 μm Cordyceps jingyuetanensis
– Part-spores less than 3 μm Cordyceps militaris

Discussion

In this study, three new species, collected from northeast China in the Cordyceps militaris group, are described. In previous work, about 38 species were recognised as belonging to the C. militaris group (Yan and Bau 2015). ML and BI analysis recognised four well-supported clades, one is Cordycipitaceae, the others are Clavicipitaceae and Ophiocordycipitaceae (Fig. 2). Moreover, the Cordycipitaceae branch is mainly divided into three clades, the Akanthomyces clade near the Cordyceps clade, implies a closer biological relationship.

The previous studies have revealed that the genus Cordyceps was not monophylic (Artjariyasripong et al. 2001), the species of Isaria was nested within Cordyceps (Kepler et al. 2017) and our phylogenetic analysis also shows a similar result. Cordyceps clade consisted of three major subclades designated as clade 1, clade 2 and clade 3 (Fig. 2). Nearly all the subclades in Cordyceps clade were strongly supported.

Clade 1, including nine Cordyceps spp. and two Isaria spp. I. cicadae, based on Chinese sequences, gathers into one branch with Cordyceps species. What is known as I. cicadae in China, named on a Brazilian specimen, is of confused classification status, due to the teleomorph having remained undiscovered. In China, C. cicadae Massee has been regarded as a teleomorph of I. cicadae as well as a teleomorph of O. sobolifera (Hill ex Watson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora and referred to as C. sobolifera (Hill ex Watson) Berk. & Broome. Until recently, the teleomorph was discovered in Mt. Jinggang, Jiangxi Province, China and both teleomorph and anamorph existed on some specimens, with the morphology of the anamorph consistent with those, “I. cicadae”, harvested throughout southern China, significantly different from the type specimen of I. cicadae. For this reason, it was published as a new species named C. chanhua Z.Z. Li, F.G. Luan, Hywel-Jones, C.R. Li & S.L. Zhang (Zhi et al. 2021). Furthermore, I. japonica Yasuda reported from Japan, exhibits exceptionally high affinity with the genus Cordyceps. The teleomorph, however, still remains a mystery and a more intensive study is needed. Clade 2 consists of C. scarabaeicola Kobayasi, C. bassiana Z.Z. Li, C.R. Li, B. Huang & M.Z. Fan and C. brongniartii Shimazu. Yellow stromata seem to be a synapomorphic character of clade 2. Clade 3 included 15 Cordyceps spp. However, clade 3 did not form a monophyletic group. C. ninchukispora (C.H. Su & H.H. Wang) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, C. chiangdaoensis Tasan., Thanakitp., Khons. & Luangsa-ard, C. pruinosa Petch and C. morakotii Tasan., Thanakitp. & Luangsa-ard gather into one branch. Cordyceps spp. of clade 3A all arise from pupae. Clade 3B includes 11 Cordyceps spp., seven known Cordyceps spp., one unidentified Cordyceps sp. and our three new species. Being visually similar to Cordyceps militaris seems to be a synapomorphic character of clade 3B.

About 60% of Cordyceps sensu lato species are recorded on two insect orders–Coleoptera and Lepidoptera (Shrestha et al. 2016). Host preferences have been variously implemented in taxonomic work, so this is also in Cordyceps. Host associations, when superimposed on phylogeny, suggested that some groups of taxa have conserved the endoparasite-host interactions to some extent; however, several host shifts have occurred during the evolution of Cordyceps (Stensrud et al. 2005). In Cordyceps species, hosts were considered as having low significance as a phylogenetic character, but are the most crucial feature in morphological aspects (Torres et al. 2005).

Acknowledgements

This study is funded by the National Key R & D of Ministry of Science and Technology (2018YFE0107800), the National Key R & D of Ministry of Science and Technology (2019YFD1001905-33), Jilin Province Science and Technology Development Plan Project (20190201256JC), Scientific and Technological Tackling Plan for the Key Fields of Xinjiang Production and Construction Corps (No. 2021AB004) and “111” programme (No. D17014). The authors are very thankful to Dr. Xue-Fei Li, Miss Hui-Ze Hu and Miss Fang-Fang Zhang from Engineering Research Centre of Edible and Medicinal Fungi, Ministry of Education, for help during this study.

References

  • Artjariyasripong S, Mitchell JI, HyweI-Jones NL, Jones EBG (2001) Relationship of the genus Cordyceps and related genera, based on parsimony and spectral analysis of partial 18S and 28S ribosomal gene sequences. Mycoscience 42: 503–517. https://doi.org/10.1007/BF02460949
  • Ban S, Sakane T, Nakagiri A (2015) Three new species of Ophiocordyceps and overview of anamorph types in the genus and the family Ophiocordyceptaceae. Mycological Progress 14: 1–12.
  • Castillo LP, Osorio A, Vargas N, Sanjuan T, Grajales A, Restrepo S (2018) Genetic diversity of the entomopathogenic fungus Cordyceps tenuipes in forests and butterfly gardens in Quindío, Colombia. Fungal Biology 122: 891–899. https://doi.org/10.1016/j.funbio.2018.05.003
  • Castlebury LA, Rossman AY, Sung G-H, Hyten AS, Spatafora JW (2004) Multigene phylogeny reveals new lineage for Stachybotrys chartarum, the indoor air fungus. Mycological Research 108: 864–872.
  • Chan WH, Ling KH, Chiu SW, Shaw PC, But PPH (2011) Molecular analyses of Cordyceps gunnii in China. Journal of Food and Drug Analysis 19: 18–25. https://doi.org/10.1310/hct1202-118
  • Edler D, Klein J, Antonelli A, Silvestro D (2020) raxmlGUI 2.0: a graphical interface and toolkit for phylogenetic analyses using RAxML. Methods in Ecology and Evolution 12: 373–377. https://doi.org/10.1111/2041-210X.13512
  • Eriksson O (1982) Cordyceps bifusispora spec. nov. Mycotaxon 15: 185–188.
  • Guo Y, Li C (2000) Cordyceps kyushuensis new to China. Mycosystem 19: 296.
  • Jiang X, Yu H, Xiang M, Liu X, Liu X (2011) Echinochlamydosporium variabile, a new genus and species of Zygomycota from soil nematodes. Fungal Diversity 46: 43–51. https://doi.org/10.1007/s13225-010-0076-7
  • Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772–780. https://doi.org/10.1093/molbev/mst010
  • Kepler R, Sung GH, Ban S, Nakagiri A, Chen MJ, Huang B, Li Z, Spatafora J (2012) New teleomorph combinations in the entomopathogenic genus Metacordyceps. Mycologia 104: 182–197. https://doi.org/10.3852/11-070
  • Kepler RM, Luangsa-Ard JJ, Hywel-Jones NL, Quandt CA, Sung G-H, Rehner SA, Aime MC, Henkel TW, Sanjuan T, Zare R (2017) A phylogenetically-based nomenclature for Cordycipitaceae (Hypocreales). IMA Fungus 8: 335–353.
  • Khonsanit A, Luangsa-ard JJ, Thanakitpipattana D, Noisripoom W, Chaitika T, Kobmoo N (2020) Cryptic diversity of the genus Beauveria with a new species from Thailand. Mycological progress 19: 291–315.
  • Kobayasi Y (1941) The genus Cordyceps and its allies. Tokyo Bunrika Daigaku. Science Report 84: 73–75.
  • Kobayasi Y (1982) Keys to the taxa of the genera Cordyceps and Torrubiella. Transactions of the Mycological Society of Japan 23: 329–364.
  • Kobayasi Y (1983) Cordyceps species from Japan 6. Bulletin of the National Science Museum Tokyo Series B 9: 1–21.
  • Kuo HC, Su YL, Yang HL, Chen TY (2005) Identification of Chinese medicinal fungus Cordyceps sinensis by PCR-single-stranded conformation polymorphism and phylogenetic relationship. Journal of Agricultural and Food Chemistry 53: 3963–3968. https://doi.org/10.1021/jf0482562
  • Li Y, Li TH, Yang ZL, Bau T, Dai YC (2015) Atlas of Chinese macrofungal resources. Central China Farmers Publishing House, ZhengZhou, China, 93–109.
  • Li Z, Li C, Huang B, Fan M (2001) Discovery and demonstration of the teleomorph of Beauveria bassiana (Bals.) Vuill., an important entomogenous fungus. Chinese Science Bulletin 46: 751–753.
  • Luangsa-ard JJ, Tasanathai K, Mongkolsamrit S, Hywel-Jones N (2007) Atlas of invertebrate-pathogenic fungi of Thailand. National Center of Genetic Engineering and Biotechnology, National Science and Technology Development, Thailand, 75 pp.
  • Mains E (1958) North American entomogenous species of Cordyceps. Mycologia 50: 169–222.
  • Mongkolsamrit S, Noisripoom W, Tasanathai K, Khonsanit A, Thanakitpipattana D, Himaman W, Kobmoo N, Luangsa-ard JJ (2020) Molecular phylogeny and morphology reveal cryptic species in Blackwellomyces and Cordyceps (Cordycipitaceae) from Thailand. Mycological Progress 19: 957–983.
  • Mongkolsamrit S, Noisripoom W, Thanakitpipattana D, Wutikhun T, Spatafora JW, Luangsa-Ard J (2018) Disentangling cryptic species with isaria-like morphs in Cordycipitaceae. Mycologia 110: 230–257. https://doi.org/10.1080/00275514.2018.1446651
  • Ranwez V, Douzery EJ, Cambon C, Chantret N, Delsuc F (2018) MACSE v.2: toolkit for the alignment of coding sequences accounting for frameshifts and stop codons. Molecular Biology and Evolution 35: 2582–2584.
  • Royal Botanic Garden E (1969) Flora of British Fungi: colour identification chart. HM Stationery Office.
  • Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Consortium FB (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences 109: 6241–6246. https://doi.org/10.1073/pnas.1117018109
  • Shrestha B, Tanaka E, Hyun MW, Han J-G, Kim CS, Jo JW, Han S-K, Oh J, Sung G-H (2016) Coleopteran and Lepidopteran hosts of the entomopathogenic genus Cordyceps sensu lato. Journal of Mycology 2016: e7648219. https://doi.org/10.1155/2016/7648219
  • Stensrud Ø, Hywel-Jones NL, Schumacher T (2005) Towards a phylogenetic classification of Cordyceps: ITS nrDNA sequence data confirm divergent lineages and paraphyly. Mycological Research 109: 41–56. https://doi.org/10.1017/S095375620400139X
  • Sung GH, Hywel-Jones NL, Sung JM, Luangsa-Ard JJ, Shrestha B, Spatafora JW (2007) Phylogenetic classification of Cordyceps and the clavicipitaceous fungi. Studies in Mycology 57: 5–59. https://doi.org/10.3114/sim.2007.57.01
  • Sung GH, Spatafora JW, Zare R, Hodge KT, Gams W (2001) A revision of Verticillium sect. Prostrata. II. Phylogenetic analyses of SSU and LSU nuclear rDNA sequences from anamorphs and teleomorphs of the Clavicipitaceae. Nova Hedwigia 72: 311–328. https://doi.org/10.1111/j.1756-1051.2001.tb00824.x
  • Tasanathai K, Thanakitpipattana D, Noisripoom W, Khonsanit A, Kumsao J, Luangsa-ard JJ (2016) Two new Cordyceps species from a community forest in Thailand. Mycological Progress 15: 28.
  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876–4882. https://doi.org/10.1093/nar/25.24.4876
  • Vega FE, Goettel MS, Blackwell M, Chandler D, Jackson MA, Keller S, Koike M, Maniania NK, Monzon A, Ownley BH (2009) Fungal entomopathogens: new insights on their ecology. Fungal Ecology 2: 149–159. https://doi.org/10.1016/j.funeco.2009.05.001
  • Vizzini A, Antonin V, Sesli E, Contu M (2015) Gymnopus trabzonensis sp. nov. Omphalotaceae and Tricholoma virgatum var. fulvoumbonatum var. nov. Tricholomataceae, two new white-spored agarics from Turkey. Phytotaxa 226: 119–130. https://doi.org/10.11646/phytotaxa.226.2.2
  • Vu D, Groenewald M, De Vries M, Gehrmann T, Stielow B, Eberhardt U, Al-Hatmi A, Groenewald J, Cardinali G, Houbraken J (2019) Large-scale generation and analysis of filamentous fungal DNA barcodes boosts coverage for kingdom fungi and reveals thresholds for fungal species and higher taxon delimitation. Studies in Mycology 92: 135–154. https://doi.org/10.1016/j.simyco.2018.05.001
  • Wang L, Zhang W, Hu Ba, Chen Y, Qu L (2008) Genetic variation of Cordyceps militaris and its allies based on phylogenetic analysis of rDNA ITS sequence data. Fungal Diversity 31: 147–155. https://doi.org/10.1002/yea.1604
  • White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications 18: 315–322.
  • Zha LS, Huang SK, Xiao YP, Boonmee S, Eungwanichayapant PD, McKenzie EH, Kryukov V, Wu XL, Hyde KD, Wen TC (2018) An evaluation of common Cordyceps (Ascomycetes) species found in Chinese markets. International Journal of Medicinal Mushrooms 20: 1149–1162. https://doi.org/10.1615/IntJMedMushrooms.2018027330
  • Zhi LZ, Gang LF, L H-JN, Li ZS, Jun CM, Bo H, Sheng SC, An CZ, Ru LC, Jiu TY, Fei DJ (2021) Biodiversity of cordycipitoid fungi associated with Isaria cicadae Miquel II: Teleomorph discovery and nomenclature of chanhua, an important medicinal fungus in China. Mycosystema 40: 95–107. https://doi.org/10.13346/j.mycosystema.200119
  • Zhong X, Peng Q, Qi L, Lei W, Liu X (2010) rDNA-targeted PCR primers and FISH probe in the detection of Ophiocordyceps sinensis hyphae and conidia. Journal of Microbiological Methods 83: 188–193. https://doi.org/10.1016/j.mimet.2010.08.020
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