MycoKeys 33: | 6 II 380 (202 | ) er-reviewed open-access journal doi: 10.3897/mycokeys.83.72325 RESEARCH ARTICLE . 03 Mycokeys https://mycokeys.pensoft. net Launched to accelerate biodiversity research Morphology and molecular study of three new Cordycipitoid fungi and its related species collected from Jilin Province, northeast China 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” I School of Life Science, Northeast Normal University, Changchun City, 130024, Jilin Province, China 2 Engineering Research Centre of Edible and Medicinal Fungi, Ministry of Education, Jilin Agricultural University, Changchun City, 130118, Jilin Province, China 3 Hebei Normal University of Science and Technology, Qinghuangdao City, 066004, Hebei Province, China Corresponding authors: Bo Zhang (zhangbofungi@126.com), Yu Li (yuli966@126.com) Academic editor: Thorsten Lumbsch | Received 29 July 2021 | Accepted 29 August 2021 | Published 27 September 2021 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 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-1a 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 Copyright Jia-Jun Hu et al. 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. 162 Jia-Jun Hu et al. / MycoKeys 83: 161-180 (2021) 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 Cordycipi- taceae 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 be- ing 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 view- point, two subgenera, C. subg. Cryptocordyceps and C. subg. Racemella, were added (Mains 1958). Based on nrI TS, nrSSU, nrLSU, EF-1a, 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, Ophio- cordyceps 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 speci- mens were dried in an electric drier at 40-45 °C. Three new Cordycipitoid fungi from China 163 Descriptions of macroscopic characters were based on field notes and photo- graphs. 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. Al microscope for observation. For each species, a minimum of 40 part-spores was measured from two different ascocarps, part-spores are given as length x width (I x 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). Se- quences of the internal transcribed spacer region (ITS), nuclear large ribosomal subu- nits (LSU) and translation elongation factor 1-alpha (EF-1«) were used for phyloge- netic 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-1« 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 elec- trophoresis 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, Migelia martiale (Speg.) Luangsa-ard & Thanakitp., Ophiocordyceps spp. and Tolypocladium ophioglossoides (J.F. Gmoel.) 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-1a« sequences were com- bined with Mesquite. The tree construction procedure was performed in PAUP* ver- 164 Table |. Voucher information and GenBank accession numbers of ITS, LSU and EF-1« DNA sequences of Cordyceps changchunensis, Cordyceps changbaiensis, Cordyceps jingyuetanensis and related species used in this study. Species name Akanthomyces lecanii A, tuberculatus Blackwellomyces cardinalis B. pseudomilitaris Cordyceps bassiana C. bifusispora C. brongniartii C. cateniobliqua C. changbaiensis C. changbaiensis C. changchunensis C. changchunensis C. changchunensis C. chiangdaoensis C. coleopterorum C. exasperata C. farinosa C. fumosorosea C. hepialidicola C. jingyuetanensis C. jingyuetanensis C. kyushuensis C. militaris C. militaris C. morakotii C. ninchukispora C. ningxiaensis C. polyarthra C. pruinosa C. gingchengensis C. rosea C. roseostromata C. scarabaeicola C. scarabaeicola Cordyceps sp. Jia-Jun Hu et al. / MycoKeys 83: 161-180 (2021) Specimen/Strain number CBS101247 NBRC106949 CBS113414 NBRC101411 IFO4848 ARS5690/EFCC8260 NBRC101395 CBS153.83 HMJAU48255 HMJAU48260 HMJAU48251 HMJAU48252 HMJAU48259 Host/Substratum Homopteran Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidopteran pupae Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera BCC75734/TBRC7274 Coleopteran larvae CBS110.73 MCA2155 CBS111113 CBS244.31 HMJAU48253 HMJAU48261 HMAS78115 OSC93623 HMJAU48256 BCC55820/TBRC7276 BCC30937 HMJAU25074 6578 ARSEF5413 MEFLU17-1022 Spat09-053 ARSEF4870 ARSEF5689 Arsef5689 HMJAU48254 Coleoptera Lepidoptera Lepidoptera Coleoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidopteran pupae Lepidopteran pupae Hymenoptera Lepidoptera Diptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Larva, not specified Coleoptera Coleoptera Lepidoptera GenBank accession numbers ITS JN049836 JN943318 MH862930 JN943308 AB027382 AY245627 JN943298 MH861560 MW893252 MW893270 MW893249 MW893250 MW893251 KT261394 AY624177 AY624181 AY624182 AF315649 MW893253 MW893271 EF368021 JN049825 MW888227 KT261389 FJ765274 KF309668 AJ536548 JN049826 KY423506 AY245637 JN049827 JN049827 MW888228 LSU AF339555 JN941400 MH874497 JN941395 ABO027382 EF468807 JN941382 MW893277 MW893272 MW893274 MW6893275 MW893276 MF140732 JF415988 MF416542 MF416554 MF416557 MW893278 MW893273 EF468813 AY184966 MW893279 MF140731 FJ765242 KF309671 MK761215 MK761211 MF416536 AF339523 AF339524 AF339524 MW893280 EF-le DQ522359 MF416490 EF469059 MT017849 MN401498 EF468747 JF416009 MT017860 MZ616772 MZ616774 MZ616769 MZ616775 MZ616773 KT261404 JF416028 MF416486 MF416499 MF416503 MZ616770 EF468754 DQ522332 KT261399 MF416477 DQS522351 MK770630 MF416480 DQ522335 MZ616771 References (Kepler et al. 2012) (Kepler et al. 2017; Schoch et al. 2012) (Sung et al. 2007; Vu et al. 2019) (Mongkolsamrit et al. 2020; Schoch et al. 2012) (Khonsanit et al. 2020; Nikoh and Fukatsu 2000) (Kuo et al. 2005; Sung et al. 2007) (Kepler et al. 2012; Schoch et al. 2012) (Vu et al. 2019) This study This study This study This study This study (Mongkolsamrit et al. 2018; Tasanathai et al. 2016) (Kepler et al. 2012; Luangsa- Ard et al. 2005) (Kepler et al. 2017) (Kepler et al. 2017; Luangsa- Ard et al. 2005) (Kepler et al. 2017; Luangsa-Ard et al. 2005) Unpublished This study This study (Sung et al. 2007; Wang et al. 2008) (Sung et al. 2007) This study (Mongkolsamrit et al. 2018; Tasanathai et al. 2016) (Kepler et al. 2017) (Yan and Bau 2015) Unpublished (Kepler et al. 2012; Zha et al. 2019) (Zha et al. 2019) (Kepler et al. 2017) (Kuo et al. 2005; Sung et al. 2001) (Kepler et al. 2012; Sung et al. 2007) (Kepler et al. 2012; Sung et al. 2001) This study Three new Cordycipitoid fungi from China 165 Species name Specimen/Strain Host/Substratum GenBank accession numbers References number ITS LSU EF-lu C. spegazzinii ARSEF7850 Diptera DQ196435 DQ196435 GU734752 (Torres et al. 2005) C. taishanensis A-l Lepidoptera FJ008927 Unpublished C. tenuipes TBRC7266 Lepidoptera MF140742 MF140828 (Mongkolsamrit et al. 2018; Vu et al. 2019) Tsaria cicadae GACP07071701 Hemiptera KX017277. 9MK761212 MT268245 (Zhi et al. 2021) L. japonica BCC2808 Lepidoptera AY624199 (Luangsa-Ard et al. 2005) Metarhizium yong- EFCC2131 Lepidoptera JN049856 EF468833 EF468770 (Kepler et al. 2012; munense 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 OSC12858/ Coleoptera JN049820 =DQ518757 DQ522326 (Kepler et al. 2012) acicularis OSC110987 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. rubiginosoperi- NBRC106966 Coleoptera JN943344 JN941437 (Schoch et al. 2012) theciata O. sinensis ARSEF6282 Lepidopteran pupae HM595981 HM595885 EF468767 (Chan et al. 2011; Sung et al. 2007) Tolypocladium NBRC106331 Elaphomyces sp. JN943320 JN941408 (Schoch et al. 2012) ophioglossoides sion 4.0b10 (Swofford 2002) as described by Jiang et al. (Jiang et al. 2011). All char- acters were equally weighted and gaps were treated as missing data. 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 vari- ation 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 GIRGAMMA 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. Accord- ing 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. 166 Jia-Jun Hu et al. / MycoKeys 83: 161-180 (2021) 84 Blackwellomyces cardinalis CBS113414 Blackwellomyces pseudomilitari NBRC101411 68 Akanthomyces tuberculatus NBRC106949 Akanthomyces lecanii CBS101247 100 80 Cordyceps chiangdaoensis BCC75734/TBRC7274 100 Cordyceps morakotii BCC55820/TBRC7276 95 Cordyceps ninchukispora BCC30937 100 Cordyceps pruinosa ARSEF5413 92 64 Cordyceps cateniobliqua CBS153.83 92 Cordyceps brongniartii NBRC101395 100 Cordyceps scarabaeicola Arsef5689 Cordyceps bassiana 1FO4848 35 Isaria cicadae GACP07071701 Cordyceps bifusispora ARS5690/EFCC8260 100f Isaria japonica BCC2808 97 81 Cordyceps tenuipes TBRC7266 100 Cordyceps coleopterorum CBS110.73 99 Cordyceps qingchengensis MFLU17-1022 100 Cordyceps fumosorosea CBS244.31 Cordyceps farinosa CBS111113 Cordyceps spegazzinii ARSEF7850 Cordyceps ningxiaensis HMJAU25074 80 Cordyceps militaris BCC55820/TBRC7276 Cordyceps militars HMJAU 48256 Cordyceps roseostromata ARSEF4870 Cordyceps sp. HMJAU48254 Cordyceps taishanensis A-1 Cordyceps kyushuensis HMAS78115 Cordyceps hepialidicola AF315649 Cordyceps jingyuetanensis HMJAU48261 100 Cordyceps jingyuetanensis HMJAU48253 Cordyceps changbaiensis HMJAU48255 Cordyceps changbaiensis HMJAU48260 Cordyceps changchunensis HMJAU48251 70 Cordyceps changchunensis HMJAU48252 Cordyceps changchunensis HMJAU48259 75 Ophiocordyceps rubiginosiperit NBRC 106966 99 Ophiocordyceps sinensis ARSEF6282 100 65 Ophiocordyceps clavata NBRC106961 Ophiocordyceps gracilis EFCC8572 Ophiocordyceps acicularis OSC12858/OSC110987 92 Metacordyceps taii_ ARSEF5714 100 88 Metarhizium yongmunense EFCC2131 Nigelia martiale HMAS197472(S) Tolypocladium ophioglossoides NBRC106331 92 0.05 Figure |. 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-1« 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. milita- ris complex, C. jingyuetanensis was close to C. hepialidicola Kobayasi & Shimizu and is dif- ferent 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. Taxonomy Cordyceps changchunensis J.J. Hu, Bo Zhang & Y. Li, sp. nov. MycoBank No: 839249 Figs 3, 4 Holotype. Curna. 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 (HM- JAU 48251, holotype, GenBank Acc. nos.: ITS = MW893249, LSU = MW893274, EF-la = MZ616769). Three new Cordycipitoid fungi from China 100/1 100/1 100/1 100/1 100/1 100/1 100/1 97/1 100/0.99 100/1_ }100/1 97/0.99 AA Lioo_} 104 100/0.98 100/1 100/1 100/0.99 97/0.98 100/1 100/0.99 100/1 5310.85 100/1 100/0.99 42/0.76 00/1 100/1 99/1 100/1 97/0.95 “TI 68/0.59 100/1 78/0.91 81/0.93 63/0.50 79/0.92 Metarhizium yongmunense EFCC2131 Metacordyceps taii ARSEF5714 Tolypocladium ophioglossoides NBRC106331 Nigelia martiale HMAS197472 (S) Ophiocordyceps gracilis EFCC8572 Blackwellomyces pseudomilitaris NBRC101411 Blackwellomyces cardinalis CBS113414 Akanthomyces tuberculatus NBRC106949 Akanthomyces lecanii CBS101247 Cordyceps cateniobliqua CBS153.83 Cordyceps spegazzinii ARSEF7850 Cordyceps polyarthra 6578 Cordyceps exasperata MCA2155 Cordyceps farinosa CBS111113 Cordyceps fumosorosea CBS244.31 Cordyceps qingchengensis_ MFLU17-1022 Cordyceps bifusispora ARS5690/EFCC8260 Isaria cicadae “Chanhua” GACP07071701 Cordyceps coleopterorum CBS110.73 Isaria japonica BCC2808 Cordyceps tenuipes TBRC7266 Cordyceps brongniartii NBRC101395 Cordyceps bassiana 1FO4848 Cordyceps scarabaeicola Arsef5689 Cordyceps scarabaeicola ARSEF5689 Cordyceps ninchukispora BCC30937 Cordyceps chiangdaoensis BCC75734/TBRC7274 Cordyceps pruinosa ARSEF5413 Cordyceps morakotii_ BCC55820/TBRC7276 Cordyceps ningxiaensis HMJAU25074 Cordyceps rosea Spat09-053 Cordyceps sp. HMJAU48254 Cordyceps taishanensis A-| Cordyceps roseostromata ARSEF4870 Cordyceps kyushuensis HMAS78115 Cordyceps changbaiensis HMJAU48255 Cordyceps changbaiensis HMJAU48260 Cordyceps militaris OSC93623 Cordyceps militaris HMJAU48256 Cordyceps changchunensis HMJAU48251 Cordyceps changchunensis HMJAU48252 Cordyceps changchunensis HMJAU48259 Cordyceps hepialidicola Cordyceps jingyuetanensis HMJAU48253 Cordyceps jingyuetanensis HMJAU48261 Clavicipitaceae Cordycipitaceae 167 Clade3 A Clade 2 Clade 1 Clade 3B Ophiocordyceps clavata NBRC 106961 Ophiocordyceps sinensis ARSEF6282 Ophiocordyceps rubiginosiperitheciata NBRC106966 Ophiocordyceps acicularis OSC12858/OSC110987 Ophiocordycipitaceae 100/1 100/1 100/1 0.01 Figure 2. Phylogenetic tree of Cordycepitiod fungi, based on concatenated ITS, LSU and EF-1a from Bayesian analysis and Maximum Likelihood analysis; self-examined sequences are shown in bold. Etymology. changchunensis: referring to Changchun, the location of the holotype. Diagnosis. Cordyceps changchunensis can be easily differentiated from closely-relat- ed species C. militaris by its unique host, smaller stromata, immersed perithecia and larger part-spores (2.6—6 x 1.0—1.4 um). 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, some- times 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 x 180-520 um, with a thick wall about 10-15 um. Asci cylindrical, 80-300 x 2.5—5 um, 8-spored, apex of ascus hemispherical, 3.0—4.0 x 2.0-3.0 um. 168 Jia-Jun Hu et al. / MycoKeys 83: 161-180 (2021) Tf _ i> le A : a { = L i Figure 3. Morphological characters of Cordyceps changchunensis (HMJAU 48251, holotype) a, b, e stromata and host of Cordyceps changchunensis ¢ surface of fertile apex of ascostroma d host of Cordyceps changchunensis fh apex of ascus i-K ascus I=n part-spores. Scale bars: 1 cm (a,b); 2 mm (c,e); 1 mm (d); 10 um (f-h); 50 um (i-k); 5 um (I=n). Part-spores oblong, 2.6-6 x 1.0—1.4 um, smooth, hyaline in 3% KOH, thin- walled, inamyloid. Asexual Morph. Unknown. Host. Growing on pupae of Lepidoptera. Other specimens examined. Curna. Jilin Province: Changchun City, Jingyuetan National Forest Park, 20 August 2015, Bo Zhang (HMJAU 48259, GenBank Acc. nos.: ITS = MW893251, LSU = MW893276, EF-1a = MZ616773); Changchun City, Jingyuetan National Forest Park, 18 August 2018, Bo Zhang (HMJAU 48252, isotype, GenBank Acc. nos.: ITS = MW893250, LSU = MW893275, EF-la = 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 Three new Cordycipitoid fungi from China 169 Figure 4. Microscopical characters of Cordyceps changchunensis (HMJAU 48251, holotype) a perithecia b apex of ascus € part-spores. Scale bars: 100 um (a); 5 pm (b, Cc). (500-1089 x 132-264 um) and smaller part-spores (2-4 x 1 um) (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%). Cordyceps changbaiensis J.J. Hu, Bo Zhang & Y. Li, sp. nov. MycoBank No: 839250 Figs 5, 6 Holotype. Curna. 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-la = 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 x 1.0—1.4 pm). Description. Sexual Morph. Stromata 2.4—5.2 cm long, single or multiple, soli- tary, 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, obvi- ously distinguishable from the stipe. Sterile stipe fleshy, white to light yellow, cylin- drical, 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 x 90-170 um, with a thick wall about 15 um. Asci cylindrical, 225-625 x 4—5 um, 8-spored, apex of ascus hemispherical, 3.0-4.0 x 2.2—3.2 um. Part-spores oblong, 3.0—7.0 x 1.0—1.4 um, smooth, hyaline in 3% KOH, thin-walled, inamyloid. Asexual Morph. Unknown. Host. Growing on larvae of Lepidoptera. Distribution. China (Jilin Province). Other specimen examined. Curna. Jilin Province: Baishan City, Fusong County, Quanyang Town, 42.30°N, 127.29°E, 22 August 2021, Jia-Jun Hu, Bo Zhang & Gui- 170 Jia-Jun Hu et al. / MycoKeys 83: 161-180 (2021) Figure 5. Morphological characters of Cordyceps changbaiensis (HMJAU 48255, holotype) a stromata and host of Cordyceps changbaiensis b host of Cordyceps changbaiensis €, d surface of fertile apex of ascos- troma e=h ascus i=k apex of ascus =m part-spores. Scale bars: 1 cm (a); 5 mm (b=c); 200 um (d); 20 um (e-h); 10 um (i-k); 5 pm (I-m). Ping Zhao (HMJAU 482260, isotype, GenBank Acc. nos.: ITS = MW893270, LSU = MW893272, EF-la = MZ616774) Note. C. changbaiensis has orange to white and branched stromata. Morphologi- cally, 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. rose- ostromata and C. kyushuensis have a larger perithecia and smaller part-spores. Further- more, 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. chang- baiensis was placed in separate monophyletic lineages (BPP = 0.95, MLBS = 97%) and formed a sister relationship with C. rosea. Three new Cordycipitoid fungi from China 171 a eure 6. Microscopical characters of Deicak changbaiensis (HMJAU 48255, holotype) a perithecia b apex of ascus € part-spores. Scale bars: 100 um (a); 5 pm (b, Cc). Cordyceps jinyuetanensis J.J. Hu, Bo Zhang & Y. Li, sp. nov. MycoBank No: 839251 Figs 7, 8 Holotype. Curna. 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 (HM- JAU 48253, holotype, GenBank Acc. nos.: ITS = MW893253, LSU = MW893278, EF-1a = MZ616770). Etymology. jinyuetanensis: referring to Jingyuetan National Forest Park, the loca- tion 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. Description. Sexual Morph. Stromata 4—4.5 cm long, multiple, solitary, aris- ing 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. Ster- ile 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 x 110-220 um, with a thick wall about 15-20 um. Asci cylindrical, 225-475 x 3-5 um, 8-spored, apex of as- cus hemispherical to irregular, 3.0-4.0 x 1.4-2.8 um. Part-spores oblong, 2.8—5.0 x 1.0—1.4 um, smooth, hyaline in 3% KOH, thin-walled, inamyloid. Asexual Morph. Unknown. Host. Growing on pupae of Lepidoptera. Distribution. China (Jilin Province). Other specimen examined. Curna. 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 Cordy- cipitiod fungi growing on pupae, like the unusual medicinal fungi O. sinensis (Berk.) 172 Jia-Jun Hu et al. / MycoKeys 83: 161-180 (2021) 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 @, f part-spores g—K ascus I-o apex of ascus. Scale bars: 1 cm (a); 2 mm (b, €); 500 um (d); 5 um (e, f); 20 um (g—k); 10 um (i-o). G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, C. militaris, I. cicadae Mig. and also like the two new species, C. ningxiaensis T. Bau & J.Q. Yan and C. gingchengen- sis 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 Three new Cordycipitoid fungi from China Wes: an B Ea & Figure 8. Microscopical characters of Cordyceps jingyuetanensis (HMJAU 48253, holotype) a perithecia b apex of ascus € part-spores. Scale bars: 100 um (a); 5 pm (b, Cc). on the pupae of Diptera, 1. cicadae grows on the pupae of Hemiptera and the stro- mata of C. gingchengensis 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 x 500 ym) and smaller part-spores (3—4 x 1 um) (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. Curna. Yunnan Province: Qujin City, Huize County, 26.24°N, 103.25°E, 30 July 2019, Jia-Jun Hu, Bo Zhang & Di-Zhe Guo (HM- JAU 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, Jian 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 (HM- JAU 48263). 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. 174 Jia-Jun Hu et al. / MycoKeys 83: 161-180 (2021) Table 2. Morphological comparisons of sexual states of Cordyceps changchunensis, Cordyceps changbaiensis and Cordyceps jingyuetanensis. Species Host Stromata Fertile part Perithecia Asci Reference Beauveria | Larvae of | Single or sev- Elliptical, 610-720 x Cylindrical, 230- Filamentous, (Li et al. bassiana | Lepidop- | eral, unbranched, 230-320 um, immersed | 590 x 3.5-4.0 um | 300-570 x 1.0 um, 2001) tera slender and to surface with ascus cap not broken into part- cylindrical, 3.6—4.0 um in spores brownish- yellow diameter to yellowish Black- Larvae of Single or Elongate-ellipsoid or —_| Filiform, 290-410 x | Filiform, 280-390 | (Hywel- wellomyces | Lepidop- | cluster, simple elongate-ovoid, 290-570 5-6 pm x 1 um, not broken Jones pseudomili-| tera or branched, x 120-245 um, superficial into part-spores 1994) taris cylindrical, white to white-orange Cordyceps | Larvae of | Simple, cylin- | 6 x 1.3 mm | Pyriform, with protruding | Cylindrical, 200- Bifusiform, 145— (Eriksson bifusispora | Lepidop- | drical clavate, apices, yellowish, 300 x 220 x 3—4.5 ym 220 um in length, 1982) tera whitish 150-170 um, immersed central part filiform about 0.4 um wide, terminal parts nar- rowly fusiform, about 30 x 1.6 pm and 3 septate C. kyush- | Larvae of Cluster, Elliptical, 300-500 x Cylindrical, Short cylindrical, (Guo and uensis Lepidop- | cylindrical, Light 200-300 um, half-buried 3-4.5 um wide part-spores 5—7 x Li 2000; tera yellow to orange 0.7-1 um Li et al. red 2015) C. militaris| Lepi- Single or several, Conical, half-buried Clavate, 300-400 x | Filiform, part spores | (Liet al. dopteran | clavate, orange 4-5 um 2-3 x 1 pm 2015) pupae C. ningxi- | Fly pupae] One to two ina Ellipsoid to ovoid, 288— | Cylindrical, 168— | Filiform, irregularly | (Yan and aensis (Diptera) | group, clavate, | 1.2-2.8 mm |} 400 x 103-240 um, with | 205 x (3.7-)4.1- multiseptate, part- | Bau 2015) orange a wall about 10 um thick, | 5.5(—6.6) pm, with spores 3.6—7.8 x loosely embedded at right | oblate spheroid or 1.0-1.4 pm angles to the surface hemispherical refrac- tive cap 3.4-3.8 x 2.9-3.4 um at apex C. polyar- Cespitose, nar- Ovoid, 250-450 x Cylindrical, Filiform, part-spores | (Mains thra rowly clavate, 125-250 pm, brown, 150-260 x 3-4 um, 4-6 x 0.75-1 um 1958) light yellow to with a definite wall 25 pm | with a 1.5-2 pm reddish-brown thick, embedded at right thick cap angles to the surface C. prui- | Larvae of Solitary or Ovoid to fusiform, Cylindrical, 100— | Filiform, part-spores | (Liet al. nosa Lepidop- | several, clavate, 360-400 x 130-200 pm, 200 x 2.5-4 um 4-6 x lum 2015) tera orange to red crowded, red, ordinal in orientation, immersed C. qin- Lepi- | Branched, yellow 7-9 x Ovoid but apex sharply | Cylindrical, 180- Filiform, 180-220 | (Zha et al. gchengensis | dopteran 2.0-2.5 mm pointed, 335-490 x 200 x 2.44.0 um x 0.45—0.65 pm, 2019) pupae 145-240 um, partially wide, caps hemi- not at all bifusiform immersed at right angle to | spherical, 1.8-2.2 x | and not broken into the surface 2.5-3.2 um part-spores C. roseo- Larva, Single or 1.2-5 x Pyriform, 280-300 x 3-3.5 x 2.5-3 pm 4-5x lum (Kobayasi stromata | not speci- branched 1.5-2.2 mm | 140-160 um, Superficial 1983) fied C. change- Lepi- Single or mul- 2.0-3.5 x Globose to ovoid, 180— | Cylindrical, 80-300 Oblong, 2.6-6 x This study hunensis | dopteran | tiple, clavate, | 0.4-0.6 mm | 600 x 180-520 um, with | x 2.5—5 um, caps 1.0-1.4 pm pupae orange a thick wall about 10-15 | hemispheric, 3.0-4.0 um, partially immersed at | x 2.0-3.0 um at right angles to the surface apex C. chang- | Larvae of | Single or multi- Globose to ovoid, 120— Cylindrical, Oblong, 3.0-7.0 x | This study baiensis Lepidop- | ple, clavate, white 230 x 90-170 um, with | 225-625 x 4-5 um, 1.0-1.4 um tera to orange a thick wall about 15 jm, | caps hemispherical, immersed to surface 3.0-4.0 x 2.2— 3.2 um at apex C. jingyu- | Lepi- Single or mul- | 0.8-1.3x | Almond-shaped to ovoid, | Cylindrical, 225— | Oblong, 2.8-5.0 x | This study etanensis dopteran tiple, clavate, 0.1-0.2 mm | 220-340 x 110-220 um, | 475 x 3—5 um, caps 1.0-1.4 um pupae orange to light red with a thick wall about 15-20 um, immersed to surface hemispherical to irregular, 3.0-4.0 x 1.42.8 um at apex Three new Cordycipitoid fungi from China 175 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 ¢, e collected from Changchun City, Jilin Province d collected from Qujin City, Huize County, Yunnan Province). Scale bars: 1 cm (a=e). Key to reported species in this study 1 SEOMatarariseh Le mig Dae yal Se Rea Wren. a Mere Lat Ode Ph all se oe 2 - SLOMAtAta Se LOMA Aes. Poca: acs weirs eh eee Cordyceps changbaiensis 2 Stromata branched into two forks sometimes...... Cordyceps changchunensis — SOME MO branche ker: mm. weominnd en Ise tER PEN ON, ok eae rennet ane aT 3 3 ATES POLES OVC) MINH A528 ras Birds hase Rahat ane! Cordyceps jingyuetanensis - Patrsspores.léssithani Sypiiiy eeas-8. eee k ot ERD. cco 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 176 Jia-Jun Hu et al. / MycoKeys 83: 161-180 (2021) belonging to the C. militaris group (Yan and Bau 2015). ML and BI analysis recog- nised 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 Jsaria 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 Lsaria 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 J. 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 an- amorph existed on some specimens, with the morphology of the anamorph consistent with those, “J. 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, RG. Luan, Hywel-Jones, C.R. Li & S.L. Zhang (Zhi et al. 2021). Furthermore, /. japonica Yasuda reported from Japan, exhibits exception- ally 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—Co- leoptera 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 oc- curred 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). Three new Cordycipitoid fungi from China 177 Acknowledgements This study is funded by the National Key R & D of Ministry of Science and Technol- ogy (2018YFE0107800), the National Key R & D of Ministry of Science and Tech- nology (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, Hywel-Jones NL, Jones EBG (2001) Relationship of the ge- nus 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 anamo- rph 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 Quindio, 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/ het1202-118 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. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis pro- gram for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98. https://doi. org/10.1021/bk-1999-0734.ch008 Hywel-Jones NL (1994) Cordyceps khaoyaiensis and C. pseudomilitaris, two new pathogens of lepi- dopteran larvae from Thailand. Mycological Research 98: 939-942. https://doi.org/10.1016/ S0953-7562(09)80267-0 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/s13225-010-0076-7 178 Jia-Jun Hu et al. / MycoKeys 83: 161-180 (2021) Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: im- provements 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 Cordy- cipitaceae (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. Myco- logical 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, LiTH, 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 Beau- veria bassiana (Bals.) Vuill., an important entomogenous fungus. Chinese Science Bulletin 46: 751-753. Luangsa-Ard JJ, Hywel-Jones NL, Manoch L, Samson RA (2005) On the relationships of Paecilomyces sect. Isarioidea species. Mycological Research 109: 581-589. https://doi. org/10.1017/S0953756205002741 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 cryp- tic species in Blackwellomyces and Cordyceps (Cordycipitaceae) from Thailand. Mycological Progress 19: 957-983. Mongkolsamrit $, 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 Three new Cordycipitoid fungi from China Les) Nikoh N, Fukatsu T (2000) Interkingdom host jumping underground: phylogenetic analysis of entomoparasitic fungi of the genus Cordyceps. Molecular Biology and Evolution 17: 629-638. https://doi.org/10.1093/oxfordjournals.molbev.a026341 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. Ronquist FE, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574. https://doi.org/10.1093/bioinformatics/btg180 Royal Botanic Garden E (1969) Flora of British Fungi: colour identification chart. HM Sta- tionery Office. Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Consor- tium 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 ©, Hywel-Jones NL, Schumacher T (2005) Towards a phylogenetic classification of Cordyceps: ITS nrDNA sequence data confirm divergent lineages and paraphyly. Mycologi- cal 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 Mycol- ogy 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 anamo- rphs and teleomorphs of the Clavicipitaceae. Nova Hedwigia 72: 311-328. https://doi. org/10.1111/j.1756-1051.2001.tb00824.x Swofford DL (2002) PAUP*: phylogenetic analysis using parsimony (*and other methods), version 4.0b10. Sinauer Associates, Sunderland. https://doi.org/10.1002/0471650129. dob0522 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 Pro- gress 15: 28. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin EK Higgins DG (1997) The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by qual- ity analysis tools. Nucleic Acids Research 25: 4876-4882. https://doi.org/10.1093/ nar/25.24.4876 Torres MS, White J, Bischoff JF (2005) Cordyceps spegazzinii sp. nov., a new species of the C. militaris group. Mycotaxon 94: 253-263. https://doi.org/10.1016/j.microrel.2005.07.047 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 ecol- ogy. Fungal Ecology 2: 149-159. https://doi.org/10.1016/j.funeco.2009.05.001 180 Jia-Jun Hu et al. / MycoKeys 83: 161-180 (2021) Vizzini A, Antonin V, Sesli E, Contu M (2015) Gymnopus trabzonensis sp. nov. Omphalota- ceae and Tricholoma virgatum vat. 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, Groe- newald J, Cardinali G, Houbraken J (2019) Large-scale generation and analysis of filamen- tous 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 ribo- somal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications 18: 315-322. Yan JQ, Bau T (2015) Cordyceps ningxiaensis sp. nov., a new species from Dipteran pupae in Ningxia Hui Autonomous Region of China. Nova Hedwig 100: 251-258. https://doi. org/10.1127/nova_hedwigia/2014/0222 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 (Ascomy- cetes) species found in Chinese markets. International Journal of Medicinal Mushrooms 20: 1149-1162. https://doi.org/10.1615/IntJ MedMushrooms.2018027330 Zha LS, WEN TC, Huang SK, Boonmee S, Eungwanichayapant PD (2019) Taxonomy and biology of Cordyceps gingchengensis sp. nov. and its allies. Phytotaxa 416: 14—24. https:// doi.org/10.11646/phytotaxa.416.1.2 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 lsaria cicadae Miquel II: Teleo- morph 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