Front. Microbiol. Frontiers in Microbiology Front. Microbiol. 1664-302X Frontiers Media S.A. 10.3389/fmicb.2022.846909 Microbiology Original Research Morphology and Phylogeny Reveal Five Novel Species in the Genus Cordyceps (Cordycipitaceae, Hypocreales) From Yunnan, China Dong Quan-Ying 1 2 Wang Yao 1 2 Wang Zhi-Qin 1 2 Tang De-Xiang 1 2 Zhao Zhi-Yuan 1 2 Wu Hui-Juan 1 2 Yu Hong 1 2 * 1Yunnan Herbal Laboratory, College of Ecology and Environmental Sciences, Yunnan University, Kunming, China 2The International Joint Research Center for Sustainable Utilization of Cordyceps Bioresources in China and Southeast Asia, Yunnan University, Kunming, China

Edited by: Rajesh Jeewon, University of Mauritius, Mauritius

Reviewed by: Sanjay K. Singh, Agharkar Research Institute, India; Samantha Chandranath Karunarathna, Qujing Normal University, China; Tingchi Wen, Guizhou University, China

 *Correspondence: Hong Yu, hongyu@ynu.edu.cn

This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology

 13 04 2022 2022 13 846909 31 12 2021 16 02 2022 Copyright © 2022 Dong, Wang, Wang, Tang, Zhao, Wu and Yu. 2022 Dong, Wang, Wang, Tang, Zhao, Wu and Yu

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The current study was aimed to introduce five new species of Cordyceps from Yunnan, with morphological descriptions, illustrations, color photographs, phylogenetic placement, associated host, and a comparison with allied taxa. The five new species were morphologically distinct from all other Cordyceps sensu lato species, and it was also suggested that they should differ from other species in the genus Cordyceps based on combined multigene analyses. Employing DNA nucleotide sequences of the nrLSU, nrSSU, tef-1α, rpb1, and rpb2, the five new species were recognized in the clade of Cordyceps by using molecular phylogenetic analyses, including five well-supported subclades: three new species, Cordyceps bullispora, Cordyceps longiphialis, and Cordyceps nabanheensis, were found in the subclade of C. pruinosa, and two new species, Cordyceps pseudotenuipes and Cordyceps simaoensis, were located in the subclade of C. tenuipes. The five novel species shared similar morphologies to other species in the genus Cordyceps, with fleshy and brightly pigmented stromata; perithecia superficial to completely immersed, ordinal in arrangement; and hyaline asci, with thickened cylindrical ascus apex. The morphological characteristics of 66 species in Cordyceps sensu stricto, namely, 5 novel species and 61 known taxa, were also compared.

 entomopathogenic fungi multilocus phylogeny new taxon species diversity taxonomy No. 31870017 National Natural Science Foundation of China10.13039/501100001809 Yunnan Provincial Science and Technology Department10.13039/501100008871 Introduction

Cordyceps Fr. is a well-known genus of arthropod-pathogenic fungi. It was shown that many species of Cordyceps played a significant role in the cycling of matter in an ecological system, had a high ecological and economic value for biocontrol and bioactive compounds, and served as a model system for research on fungal insect pathology (Zha et al., 2018; Chen W. H. et al., 2019). Complexes of some cordycipitoid fungal species and their natural host, such as C. militaris Fr., C. chanhua Z. Z. Li, F. G. Luan, Hywel-Jones, C. R. Li and S. L. Zhang, and C. kyusyuensis Kawam, have received significant attention in traditional medicine industry due to the detection of bioactive compounds with anti-aging, anti-tumor, antioxidant, anti-inflammatory, and immuno-modulatory effects (Castillo et al., 2018; Zhao et al., 2018; Lou et al., 2019; Li et al., 2021; Zhang et al., 2021). Cordyceps tenuipes (Peck) Kepler, B. Shrestha and Spatafora has been applied in a variety of functional foods in Japan and South Korea, possessing nutritional, immune-regulatory, antitumor, analgesic, antibacterial, and anti-malaria effects (Wang Y. et al., 2020). Cordyceps species reproduce via sexual (ascospores) or asexual (conidia) spores, or both (Mora et al., 2017). The host range of Cordyceps embraces 7 orders of Arthropoda, namely, Araneae, Coleopteran, Dermaptera, Hemiptera, Hymenoptera, Lepidoptera, and Orthoptera, where Coleoptera and Lepidoptera are the two significant orders to host beyond the estimated 200 Cordyceps spp. as recorded (Shrestha et al., 2016; Kepler et al., 2017; Mongkolsamrit et al., 2020; Wang Y. B. et al., 2020).

Fries (1818) was credited with coining Cordyceps as a genus in Pyrenomycetes from a hybrid of the Greek word cordyle and the Latin word caput, meaning a club and a head, respectively. However, this genus was then treated as a tribe level of Sphaeria and described as stroma erect, stipe simple or branching, a sterile stalk supporting the perithecia at the periphery, and projecting openings at the apex (Shrestha et al., 2014). Nearly 20 different genera have been accounted as synonyms of Cordyceps in various sources (Shrestha et al., 2014). Shrestha et al. (2014) provided a comprehensive review regarding the taxonomic history of Cordyceps and concluded that the genus is the oldest valid genus in Cordycipitaceae (Hypocreales, Ascomycota) and is typified by a sexual morph. Owing to the cylindrical shape of the stroma, C. militaris, the type species of Cordyceps, was already described in the 17th- and early 18th-century literature (Shrestha et al., 2014).

Species of Cordycipitaceae produce three types of ascospore, namely disarticulating ascospores (e.g., C. militaris), intact ascospores [e.g., Blackwellomyces cardinalis (G. H. Sung and Spatafora) Spatafora and Luangsa-ard and Blackwellomyces pseudomilitaris Hywel-Jones and Sivichai], and bola-ascospores (e.g., C. bifusispora O. E. Eriksson), with superficial to partially immersed perithecia on fleshy stromata that are pallid to brightly pigmented. Currently, Cordyceps s. l. (Fr) Link (1833) consists of approximately 1,300 known species assigned to three families (Cordycipitaceae, Ophiocordycipitaceae, and partial Clavicipitaceae) in the order Hypocreales (Sung et al., 2007; Zha et al., 2018). Progressive works from researchers worldwide have enriched the account of species diversity within Cordycipitaceae and revealed new genera (Kepler et al., 2017; Mongkolsamrit et al., 2018, 2020; Wang Y. B. et al., 2020). Up to now, the Cordycipitaceae has twenty genera: Akanthomyces Lebert, Amphichorda Fr., Ascopolyporus Möller, Beauveria Vuill, Blackwellomyces Spatafora and Luangsa-ard, Cordyceps s. s., Engyodontium de Hoog, Flavocillium H. Yu, Y. B. Wang, Y. Wang, Q. Fan and Zhu L. Yang, Gamszarea Z. F. Zhang and L. Cai, Gibellula Cavara, Hevansia Luangsa-ard, Hywel-Jones and Spatafora, Hyperdermium J. F. White, R. F. Sullivan, Bills and Hywel-Jones, Lecanicillium W. Gams and Zare, Leptobacillium Zare and W. Gams, Liangia H. Yu, Y. B. Wang, Y. Wang, Z. H. Chen and Zhu L. Yang, Neotorrubiella Tasan., Thanakitp. and Luangsa-ard, Parengyodontium C. C. Tsang, J. F. W. Chan, W. M. Pong, J. H. K. Chen, A. H. Y. Ngan, M. Cheung, C. K. C. Lai, D. N. C. Tsang, S. K. P. Lau and P. C. Y. Woo, Samsoniella (G. Sm.) Mongkols, Noisrip, Thanakitp, Spatafora and Luangsa-ard, Simplicillium W. Gams and Zare, and Torrubiella W. Gams and Zare.

The unispecific genus Phytocordyceps possesses bola-ascospores, and because of its phylogenetic placement, it is also recognized as a member of Cordycipitaceae and thus transferred to Cordyceps s. s. Based on molecular phylogenetic studies or morphological descriptions, 35 names and new combinations were accepted for Cordyceps s. s. (Sung et al., 2007). Concerning the application of Isaria, in an effort to avoid the confusion with Cordyceps, Kepler et al. (2017) proposed the rejection of Isaria and combined 13 species into Cordyceps s. s. To date, together with the species as reported in recent years, there are about 70 species whose phylogenetic positions are known in Cordyceps worldwide (Tasanathai et al., 2016; Chirivi et al., 2017; Catania et al., 2018; Mongkolsamrit et al., 2018, 2020; Olatunji et al., 2018; Crous et al., 2019; Hyde et al., 2019; Li et al., 2020; Wang Y. B. et al., 2020; Hu et al., 2021).

Harvesting wild specimens have become a challenge as natural fungi populations are declining and large-scale cultivation has not been achieved. Recently, the demand for cultivable fungal species with medicinal applications has been increasing worldwide. Yunnan Province and the Tibetan plateau are home to a large diversity of entomogenous fungi (Chen Z. H. et al., 2019). C. chaetoclavata H. Yu, Y.B. Wang, Y. Wang, Q. Fan and Zhu L. Yang, C. cocoonihabita H. Yu, Y.B. Wang, Y. Wang, Q. Fan and Zhu L. Yang, C. shuifuensis H. Yu, Y. B. Wang, Y. Wang and Zhu L. Yang, and C. subtenuipes H. Yu, Y. B. Wang, Y. Wang, D. E. Duan and Zhu L. Yang have been described from this area (Wang Y. B. et al., 2020). In this study, based on macro- and micro-morphological characteristics, ecological data together with DNA nucleotide sequence analyses of the nuclear ribosomal large subunit (nrLSU) and small subunit (nrSSU), the genes encoding translation elongation factor 1-α (tef-1α), the largest subunit of RNA polymerase II (rpb1), and the second-largest subunit of RNA polymerase II (rpb2), the fungus’ phylogenetic position was assessed. Furthermore, we have also compared the morphological characteristics of 66 species in Cordyceps s. s., consisting of 5 novel species and 61 known taxa.

 Materials and Methods Sampling

Cordyceps samples were newly collected from Kunming, Weishan, Jinghong, and Pu’er of Yunnan, southwestern China. Voucher specimens were deposited in the YHH (Yunnan Herbal Herbarium) of Yunnan University. The isolated strains were deposited in YFCC (Yunnan Fungal Culture Collection) of Yunnan University.

Fungal materials, including the hosts, were photographed and recorded. Isolation of the fungi was achieved as per Wang Y. B. et al. (2020). The stromata or synnemata were cut into 5-mm-long segments, followed by surface sterilization with 30% H2O2 for 30 s to 1 min, and then rinsed with sterile water five times, dried with sterilized filter paper. Then, a part of the insect body was cut off, and the resulting segments and insect body were inoculated onto potato dextrose agar (PDA: potato 200 g/L, dextrose 20 g/L, and agar 20 g/L) plates containing 0.1 g/L streptomycin and 0.05 g/L tetracycline.

 Morphological Studies

Given the field notes, color images of the materials, and complementary literature data, macro-morphological characteristics, such as the host, fungi location, color, and shape of the stromata, and perithecial orientation (superficial, immersed, semi-immersed; ordinal or oblique) were examined under a dissecting microscope (Olympus SZ61), where the insect hosts were recognized with the support from professional entomologists.

The sexual characteristics, such as perithecia, asci, and ascospores, were firstly mounted on glass slides with lactophenol cotton blue solution after removing from the stroma, whereas the asexual characteristics, such as phialides and conidia, were firstly inoculated on glass slides with a thin layer of PDA medium block (5–10 mm in diameter) and overlaid by a cover slip, and cultivated in Petri dishes with a small amount of water under room temperature until further development of phialides and conidia. Then, the micro-morphological descriptions in both size and shape were determined with Olympus CX40 and BX53 microscopes, and FEI Quanta 200 scanning electron microscope. Twenty to thirty individual length and width measurements were taken, with absolute minima and maxima. Mycelia were inoculated on PDA plates and incubated at 25°C for a couple of days, and the colonies were photographed and measured every week.

 Molecular Studies DNA Extraction and PCR Amplification

Total DNA was extracted from the fungal mycelia on PDA plates or herbarium materials using the modified CTAB procedure (Doyle and Doyle, 1987). For DNA amplification, the primer pairs nrSSU-CoF and nrSSU-CoR (Wang Y. B. et al., 2015) and LR5 and LR0R (Vilgalys and Hester, 1990; Rehner and Samuels, 1994) were used for the nrSSU and nrLSU, EF1α-EF and EF1α-ER (Bischoff et al., 2006; Sung et al., 2007) were used to amplify the translation elongation factor 1α (tef-1α), and primers RPB1-5’F and RPB1-5’R, and RPB2-5’F and RPB2-5’R (Bischoff et al., 2006; Sung et al., 2007) were used to amplify the largest and second-largest subunits of RNA polymerase II (rpb1 and rpb2), respectively.

The polymerase chain reaction (PCR) matrix was composed of 2.5 μl of PCR 10 × Buffer (2 mmol/L Mg2+) (Transgen Biotech, Beijing, China), 0.25 μl of Taq DNA polymerase (Transgen Biotech, Beijing, China), 2 μl of dNTP (2.5 mmol/L), 1 μl of DNA template (500 ng/μl), 1 μl of forward primers (10 μmol/L), 1 μl of reverse primers (10 μmol/L), and 17.25 μl of sterile ddH2O. Amplification reactions were performed in a BIO-RAD T100™ thermal cycler (BIO-RAD Laboratories, Hercules, CA, United States). The PCR program was performed as described by Sung et al. (2007) and Wang Y. B. et al. (2020). Products of PCR were purified with the Gel Extraction and PCR Purification Combo Kit Beijing Genomics Institute (Shenzhen, China) and then sequenced on an automatic sequence analyzer (BGI Co., Ltd, Shenzhen, China) using the same primers as those used in amplification.

 DNA Sequence Alignments

The samples’ nrSSU, nrLSU, tef-1α, rpb1, and rpb2 nucleotide sequences were compared with those deposited in the GenBank database. To understand the relationship of our sample with those in the GenBank, nrSSU, nrLSU, tef-1α, rpb1, and rpb2 sequences of the representative Cordyceps s. s. species available in GenBank were retrieved and combined with our sequences (Table 1). Five datasets, the nrSSU, nrLSU, tef-1α, rpb1, and rpb2 sequences, were aligned and manually checked on Bioedit v7.0.9 (Hall, 1999). In order to examine phylogenetic conflicts among these datasets, the partition homogeneity (PH) test was performed with 1,000 randomized replicates, using heuristic searches with simple addition of sequences in PAUP* 4.0b10 (Swofford, 2002); the phylogenetic signals in the five gene markers showed no conflict.

Names, voucher information, host, and corresponding GenBank accession numbers of the taxa used in this study.

Taxon Voucher information Host GenBank Accession Number
 References
nrSSU nrLSU tef-1α rpb1 rpb2
Cordyceps albocitrina spat 07-174 MF416575 MF416467 MF416629 Kepler et al., 2017
Cordyceps amoene-rosea CBS 107.73 Coleoptera (pupa) AY526464 MF416550 MF416494 MF416651 MF416445 Luangsa-ard et al., 2005
Cordyceps amoene-rosea CBS 729.73 Coleoptera; Nitidulidae MF416604 MF416551 MF416495 MF416652 MF416446 Luangsa-ard et al., 2005
Cordyceps araneae BCC 85065 Arachnid; Araneae MT003037 MT017850 MT017810 MT017828 Mongkolsamrit et al., 2020
Cordyceps araneae BCC 85066 Arachnid; Araneae MT003038 MT017851 MT017811 MT017829 Mongkolsamrit et al., 2020
Cordyceps araneae BCC 88291 Arachnid; Araneae MT003039 MT017852 MT017812 MT017830 Mongkolsamrit et al., 2020
Cordyceps bifusispora spat 08-129 MF416576 MF416523 MF416468 MF416630 Kepler et al., 2017
Cordyceps bifusispora spat 08-133.1 MF416577 MF416524 MF416469 MF416631 MF416434 Kepler et al., 2017
Cordyceps bifusispora EFCC 5690 Lepidopteran (pupa) EF468952 EF468806 EF468746 EF468854 EF468909 Sung et al., 2007
Cordyceps bifusispora EFCC 8260 Lepidopteran (pupa) EF468953 EF468807 EF468747 EF468855 EF468910 Sung et al., 2007
Cordyceps blackwelliae TBRC 7255 Coleoptera (larva) MF140703 MF140823 MF140772 MF140796 Mongkolsamrit et al., 2018
Cordyceps blackwelliae TBRC 7256 Coleoptera (larva) MF140702 MF140822 MF140771 MF140795 Mongkolsamrit et al., 2018
Cordyceps blackwelliae YFCC 856 Lepidoptera (larva) MW181780 MW173992 MW168233 MW168199 MW168216 Unpublished
Cordyceps brevistroma BCC 78209 Lepidoptera (larva) MT003044 MT017855 MT017817 MT017835 Mongkolsamrit et al., 2020
Cordyceps brevistroma BCC 79253 Lepidoptera (larva) MT003045 MT017856 MT017836 Mongkolsamrit et al., 2020
Cordyceps bullispora YFCC 8400 Lepidopteran (pupa) OL468555 OL468575 OL473523 OL739569 OL473534 This study
Cordyceps bullispora YFCC 8401 Lepidopteran (pupa) OL468556 OL468576 OL473524 OL739570 OL473535 This study
Cordyceps caloceroides MCA 2249 Araneae MF416578 MF416525 MF416470 MF416632 Kepler et al., 2017
Cordyceps cateniannulata CBS 152.83 Coleoptera (adult) AY526465 MG665226 JQ425687 Luangsa-ard et al., 2004; Mongkolsamrit et al., 2018
Cordyceps cateniobliqua YFCC 3367 Coleopteran adult MN576765 MN576821 MN576991 MN576881 MN576935 Wang Y. B. et al., 2020
Cordyceps cateniobliqua YFCC 5935 MN576766 MN576822 MN576992 MN576882 MN576936 Wang Y. B. et al., 2020
Cordyceps cateniobliqua CBS 153.83 Adoxophyes privatana AY526466 JQ425688 MG665236 Luangsa-ard et al., 2004; Mongkolsamrit et al., 2018
Cordyceps cf. ochraceostromata ARSEF 5691 EF468964 EF468819 EF468759 EF468867 EF468921 Kepler et al., 2012
Cordyceps cf. pruinosa spat 08-115 MF416586 MF416532 MF416476 MF416635 MF416439 Kepler et al., 2017
Cordyceps cf. pruinosa spat 09-021 MF416587 MF416533 MF416477 MF416636 Kepler et al., 2017
Cordyceps cf. pruinosa NHJ 10627 Limacodid pupa (Lepidoptera) EF468967 EF468822 EF468763 EF468870 Sung et al., 2007
Cordyceps cf. pruinosa NHJ 10684 Limacodid pupa (Lepidoptera) EF468968 EF468823 EF468761 EF468871 Sung et al., 2007
Cordyceps cf. pruinosa EFCC 5693 EF468966 EF468821 EF468762 EF468869 Sung et al., 2007
Cordyceps cf. pruinosa EFCC 5197 EF468965 EF468820 EF468760 EF468868 Sung et al., 2007
Cordyceps cf. takaomontana NHJ 12623 Lepidoptera EF468984 EF468838 EF468778 EF468884 EF468932 Sung et al., 2007
Cordyceps chaetoclavata YHH 15101 MN576722 MN576778 MN576948 MN576838 MN576894 Wang Y. B. et al., 2020
Cordyceps chiangdaoensis BCC 68469 Coleoptera MF140732 KT261403 Tasanathai et al., 2016; Mongkolsamrit et al., 2018
Cordyceps chiangdaoensis YFCC 857 Coleoptera: Elateridae MW181781 MW173993 MW168234 MW168200 MW168217
Cordyceps cicadae GACP 07071701 Hemiptera MK761207 MK761212 MK770631 Zha et al., 2019
Cordyceps cicadae RCEF HP090724-31 Hemiptera: Cicadidae MF416605 MF416552 MF416496 MF416653 MF416447 Kepler et al., 2017
Cordyceps cocoonihabita YFCC 3415 MN576723 MN576779 MN576949 MN576839 MN576895 Wang Y. B. et al., 2020
Cordyceps cocoonihabita YFCC 3416 MN576724 MN576780 MN576950 MN576840 MN576896 Wang Y. B. et al., 2020
Cordyceps coleopterorum CBS 110.73 Coleoptera (larva) JF415965 JF415988 JF416028 JN049903 JF416006 Kepler et al., 2012
Cordyceps exasperata MCA 2288 Lepidoptera (larva) MF416592 MF416538 MF416482 MF416639 Kepler et al., 2017
Cordyceps farinosa CBS 111113 AY526474 MF416554 MF416499 MF416656 MF416450 Luangsa-ard et al., 2004; Kepler et al., 2017
Cordyceps fumosorosea YFCC 4561 Lepidoptera MN576761 MN576817 MN576987 MN576877 MN576931 Wang Y. B. et al., 2020
Cordyceps fumosorosea CBS 244.31 Butter MF416609 MF416557 MF416503 MF416660 MF416454 Kepler et al., 2017
Cordyceps fumosorosea CBS 375.70 Food MF416501 MF416658 MF416452 Kepler et al., 2017
Cordyceps fumosorosea CBS 107.10 MG665227 HM161735 MG665237 Luangsa-ard et al., 2005
Cordyceps grylli MFLU 17-1023 MK863048 MK863055 MK860193 Unpublished
Cordyceps grylli MFLU 17-1024 MK863049 MK863056 MK860194 Unpublished
Cordyceps inthanonensis BCC 79828 Lepidoptera (pupa) MT017854 MT017816 MT017833 Mongkolsamrit et al., 2020
Cordyceps inthanonensis BCC 56302 Lepidoptera (pupa) MT003040 MT017853 MT017814 MT017831 Mongkolsamrit et al., 2020
Cordyceps inthanonensis BCC 55812 Lepidoptera (larva) MT003041 – MT017815 MT017832 Mongkolsamrit et al., 2020
Cordyceps jakajanicola BCC 79816 Hemiptera MN275696 MN338479 MN338484 MN338489 Crous et al., 2019
Cordyceps jakajanicola BCC 79817 Hemiptera MN275697 MN338480 MN338485 MN338490 Crous et al., 2019
Cordyceps javanica TBRC 7259 Lepidoptera MF140711 MF140831 MF140780 MF140804 Mongkolsamrit et al., 2018
Cordyceps javanica CBS 134.22 Coleoptera MF416610 MF416558 MF416504 MF416661 MF416455 Kepler et al., 2017
Cordyceps kuiburiensis BCC 90322 Araneidae MK968816 MK988032 MK988030 Crous et al., 2019
Cordyceps kuiburiensis BCC 90323 Araneidae MK968817 MK988033 MK988031 Crous et al., 2019
Cordyceps kyusyuensis EFCC 5886 Lepidoptera (pupa) EF468960 EF468813 EF468754 EF468863 EF468917 Sung et al., 2007
Cordyceps lepidopterorum TBRC 7263 Lepidoptera (larva) MF140699 MF140819 MF140768 MF140792 Mongkolsamrit et al., 2018
Cordyceps lepidopterorum TBRC 7264 Lepidoptera (larva) MF140700 MF140820 MF140769 MF140793 Mongkolsamrit et al., 2018
Cordyceps longiphialis YFCC 8402 Rotten wood OL468557 OL468577 OL473525 OL739571 OL473536 This study
Cordyceps longiphialis YFCC 8403 Rotten wood OL468558 OL468578 OL473526 OL739572 OL473537 This study
Cordyceps militaris YFCC 6587 Lepidoptera (pupa) MN576762 MN576818 MN576988 MN576878 MN576932 Wang Y. B. et al., 2020
Cordyceps militaris YFCC 5840 Lepidoptera (pupa) MN576763 MN576819 MN576989 MN576879 MN576933 Wang Y. B. et al., 2020
Cordyceps morakotii BCC 55820 Hymenoptera (ant pupa) MF140730 KT261399 Tasanathai et al., 2016
Cordyceps morakotii BCC 68398 Hymenoptera (ant pupa) MF140731 KT261398 Tasanathai et al., 2016
Cordyceps nabanheensis YFCC 8409 Lepidopteran OL468564 OL468584 OL473532 OL739578 OL473543 This study
Cordyceps nabanheensis YFCC 8410 Lepidopteran OL468565 OL468585 OL473533 OL739579 OL473544 This study
Cordyceps neopruinosa BCC 91361 Lepidoptera (pupa) MT003047 MT017858 MT017838 Mongkolsamrit et al., 2020
Cordyceps neopruinosa BCC 91362 Lepidoptera (pupa) MT003048 MT017859 MT017818 MT017839 Mongkolsamrit et al., 2020
Cordyceps nidus HUA 186125 Araneae (Mygalomorphae) KC610778 KC610752 KC610722 KC610711 Chirivi et al., 2017
Cordyceps nidus HUA 186186 Araneae (Mygalomorphae) KY360301 KC610753 KC610723 KY360297 Chirivi et al., 2017
Cordyceps ninchukispora EGS 38.165 Plant (Beilschmiedia erythrophloia) EF468991 EF468846 EF468795 EF468900 Sung et al., 2007
Cordyceps ninchukispora EGS 38.166 Plant (Beilschmiedia erythrophloia) EF468992 EF468847 EF468794 EF468901 Sung et al., 2007
Cordyceps ningxiaensis HMJAU 25074 KF309671 Yan and Bau, 2015
Cordyceps ningxiaensis HMJAU 25076 KF309673 Yan and Bau, 2015
Cordyceps pseudotenuipes YFCC 8404 Lepidoptera OL468559 OL468579 OL473527 OL739573 OL473538 This study
Cordyceps pseudotenuipes YFCC 8405 Lepidoptera OL468560 OL468580 OL473528 OL739574 OL473539 This study
Cordyceps oncoperae ARSEF 4358 Lepidoptera; Oncopera intricate AF339581 AF339532 EF468785 EF468891 EF468936 Sung et al., 2007
Cordyceps polyarthra MCA 996 MF416597 MF416543 MF416487 MF416644 Kepler et al., 2017
Cordyceps polyarthra MCA 1009 Lepidoptera MF416598 MF416544 MF416488 MF416645 Kepler et al., 2017
Cordyceps pruinosa ARSEF 5413 Lepidoptera: Limacodidae AY184979 AY184968 DQ522351 DQ522397 DQ522451 Spatafora et al., 2007
Cordyceps qingchengensis MFLU 17-1022 Lepidoptera; Bombycidae MK761206 MK761211 MK770630 Zha et al., 2019
Cordyceps rosea spat 09-053 Lepidopteran larva MF416590 MF416536 MF416480 MF416637 MF416442 Kepler et al., 2017
Cordyceps roseostromata ARSEF 4871 AF339573 AF339523 Sung et al., 2001
Cordyceps shuifuensis YFCC 5230 MN576721 MN576777 MN576947 MN576837 MN576893 Wang Y. B. et al., 2020
Cordyceps simaoensis YFCC 8406 Lepidoptera OL468561 OL468581 OL473529 OL739575 OL473540 This study
Cordyceps simaoensis YFCC 8407 Lepidoptera OL468562 OL468582 OL473530 OL739576 OL473541 This study
Cordyceps simaoensis YFCC 8408 Lepidoptera OL468563 OL468583 OL473531 OL739577 OL473542 This study
Cordyceps sp. CBS 102184 Spider (Arachnida) AF339613 AF339564 EF468803 EF468907 EF468948 Kepler et al., 2012
Cordyceps sp. EFCC 2535 EF468980 EF468835 EF468772 Sung et al., 2007
Cordyceps sp. YFCC 5833 MN576764 MN576820 MN576990 MN576880 MN576934 Wang Y. B. et al., 2020
Cordyceps spegazzinii ARSF 7850 DQ196435 Torres et al., 2005
Cordyceps subtenuipes YFCC 6051 MN576719 MN576775 MN576945 MN576835 MN576891 Wang Y. B. et al., 2020
Cordyceps subtenuipes YFCC 6084 MN576720 MN576776 MN576946 MN576836 MN576892 Wang Y. B. et al., 2020
Cordyceps succavus MFLU 18-1890 MK086058 MK086062 MK084616 MK079353 Unpublished
Cordyceps tenuipes TBRC 7265 Lepidopteran (pupa) MF140707 MF140827 MF140776 MF140800 Mongkolsamrit et al., 2018
Cordyceps tenuipes TBRC 7266 Lepidopteran (pupa) MF140708 MF140828 MF140777 MF140801 Mongkolsamrit et al., 2018
Cordyceps tenuipes ARSEF 5135 Lepidopteran (pupa) MF416612 JF415980 JF416020 JN049896 JF416000 Kepler et al., 2012, 2017
Cordyceps tenuipes YFCC 4266 MN576774 MN576830 MN577000 MN576890 MN576944 Wang Y. B. et al., 2020
Cordyceps yinjiangensis YJ 06221 Ant MT577003 MT577002 Li et al., 2020
Liangia sinensis YFCC 3103 MN576726 MN576782 MN576952 MN576842 MN576898 Wang Y. B. et al., 2020
Liangia sinensis YFCC 3104 MN576727 MN576783 MN576953 MN576843 MN576899 Wang Y. B. et al., 2020

Boldface: data generated in this study.

 Phylogenetic Analyses

Phylogenetic trees were visualized with FigTree v1.4.0 (Rambaut, 2006) and edited in Microsoft PowerPoint, then saved as .PDF format and finally converted to .JPG format using Adobe Illustrator CS6 (Adobe Systems Inc., United States). The finalized alignments and trees were submitted in TreeBASE (Submission ID: 29339).

Phylogenetic analyses of the concatenated five-gene datasets were conducted using ML and BI methods. The GTR + I + G were chosen as the best models for nrSSU-nrLSU-tef-1α-rpb1-rpb2, using the Akaike Information Criterion (AIC) implemented in MrModeltest v 2.3 (Nylander, 2004), and then the partitioned analyses were separately conducted. For ML analyses, raxml v 8.2.7 was employed. All parameters were kept as default with an exception that the model was chosen as GTRGAMMAI. The statistic supports were calculated using 1,000 replicates of non-parametric bootstrapping. BI analysis was carried out with MrBayes v 3.2.6 using the selected models for 5 million generations with the value of stopval set to 0.01 via the stoprul command. At the same time, other parameters were kept as default and trees were summarized. Statistic supports were obtained using sumt command complemented in MrBayes by discarding the first 25% generations as burn-ins. The Bayesian trees were sampled every 100 generations. The first 25% trees were discarded as burn-ins, and the remaining trees were employed to create a consensus tree using sumt command.

 Results Sequence Alignment

The combined 101-taxon 5-gene dataset consisted of 4,627 base pairs of sequence data (nrSSU 1060 bp, nrLSU 877 bp, tef-1α 999 bp, rpb1 719 bp, and rpb2 972 bp). A total of 1,039 were parsimony-informative (nrSSU 45 bp, nrLSU 77 bp, tef-1α 370 bp, rpb1 234 bp, and rpb2 313 bp). A total of 101 taxa were complete for all five genes, and the number of taxa for each gene was as follows: nrSSU 71 taxa, nrLSU 97 taxa, tef-1α 95 taxa, rpb1 82 taxa, and rpb2 72 taxa (Table 1).

 Molecular Phylogeny

In this study, we generated nrSSU, nrLSU, tef-1α, rpb1, and rpb2 sequences by ten living cultures and one wild material, and their accession numbers are shown in Table 1. Sequences of Liangia sinensis YFCC 3103 and YFCC 3104 in the Cordycipitaceae were chosen as outgroups in the phylogenetic analyses.

Five major (I–IV) clades and five new species could be recognized in Cordyceps s. s. (Figure 1); collections from southwestern China were grouped into five separate species (in boldface, see below) (C. bullispora, C. longiphialis, and C. nabanheensis in clade I, and C. pseudotenuipes and C. simaoensis in clade III). Clade I included C. pruinose and 19 other species, with 98% bootstrap support and 1 Bayesian PP support (Figure 1). In clade II, C. militaris and eight other species were grouped together (BS = 70%, PP = 1) (Figure 1). Clade III harbored C. tenuipes and 18 other taxa (BS = 97%, PP = 1) (Figure 1). Clade IV included C. cf. takaomontana NHJ 12623, C. javanica, C. amoenerosea, and C. cateniobliqua (BS = 91%, PP = 1) (Figure 1). Clade V included only two exemplars of the species, C. grylli (Figure 1).

Both ML and BI analyses generate a phylogenetic tree from the concatenated nrSSU, nrLSU, tef-1α, rpb1, and rpb2 datasets. There were no discrepancies between the topology resulting from Bayesian and ML analysis for supported nodes. Bootstrap values (≥60%) derived from ML analyses and posterior probabilities from Bayesian inference (≥0.50) are shown either above or beneath the branches of the nodes. Isolates in bold type are those analyzed in this study.

 Taxonomy

The key morphological characteristics that distinguish current Cordyceps s. s. species are summarized in the literature (Tables 2, 3). Including the five new species, there are 66 species of Cordyceps s. s. involved in the current study, among which we have compared 51 species of the sexual morphs in Cordyceps s. s. in Table 2 and 38 species of the asexual morphs in Cordyceps s. s. in Table 3.

Comparison between the sexual morphs in Cordyceps.

Species Stromata (mm) Fertile part (mm) Perithecia (μm) Asci (μm) Ascospores (μm) Part-spores (μm) References
Cordyceps araneae Solitary or gregarious, 4–8 × 0.5 Clavate, elliptical to fusiform, 2.5– 5 × 1–2 Semi-immersed, 450–500 × 150–200 Cylindrical, 8-spored, (60)145–220(250) × 2–2.5 Bola-shaped, (250)280–340(400), central part filiform, 0.3 μm broad, 3 or 4-septa Mongkolsamrit et al., 2020
Cordyceps belizensis 100 20 × 9 Immersed, 480–570 × 260–300 Cylindrical, 260–300 × 6 Filiform 4–8 × 1.5 Mains, 1940
Cordyceps bifusispora Simple, 13 × 0.5–0.7 Cylindrical, 6 × 1.3 Immersed, 300 × 150–170 Cylindrical, 8-spored, 200–300 × 3–4.5 Bifusiform, 3-septa, 145–220 × 0.4–1.6 Eriksson, 1982
Cordyceps blackwelliae Gregarious, cylindrical to clavate, 8–10 × 1–1.5 4–6 × 1.5–2 Superficial, (300–)302–332 (–350) × (150–)155–189 (–200) Cylindrical, 8-spored, 300 × 2.5–3 Bola-shaped, 3- or 4-septa, (250–)259–283.5 (–290) × 1 Mongkolsamrit et al., 2018
Cordyceps brasiliensis Sub-solitary or branch, 60–73 × 1.5–2 15–20 × 3–5 Immersed, 350–700 × 175–315 Worm-form, 70–333 × 3.5–5 Filiform 3.5–7 × 0.7 Fang et al., 1995
Cordyceps brevistroma Solitary or gregarious, 2.5–10 × 0.5 Clavate, subglobose, 2–5 × 1–1.5 Semi-immersed, ovoid, 250–330(350) × 130–185(200) Cylindrical, 8-spored, (110)140–220(250) × 2– 5 Whole, bola-shaped, 150– 200 × 0.5 Mongkolsamrit et al., 2020
Cordyceps bullispora Solitary, 10–20, rhizoids flexuous Clavate, 3.5–7.4 × 0.5–1.5 This study
Cordyceps chaetoclavata Solitary, 23 × 0.8 Clavate, 5.6 × 0.7–1.1 Superficial, 402–610 × 280–427 Cylindrical, 8-spored, 274–385 × 3.7–4.8 Filiform, 127–260 × 0.9–1.2 Cylindrical, 3–6 μm long Wang Y. B. et al., 2020
Cordyceps chanhua Solitary, simple Partly immersed, 475–602 × 222–319 Cylindrical, 8-spored, 235–380 × 2.1–3 Filiform, 246–360 × 1.5–1.8 6.4–13.8 × 2.1–3.1 Li et al., 2021
Cordyceps chiangdaoensis Solitary to gregarious, simple or compound 2–7 × 1–1.5 Superficial, 200–450 × 70–170 Cylindrical, 8-spored, 175–315 × 2–3 Bola-shaped, 3-septa, 200–300 × 1 Tasanathai et al., 2016
Cordyceps chichibuënsis Solitary, 13–15 × 1–1.5 Semi-immersed, 400 × 230–260 5.5 μm wide 1.5–2 × 1.5 Kobayasi and Shimizu, 1980
Cordyceps chishuiensis Solitary or 2–3, 10–20 × 1–2 Elliptical, 5–6 × 3–4 Superficial, 340–440 × 200–240 Clavate, 165 × 6 Cylindrical, 3.6–5.4 × 1.2 Liang et al., 2002; Zha et al., 2021
Cordyceps coccinea Gregarious, 4–35 × 0.3–0.5 Cylindrical or clavate, 3–4 × 1.25–1.75 Immersed, 350 × 100 Cylindrical, 2–4 × 1 Petch, 1924; Shrestha, 2011
Cordyceps cocoonihabita Two or gregarious, 15.2–57.8 Clavate, 3.5–17.4 × 0.3–1.5 Superficial, 346–435 × 125–199 Cylindrical, 8-spored, 205–330 × 2.1–3.3 Cylindrical, septate, 140–269 × 1.4–2.1 Cylindrical, 2.9–8.0 μm long Wang Y. B. et al., 2020
Cordyceps cuncunae Solitary, rarely two, 59–92–105 × 7–8–10 Ovalis to subglobose, 15–18–21 × 12–15–18 Immersed, 772–793–829 × 257–279–314 Cylindrical, poriferous, 8-spored, 364–391–422 × 6–7–8 Filiform, 3–septate, 340–375–414 × 1.5–2 Cylindrical to subfusiform, 4.3–6.3–8.6 Palfner et al., 2012
Cordyceps cylindrica Single, 36 × 2–2.6 Cylindrical, clavate with obtuse top, 13 × 3.7–4 Immersed, fusiform to elliptical or flask-shaped, 850–1000 × 200–225 4.5–5.5 μm wide Truncate ends, 3–4 × 1.2 Kobayasi and Shimizu, 1977
Cordyceps dermapteoigena Solitary, simple, 15 × 1 Cylindrical, 7 × 1.5, sterile apex 5 mm long Embedded, 405–450 × 180–230 6–7.2 μm wide Filiform, multi-septate, (4.8–)6–15 × 2–3 Liang et al., 2003
Cordyceps doiana Solitary, simple, 30 × 0.3–0.4 Cylindrical, 9 × 0.7–0.8 Semisuperficial, 2/3 embedded, 250 × 170 125–135 × 6–7 4–5 × 1 Kobayasi, 1981
Cordyceps formosana Cylindrical or branched, 8.7–21.6 5.3–8.0 × 1.5–4.0 Semi-immersed, 360–520 × 230–330 Linear, 230–335 × 6.0–7.2 5.0–9.8 × 1.4–2.0 Olatunji et al., 2018
Cordyceps inthanonensis Multiple, 6–25 mm long Cylindrical to clavate, half of stroma, 3–5 mm wide Semi-immersed, ovoid, 600–720 × 220–420 Cylindrical, 450–600 × 4–6 Cylindrical, 3–4 × 1–1.5 Mongkolsamrit et al., 2020
Cordyceps ishikariensis Gregarious, 45–65 × 1.5–2 Fertile part is slightly wider than stipe Semi-immersed, 500–570 × 240–300 250–360 × 4 Shrestha, 2011
Cordyceps jakajanicola Gregarious, simple, 32–45 On the terminal end c. 1/3 of the stroma Semi-immersed, 400–650 × 300–400 Cylindrical, 265–360 × 4–5 Bola-shaped, 250–310 × 1 Crous et al., 2019
Cordyceps kuiburienis Solitary, 8 × 1–1.5, rhizoids flexuous Cavate to subglobose, 1.5–5 × 1–2.5 Pseudo-immersed, obpyriform, (350–)370–460(–550) × (120–) × 140–190(–240) Cylindrical, 280 × 3–5 Filiform, 250 × 1 Crous et al., 2019
Cordyceps kyusyuensis Multiple, 15–20 mm long Cylindrical, 10–12 mm long Semi-superficial, ovoid, 410–580 × 210–330 4 μm wide 4–5 × 1 Kobayasi, 1981
Cordyceps longiphialis Two, 13–25 Clavate, 4–15 × 2.0–2.5 Superficial, 380–612 × 167.5–268.3 Fusiform to cylindrical. 8-spored, 113–200 × 1.1–2.7 Bola-shaped, 110–184 × 0.8–1.3 This study
Cordyceps militaris Solitary or gregarious, 8–70 mm long Clavate, half of stroma Immersed to semi-immersed, 500–720 × 300–480 300–510 × 3.5–5 Filiform, multi-septate 2–4.5 × 1–1.5 Mains, 1958; Liang et al., 2007
Cordyceps morakotii Simple or compound, 3–7 × 0.5–1 Cylindrical to obovoid Superficial, 200–300 × 70–120 Cylindrical, 8-spored, 150–200 × 3–5 Bola-shaped, 200–250 × 1, 3-septate Tasanathai et al., 2016
Cordyceps nabanheensis Solitary or gregarious, 14–23, rhizoids flexuous, Clavate, 2.0–6.5 × 0.7–1.5 Superficial, 224–322.6 × 71.2–317.3 This study
Cordyceps nidus Gregarious, simple, 10–42 × 0.5–2 Subcylindrical, 2.5–18 × 0.5–3 Pseudoimmersed, 300–500 (–630) × 110–190 (–205) Cylindrical, (145–) 190–360 × 2–4 Filiform, 100–120 × 1.0 (4–)6–10 × 1 Chirivi et al., 2017
Cordyceps ninchukispora 1–6 branches, 13.8–22.4 × 0.3–0.9 5.7–14.2 × 0.8–0.9 Superficial, 95–145 × 50–60 Long cylindrical, 75–105 × 2.1–3.1 Ninchukiform, 90–110 × 1.2, 3–4 septate Su and Wang, 1986
Cordyceps ningxiaensis 1–2 branches, 5–15 × 0.3–1.2 Spherical to ovoid, 1.2–3 × 1.2–2.8 Immersed, 288–400 × 103–240 Cylindrical, 8-spored, 168–205 × (3.7–)4.1–5.5(–6.6) Filiform, multi-septate 3.6–7.8 × 1.0–1.4 Yan and Bau, 2015
Cordyceps oncoperae 1–4 branches, 35 mm long, Acute apex, 4–10 × 2–3 Ovoid, 350–410 × 180–230(–380) Cylindrical, 8-spored, (168–)200–224 (–256) × (5–)6–6.5 Filiform, 104–139 × 1.5–2 Wright, 1993
Cordyceps parvula Superficial, 500–650 × 250–300 Cylindrical, 400–500 × 5–6 Filiform 8–10 × 1 Mains, 1959
Cordyceps parvistroma Solitary, 5 mm long Subglobose, clavate, 4–10 × 2–3 Superficial, ovoid, (320)330–390(410) × (175)180–270(350) Cylindrical, (150)175–260(300) × 2–2.5 Bola-shaped, (180)210–225(250) × 0.5 Mongkolsamrit et al., 2020
Cordyceps polyarthra Gregarious, 30–44 Cylindrical to narrowly clavate, 12–14 Semi-immersed, 220–300 × 180–200 Cylindrical, 167–217.5 × 3.5–4.5 Filiform, multi-septate Cylindrical, 4.5–11 × 0.5–1.0 Catania et al., 2018
Cordyceps polystromata Gregarious, 11–37 × 3.0–9.8 Cylindrical to clavate, 5–17 × 2.3–8.6 Superficial, 522.3–663.4 × 296.4–576.7 (583.5 × 412.2) Cylindrical, 34.2–172.8 × 4.1–6.5 (68.4 × 5.2) Linear, 34.2–172.8 × 0.9–2.6 (68.4 × 1.7) Cylindrical, 1.3–3.0 × 1.1–2.2 (2.1 × 1.6) Duan, 2019
Cordyceps pruinosa Solitary, 15–47.5 Narrow-clavate or subcylindrical, 7 × 1.5 Superficial, narrow-oval or ovoid-cylindrical, 400 × 100 185–200 × 2 Bifusiform and filiform, 4–6 μm wide Cylindrical, 6 × 1 Petch, 1924; Liang, 1983; Liang et al., 2007
Cordyceps pseudomilitaris 1-3, simple, 12–25 × 1–2 2.5–9(–10) × 1.5–2.5 Semi-immersed, 320–500 × 225–350 Cylindrical, 210–395 × 5–6 Filiform, multi-septate, 200–380 × 1 Catania et al., 2018; Olatunji et al., 2018
Cordyceps qingchengensis 1-3 branches, 25 mm long 7–9 × 2.0–2.5 Partially immersed, sharply pointed, 335–490 × 145–240 Cylindrical, 8-spored, 180–200 × 2.4–4 Filiform, partly bifusiform, 180–220 × 0.45–0.65 Zha et al., 2019
Cordyceps racemosa Gregarious, branched Lanceolate, not much differentiated from the stipe Zang and Kinjo, 1998
Cordyceps rosea Solitary, 11 mm long Cavate Immersed, ovoid, 330–380 × 160–230 100 × 3–4 Whole, multi-septate Kobayasi and Shimizu, 1982
Cordyceps rostrata Solitary, simple, 35 mm long Cylindrical, 10 × 2 Superficial, subglobose, 420–525 × 255–375 Cylindrical, 6 μm wide Cylindrical, (3.6–) 4.8–6 (–7.2) × (1.2–)1.5–2 Liang et al., 2003
Cordyceps simaoensis Solitary or gregarious, 7–25.1 Elliptical to fusiform, 1–4.2 × 1.5–3 Immersed, 638.4–757.6 × 371–531.1 Clavate to nearly cylindrical, 8-spored, 66.9–126.1 × 1.9–2.7 This study
Cordyceps singeri 1-2, simple, 10–20 × 0.5–1.5 Cylindrical to clavate, 1 × 3–4 Embedded, 325–520 × 220–475 Cylindrical, (187–)425–475 × 3–4(–4.5) Filiform, multi-septate 3.0–4.0 × 1.0 Catania et al., 2018
Cordyceps spegazzinii Solitary, simple, 7–9 mm long Cylindrical to clavate, 1 × 3–4 Superficial to partially immersed, 400–460 × 200–240 Cylindrical, 8-spored, 200–250 × 2.5–3 Filiform, multi-septate, 100–250 × 0.5–1 Torres et al., 2005
Cordyceps submilitaris 20–30 × 1–1.5 10–25 × 2 Embedded Cylindrical, 300–420 × 3–4 Filiform 2.0–4.0 × 0.5 Mains, 1940
Cordyceps shuifuensis Solitary, 25 mm long Clavate,4 × 1.5 Pseudoimmersed, 450–620 × 300–430 Cylindrical, 275–510 × 3.5–5.2 Filiform, multiseptate, 180–410 × 1.2–1.7 Cylindrical, 2.8–6.5 μm long Wang Y. B. et al., 2020
Cordyceps succavus Solitary, 40–50 × 3–6 Cylindrical, 15–20 × 4–5 Semi-immersed, 534–655 × 179–278 Cylindrical, 8-spored, 486–600 × 3.6–4.9 Filiform, 466–594 × 0.9–1.2 Cylindrical, 2.8–4.9 × 0.9–1.2 Hyde et al., 2019
Cordyceps suoluoensis Gregarious (2–4), 15 × 2–3 Cylindrical, 7 × 3 Pseudoimmersed, 400–500 × 260–300 3.6–4.8 μm wide Filiform Cylindrical,(6–)9–12 × 1.8–2.0 Liang et al., 2002
Cordyceps takaomontana Solitary or gregarious Cylindrical, 8–10 × 1.5–2 mm Superficial, 375–450 × 145–195 Filiform, 1200 × 2.4–3 Filiform Cylindrical, 6–8 × 0.5–0.8 Liang et al., 2003, 2007
Cordyceps translucens 10 × 1 Globose or ovoid, 2.5 × 2 Superficial, 0.5 × 0.3 Cylindrical, 8-spored Cylindrical, 6 × 1 Petch, 1924

Boldface: data generated in this study.

Comparison between the asexual morphs in Cordyceps.

Species Conidiophores (μm) Phialides Phialides size (μm) Conidia (μm) Other key characteristics References
Cordyceps albocitrinus 3.8–10.4 × 1.1–1.4 (7.1 × 1.2) 3.3–11.3 × 0.9–1.2 (7.3 × 1.1) In chains 1–5, ellipsoidal or cylindrical, 0.7–3.5 × 0.6–1.8 (2.1 × 1.2) Duan, 2019
Cordyceps amoene-rosea 90–150 × 2.0–2.5 Verticillate with whorls of 2 to 4 4.0–7.5 × 1.5–3.0, basal portion globose, neck 0.3–0.5 Subglobose to ellipsoidal, irregularly cylindrical, 2.5–3.5 × 1.7–2.2 Synnemata up to 8–10 × 0.3–0.8 mm Samson, 1974
Cordyceps araneae Solitary or verticillate with whorls of 2–3 5–8 × 1.5–2, basal portion swollen tapering into the apex Fusoid to ovoid, 3–5 × 1–2 Mongkolsamrit et al., 2020
Cordyceps bifusispora Solitary or verticillate 9–50 × 1.5–2, flask shaped Globose, ovoid or cylindrical, 1- to 8-celled, 2.5–35 × 2.4–4.5 Liang et al., 2007
Cordyceps blackwelliae Verticillate with whorls of 2–5 (6–)6.5–8(–9) × (2–)2.5–3.5(–4), basal portion globose, neck 2–3 × 1 Cylindrical to ellipsoidal or reniform, (3–)5–7(–8) × 2–3.5 Synnemata numerous, up to 1.5 × 0.5 mm, powdery Mongkolsamrit et al., 2018
Cordyceps brevistroma Solitary or verticillate with whorls of 2–3 6–9.5(12) × 1.5–2, basal portion swollen or cylindrical tapering into the apex Ovoid to fusiform, 3–4 × 1–2 Mongkolsamrit et al., 2020
Cordyceps bullispora Solitary 5.6–20.7 × 1.8–3.3 Cylindrical or slightly allantoid, oblong-elliptical to ellipsoidal, 4.9–11.1 × 1.9–4.5 Asexual morph: Acremonium-like and Mariannaea-like This study
Cordyceps cateniannulata 3–8 × 1.5–3, basal portion bola, neck 0.5 In chain, ovate to ellipsoidal, 2–3.5 × 1–1.5 Liang, 1981
Cordyceps cateniobliqua 90–150 × 1–1.5 Verticillate with whorls of 2–4 8.5–12 × 1 –1.5 and 5–8 × 2–2.5, basal portion ellipsoidal In chains, long-ovoid and long-ellipsoidal 2.5–7(–12) × 1.5–2 Synnemata unbranched, red Liang, 1981
Cordyceps chanhua Verticillate with whorls of 2–5 4.2–7 (–13.5) × 2.3–3.5(–5.2) 4.2–7(13.5) × 2.3–3.5(5.2) Chlamydospores 13–26.5 × 3–12 μm Li et al., 2021
Cordyceps chiangdaoensis 5–22.5 × 1–2 Ovoid to cylindrical, 4–10 × 1.5–2 Tasanathai et al., 2016
Cordyceps cocoonihabita 5.8–8.3 × 1.4–2.0 Solitary, alternate or whorled 4.0–16.7 μm long, basal portion 1.5–2.7, neck 0.5–1.2 In chains or solitary, oval to fusiform, 1.6–3.0 × 0.7–1.5 Wang Y. B. et al., 2020
Cordyceps farinosa 60–15(300) × 1–1.5 Cylindrical, 7–14 × 1–2 Ovoid or spindle-shaped, 2–2.5(3) × 1–2 Yellow-white powdery spores Liang et al., 2007
Cordyceps formosana 6.0–22.5 × 1.5–2.6 Solitary or verticillate with whorls of 2–3 Ovoid, 2 × 1.5 Hyphae septate, hyaline, 1.9–2.5 μm wide Olatunji et al., 2018
Cordyceps fumosorosea 100 × 1.5–2 Verticillate with whorls of 4–6 5.7–8 × 1–2, basal portion globose or ellipsoidal, neck 0.5 Cylindrical to fusiform, 3–4 × 1–2 Synnemata branched, up to 30 × 0.4 mm, powdery Liang, 1981
Cordyceps ghanensis 90–180 × 2.5–3.5 Verticillate with whorls of 2–4 5.5–8 × 2–3.5, basal portion ellipsoidal, neck, 0.5–0.75 Fusiform, 3.5–5.0 × 2.5–3.0 Synnemata branched, 15–30 × 0.3–0.5 mm, powdery Liang, 1981
Cordyceps inthanonensis Solitary (12)14–18.5(20) × 1.5–3, basal portion cylindrical, Cylindrical, 4–7(9) × 1.5–2 Mongkolsamrit et al., 2020
Cordyceps jakajanicola Verticillate 4–5.3(–6) × 2–3.5(–4), basal portion globose, oval or occasionally conical swollen, neck 0.5 Ellipsoid or cylindrical, (4–)4.5–6(–7) × (1.5–)2–2.5(–3) Synnemata branched, powdery and floccose Crous et al., 2019
Cordyceps javanica Verticillate with whorls of 2–5 (6–)9(–10) × 2–2.5, basal portion cylindrical, neck (1–)1.5–3(–4) × 0.5 In chains, fusiform, (3.5–)4(–5) × 2–2.5 Mongkolsamrit et al., 2018
Cordyceps kuiburienis Verticillate with whorls of 2–5 (3–)4–8(–10) × 1.5–2, basal portion swollen, ellipsoidal, necks 1–3 × 1 Ellipsoidal, fusiform, 3–4 × 1.5 –2 Asexual morph: Evlachovaea-like Crous et al., 2019
Cordyceps lepidopterorum Verticillate with whorls of 2–3 5–5.5–8 × 4–5, basal portion globose to flask shaped, neck 2–3 × 1 In chains, ellipsoidal or slipper-shaped, (6–)8–9.5(–10) × 3–4 Mongkolsamrit et al., 2018
Cordyceps longiphialis Solitary 7.0–70.8 × 0.9–2.1 Cylindrical, 2.1–6.0 × 0.8–2.5 Apical conidia more prominent than other conidia, 4.6–10.0 × 1.4–2.3 μm This study
Cordyceps militaris Solitary or verticillate Paecilomyces-type: cylindrical, (0.5–)0.8–1.5 × 6–15(–20), Verticillium-type: 0.8–1.2 × (8–)14–20(–25) Subglobose, 1.5–2 μm, ellipsoidal, 1–2 × 1.5–3 Liang et al., 2007
Cordyceps nabanheensis Solitary, cylindrical, 4.6–11.5 × 1.6–3.2 Solitary or verticillate with whorls of 2 to 3 5.6–19.1 × 1.3–3.5 Elliptical to oblong, 2.1–4.3 × 1.1–2.7 This study
Cordyceps neopruinosa Solitary or verticillate with whorls of 2–4 (8)10–17.5(20) × 1–2, basal portion cylindrical to ellipsoidal Irregularly ellipsoidal, cylindrical, (3)4.5–8.5(10) × 1–2 Synnemata awl-shaped, 2–10 × 1 mm Mongkolsamrit et al., 2020
Cordyceps ninchukispora Solitary or verticillate 30–50 × 2.0–3.0 Ellipsoidal, cylindrical, 0–1 septate, 2.5–10 × 1.5–3 Liang et al., 2007
Cordyceps parvistroma Solitary 10 × 0.5 Cylindrical, (5)7–10(11) × 1– 2(2.5) Mycelium density Mongkolsamrit et al., 2020
Cordyceps polyarthra Ellipsoid, 2–2.5 × 1–1.5 Synnemata 30 × 1.0–1.5 mm Catania et al., 2018
Cordyceps polystromata 6.1–43.7 × 1.5–2.9 (27.2–2.1) Solitary or verticillate 6.2–17.2 × 0.9–2.7 (10.5 × 1.9), basal portion cylindrical or globose Near-spherical or pseudo-oval, 1.5–3.7 × 1.1–2.5 (2.1 × 1.7) Duan, 2019
Cordyceps poprawskii Verticillate 5.4–5.6 × 2.4–2.6, basal portion cylindrical to bottle-shaped Cylindrical to fusiform, 3.9 (2.9–4.6) × 1.6 (1.4–2.1) Synnemata 5 (2.0–7.0) × 6 (4.0–8.0) mm Cabanillas et al., 2013
Cordyceps pruinosa 6.1–21.6 × 0.9–1. 7 (7.6 × 1.2) Verticillate 3.7–12.5 × 0.9–1.6 (8.1 × 1.2) Ellipsoidal, cylindrical, 0.6–1.3 × 0.5–1.0 (1.0 × 0.8, apical conidia two-celled, 7.6–16.3 × 2.0 – 2.5 (11.4 × 2.2) Liang, 1991; Duan, 2019
Cordyceps pseudotenuipes Cylindrical, 17.9–25.9 × 1.7–2.1 Solitary or verticillate with whorls of 2–5 6.8–31.8 × 1.2–3.3 Ovoid to ellipsoidal, 3.4–6.5 × 2.2–4.0 Chlamydospores 9.2–18.5 × 3.4–7.5 μm This study
Cordyceps shuifuensis Solitary, cylindrical, 5.5–9.2 × 1.6–2.7 Cylindrical or subulate, 4.7–20 × 0.4–2.1 Macroconidia clavate to oblong-ovate, 5.1–11.8 × 1.3–2.4. Microconidia globose to ellipsoidal, 1.8–3.0 × 1.6–2.5 Asexual morph: Verticillium-like Wang Y. B. et al., 2020
Cordyceps simaoensis Solitary or verticillate, cylindrical, 17.1–25.2 × 1.4–1.6 Solitary or verticillate with whorls of 2–3 11.7–50.2 × 3.4–4.0 In chains, fusiform or oval, 2.0–4.9 × 2.0–3.3 This study
Cordyceps spegazzinii 7–15 × 3 4–5 × 2 Asexual morph: Evlachovaea-like Torres et al., 2005
Cordyceps subtenuipes Biverticillate, 3.5–8.6 × 1.5–2.9 Solitary or verticillate with whorls of 3–6 5.3–42.5 × 1.6–3.4, basal portion cylindrical or flask-shaped Ellipsoidal or fusiform, 1.9–3.4 × 1.7–2.5 Wang Y. B. et al., 2020
Cordyceps tenuipes Verticillate with whorls of 2–4 6.0–6.5 × 2.5, basal portion subglobose 2–7 × 1.2–2.5 Samson, 1974
Cordyceps yinjiangensis Erect Verticillate with whorls of 2–3 11.6–18.9 × 1.7–2.2 Cylindrical, multiple-septate, (3.1–)5.3–7.2(–16.1) × (1.6–)2.1(–4.4) Li et al., 2020

Boldface: data generated in this study.

 <italic>Cordyceps bullispora</italic> H. Yu, Q. Y. Dong and Z. Y. Zhao, sp. nov. (<xref ref-type="fig" rid="F2">Figure 2</xref>)

Mycobank: MB 842328

Cordyceps bullispora. (A) Fungus on the pupa of Lepidoptera. (B,C) Colony obverse (B) and reverse (C) on PDA at 21 days. (D–G,I–L) Phialides. (H) Conidia. Scale bars: (A) = 10 mm; (B,C) = 10 mm; (D) = 25 μm; (E) = 50 μm; (F,G) = 15 μm; (H) = 3 μm; (I) = 5 μm; (J) = 15 μm; (K,L) = 10 μm.

Etymology: Referring to button-like structures on the spores.

Type: The Taiji Mountains Nature Reserve, Mizhi Town, Midu County, Yunnan, China. September 20, 2019, H. Yu (YHH 20011, holotype; YFCC 8400, ex-holotype living culture).

Teleomorph: Stromata solitary, 10–20 mm long, unbranched, orange-yellow, cylindrical to enlarging apically. The host is covered by a white mycelial surface. Rhizoids flexuous, arising from the head region of host larva buried in soil, 7–10 mm deep under the ground. Stipes cylindrical, white to reddish-orange, 0.1–1.2 mm wide. Fertile parts clavate, orange to reddish-orange, 3.5–7.4 × 0.5–1.5 mm.

Anamorph: Two types of conidial arrangement. Acremonium-like conidia aggregated in heads at the apex of phialides; Mariannaea-like, conidia in imbricate chains, connected laterally.

Colonies on PDA are moderately fast-growing, attaining a diameter of 31–34 mm in 21 days at 25°C, pulvinate, with high mycelial density, Whitish to orange-yellow, reverse deep yellow. Hyphae smooth-walled, branched, septate, hyaline, 1.1–2.7 μm wide. Cultures readily produced phialides and conidia after 2 weeks on potato dextrose agar at room temperature. Phialides arising from aerial hyphae, solitary, 5.6–20.7 × 1.8–3.3 μm, cylindrical, tapering gradually or abruptly toward the apex. Conidia hyaline, one-celled, cylindrical or slightly allantoid, oblong-elliptical to ellipsoidal, 4.9–11.1 × 1.9–4.5 μm.

Habitat and known distribution: On larva of Lepidoptera buried below ground at elevation 2000 m in northwestern Yunnan, China.

Additional specimens examined: The Taiji Mountains Nature Reserve, Mizhi Town, Midu County, Yunnan, China. On pupae of Lepidoptera, September 20, 2019. YHH 20012, YFCC 8401.

Comments: Cordyceps bullispora was characterized by unbranched stromata, with cylindrical and orange to reddish-orange fertile parts, Rhizoids flexuous, and the host was the lepidopteran larva. For timing reasons, the fertile part of the specimen was not yet mature at the time of collection in the field. The asexual morph of PDA culture produces cylindrical phialides, which are monothetic, oblong-elliptical to ellipsoidal conidia with a button-like shape.

Based on nrLSU, nrSSU, tef-1α, rpb1, and rpb2 multigene analyses, C. cocoonihabita was revealed to have a close relationship with C. pruinosa and C. ninchukispora (Wang Y. B. et al., 2020). Multigene analyses of ITS, nrLSU, rpb1, rpb2, and tef-1α revealed that C. neopruinosa had a close relationship with C. pruinosa and C. ninchukispora (Mongkolsamrit et al., 2020). C. cocoonihabita, C. neopruinosa, C. pruinosa, and C. ninchukispora all had close relationships, where they shared many similar morphological characteristics, such as they were all characterized by orange- to red-colored stromata and superficial perithecia. C. bullispora shared such features, and our phylogenetic analysis indeed indicated that C. bullispora was closely relevant to a previously undescribed taxon C. cf. pruinosa (spat 08-115, spat 09-221) and was separated from C. cocoonihabita, C. neopruinosa. C. pruinosa, and C. ninchukispora in this subclade. However, the perithecia in C. neopruinosa were more prolonged and broader than those reported in Cordyceps pruinosa and C. ninchukispora (330–450 × 150–240 μm vs. 400 × 100 μm vs. 95–145 × 50–60 μm, respectively). C. cocoonihabita had a longer stroma. The micromorphological arrangement of conidia was Isaria-like characteristics and was significantly different from C. pruinosa and C. ninchukispora, which had respective morphs of Mariannaea G. Arnaud and Acremonium Link. C. bullispora had rhizoid stromata, superficial perithecia, wider phialides, and longer conidia 4.9–11.1 × 1.9–4.5 μm. The insect host of C. bullispora and C. neopruinosa all occurred on lepidopteran pupae, and the host of C. ninchukispora was the seed of Beilschmiedia Nees (Liang, 1983, 1991; Su and Wang, 1986; Mongkolsamrit et al., 2020).

Cordyceps yinjiangensis Li et al. (2020) was recently described from Guizhou. Morphologically, it differed from C. bullispora by cylindrical and orange to reddish-orange fertile parts, rhizoids several, flexuous, and the host was the lepidopteran larva. The asexual morph from PDA culture produced cylindrical phialides, which were monothetic and oblong-elliptical to ellipsoidal conidia with a button-like character. Tasanathai et al. (2016) reported an anti-pathogenic species, C. morakotii. C. yinjiangensis had a close relationship with C. morakotii with ant host and conidia formed in an imbricate chain. However, C. yinjiangensis was distinct from C. morakotii, which had longer phialides (16–20 × 2–3 μm) and bigger aseptate conidia (4–12 × 1–2 μm) (Li et al., 2020).

 <italic>Cordyceps longiphialis</italic> H. Yu, Q. Y. Dong and D. X Tang, sp. nov. (<xref ref-type="fig" rid="F3">Figure 3</xref>)

MycoBank: MB 842329

Cordyceps longiphialis. (A,B) Fungus on the host of Lepidoptera. (C) Fertile part. (D) Perithecia. (E) Asci. (F,G) Ascospore. (H,I) Colony obverse (H) and reverse (I) on PDA at 14 days. (J–P) Phialide. (Q) Conidia. Scale bars: (A) = 5 mm; (B) = 2 mm; (C) = 1 mm; (D) = 200 μm; (E–G,J–M) = 20 μm; (H,I) = 10 mm; (N–P) = 10 μm; (Q) = 5 μm.

Etymology: Referring to its longer phialide than close relationship species in this genus.

Type: Xinfang Reservoir, Simao District, Pu’er City, Yunnan, China. September 28, 2020, H. Yu (YHH 20013, holotype; YFCC 8402, ex-holotype living culture).

Teleomorph: Stromata (Figures 3A,B) arising from rotten wood, two, unbranched, 13–25 mm long. Stipe cylindrical, 4–10 mm long, 1.5–2 mm in diameter, orange-red to crimson, fleshy, glabrous, smooth. Fertile part single, clavate, covered by a spinous surface, 4–15 mm long, 2.0–2.5 mm in diameter, reddish-orange. Perithecia (Figure 3D) crowded or sparse, crimson-yellowish, superficial, vase-form to oblong, 380–612 × 167.5–268.3 μm. Asci (Figure 3E) 8-spored, fusiform to cylindrical, 113–200 × 1.1–2.7 μm when mature; Ascus caps hemispherical, 1.6–3.6 μm in height, 1.7–3.3 μm in width. Ascospores hyaline, bola-shaped, septate, 110–184 × 0.8–1.3 μm, central region filiform, terminal region narrowly fusiform, do not disarticulate into part-spores.

Anamorph: Conidial arrangement Mariannaea-like. Colonies on PDA are fast-growing, attaining a diameter of 35–37 mm in 14 days at 25°C, white to pale yellow, cottony, with high mycelial density at the centrum, reverse white to pale yellow. Hyphae smooth-walled, septate, hyaline, 0.8–2.5 μm wide. Cultures readily produced phialides and conidia after 2 weeks on potato dextrose agar at room temperature. Phialides usually solitary on hyphae, basal portion cylindrical to clavate, tapering gradually toward the apex; 7.0–70.8 μm long, 0.6–2.1 μm wide at the base, 0.9–2.1 μm at the middle, and 0.6–1.8 μm wide at the apex. Conidia one-celled, smooth-walled, hyaline, cylindrical, 2.1–6.0 × 0.8–2.5 μm, often formed in an imbricate chain, the size of apical conidia significantly more prominent than other conidia in the chain, 4.6–10.0 × 1.4–2.3 μm.

Habitat and known distribution: Buried in rotten logs below ground, in northwestern Yunnan, China.

Additional specimens examined: Xinfang Reservoir, Simao District, Pu’er City, Yunnan, China, isolated from stromata of rotten wood at elevation 1,350 m. September 28, 2020, H. Yu (YFCC 8403, ex-holotype living culture).

Comments: Phylogenetic analyses showed that C. longiphialis was closely related to C. bullispora; however, the independent phylogenetic position and different physiological characteristics could distinguish C. longiphialis from its sister species, C. bullispora (as mentioned above). The distinctive characteristics of Cordyceps longiphialis were the cylindrical stromata with a spinous surface, superficial perithecia, much shorter ascospores (110–184 × 0.8–1.3 μm), and much longer phialides (7.0–70.8 μm long). Ascospores of C. longiphialis were shorter than C. neopruinosa (135–275 × 0.5 μm) and C. pruinose (185–200 × 2 μm), but longer than C. ninchukispora (90–110 × 1.2 μm). The size of the conidia in C. longiphialis was relatively shorter than C. bullispora.

 <italic>Cordyceps nabanheensis</italic> H. Yu and Q. Y. Dong, sp. nov. (<xref ref-type="fig" rid="F4">Figure 4</xref>)

MycoBank: MB 842341

Cordyceps nabanheensis. (A–C) Fungus on the pupa of Lepidoptera. (D) Fertile part. (E) Perithecia. (F,G) Colony obverse (F) and reverse (G) on PDA at 14 days. (I) Solitary phialide on hyphae. (H,J–N) Opposite conidiophore and verticillate phialides. (O) Conidia. Scale bars: (A–C) = 10 mm; (D) = 1 mm; (E) = 100 μm; (F,G) = 10 mm; (H,J,K) = 20 μm; (I) = 10 μm; (L–O) = 5 μm.

Etymology: The location in Nabanhe National Nature Reserve where the species was collected.

Type: Manlv village, Nabanhe National Nature Reserve, Jinghong City, Yunnan, China. August 16, 2020, H. Yu (YHH 20019, holotype; YFCC 8409, ex-holotype living culture).

Teleomorph: Stromata arising from the pupa of Lepidoptera buried in soil, the host covered by a white mycelial surface, solitary or gregarious, cylindrical to enlarging apically, reddish-orange to crimson, 1.4–2.3 cm long. Rhizoids flexuous, arising from the head region of host larva buried in soil, 7–12 mm deep under the ground. Stipes cylindrical, 12.0–20.9 × 0.6–1.3 mm, fertile parts clavate, 2.0–6.5 × 0.7–1.5 mm. Perithecia superficial, oblong-ovate, 224–322.6 × 71.2–317.3 μm. Asci and ascospores were not observed.

Anamorph: Conidial arrangement Evlachovaea-like. Colonies on PDA moderately fast-growing, 48–51 mm diameter in 14 days at 25°C, floccose, with high mycelium density; white to orange pinkish, reverse orange-brown. Hyphae smooth-walled, branched, septate, hyaline, septate, 3.5–9.3 μm wide. Cultures readily produced phialides and conidia after 1 week on potato dextrose agar at room temperature showing a powdery appearance due to profuse conidiation. Conidiophores smooth-walled, solitary, cylindrical, 4.6–11.5 × 1.6–3.2 μm. Phialides arising from aerial hyphae, cylindrical or clavate, solitary or in whorls of two to three, tapering abruptly into a narrow neck, 5.6–19.1 × 1.3–3.5 μm. Conidia one-celled, smooth-walled, hyaline, elliptical to oblong, 2.1–4.3 × 1.1–2.7 μm, often formed in an imbricate chain.

Habitat and known distribution: On larvae of Lepidoptera buried below ground at elevation 600 m in northeastern Yunnan, China.

Additional specimens examined: Manlv village, Nabanhe National Nature Reserve, Jinghong City, Yunnan, China, on larvae of Lepidoptera. August 16, 2020 (YHH 20020, paratype; YFCC 8410 ex-paratype living culture).

Comments: Cordyceps araneae was firstly reported from Khon Kaen Province, northeastern Thailand by Mongkolsamrit et al. (2020); C. araneae was a spider cocoon pathogenetic fungus producing pale orange stromata, perithecia semi-immersed, narrowly ovoid, 450–500 × 150–200 μm with whole bola-shaped ascospores breaking into part-spores 30–65 × 0.5 μm, and developed the Evlachovaea-like anamorph, phialides solitary or in whorls of two to three, 5–8 × 1.5–2 μm, conidia fusoid to ovoid, 3–5 × 1–2 μm (Mongkolsamrit et al., 2020).

Based on the ITS, ribosomal large subunit, rpb1, rpb2, and tef-1α genes, multigene analyses revealed that C. araneae had a close relationship with C. kuiburiensis, C. brevistroma, and C. nidus, and they were all characterized by orange to reddish-orange, cylindrical to enlarging apically stromata and a conidial arrangement Evlachovaea-like (Chirivi et al., 2017; Crous et al., 2019; Mongkolsamrit et al., 2020). Interestingly, C. nabanheensis shared such features, and our phylogenetic analysis indeed indicated that C. nabanheensis had a close relationship to C. araneae and C. brevistroma. However, C. nabanheensis and C. brevistroma differed from C. araneae and C. kuiburiensis regarding their hosts. Both C. nabanheensis and C. brevistroma occur on Lepidoptera larvae, whereas both C. araneae and C. kuiburiensis occurred on spiders. C. brevistroma had bola-shaped whole ascospores, which was the same shape but shorter than C. araneae reported (150–200 μm vs. 250–400 μm). In the natural specimen, C. kuiburiensis developed the anamorph.

 <italic>Cordyceps pseudotenuipes</italic> H. Yu, Q. Y. Dong, and Y. Wang, sp. nov. (<xref ref-type="fig" rid="F5">Figure 5</xref>)

Mycobank: MB 842330

Cordyceps pseudotenuipes. (A,B) Fungus on the host of Coleopteran. (C,D) Colony obverse (C) and reverse (D) on PDA at 14 days. (E,G,H,L) Solitary phialides on hyphae. (F,J,K) Conidiophore and verticillate phialides. (I) Chlamydospore. Scale bars: (C,D) = 5 mm; (E,F,G) = 25 μm, (H,I) = 20 μm; (J,L) = 5 μm; (K) = 10 μm.

Etymology: Referring to morphological resemblance of Cordyceps tenuipes and Cordyceps subtenuipes but phylogenetically distinct, “not” C. tenuipes.

Type: Wild Duck Lake Forest Park, Shuanglong Town, Panlong District, Kunming City, Yunnan, China. September 10, 2019, H. Yu and Y. Wang (YHH 20014, holotype; YFCC 8404, ex-holotype living culture).

Teleomorph: Undetermined.

Anamorph: Conidial arrangement Isaria-like. Synnemata arising from the pupae of Lepidoptera buried in soil, synnemata erect, solitary, flexuous, white, up to 0.5 cm long. Stipes cylindrical, 0.5 mm wide, producing a mass of conidia at the branches of synnemata, powdery.

Colonies on PDA attaining a diameter of 53–55 mm in 14 days at 25°C, white to cream-colored, soft cottony aerial mycelium, reverse pale yellow. Hyphae smooth-walled, branched, septate, hyaline. Synnemata arising from the entire body of larvae were irregularly branched, 0.2–1.0 cm long, 0.1–0.3 mm wide; cylindrical or clavate stipes with powdery white heads. Cultures readily produced phialides and conidia after 2 weeks on potato dextrose agar at room temperature showing a granular appearance due to profuse conidiation. Conidiophores cylindrical, hyaline, smooth-walled, 17.9–25.9 × 1.7–2.1 μm. Phialides from aerial mycelium straight to slightly flexuose, solitary or in whorls of two to five on each branch, cylindrical, usually with a slightly swollen basal part, tapering into the apex, 6.8–31.8 × 1.2–3.3 μm. Conidia hyaline, ovoid to ellipsoidal, smooth, one-celled, 3.4–6.5 × 2.2–4.0 μm. Chlamydospores present, one-celled, solitary, eggplant shape or oval to pyriform, 9.2–18.5 × 3.4–7.5 μm, hyaline becoming brown, thick and smooth-walled.

Habitat and known distribution: On the pupa of Lepidoptera buried in the soil. Kunming City, China.

Additional specimens examined: Wild Duck Lake Forest Park, Shuanglong Town, Panlong District, Kunming City, Yunnan, China. September 10, 2019, H. Yu (YHH 20015, paratype; YFCC 8405, ex-paratype living culture).

Comments: Phylogenetically, C. pseudotenuipes formed a separate subclade from the other species of Cordyceps with high credible support (100%). C. pseudotenuipes was similar to C. tenuipes (Peck) Kepler et al. (2017) based on its conspicuous synnemata and Isaria-like asexual conidiogenous structure forming phialides with a swollen basal portion. It differed from C. tenuipes by its unbranched synnemata, white color, phialides with a globose basal part, and smaller ovoid to ellipsoidal wider conidia measuring 3.4–6.5 × 2.2–4.0 μm. C. tenuipes had multiple synnemata, more giant cylindrical to botuliform conidia with a size of 2.0–7.0 × 1.2–2.5 μm (Samson, 1974). The sexual morph of C. tenuipes was proposed as C. takaomontana Yakush and Kumaz, with yellowish stromata and being often concurrent with its asexual morph (Liang et al., 2007). However, the sexual morph of C. pseudotenuipes was not found in the field.

 <italic>Cordyceps simaoensis</italic> H. Yu, Q. Y. Dong and Z. Q. Wang, sp. nov. (<xref ref-type="fig" rid="F6">Figure 6</xref>)

MycoBank: MB 842331

Cordyceps simaoensis. (A–C) Fungus on the host of Lepidoptera. (D) Fertile part. (E,F) Perithecia. (G–J) Asci. (K,L) Colony obverse (K) and reverse (L) on PDA at 21 days. (M–S) Conidiophore and phialide. Scale bars: (A,B) = 10 mm; (C) = 5 mm; (D) = 2 mm; (E) = 400 μm; (F) = 100 μm; (G–J) = 10 μm; (K,L) = 10 mm; (M–S) = 20 μm.

Etymology: The location in Simao District where the species was collected.

Type: Xinfang Reservoir, Simao District, Pu’er City, Yunnan, China. September 28, 2020. H. Yu (YHH 20016, holotype; YFCC 8406, ex-holotype living culture).

Teleomorph: Stroma arising from the host’s head, solitary or gregarious, mace-shaped, unbranched, 7–25.1 mm in height. Stipe cylindrical, 22 mm long, 1.1–1.2 mm in diameter, bright yellow, fleshy, glabrous, smooth, enlarging abruptly at fertile portion. Fertile portion single, elliptical to fusiform, 1–4.2 mm long, 1.5–3 mm in diameter, bright yellow. Perithecia (Figure 6E) crowded, nearly fully immersed, vase-form, oval to oblong, 638.4–757.6 × 371–531.1 μm, ostioles protruding. Asci (Figures 6G–J) 8-spored, narrowly clavate to nearly cylindrical, 66.9–126.1 × 1.9–2.7 μm; cap 1.2–2.3 μm in height, 2.1–3.5 μm in width. Ascospores not observed.

Anamorph: Conidial arrangement Isaria-like. Colonies on PDA are fast-growing, attaining a diameter of 40–43 mm at 25°C in 21 days, white to bright yellow, cottony, with high mycelial density at the centrum, forming concentric rings around the inoculum, reverse pale yellow to deep yellow. Hyphae smooth-walled, branched, septate, hyaline, 2.1–2.9 μm wide. Cultures produced phialides and conidia after 45 days on potato dextrose agar at room temperature. Conidiophores are smooth-walled, cylindrical, solitary, or verticillate, 17.1–25.2 × 1.4–1.6 μm. Phialides solitary or verticillate, in whorls of two to three, usually solitary on hyphae, basal portion cylindrical to narrow lageniform, gradually or abruptly tapering toward the apex; 11.7–50.2 μm long, 1.5–3.1 μm wide at the base, 3.4–4.0 μm at the middle, and 0.9–2.0 μm wide at the apex. Conidia is one-celled, hyaline, smooth-walled, fusiform or oval, 2.0–4.9 × 2.0–3.3 μm, often in chains.

Habitat and known distribution: On the pupa of Lepidoptera buried in soil.

Additional specimens examined: Xinfang Reservoir, Simao District, Pu’er City, Yunnan, China. October 6, 2019, H. Yu (YHH 20017 paratype, YFCC 8407 ex-paratype living culture; YHH 20018 paratype, YFCC 8408 ex-paratype living culture).

Comments: Our phylogenetic results demonstrated that the isolated position of this collection was within the Cordyceps genus. C. simaoensis formed a separate subclade from other species of Cordyceps with moderately high credible support (100%). This species was mainly isolated from soil and produced Isaria-like asexual morphs and fusiform or oval conidia. C. simaoensis was closely related to C. tenuipes (Peck) Kepler et al. and might be identified by the size and presence of conidia and its distinct sexual morph. The conidial size of Cordyceps tenuipes was 2.0–7.0 × 1.2–2.5 μm, the wider conidia of Cordyceps pseudotenuipes was 3.4–6.5 × 2.2–4.0 μm, that of Cordyceps subtenuipes was 1.9–3.4 × 1.5–2.7 μm, and that of Cordyceps simaoensis was 2.0–4.9 × 2.0–3.3 μm. Cultures of C. simaoensis produced phialides and conidia, and it took more days than C. tenuipes. Our field observations and herbaria record also indicated that the C. tenuipes was widely distributed and significantly ecologically diverse in China, with small bright yellow fleshy stromata as teleomorph and Isaria-like anamorph.

 Discussion

Considerable changes to the taxonomy of Cordyceps have occurred since the research on entomogenous fungi entered the molecular era. At present, multi-locus phylogenetic analyses have gained importance in delimiting Cordyceps species (Tasanathai et al., 2016; Zha et al., 2019; Li et al., 2020, 2021; Wang Y. B. et al., 2020). In this study, most species of the newly circumscribed genus Cordyceps were analyzed based on phylogenetic inferences of five nuclear molecular markers (nrSSU, nrLSU, tef-1α, rpb1, and rpb2). Both ML and BI analyses produced trees with similar topologies that resolved most Cordyceps lineages in separate terminal branches. Cordyceps s. s. was recognized by five statistically well-supported clades, designated as clade I, clade II, clade III, clade IV, and clade V (Figure 1). There were 20 species in clade I. Morphologically, the 20 species shared relatively complicated host, such as spider, Coleoptera, Lepidoptera, Limacodidae, ant, and even plants. They were also complex and varied in shape (ascospores bola-shaped, filiform, ninchukiform, or bifusiform). Clade II was made up of C. militaris and other closely related species. With the exception of C. kyusyuenis and C. rosea, all other species had filiform ascospores; in addition, unlike C. oncoperae and C. rosea, the rest of the species could easily disarticulate into part-spores. It was found that all the members in clade III had Isaria-like anamorphs and all four species in clade IV were described as conidiophores verticillate with phialides in whorls of 2 to 4 or 5, conidia in chains (Table 3). Clade V consisted solely of Cordyceps grylli, characterized as pathogenic on Gryllidae adults and relatively larger perithecia (up to 650–810 × 270–370 μm) (Teng, 1963; Liang et al., 2007).

Phylogenetic classifications of cordycepitoid fungi showed that most diagnostic characteristics used in current classifications of Cordyceps species (e.g., host, arrangement of perithecia, ascospores fragmentation, conidiogenous structures, conidial shape and size) were not phylogenetically informative (Sung et al., 2007; Kepler et al., 2017; Mongkolsamrit et al., 2018; Wang Y. B. et al., 2020). Cordyceps lepidopterorum Mongkolsamrit, Noisripoom, Thanakitpipattana, Spatafora and Luangsa-ard was firstly reported from Chiang Mai Province Thailand by Mongkolsamrit et al. (2018). Cordyceps chanhua, which had long been mistaken as Isaria (Paecilomyces) cicadae of a Brazilian specimen, was recently reported as a new species in the genus Cordyceps s. s., for the discovery of its teleomorph in Mt. Jinggang, Jiangxi, China and analyses of both morphological and phylogenetical evidence (Li et al., 2021). Our phylogenetic trees suggested that C. chanhua (see Cordyceps cicadae in Figure 1) could not be distinguished from C. lepidopterorum (Figure 1). Regarding the morphology, there were no significant differences in the morphological characteristics of anamorph between the two species except for their host (Table 3). Because C. lepidopterorum was described earlier than C. chanhua, C. lepidopterorum should be recommended as the scientific name for this species.

 Data Availability Statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

 Author Contributions

Q-YD and HY: conceptualization. Q-YD: methodology, writing—original draft preparation, and formal analysis. Q-YD and YW: software and resources.. Z-QW and H-JW: validation. Q-YD, YW, Z-QW, D-XT, Z-YZ, H-JW, and HY: investigation. HY: writing—review and editing and funding acquisition. All authors reviewed and approved the final manuscript.

 Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

 Funding

This work was supported by the National Natural Science Foundation of China (Nos. 31870017 and 32060007) and the Department of Science and Technology of Yunnan Province [No. 2018FY001(-006)].

We are very grateful to Yuan-Bing Wang and Nian-Kai Zeng (College of Pharmacy, Hainan Medical University, Haikou, China) for help in naming the new fungal species. Special thanks are due to the reviewers for constructive comments and suggestions for improving our work.

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