Research Article |
Corresponding author: Yu Li ( fungi966@126.com ) Corresponding author: Bo Zhang ( zhangbofungi@126.com ) Academic editor: Thorsten Lumbsch
© 2021 Jia-Jun Hu, Gui-Ping Zhao, Yong-Lan Tuo, Dan Dai, Di-Zhe Guo, Gu Rao, Zheng-Xiang Qi, Zhen-Hao Zhang, Yu Li, Bo Zhang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Hu J-J, Zhao G-P, Tuo Y-L, Dai D, Guo D-Z, Rao G, Qi Z-X, Zhang Z-H, Li Y, Zhang B (2021) Morphology and molecular study of three new Cordycipitoid fungi and its related species collected from Jilin Province, northeast China. MycoKeys 83: 161-180. https://doi.org/10.3897/mycokeys.83.72325
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Cordyceps species are notable medicinal fungi in China, which are pathogenic on insects and exhibit high biodiversity in tropical and subtropical regions. Recently, three new Cordyceps species, Cordyceps changchunensis and Cordyceps jingyuetanensis growing on pupae of Lepidoptera and Cordyceps changbaiensis growing on larvae of Lepidoptera, were found in Jilin Province, China and are described, based on morphological and ecological characteristics. These three new species are similar to the Cordyceps militaris group, but are distinctly distinguishable from the known species. Cordyceps changchunensis, characterised by its small and light yellow to orange stromata which is occasionally forked, covered with white mycelium at the base of stipe, globose to ovoid perithecia, is macroscopically similar to Cordyceps militaris. Cordyceps changbaiensis is clearly discriminated from other Cordyceps species by its white to orange and branched stromata, clavate to cylindrical fertile apical portion, immersed and globose to ovoid perithecia. Moreover, unbranched, clavate and orange to light red stromata, almond-shaped to ovoid and immersed perithecia separate Cordyceps jingyuetanensis from other Cordyceps species. nrITS, nrLSU and EF-1α sequences were undertaken and phylogenetic trees, based on Maximum Likelihood and Bayesian Inference analysis showed that the three new species clustered with Cordyceps militaris, but formed individual clades, as well as confirmed the results of our morphological study.
Cordyceps, host, new species, phylogenetic study, relationship
The family Cordycipitaceae belongs to Hypocreales with plant-, animal- and fungus-based nutrition modes (
Cordycipitoid fungi were first described in 1753 as Clavaria militaris L., later being recognised as Cordyceps militaris (L.) Fr. The genus Cordyceps Fr. was established by Fries in 1818, encompassing over 450 species (
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 (
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.
The specimens were photographed in situ. The size of the stromata was measured when fresh. After examination and description of the fresh macroscopic characters, the specimens were dried in an electric drier at 40–45 °C.
Descriptions of macroscopic characters were based on field notes and photographs. The colours correspond to the “Flora of British fungi: colour identification chart” (Royal Botanic Garden 1969). The dried specimens were rehydrated in 94% ethanol for microscopic examination and then mounted in 3% potassium hydroxide (KOH), 1% Congo Red, Cotton Blue and Melzer’s Reagent (
Total DNA was extracted from dried specimens using the NuClean Plant Genomic DNA Kit (Kangwei Century Biotechnology Company Limited, Beijing, China). Sequences of the internal transcribed spacer region (ITS), nuclear large ribosomal subunits (LSU) and translation elongation factor 1-alpha (EF-1α) were used for phylogenetic analysis. The ITS sequence was amplified using the primer pair ITS4 and ITS5 (
Reaction programmes followed
Based on the results of BLAST and morphological similarities, the sequences obtained and related to these samples are listed in Table
Voucher information and GenBank accession numbers of ITS, LSU and EF-1α DNA sequences of Cordyceps changchunensis, Cordyceps changbaiensis, Cordyceps jingyuetanensis and related species used in this study.
Species name | Specimen/Strain number | Host/Substratum | GenBank accession numbers | References | ||
---|---|---|---|---|---|---|
ITS | LSU | EF-1α | ||||
Akanthomyces lecanii | CBS101247 | Homopteran | JN049836 | AF339555 | DQ522359 | ( |
A. tuberculatus | NBRC106949 | Lepidoptera | JN943318 | JN941400 | MF416490 | ( |
Blackwellomyces cardinalis | CBS113414 | Lepidoptera | MH862930 | MH874497 | EF469059 | ( |
B. pseudomilitaris | NBRC101411 | Lepidoptera | JN943308 | JN941395 | MT017849 | ( |
Cordyceps bassiana | IFO4848 | Lepidoptera | AB027382 | AB027382 | MN401498 | ( |
C. bifusispora | ARS5690/EFCC8260 | Lepidoptera | AY245627 | EF468807 | EF468747 | ( |
C. brongniartii | NBRC101395 | Lepidopteran pupae | JN943298 | JN941382 | JF416009 | ( |
C. cateniobliqua | CBS153.83 | Lepidoptera | MH861560 | MT017860 | ( |
|
C. changbaiensis | HMJAU48255 | Lepidoptera | MW893252 | MW893277 | MZ616772 | This study |
C. changbaiensis | HMJAU48260 | Lepidoptera | MW893270 | MW893272 | MZ616774 | This study |
C. changchunensis | HMJAU48251 | Lepidoptera | MW893249 | MW893274 | MZ616769 | This study |
C. changchunensis | HMJAU48252 | Lepidoptera | MW893250 | MW893275 | MZ616775 | This study |
C. changchunensis | HMJAU48259 | Lepidoptera | MW893251 | MW893276 | MZ616773 | This study |
C. chiangdaoensis | BCC75734/TBRC7274 | Coleopteran larvae | KT261394 | MF140732 | KT261404 | ( |
C. coleopterorum | CBS110.73 | Coleoptera | AY624177 | JF415988 | JF416028 | ( |
C. exasperata | MCA2155 | Lepidoptera | MF416542 | MF416486 | ( |
|
C. farinosa | CBS111113 | Lepidoptera | AY624181 | MF416554 | MF416499 | ( |
C. fumosorosea | CBS244.31 | Coleoptera | AY624182 | MF416557 | MF416503 | ( |
C. hepialidicola | Lepidoptera | AF315649 | Unpublished | |||
C. jingyuetanensis | HMJAU48253 | Lepidoptera | MW893253 | MW893278 | MZ616770 | This study |
C. jingyuetanensis | HMJAU48261 | Lepidoptera | MW893271 | MW893273 | This study | |
C. kyushuensis | HMAS78115 | Lepidoptera | EF368021 | EF468813 | EF468754 | ( |
C. militaris | OSC93623 | Lepidopteran pupae | JN049825 | AY184966 | DQ522332 | ( |
C. militaris | HMJAU48256 | Lepidopteran pupae | MW888227 | MW893279 | This study | |
C. morakotii | BCC55820/TBRC7276 | Hymenoptera | KT261389 | MF140731 | KT261399 | ( |
C. ninchukispora | BCC30937 | Lepidoptera | FJ765274 | FJ765242 | MF416477 | ( |
C. ningxiaensis | HMJAU25074 | Diptera | KF309668 | KF309671 | ( |
|
C. polyarthra | 6578 | Lepidoptera | AJ536548 | Unpublished | ||
C. pruinosa | ARSEF5413 | Lepidoptera | JN049826 | MK761215 | DQ522351 | ( |
C. qingchengensis | MFLU17-1022 | Lepidoptera | KY423506 | MK761211 | MK770630 | ( |
C. rosea | Spat09-053 | Lepidoptera | MF416536 | MF416480 | ( |
|
C. roseostromata | ARSEF4870 | Larva, not specified | AY245637 | AF339523 | ( |
|
C. scarabaeicola | ARSEF5689 | Coleoptera | JN049827 | AF339524 | DQ522335 | ( |
C. scarabaeicola | Arsef5689 | Coleoptera | JN049827 | AF339524 | ( |
|
Cordyceps sp. | HMJAU48254 | Lepidoptera | MW888228 | MW893280 | MZ616771 | This study |
C. spegazzinii | ARSEF7850 | Diptera | DQ196435 | DQ196435 | GU734752 | ( |
C. taishanensis | A-1 | Lepidoptera | FJ008927 | Unpublished | ||
C. tenuipes | TBRC7266 | Lepidoptera | MF140742 | MF140828 | ( |
|
Isaria cicadae | GACP07071701 | Hemiptera | KX017277 | MK761212 | MT268245 | ( |
I. japonica | BCC2808 | Lepidoptera | AY624199 | ( |
||
Metarhizium yongmunense | EFCC2131 | Lepidoptera | JN049856 | EF468833 | EF468770 | ( |
Metacordyceps taii | ARSEF5714 | Lepidoptera | JN049829 | AF543787 | AF543775 | ( |
Nigelia martiale | HMAS197472(S) | Coleoptera | JN049881 | JF415975 | JF416016 | ( |
Ophiocordyceps acicularis | OSC12858/OSC110987 | Coleoptera | JN049820 | DQ518757 | DQ522326 | ( |
O. clavata | NBRC106961 | Coleoptera | JN943327 | JN941414 | MH879672 | ( |
O. gracilis | EFCC8572 | Lepidoptera | HM142942 | EF468811 | EF468751 | ( |
O. rubiginosoperitheciata | NBRC106966 | Coleoptera | JN943344 | JN941437 | ( |
|
O. sinensis | ARSEF6282 | Lepidopteran pupae | HM595981 | HM595885 | EF468767 | ( |
Tolypocladium ophioglossoides | NBRC106331 | Elaphomyces sp. | JN943320 | JN941408 | ( |
MrModeltest 2.3 was used to determine the best fitting substitution model for each dataset for Bayesian Inference, which was calculated with MrBayes 3.2.6 with a general time-reversible DNA substitution model and a gamma distribution rate variation across sites (
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.
For these reasons, the combined ITS, LSU and EF-1α dataset including 121 fungal samples representing 48 taxa was used for analysis and the results are shown in Fig.
changchunensis: referring to Changchun, the location of the holotype.
Cordyceps changchunensis can be easily differentiated from closely-related species C. militaris by its unique host, smaller stromata, immersed perithecia and larger part-spores (2.6–6 × 1.0–1.4 μm).
Sexual Morph. Stromata 2.4–4.5 cm long, single or multiple, solitary to gregarious, arising from pupa; branched, sometimes single at base, then branched into two forks. Fertile apical portion, orange, clavate to globose, sometimes irregular, 2.0–3.5 cm long and 0.4–0.6 cm wide, distinctly distinguishable from the stipe. Sterile stipe fleshy, light yellow to orange, cylindrical, 1.3–3.3 cm long and ca. 0.4 cm wide, usually with white mycelium at the base. Perithecia immersed at right angles to the surface of the fruiting body, globose to ovoid, 180–600 × 180–520 μm, with a thick wall about 10–15 μm. Asci cylindrical, 80–300 × 2.5–5 μm, 8–spored, apex of ascus hemispherical, 3.0–4.0 × 2.0–3.0 μm. Part-spores oblong, 2.6–6 × 1.0–1.4 μm, smooth, hyaline in 3% KOH, thin-walled, inamyloid.
Morphological characters of Cordyceps changchunensis (
Asexual Morph. Unknown.
Growing on pupae of Lepidoptera.
China. Jilin Province: Changchun City, Jingyuetan National Forest Park, 20 August 2015, Bo Zhang (
China (Jilin Province).
C. changchunensis is easily confused with C. militaris due to highly similar morphology and sharing the same habitat. Morphologically, the stromata of C. militaris are larger than C. changchunensis, single or gregarious, larger perithecia (500–1089 × 132–264 μm) and smaller part-spores (2–4 × 1 μm) (
changbaiensis: referring to Mt. Changbai, the location of the holotype.
The species is characterised by orange to white and branched stromata, globose to ovoid perithecia and larger part-spores (3.0–7.0 × 1.0–1.4 μm).
Morphological characters of Cordyceps changbaiensis (
Sexual Morph. Stromata 2.4–5.2 cm long, single or multiple, solitary, arising from the head of the host insect covered with white mycelia. Fertile apical portion, orange, clavate to cylindrical, 0.6–1.5 cm long and 0.2–0.6 cm wide, obviously distinguishable from the stipe. Sterile stipe fleshy, white to light yellow, cylindrical, 1.8–3.7 cm long and 0.2–0.5 cm wide. Perithecia immersed to the surface of the fruiting body, globose to ovoid, 120–230 × 90–170 μm, with a thick wall about 15 μm. Asci cylindrical, 225–625 × 4–5 μm, 8–spored, apex of ascus hemispherical, 3.0–4.0 × 2.2–3.2 μm. Part-spores oblong, 3.0–7.0 × 1.0–1.4 μm, smooth, hyaline in 3% KOH, thin-walled, inamyloid.
Asexual Morph. Unknown.
Growing on larvae of Lepidoptera.
China (Jilin Province).
C. changbaiensis has orange to white and branched stromata. Morphologically, C. roseostromata Kobayasi & Shimizu is similar to C. changbaiensis due to the single or branched stromata. C. kyushuensis A. Kawam. is also close to C. changbaiensis because of the host and the stromata being similar in colour. However, both C. roseostromata and C. kyushuensis have a larger perithecia and smaller part-spores. Furthermore, the stromata of C. kyushuensis is gregarious or fascicled and grows from the head or abdomen of the host (
jinyuetanensis: referring to Jingyuetan National Forest Park, the location of the holotype.
C. jingyuetanensis is different from other species by growing on pupae, orange to light red stromata, immersed and almond-shaped to ovoid perithecia.
Morphological characters of Cordyceps jingyuetanensis (
Sexual Morph. Stromata 4–4.5 cm long, multiple, solitary, arising from pupae of Lepidoptera. Fertile apical portion, orange to light red, clavate, 0.8–1.3 cm long and 0.1–0.2 cm wide, obviously distinguishable from the stipe. Sterile stipe fleshy, light yellow to orange, cylindrical, 2.7–3.7 cm long and 0.1–0.2 cm wide, usually with white mycelium at the base. Perithecia immersed to the surface of the fruiting body, almond-shaped to ovoid, 220–340 × 110–220 μm, with a thick wall about 15–20 μm. Asci cylindrical, 225–475 × 3–5 μm, 8-spored, apex of ascus hemispherical to irregular, 3.0–4.0 × 1.4–2.8 μm. Part-spores oblong, 2.8–5.0 × 1.0–1.4 μm, smooth, hyaline in 3% KOH, thin-walled, inamyloid.
Asexual Morph. Unknown.
Growing on pupae of Lepidoptera.
China (Jilin Province).
A review of literature revealed that there are about 20 species of Cordycipitiod fungi growing on pupae, like the unusual medicinal fungi O. sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, C. militaris, I. cicadae Miq. and also like the two new species, C. ningxiaensis T. Bau & J.Q. Yan and C. qingchengensis L.S. Zha & T.C. Wen, reported from China in 2015 and 2019. Nevertheless, C. jingyuetanensis is different from these Cordycipitiod species; C. ningxiaensis grows on the pupae of Diptera, I. cicadae grows on the pupae of Hemiptera and the stromata of C. qingchengensis are yellow, single or branched on the top. C. hepialidicola Kobayasi & Shimizu from Japan is similar to C. jingyuetanensis in its phylogenetic relationship, but there are distinct morphological differences. Morphologically, the stromata of C. hepialidicola are multiple, branched on the top sometimes, grow from the head of larva of Hepialida or Lepidoptera, have larger perithecia (300–350 × 500 μm) and smaller part-spores (3–4 × 1 μm) (
China. Yunnan Province: Qujin City, Huize County, 26.24°N, 103.25°E, 30 July 2019, Jia-Jun Hu, Bo Zhang & Di-Zhe Guo (
Macrocharacter of Cordyceps militaris a–e stromata and host of Cordyceps militaris (a collected from Daxing’an Mountains, Heilongjiang Province b collected from Ji’an County, Tonghua City, Jilin Province c, e collected from Changchun City, Jilin Province d collected from Qujin City, Huize County, Yunnan Province). Scale bars: 1 cm (a–e).
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.
Morphological comparisons of sexual states of Cordyceps changchunensis, Cordyceps changbaiensis and Cordyceps jingyuetanensis.
Species | Host | Stromata | Fertile part | Perithecia | Asci | Ascospores | Reference |
---|---|---|---|---|---|---|---|
Beauveria bassiana | Larvae of Lepidoptera | Single or several, unbranched, slender and cylindrical, brownish- yellow to yellowish | 18.7–33. 3 × 2.8–8.0 mm | Elliptical, 610–720 × 230–320 µm, immersed to surface | Cylindrical, 230–590 × 3.5–4.0 µm with ascus cap 3.6–4.0 µm in diameter | Filamentous, 300–570 × 1.0 µm, not broken into part-spores | ( |
Blackwellomyces pseudomilitaris | Larvae of Lepidoptera | Single or cluster, simple or branched, cylindrical, white to white-orange | 15–30 × 0.9–3 mm | Elongate-ellipsoid or elongate-ovoid, 290–570 × 120–245 µm, superficial | Filiform, 290–410 × 5–6 µm | Filiform, 280–390 × 1 µm, not broken into part-spores | ( |
Cordyceps bifusispora | Larvae of Lepidoptera | Simple, cylindrical clavate, whitish | 6 × 1.3 mm | Pyriform, with protruding apices, yellowish, 300 × 150–170 µm, immersed | Cylindrical, 200–220 × 3–4.5 µm | Bifusiform, 145–220 µm in length, central part filiform about 0.4 µm wide, terminal parts narrowly fusiform, about 30 × 1.6 µm and 3 septate | ( |
C. kyushuensis | Larvae of Lepidoptera | Cluster, cylindrical, Light yellow to orange red | 20–30 × 5–8 mm | Elliptical, 300–500 × 200–300 µm, half-buried | Cylindrical, 3–4.5 µm wide | Short cylindrical, part-spores 5–7 × 0.7–1 µm | ( |
C. militaris | Lepidopteran pupae | Single or several, clavate, orange | 10–20 × 3–5 mm | Conical, half-buried | Clavate, 300–400 × 4–5 µm | Filiform, part spores 2–3 × 1 µm | ( |
C. ningxiaensis | Fly pupae (Diptera) | One to two in a group, clavate, orange | 1.2–3 × 1.2–2.8 mm | Ellipsoid to ovoid, 288–400 × 103–240 μm, with a wall about 10 μm thick, loosely embedded at right angles to the surface | Cylindrical, 168–205 × (3.7–)4.1–5.5(–6.6) μm, with oblate spheroid or hemispherical refractive cap 3.4–3.8 × 2.9–3.4 μm at apex | Filiform, irregularly multiseptate, part-spores 3.6–7.8 × 1.0–1.4 μm | ( |
C. polyarthra | Larvae of Lepidoptera | Cespitose, narrowly clavate, light yellow to reddish-brown | Ovoid, 250–450 × 125–250 μm, brown, with a definite wall 25 μm thick, embedded at right angles to the surface | Cylindrical, 150–260 × 3–4 μm, with a 1.5–2 μm thick cap | Filiform, part-spores 4–6 × 0.75–1 μm | ( |
|
C. pruinosa | Larvae of Lepidoptera | Solitary or several, clavate, orange to red | 2–8 × 1–3 mm | Ovoid to fusiform, 360–400 × 130–200 μm, crowded, red, ordinal in orientation, immersed | Cylindrical, 100–200 × 2.5–4 μm | Filiform, part-spores 4–6 × 1 μm | ( |
C. qingchengensis | Lepidopteran pupae | Branched, yellow | 7–9 × 2.0–2.5 mm | Ovoid but apex sharply pointed, 335–490 × 145–240 μm, partially immersed at right angle to the surface | Cylindrical, 180–200 × 2.4–4.0 μm wide, caps hemispherical, 1.8–2.2 × 2.5–3.2 μm | Filiform, 180–220 × 0.45–0.65 μm, not at all bifusiform and not broken into part-spores | ( |
C. roseostromata | Larva, not specified | Single or branched | 1.2–5 × 1.5–2.2 mm | Pyriform, 280–300 × 140–160 μm, Superficial | 3–3.5 × 2.5–3 μm | 4–5 × 1 μm | ( |
C. changchunensis | Lepidopteran pupae | Single or multiple, clavate, orange | 2.0–3.5 × 0.4–0.6 mm | Globose to ovoid, 180–600 × 180–520 μm, with a thick wall about 10–15 μm, partially immersed at right angles to the surface | Cylindrical, 80–300 × 2.5–5 μm, caps hemispheric, 3.0–4.0 × 2.0–3.0 μm at apex | Oblong, 2.6–6 × 1.0–1.4 μm | This study |
C. changbaiensis | Larvae of Lepidoptera | Single or multiple, clavate, white to orange | 0.6–1.5 × 0.2–0.6 mm | Globose to ovoid, 120–230 × 90–170 μm, with a thick wall about 15 μm, immersed to surface | Cylindrical, 225–625 × 4–5 μm, caps hemispherical, 3.0–4.0 × 2.2–3.2 μm at apex | Oblong, 3.0–7.0 × 1.0–1.4 μm | This study |
C. jingyuetanensis | Lepidopteran pupae | Single or multiple, clavate, orange to light red | 0.8–1.3 × 0.1–0.2 mm | Almond-shaped to ovoid, 220–340 × 110–220 μm, with a thick wall about 15–20 μm, immersed to surface | Cylindrical, 225–475 × 3–5 μm, caps hemispherical to irregular, 3.0–4.0 × 1.4–2.8 μm at apex | Oblong, 2.8–5.0 × 1.0–1.4 μm | This study |
1 | Stromata arise from pupae | 2 |
– | Stromata arise from larvae | Cordyceps changbaiensis |
2 | Stromata branched into two forks sometimes | Cordyceps changchunensis |
– | Stromata not branched | 3 |
3 | Part-spores over 3 μm | Cordyceps jingyuetanensis |
– | Part-spores less than 3 μm | Cordyceps militaris |
In this study, three new species, collected from northeast China in the Cordyceps militaris group, are described. In previous work, about 38 species were recognised as belonging to the C. militaris group (
The previous studies have revealed that the genus Cordyceps was not monophylic (
Clade 1, including nine Cordyceps spp. and two Isaria spp. I. cicadae, based on Chinese sequences, gathers into one branch with Cordyceps species. What is known as I. cicadae in China, named on a Brazilian specimen, is of confused classification status, due to the teleomorph having remained undiscovered. In China, C. cicadae Massee has been regarded as a teleomorph of I. cicadae as well as a teleomorph of O. sobolifera (Hill ex Watson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora and referred to as C. sobolifera (Hill ex Watson) Berk. & Broome. Until recently, the teleomorph was discovered in Mt. Jinggang, Jiangxi Province, China and both teleomorph and anamorph existed on some specimens, with the morphology of the anamorph consistent with those, “I. cicadae”, harvested throughout southern China, significantly different from the type specimen of I. cicadae. For this reason, it was published as a new species named C. chanhua Z.Z. Li, F.G. Luan, Hywel-Jones, C.R. Li & S.L. Zhang (
About 60% of Cordyceps sensu lato species are recorded on two insect orders–Coleoptera and Lepidoptera (
This study is funded by the National Key R & D of Ministry of Science and Technology (2018YFE0107800), the National Key R & D of Ministry of Science and Technology (2019YFD1001905-33), Jilin Province Science and Technology Development Plan Project (20190201256JC), Scientific and Technological Tackling Plan for the Key Fields of Xinjiang Production and Construction Corps (No. 2021AB004) and “111” programme (No. D17014). The authors are very thankful to Dr. Xue-Fei Li, Miss Hui-Ze Hu and Miss Fang-Fang Zhang from Engineering Research Centre of Edible and Medicinal Fungi, Ministry of Education, for help during this study.