Phylogenetic classification of Cordyceps and the clavicipitaceous fungi

Abstract

Cordyceps, comprising over 400 species, was historically classified in the Clavicipitaceae, based on cylindrical asci, thickened ascus apices and filiform ascospores, which often disarticulate into part-spores. Cordyceps was characterized by the production of well-developed often stipitate stromata and an ecology as a pathogen of arthropods and Elaphomyces with infrageneric classifications emphasizing arrangement of perithecia, ascospore morphology and host affiliation. To refine the classification of Cordyceps and the Clavicipitaceae, the phylogenetic relationships of 162 taxa were estimated based on analyses consisting of five to seven loci, including the nuclear ribosomal small and large subunits (nrSSU and nrLSU), the elongation factor 1alpha (tef1), the largest and the second largest subunits of RNA polymerase II (rpb1 and rpb2), beta-tubulin (tub), and mitochondrial ATP6 (atp6). Our results strongly support the existence of three clavicipitaceous clades and reject the monophyly of both Cordyceps and Clavicipitaceae. Most diagnostic characters used in current classifications of Cordyceps (e.g., arrangement of perithecia, ascospore fragmentation, etc.) were not supported as being phylogenetically informative; the characters that were most consistent with the phylogeny were texture, pigmentation and morphology of stromata. Therefore, we revise the taxonomy of Cordyceps and the Clavicipitaceae to be consistent with the multi-gene phylogeny. The family Cordycipitaceae is validated based on the type of Cordyceps, C. militaris, and includes most Cordyceps species that possess brightly coloured, fleshy stromata. The new family Ophiocordycipitaceae is proposed based on Ophiocordyceps Petch, which we emend. The majority of species in this family produce darkly pigmented, tough to pliant stromata that often possess aperithecial apices. The new genus Elaphocordyceps is proposed for a subclade of the Ophiocordycipitaceae, which includes all species of Cordyceps that parasitize the fungal genus Elaphomyces and some closely related species that parasitize arthropods. The family Clavicipitaceaes. s. is emended and includes the core clade of grass symbionts (e.g., Balansia, Claviceps, Epichloë, etc.), and the entomopathogenic genus Hypocrella and relatives. In addition, the new genus Metacordyceps is proposed for Cordyceps species that are closely related to the grass symbionts in the Clavicipitaceaes. s.Metacordyceps includes teleomorphs linked to Metarhizium and other closely related anamorphs. Two new species are described, and lists of accepted names for species in Cordyceps, Elaphocordyceps, Metacordyceps and Ophiocordyceps are provided.

Fig. 1.

Phylogenetic relationships among 162 taxa in the Clavicipitaceae and other families in the Hypocreales. One of 156 equally parsimonious trees is shown based on maximum parsimony analyses with combined data set of five genes (i.e., nrSSU, nrLSU, tef1, rpb1 & rpb2). Bootstrap proportions (MP-BP) of ≥ 70 % are provided above corresponding nodes and in a thicker line. Internodes that are collapsed in strict consensus tree are marked with an asterisk (*).

Fig. 1.

Phylogenetic relationships among 162 taxa in the Clavicipitaceae and other families in the Hypocreales. One of 156 equally parsimonious trees is shown based on maximum parsimony analyses with combined data set of five genes (i.e., nrSSU, nrLSU, tef1, rpb1 & rpb2). Bootstrap proportions (MP-BP) of ≥ 70 % are provided above corresponding nodes and in a thicker line. Internodes that are collapsed in strict consensus tree are marked with an asterisk (*).

Fig. 2.

Phylogenetic relationships among 162 taxa in the Clavicipitaceae and other families in the Hypocreales. A 50 % majority consensus tree is shown based on Bayesian analyses with combined data set of five genes (i.e., nrSSU, nrLSU, tef1, rpb1 & rpb2). Outgroups (Glomerella cingulata and Verticillium dahliae) are not shown. Posterior probabilities (PP) of ≥ 0.95 are provided in percentage below corresponding nodes. Bootstrap proportions (ML-BP) are obtained in maximum likelihood analyses and shown above corresponding nodes for ≥ 70 %. Internodes that are supported with both bootstrap proportions (ML-BP ≥ 70 %) and posterior probabilities (PP ≥ 0.95) are considered strongly supported and drawn in a thicker line.

Fig. 2.

Phylogenetic relationships among 162 taxa in the Clavicipitaceae and other families in the Hypocreales. A 50 % majority consensus tree is shown based on Bayesian analyses with combined data set of five genes (i.e., nrSSU, nrLSU, tef1, rpb1 & rpb2). Outgroups (Glomerella cingulata and Verticillium dahliae) are not shown. Posterior probabilities (PP) of ≥ 0.95 are provided in percentage below corresponding nodes. Bootstrap proportions (ML-BP) are obtained in maximum likelihood analyses and shown above corresponding nodes for ≥ 70 %. Internodes that are supported with both bootstrap proportions (ML-BP ≥ 70 %) and posterior probabilities (PP ≥ 0.95) are considered strongly supported and drawn in a thicker line.

Fig. 3.

Schematic diagrams of phylogenetic relationships from MP, ML, and Bayesian analyses that differ in character or taxon sampling. In addition to 162-taxon 5-gene and 7-gene data sets, 107-taxon and 152-taxon 5-gene data sets were generated with taxa complete for five genes (i.e., nrSSU, nrLSU, tef1, rpb1 and rpb2) and at least three genes, respectively. To address the impact of C. sphecocephala clade to nodal support of C. unilateralis clade in Fig. 1, a 147-taxon 5-gene data set was constructed after members of C. sphecocephala clade were excluded. Bootstrap proportions (BP ≥ 70 %) or posterior probabilities (PP ≥ 0.95 in percentage) are shown above corresponding nodes and in a thicker line.

Fig. 4.

Enlargement of Bayesian consensus tree in Fig. 2, showing Clavicipitaceae clade A, to emphasize relationships within the clade. Respective subgenera of Cordyceps species in current classification are provided to the right of species. Known anamorphic genera of Cordyceps species are in parentheses. Tree description is the same as in Fig. 2.

Fig. 5.

A–E. Representative species of Cordyceps and its allies in Clavicipitaceae clade A. F-K. Morphology of Cordyceps sp. (described here as Metacordyceps yongmunensis sp. nov. below). A. C. liangshanensis on lepidopteran larva, EFCC 1452. B. Cordyceps sp. on lepidopteran pupa, EFCC 1228. C. Hypocrella schizostachyi on scale insect (Hemiptera). D. Shimizuomyces paradoxus on seed of plant (Smilax sieboldii: Smilacaceae). E. Metarhizium sp. on adult of cicada. F. Section of perithecium, EFCC 2131. G. Asci and fascicle, EFCC 2131. H. Asci showing prominent ascus cap, EFCC 2131. I. Asci showing ascus foot, EFCC 2131. J. Ascospores showing indistinct septation, EFCC 2131. K. Discharged intact ascospores on SDAY agar, EFCC 2131. Scale bars: A–E = 10 mm, F = 200 μm, G = 100 μm, H–J = 10 μm, K = 100 μm.

Fig. 6.

Enlargement of Bayesian consensus tree in Fig. 2, showing Clavicipitaceae clade B, to emphasize relationships within the clade. Respective subgenera of Cordyceps species in current classification are provided to the right or below of species. Known anamorphic genera of Cordyceps species are in parentheses. Numbers above corresponding nodes are bootstrap proportions of ML analyses (before the backslash) and posterior probabilities (after the backslash) from 147-taxon 5-gene data set in Fig. 3. Numbers below corresponding nodes are bootstrap proportions of ML analyses (before the backslash) and posterior probabilities (after the backslash) from 162-taxon 5-gene data set in Fig. 2. Bootstrap proportions of ≥ 70 % or posterior probabilities of ≥ 0.95 (in pergentage) are shown in corresponding nodes. Internodes in a thicker line are supported by the bootstrap proportions and posterior probabilities from either 147-taxon or 162-taxon 5-gene data sets. Numbers in a circle correspond to internode that is informative for placing the C. sphecocephala clade.

Fig. 7.

A–S. Representative species of Cordyceps and its allies in Clavicipitaceae clade B. T–X. Ascus and ascospore of Cordyceps species in this clade. A. C. ophioglossoides on truffle (Elaphomyces sp.: Eurotiomycetes). B. C. japonica on truffle (Elaphomyces muricatus: Eurotiomycetes), OSC 110991. C. C. subsessilis on scarabaeid beetle in decaying wood (Coleoptera), OSC 128581. D. C. gracilis on lepidopteran larva, EFCC 10121. E. C. heteropoda on nymph of cicada (Hemiptera), EFCC 1012. F. C. nigrella on coleopteran larva, EFCC 3438. G. C. sobolifera on nymph of cicada (Hemiptera), EFCC 7768. H. C. longissima on nymph of cicada (Hemiptera), EFCC 8576. I. C. unilateralis on ant (Hymenoptera). J. C. cochlidiicola on lepidopteran larva, EFCC 377. K. C. agriotidis on coleopteran larva, EFCC 5274. L. C. sinensis on larva of Hepialus sp. (Lepidoptera), EFCC 3248. M. C. brunneipunctata on coleopteran larva. N. C. sphecocephala on wasp (Hymenoptera). _O. C. nutans on stink bug (Hemiptera). _P. C. tricentri on adult of Tricentrus sp. (Hemiptera), EFCC 1001; bar = 10 mm. Q. Hymenostilbe odonatae on adult of dragonfly (Odonata), EFCC 12459; bar = 10 mm. R. Hirsutella sp. on wasp (Hymenoptera). S. Paecilomyces lilacinus. T. C. robertsii, ascus with disarticulating ascospores, MICH 2874. U. C. acicularis, ascus and nondisarticulating ascospores, OSC 110987. V. C. paludosa, non-disarticulating ascospores, MICH 14366. W. C. variabilis, disarticulated part-spores in ascus, and X. Part-spores, OSC 128581. Scale bars: A–B = 10 mm, C = 1 mm, D–H = 10 mm, I = 5 mm, J–S = 10 mm, T–X = 10 μm.

Fig. 8.

Enlargement of Bayesian consensus tree in Fig. 2, showing Clavicipitaceae clade C, to emphasize relationships within the clade. Respective subgenera of Cordyceps species in previous classification are provided to the right of the species. Known anamorphic genera of Cordyceps species are in parentheses. Tree description is the same as in Fig. 2.

Fig. 9.

A–M. Representative species of Cordyceps and its allies in Clavicipitaceae clade C. N–S. Perithecia, asci, and ascospores. A. C. militaris on lepidopteran pupa, EFCC 5192. B. C. kyusyuënsis on lepidopteran larva, EFCC 10985. C. C. chichibuënsis on coleopteran pupa, EFCC 422. D. C. cf. ochraceostromata on lepidopteran larva, EFCC 11846. E. C. scarabaeicola on scarabaeid beetle (Coleoptera), EFCC 5014. F. C. staphylinidicola on coleopteran larva, EFCC 783. G. C. bifusispora on lepidopteran pupa, EFCC 2626. H. C. cf. pruinosa on lepidopteran pupa (Limacodidae), EFCC 11756. I. C. cardinalis on lepidopteran larva, EFCC 12212. J. C. tuberculata on adult of moth (Lepidoptera), EFCC 2067. K. Torrubiella sp. on spider (Arachnida), EFCC 10882. L. Beauveria sp. on adult of beetle (Coleoptera), EFCC 1357. M. Isaria tenuipes on lepidopteran pupa, EFCC 1497. N. C. cardinalis, section of perithecia in stroma, OSC 93609. O. C. militaris, ascus with disarticulating ascospores, OSC 93623. P. C. cardinalis, ascus with nondisarticulating ascospores, OSC 93609. Q. C. cf. pruinosa, fusiform terminal parts of ascospores in ascus, EFCC 7481. R. C. militaris, multiseptated ascospores in ascus, OSC 93623. S. C. cf. pruinosa, thread-like structures connecting fusiform terminal parts of ascospores, EFCC 7481. Scale bars: A–M = 10 mm, N = 100 μm, O–S = 5 μm.

Fig. 10.

New classification of Cordyceps and clavicipitaceous fungi based on Bayesian consensus tree in Fig. 2. Portions of Bionectriaceae and Nectriaceae are not shown. Tree description is the same as in Fig. 2. For internodes that are related with nomenclatural changes, bootstrap proportions of MP analyses (MP-BP) in Fig. 1 are shown above corresponding nodes before the backslash. Bootstrap proportions of ML analyses (ML-BP) and posterior probabilities (PP) in Fig. 2 are shown above internodes after backslash and below internodes, respectively. For the corresponding internode of Ophiocordyceps, bootstrap proportions (MP-BP & ML-BP) and posterior probabilities (PP) were obtained from analyses based on the 147-taxon 5-gene data set in Fig. 3. Portions of the tree in grey rectangular boxes indicate nomenclatural changes of Cordyceps.

Fig. 11.

A–B. Line drawings of morphology of Metacordyceps yongmunensis. C–G. Line drawings of pochonia-like anamorph of M. yongmunensis. A. Non-disarticulating ascospore and ascus. B. Oblique arrangement of perithecia in stroma. C. Conidia and phialides. D. Developing conidia germinated from ascospore. E. Chlamydospores submerged in SDAY agar. F. Developing chlamydospores submerged in SDAY agar. G. Intercalary swollen hyphae. Scale bars: A, C–G = 10 μm, B = 200 μm.

Fig. 12.

A–G. Morphology of Ophiocordyceps communis. A. Stromata, bar = 10 mm. B. Arrangement of perithecia. C. Ascus with ascospores. D. Ascus and ascus apex. E. Non-disarticulating ascospores. F. Conidiophores (Hymenostilbe/Hirsutella anamorph). G. Denticles of phialide (Hymenostilbe/Hirsutella anamorph). Scale bars: A, C–G = 10 μm, B = 00 μm.