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Stud Mycol 57(1): 5-59 2007
DOI: 10.3114/sim.2007.57.01
Copyright © 2007 CBS Fungal Biodiversity Centre
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Phylogenetic classification of Cordyceps and the clavicipitaceous fungi

Gi-Ho Sung1, Nigel L. Hywel-Jones2, Jae-Mo Sung3, J. Jennifer Luangsa-ard4, Bhushan Shrestha3 and Joseph W. Spatafora1

1 Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331–2902, U.S.A.
2 Mycology Laboratory, National Center for Genetic Engineering and Biotechnology, Science Park, Pathum Thani, Thailand
3 Department of Applied Biology and Entomopathogenic Fungal Culture Collection (EFCC), Kangwon National University, Chuncheon 200-701, Republic of Korea
4 Phylogenetics Laboratory, National Center for Genetic Engineering and Biotechnology, Science Park, Pathum Thani, Thailand.

*Correspondence: Gi-Ho Sung, sungg{at}science.oregonstate.edu


   Abstract
TOP
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 TAXONOMIC REVISION
 KEY TO THE GENERA...
 References
 
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 1{alpha} (tef1), the largest and the second largest subunits of RNA polymerase II (rpb1 and rpb2), β-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 Clavicipitaceae s. 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 Clavicipitaceae s. 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.

Taxonomic novelties: New family: Ophiocordycipitaceae G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora. New genera: Elaphocordyceps G.H. Sung & Spatafora, Metacordyceps G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora. New species: Metacordyceps yongmunensis G.H. Sung, J.M. Sung & Spatafora; Ophiocordyceps communis Hywel-Jones & Samson. New combinations: Cordyceps confragosa (Mains) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, C. ninchukispora (C.H. Su & H.-H. Wang) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora; Elaphocordyceps capitata (Holmsk.) G.H. Sung, J.M. Sung & Spatafora, E. delicatistipitata (Kobayasi) G.H. Sung, J.M. Sung & Spatafora, E. fracta (Mains) G.H. Sung, J.M. Sung & Spatafora, E. inegoënsis (Kobayasi) G.H. Sung, J.M. Sung & Spatafora, E. intermedia (S. Imai) G.H. Sung, J.M. Sung & Spatafora, E. japonica (Lloyd) G.H. Sung, J.M. Sung & Spatafora, E. jezoënsis (S. Imai) G.H. Sung, J.M. Sung & Spatafora, E. longisegmentis (Ginns) G.H. Sung, J.M. Sung & Spatafora, E. minazukiensis (Kobayasi & Shimizu) G.H. Sung, J.M. Sung & Spatafora, E. miomoteana (Kobayasi & Shimizu) G.H. Sung, J.M. Sung & Spatafora, E. ophioglossoides (Ehrh.) G.H. Sung, J.M. Sung & Spatafora, E. paradoxa (Kobayasi) G.H. Sung, J.M. Sung & Spatafora, E. ramosa (Teng) G.H. Sung, J.M. Sung & Spatafora, E. rouxii (Cand.) G.H. Sung, J.M. Sung & Spatafora, E. subsessilis (Petch) G.H. Sung, J.M. Sung & Spatafora, E. szemaoënsis (M. Zang) G.H. Sung, J.M. Sung & Spatafora, E. tenuispora (Mains) G.H. Sung, J.M. Sung & Spatafora, E. toriharamontana (Kobayasi) G.H. Sung, J.M. Sung & Spatafora, E. valliformis (Mains) G.H. Sung, J.M. Sung & Spatafora, E. valvatistipitata (Kobayasi) G.H. Sung, J.M. Sung & Spatafora, E. virens (Kobayasi) G.H. Sung, J.M. Sung & Spatafora; infraspecific: E. intermedia f. michinokuënsis (Kobayasi & Shimizu) G.H. Sung, J.M. Sung & Spatafora, E. ophioglossoides f.alba (Kobayasi & Shimizu ex Y.J. Yao) G.H. Sung, J.M. Sung & Spatafora, E. ophioglossoides f. cuboides (Kobayasi) G.H. Sung, J.M. Sung & Spatafora; Metacordyceps brittlebankisoides (Z.Y. Liu, Z.Q. Liang, Whalley, Y.J. Yao & A.Y. Liu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, M. campsosterni (W.M. Zhang & T. H. Li) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, M. chlamydosporia (H.C. Evans) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, M. liangshanensis (M. Zang, D. Liu & R. Hu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, M. taii (Z.Q. Liang & A.Y. Liu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora; Ophiocordyceps agriotidis (A. Kawam.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. ainictos (A. Möller) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. amazonica (Henn.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. aphodii (Mathieson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. appendiculata (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. arachneicola (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. arbuscula (Teng) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. armeniaca (Berk. & M.A. Curtis) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. asyuënsis (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. aurantia (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. australis (Speg.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. barnesii (Thwaites) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. bicephala (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. bispora (Stifler) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. brunneipunctata (Hywel-Jones) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. cantharelloides (Samson & H.C. Evans) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. carabidicola (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. cicadicola (Teng) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. clavata (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. coccidiicola (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. coccigena (Tul. & C. Tul.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. cochlidiicola (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. corallomyces (A. Möller) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. crassispora (M. Zang, D. R. Yang & C.D. Li) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. crinalis (Ellis ex Lloyd) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. cucumispora (H.C. Evans & Samson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. curculionum (Tul. & C. Tul.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. cusu (Pat.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. cylindrostromata (Z.Q. Liang, A.Y. Liu & M.H. Liu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. dayiensis (Z.Q. Liang) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. dermapterigena (Z.Q. Liang, A.Y. Liu & M.H. Liu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. dipterigena (Berk. & Broome) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. discoideicapitata (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. ditmarii (Quél.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. dovei (Rodway) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. elateridicola (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. elongata (Petch) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. elongatiperitheciata (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. elongatistromata (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. emeiensis (A.Y. Liu & Z.Q. Liang) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. engleriana (Henn.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. entomorrhiza (Dicks.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. evdogeorgiae (Koval) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. falcata (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. falcatoides (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. fasciculatistromata (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. ferruginosa (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. filiformis (Moureau) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. formicarum (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. forquignonii (Quél.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. furcicaudata (Z.Q. Liang, A.Y. Liu & M.H. Liu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. gansuënsis (K. Zhang, C. Wang & M. Yan) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. geniculata (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. gentilis (Ces.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. glaziovii (Henn.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. goniophora (Speg.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. gracilioides (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. gracilis (Grev.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. heteropoda (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. hiugensis (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. huberiana (Henn.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. humbertii (C.P. Robin) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. insignis (Cooke & Ravenel) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. irangiensis (Moureau) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. japonensis (Hara) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. jiangxiensis (Z.Q. Liang, A.Y. Liu & Y.C. Jiang) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. jinggangshanensis (Z.Q. Liang, A.Y. Liu & Y.C. Jiang) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. kangdingensis (M. Zang & Kinjo) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. kniphofioides (H.C. Evans & Samson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. koningsbergeri (Penz. & Sacc.) G. H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. konnoana (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. lachnopoda (Penz. & Sacc.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. larvarum (Westwood) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. lloydii (H.S. Fawc.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. longissima (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. lutea (Moureau) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. melolonthae (Tul. & C. Tul.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. michhganensis (Mains) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. minutissima (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. monticola (Mains) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. mrciensis (J.C. Jung, Z.Q.Liang, Soytong & K.D. Hyde) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. multiaxialis (M. Zang & Kinjo) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. myrmecophila (Ces.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. neovolkiana (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. nepalensis (M. Zang & Kinjo) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. nigra (Samson, H.C. Evans & Hoekstra) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. nigrella (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. nigripes (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. nutans (Pat.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. obtusa (Penz. & Sacc.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. octospora (M. Blackwell & Gilb) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. odonatae (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. osuzumontana (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. ouwensii (Höhn.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. owariensis (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. oxycephala (Penz. & Sacc.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. pentatomae (Koval) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. petchii (Mains) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. proliferans (Henn.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. pseudolloydii (H.C. Evans & Samson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. pseudolongissima (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. purpureostromata (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. ravenelii (Berk. & M.A. Curtis) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. robertsii (Hook.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. rubiginosiperitheciata (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. rubripunctata (Moureau) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. ryogamiensis (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. salebrosa (Mains) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. scottiana (Olliff) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. selkirkii (Olliff) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. sichuanensis (Z.Q. Liang & B. Wang) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. smithii (Mains) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. sobolifera (Hill ex Watson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. sphecocephala (Klotzsch ex Berk.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. stipillata (Z.Q. Liang & A.Y. Liu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. stylophora (Berk. & Broome) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. subflavida (Mains) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. superficialis (Peck) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. takaoënsis (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. taylorii (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. thyrsoides (A. Möller) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. tricentri (Yasuda) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. uchiyamae (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. variabilis (Petch) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. voeltzkowii (Henn.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. volkiana (A. Möller) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. wuyishanensis (Z.Q. Liang, A.Y. Liu & J.Z. Huang) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. yakusimensis (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. zhangjiajiensis (Z.Q. Liang & A.Y. Liu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora; infraspecific: O. amazonica var. neoamazonica (Kobayasi & Hara) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. cucumispora var. dolichoderi (H.C. Evans & Samson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. kniphofioides var. dolichoderi (H.C. Evans & Samson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. kniphofioides var. monacidis (H.C. Evans & Samson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. kniphofioides var. ponerinarum (H.C. Evans & Samson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. lloydii var. binata (H.C. Evans & Samson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. melolonthae var. rickii (Lloyd) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. owariensis f. viridescens (Uchiyama & Udagawa) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. purpureostromata f. recurvata (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, O. superficialis f. crustacea (Kobayasi & Shimizu) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora; Pochonia parasitica (G.L. Barron) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora.

Keywords Clavicipitaceae / Cordyceps / Cordycipitaceae / Elaphocordyceps / Metacordyceps / multigene phylogeny / Ophiocordyceps / Ophiocordycipitaceae


   INTRODUCTION
TOP
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 TAXONOMIC REVISION
 KEY TO THE GENERA...
 References
 
Cordyceps Fr. is the most diverse genus in the family Clavicipitaceae in terms of number of species and host range (Kobayasi 1941, 1982, Mains 1957, 1958). There are estimated to be more than 400 species (Mains 1958, Kobayasi 1982, Stensrud et al. 2005) although this is expected to be an underestimation of the extant global diversity (Hawksworth & Rossman 1997). Its host range is broad, ranging from ten orders of arthropods to the truffle-like genus Elaphomyces, although most species are restricted to a single host species or a set of closely related host species (Kobayasi 1941, 1982, Mains 1957, 1958). The distribution is cosmopolitan, including all terrestrial regions except Antarctica, with the height of known species diversity occurring in subtropical and tropical regions, especially East and Southeast Asia (Kobayasi 1941, 1982, Samson et al. 1988). The genus is generally included in the family Clavicipitaceae, based on its cylindrical asci, thickened ascus apices, and filiform ascospores that often disarticulate into part-spores (Mains 1958, Kobayasi 1982, Rossman et al. 1999, Hywel-Jones 2002). Cordyceps is characterized and distinguished from other genera of the family by its production of superficial to completely immersed perithecia on stipitate and often clavate to capitate stromata and its ecology as a pathogen of arthropods and the fungal genus Elaphomyces (Kobayasi 1941, Mains 1957, 1958, Kobayasi & Shimizu 1960, Rogerson 1970).

Modern infrageneric classifications of Cordyceps have been based primarily on the taxonomic studies of Kobayasi (1941, 1982) and Mains (1958) (but see Massee 1895). Kobayasi (1941, 1982) recognized three subgenera (C. subg. Cordyceps, C. subg. Ophiocordyceps, and C. subg. Neocordyceps), emphasizing arrangement of perithecia and morphology of asci, ascospores and part-spores. Species of C. subg. Cordyceps (type C. militaris) were characterized by the production of either immersed or superficial perithecia produced at approximately right angles (ordinal) to the surface of the stroma and ascospores that disarticulate into part-spores at maturity. Cordyceps subg. Ophiocordyceps (Petch) Kobayasi (type C. blattae Petch) was distinguished by the production of whole ascospores that do not disarticulate into part-spores and, in some species, asci lacking pronounced apical hemispheric caps. Cordyceps subg. Neocordyceps Kobayasi (type C. sphecocephala (Klotzsch ex Berk.) Berk. & M.A. Curtis) was characterized by perithecia immersed at oblique angles in the clava region of the stroma and ascospores that disarticulate into part-spores upon maturity.

Mains (1958) expanded the infrageneric classification with a different emphasis on diagnostic characters and recognized two additional subgenera, C. subg. Racemella (Ces.) Sacc. and C. subg. Cryptocordyceps Mains. Cordyceps subg. Racemella (type C. memorabilis (Ces.) Sacc.) included species that produce superficial perithecia and asci with hemispheric to short cylindrical caps. Cordyceps subg. Cryptocordyceps (type C. ravenelii Berk. & M.A. Curtis) was diagnosed by the production of brown, partly immersed to superficial perithecia in a palisade-like layer at more or less right angles to the surface of the stroma. Kobayasi and Mains also differed in their treatments of C. subg. Ophiocordyceps and C. subg. Neocordyceps. In contrast to Kobayasi (1941), who essentially adopted the diagnosis of Petch (1931) but at the rank of subgenus, Mains (1958) placed only C. blattae and C. peltata Wakef. in C. subg. Ophiocordyceps based on their lack of a thickened ascus apex, thus deemphasizing the importance of ascospore disarticulation at the subgeneric level. Furthermore, Mains (1958) did not recognize C. subg. Neocordyceps, rather he included species with oblique perithecia in C. subg. Cordyceps sect. Cremastocarpon subsect. Entomogenae. Currently, the subgenera C. subg. Cordyceps, C. subg. Ophiocordyceps, and C. subg. Neocordyceps sensu Kobayasi (1941) have been arguably the most widely used infrageneric taxa of Cordyceps (Zang & Kinjo 1998, Artjariyasripong et al. 2001, Hywel-Jones 2002, Sung & Spatafora 2004, Stensrud et al. 2005) with the relatively recent addition of C. subg. Bolacordyceps O.E. Erikss., which is characterized by the production of bola-ascospores (Eriksson 198). Although this ascospore form has been likened to the South American bola or the East Asian ninchuk (martial arts weapon), the overall form is best likened to that of a skipping rope. The two handles of the skipping rope are two terminal sets of four cells. The `rope' is a slender hyphal thread, which appears to lack cytoplasm or, at most, has relic quantities.

In addition to the morphological characters discussed above, host affiliation has played an important role in the classification of Cordyceps (Massee 1895, Kobayasi 1982). Cordyceps species that parasitize the truffle genus Elaphomyces have been recognized as a unique taxon. The genus Cordylia Fr. (1818) was once assigned for the mycogenous Cordyceps species (Massee 1895) although it is a homonym of Cordylia Pers. 1807. Kobayasi (1941, 1982) also recognized the mycogenous Cordyceps species as taxonomic units (e.g., C. subg. Cordyceps sect. Cystocarpon subsect. Eucystocarpon ser. Mycogenae) and emphasized the utility of host affiliations in delimiting closely related species of arthropod pathogens. Mains (1958) adopted Kobayasi's treatment of the parasites of Elaphomyces, but questioned whether morphologically similar species on different insect hosts (e.g., C. irangiensis Moureau and C. sphecocephala attacking ants and wasps, respectively) are conspecific. The applicability of hosts as a taxonomic character is complicated, however, due to the difficulty in identifying immature hosts (e.g., larvae and pupae) and insufficient host identification for many herbarium collections.

Several phylogenetic studies employing ribosomal DNA (Artjariyasripong et al. 2001, Sung et al. 2001, Stensrud et al. 200) have been conducted to test and refine the classification of Cordyceps. Such studies were restricted by both limited taxon sampling and the inadequate resolution power of ribosomal DNA, resulting in limited conclusions regarding systematics of the genus. Recent phylogenetic studies (Spatafora et al. 2007, Sung et al. 2007) based on multiple independent loci provided a greater level of resolution and support, and revealed that neither Cordyceps nor the family Clavicipitaceae is monophyletic. Three monophyletic groups of the clavicipitaceous fungi were recognized, all of which include species of Cordyceps. These results reject the current infrafamilial classification (Diehl 1950) and indicate that the phylogenetic diversity of Cordyceps is representative of the entire family Clavicipitaceae (Spatafora et al. 2007, Sung et al. 2007). Therefore, a new classification of Cordyceps and the Clavicipitaceae is necessary to reflect the current hypotheses of phylogenetic relationships and to be predictive in nature.

Here, we conducted the most extensive multi-gene phylogenetic analyses to provide a basis for the phylogenetic classification of Cordyceps and the clavicipitaceous fungi. The main objectives of this study are to 1) reassess the morphological traits used in the current classifications of Cordyceps in the context of phylogeny, 2) investigate the taxonomic utility of the anamorphic forms in classification of Cordyceps and better understand the teleomorph–anamorph connections, and 3) revise the classification of Cordyceps and Clavicipitaceae to be consistent with phylogenetic relationships.


   MATERIALS AND METHODS
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 TAXONOMIC REVISION
 KEY TO THE GENERA...
 References
 
Taxon and character sampling
A total of 162 taxa were sampled from Clavicipitaceae and other families of Hypocreales with Glomerella cingulata (Stoneman) Spauld. & H. Schrenk (Glomerellaceae) and Verticillium dahliae Kleb. (Plectosphaerellaceae) included as outgroups (Table 1). DNA extractions from cultures or herbarium specimens were conducted using a FastDNA kit (Qbiogene) following the manufacturer's instruction, with minor modifications. Polymerase chain and sequencing reactions were performed as previously described (Sung et al. 2007). DNA sequence data unique to this study were determined from five genes, including the nuclear ribosomal small and large subunits (nrSSU and nrLSU), the elongation factor 1{alpha} (tef1), and the largest and second largest subunits of RNA polymerase II (rpb1 and rpb2). These sequences were combined with data from 91 taxa, which were obtained from Sung et al. (2007). Information pertaining to voucher numbers concerning the sequences is provided in Table 1.


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  		Table 1. Taxa used in molecular phylogenetic analyses. (AUTAuthentic material, Tex-type culture).

 

Sequence alignment and phylogenetic analyses
Sequences were edited using SeqEd 1.0.3 (Applied Biosystems Inc.) and contigs were assembled using CodonCode Aligner 1.4 (CodonCode Inc.). Sequences of each gene partition were initially aligned with Clustal W 1.64 (Thompson et al. 1994) and appended to an existing alignment (Sung et al. 2007). This initial alignment was manually edited as necessary in MacClade 4.0 (Maddison & Maddison 2000). All five gene regions sampled in this study were concatenated into a single, combined data set (162-taxon -gene data set) with ambiguously aligned regions excluded from phylogenetic analyses. Sequences from two additional gene regions, β-tubulin (tub) and mitochondrial ATP6 (atp6), from Sung et al. (2007) were also combined with the 162-taxon 7-gene data set to generate a supermatrix of 162-taxon 7-gene data set.

In order to detect incongruence among the five individual gene regions sampled in this study, bootstrap proportions were used for each individual data set with the 107 taxa that was complete for all five genes (Table 1). Bootstrap proportions (BP) were determined in a maximum-parsimony framework using the program PAUP* 4.0b10 (Swofford 2002). Only parsimony-informative characters were used with the following search options: 100 replicates of random sequence addition, TBR branch swapping, and MulTrees OFF. The incongruence was assumed to be significant if two different relationships for the same set of taxa were both supported with greater than 70 % bootstrap proportions by different genes (Mason-Gamer & Kellogg 1996, Wiens 1998). Previous studies revealed that tub was double copy in some clavicipitaceous species (Spatafora et al. 2007), and Sung et al. (2007) also showed that while atp6 possessed conflicting data for a limited number of taxa, the conflict was localized and the locus simultaneously provided increased level of support for other nodes. Thus, although we focused our sampling and analyses of the five aforementioned loci, we also conducted phylogenetic supermatrix analyses with tub and atp6 (162-taxon 7-gene) to detect any increased nodal support provided by those two loci.

Maximum parsimony (MP) analyses were conducted on the 162-taxon 5-gene and the 162-taxon 7-gene data set (Table 1, Fig. 3). All characters were equally weighted and unordered. MP analyses were performed using only parsimony-informative characters with the following settings: 100 replicates of random sequence addition, TBR branch swapping, and MulTrees ON. Phylogenetic confidence was assessed by nonparametric bootstrapping (Felsenstein 198). A total of 200 bootstrap replicates were used to calculate bootstrap proportions; bootstrapping used the same search options with five replicates of random sequence addition per bootstrap replicate.


Figure 3
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  		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.

 
Maximum likelihood (ML) analyses were performed with RAxML-VI-HPC v2.2. using a GTRCAT model of evolution with 25 rate categories (Stamatakis et al. 2005). The model was separately applied to each of the eleven partitions, which consisted of nrSSU, nrLSU and the nine codon positions of three protein-coding genes (tef1, rpb1, and rpb2). Nodal support was assessed with nonparametric bootstrapping using 200 replicates. Bayesian Metropolis coupled Markov chain Monte Carlo (B-MCMCMC) analyses were performed on combined datasets using MrBayes 3.0b4 (Huelsenbeck & Ronquist 2001). In estimating the likelihood of each tree, we used the general time-reversible model, with invariant sites and gamma distribution (GTR+I+{Gamma}) and employed the model separately for each partition. In an initial analysis, a B-MCMCMC analysis with five million generations and four chains was conducted in order to test the convergence of log-likelihood. Trees were sampled every 100 generations, for a total of 50,000 trees. For a second analysis, five independent Bayesian runs with two million generations and random starting trees were conducted to reconfirm log-likelihood convergence and mixing of chains.

In addition to the analyses with 162-taxon 5-gene data set, a series of analyses were conducted in MP, ML, and Bayesian frameworks with different taxon samplings (107- and 152-taxon 5-gene data sets) to address the potential topological effects of missing data. Previous phylogenetic and simulation studies demonstrated that the phylogenetic analyses are often not negatively affected if less than 50 % characters are missing for each taxon in the phylogenetic analyses (Wiens 2003, Phylippe et al. 2004). In this study, we assumed that the phylogenetic analysis is not confounded if the taxa were complete for at least three out of five gene partitions. Therefore, ten taxa (Table 1) in the 162-taxon 5-gene data set that were complete for only two gene partitions were excluded to generate the 162-taxon 5-gene data set. A 107-taxon 5-gene data set that does not contain any missing data in gene partitions was also prepared to compare the phylogenetic relationships between 107-taxon and 152-taxon 5-gene analyses. MP, ML, and Bayesian analyses based on the 162-taxon 5-gene data set (Figs 1, 2) showed that the C. sphecocephala clade is characterized by long-branch lengths relative to the rest of the clavicipitaceous fungi. To address the impact of the C. sphecocephala clade on the phylogenetic resolution, we excluded all members of the C. sphecocephala clade from the 152-taxon 5-gene data set and constructed a 147-taxon 5-gene data set.


Figure 1
Figure 1
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  		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 (*).

 

Figure 2
Figure 2
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  		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.

 

   RESULTS
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 TAXONOMIC REVISION
 KEY TO THE GENERA...
 References
 
Sequence alignment
The combined 162-taxon 5-gene dataset consisted of 4927 base pairs of sequence data (nrSSU 1102 bp, nrLSU 954 bp, tef1 1020 bp, rpb1 803 bp, rpb2 1048 bp). As a result of excluding ambiguously aligned regions, the final alignment comprised 4600 base pairs (nrSSU 1088 bp, nrLSU 767 bp, tef1 1020 bp, rpb1 677 bp, rpb2 1048 bp), 1882 of which were parsimony-informative (nrSSU 233 bp, nrLSU 220 bp, tef1 466 bp, rpb1 382 bp, rpb2 581 bp). A total of 107 taxa were complete for all five genes and the number of complete taxa for each gene was as follows: nrSSU 158 taxa, nrLSU 157 taxa, tef1 149 taxa, rpb1 143 taxa, rpb2 122 taxa (Table 1).

Phylogenetic analyses
The reciprocal comparisons of 70 % bootstrap trees from individual data sets of the 162-taxon 5-gene dataset did not reveal any significantly supported contradictory nodes (data not shown). These results were interpreted as indicating that no strong incongruence existed among the individual data sets that would be indicative of different phylogenetic gene histories (e.g., lineage sorting or horizontal gene transfer). As a result, all five individual data sets were combined in simultaneous analyses.

MP analyses of the 162-taxon 5-gene data set resulted in 156 equally parsimonious trees. These trees were 21,323 steps with a consistency index (CI) of 0.1598 and a retention index (RI) of 0.6131. One of 156 equally parsimonious trees is shown in Fig. 1. Nodes that collapse in the strict consensus tree are denoted with asterisks. ML analyses of the 162-taxon 5-gene data set resulted in a tree with a log-likelihood (–ln) of 92019.95. In the Bayesian analyses, the five-million generation analysis converged on the log-likelihood (harmonic mean = –ln 9951.22) at approximately around 250,000 generations. The results from five of two-million generation analyses also showed a convergence on the log-likelihood at approximately 2 0,000 generations and the topologies were identical. As a result, the 3,000 trees from the first 300,000 generations were deleted from the five million generation analysis to generate a 50 % majority-rule consensus tree.

A 50 % majority consensus tree (Fig. 2) was generated from the million generation analysis. Since the topology of ML analyses (tree not shown) was nearly identical to that of the Bayesian consensus tree of Fig. 2, the bootstrap proportions of ML analyses are provided above the corresponding nodes in Fig. 2. Previous studies have shown that in interpreting the supports of the phylogenetic estimates of relationships, the posterior probability tends to overestimate the phylogenetic confidence (Doaudy et al. 2003, Lutzoni et al. 2004, Reeb et al. 2004). As a result, the posterior probabilities were used as a supplementary indicator to bootstrap proportions. In this study, nodes were considered strongly supported when supported by both bootstrap proportions (BP ≥ 70 %) and posterior probabilities (PP ≥ 0.95) (Lutzoni et al. 2004).

Phylogenetic relationships of the clavicipitaceous fungi
All MP, ML, and Bayesian analyses of the five-gene 162-taxon 5-gene data set recognized three well-supported clades of clavicipitaceous fungi (Figs 1, 2), designated here as Clavicipitaceae clades A, B, and C (Figs 1, 2), following the convention of the previous phylogenetic studies (Spatafora et al. 2007, Sung et al. 2007). These clades were statistically well supported by the bootstrap proportions of the MP (MP-BP) and ML (ML-BP) analyses and posterior probabilities (PP) of the Bayesian analyses (clade A: MP-BP = 98 %, ML-BP = 99 %, PP = 1.00; clade B: MP-BP = 93 %, ML-BP = 98 %, PP = 1.00; clade C: MP-BP = 100 %, ML-BP = 100 %, PP = 1.00). A sister-group relationship between clades A and B was also strongly supported (MP-BP = 72 %, ML-BP = 90 %, PP = 1.00). The monophyletic group of clade C and Hypocreaceae was moderately to strongly supported (MP-BP = 63 %, ML-BP = 92 %, PP = 1.00).

Clavicipitaceae clade A comprised five statistically well-supported subclades (Figs 1, 2, 4). These were labelled in Figs 1, 2, and 4 as the C. taii clade (MP-BP = 73 %, ML-BP = 78 %, PP = 1.00), the Claviceps clade (MP-BP = 95 %, ML-BP = 98 %, PP = 1.00), the Hypocrella clade (MP-BP = 99 %, ML-BP = 99 %, PP = 1.00), the Shimizuomyces clade (MP-BP = 100 %, ML-BP = 100 %, PP = 1.00), and the Torrubiella luteorostrata clade (MP-BP = 100 %, ML-BP = 100 %, PP = 1.00). As indicated previously by Sung et al. (2007), internal relationships among these five subclades were not strongly supported in MP and ML analyses (Figs 1, 2, 4).


Figure 4
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  		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.

 
Clavicipitaceae clade B consisted of five major subclades designated as the C. gunnii, C. ophioglossoides, C. sphecocephala, C. unilateralis, and Pa. lilacinus clades (Figs 1, 2, 6). Nearly all of the subclades in clade B were strongly supported by bootstrap proportions and posterior probabilities (C. gunnii clade: MP-BP = 97 %, ML-BP = 100 %, PP = 1.00; C. ophioglossoides clade: MP-BP = 71 %, ML-BP = 88 %, PP = 1.00; C. sphecocephala clade: MP-BP = 100 %, ML-BP = 100 %, PP = 1.00, Pa. lilacinus clade: MP-BP = 64 %, ML-BP = 76 %, PP = 1.00). It should be noted, however, that the C. unilateralis subclade was not resolved in the MP analyses (Fig. 1). This lack of resolution was due to the instability of the C. sphecocephala clade, which is characterized by long-branch lengths relative to the rest of the clavicipitaceous fungi. Multiple placements of the C. sphecocephala subclade, ranging from a basal lineage of the Clavicipitaceae clade B to a terminal clade nested within the C. unilateralis subclade, were present among the most parsimonious trees (data not shown). Our ML and Bayesian results (Fig. 3) indicate that the C. sphecocephala subclade is either a sister-group of the C. unilateralis subclade (107-taxon 5-gene data set) or in the terminal group of the C. unilateralis subclade (152-taxon 5-gene data set). In MP, ML, and Bayesian analyses with a supermatrix of 162-taxon 7-gene data set (Fig. 3), the C. sphecocephala subclade was placed as a terminal group of the C. unilateralis subclade with strong support (MP-BP = 89 %, ML-BP = 94 %, PP = 1.00) as seen in the previous analyses (Sung et al. 2007). In the light of long-branch attraction problems associated with the MP analyses (Fig. 1), we use the Bayesian tree (Fig. 2) to further discuss the relationships in clade B and we conclude that the C. sphecocephala subclade was best included as a member of the C. unilateralis subclade (Figs 2, 6). In interpreting the C. unilateralis subclade in terms of statistical support, we used the bootstrap proportions and posterior probabilities (MP-BP = 88 %, ML-BP = 88 %, PP = 1.00) based on the results of 147-taxon 5-gene data set (Fig. 3).


Figure 6
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  		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.

 

Clavicipitaceae clade C comprised two well-supported subclades (Figs 1, 2, 8). The Simplicillium subclade (MP-BP = 100 %, ML-BP = 100 %, PP = 1.00) consisted of isolates of the anamorph genus Simplicillium, most of which were isolated as parasites of other fungi. The Cordyceps subclade (MP-BP = 98 %, ML-BP = 100 %, PP = 1.00) included numerous species of Torrubiella and species of Cordyceps that produce pallid to brightly coloured stromata with ascospore morphologies ranging from whole ascospores to part-spores to bola-ascospores according to species. Importantly, the Clavicipitaceae clade C included C. militaris and represents the core Cordyceps clade. The remaining species, Torrubiella wallacei H.C. Evans, was also a member of the Cordyceps clade with strong support (ML-BP = 91 %, PP = 1.00) in ML and Bayesian analyses (Figs 2, 8), but could not be confidently assigned to either subclade in MP analyses (Fig. 1).


Figure 8
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  		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.

 

   DISCUSSION
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
TAXONOMIC REVISION
 KEY TO THE GENERA...
 References
 
Phylogenetic implications on the systematics of the genus Cordyceps
The present and previous phylogenetic analyses (Spatafora et al. 2007, Sung et al. 2007) have revealed that species in the Clavicipitaceae form three strongly supported monophyletic groups based on combined data sets of six or seven genes (the genes analyzed herein with and without atp6). Although more taxa were used in our study, these results were consistent with the previous studies, recognizing three monophyletic groups designated as Clavicipitaceae clades A–C (Figs 1, 2). In addition, our results also support the paraphyly of the Clavicipitaceae as defined by the monophyly of Clavicipitaceae clade C and Hypocreaceae (Figs 1, 2). Although the paraphyly of the Clavicipitaceae (clade C + Hypocreaceae) was moderately supported (MP-BP = 63 %) in the 162-taxon 5-gene MP analyses (Fig. 1), it was strongly supported (ML-BP = 92 %, PP = 1.00) in the ML and Bayesian analyses (Fig. 2) and more robustly addressed in the previous MP analyses, which investigated localized conflicts among gene partitions and compared bootstrap proportions among alternative sampling strategies (Sung et al. 2007).

The phylogenetic hypothesis presented here contradicts current infrafamilial classification of the Clavicipitaceae. Diehl (1950) proposed three subfamilies, Oomycetoideae, Clavicipitoideae, and Cordycipitoideae, based on the development of stromata, anamorphic characters and host affiliations. However, these three subfamilies do not coincide with the three clades of the Clavicipitaceae inferred in the present analyses (Figs 1, 2). Clavicipitaceae clade A includes members of all three subfamilies (e.g., Claviceps of Clavicipitoideae, Cordyceps of Cordycipitoideae, and Hypocrella of Oomycetoideae), whereas the remaining clades only comprise members of Cordycipitoideae (e.g., Cordyceps and Torrubiella). Importantly, all three major clades include members of Cordyceps, indicating that Cordyceps, like Clavicipitaceae, is not monophyletic (Figs 1, 2). As a result, the three recognized well-supported clades (clades A–C) of the clavicipitaceous fungi represent a robust phylogenetic framework for the taxonomic revision of Cordyceps and the Clavicipitaceae.

In the current infrageneric classification of the genus, Cordyceps comprises four subgenera (C. subg. Bolacordyceps, C. subg. Cordyceps, C. subg. Neocordyceps, and C. subg. Ophiocordyceps) based on ascospore morphology and arrangement of the perithecia in the stromata (Kobayasi 1941, 1982, Eriksson 1986). However, most of these characters are not consistent with the new phylogenetic hypothesis and are not diagnostic of monophyletic taxa (e.g., subgenera and genera) (Figs 1, 2). For example, Kobayasi (1941, 1982) emphasized ascospore morphology and the lack of ascospore disarticulation into part-spores to delimit C. subg. Ophiocordyceps from the other subgenera. Species with non-disarticulating ascospores, however, are included in all three major clades (C. acicularis Ravenel of clade B, C. cardinalis G.H. Sung & Spatafora of clade C, and Cordyceps sp. EFCC 2131 and 2135 of clade A described below as Metacordyceps yongmunensis) (Figs 1, 2), indicating that non-disarticulating ascospores are not phylogenetically informative at this level (Figs 1, 2). Therefore, a reassessment of diagnostic characters, in the previous and current classifications of Cordyceps, is necessary for the three major clades to provide a basis for taxonomic revisions of Cordyceps and the Clavicipitaceae.

Species in Clavicipitaceae clade A
Clavicipitaceae clade A comprises five well-supported subclades (Fig. 4). All known species of Cordyceps in the clade are included in the C. taii clade. Species of Cordyceps in the clade possess partially or completely immersed perithecia on clavate to cylindrical fertile parts of the stromata (Zang et al. 1982, Liang et al. 1991, Zare et al. 2001). They produce ascospores that either disarticulate or remain intact at maturity and include species that possess ordinal and obliquely embedded perithecia. In the current classification, clade A includes species of Cordyceps that were formerly classified in three subgenera of Cordyceps. Cordyceps liangshanensis M. Zang, D. Liu & R. Hu forms ordinal perithecia and possess disarticulating ascospores, consistent with C. subg. Cordyceps (Kobayasi 1982, Zang et al. 1982). Cordyceps chlamydosporia H.C. Evans possesses nondisarticulating ascospores, consistent with C. subg. Ophiocordyceps (Zare et al. 2001). Cordyceps taii Z.Q. Liang & A.Y. Liu, a known teleomorph species linked to the anamorph genus Metarhizium Sorokin, produces disarticulating ascospores and obliquely embedded perithecia in the stromata, a trait used to recognize C. subg. Neocordyceps (Liang et al. 1991). Importantly, Cordyceps sp. EFCC 2131 and 2135 (described below as Metacordyceps yongmunensis) produce non-disarticulating ascospores and obliquely embedded perithecia in the stromata, characters inconsistent with any of the subgenera in the current classification.


Figure 5
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  		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.

 
These results suggest that ascospore morphology and arrangement of perithecia are not phylogenetically informative in recognizing either the C. taii clade, or higher clades of clavicipitaceous fungi. Rather, they are more useful at species level classification. For example, our phylogenetic analyses revealed that C. taii is closely related to C. brittlebankisoides Z. Y. Liu, Z.Q. Liang, Whalley, Y.J. Yao & A.Y. Liu, the purported teleomorph of M. flavoviride (Huang et al. 2005). Although these species are similar to each other in macromorphology (e.g., greenish clavate stromata), they differ in the arrangement of the perithecia. C. brittlebankisoides possesses perithecia that are ordinally placed in the stromata, whereas C. taii has obliquely embedded perithecia. These results therefore suggest that arrangement of the perithecia in the stromata is useful in delimiting these closely related species in the C. taii clade (Fig. 4).

Species in Clavicipitaceae clade B
Species of Cordyceps in Clavicipitaceae clade B possess disarticulating or non-disarticulating ascospores and produce superficial to completely immersed perithecia that are ordinally or obliquely inserted in the stromata. As with the Cordyceps species of clade A, this clade also includes members of the former C. subg. Cordyceps (e.g., C. ophioglossoides (Ehrh.) Link and C. variabilis Petch), C. subg. Ophiocordyceps (e.g., C. acicularis and C. unilateralis (Tul. & C. Tul.) Sacc.), and C. subg. Neocordyceps (e.g., C. nutans Pat. and C. sphecocephala). The majority of Cordyceps species in this clade produce wiry to pliant or fibrous stromata that typically are completely or partially darkly pigmented and parasitize subterranean or wood-inhabiting hosts, which are buried in soil or embedded in decaying wood. Exceptions to this morphology and ecology do exist, however; for example, C. melolonthae (Tul. & C. Tul.) Sacc. is pigmented bright yellow but stains darkly upon handling, and members of the C. sphecocephala clade parasitize adult insects.

Clade B consists of five subclades. All subclades include either species of Cordyceps or anamorphs with potential links to Cordyceps (e.g., Nomuraea atypicola (Yasuda) Samson linked to C. cylindrica Petch) (Fig. 6, Evans & Samson 1987). The well-resolved tree in the present study (Fig. 6) provides the basis to characterize three of the five subclades of clade B. Due to insufficient taxon sampling, it is not possible to characterize the members of the Cordyceps species in the C. gunnii and Pa. lilacinus subclades. In the light of this, we focus on the remaining three subclades that include sufficient numbers of Cordyceps species.

The C. ophioglossoides subclade primarily consists of Cordyceps species that parasitize species of the genus Elaphomyces (e.g., C. ophioglossoides and C. capitata (Holmsk.) Link) and the nymphs of cicadas (e.g., C. inegoënsis Kobayasi and C. paradoxa Kobayasi) buried in soil (Kobayasi 1939, Mains 1957, Kobayasi & Shimizu 1960, 1963). Species in this subclade produce partially or completely immersed perithecia, in clavate to capitate fertile parts of stromata that are darkly pigmented with olivaceous tints (Kobayasi & Shimizu 1960, 1963). Because they produce disarticulating ascospores and ordinal perithecia, all known species of this clade are classified in C. subg. Cordyceps.

Cordyceps subsessilis Petch is unique to the C. ophioglossoides subclade (Fig. 6). It produces perithecia on white or pallid reduced stromata, arising from a rhizomorph-like structure from scarabaeid beetle larvae (Hodge et al. 1996). It is the only member of the subclade that parasitizes beetles embedded in decaying wood (Hodge et al. 1996). Therefore, C. subsessilis differs greatly in ecology and morphology of its stromata from most other taxa in the C. ophioglossoides clade, but it possesses several characters shared by its close relative, C. ophioglossoides (Kobayasi & Shimizu 1960, Hodge et al. 1996). Both species grow axenically on simple media, produce verticilliate anamorphs (C. subsessilis has a Tolypocladium anamorph, whereas C. ophioglossoides has verticillium-like conidiophores), possess nearly identical part-spore morphologies, and produce stromata that are connected to their hosts via rhizomorph-like structures. In contrast, C. capitata/C. longisegmentis have not successfully been grown in culture, they are attached directly to the host, and an anamorph is unknown.

The C. ophioglossoides subclade (Fig. 6) also includes parasites of subterranean cicada nymphs (e.g., C. inegoënsis and C. paradoxa), which are grouped with their close relatives (e.g., C. jezoënsis S. Imai and C. ophioglossoides) that parasitize subterranean truffles of Elaphomyces. Despite low support of inter-species relationships within the C. ophioglossoides subclade due to short branch lengths, C. paradoxa and C. inegoënsis are morphologically more similar to C. jezoënsis and C. ophioglossoides than to any other members of the clade. These taxa produce clavate fertile parts of the stromata rather than capitate stromata like other members of the clade (e.g., C. capitata and C. fracta Mains). Many of these species (e.g., C. jezoënsis and C. paradoxa) are also known to connect to their hosts via rhizomorph-like structures (Kobayasi & Shimizu 1960, 1963), supporting a close phylogenetic relationship.

The C. unilateralis subclade includes the most morphologically diverse assemblages of Cordyceps species (Fig. 6). Most of the species in the clade parasitize larval, pupal or nymph stages of arthropods (Kobayasi 1941, Mains 1958). Species of this clade produce superficial to completely immersed perithecia on the stromata with morphologies ranging from capitate to clavate to filiform (Kobayasi 1941, Mains 1958). They typically possess tough, pliant, or fibrous stromata that are entirely or partially darkly pigmented, although some exceptions (e.g., C. melolonthae and C. variabilis) do exist, which produce brightly pigmented stromata (Mains 1958). Many species in the clade (e.g., C. brunneipunctata Hywel-Jones, C. stylophora Berk. & Broome, and C. unilateralis) are also differentiated by aperithecial stromatal apices while the production of perithecia occurs in subterminal regions of the stroma.

Similar to Cordyceps species in clade A, the C. unilateralis subclade includes species that produce disarticulating or non-disarticulating (intact) ascospores. For example, some species in the C. unilateralis subclade (e.g., C. sinensis (Berk.) Sacc. and C. unilateralis) were formerly classified in C. subg. Ophiocordyceps. But these species are interspersed among other species (e.g., C. agriotidis A. Kawam. and C. robertsii (Hook.) Berk.) that are classified in C. subg. Cordyceps. This indicates that, while ascospore morphology is useful in delimiting closely related Cordyceps species and uniting others in species complexes, it is not diagnostic of the C. unilateralis subclade itself (Fig. 6).

Most members of C. subg. Neocordyceps, as classically treated by Kobayasi (1941, 1982) and others (e.g., Artjariyasripong et al. 2001, Stensrud et al. 2005), form a monophyletic group labelled as the C. sphecocephala subclade within the C. unilateralis group (Fig. 6). The majority of species in the C. sphecocephala subclade produce long, thin, pliant, brightly coloured (or dark marasmioid in a few species) stromata, which terminate in clavate to elongated fertile parts, and possess ascospores that disarticulate into sixty-four part-spores (Kobayasi 1941, 1982, Hywel-Jones 2002). Species in this clade produce perithecia, which are partially or completely immersed in the stromata at strongly oblique angles (Kobayasi 1941, 1982, Mains 1958, Hywel-Jones 1996). This clade is one of the best characterized by its morphology (obliquely embedded perithecia in a well-defined clava) and its ecology of parasitizing adult stages of insects.


Figure 7
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  		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.

 
Species in Clavicipitaceae clade C
Clavicipitaceae clade C includes C. militaris, the type species of the genus Cordyceps (Fig. 8). Most Cordyceps species in this clade are currently classified in C. subg. Cordyceps (Kobayasi 1941, 1982). This clade also contains the members of the former C. subg. Ophiocordyceps and C. subg. Bolacordyceps, resulting in C. subg. Cordyceps being paraphyletic within clade C (Eriksson 1982, Hywel-Jones 1994, Sung & Spatafora 2004). Species of Cordyceps in this clade produce three ascospore types, including disarticulating ascospores (e.g., C. militaris), intact ascospores (e.g., C. cardinalis and C. pseudomilitaris Hywel-Jones & Sivichai), and bola-ascospores (e.g., C. bifusispora O.E. Erikss.). Of particular note, this clade includes Phytocordyceps ninchukispora C.H. Su & H.-H. Wang in the unispecific genus Phytocordyceps C.H. Su & H.-H. Wang. The genus Phytocordyceps was originally described based on bola-ascospores and its host affiliation as a pathogen of Beilschmiedia erythrophloia Hayata (Lauraceae) plant seeds (Su & Wang 198). Morphologically, this species is most similar to C. bifusispora in that it produces bola-ascospores typical of C. subg. Bolacordyceps. However, the phylogenetic analyses in this study reveal that species producing bola-ascospores (e.g., C. bifusispora and P. ninchukispora) do not form a monophyletic group (Fig. 8). Rather, they are interspersed among other Cordyceps species possessing disarticulating ascospores, most notably C. militaris.

Species of Cordyceps in clade C produce superficial to partially immersed perithecia on fleshy stromata that are pallid to brightly pigmented. This is in contrast to Cordyceps species in clade B, which produce darkly pigmented, wiry to pliant or fibrous stromata. This suggests that pigmentation and texture of stromata may be phylogenetically informative at a higher level of classification. It should be noted, however, that some Cordyceps species in clade C are morphologically similar to distantly related Cordyceps species (e.g., C. melolonthae and C. variabilis) in stromatal pigmentation. Although these characters are useful in recognizing Cordyceps species of clade C, the utility of these characters for any future infrageneric classification is probably limited (Fig. 8). For example, C. militaris is macroscopically similar to C. cardinalis and C. pseudomilitaris. All three species produce orangish to red-coloured and fleshy stromata; however, these species differ in ascospore and anamorph morphology (Sung & Spatafora 2004). Furthermore, C. militaris is known as exhibiting considerable variability in stroma morphology (Sung & Spatafora 2004). Potentially conspecific species, such as C. roseostromata Kobayasi & Shimizu and C. kyusyuënsis A. Kawam., differ in stroma morphology, but are closely related to C. militaris and possess identical ascospore and ascus morphologies (Fig. 8, Hywel-Jones 1994, Sung & Spatafora 2004).

The variation in ascospore morphology of Clavicipitaceae clade C combined with old descriptions and unavailable type material complicates species identification for many taxa, as is the case for much of Cordyceps. For example, this study reveals a close relationship between the anamorphic species, Mariannaea pruinosa Z.Q. Liang from China, C. cf. pruinosa from Korea and Thailand, and Phytocordyceps ninchukispora from Taiwan (Fig. 8). The teleomorph of M. pruinosa is C. pruinosa Petch, which was originally described as producing disarticulating ascospores and reddish orange stromata, parasitizing lepidopteran cocoons (Petch 1924, Kobayasi 1941, Liang 1991). Although the isolate of M. pruinosa was obtained from ascospores (Liang 1991), the morphology of the ascospores was not well characterized. The species was identified primarily based on its host affiliation and macroscopic characters. In our study, C. cf. pruinosa EFCC 5197 and N.H.J. 10627 were collected from the same host family (Lepidoptera, Limacodidae) in Korea and Thailand. They are also closely related and produce reddish orange stromata (Fig. 8) and bola-ascospores and not the typical C. subg. Cordyceps part-spores. It should be noted, however, that Petch did not provide any drawings or images of ascospores and it is possible that the terminal cells of bola-ascospores could easily be interpreted as part-spores. Thus, at this time we use the name C. pruinosa for the Chinese, Korean and Thai collections and, if further attempts fail to locate type material for C. pruinosa, one of these may have to be designated a neotype. The C. pruinosa collections are closely related to and morphologically indistinguishable from P. ninchukispora with the exception of host affiliation, suggesting the possibility of host misidentification in the original description of P. ninchukispora. Because the tree topology of the C. pruinosa/P. ninchukispora complex is indicative of greater phylogenetic species diversity, i.e., the Korean, Thai, and Taiwanese material may represent unique phylogenetic species (Fig. 8), we retain the use of both names until more detailed sampling and analyses have been conducted.

Clavicipitaceae clade C not only includes members of Cordyceps but also species of the genus Torrubiella, which generally parasitize spiders and scale insects (Kobayasi & Shimizu 1982). The genus Torrubiella is morphologically characterized by the production of superficial perithecia on a mycelial subiculum that partially or completely surrounds the host (Kobayasi & Shimizu 1982, Humber & Rombach 1987). Species of Torrubiella also produce disarticulating (e.g., T. ratticaudata Humber & Rombach) and intact (e.g., T. wallacei) ascospores. Among species of Cordyceps, C. tuberculata (Lebert) Maire, a pathogen of adult Lepidoptera, has been considered an intermediate species between Torrubiella and Cordyceps (Humber & Rombach 1987, Kobayasi 1941, Mains 1958). Phylogenetic analyses in this study indicate that the members of Torrubiella do not form a monophyletic group within clade C and are interspersed among species of Cordyceps. This suggests that the stipitate stromata of Cordyceps have been gained or lost several times during the evolution of these fungi. Currently, more than 50 species of Torrubiella have been described and the members of genus Torrubiella are clearly undersampled in this study (Kobayasi & Shimizu 1982).

In summary, the characters of ascospore morphology and the arrangement of perithecia used in the current classification of the genus Cordyceps are not congruent with the three higher clades inferred in these analyses. These characters are likely to prove useful, however, in lower level classifications, such as the delimitation of closely related species and species complexes. The characters most congruent with the three higher clades of clavicipitaceous fungi are texture, pigmentation and morphology of the stromata, but with exceptions. Although we have divided Cordyceps species into three major clades, it is difficult to characterize Cordyceps species within the Clavicipitaceae clade A due to the relatively few teleomorph species that are part of this clade (see key on p. 54). They tend to produce green to white stromata, often with lilac tints, but additional sampling is needed to more definitively characterize the teleomorphs of these species. However, the majority of species within clades B and C are morphologically and/or ecologically distinct (Figs 1, 2).

The majority of Cordyceps species in clade B are characterized by darkly pigmented, wiry, pliant or fibrous stromata. The dominant form of parasitism exhibited by these species is on subterranean or wood-inhabiting hosts, buried in soil or embedded in decaying wood, such as larval and pupal stages of arthropods. In contrast, Cordyceps species of clade C have brightly pigmented and fleshy stromata and parasitize their hosts in relatively more accessible environments, such as leaf litter, moss, or the uppermost soil layer. Exceptions to these morphological and ecological traits are found in some Cordyceps species in clade B. Cordyceps melolonthae, for example, produces brightly-coloured stromata, although it bruises dark upon handling and its hosts are the larvae of cockchafers or June beetles buried in soil (Mains 1958). Cordyceps unilateralis parasitizes adult ants, but is darkly pigmented with a wiry stroma and subterminal production of perithecia, and members of the C. sphecocephala clade are at least partially brightly pigmented and are restricted to adult stages of insects. These findings suggest that the traits described above are not universally informative, but collectively useful in characterizing Cordyceps species within clade B. That is, there have been gains, losses, and diversifications of most if not all traits during the evolution of these fungi, but general trends in character state evolution are evident.

The taxonomic utility of anamorphic forms in classification of Cordyceps
The genus Cordyceps is characterized by a diverse assemblage of more than 25 anamorph genera (e.g., Beauveria Vuill., Hirsutella Pat., Hymenostilbe Petch, Isaria Fr., Lecanicillium W. Gams & Zare, Metarhizium, and Tolypocladium W. Gams) (Kobayasi 1982, Samson et al. 1988, Gams & Zare 2003, Hodge 2003). The anamorph genera of Cordyceps are hyphomycetes with conidiogenous cells that are hyaline to brightly coloured and produce conidia in dry chains or slimy drops (Samson et al. 1988). Some anamorph genera (e.g., Hymenostilbe) are known as a useful diagnostic character in recognizing monophyletic groups of Cordyceps species (Artjariyasripong et al. 2001, Kobayasi 1941, 1982), while other anamorph morphologies and genera are placed in more than one clade of the Clavicipitaceae. Therefore, the distribution of anamorphic forms is discussed to evaluate their phylogenetic utility in characterizing the three clades of Cordyceps and Clavicipitaceae and to better understand teleomorph–anamorph connections.

Anamorphs of Clavicipitaceae clade A
Clavicipitaceae clade A includes isolates of the anamorph genera Aschersonia Mont., Metarhizium, Nomuraea Maublanc, Pochonia Bat. & O.M. Fonseca, Paecilomyces s. l., Rotiferophthora