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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 G.L. Barron, Tolypocladium W. Gams, and verticillium-like (Fig. 4). Nomuraea, Paecilomyces, and Tolypocladium are found in other clades of Clavicipitaceae (Figs 1, 2). Significantly, Verticillium s. s. is known from the Plectosphaerellaceae, which is closely related with the Glomerellaceae in the Sordariomycetidae (Zare et al. 2007). Paecilomyces s. s. is in the Eurotiales (Eurotiomycetidae), but species of Paecilomyces s. l. are also present elsewhere in the Hypocreales (Luangsa-ard et al. 2004). In contrast, the anamorph genera Aschersonia, Metarhizium, Pochonia and Rotiferophthora are restricted to clade A (Figs 1, 2).

Anamorph taxa of the C. taii subclade include Nomuraea rileyi (Farl.) Samson, Paecilomyces carneus (Duché & R. Heim) A.H.S. Brown & G. Smith and Pa. marquandii (Massee) S. Hughes, Pochonia, Tolypocladium parasiticum, and Metarhizium (Fig. 4). The genera Nomuraea, Pochonia and Tolypocladium are not monophyletic, although Pochonia is restricted to clade A. Nomuraea rileyi and Metarhizium are entomogenous; Pa. carneus is a common soil fungus considered a weak insect pathogen, while Pa. marquandii, Pochonia and T. parasiticum are also common soil fungi and can be parasitic on nematodes. Metarhizium is the only monophyletic anamorph genus of clade A (Fig. 4). The conidiogenous cells in the genus Metarhizium are cylindrical to clavate without a neck and produced in candelabrum-like or palisade-like fashion (Rombach et al. 1986, Driver et al. 2000, Evans 2003). The genus is most similar to Nomuraea and differs in the compact conidiophores that form a hymenial layer (Evans 2003). Nomuraea rileyi groups with species of Metarhizium, while N. atypicola (Yasuda) Samson belongs to the Pa. lilacinus clade in clade B. Interestingly, N. rileyi produces greenish-coloured conidia, as do species of Metarhizium in the C. taii subclade, while N. atypicola possesses lavender-coloured conidia similar to those of Pa. lilacinus (Coyle et al. 1990, Hywel-Jones & Sivichai 1995, Evans 2003). Currently, three teleomorphic species of Metarhizium (C. brittlebankisoides, C. campsosterni, and C. taii) have been reported (Liang et al. 1991, Liu et al. 2001, Zhang et al. 2004). The species M. taii was described with its teleomorph species, C. taii (Liang et al. 1991) and recently synonymized with M. anisopliae var. majus (Huang et al. 2005). Cordyceps brittlebankisoides was once also considered to have the anamorph M. anisopliae var. 0majus (Liu et al. 2001), but it is likened to M. flavoviride (Huang et al. 2005). In general, Metarhizium species show extensive variation in size and colour of conidia (Driver et al. 2000, Evans 2003) and more intensive sampling of anamorphs and teleomorphs is needed for this group.

The genus Tolypocladium is characterized by producing single or whorled (verticillate) conidiogenous cells (phialides), which are flask-shaped with enlarged bases that taper into a needle-like neck usually bent from the axis of the phialides (Gams 1971, Bissett 1983). The type of the genus Tolypocladium, T. inflatum W. Gams, is linked to the teleomorph C. subsessilis (Hodge et al. 1996, Gams & Zare 2003). Tolypocladium inflatum is placed in clade B and is distantly related to T. parasiticum in the C. taii clade. Tolypocladium parasiticum was described from the rotifer host Adineta and described with underwater conidiation (Barron 1980). Morphologically, T. parasiticum differs from other species of Tolypocladium, as it is the only member of the genus that produces chlamydospores in vivo (Barron 1980) and in culture (Bissett 1983, Zare et al. 2001, Gams & Zare 2003). In a recent treatment of Verticillium sect. Prostrata W. Gams, the genus Pochonia was also reclassified based on production of dictyochlamydospores or at least swollen hyphal cells (Gams & Zare 2001, Zare et al. 2001), supporting the close phylogenetic relationship of T. parasiticum and Pochonia species demonstrated in this study (Fig. 4). Hence, T. parasiticum is transferred to Pochonia below, rendering the remaining species in Tolypocladium monophyletic. Paecilomyces marquandii also produces infrequent chlamydospores in culture, as does the anamorph of Metacordyceps yongmunensis sp. nov. (discussed below). As suggested by Barron & Onions (1966), the presence of chlamydospores can be a taxonomically informative character.

The genus Aschersonia is a monophyletic lineage labelled as Hypocrella subclade (Fig. 4). The genus Aschersonia is characterized by its pycnidial or acervular conidiomata with hymenial phialides and its ecology of parasitizing only the nymphs of scale insects and whiteflies (Petch 1921, Hywel-Jones & Evans 1993). The teleomorphs of Aschersonia have long been linked to the species of Hypocrella and more than 25 species have been reported (Petch 1921, Mains 1959). While this study does not focus on sampling of Hypocrella and Aschersonia, these findings corroborate that the unique morphology of Aschersonia is phylogenetically informative and diagnostic of a monophyletic group of clavicipitaceous fungi (Fig. 4).

Anamorphs of Clavicipitaceae clade B
Clavicipitaceae clade B includes several anamorph genera including Haptocillium W. Gams & Zare, Hirsutella, Hymenostilbe and Tolypocladium (Fig. 6). Several of the anamorphic forms in the clade are phylogenetically informative. Hirsutella and Hymenostilbe occur dominantly in the C. unilateralis subclade.

Hirsutella is characterized by its typical basally-subulate phialides, narrowing into one (usually) or more (occasionally) very slender needle-like necks, on synnemata or mononematous mycelium (Hodge 1998, Gams & Zare 2003). Hirsutella species normally produce a few (<5) conidia in mucus and the phialides are not usually bent in their needle-like necks such as in the genus Tolypocladium, but also single conidia as in Hi. thompsonii F.E. Fisher. Not all Cordyceps species in the C. unilateralis subclade are connected to Hirsutella anamorphs. Some are connected to Paecilomyces s. l., Paraisaria Samson & B.L. Brady, and Syngliocladium Petch, whereas anamorphic forms are not known for many of the Cordyceps species, especially in the C. ravenelii subclade (e.g., C. heteropoda Kobayasi). However, most Cordyceps species in the rest of the C. unilateralis subclade have been linked to Hirsutella anamorphs (Fig. 6). These results suggest that Hirsutella anamorphs are phylogenetically informative for at least part of the C. unilateralis subclade or possibly symplesiomorphic for the C. unilateralis subclade as a whole.

The taxonomic utility of Hirsutella anamorphs is exemplified by the teleomorph–anamorph connection of the genus Cordycepioideus Stifler, a termite pathogen, which does not have typical ascospore and ascus morphologies of clavicipitaceous fungi (Blackwell & Gilbertson 1984, Suh et al. 1998). It possesses thick-walled multiseptate ellipsoid ascospores and its asci lack the thickened ascus tip characteristic of most clavicipitaceous fungi (Blackwell & Gilbertson 1984, Ochiel et al. 1997). The anamorph of Cordycepioideus bisporus Stifler is a synnematous Hirsutella that is either conspecific with or closely related to Hi. thompsonii (Ochiel et al. 1997, Suh et al. 1998, Sung et al. 2001). Although Cordycepioideus bisporus differs greatly from other members of the C. unilateralis subclade in its teleomorphic characters, molecular data strongly support it as a member of the C. unilateralis subclade, a finding consistent with its Hirsutella anamorph. It should be noted that species of Cordyceps outside of clade B have been described with atypical Hirsutella anamorphs (e.g., C. pseudomilitaris), but upon further investigation were more accurately characterized in other anamorph genera (e.g., Simplicillium W. Gams & Zare).

The C. unilateralis clade includes the members of the C. sphecocephala subclade, which possess a Hymenostilbe anamorph. The genus Hymenostilbe usually produces cylindrical to clavate conidiogenous cells, which are produced in a more or less dense palisade in synnemata (Samson et al. 1988). It is differentiated from closely related genera (e.g., Akanthomyces Lebert and Hirsutella) by its polyblastic conidiogenous cells, which holoblastically produce single conidia on short denticles or scars (Samson et al. 1988, Hywel-Jones 1996). The results from the present study indicate that Hymenostilbe anamorphs may be derived from within Hirsutella (Fig. 6). The close phylogenetic relationship between Hirsutella and Hymenostilbe anamorphs is exemplified by the morphologically intermediate synnematous Hirsutella/Hymenostilbe species. For example, Hy. lecaniicola (Jaap) Mains, the anamorph of C. clavulata (Schwein.) Ellis & Everh. (Hodge 1998), was previously classified in Hirsutella although it possesses extensively polyphialidic conidiogenous cells in a discontinuous layer (Mains 1950, 1958, Samson & Evans 1975, Hodge 1998). In addition, some Hirsutella species (e.g., Hi. rubripunctata Samson, H.C. Evans & Hoekstra) produce only a single conidium without a mucous sheath on denticles of extensively polyphialidic conidiogenous cells. Therefore, the modes of asexual reproduction in Hirsutella and Hymenostilbe may overlap to some extent and additional work is necessary to address the relationships between the two genera (Hodge 1998, Gams & Zare 2003).

In addition to the C. unilateralis subclade, the remaining three subclades contain Haptocillium, Tolypocladium and verticillium-like anamorphs. The genus Haptocillium was reclassified from the former Verticillium sect. Prostrata primarily based on its adhesive conidia and its ability to parasitize free-living nematodes (Zare & Gams 2001b). This study shows that the genus is a monophyletic group in the C. gunnii subclade (Fig. 6). However, the teleomorph–anamorph connection has not been established for any of the species in the clade or its close relative, C. gunnii, and thus its taxonomic utility remains unclear. The C. ophioglossoides and Pa. lilacinus subclades include anamorphic forms of Paecilomyces s. l., Nomu-raea, Tolypocladium, and verticillium-like, all of which are polyphyletic as previously discussed (Figs 1, 2; Oborník et al. 2001, Luangsa-ard et al. 2004, 2005). Several teleomorph–anamorph connections have been reported for Cordyceps species in the C. ophioglossoides and Pa. lilacinus subclades although their taxonomic utility is limited. Cordyceps subsessilis is known to be the teleomorph of Tolypocladium inflatum (Hodge et al. 1996) and C. ophioglossoides produces a verticillium-like anamorph (Gams 1971). In the Pa. lilacinus subclade, N. atypicola is linked to C. cylindrica (Evans & Samson 1987, Hywel-Jones & Sivichai 1995).

Anamorphs of Clavicipitaceae clade C
The anamorph genera sampled that are members of clade C include Beauveria, Isaria, Lecanicillium, Microhilum H.Y. Yip & A.C. Rath, and Simplicillium. Species of Lecanicillium and Simplicillium were previously placed in Verticillium sect. Prostrata and recently reclassified based on the phylogenetic studies of Sung et al. (2001) and Zare & Gams (2001a, b). The genus Lecanicillium is characterized by producing slender aculeate phialides that are produced singly or in whorls and usually arise from prostrate aerial hyphae (Zare & Gams 2001a). Conidia are mostly produced at the tip of phialides and attached in heads or fascicles (Zare & Gams 2001a). The morphological delimitation of Simplicillium from Lecanicillium is difficult although the species of Simplicillium tend to produce phialides that more or less arise singly from prostrate aerial hyphae (Zare & Gams 2001a). This study shows again that the species of Lecanicillium form a paraphyletic group, as species of other well-delimited anamorphic genera (e.g., Beauveria, Engyodontium G.S. de Hoog, and Isaria) are interspersed among species of Lecanicillium (Fig. 8).

Some Lecanicillium species are known to be anamorphic forms of Cordyceps and Torrubiella (Petch 1932, Evans & Samson 1982, Zare & Gams 2001a). For example, C. militaris produces a Lecanicillium anamorph in culture (Zare & Gams 2001a) and the anamorph of Torrubiella alba Petch is L. aranearum (Petch) Zare & W. Gams (Petch 1932). The type species of Lecanicillium is L. lecanii (Zimm.) Zare & W. Gams, which is connected to the teleomorph T. confragosa Mains, a pathogen of scale insects (Evans & Samson 1982), which we transfer here to Cordyceps. In addition to Lecanicillium anamorphs, other genera (e.g., Akanthomyces, Gibellula Cavara, Hirsutella, Paecilomyces (Isaria), and Simplicillium) have also been linked to Torrubiella (Kobayasi & Shimizu 1982, Samson et al. 1988, 1989, Zare & Gams 2001a).

Clavicipitaceae clade C also includes the species of Isaria, the generic name of which has been conserved with I. farinosa (Holmsk.) Fr. as the type, for some of the clavicipitaceous Paecilomyces species (Gams et al. 2005, Luangsa-ard et al. 2005). The genus Paecilomyces was a diverse genus, with molecular studies indicating its polyphyletic status (Oborník et al. 2001, Luangsa-ard et al. 2004, 2005). The type species, Pa. variotii Bainier, belongs to the order Eurotiales (Ascomycota) and is distantly related to the clavicipitaceous Paecilomyces species that were previously classified in Paecilomyces sect. Isarioidea (Samson 1974, Luangsa-ard et al. 2004). The previous taxonomy of Paecilomyces was primarily based on the monographic study by Samson (1974), which included approximately 22 species in Paecilomyces sect. Isarioidea. In a recent molecular study, Luangsa-ard et al. (2005) demonstrated that species in Paecilomyces sect. Isarioidea are subdivided into four monophyletic groups, three of which are statistically supported. As a result, eleven species of Paecilomyces sect. Isarioidea were reclassified in Isaria (e.g., I. fumosorosea Wize, I. javanica (Frieder. & Bally) Samson & Hywel-Jones and I. tenuipes Peck) (Luangsa-ard et al. 2005). The present study indicates that the four isolates of Isaria do not form a monophyletic group in clade C, as they are interspersed among other anamorphic forms in the clade. Thus, the taxonomic utility of Isaria anamorph is limited to clade C, as seen with Lecanicillium and Simplicillium anamorphs. Furthermore, few connections have been made between teleomorphs of the Clavicipitaceae and species of Isaria. Kobayasi (1941) reported that the anamorph of C. takaomontana Yakush. & Kumaz. is Isaria japonica Yasuda, which Samson (1974) synonymized with Pa. tenuipes (= I. tenuipes). Isaria farinosa is the anamorph of C. memorabilis (Pacioni & Frizzi 1978), but was once mistakenly linked to C. militaris (Petch 1936). Isaria farinosa was also connected to two Torrubiella species, T. gonylepticida (A. Möller) Petch and T. pulvinata Mains. The anamorph of the latter was reported as Spicaria pulvinata Mains, and Petch described the conidial state of T. gonylepticida as Spicaria longipes Petch, two Spicaria species that Samson (1974) synonymized with Paecilomyces farinosus (= I. farinosa). Although T. gonylepticida was originally described in combination with Cordyceps, Petch (1937) transferred the species to its current combination and redescribed the species. Isaria farinosa has been reported to occur on six insect orders (Lepidoptera, Coleoptera, Hemiptera, Homoptera, Diptera, and Hymenoptera) and also on spiders (Araneae). The simplicity and plasticity in the morphology of most Isaria species make it difficult to set boundaries among and between sister-taxa and the search for better markers in species delimitation must be a goal for further studies.

The closely related species, C. scarabaeicola Kobayasi and C. staphylinidicola Kobayasi & Shimizu produce Beauveria anamorphs (Fig. 8; Sung 1996), and C. bassiana Z.Z. Li, C.R. Li, B. Huang & M.Z. Fan and C. brongniartii Shimazu are known as teleomorphs of B. bassiana (Bals.) Vuill. and B. brongniartii (Sacc.) Petch, respectively (Shimazu et al. 1988, Li et al. 2001). The genus Beauveria is morphologically well-characterized by producing basally-inflated conidiogenous cells that sympodially produce conidia on divergent denticles (MacLeod 1954, de Hoog 1972). Beauveria has a cosmopolitan distribution with quite a broad host range (Mugnai et al. 1989, Evans 2003, Rehner & Buckley 2005). A recent molecular study (Rehner & Buckley 2005) that included 87 isolates of five Beauveria species (B. amorpha (Höhn.) Samson & H.C. Evans, B. bassiana, B. brongniartii, B. caledonica Bissett & Widden, and B. vermiconia de Hoog & V. Rao) demonstrated that the genus is monophyletic and one of the more phylogenetically-informative anamorphs of clade C.

In fungal systematics, the naming of anamorphic forms is allowed for Phyla Ascomycota and Basidiomycota by Article 59 of the International Code of Botanical Nomenclature (McNeill et al. 2006) and multiple names exist for the same organisms of teleomorphic and anamorphic taxa. Recently, molecular phylogenetics has played an important role in integrating teleomorphic and anamorphic forms in a unified classification system in the clavicipitaceous fungi (Reynolds & Taylor 1993, Sung et al. 2001, Luangsa-ard et al. 2005). In such efforts, Verticillium sect. Prostrata and Paecilomyces sect. Isarioidea have recently been reclassified into several anamorphic genera (e.g., Haptocillium, Isaria, Lecanicillium, Pochonia, Rotiferophthora, and Simplicillium) to be consistent with the current hypotheses of relationships (Zare & Gams 2001a, Zare et al. 2001, Luangsa-ard et al. 2005). The phylogeny presented here further improves our understanding of the teleomorph–anamorph connections in Cordyceps and implies that several anamorphic genera (e.g., Beauveria, Hirsutella, Hymenostilbe, and Metarhizium) are more restricted in their phylogenetic distribution and therefore phylogenetically informative in characterizing Cordyceps species (Figs 4, 6, 8).


   TAXONOMIC REVISION
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 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 TAXONOMIC REVISION
KEY TO THE GENERA...
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The present phylogenetic analyses reveal three strongly supported monophyletic groups (i.e., Clavicipitaceae clades A, B, and C) of clavicipitaceous fungi (Figs 1, 2), a result consistent with studies involving fewer taxa (Spatafora et al. 2007, Sung et al. 2007). In reviewing the diagnostic characters used in previous classification schemes, most characters are not consistent with the phylogeny presented here. Therefore, the phylogenetic relationships of Cordyceps and the related clavicipitaceous fungi provide the evidence for rejecting most of the previous classifications of Cordyceps and Clavicipitaceae (Kobayasi 1941, 1982, Diehl 1950, Mains 1958). Here, we propose a new phylogenetic classification for Cordyceps and Clavicipitaceae as follows (Fig. 10).


Figure 10
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  		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.

 
Clavicipitaceae Clade A
Clavicipitaceae clade A is a well-supported monophyletic group that represents the Clavicipitaceae s. s. (MP-BP = 98 %, ML-BP = 99 %, PP = 1.00 in Figs 1, 2, 10). The name Clavicipitaceae was first used in 1901 by Earle for the former Hypocreaceae subfam. Clavicipiteae Lindau (Earle 1901). However, Earle (1901) used it without description and without reference to its basionym. The name was then invalidly used by subsequent workers, such as Nannfeldt (1932) and Diehl (1950), until it was validated by Rogerson (1970) as confirmed by Eriksson & Hawksworth (1985). Although Clavicipitaceae is well characterized by cylindrical asci, thickened ascus apices, and filiform ascospores that tend to disarticulate at maturity as in the original description, we restrict the application of Clavicipitaceae s. s. to the members of Clavicipitaceae clade A because of the non-monophyly of Clavicipitaceae s. l. (Fig. 10). These findings suggest that the character states of cylindrical asci and filiform ascospores that disarticulate at maturity are plesiomorphic for the Clavicipitaceae s. l. / Hypocreaceae clade. Importantly, the Hypocreaceae also possesses cylindrical asci and while its ascospores are subglobose to fusiform and easily distinguished from those of Clavicipitaceae s. l., they show a similarly high frequency of disarticulation (Rogerson 1970, Rossman et al. 1999).

The family Clavicipitaceae s. s. includes the grass-associated genera Balansia Speg., Claviceps, Epichloë (Fr.) Tul. & C. Tul., and Myriogenospora G.F. Atk., which were classified in Clavicipitaceae subfam. Clavicipitoideae sensu Diehl 1950 (Fig. 10). Recent molecular studies show that Aciculosporium I. Miyake, Atkinsonella Diehl, Heteroëpichloë E. Tanaka, C. Tanaka, Gafur & Tsuda, Neoclaviceps J.F. White, Bills, S.C. Alderman & Spatafora, and Parepichloë J.F. White & P.V. Reddy are also members of this clade, thus supporting their classification in the Clavicipitaceae s. s. (White & Reddy 1998, Sullivan et al. 2001, Tanaka et al. 2002). Clavicipitaceae s. s. also includes the plant-associated Shimizuomyces paradoxus Kobayasi, which occurs on seeds of Smilax (Smilacaceae). In addition to plant-associated fungi, Clavicipitaceae s. s. contains four arthropod-associated lineages. Three of the four arthropod-associated lineages are characterized as pathogens of scale insects, including Hypocrella (pathogens of scale insects and white flies; Hywel-Jones & Evans 1993, Hywel-Jones & Samuels 1998), Regiocrella P. Chaverri & K.T. Hodge (pathogen of scale insects; Chaverri et al. 2006), and Torrubiella luteorostrata Zimm. (pathogen of scale insects; Hywel-Jones 1993). The fourth lineage is described here as Metacordyceps; it comprises former species of Cordyceps and their related anamorphs and as a genus displays relatively broad arthropod host associations.


Figure 9
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  		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.

 
CLAVICIPITACEAE (Lindau) Earle ex Rogerson, Mycologia 62: 900. 1970, emend. G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora

Stromata or subiculum darkly or brightly coloured, fleshy or tough. Perithecia superficial to completely immersed, ordinal or oblique in arrangement. Asci cylindrical with thickened ascus apex. Ascospores usually cylindrical and multiseptate, disarticulating into part-spores or non-disarticulating.

Type: Claviceps Tul., Ann. Sci. Nat. Bot., Sér. 3, 20: 43. 1853.

Teleomorphic genera: Aciculosporium, Atkinsonella, Balansia, Claviceps, Epichloë, Heteroepichloë, Hypocrella, Metacordyceps gen. nov., Myriogenospora, Neoclaviceps, Parepichloë, Regiocrella, Shimizuomyces.

Anamorphic genera: Aschersonia, Ephelis Fr., Metarhizium, Neotyphodium A.E. Glenn, C.W. Bacon & Hanlin, Nomuraea, paecilomyces-like, Pochonia, Sphacelia Lév., verticillium-like.

METACORDYCEPS G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, gen. nov. MycoBank MB504182.

Stromata solitaria vel nonnulla aggregata, simplicia vel ramosa. Stipes carnosus vel tenax, albidus, viridi-luteus vel viridulus, cylindricus vel sursum dilatatus. Pars fertilis cylindrica vel clavata. Perithecia partim vel omnino in stromate immersa, perpendicularia vel oblique inserta. Asci cylindrici, apice inspissato. Ascosporae cylindricae, multiseptatae, in cellulas diffrangentes vel maturae integrae remanentes.

Stromata solitary or several, simple or branched. Stipe fleshy or tough, whitish, greenish yellow to greenish, cylindrical to enlarging in fertile part. Fertile part cylindrical to clavate. Perithecia partially or completely immersed in stromata, ordinal or oblique in arrangement. Asci cylindrical with thickened ascus apex. Ascospores cylindrical, multiseptate, disarticulating into part-spores or remaining intact at maturity.

Type: Cordyceps taii Z.Q. Liang & A.Y. Liu

Etymology: Greek meta = behind, a genus close to Cordyceps (and suggesting relationship to Metarhizium).

Anamorphic genera: Metarhizium, Nomuraea, paecilomyces-like, Pochonia.

Commentary: The genus Metacordyceps is proposed for species of Cordyceps s. l. in the Clavicipitaceae s. s. based on the phylogenetic placement of C. taii (Figs 1, 2, 10). The genus is applied to the C. taii clade, which is strongly supported (MP-BP = 73 %, ML-BP = 78 %, PP = 1.00 in Figs 1, 2, 10). Among the members of the clade, the best-known taxon is the anamorphic genus Metarhizium, because of its importance in biological control (Samson et al. 1988, Evans 2003). Currently, three species of Cordyceps (viz., C. brittlebankisoides, C. campsosterni, and C. taii) are known as teleomorphs of Metarhizium (Liang et al. 1991, Liu et al. 2001, Zhang et al. 2004). The genus name Metacordyceps is here used to emphasize that the clade includes the species of Cordyceps s. l. that produce Metarhizium anamorphs although other species of Cordyceps (e.g., C. chlamydosporia) in the clade are not connected to Metarhizium anamorphs.

Metacordyceps yongmunensis G.H. Sung, J.M. Sung & Spatafora, sp. nov. MycoBank MB504183. Figs 5B, 5F-K, 11A-G.


Figure 11
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  		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.

 
Anamorph: pochonia-like.

Stromata nonnulla vel raro singula, clavata, simplicia vel saepius ramosa, in chrysalidibus Lepidopterarum. Pars fertilis alba vel dilute lutea, a stipite haud distincta. Perithecia sparsa vel dense aggregata, partim immersa, brunneo-lutea, dilute brunnea vel aurantiobrunnea, oblique inserta, fusiformia vel clavata, 550–800 x 450–500 µm. Asci 8-spori, hyalini, cylindrici, 205–360 x 5–7 µm, apice conspicue inspissato. Ascosporae filiformes, hyalinae, inconspicue multiseptatae, haud fragmentatae, 180–345 x 1 µm. Anamorphe Pochoniae similis.

Stromata several or rarely solitary, clavate, simple or more usually branched, on pupa of Lepidoptera. Fertile area white to pale yellow, not differentiated from stipe. Perithecia scattered or crowded, loosely immersed, brownish yellow, pale brown to orangish brown, oblique in arrangement, fusiform to clavate, 550–800 x 450–500 µm. Asci 8-spored, hyaline, cylindrical, 205– 360 x 5–7 µm, possessing a prominent apical cap. Ascospores filiform, hyaline, multiseptate with indistinct septation, not fragmenting into part-spores, 180–345 x 1 µm. Conidiophores erect, produced in prostrate aerial hyphae. Phialides hyaline, solitary, awl-shaped, 20–28 x 2–2.2 µm. Conidia hyaline, elliptical to oblong, in slimy heads, 2–3.5 x 1.5–2.4 µm. Chlamydospores present.

Etymology: Yongmunensis in reference to the known locality of the first record of the species being Mt. Yongmun, Republic of Korea.

Known distribution: Republic of Korea.

Specimens examined: Mt. Yongmun, Gyunggi Province, Republic of Korea: 13 June 1998, EFCC 2131 (holotype); 13 June 1998, EFCC 2134; 13 June 1998, EFCC 2135; 30 June 1999, EFCC 3379; 30 June 1999, EFCC 3380; 29 Aug. 1999, EFCC 4342; 8 Aug. 1999, EFCC 4343; 8 June 2000, EFCC 49 1; 30 June 2004, EFCC 12287; 30 June 2004, EFCC 12288; 30 June 2004, EFCC 12291; 8 Aug. 2004, EFCC 12467. Mt. Chiak, Kangwon Province, Republic of Korea: 8 Aug. 2000, EFCC 5750. Bukbang-myun, Kangwon Province, Republic of Korea: 21 June 2002, EFCC 8808. Living culture in EFCC.

Commentary: Most specimens of M. yongmunensis possess several stromata (up to 10), on a large pupa of Lepidoptera deeply buried in soil (Fig. 5B). Stroma of the species is typically branched in a dichotomous way at its basal or upper regions (Fig. 5B). Perithecia are usually obliquely inserted in the stromata with a few exceptions that are ordinally arranged, i.e. at right angles to the surface of the stromata (Fig. 5B). While some perithecia are characterized by an acute narrowing of the perithecium at the ostiole, producing a narrow terminal end (Fig. 5F), others are not significantly narrowed (Fig. 11B). In the asci the ascospores are arranged parallel for their entire length and almost reach the ascus foot, suggesting that ascospores are of approximately the same length as the asci (Figs 5I, 11A). Unlike the distinct septation of ascospores as seen in C. militaris (Fig. 9O), the septa of the ascospores are indistinct and discharged ascospores do not disarticulate into part-spores (Figs 5K, 11A).

In the anamorph of M. yongmunensis, cultures derived from ascospores are moderately fast growing in SDAY (Sabouraud-dextrose-yeast extract agar) and the colonies reach 25–35 mm diam at 25 °C in 10 d. Colonies are slightly cottony without zonation and white with a green margin, remaining greenish brown at the reverse side of the cultures. Conidiophores are erect and produced in prostrate aerial hyphae. Phialides are solitary, not in whorls, broader at the base and tapering towards the end, measuring 20–28 x 2.0–2.2 µm (Fig. 11C). Conidia are in slimy heads (with usually 2 or 3 conidia) and ellipsoidal to oblong, measuring 2–3.5 x 1.5–2.4 µm (Fig. 11C). In submerged areas of the cultures, chlamydospores are developed in chains or reduced to intercalary swollen structures (Figs 11E-G). The anamorph of M. yongmunensis is best classified as pochonia-like because of its subulate phialides and production of chlamydospores, although verticillium-like whorls of phialides were not observed (Zare et al. 2001). In Metacordyceps, M. yongmunensis is most similar to M. chlamydosporia (= C. chlamydosporia) in the shape of perithecia and its anamorph. Both species produce brownish perithecia that possess long terminal ends in white or pale yellow stromata (Zare et al. 2001). The anamorph of M. chlamydosporia is identical with the type of Pochonia. Thus the production of chlamydospores can be informative for recognizing some species of Metacordyceps.

Accepted names and new combinations for Metacordyceps
The following taxa are accepted species of Metacordyceps based on their inclusion in molecular phylogenies presented herein1 (see Table 1) or morphological descriptions matching the characters described above2. The known anamorph connection is provided for the species of Metacordyceps.

New combinations for anamorphs associated with Metacordyceps
T. parasiticum is transferred to the genus Pochonia based on molecular phylogenies presented herein1.

CLAVICIPITACEAE Clade B
Clavicipitaceae clade B is strongly supported (MP-BP = 93 %, ML-BP = 98 %, PP = 1.00 in Figs 1, 2, 10) and the family Ophiocordycipitaceae is proposed for it with the type genus Ophiocordyceps Petch. Most species of the Ophiocordycipitaceae produce darkly pigmented stromata that are pliant to wiry, or fibrous to tough in texture. Ecologically, many species of the family are known as pathogens of subterranean or wood-inhabiting hosts, buried in soil or embedded in decaying wood. Notable exceptions do exist to these traits with brightly coloured species that may or may not attack adult stages of hosts and occur in exposed habitats.

OPHIOCORDYCIPITACEAE G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, fam. nov. MycoBank MB504190.

Stromata vel subiculum fusca vel raro laete colorata, tenacia, fibrosa vel flexibilia, raro carnosa, saepe ostiolis peritheciorum prominentibus, summa saepe peritheciis carentia. Perithecia superficialia vel omnino immersa, perpendicularia ad superficiem vel oblique inserta. Asci cylindrici, apice inspissato. Ascosporae cylindricae, multiseptatae, maturae in cellulas diffrangentes vel integrae remanentes.

Stromata or subiculum darkly pigmented or rarely brightly coloured, tough, fibrous to pliant, rarely fleshy, often with aperithecial apices or lateral pads. Perithecia superficial to completely immersed, ordinal or oblique in arrangement. Asci usually cylindrical with thickened ascus apex. Ascospores usually cylindrical, multiseptate, disarticulating into part-spores or nondisarticulating.

Type: Ophiocordyceps Petch, Trans. Brit. Mycol. Soc. 16: 74. 1931.

Teleomorphic genera: Elaphocordyceps, Ophiocordyceps

Anamorphic genera: Haptocillium, Harposporium Lohde, Hirsutella, Hymenostilbe, paecilomyces-like, Paraisaria, Syngliocladium, Tolypocladium, verticillium-like.

ELAPHOCORDYCEPS G.H. Sung & Spatafora, gen. nov. MycoBank MB504191.

Stromata singula vel nonnulla aggregata, simplicia vel ramosa. Stipes fibrosus vel tenax, raro carnosus, obscure brunneus vel olivaceo-viridulus, raro albidus, cylindricus vel sursum dilatatus. Stromata hospite insidentia vel rhizomorphis eo conjuncta. Pars fertilis clavata vel capitata, raro indistincta. Perithecia partim wel omnino in stromate immersa, perpendicularia ad superficiem. Asci cyindrici, apice inspissato. Ascosporae cylindricae, multiseptatae, maturae in cellulas diffrangentes. Anamorphe Verticillii similis vel absens.

Stromata solitary to several, simple or branched. Stipe fibrous to tough, rarely fleshy, dark brownish to greenish with olivaceous tint, rarely whitish, cylindrical to enlarging in the fertile part. Stroma connected directly to the host or indirectly through rhizomorph-like structures. Fertile part clavate to capitate, rarely undifferentiated. Perithecia partially or completely immersed in stromata, ordinal in arrangement. Asci cylindrical with thickened ascus apex. Ascospores cylindrical, multiseptate, disarticulating into part-spores.

Type: Cordyceps ophioglossoides (Ehrh.) Link

Etymology: Greek elaphe = deer, from the host fungus, Elaphomyces.

Commentary: The C. ophioglossoides clade is strongly supported (MP-BP = 71 %, ML-BP = 88 %, PP = 100 in Figs 1, 2, 10) and includes species of Cordyceps s. l. that parasitize the truffle-like genus Elaphomyces and cicada nymphs (e.g., C. inegoënsis and C. paradoxa) and beetles (e.g., C. subsessilis) (Figs 6, 10). Currently, 22 species are anticipated to be included in the C. ophioglossoides clade, of which more than 18 species are known as parasites of Elaphomyces (Mains 1957, Kobayasi & Shimizu 1960, 1963). The host affiliation of Elaphomyces parasites has long been recognized as a diagnostic character in Cordyceps classification (Massee 1895, Kobayasi 1941, 1982, Mains 1957, 1958). The oldest applicable genus name is Cordylia Fr. 1818 (Massee 1895). However, it cannot be applied to the C. ophioglossoides clade because it is a homonym of Cordylia Pers. 1807 (Mains 1958), which is also homonym of Cordyla Lour. 1790 (Leguminosae). Therefore, the genus Elaphocordyceps is proposed based on the phylogenetic placement of C. ophioglossoides and applied to the well-supported C. ophioglossoides clade. Although C. subsessilis is morphologically and ecologically distinct, the genus is well recognized by its dominant ecology as being pathogens of Elaphomyces and cicadas. The darkly pigmented, fibrous stromata with more or less olivaceous tint are also good diagnostic characters for recognizing the species of Elaphocordyceps.

Anamorphic genera: Tolypocladium, verticillium-like.

Accepted names and new combinations for Elaphocordyceps
The following taxa are accepted species of Elaphocordyceps based on their inclusion in molecular phylogenies presented herein1 (see Table 1) or morphological descriptions matching the characters described above2. Where known we provide anamorph connection for the species of Elaphocordyceps.

OPHIOCORDYCEPS Petch, Trans. Brit. Mycol. Soc. 16: 73. 1931 emend. G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora

= Cordycepioideus Stifler, Mycologia 33: 83. 1941.

Stromata or subiculum darkly pigmented or rarely brightly coloured, tough, fibrous, pliant to wiry, rarely fleshy, often with aperithecial apices or lateral pads. Perithecia superficial to completely immersed, ordinal or oblique in arrangement. Asci hyaline, cylindrical, usually with thickened ascus apex, rarely fusoid to ellipsoid. Ascospores usually cylindrical, multiseptate, disarticulating into part-spores or non-disarticulating.

Type: Cordyceps blattae Petch, Trans. Brit. Mycol. Soc. 16: 74. 1931.

Anamorphic genera: Hirsutella, Hymenostilbe, Paraisaria, Syngliocladium.

Commentary: The C. unilateralis clade is strongly supported (MP-BP = 88 %, ML-BP = 88 %, PP = 1.00 in Figs 3, 10) and includes the species of Ophiocordyceps (e.g., C. acicularis and C. unilateralis) (Petch 1931, 1933). The genus Ophiocordyceps was proposed by Petch (1931, 1933) for species of Cordyceps that produce non-disarticulating ascospores. The genus was not accepted by subsequent workers who reclassified the species of Ophiocordyceps as Cordyceps subg. Ophiocordyceps (Kobayasi 1941) or in multiple subgenera of Cordyceps (Mains 1958). The type of Ophiocordyceps Petch is O. blattae (= C. blattae), but it was not available for this taxonomic treatment. According to the morphological description, it fits in the present generic concept. Because O. unilateralis is a well-known species that was included in the original publication of Ophiocordyceps (Petch 1931) and because additional Ophiocordyceps species (e.g., O. acicularis) are members of this clade, we apply the name Ophiocordyceps based on the placement of O. unilateralis. The genus Ophiocordyceps includes the most morphologically diverse group of the species of Cordyceps s. l. including the members of C. subg. Neocordyceps (Figs 6, 10). For most of the species in Ophiocordyceps, the stromata are fibrous to tough or wiry to pliant in texture and darkly pigmented in at least some part of the stroma. The genus includes many species of Cordyceps s. l. that produce perithecia in subterminal regions of the stromata resulting in aperithecial apices. Of particular note, Ophiocordyceps is characterized by the dominant occurrence of Hirsutella and Hymenostilbe anamorphs (Fig. 6). Although the genus Cordycepioideus possesses thick-walled multiseptate ellipsoid ascospores and its asci lack the thickened ascus tip of most clavicipitaceous fungi (Blackwell & Gilbertson 1984, Ochiel et al. 1997), this study indicates that the genus Cordycepioideus can be merged with Ophiocordyceps according to its placement in molecular analyses and because of the Hirsutella anamorph (Fig. 10, Ochiel et al. 1997, Suh et al. 1998).

Ophiocordyceps communis Hywel-Jones & Samson, sp. nov. MycoBank MB504216. Figs 12A–G.


Figure 12
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  		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.

 
Anamorph: hirsutella/hymenostilbe-like.

Stromata ex duabus (tribus) termitis adultis oriunda, mycelio albo circumdata, filiformia; 50–100 mm sub superficie stramenti oriunda, 300–600 µm lata, albido-grisea, 70–130 mm super stramentum emergentia, 600–1000 µm lata, cuius 30–40 mm pars inferior hyphis sterilibus dematiaceis (luteo-brunneis) tomentosa; pars superior, ca 90 mm longa, fertilis, levis, griseo-brunnea vel grisea, conidia in strato griseo ferens et perithecia dense aggregata. Perithecia superficialia, subterminalia, 285–675 x 195–390 µm. Asci apice conspicue inspissato, 8-spori, filiformes, 215–250 x 15 µm. Ascosporae integrae, crassitunicatae, dilute pigmentatae, (100–)120–150(–180) x 5–6 µm. Cellulae conidiogenae hymenium hyalinum formantes, cylindricae, 10–14 x 2.7–3.3 µm, unum (raro duos) denticulos fertiles apicales ferentes. Blastoconidia hyalina, amygdaliformia, 8–9 x 2.5–3 µm. Anamorphe Hirsutellae vel Hymenostilbe similis.

Hosts two (rarely three) adult termites surrounded by loose, coarse white mycelium. Stromata filiform, 50–100 mm below ground, 300–600 µm wide, whitish-grey; 70–130 mm emerging above leaf litter, 600–1000 µm wide; lower 30–40 mm of above-ground portion usually hirsute with sterile, dematiaceous (yellow-brown) hairs becoming smooth, silver-brown to grey along terminal fertile (anamorph) part of ca 90 mm. Perithecia superficial subterminal; emerging through grey anamorph, tightly packed around the stipe, 285–675 x 195–390 µm. Asci with stout cap, 8-spored, filiform, 215–250 x 15 µm. Ascospores whole, stout, lightly pigmented (100–)120–150(–180) x 5–6 µm. Conidiogenous cells in a palisade, hyaline, cylindrical, 10–14 x 2.7–3.3 µm, solitary (rarely two), prominent, terminal denticle. Conidia hyaline, almond-shaped, 7–9 x 2.5–3 µm.

Etymology: refers to the communal nature of the stromata, i.e. the fact that 600–1000 Cordyceps stromata can be found in a small area (20 x 20 metres).

Type: Holotype: N.H.J. 10673, isotypes: N.H.J. 10674, N.H.J. 10675, N.H.J. 10676, N.H.J. 10677, all on termites; coll. R. Nasit; Khao Yai National Park, Gong Giao Nature Trail; 13 June 2000.

Commentary: Most collections of O. communis were from Khao Yai National Park with the type locality (Gong Giao Nature Trail) regularly having epizootics containing (in any one season) several hundred stromata over a 20 x 20 metre area. A few other collections were from Khao Soi Dao Wildlife Sanctuary (N.H.J. 6422 and N.H.J. 6452) and Sam Lan National Park (N.H.J. 6332). All collections of the species were from adult termites. Although surveys were made over an eighteen-year period from the far north of Thailand to the far south and from sea level to over 2500 metres, O. communis is only known from these three sites in central Thailand below 800 metres elevation.

In any year there appeared to be a single `flush' with O. communis first appearing at the start of the rainy season in May/June. The earliest collections were made in May (10 May 1994: N.H.J. 3687, N.H.J. 3681 and N.H.J. 3683, Heo Sawat Waterfall; 23 May 1996; N.H.J. 6330, Gong Giao Nature Trail). In the first 2–3 weeks after appearance, the stromata appeared slender and acicular with the lower part having a shiny silken appearance and the terminal part dull greyish. The terminal grey region consisted of a palisade of tightly packed conidiogenous cells with typically a stout elongate denticle, giving rise to a single conidium.

This anamorph is intermediate between a typical Hirsutella (e.g., Hi. formicarum, Hi. citriformis, and Hi. saussurei) and a typical Hymenostilbe (e.g., Hy. dipterigena – closer to the latter) (Figs 12F–G). The palisade of crowded conidiogenous cells is indicative of Hymenostilbe rather than Hirsutella, where conidiogenous cells are sparse and mostly immature at any given time (Fig. 12F). The denticulate nature of the conidiogenous cell also is indicative of Hymenostilbe. However, in all specimens examined to date there is no evidence of multiple denticles (five or more) usually associated with Hymenostilbe; only a few conidiogenous cells were seen with two denticles (Figs 12F–G).

The anamorph of O. communis is closest to Hy. ventricosa Hywel-Jones (Hywel-Jones 1995). That species infects cockroaches and is found attached to the under side of leaves. As with the anamorph of O. communis, Hy. ventricosa produces conidiogenous cells with only a single terminal stout denticle. Conidia of Hy. ventricosa have a pronounced point and are not typical of the clavate shape usually associated with Hymenostilbe. Similarly, the conidia of the O. communis anamorph are also fattened naviculate, appearing similar to those of Hy. ventricosa but without the processed tip.

The perithecia erupt through the dull greyish anamorph spike appearing first as longitudinal splits in the palisade of conidiogenous cells at the base of the anamorph spike. Each develops as a superficial perithecium, but they become crowded and give the overall appearance of a brown subterminal fertile region (Kobayasi 1941; Figs 12A–B). The ascus shape and the form of the ascus cap comes close to Kobayasi's Figs 12C–D (Kobayasi 1941) being typical of species in the C. unilateralis clade (with Hirsutella as an anamorph). Mature perithecia eject pigmented, whole ascospores (Fig. 12E) and often the ostiole becomes blocked with these half-emerged ascospores.

Only a few species of Cordyceps sensu Kobayasi and Mains have been reported from termites. Currently accepted species include O. koningsbergeri (= C. koningsbergeri Penz. & Sacc.), which is known only from the type locality (Java, Indonesia) (Kobayasi 1941), and C. termitophila Kobayasi & Shimizu) which is known from Japan and Taiwan (Kobayasi & Shimizu 1976, 1978). Penzig & Saccardo (1904) found O. koningsbergeri to be similar to O. myrmecophila in that it had a terminal, globose head with immersed perithecia. In this feature alone it differs significantly from O. communis with its subterminal and superficial perithecia. However, as with O. communis, Penzig & Saccardo (1904) described whole ascospores of O. koningsbergeri, which were 150 x 1 µm compared with 120–150 x 5–6 µm for O. communis. Cordyceps termitophila differs from O. communis in having a `pale rosy-grey' stroma, much smaller perithecia (280–320 x 175–190 µm for C. termitophila versus 285–675 x 195–390 µm for O. communis) and smaller ascospores (100–125 x 3 µm).

Accepted names and new combinations for Ophiocordyceps
The following taxa are accepted species of Ophiocordyceps based on their inclusion in molecular phylogenies presented herein1 or morphological descriptions matching the characters described above2. Where known, the anamorph connection is provided for the species of Ophiocordyceps.

CLAVICIPITACEAE Clade C
Clavicipitaceae clade C is a strongly supported group that includes the type species, C. militaris, of Cordyceps (MP-BP = 100 %, ML-BP = 100 %, PP = 1.00 in Figs 1, 2). Because of the non-monophyly of Cordyceps, we reintroduce the preexisting family name Cordycipitaceae for Clavicipitaceae clade C. This family name was not validly published and it is validated herein based on the type genus Cordyceps. Most of the species in the family parasitize hosts in leaf litter, moss, or upper soil layers and produce superficial to partially immersed to completely immersed perithecia on a fleshy stroma or subiculum that is pallid or brightly coloured. The family contains species of Cordyceps and Torrubiella (Figs 5, 7). The unispecific genus Phytocordyceps is also recognized as a member of this family and transferred to Cordyceps (Fig. 6). In addition, the recent molecular study shows that species of the genera Ascopolyporus A. Möller and Hyperdermium J. White, R. Sullivan, G. Bills & N. Hywel-Jones 2000 [non Link], both pathogens of scale insects, are also inferred to be members of the family (Sullivan et al. 2000, Bischoff et al. 2005).

CORDYCIPITACEAE Kreisel 1969 ex G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora, fam. nov. MycoBank MB504360.

Cordycipitaceae Kreisel, Grundz. Natürl. Syst. Pilze: 112. 1969 [nom. inval., Art. 36].

Stromata vel subiculum pallida vel laete colorata, carnosa. Perithecia superficialia vel omnino immersa, perpendicularia ad superficiem. Asci cylindrici, apice inspissato. Ascosporae cylindricae, multiseptatae, maturae diffrangentes vel integrae remanentes.

Stromata or subiculum pallid or brightly pigmented, fleshy. Perithecia superficial to completely immersed, oriented at right angles to the surface of the stroma. Asci cylindrical with thickened ascus apex. Ascospores usually cylindrical, multiseptate, disarticulating into part-spores or remaining intact at maturity.

Type: Cordyceps Fr.

Teleomorphic genera: Ascopolyporus, Cordyceps, Hyperdermium, Torrubiella.

Anamorphic genera: Beauveria, Engyodontium, Isaria, Lecanicillium, mariannaea-like, Microhilum, Simplicillium.

CORDYCEPS Fr., Observ. Mycol. 2 (revis.): 316. 1818 emend. G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora

= Phytocordyceps C.H. Su & H.-H. Wang, Mycotaxon 2: 338. 1986.

Stromata or subiculum pallid or brightly pigmented, fleshy. Perithecia superficial to completely immersed, ordinal in arrangement. Asci hyaline, cylindrical with thickened ascus apex. Ascospores hyaline, cylindrical, multiseptate, disarticulating into part-spores or nondisarticulating, rarely possessing a thread-like structure connecting the fusiform ends.

Type: Cordyceps militaris (L.: Fr.) Fr., Observ. Mycol. 2(revis.): 317. 1818.

Anamorphic genera: Beauveria, Isaria, Lecanicillium, mariannaea-like, Microhilum, Simplicillium.

Commentary: Species of Cordyceps s. s. are characterized by possessing fleshy stromata that are pallid or brightly coloured. Because species of Torrubiella are interspersed among Cordyceps species in the basal part of the Cordycipitaceae, its ultimate application to a monophyletic taxon within the Cordycipitaceae is not clear, however (Fig. 10). The genus Torrubiella was erected in 1885 by Boudier with the type species T. aranicida Boud. (Kobayasi & Shimizu 1982). Our sampling included several species of Torrubiella that were interspersed amongst species of Cordycipitaceae, but we could not get hold of T. aranicida. Thus, Cordyceps s. s. is narrowly applied to the strongly supported clade (MP-BP = 98 %, ML-BP = 98 %, PP = 1.00 in Figs 1, 2, 10) that includes Cordyceps species closely related to C. militaris. Cordyceps species that are placed outside of the Cordyceps s. s. node, but within the Cordycipitaceae, are provisionally retained within Cordyceps s. l. Torrubiella species that are part of the Cordyceps s. s. are transferred accordingly. The full extent to which the names Cordyceps and Torrubiella will ultimately be applied awaits additional sampling of Torrubiella, especially that of T. aranicida with the possibility that Torrubiella will need to be synonymized with Cordyceps. Although Phytocordyceps is characterized by its possession of bola-ascospores, it is also synonymized with Cordyceps because of its phylogenetic placement (Figs 8, 10).

Accepted names and new combinations for Cordyceps s. s.
The following taxa are accepted species of Cordyceps s. s. based on their inclusion in molecular phylogenies presented herein1 (see Table 1) or morphological descriptions matching the characters described above2. Where known we provide the anamorph connection for the species of Cordyceps s. s.

Clavicipitaceae incertae sedis
The following teleomorph genera could not be confidently assigned in the new classification because they were either not sampled as part of this study, were not sampled as part of other molecular phylogenetic studies, or the assessment of their morphology and ecology was inconclusive: Berkelella (Sacc.) Sacc., Cavimalum Yoshim. Doi, Dargan & K.S. Thind, Dussiella Pat., Epicrea Petr., Helminthascus Tranzschel, Konradia Racib., Moelleriella Bres., Mycomalus A. Möller, Neobarya Lowen, Neocordyceps Kobayasi, Podocrella Seaver, Romanoa Thirum., Sphaerocordyceps Kobayasi, and Stereocrea Syd. & P. Syd.

Residual species of Cordyceps
The following species of Cordyceps s. l. could not be confidently assigned in the new classification because they were either not assigned in any of the proposed genera in this study, were not sampled as part of this or other molecular phylogenetic studies, or the assessment of their morphology and ecology was inconclusive. These species are provisionally retained within Cordyceps s. l. until further phylogenetic analyses are conducted to classify them in a phylogenetic system. Where known we provide the anamorph connection for the species of Cordyceps s. l.


   KEY TO THE GENERA OF FUNGI FORMERLY CLASSIFIED IN CORDYCEPS
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 TAXONOMIC REVISION
 KEY TO THE GENERA...
References
 
This key is designed to emphasize the most conspicuous field-, host-, and macroscopic characters available to the user for Cordyceps sensu Kobayasi and Mains. It is a key to the monophyletic genera described herein and is not a key to the species. As relatively few species occur on Elaphomyces and adult stages of Arthropoda, the key begins with these characters so as to expeditiously highlight or remove these taxa from consideration. Host is an exceedingly important character in most species descriptions of arthropod-pathogenic fungi. The host should be collected with the fungal specimen whenever possible, but this often proves problematic. The vast majority of arthropod-pathogenic fungi occur on immature stages (e.g., larvae, pupae) of arthropods. Therefore, if the host is lacking from a particular specimen or collection, we suggest the user to begin with couplet (6). The multigene phylogeny reveals that colour, texture, and shape of stromata are particularly phylogenetically informative, thus we place special emphasis on these characters where possible but emphasize that, as with most fungal taxa, exceptions are to be expected.

To assist the user we briefly define some characters of stromatal texture and morphology that may not be intuitive:


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Cordyceps s. s. consists almost entirely of pallid to brightly coloured species that produce soft fleshy stromata (e.g., C. militaris). The majority of species attack larvae and pupae of Lepidoptera and Coleoptera in leaf litter, moss or upper soil layers. Numerous species that produce highly reduced stromata, loosely organized hyphae, or a subiculum on the host also occur in this genus (e.g. C. tuberculata), some of which were previously classified in Torrubiella (e.g., T. confragosa).

Elaphocordyceps includes all species that parasitize Elaphomyces and closely related species that attack nymphs of cicadas. The morphology of the Elaphomyces parasites and the cicada pathogens are remarkably similar and attest to the recent history of inter-kingdom host-jumps in a common subterranean environment (Nikoh & Fukatsu 2000). The exception to this genus is E. subsessilis, which macroscopically and ecologically is distinct from the rest of the species, but is well supported as being a member of the genus based on molecular data and micromorphology.

Metacordyceps includes only a limited number of described species, of which all but one are only known from East Asia. The stromatal colour of fresh specimens ranges from white to lilac, purple or green, and the darker pigments are almost black in dried specimens. The texture of the stromata is fibrous and not fleshy like Cordyceps s. s., and the hosts are almost always buried in soil.

Ophiocordyceps is the largest genus of arthropod-pathogenic fungi. Many species are darkly pigmented and occur on immature stages of hosts buried in soil or in decaying wood. Notable exceptions exist for both of these traits among species that attack adult stages of hosts, however. For example, O. unilateralis is common on adult ants and occurs on the under sides of leaves, and O. sphecocephala is common on adult wasps and is found in leaf litter. Stromatal morphology is diverse, ranging from filiform and wiry to clavate and fibrous, according to species, and many species produce their perithecia in nonterminal regions of the stroma, either distinctly superficial, or in broad irregular patches, or in lateral pads.


   Acknowledgments
 
The authors wish to thank Dr Walter Gams for assistance with Latin diagnoses for new taxa and for providing editorial comments to early drafts of this manuscript. This research was supported by grants from the National Science Foundation (DEB-0129212 and DEB-0 297 2 to J.W.S.), the Korea Science and Engineering Foundation (to J-M Sung), and continuing support from Morakot Tanticharoen and BIOTEC (to N.H.J. and J.J.L.).


   References
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 TAXONOMIC REVISION
 KEY TO THE GENERA...
 References
 

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