Mycol. Res. 109 (1): 41–56 (January 2005). f The British Mycological Society 41 DOI: 10.1017/S095375620400139X Printed in the United Kingdom. Towards a phylogenetic classification of Cordyceps: ITS nrDNA sequence data confirm divergent lineages and paraphyly Øyvind STENSRUD1, Nigel L. HYWEL-JONES2 and Trond SCHUMACHER1 1 Division of Botany and Plant Physiology, Department of Biology, University of Oslo, P.O. Box 1045, Blindern, 0316 Oslo, Norway. 2 BIOTEC (Yothi-Research Unit), National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency Building, 73/1 Rama VI Road, Rajdhevee, Bangkok 10400, Thailand. E-mail : [email protected] Received 1 June 2003; accepted 3 September 2004. The ascomycetous genus Cordyceps accommodates endoparasitic species that attack arthropods or other fungi. Analyses of ITS nrDNA sequence data of 72 taxa from the teleomorph genera Cordyceps, Claviceps, Epichloe¨, and the anamorph genera Akanthomyces, Beauveria, Metarhizium, Hirsutella, Hymenostilbe, Paecilomyces, Polycephalomyces, and Tolypocladium assigned the taxa to four main evolutionary lineages not reflected in the current classification of Cordyceps. Ten subclades were recognized from separate analyses of data subsets. Judged from the ITS phylogenies, Cordyceps spp. with branched stromata were highly supported as a divergent lineage. Host specificity was found to be of limited phylogenetic significance, and several host shifts are suggested to have occurred during the evolution of Cordyceps. Similar ascospore morphology was not reflected in the phyletic groups, and closely related taxa showed large interspecific variation with respect to the number of segments in which the ascospores are divided. However, combinations of selected characters were found to delimitate some lineages, e.g. all Cordyceps spp. that attack hosts in the insect orders Coleoptera and Lepidoptera, and with non-immersed perithecia and clavate to acicular, brightly yellowish to reddish stromata, constituted a separate clade. Furthermore, all Cordyceps spp. with perithecia obliquely immersed in the stroma were recognized as a distinct monophyletic group. This clade is additionally characterized by the formation of anamorphs ascribable to the genus Hymenostilbe. The mycogenous Cordyceps spp. grouped in a separate subclade, interspersed by two cicadaen parasites and all Tolypocladium spp. except T. parasiticum. Tolypocladium and Beauveria were found to be polyphyletic. The included Claviceps and Epichloe¨ taxa appeared to be derived within Cordyceps, thus making Cordyceps paraphyletic as suggested in other studies. INTRODUCTION capitate-stipitate stromata. The non-amyloid and apically thickened asci generally contain eight (or four Morphologically and ecologically the megagenus in C. sinensis; Kobayasi 1941) more or less filiform Cordyceps constitutes a well-defined group of patho- multiseptate ascospores that in most species undergo gens and parasites on arthropods (insects, spiders and fragmentation just before or after discharge. Relative mites) and hypogeous fungi (Elaphomyces spp.) to the large number of species, subdivisions into (Kobayasi 1941, 1982, Mains 1954, 1957, Kobayasi & infrageneric groups, e.g. subgenera and sections, have Shimizu 1960, 1977, Evans 1982). The genus is cosmo- been proposed in the classification of Cordyceps, politan in distribution (Hawksworth et al. 1995), and traditionally based on morphological and ecological to date approx. 450 taxa have been accommodated in (host relations) characters. Massee (1895) referred the Cordyceps. However, quite a number are expected to mycogenous species of Cordyceps to the genus Cordylia. represent mere taxonomic synonyms. Kobayasi (1982) In erecting the genus Ophiocordyceps, Petch (1931b) recognized y280 species in his synopsis of the genus, emphasized the microanatomy of clavate asci and several of which are depicted by Imazeki, Otani, & elongate-fusoid, non-fragmentating ascospores as diag- Hongo (1988). nostic characters. Kobayasi (1982) divided Cordyceps Cordyceps spp. are characterized by the development into three subgenera, Ophiocordyceps, Eucordyceps, of perithecioid ascomata that develop superficially or and Neocordyceps, putting emphasis on the direction are embedded in more or less acicular, clavate or of perithecial placement in the stroma, and type of Phylogenetic classification of Cordyceps 42 ascospore shape and septation. Within subgen. provided by Julian Mitchell). Taxon names and refer- Eucordyceps sect. Cystocarpon subsect. Eucystocarpon, ences to the included ITS sequences are listed in Table 1. Kobayasi (1982) recognized ser. Mycogenae that ac- Axenic culture isolates were maintained on water commodated the Elaphomyces inhabiting taxa. agar added antibiotics (tsWA; 1.5% agar sup- More recently, nuclear ribosomal DNA (nrDNA) plemented with 12.5 mg lx1 tetracycline and 25.0 mg lx1 sequence data have proved advantageous in studying streptomycin), 2.0% potato dextrose agar (PDA; species delimitation, anamorph–teleomorph connec- Difco Laboratories, Detroit, MI), and 2.0% potato tions, and evolutionary relationships in fungi (e.g. dextrose agar added antibiotics (tsPDA; PDA supple- Guadet et al. 1989, Hibbett 1992, LoBuglio, Pitt & mented with 12.5 mg lx1 tetracycline and 25.0 mg lx1 Taylor 1993, Holst-Jensen & Schumacher 1997, streptomycin). A piece of fertile stroma with the Schumacher & Holst-Jensen 1997, Hansen, Læssøe & perithecia directed downwards was glued inside a lid of Pfister 2001). In Cordyceps, several studies that also a 9 cm Petri dish containing tsPDA or tsWA, and include associated mitosporic anamorphs have been ascospores were allowed to discharge directly on the made; Rakotonirainy et al. (1991) compared LSU agar. Single, germinating, complete ascospores from rDNA sequences of Beauveria bassiana, Tolypocladium tsWA were transferred to PDA for colony growth. cylindrosporum,andT. extinguens and found the level Somatic cultures were obtained by transferring small of variation sufficient to discriminate among genera pieces (approx. 0.5 cm2) from internal tissues of stroma and species. In Metarhizium, the internal transcribed to tsPDA for colony growth, after submersion in 70% spacer (ITS) region of nrDNA resolved intra- and ethanol (20 s), household bleach (20 s), and dsH2O inter-species relationships, and efficiently demarcated (20 s). the genus from the hyphomycete genera Beauveria and Paecilomyces (Curran et al. 1994). Engh (1999) DNA extraction and PCR inferred three main evolutionary lineages in the study sample of twenty-one taxa of Cordyceps, Beauveria, Various protocols for DNA extraction were used in and Tolypocladium using ITS nrDNA sequences. this study, including the 2% CTAB miniprep method Nikoh & Fukatsu (2000) used nrDNA and mitochon- described by Murray & Thompson (1980), the micro- drial rDNA sequence data in inferring phyletic groups wave miniprep procedure described by Goodwin & among 22 representatives of Cordyceps, Beauveria, Lee (1993) and the Dynabeads R (Dynal, Oslo) Metarhizium, and Paecilomyces. Sung et al. (2001) DNA DirectTM System 1 extraction kit (Rudi et al. concluded that the genus Cordyceps was paraphyletic, 1997). based on phylogenetic analyses of SSU and LSU To obtain target DNA, PCR amplification was nrDNA sequence data in nine Cordyceps spp., includ- performed on a Genius Operator (Techne, Cambridge, ing other clavicipitaceous fungi as well. Cordyceps was UK) or a Biometra T gradient PCR machine observed to be paraphyletic also in the study by (Whatman Biometra, Go¨ ttingen), using the primer Artjariyasripong et al. (2001), who recognized four pairs ITS5/ITS4 or ITS1/ITS4 (White et al. 1990), and groups (clades) of taxa within the Clavicipitaceae based 40.0 ml totvols, consisting of 20.5 ml reaction mix on SSU and LSU nrDNA sequence data. [5 ml5mM ITS5 (or ITS1) primer, 5 ml5mM ITS4 In this study our goal was to infer the main evol- primer, 5 ml2mM dNTPs, 5 ml reaction buffer, 0.5 ml utionary lineages in Cordyceps, based on a broad (1 U) DyNAzymeTM II polymerase (Finnzymes Oy, sample of teleomorphic as well as anamorphic taxa Espoo)] and 19.5 ml20r diluted DNA template. A world-wide, using cladistic analyses of ITS nrDNA cyclic thermal program of an initial 4 min denaturation sequences. Secondly we wanted to see whether the step (94 xC), followed by 35 cycles of 40 s at 94 x current classification and previously proposed classifi- (denaturation), 40 s at 50 x (annealing), and 45 s at 72 x cation schemes reflect phylogenetically meaningful (synthesis), and termination with a 10 min elongation groups. step at 72 x was performed. A negative control (dsH2O) was included. Gel electrophoresis of PCR products was performed on a 1.5% agarose gel added 3 ml ethidium bromide MATERIALS AND METHODS per 50 ml Tris-Acetate-EDTA (TAE) buffer, using Morphological and molecular data were obtained from HaeIII digested bacteriophage WX174 as a standard fresh collections, axenic culture isolates and dried size marker. The gels were photographed under an specimens deposited in the fungal herbaria of Oslo (O), UV-trans-illuminator. Helsinki (H), Copenhagen (C), and ARON (Asco- mycete Research group of Oslo, Norway). Fresh DNA sequencing specimens were collected in 1997–2001 in Norway and Denmark. 26 complete ITS sequences (ITS1–5.8S- The forward and reverse strands of ITS nrDNA were ITS2) were obtained from herbarium specimens and manually and/or automatically sequenced, using one of axenic cultures. 73 additional ITS sequences were re- the