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 primers ITS1/ITS2/ITS3/ITS4/ITS5 (White et al. trieved from other sources (including eight sequences 1990), adjusted to the material. Manual sequencing was Ø. Stensrud, N. L. Hywel-Jones and T. Schumacher 43 performed using the ThermoSequenase radio-labeled Phylogenetic analyses terminator cycle sequencing kit (Amersham Pharmacia The MolecularALL was submitted to parsimony Biotech, Cleveland, OH) following the manufacturer’s analyses with all characters equally weighted in PAUP* instructions, and completed reaction mixtures were version 4.10b (Swofford 2000). All transformations loaded on 6% polyacrylamide gels and electrophoresed were coded as unordered, and gaps treated as ‘new at 65 V. A fixation solution (5% methanol and 5% state’ or ‘missing data’ in two parallel analyses. For the acetic acid) was applied to the gels for approximately large data sets we employed the heuristic tree search 15 min before drying the gels under vacuum at 80 x. option, with the tree bisection-reconnection (TBR) The radioactive radiation was measured with a Geiger branch swapping algorithm and random addition se- counter before the exposure of the gels to a Kodak AR quence with 1,000 replicates switched on in order to X-ray film for 2–10 d. find multiple islands (Maddison 1991) for all searches For automatic sequencing, PCR products and cycle- for the MPT(s). Furthermore, we performed successive sequencing products were cleaned with the ExoSAP-IT weighting (Farris 1969) on the MolecularALL data set and AutoSeq96 Dye Terminator Clean-up Kits, re- (with gaps treated as ‘new state’), based on the maxi- spectively, according to manufacturer’s instructions mum value of the rescaled consistency index (Farris (Amersham Biosciences, Cleveland, OH). Sequencing 1989) for each character over all MPTs, in order to reactions were performed using one of the primers weight down homoplastic characters and reduce the ITS1/ITS4/ITS5 and a DYEnamicTM ET Dye Termin- number of competing topologies. After reweighting, ator Cycle Sequencing kit (Amersham Biosciences, the heuristic tree search option, with the TBR branch Chalfont), following the manufacturer’s instructions, swapping algorithm and random addition sequence and subjected to capillary electrophoresis on a Mega- with 1000 reps was switched on, was employed for all BACETM 500 DNA Analysis System (Amersham searches. Bioscience). The realigned matrices of the clade II, III and IV data subsets were imported to PAUP* and submitted Data handling to parsimony analyses with all characters equally weighted and scored as above. For the clade II and The ITS sequences were imported into the programme III data subsets the branch-and-bound tree searching BioEdit Sequence Alignment Editor version 4.8.6. algorithm was used. For the larger clade IV data (Hall 1999), aligned in CLUSTALW and then the subset, the heuristic tree search option, with the TBR matrix was adjusted by eye. Sequences were read with branch swapping algorithm and the random addition valid character state symbols A, C, G, T and gap (-). sequence with 1000 replicates were employed for all Ambiguous sites were recorded using the standard searches for the MPT(s). IUPAC codes. Due to uncertainty about the validity All matrices were also submitted to Neighbour- of some indels observed in the 5.8 S region in some Joining (NJ) distance analyses with default settings in sequences, these sites were replaced with question PAUP*. marks. For taxa where two or more variable sequences Branch robustness was estimated by bootstrap were included, a consensus (‘cons.’) sequence was analyses (Felsenstein 1985), with 2000 search replicates computed, and ambiguous characters scored as (Hedges 1992). For the MolecularALL data matrix and described above. the clade IV data subset, bootstrap support for the Hypocrea lutea and H. pilulifera were chosen as out- branching topologies was examined by using the group taxa in the analyses, based on published reports heuristic tree search option, with the TBR branch on the close relationship of Hypocrea (Hypocreaceae) swapping algorithm, and with the simple sequence and Cordyceps/Clavicipitaceae (Spatafora & Blackwell addition switched on. For the clade II and III data 1993, Glenn et al. 1996). subsets, bootstrap support for the branching topologies A data matrix, hereafter referred to as Molecular- was examined by using the branch-and-bound tree ALL, including 99 ITS sequences from 74 taxa was searching algorithm. Bootstrap support o50% is constructed. Eight segments of ambiguously aligned superimposed in all tree constructions, and tree stat- characters or insertions (bases 85–105, 113–171, istics (tree length, S; CI; ensemble consistency index, 215–240, 482–530, 535–549, 555–565, 580–627 and CIx; RI; and rescaled consistency index, RC) are 748–799) were identified and excluded prior to the superimposed in Figs 1–5. analyses. Based on the analyses, three new subsets of the ITS sequences, referring to as the clade II, clade III and clade IV subsets of data, were constructed. Prior to analyses of these new subsets, five segments (bases RESULTS 77–97, 113–146, 448–462, 468–478 and 490–505) of the Intraspecific variation clade III data subset, and four segments (bases 85–96, 400–429, 476–501 and 648–656) of the clade IV data ITS sequences from thirty-two specimens, representing subset consisting of ambiguously aligned characters or geographically separate localities of seven Cordyceps insertions, were identified and excluded. spp., i.e. C. capitata (4), C. gracilis (3), C. entomorrhiza Phylogenetic classification of Cordyceps 44

Table 1. DNA sequences included in this study.

Geographic Reference GenBank Taxon origina no.b Source of sequencec accession no.d

Akanthomyces pistillariiformis Thailand/BIOTEC NHJ1 This study AJ786552 Beauveria amorpha GenBank; Coates et al., unpubl. AF322933 B. bassiana GenBank; Coates et al., unpubl. AF322924 B. brongniartii GenBank; Nikoh & Fukatsu (2000) AB027381 B. caledonica GenBank; Shih et al., unpubl. U35284 B. sobolifera GenBank; Liu et al. (2001a) AJ309325 B. velata GenBank; Shih et al., unpubl. U35289 B. vermiconia GenBank; Shih et al., unpubl. U35288 Claviceps purpurea GenBank; Pazoutova´ (2001) AJ133401 Cordyceps bassiana GenBank; Huang et al. (2002) AF347612 C. bicephala GenBank; Liu, unpubl. AJ309365 C. bifusispora Norway/Oslo UoO-88476 This study AJ786553 C. brittlebankisoides GenBank; Liu, unpubl. AJ309332 C. cantharelloides GenBank; Liu, unpubl. AJ309363 C. capitata Norway/ARON 2730.S Engh (1999) AJ786554 C. capitata Norway/ARON 3001.H This study AJ786555 C. capitata Norway/ARON 3086.1 This study AJ786556 C. capitata Norway/ARON 3087.P This study AJ786557 C. coccidiicola GenBank; Nikoh & Fukatsu (2000) AB031196 C. cochlidiicola GenBank; Nikoh & Fukatsu (2000) AB027377 C. cylindrica Thailand/BIOTEC NHJ2 This study AJ786558 C. emeiensis fide GenBank; Liu, unpubl. AJ309347 C. entomorrhiza Norway/ARON 2709.H Engh (1999) AJ786559 C. entomorrhiza Norway/ARON 3070.2 This study AJ786560 C. entomorrhiza Norway/ARON 3070.4 This study AJ786561 C. forquignoni Denmark/Copenhagen UoC-17.06.84 This study AJ786562 C. gracilis Norway/ARON 2684.S Engh (1999) AJ786563 C. gracilis Norway/ARON 2685.4 Engh (1999) AJ786564 C. gracilis Norway/ARON 3069.3 This study AJ786565 C. hawkesii GenBank; Liu, unpubl. AJ309341 C. hepialidicola GenBank; Kwang-Soo et al., unpubl. AF315649 C. heteropoda GenBank; Nikoh & Fukatsu (2000) AB027373 C. inegoensis GenBank; Nikoh & Fukatsu (2000) AB027368 C. irangiensis Thailand/BIOTEC NHJ3 This study AJ786566 C. japonica GenBank; Nikoh & Fukatsu (2000) AB027366 C. jezoensis GenBank; Nikoh & Fukatsu (2000) AB027365 C. kanzashiana GenBank; Nikoh & Fukatsu (2000) AB027371 C. khaoyaiensis Thailand/BIOTEC NHJ4 This study AJ786567 C. konnoana GenBank; Nikoh & Fukatsu (2000) AB031193 C. longisegmentis Norway/ARON 2731.S Engh (1999) AJ786568 C. militaris Norway/ARON 2724.S Engh (1999) AJ786569 C. militaris Norway/ARON 2726.S Engh (1999) AJ786570 C. militaris Norway/ARON 2729.S Engh (1999) AJ786571 C. militaris Norway/ARON 2725.H This study AJ786572 C. militaris Norway/ARON 3856.H This study AJ786573 C. multiaxialis GenBank; Liu, unpubl. AJ309359 C. myrmecophila Denmark/Copenhagen UoC-35517 This study AJ786574 C. myrmecophila Finland/Helsinki UoH-13783 This study AJ786575 C. myrmecophila Finland/Helsinki UoH-17782 This study AJ786576 C. myrmecophila Finland/Helsinki UoH-17982 This study AJ786577 C. myrmecophila Finland/Helsinki UoH-22580 This study AJ786578 C. myrmecophila Finland/Helsinki UoH-24680 This study AJ786579 C. myrmecophila Finland/Helsinki UoH-27480 This study AJ786580 C. myrmecophila Finland/Helsinki UoH-27582 This study AJ786581 C. myrmecophila Finland/Helsinki UoH-29280 This study AJ786582 C. nepalensis GenBank; Liu, unpubl. AJ309358 C. nutans Thailand/Oslo UoO-113/78 This study AJ786583 C. ophioglossoides Norway/ARON 2721.P Engh (1999) AJ786584 C. ophioglossoides Norway/ARON 2723.S Engh (1999) AJ786585 C. ophioglossoides Norway/ARON 2732.H This study AJ786586 C. ophioglossoides Norway/Oslo UoO-86175 This study AJ786587 C. ophioglossoides Norway/Oslo UoO-07.09.87 This study AJ786588 C. paradoxa GenBank; Nikoh & Fukatsu (2000) AB027369 C. prolifica GenBank; Nikoh & Fukatsu (2000) AB027370 C. pruinosa GenBank; Nikoh & Fukatsu (2001) AB044635 C. pseudomilitaris Thailand/BIOTEC NHJ6 This study AJ786589 Ø. Stensrud, N. L. Hywel-Jones and T. Schumacher 45

Table 1. (Cont.)

Geographic Reference GenBank Taxon origina no.b Source of sequencec accession no.d

C. ramosopulvinata GenBank; Nikoh & Fukatsu (2000) AB027372 C. robertsii GenBank; Liu, unpubl. AJ309335 C. scarabaeicola GenBank; Park et al., unpubl. AF199592 C. sinensis China/ARON 2868.H Engh (1999) AJ786590 C. sobolifera GenBank; Nikoh & Fukatsu (2000) AB027374 C. sphecocephala Thailand/BIOTEC NHJ7 This study AJ786591 C. subsessilis USA, MI/USA, NY 2747.S Engh (1999) AJ786592 C. taii GenBank; Huang et al., unpubl. AF348393 C. takaomontana GenBank; Nikoh & Fukatsu (2001) AB044637 C. tricentri GenBank; Nikoh & Fukatsu (2000) AB027376 C. yakusimensis GenBank; Nikoh & Fukatsu (2001) AB044643 Cordyceps tax. sp. 1 Norway/ARON 2870.H Engh (1999) AJ786593 Cordyceps tax. sp. 2 Norway/ARON 3002.H This study AJ786594 Cordyceps tax. sp. 3 Norway/ARON 3083.H This study AJ786595 Epichloe¨ typhina GenBank; Schardl et al. (1991) L20306 Hirsutella rhossiliensis GenBank; Gernandt & Stone (1999) AF169197 H. sinensis GenBank; Zhao et al. (1999) AJ243980 Hymenostilbe aurantiaca Thailand/BIOTEC NHJ8 This study AJ786596 H. sphecocephala nomen Thailand/BIOTEC NHJ9 This study AJ786597 provisorium Hypocrea lutea GenBank; Nikoh & Fukatsu (2000) AB027384 H. pilulifera GenBank; Kuhls, unpubl. Z48813 Metarhizium album GenBank; Driver, Milner & Trueman (2000) AF137067 M. anisopliae GenBank; Nikoh & Fukatsu (2000) AB027383 M. taii GenBank; Huang et al., unpubl. AF348394 Paecilomyces farinosus GenBank; Park et al., unpubl. AF237664 P. tenuipes GenBank; Nikoh & Fukatsu (2000) AB027380 Polycephalomyces ramosus Norway/ARON 3078.H This study AJ786598 Tolypocladium GenBank; Jacobsen, unpubl. AJ303055 cylindrosporum T. geodes GenBank; Shih & Tzean, unpubl. U19037 T. inflatum Norway/Switzerland 2869.S Engh (1999) AJ786599 (Novartis) T. nubicola GenBank; Shih & Tzean, unpubl. U19040 T. parasiticum GenBank; Shih et al., unpubl. U35305 T. tundrense GenBank; Shih & Tzean, unpubl. U19041

a Voucher specimen collected/deposited (ARON, Ascomycete Research group of Oslo, Norway). b Unique digit and letter reference numbers, used in the phylogenetic trees in the following, for sequences not retrieved from EMBL/ GenBank/DDBJ. NHJ1–NHJ9, Code for sequences provided by Julian S. Mitchell. Specimens of the ARON collection have a four digit voucher number, followed by a digit (single ascospore culture) or letter (P, mass ascospore culture; S, somatic culture; H, herbarium specimen) indicating the source of inoculum. c For sequences retrieved from EMBL/GenBank/DDBJ, the original source is cited. d Accession numbers in EMBL/GenBank/DDBJ (http://www.ncbi.nlm.nih.gov).

(3), C. militaris (5), C. myrmecophila (9), C. ophioglos- is shown in Fig. 1. A distance analysis of the soides (5), and Cordyceps sp. (3), were found invariable MolecularALL data set yielded a tree 1637 steps long, with one exception: in C. gracilis a single substitution with only the main lineages (cfr Fig. 1) shown in Fig. 2. in the ITS2 separated sample 2684.S from the samples Although the inter-relationships of groups (clades I 2685.4 and 3069.3. to IV) changed to some extent, the parsimony analyses all recognized the same four main evolutionary lineages (cfr Fig. 1): Clade I consisting of Cordyceps prolifica, C. Phylogenies kanzashiana, and C. ramosopulvinata; clade II including Parsimony analysis of the MolecularALL data set with eight Cordyceps spp., Akanthomyces pistillariiformis, gaps treated as ‘new state’ yielded 25480 MPTs, 2002 six Beauveria spp., and two Paecilomyces spp.; clade III steps long (data not shown). An analysis with gaps including eleven Cordyceps spp., Claviceps purpurea, treated as ‘missing data’, yielded 9034 MPTs, 1610 Epichloe¨ typhina, two Hymenostilbe spp., three steps long (data not shown). Successive weighting was Metarhizium spp., and Tolypocladium parasiticum; performed on the data set, and after reweighting a and clade IV including 24 Cordyceps spp., Beauveria parsimony analysis with gaps treated as ‘new state’ sobolifera, two Hirsutella spp., Polycephalomyces yielded 117 MPTs, 621.13 steps long, one of which ramosus, and five Tolypocladium spp. In the distance Phylogenetic classification of Cordyceps 46

Hyp. lutea Hyp. pilulifera 100 100 Cor. kanzashiana # Trees: 117 I Cor. ramosopulvinata Length: 621.13 Cor. prolifica CI: 0.6298 Cix: 0.5328 Bea. amorpha RI: 0.8313 Bea. caledonica RC: 0.5122 Bea. vermiconia Bea. bassiana Bea. brongniartii Cor. bassiana Cor. scarabaeicola Aka. pistillariiformis Cor. bifusispora Cor. takaomontana Pae. farinosus 100 Pae. tenuipes Clavici- Cor. pruinosa Ipitaceae 100 II Cor. militaris cons. Cor. hepialidicola Bea. velata Cor. pseudomilitaris Cla. purpurea Epi. typhina

Cor. brittlebankisoides Group 1 Tol. parasiticum Cor. hawkesii Met. album Cor. khaoyaiensis 75 Met. anisopliae III 99 Met. taii Cor. taii Cor. bicephala Cor. myrmecophila cons. Cor. tricentri Group 2 Hym. sphecocephala Cor. sphecocephala Cor. irangiensis Hym. aurantiaca Cor. forquignoni Cor. nutans Cor. cantharelloides Cor. yakusimensis 94 Bea. sobolifera Cor. sobolifera Cor. heteropoda Cor. gracilis cons. Cor. robertsii Hir. rhossiliensis Cor. coccidiicola Cor. emeiensis Hir. sinensis Cor. multiaxialis Cor. sinensis Cor. nepalensis Cor. cochlidiicola 88 Cor. cylindrica IV Cor. konnoana Pol. ramosus Cor. entomorrhiza cons. Cor. japonica Cor. jezoensis Tol. geodes Cor. ophioglossoides cons. Tol. cylindrosporum Tol. nubicola Tol. inflatum Cor. subsessilis Tol. tundrense Cor. paradoxa Cor. inegoensis Cor. longisegmentis Cordyceps sp. cons. Cor. capitata cons. 5 changes

Fig. 1. One of 117 MPTs resulting from parsimony analysis of the reweighted MolecularALL data set with gaps treated as ‘new state’. Bootstrap support (BS) is given for branches of higher order. Roman numerals denote the inferred main evolutionary lineages (clades I to IV) discussed in the text. Hyp., Hypocrea; Cor., Cordyceps; Cla., Claviceps; Epi., Epichloe¨; Aka., Akanthomyces; Bea., Beauveria; Hir., Hirsutella; Hym., Hymenostilbe; Met., Metarhizium; Pae., Paecilomyces; Pol., Polycephalomyces; and Tol., Tolypocladium. The inference of Claviceps purpurea and Epichloe¨ typhina of clade III is highlighted. Cordyceps militaris, the generic type, is shaded. The anamorph genera are assigned separate colours. Ø. Stensrud, N. L. Hywel-Jones and T. Schumacher 47

Hyp. lutea and H. sphecocephala. Differences in tree topologies of the two MPTs were restricted to the terminal grouping Hyp. pilulifera of C. irangiensis, C. sphecocephala,andHymenostilbe 100 Clade I aurantiaca of subclade III-F. C. brittlebankisoides, C. 68 100 hawkesii, C. khaoyaiensis, Metarhizium album, and 66 65 Clade IV 83 Tolypocladium parasiticum were not included in either of the subclades, due to low BS and/or contradictory 64 77 Clade III - Group 1 51 89 grouping in the parsimony and distance analyses. 100 100 Clade II The 33 taxa which constituted clade IV were aligned 100 100 with C. khaoyaiensis and C. taii as the outgroup. 100 100 Clade III - Group 2 Parsimony analysis of the clade IV data subset yielded 14 MPTs, 1394 steps long, one of which is shown in Fig. 2. Distance tree obtained from analysis of the Fig. 5. A distance analysis yielded a tree 1004 steps long MolecularAll data set, with the main lineages (cfr Fig. 1) (data not shown). Four subclades were recognized shown. Numbers above branches indicate BS. Hyp., (cfr Fig. 5): Subclade IV-G consisting of C. cocci- Hypocrea. diicola, C. cochlidiicola, C. emeiensis, C. multiaxialis, C. nepalensis, C. robertsii, C. sinensis, Hirsutella rhossi- analyses, clade III did not form a monophyletic group liensis, and H. sinensis; subclade IV-H consisting of C. (cfr Fig. 2), however, the support for the three nodes cylindrica, C. gracilis, and C. heteropoda; subclade IV-J that defined the topology of the ingroup was low consisting of C. capitata, C. inegoensis, C. japonica, C. (<70% BS). jezoensis, C. longisegmentis, C. ophioglossoides, C. In order to infer a confident internal phylogeny, paradoxa, Cordyceps sp., C. subsessilis, Tolypocladium and minimize the noise of homoplasy, the taxa accom- cylindrosporum, T. geodes, T. inflatum, T. nubicola, modated in clades II, III, and IV were aligned separ- and T. tundrense; and subclade IV-K consisting of C. ately and the re-aligned matrices submitted to sobolifera, C. yakusimensis, and Beauveria sobolifera. parsimony and distance analyses in isolation, using the Differences in tree topologies of the 14 MPTs were gap mode option ‘new state’ in the parsimony analyses. restricted to the grouping of the terminal taxa of sub- Cordyceps prolifica and C. ramosopulvinata were clade IV-J. C. cantharelloides, C. entomorrhiza, C. chosen as outgroup to the 17 taxa constituting clade II. konnoana, and Polycephalomyces ramosus were not in- The parsimony analysis yielded eight MPTs, 662 steps cluded in either of the subclades, due to low BS and/or long, one of which is shown in Fig. 3. The NJ distance contradictory grouping in the parsimony and distance analysis yielded a tree 448 steps long (data not shown). analyses. Three subclades were recognized from the analyses of the clade II data subset (cfr Fig. 3): Subclade II-A consisting of Cordyceps bassiana, C. scarabaeicola, DISCUSSION Beauveria amorpha, B. bassiana, B. brongniartii, B. caledonica, and B. vermiconia; subclade II-B consisting Analyses of sequence data have become a widely used of C. hepialidicola,andC. militaris; and subclade approach in studying species delimitation and phylo- II-C consisting of C. bifusispora, C. takaomontana, genetic relationships. Our observations that intra- Paecilomyces farinosus, and P. tenuipes. Differences in specific ITS nrDNA variation was non-existent or topologies among the MPTs were restricted to the negligible among individuals of a species, but diverged grouping of terminal taxa of subclade II-A and II-C. C. between species, demonstrated the suitability of the ITS pruinosa, C. pseudomilitaris, Akanthomyces pistillarii- region of nrDNA in discriminating specific taxa in this formis, and Beauveria velata were not included in group of fungi. either of the subclades due to low BS and/or contradic- The grouping of the three included cicada parasites tory grouping of the parsimony and distance analyses. of clade I (Fig. 1), i.e. Cordyceps kanzashiana Similarly, the 19 taxa constituting clade III were re- (Kobayasi & Shimizu 1982), C. prolifica (Kobayasi & aligned and submitted to parsimony and distance Shimizu 1963), and C. ramosopulvinata (Kobayasi & analyses, using C. bifusispora and C. pruinosa of clade Shimizu 1983), is in agreement with the observations by II as outgroup. The parsimony analysis yielded two Nikoh & Fukatsu (2000), who referred these taxa to a MPTs, 1305 steps long, one of which is shown in Fig. 4. separate ‘cicada clade’. The presence of branched The NJ distance analysis yielded a tree 997 steps long stromata seems to be a synapomorphic character of (data not shown). Three subclades were recognized clade I. So far, none of the taxa of clade I have been (cfr Fig. 4): Subclade III-D consisting of Claviceps linked to anamorph genera, however, Kobayasi & purpurea and Epichloe¨ typhina; subclade III-E consist- Shimizu (1976) reported that conidial formation might ing of Cordyceps taii, Metarhizium anisopliae, and M. occur associated with stromata of C. prolifica. taii; and subclade III-F consisting of C. bicephala, C. Cordyceps spp. of clade II (Figs 1, 3) all have peri- forquignoni, C. irangiensis, C. myrmecophila, C. nutans, thecia born in brightly coloured (yellow to orange) C. sphecocephala, C. tricentri, Hymenostilbe aurantiaca, acicular to clavate stromata. In C. bifusispora the Phylogenetic classification of Cordyceps 48

Cor. prolifica Cor. ramosopulvinata 73 Bea. amorpha # Trees: 8 100 Bea. caledonica Length: 662 CI: 0.7885 Bea. vermiconia Cix: 0.6854 Bea. brongniartii RI: 0.7771 98 Bea. bassiana RC: 0.6127 A Cor. scarabaeicola 93 Cor. bassiana 100 Cor. militaris cons. B Cor. hepialidicola Aka. pistillariiformis Cor. pseudomilitaris Bea. velata 57 100 Cor. bifusispora II 100 Cor. takaomontana C 99 Pae. farinosus Pae. tenuipes Cor. pruinosa 10 changes Fig. 3. One of eight MPTs resulting from parsimony analysis of the clade II data subset, with gaps treated as ‘new state’. Numbers indicate BS. Uppercase letters denote the subclades commented upon in the text. Cor., Cordyceps; Aka., Akanthomyces; Bea., Beauveria; and Pae., Paecilomyces. The anamorph genera are assigned separate colours.

Cor. pruinosa Cor. bifusispora # Trees: 2 94 Cla. purpurea D Length: 1,305 Epi. typhina CI: 0.5969 75 Tol. parasiticum Cix: 0.5561 RI: 0.6856 Cor. hawkesii RC: 0.4093 100 Met. album III Cor. brittlebankisoides Cor. khaoyaiensis 100 Met. anisopliae E Met. taii 94 52 Cor. taii Cor. bicephala 100 80 Cor. forquignoni F Cor. nutans 90 Cor. myrmecophila cons. Cor. tricentri 78 Hym. sphecocephala 92 58 Cor. sphecocephala 100 Cor. irangiensis Hym. aurantiaca 50 changes

Fig. 4. One of two MPTs resulting from parsimony analysis of the clade III data subset with gaps treated as ‘new state’. Numbers indicate BS. Uppercase letters denote the subclades commented upon in the text. Cor., Cordyceps; Cla., Claviceps; Epi., Epichloe¨; Hym., Hymenostilbe; Met., Metarhizium; and Tol.=Tolypocladium. The clade consisting of Claviceps purpurea and E. typhina is highlighted. The anamorph genera are assigned separate colours. ascospores are bifusiform, with septated end segments; closest relatives among taxa of this clade (Sung et al. in the closely linked C. takaomontana (Kobayasi 1941) 2001). The hypothesis that Hirsutella is the most it is of the typical filiform and multiseptate type. appropriate place to accommodate the anamorph of C. The anamorph genera of clade II include Akantho- pseudomilitaris (Hywel-Jones 1994) receives no support myces, Beauveria, Lecanicillium (represented by its from our study. The two included Hirsutella taxa are teleomorph C. militaris; Zare & Gams 2001), Mar- both positioned in clade IV. Akanthomyces, erected by iannaea (represented by its teleomorph C. pruinosa; Lebert (1858) to include the moth parasite A. aculeatus, Liang 1991b), Paecilomyces, and Septofusidium (rep- was later emended to include representatives attacking resented by its teleomorph C. bifusispora; Liu, Liang the insect orders Coleoptera, Diptera, Heteroptera, & Liu 1996). Additionally, the lepidopteran parasite Homoptera, Hymenoptera and Lepidoptera as well Microhilum oncoperae has been shown to have its as Arachnida (Mains 1950, Samson & Evans 1974, Ø. Stensrud, N. L. Hywel-Jones and T. Schumacher 49

Cor. khaoyaiensis Cor. taii Cor. cantharelloides Cor. robertsii Cor. emeiensis 68 Cor. cochlidiicola 71 Cor. coccidiicola 83 G Hir. rhossiliensis Hir. sinensis 50 79 69 Cor. multiaxialis 100 Cor. nepalensis Cor. sinensis 100 Cor. heteropoda 76 H Cor. gracilis cons. Cor. cylindrica 59 Cor. japonica Cor. jezoensis 56 Cor. ophioglossoides cons. Cor. inegoensis Cor. paradoxa Tol. cylindrosporum 86 Tol. tundrense Tol. nubicola Tol. inflatum 51 94 Cor. subsessilis 70 J Tol. geodes Cordyceps sp. cons. 100 Cor. capitata cons. IV Cor. longisegmentis # Trees: 14 59 Cor. konnoana Length: 1,394 100 Pol. ramosus CI: 0.5416 Cor. entomorrhiza cons. Cix: 0.4992 RI: 0.6651 100 Cor. yakusimensis RC: 0.3602 K 100 Bea. sobolifera Cor. sobolifera 10 changes

Fig. 5. One of 14 MPTs resulting from parsimony analysis of the clade IV data subset with gaps treated as ‘new state’. Numbers indicate BS. Uppercase letters denote the subclades commented upon in the text. Cor., Cordyceps; Bea., Beauveria; Hir., Hirsutella; Pol., Polycephalomyces; and Tol., Tolypocladium. The anamorph genera are assigned separate colours.

Samson & Brady 1982, Hywel-Jones 1996a, Hsieh, disparate positions of these two anamorph genera, that Tzean & Wu 1997). Mains (1950) emphasized the comes out in separate clades (II and IV). cylindrical synnemata and the tightly compacted longi- Clade II also includes the Beauveria spp. Beauveria tudinal conidiophores, producing one-celled catenulate was established by Vuillemin (1912) and is typified by conidia as typical for Akanthomyces. Taxa with clavate B. bassiana, a specimen growing parasitically on larvae synnemata, such as in A. pistillariiformis (cfr clade II), of the silkworm Bombyx mori. Conidia in Beauveria are were referred to the genus Insecticola (Mains 1950). borne in loose, globose heads from phialides located on This latter genus was not accepted by Samson & Evans main hyphae or on short lateral conidiophores. The (1974), who noted that the ‘synnematous habit does conidiophores terminate in elongated, flask-shaped to not allow a clear delimitation between Akanthomyces cylindric phialides, and new conidia are produced from and Insecticola’, an opinion also concurred by Hywel- the same sympodially elongated phialides. After the Jones (1996a). The conidial chains in Akanthomyces formation of the first conidium, a zigzag or flexuose are short and often difficult to observe, resulting in apical sterigma gives off a subapical branch where a their frequently wrong placement in Hymenostilbe new conidium is produced. In its present circumscrip- (Samson & Evans 1975). While Hymenostilbe resembles tion, Beauveria attacks a wide range of hosts, especially Akanthomyces, however, a re-examination of the type including coleopteran and lepidopteran hosts (de Hoog specimens of several Hymenostilbe and Akanthomyces 1972). A similar broad host range is also observed spp. has shown that the conidiogenous cells of the among the related teleomorphic (Cordyceps) species of former are polyblastic, bearing solitary conidia on clade II. Beauveria vermiconia, originally suspected as a short denticles, whilst Akanthomyces is phialidic with non-entomogenous fungus isolated from volcanic ash conidia borne in basipetal chains (Samson & Evans (Mugnai, Bridge & Evans 1989), was later found to 1974, 1975). The ITS phylograms clearly support the attack Scarabaeidae (Glare, Jackson & Cisternas 1993). Phylogenetic classification of Cordyceps 50

Three Cordyceps spp. are reported to form Beauveria Obornı´ k, Jirku & Dolezel (2001) suggested Paecilo- anamorphs, i.e. C. brongniartii (Shimazu, Mitsuhashi myces to be polyphyletic. Our study supports a close & Hashimoto 1988), C. sobolifera (Liu et al. 2001a) connection between the two genera, accommodated in and C. bassiana (Li et al. 2001, Huang et al. 2002). The clade II. Isaria japonica is the anamorph of C. takao- conidial state of C. scarabaeicola (Kobayasi & Shimizu montana (Kobayasi & Shimizu 1976, Kobayasi 1981, 1978) has not been accommodated in any anamorph Ito & Hirano 1997). Samson (1974) treated Isaria genus yet, however, its close relationship to the japonica as a synonym to Paecilomyces tenuipes. This ‘Beauveria-clade’ (II-A) may be indicative of an ana- disposition was also concurred with by Fukatsu, Sato morph affiliation to Beauveria. Beauveria velata did & Kuriyama (1997) and Liang (1997), and a retrieved not group with the other Beauveria spp. (cfr Fig. 3), sequence of Isaria japonica (AF200370) from EMBL/ and showed a loose affinity to subclade II-C in the GenBank/DDBJ showed 100% ITS similarity with the parsimony analysis. However, in common with other sequence of P. tenuipes presented here. Judged from Beauveria spp., B. velata is entomogenous, and lepi- ITS nrDNA sequence data, the three holomorphs C. dopteran hosts are commonly reported (Mugnai et al. bifusispora (anamorph Septofusidium bifusisporum), 1989). C. memorabilis (anamorph Paecilomyces farinosus) and Cordyceps militaris, the conserved type of Cordyceps, C. takaomontana (anamorph P. tenuipes) are certainly is closely related to Beauveria spp. (Figs 1, 3). The closely related. Chen & Xu (1989), on the other hand, imperfect state of C. militaris has recently been referred stated that P. tenuipes is the anamorph of Cordyceps to the new anamorph genus Lecanicillium (Zare & polyarthra, a taxon not included in this study. How- Gams 2001), a genus erected for some species formerly ever, Fukatsu et al. (1997) considered the taxonomic assigned to Aphanocladium, Cephalosporium, Engyo- relationship between C. takaomontana and C. poly- dontium and Verticillium (Zare & Gams 2001). Torru- arthra as unclear. biella alba (Petch 1932) and T. confragosa (Mains 1949) Again, very little phylogenetic support is to be gained also have anamorphs in Lecanicillium (Zare & Gams from ascospore characteristics and ecology of this 2001). group; C. bifusispora has bifusiform ascospores and Liu et al. (2001a) described Beauveria sobolifera as is restricted to lepidopteran hosts, whereas C. memor- the anamorph of Cordyceps sobolifera, and the inclusion abilis and C. takaomontana have filiform ascospores of B. sobolifera in clade IV makes Beauveria poly- and occur on coleopteran and lepidopteran hosts, phyletic. Petch (1942) described Isaria cicadae as respectively. the anamorph of Cordyceps sobolifera. The name The hosts for Cordyceps spp. of clade II are to be I. cicadae, however, is currently obsolete due to its found among the insect orders Coleoptera and Lepi- transference to Paecilomyces (Samson 1974). The doptera. The coleopteran parasites include C. bassiana measurements of the large, narrowly cylindrical conidia (Li et al. 2001), C. scarabaeicola (Kobayasi & Shimizu in B. sobolifera (7.5–9.5r1.9–3.2 mm; Liu et al. 2001a) 1976), C. brongniartii, here represented by its ana- are in agreement with the measurements given for morph Beauveria brongniartii (Shimazu et al. 1988), P. cicadae (7–11r2.5–3 mm; Petch 1942). Thus, B. and C. memorabilis, the stated teleomorph of Paecilo- sobolifera is apparently a taxonomic synonym of myces farinosus (Pacioni & Frizzi 1978). Noteworthy, P. cicadae. the three former taxa, accommodated in subclade II-A The anamorph genus Paecilomyces (Bainier 1907) of clade II, attack Scarabaeidae, whereas the latter, is characterized by synnematous or mononematous accommodated in subclade II-C, attacks Carabidae conidiogenous structures that mostly consist of ver- or Coccinellidae hosts. The lepidopteran parasites in- ticillate or irregularly branched conidiophores produc- clude C. bifusispora, C. hepialidicola (Kobayasi & ing whorls of solitary or multiple conidiogenous cells Shimizu 1983), C. militaris, C. pruinosa (Petch 1924), C. terminally on each branch. The cylindrical or swollen pseudomilitaris (Hywel-Jones 1994), C. takaomontana basal portion of the conidiogenous cells often taper (Kobayasi 1941), and C. tuberculata, the latter rep- abruptly into a long distinct neck. The one-celled resented in this study by its suggested anamorph (rarely two-celled) conidia are produced in dry, diver- Akanthomyces pistillariiformis (Samson & Evans 1974). gent or tangled basipetal, hyaline or slightly pigmented, Cordyceps spp. of clade III (cfr Figs 1, 4) are also smooth-walled or echinulate chains. Chlamydospores, highly diverse in morphology, ecology and anamorph when present, are usually thick-walled, smooth-walled affiliation. The analysis of the reweighted Molecular- or ornamented and borne singly or in short chains ALL data set suggests a relationship (75% BS) among (Brown & Smith 1957, Samson 1974). Samson (1974) the taxa of this evolutionary lineage (cfr Fig. 1), how- recognized two sections, i.e. Paecilomyces and ever, the NJ distance analysis did not recognize group 1 Isarioidea. Paecilomyces farinosus is the type species (including subclade III-D and III-E) and group 2 of sect. Isarioidea. Samson (1974) pointed out the (subclade III-F) of clade III to share a common ances- morphological similarity between Paecilomyces sect. tor (cfr Figs 1, 4). This may well be a result of long Isarioidea and Akanthomyces and stated that A. gracilis, branch attraction in the parsimony analyses; the high a species particularly common on ants (Samson & number of synapomorphic characters supporting sub- Evans 1974), may form a link between the two genera. clade III-F contemporaneously creates a long branch Ø. Stensrud, N. L. Hywel-Jones and T. Schumacher 51 length. Nevertheless, the 100% BS, and additionally evolution of Cordyceps. We conclude that presence the fact that subclade III-F is delimitated by mor- of obliquely immersed perithecia is a phylogenetically phology and anamorph affiliation (discussed in the significant character that separates Neocordyceps and following), implies that this group of Cordyceps spp. Eucordyceps sect. Cremastocarpon from other Cordy- possibly deserve rank as a distinct genus. ceps spp. In this emended concept of Neocordyceps, The common ancestry of the plant pathogenic taxa the host range would include a range of insect orders, Claviceps purpurea and Epichloe¨ typhina (cfr subclade which further demonstrates the generally low signifi- III-D) is highly supported (94% BS), and the inclusion cance of host preference as a phylogenetically signifi- of Claviceps/Epichloe¨ in clade III therefore makes cant character in Cordyceps. Cordyceps paraphyletic. A paraphyly of Cordyceps The two included Hymenostilbe taxa were accom- was also suggested by Suh et al. (1998), Sung et al. modated in subclade III-F of clade III. All known (2001), and Artjariyasripong et al. (2001), and teleo- anamorphs of Cordyceps spp. of this subclade are morph genera that affect the appraisal of monophyly ascribable to Hymenostilbe (Petch 1931a, 1932, 1937, in Cordyceps also include Atkinsonella, Atricordyceps, Samson & Evans 1975, Hywel-Jones 1996b). The for- Balansia, Cordycepioideus, Hypocrella, Myriogenospora mation of a Hymenostilbe anamorph, in addition and Torrubiella. to the character of obliquely immersed perithecia in the Metarhizium anisopliae, M. taii and its teleomorph stroma, seems to characterize this Cordyceps lineage. Cordyceps taii (Liang et al. 1991) constituted a 100% Hymenostilbe was proposed by Petch (1931a) to ac- supported subclade (III-E). Huang et al. (2002) placed commodate H. muscaria, the imperfect state of C. M. taii in synonymy with M. anisopliae var. anisopliae, forquignoni. Petch (1932, 1933, 1937, 1944) later estab- based on ITS nrDNA sequence similarity. Other lished a number of new Hymenostilbe (anamorph) – Metarhizium taxa in our study, i.e. Metarhizium album Cordyceps (teleomorph) connections. Moureau (1949) and Cordyceps brittlebankisoides (a newly described regarded Cordyceps nutans a subspecies of C. bicephala taxon with a Metarhizium anamorph; Liu et al. 2001b) (C. bicephala subsp. nutans), a disposition that gains no were also accommodated in clade III. Metarhizium was support from the ITS sequence data. erected by Sorokin (1883) for mycelia often wholly The broad assemblance of Cordyceps taxa of clade covering the host individuals (Humber 1998), produc- IV (Figs 1, 5) represents mainly tropical and subtropi- ing compact patches of branched, candelabrum-like cal taxa plus some more widespread mycogenous and densely intertwined conidiophores. The conidio- taxa (subclade IV-J). The group shows morphological genous cells have rounded to conical apices, and the heterogeneity, spanning from the clavate C. ophioglos- conidia are aseptate, cylindrical to ovoid, and produced soides (to 120 mm high) to the capitate C. clavulata in a solid mass of parallel chains. Curran et al. (1994) (to 3 mm high) and to the peltate C. cantharelloides and Driver, Milner & Trueman (2000) revised the (Samson & Evans 1985). However, this somewhat genus, including nrDNA sequence data, and found heterogenous group was well supported (88% BS) the level of intra- and interspecific nrDNA variation from the parsimony analysis of the reweighted to be high. The apparent aberrant placement of MolecularALL data set, and the separate analyses of Tolypocladium parasiticum in clade III, separated from the data subset of clade IV inferred the same subclades the rest of the Tolypocladium taxa (clade IV), calls for a with minor differences. One group of taxa consisting of re-assessment of the diagnostic characters of this taxon. Cordyceps multiaxialis, C. nepalensis, C. sinensis and Bissett (1983) noticed that T. parasiticum produced Hirsutella sinensis, was recognized (subclade IV-G), chlamydospores in culture, a feature not observed in of which the two former were described recently from other Tolypocladium spp. T. parasiticum also differs alpine areas of China and Nepal (Zang & Kinjo 1998). from other Tolypocladium spp. in being the only species The conclusion of Liu et al. (2001c) that C. multiaxialis that is parasitic on bdelloid rotifers (Barron 1980). and C. nepalensis are synonyms of C. sinensis, is not Efrapeptins, which are key substances in Tolypocla- contradicted by the level of ITS divergence observed dium spp., are not detected in T. parasiticum (Krasnoff among these taxa in our study. Here, the long branch of & Gupta 1992). Accordingly, there are many reasons C. sinensis is largely due to a number of unique CpT to question whether T. parasiticum represents a ‘true’ transitions, of which several were located in the con- Tolypocladium. served 5.8S gene. Similar A-T-biased sequences have Artjariyasripong et al. (2001) and Hywel-Jones (2002) been observed in other fungal taxa as well, but remain suggested species of Cordyceps subgenus Neocordyceps, unexplained. A number of anamorph taxa have been all of which have perithecia obliquely immersed in the attributed to C. sinensis, e.g. Paecilomyces sinensis stroma, to be closely related. Our results indicate that (Chen, Xiao & Shi 1984), Synnematium sinense (Yin & all Cordyceps spp. with obliquely immersed perithecia, Shen 1990) and Chrysosporium sinense (Liang 1991a). i.e. subgenus Neocordyceps and in addition subgenus An anamorph-teleomorph connection between Hirsu- Eucordyceps sect. Cremastocarpon (Kobayasi 1982), tella sinensis and Cordyceps sinensis was suggested by are closely related (100% BS, cfr subclade III-F). The Liu et al. (1989) and has later been confirmed by others MPTs suggest that a redirection of perithecia from (Zhao et al. 1999, Li, Huang & Fan 2000, Chen et al. square to oblique in the stroma is a single event in the 2001, Liu et al. 2001c). Phylogenetic classification of Cordyceps 52

Noteworthy, scale insects attacked by Cordyceps Spatafora & Blackwell (1993) suggested that spp. seem to have a Hirsutella-anamorph. Patouillard Cordyceps capitata was basal in the Clavicipitales based (1892), in describing Hirsutella, erroneously placed the on 18S nrDNA sequence data, and concluded that genus in Basidiomycota, a misconception later cor- ‘if the Clavicipitales and Hypocrea clades are sister rected by Speare (1920). Petch (1924) was the first to groups, one is justified in polarizing the fungicolous recognize an association between Hirsutella and tendency within the Clavicipitales as primitive’. Others Cordyceps, noting that the ant pathogen C. unilateralis have contradicted this opinion, e.g. Suh et al. (1998) had a Hirsutella anamorph, i.e. H. formicarum (Petch and Engh (1999), who recognized the mycogenous 1934). Mains (1951) revised Hirsutella to include species as derived within Cordyceps. Nikoh & Fukatsu synnematous as well as mononematous species (Minter (2000) stated that in the evolution of Cordyceps, & Brady 1980, Samson, McCoy & O’Donnell 1980, parasites of Elaphomyces spp. probably originated Samson & Evans 1991, Humber 1998). Synnemata from parasites of cicada nymphs. The suggestion that (when present) of Hirsutella are erect, cylindrical to the mycogenous species ‘have something in common’ slightly tapered, simple or branched, varying from with cicada parasites was supported from a recent short and verrucose to long and filiform. Phialides are observation by Spatafora (2002). He found that four scattered to crowded with inflated lower portions and stromata of the usually mycogenous species Cordyceps long slender sterigma, projecting laterally from synne- ophioglossoides were growing separately in the same matous or non-synnematous hyphae that emerge from locality on four adjacent host specimens of the host body. The variously shaped conidia are one- or Elaphomyces sp. (3) and a cicada host (1). In our two-celled and borne singly or in small groups with analyses, the capitate representatives C. capitata, C. or without mucus (Evans & Samson 1982, 1984). longisegmentis and Cordyceps sp. branched off basally Hirsutella resembles Akanthomyces and Hymenostilbe to the rest of the taxa in subclade IV-J (70% BS). The in synnematal structures and in the origin and location cicada parasites C. inegoensis and C. paradoxa, which of the phialides, however, the two latter genera have have clavate stromata (Kobayasi 1939, Kobayasi & short or wanting sterigma and the conidia are produced Shimizu 1963), appear closely related to the clavate in scanty or no mucus. The ITS phylogenies un- Elaphomyces parasites, i.e. C. japonica (Lloyd 1920), ambiguously distinguished between these anamorph C. jezoensis (Imai 1929) and C. ophioglossoides. genera, in referring Hirsutella spp. to subclade IV-G Five Tolypocladium spp. comprised a major portion and Hymenostilbe spp. to subclade III-F. According of subclade IV-J. Tolypocladium was established by to Suh et al. (1998), a Hirsutella anamorph was also Gams (1971) for anamorph taxa producing conidia observed in the termite pathogen Cordycepioideus bis- terminally or laterally from aerial hyphae and verticil- porus, a species found to be closely related to Cordyceps late phialides, sometimes supported by short lateral coccidiicola and C. cochlidiicola (Sung et al. 2001). cells. The characteristic ‘flask-shaped’ phialides consist Cordyceps taxa of subclade IV-G, except in C. emeien- of a swollen base and a narrowly tapering, frequently sis where the host range is uncertain, are all associated bent neck. The small, one-celled conidia are borne with scale insects and lepidopteran hosts (Berkeley in slimy heads (Gams 1971). Arx (1986) referred 1843, Kobayasi & Shimizu 1978, 1980b). Tolypocladium to Beauveria, based on a re-investi- In the analyses of the clade IV data subset, the spider gation of the type specimens of the two genera, i.e. parasite Cordyceps cylindrica (Petch 1937), the lepi- T. inflatum and B. bassiana. Subsequent workers have dopteran parasite C. gracilis and the cicada parasite strongly opposed this solution, e.g. Gams et al. (1998), C. heteropoda (Kobayasi 1939) were accommodated in who pointed out the fundamental difference in the the same subclade (IV-H) (76% BS), with Cordyceps conidiogenesis of the two genera, i.e. Tolypocladium gracilis and C. heteropoda found to be very closely re- with phialidic, and Beauveria with polyblastic conidio- lated (100% BS). In these two species a palisade layer genesis. Tolypocladium and Beauveria also differ of erect hyphal tips occur in the outer cortex, a feature in secondary metabolite production and rRNA geno- not observed in C. cylindrica (Petch 1937, Mains 1954, types (Mugnai et al. 1989, Rakotonirainy et al. 1991, Kobayasi & Shimizu 1977). A similar structure is ob- Kadlec et al. 1994, Todorova, Cote & Coderre 1998). served in C. longisegmentis, positioned in subclade IV- The inferred ITS phylogenies clearly demonstrate J. The suggestion that Nomuraea atypicola represents that Beauveria and Tolypocladium represent divergent the conidial state of C. cylindrica (Petch 1937, 1939) groups that should be maintained as separate genera was confirmed by Evans (1982) and Hywel-Jones & (Engh 1999; this study, cfr Fig. 1). Hodge, Krasnoff & Sivichai (1995). The monotypic genus Paraisaria Humber (1996) demonstrated an anamorph-teleo- (Samson & Brady 1983) accommodates the anamorph morph connection between Tolypocladium inflatum, the of C. gracilis, while C. heteropoda has not been linked alleged type of the genus Tolypocladium Gams, and to any anamorph genus. The coleopteran parasite C. Cordyceps subsessilis, using biochemical profiles. This konnoana (Kobayasi & Shimizu 1980a) was included in conclusion was also supported by comparing ITS subclade IV-H in the distance analysis of the clade IV nrDNA sequences from the type specimen of T. in- data subset, but grouped with C. entomorrhiza, also flatum and North-American material of C. subsessilis attacking Coleoptera, in the parsimony analysis. (Engh 1999). Although weakly supported (51% BS), Ø. Stensrud, N. L. Hywel-Jones and T. Schumacher 53 the ‘Tolypocladium’-clade (except T. parasiticum) considered host preference to be of little importance formed a monophyletic group derived within subclade and referred the mycogenous species to a separate IV-J. The low BS support may be attributed to the high series of taxa, i.e. subgen. Eucordyceps sect. Cystocarpon sequence similarity in this taxon, rather than doubtful subsect. Eucystocarpon ser. Mycogenae. Host associ- relationships (cfr James et al. 2001). Even though a ations, when superimposed on the ITS phylogeny, sug- 100% sequence similarity was observed between speci- gest, however, that some groups of taxa have conserved mens of T. inflatum and C. subsessilis, their grouping the endoparasite-host interactions to some extent; taxa did not achieve 100% BS in our analyses. This dem- of clade I and subclade IV-K all attack cicadae, taxa of onstrates what was pointed out by Berbee, Carmean & clade II are restricted to the insect orders Coleoptera Winka (2000), that more data is often required in order and Lepidoptera, taxa of subclade IV-G include para- to achieve high BS for a node when the percentage of sites on scale insects and lepidopteran hosts, and the informative characters supporting the node is low. mycogenous taxa are referred to subclade IV-J. Efrapeptins, which have insecticidal activity (Krasnoff Until now, anamorph affiliation as a contributing et al. 1991), are detected in all Tolypocladium spp. of factor in the subdivision of Cordyceps has been mostly subclade IV-J (Krasnoff & Gupta 1992). These taxa neglected. Although closely related species may pro- have been maintained as either soil inhabitants or in- duce conidial states ascribable to different anamorph sect pathogens (Bissett 1983). However, Carlsen (2002) genera (e.g. clade II), the opposite situation, that dis- recently isolated T. cylindrosporum from a sterilized tantly related teleomorph species produce conidial root of Carex capillaris, indicating that this taxon may states attributed to the same anamorph genus, is quite have an endophytic life strategy as well. exceptional. Although a sexual state is unknown in In agreement with Nikoh & Fukatsu (2001), the most anamorph taxa included in this study, those ana- grouping of the cicada parasites Cordyceps sobolifera morphs that have a proven teleomorph all produced and C. yakusimensis (Kobayasi 1939, Kobayasi & teleomorphs in Cordyceps. The fact that teleomorphs Shimizu 1963) got high BS support (100%) in our produce similar anamorphs of the same generic affili- study (subclade IV-K). Although speculative when ation (e.g. subclade III-F and IV-G) is a strong indi- based on 2(3) taxa, this may imply a conservation of an cation of genetic relatedness in these groups. endoparasite-host connection in this subclade. The genus Polycephalomyces, with P. formosus as the type, was erected by Kobayasi (1941). Later, Mains (1948) included Polycephalomyces ramosus, a taxon ACKNOWLEDGEMENTS that has been variously interpreted in the literature This study was financially supported by the University of Oslo. We (Petch 1933, 1939) with respect to a connection with acknowledge Ingeborg B. Engh and Julian Mitchell for providing Cordyceps entomorrhiza. A 100% ITS sequence simi- sequence data. We thank Anna M. Bendiksby, Kjell T. Hansen, larity between P. ramosus and C. entomorrhiza suggests Ha˚ vard Kauserud, and Kenneth Weierud for technical assistance. Herbaria and collegues in the Nordic countries are acknowledged for conspecificity of the two taxa. providing specimens. 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