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Cordyceps cardinalis sp. nov., a new species of Cordyceps with an east Asian-eastern North American distribution

     Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331-2902

	ABSTRACT

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Cordyceps cardinalis, a pathogen of lepidopteran larva, is described as a new species from the southern Appalachian Mountains of the eastern United States and from southeastern Japan. It is macroscopically similar to both C. militaris and C. pseudomilitaris, however, microscopic, cultural and molecular evidence support it being a phylogenetically distinct species. Cordyceps cardinalis is most similar to C. pseudomilitaris in the microscopic characters of non-disarticulating ascospore morphology and its host affinity for lepidopteran larvae, which contrast with the characters of C. militaris. Aspects of morphology, host association, phylogeny and biogeography are discussed.

Key words: Clavicipitaceae, Cordyceps, Lepidoptera, pathogen, systematics

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
Cordyceps (Fr.) Link includes pathogens of species from nine orders of arthropods and parasites of one genus of fungi, Elaphomyces (Kobayashi 1941, 1982; Mains 1957, 1958). The type of the genus, and possibly best known species, is C. militaris (L. : Fr.) Link. It is characterized by the production of orange to orangish-red colored stromata that erupt from the carcass of the dead host (Mains 1958, Kobayasi 1982). C. militaris is one of the most frequently collected species with collections from all major continents except Antarctica; however, it is also one of the most variable species in the genus with respect to morphology and host affiliation. It is most commonly reported from numerous families of lepidopteran pupae with less frequent reports from lepidopteran larvae and coleopteran pupae (Kobayashi 1941, Mains 1958). Therefore, the species generally is recognized as a cosmopolitan taxon with a relatively broad host range that is characterized by a propensity for attacking lepidopteran pupae but with the potential to attack multiple stages and hosts from more than one order of arthropods (Kobayashi 1941, Mains 1958).

A number of macroscopically similar species, which either had been considered conspecific with C. militaris, close relatives, or simply had gone unnoticed among large collections from a variety of hosts and life-cycle stages, have been described (Kobayashi 1982, Mains 1947, Hywel-Jones 1994). Some examples include Cordyceps washingtonensis Mains from lepidopteran pupae (Mains 1947), C. rosea Kobayasi & Shimizu from lepidopteran larvae (Kobayasi & Shimizu 1982), C. roseostromata Kobayasi & Shimizu from coleopteran larvae (Kobayasi & Shimizu 1983), and C. pseudomilitaris Hywel-Jones & Sivichai from lepidopteran larvae (Hywel-Jones 1994). In the example of C. pseudomilitaris, Hywel-Jones (1994) noted its macroscopic similarity to C. militaris but also recognized diagnostic differences between the two species. Cordyceps pseudomilitaris possesses nondisarticulating ascospores in contrast to C. militaris, which produces ascospores that disarticulate into partspores at the septations. The anamorph of C. militaris is a Lecanicillium Gams & Zare (= Verticillium Nees), whereas C. pseudomilitaris mainly produces simple, unbranched phialides that give rise to a single conidiogenous cell and conidium (Hywel-Jones 1994), a morphology that is more appropriately accommodated in the recently described taxon Simplicillium Gams & Zare (Zare and Gams 2001). Finally, C. pseudomilitaris is known only from lepidopteran larvae of undetermined familial affinity whereas C. militaris most frequently is collected from pupae of a number of different families of moths. Recent molecular data for C. pseudomilitaris supported its distinction from C. militaris (Artjariyasripong et al 2001) and that these characters may be informative in recognizing unique and possibly cryptic species of the C. militaris complex.

For the past several years we have been conducting a phylogenetic investigation of the genus Cordyceps and related entomopathogenic fungi. This research has involved several collecting trips to the southern Appalachian Mountains of North America, the richest region of the United States with respect to Cordyceps species diversity (Mains 1958). As part of this survey, we made numerous collections of C. militaris sensu lato from both lepidopteran pupae and larvae. Here we report that morphological, cultural, and molecular data all are consistent with there being two phylogenetic species present among these collections. One is specific to lepidopteran pupae and is consistent with C. militaris sensu stricto. The second is specific to lepidopteran larvae and is more closely related to C. pseudomilitaris but represents a previously undescribed phylogenetic species of the genus Cordyceps. During this investigation, specimens of C. militaris-like, lepidopteran pathogen from Japan were made available to us. After morphological and molecular investigations, these specimens were found to be arguably conspecific with the undescribed pathogen of lepidopteran from the southern Appalachians, supporting an east Asian-eastern Northern American disjunct distribution.

	MATERIALS AND METHODS

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Field collections. – Collecting expeditions were conducted during Jul 1999 and Jul 2000. Specimens were collected with host when possible. Notes of stromatal color and size were taken on fresh specimens. Individual perithecia were dissected from the stromata, and ascospores were shot onto potato-dextrose agar (PDA) amended with Ampicillin to a final concentration of 100 µg/ml. All cultures were deposited in the ARS entomopathogenic fungus collection (AR-SEF) and the Centraal Bureau voor Schimmelcultures (CBS). Specimens were dried on a food dehydrator on low heat with air movement. Dried specimens were placed in sealed plastic bags with silica gel and stored in specimen boxes for transport and shipment. Four specimens from Zaitacho, Mitoyogun, Kagawa Prefecture, Shikoku Island, Japan, of a C. militaris-like fungi also were sampled. These latter specimens also are pathogens of lepidopteran larvae and were selected due to their morphological similarity to the C. militaris-like species from the southern Appalachians and existing preliminary molecular data from one specimen. All specimens were deposited in the Mycological Collection of the Oregon State University Herbarium (OSC).

Morphological observations. – For examination of ascus and ascospore characteristics, dried specimens were rehydrated and mounted in sterilized water. Microscopic examinations of anamorphic characters were made from cultures maintained on potato-carrot agar (PCA) at 24 C in the dark for 10 d. Conidiophore branching and conidial arrangement were observed as described by Zare and Gams (2001), and slide preparations were mounted in lactic acid-cotton blue. Twenty-five measurements were made for asci, ascospores, phialides and conidia. To examine the structure of perithecia and their attachment to stromata, dried specimens were fixed in FAA (five parts stock formalin:five parts glacial acetic acid:90% parts 50% ethanol) and dehydrated in a graded ethanol series. Samples were transferred from 95% ethanol into 1:1, plastic infiltration solution: 95% ethanol under vacuum. After 12 h, samples were vacuum-infiltrated with straight solution. Samples were embedded in Historesin (Leica) GMA (glycol methacrylate) plastic and sectioned (5–6 µm) on a rotary microtome with steel knives. Sections were stained in 0.5% TBO (Toluidine Blue O). All microscopic examinations were made using either bright field or Normaski DIC on a Leica DMRB compound microscope.

DNA extraction, PCR and sequencing. – DNA was extracted from cultures by a modified CTAB method as previously described in Sung et al (2001). Approximately 1150 base pairs (bp) of the nuclear small-subunit (SSU) ribosomal DNA (rDNA) and ca. 950 bp of nuclear large-subunit (LSU) rDNA were amplified using PCR. Amplifications and sequencing reactions were performed as described in Sung et al (2001).

Phylogenetic analyses. – Sequences of nuclear SSU rDNA and LSU rDNA from 35 taxa were selected from the major phylogenetic groups of the Clavicipitaceae based on the result of Nikoh and Fukatsu (2000) and Sung et al (2001). Three representative taxa of the Hypocreaceae were used as out-group. Species included in this study and their GenBank accession numbers of rDNA sequences (SSU rDNA; LSU rDNA) weres: Aphysiostroma stercorarium Barassa et al (U32398; U47820), Hypocrea schweinitzii (Fr.) Sacc. (L36986; U47833), Sphaerostilbella aureonitens (Tul.) Seifert et al (U32415; U00755), Atkinsonella hypoxylon (Peck) Diehl (U44034; U57087), Balansia aristidae Atkinson (U44035; U57677), Balansia henningsiana (Moell.) Diehl (U44036; U57678), Beauveria bassiana (Bals.) Vuill. (AB027336; AB027382), Claviceps paspali F. Stevens & J. G. Hall (U32401; U47826), Claviceps purpurea (Fr. : Fr.) Tul. (U44040; U57085), Cordycepioideus bisporus Stifler (AF009651; AF009654), Cordyceps bifusispora O. E. Eriksson (AF339571; AF339521), C. capitata (Fr.) Link (AB027318; AB027364), C. coccidiicola Kobayasi & Shimizu (AB031195; AB031196), C. cochlidiicola Kobayasi & Shimizu (AB027331; AB027377), C. heteropoda Kobayasi (AB027327; AB027373), C. militaris (AF049146; AF049166), C. militaris (AB027333; AB027379), C. ophioglossoides (Ehrh.) Link (AB027321; AB027367), C. pseudomilitaris (AF327394; AF327376), C. roseostromata Kobayasi & Shimizu (AF339573; AF339523), C. scarabaeicola Kobayasi & Shimizu (AF339574; AF339524), C. sobolifera Berk (AB027328; AB027374), C. takaomontana Kobayasi (AB044631; AB044637), Cordyceps sp. (AB027332; Ab027378), Epichloe amarillans J. F. White (U35034; U57680), Epichloe typhina (Pers.) Tul. (U32405; U17396), Lecanicillium psalliotae (Treschow) Zare & W. Gams (AF339607; AF339558), Myriogenospora atramentosa (Berk. & Curt.), Diehl (U44155; U57084), Paecilomyces lilacinus (Thom) Samson (AF339583; AF339534), Paecilomyces javanicus (Friderichs & Ballly) Brown & Smith (AF339582; AF339533), Paecilomyces tenuipes (Peck) Samson (AB027334; AB027380), Simplicillium lamellicola (F. E. W. Smith) Zare & W. Gams (AF339601; AF339552), S. lanosoniveum (van Beyma) Zare & W. Gams (AF339603; AF339554), S. obclavatum (W. Gams) Zare & W. Gams (AF339567; AF339517), Torrubiella confragosa Mains (AF339604, AF339555). GenBank accession numbers of sequences determined in this study are listed in Results.

Sequences were edited in SeqEd version 1.0.3 and manually aligned in PAUP* 4.0 (Swofford 2002) using a color font. Intron sequences for nSSU rDNA of fours species (AB027331, AB031195, AB027328 and AB027327) were deleted, and the final alignment was deposited in TreeBase SN1280. PAUP* 4.0 was used to perform the maximum parsimony analyses on the combined dataset of nSSU and nLSU rDNA. Heuristic searches were conducted using these options: 100 replicates of random sequence addition, TBR branch swapping and MulTrees option in effect. Insertions and deletions are minimized and treated as missing data. Ambiguously aligned sequence regions were excluded from the data matrix before analysis. Relative support of the resulting trees was determined by 1000 bootstrap replications on informative characters only with the previously mentioned search options (Felsenstein 1985). The phylogenetic trees generated from the combined dataset with nSSU and nLSU rDNA datasets were rooted with A. stercorarium, H. schweinitzii and S. aureonitens.

	TAXONOMY

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Examination of perithecium, ascus, ascospore, and anamorph characters combined with phylogenetic analyses of molecular data revealed that the C. militaris-like fungus which parastizes lepidopteran larva in the southern Appalachians of the eastern United States and in Japan does not match C. militaris, C. pseudomilitaris or other closely related species. Therefore, a new species, C. cardinalis, is proposed.

Cordyceps cardinalis G.-H. Sung & J. W. Spatafora, sp. nov.

Stromata solitaria vel plures, simplicia vel raro ramosa, in larvis Lepidopterorum. Stipes carnosus, ochraceo-aurantius vel cardinalis, cylindricus vel versus apicem amplificatus, 4–50 x 0.5–1.5 mm. Area fertilis terminalis, armeniaca vel cardinalula, cylindrica, ellipsoidea vel fusiformis, 2–9 x 1–4 mm. Perithecia congesta, laxe inclusa, in orientatione ordinalia, ellipsoidea, fusiformia vel obclavata, 230–540 x 110–240 µm. Asci 8-spori, hyalini, cylindrici, 175–330 x 3–5 µm, apice prominenti. Ascosporae laeves, filiformes, hyalinae, irregulariter multiseptatae, into partisporas non rumpentes, 160–320 x 1 µm.

Stromata solitary or occasionally several, simple or rarely branched, on larva of Lepidoptera. Stipe fleshly, ochraceous orange to red, cylindrical to enlarging apically, 4–50 x 0.5–1.5 mm. Fertile area terminal, reddish orange to reddish, cylindrical, elliptical to fusiform, 2–9 x 1–4 mm. Perithecia crowded, loosely embedded, ordinal in orientation, elliptical to fusiform to obclavate, 230–540 x 110–240 µm (FIG. 1E, F). Asci 8-spored, hyaline, cylindrical 175–330 x 3–5 µm, possessing a prominent apex (FIG. 1B). Ascospores smooth, filiform, hyaline, irregularly multiseptate, not fragmenting into part-spores, 160–320 x 1 µm (FIG. 1A).

View larger version (79K): [in this window] [in a new window]   FIG. 1. Morphology and ecology of C. cardinalis. A. Irregular septation of ascospores. B. Distal end of ascus showing pronounced ascus cap. C. Proximal end of ascus showing ascus foot characteristic of the basal fascicle. D. Host of C. cardinalis, larval stage of Archolophinae lying in its web. E. Cross section of stroma, showing presentation of perithecia on a stroma. F. Enlargement of single perithecium with an ascus projecting through the ostiole. G. Typical stromata of C. cardinalis fruiting from a larva among moss and forest litter. (FIG. 1A–C, FIG. 1E, F) in Nomarski interference contrast, FIG. 1D with a digital Nikon 990 COOLPIX, FIG. 1G with a 35 mm Cannon EOS-1D with a 100 mm macro lens. Scale bars in FIG. 1A–C = 4.5 µm; FIG. 1D = 1.5 mm; FIG. 1E = 87.5 µm; FIG. 1F = 30.5 µm; FIG. 1G = 2.25 mm.

Specimen examined. – USA. TENNSSEE: Great Smoky Mountains National Park, Hen Wallow Trail, 19 Jul 2000, OSC 93609 (HOLOTY PE), OSC 93610, OSC 93611, OSC 93612, OSC 93613; Roan Mountain State Park, 25 Jul 2000, OSC 93614, OSC 93615, OSC 93616, OSC 93617, OSC 93618, OSC 93619. JAPAN. KAGAWA: Shikoku, 23 Jun 1997, OSC 93619, OSC 93620, OSC 93621, OSC 93622.

Etymology. – Red, in reference to both the color of the stroma and the pigment produced in culture.

Commentary. – Cordyceps cardinalis is collected most frequently as a single, small, reddish stroma emerging from lepidopteran larvae buried in the upper 1–2 cm of soil or from under well-developed moss mats (FIG. 1G). The host frequently is enclosed in a web or silken matrix (FIG. 1D). In ascus maturation, young asci are characterized by a pronounced apical cap or apex that possesses a distinct canal. In the mature ascus, the apical cap becomes less pronounced, flattened and the canal is usually not as apparent (FIG. 1B). The arrangement of ascospores within asci was more or less parallel for the entire length of the asci, indicating that the ascospores were of approximately the same length as the asci (FIG. 1C). The nondisarticulating ascospores of C. cardinalis are irregularly septate with the size of each segment ranging from 4–13 µm long (FIG. 1A). In the conidial stage of C. cardinalis, cultures are moderately fast growing in PDA, attaining approximately 25 ± 8 mm at 18 C for 10 d. Aerial mycelium is cottony in texture and whitish to whitish yellow and the reverse side of cultures is reddish. Phialides are solitary or in whorls of 2 or 3 and swollen at the base or slightly flask-shaped, measuring 7–25 x 1.5–3 µm wide near the base and tapering to 0.5–1.3 µm at the apex (FIG. 2). Conidia are ellipsoidal to elliptical, measuring 4–6 x 1.5–2.5 µm, and are produced in long divergent sympodially imbricate chains (FIG. 2). Chlamydospores are absent. The mitotic stage is best described as being Clonostachys-like or Mariannaea-like. In comparison with other species, C. cardinalis differs from C. militaris in color and size of stromata, disarticulation of ascospores, type of anamorph produced in culture, and developmental stage of host in which stromata are produced. Cordyceps cardinalis is most similar to C. pseudomilitaris in both morphology and ecology but differs in size of asci, ascospores, type of anamorph and in their color characteristics in culture.

View larger version (16K): [in this window] [in a new window]   FIG. 2. Line drawing of variation in anamorph of C. cardinalis. Variation in phialide morphology ranges from simple Simplicillium-like phialides to branched verticillate Lecanicillium-like phialides. Conidium ontogeny is basipetal with conidia arranged in partially imbricate chains. Scale bar in FIG. 2 = 10.0 µm.

	RESULTS

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Host identification. – Intact or nearly intact hosts were collected with approximately one-quarter of the specimens. Hosts that retained enough characters for identification were identified as members of the subfamily Archolophinae (Lepidoptera: Tineidae) and often were enclosed in a loosely woven web or silken matrix. In contrast, the hosts of C. militaris s.s., which were collected throughout the same region, all were pupae of the lepidopteran families Arctiidae and Hesperiidae.

Phylogenetic analyses. – The combined alignment of the nuclear SSU and LSU rDNA dataset is 2017 bases in length, 1099 from the NS1/NS4 region of the SSU rDNA and 918 from the LR0R/LR5 region of LSU rDNA. Of these, 111 SSU rDNA positions and 86 LSU rDNA positions were ambiguously aligned or contain an excess of missing data near the 5' and 3' ends. These positions were excluded and the final dataset included 1820 nucleotide positions. One hundred eighty-seven characters were parsimony informative, 75 from the SSU rDNA and 112 from the LSU rDNA. Maximum parsimony analysis of the 42 taxa dataset yielded six equally most-parsimonious trees of 613 steps with consistency indices (CI) of 0.4078 and retention indices (RI) of 0.7105. One of six equally parsimonious trees is shown in FIG. 3 with nodes that collapse in the strict consensus denoted with asterisks.

View larger version (44K): [in this window] [in a new window]   FIG. 3. One of six most-parsimonious trees from the maximum parsimony analysis of combined SSU and LSU rDNA data. Asterisks denote branches that collapse in a strict consensus. Numbers above nodes are nonparametric bootstrap values from 1000 replications.

	DISCUSSION

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Morphological comparison of C. cardinalis with other C. militaris-like species. – Cordyceps cardinalis macroscopically is similar to numerous species of Cordyceps with the two best examples for purpose of comparison being C. militaris and C. pseudomilitaris. These three species differ, however, in several microscopic, cultural and ecological characteristics, which in this case, are indicative of phylogenetic species. The characteristics of C. militaris and C. pseudomilitaris are summarized according to the previous studies of Mains (1958) and Hywel-Jones (1994), with the characteristics for differentiating these three species listed in TABLE I. In overall size of stromata, C. militaris is relatively large compared to C. cardinalis and C. pseudomilitaris and there is slight, but observable, difference in the color of stromata, at least in the southern Appalachians. C. militaris is more of an orange to orangish-red, whereas C. cardinalis and C. pseudomilitaris are orangish-red to red. We previously had interpreted the characters of stromatal size and color to be variable traits within C. militaris that might have been linked to ecology (e.g., host) or simply represented intraspecific variation. This interpretation of C. militaris in the southern Appalachians was not supported by the data presented here (FIG. 3). However, the integration of C. roseostromata among C. militaris isolates in the molecular analyses (FIG. 3) suggests that there may well be greater color variation within C. militaris in other parts of the world (e.g., in eastern Asia).

Anamorphic and cultural differences among C. cardinalis, C. militaris and C. pseudomilitaris. – The anamorph of C. militaris in culture is a Lecanicillium, which exhibits fairly standard verticillate awl-shaped phialides and conidium production. The anamorph of C. pseudomilitaris consists of simple phialides resembling a solitary phialide of Lecanicillium or Simplicillium-like species. In contrast, the anamorph of C. cardinalis exhibits a solitary or verticillate phialide that is swollen at the base and its conidia are produced in partially imbricate chains (FIG. 2). This latter morphology is somewhat intermediate between the anamorph genera Clonostachys Corda and Mariannaea Arnaud & Samson (Samson 1974), which are described as possessing similarly shaped phialides with swollen basal portions and producing conidia in imbricate chains. While the anamorph of C. cardinalis also produces imbricate chains of conidia it differs from Mariannaea species due to its partially imbricate arrangement of conidia (FIG. 2). The phylogenetic analyses are consistent with these differences and indicate that C. cardinalis does not form a monophyletic group with other clavicipitaceous species of Mariannaea (e.g., M. pruinosa). The anamorph of C. cardinalis differs from typical species of Clonostachys in that the phialides of C. cardinalis are produced more sparsely and do not form dense heads of phialides and chains typical of Clonostachys (Schroers 2001).

While C. cardinalis and C. pseudomilitaris do differ in anamorph morphology, the most distinguishing nonmolecular trait is the color of the reverse side of culture (TABLE I). Cordyceps cardinalis imparts a red pigment to the medium, whereas the reverse side of C. militaris and C. pseudomilitaris cultures are cream or yellowish white. Among other clavicipitaceous species, L. psalliotae (Treschow) Zare & W. Gams exhibits the same color in culture (Zare and Gams 2001) as that of C. cardinalis. However, L. psalliotae possesses awl-shaped phialides and molecular data do not support its conspecificity with C. cardinalis or any of the other known teleomorphs of Cordyceps (FIG. 3).

Phylogenetics of C. cardinalis. – The main objectives of the phylogenetic analyses presented here were to determine the phylogenetic affinity of C. cardinalis to C. militaris and C. pseudomilitaris, to test the taxonomic utility of the aforementioned morphological and ecological traits in distinguishing closely related species of Cordyceps, and to determine if the morphologically and ecologically similar specimens from eastern North America and Japan were conspecific. Although the Clavicipitaceae is a morphologically and ecologically unique family of fungi, molecular phylogenetic analyses have never strongly supported or rejected its monophyly (Spatafora and Blackwell 1993, Suh et al 1998, Gams et al 1998, Sung et al 2001). These analyses presented here are not designed to test the monophyly of the family and should not be interpreted as such. The three main clades of clavicipitaceous fungi inferred as part of this study are consistent with phylogenetic hypotheses based on more extensive taxon sampling, but the interrelationships of the three clades remains equivocal (Sung et al 2001). These data do reject the monophyly of Cordyceps, which includes species that are members of at least two clades of the Clavicipitaceae (FIG. 3): the Cordyceps s.s. clade and the C. ophioglossoides clade (Sung et al 2001). Cordyceps s.s. clade is defined by the inclusion of C. militaris, the type of the genus, and numerous closely related and morphologically similar species. The C. ophioglossoides clade includes many well-known fungal and arthropod pathogens that cannot be accommodated in a monophyletic generic concept of Cordyceps. A generic revision of Cordyceps ultimately is needed. However it is a problem beyond the scope of this study and will require a considerable amount of data from independent genes to confidently address the issue.

Cordyceps cardinalis is well supported as a member of the monophyletic Cordyceps s.s. clade and thus it is appropriate for its nomenclature to reflect this phylogenetic affinity. The molecular data and analyses also support a clear separation of C. cardinalis from C. militaris and a close relationship of it with C. pseudomilitaris. These findings are consistent with stromatal color, disarticulation and morphology of ascospores, cultural characteristics and stage of host attacked by the pathogen as all being informative traits in distinguishing the three phylogenetic species. While C. cardinalis and C. pseudomilitaris are morphologically and ecologically similar, and while they are each other’s closest relatives in these analyses, these data are consistent with two separate species. Although sequence from only one specimen of C. pseudomilitaris is available for comparison, the monophyly of the isolates sampled from C. cardinalis, the amount of sequence divergence between C. cardinalis and C. pseudomilitaris as compared to other species of Cordyceps, and the distinguishing morphological traits are sufficient evidence for recognition of distinct species. Cordyceps pseudomilitaris is known only from Thailand, and C. cardinalis is only known from the southern Appalachians of eastern United States and southeastern Japan. The molecular divergence between these two taxa is in stark contrast to C. militaris s.s., which also includes specimens from Asia and eastern North America as well as other continents (FIG. 3).

Biogeography of C. cardinalis. – The east Asian-eastern North American disjunct species distribution is well documented in plants (reviewed in: Qian and Ricklefs 2000, Tiffney 1985, Wen 1999) but relatively understudied in fungi (Vilgalys and Sun 1994, Wu and Mueller 1997, Wu et al 2000). The current known distribution of C. cardinalis ( Japan and the southern Appalachians of United States) is consistent with that of an east Asian-eastern North American disjunct species distribution; however, for two main reasons, we offer this as a hypothesis that is in need of further testing. First, additional data is needed from existing ‘‘C. militaris’’ collections because numerous cryptic or previously unrecognized species are likely to house in herbaria under the name C. militaris. Cordyceps militaris is one of the more frequently collected species of Cordyceps, and many collections were placed in herbaria without having been subjected to microscopic examination. Second, much of what we know about fungal geographic distributions is more reflective of where mycologists have collected and not a systematic survey of geographic regions of the planet. There are numerous regions where Cordyceps has not been collected in a systematic manner, and our current understandings of the biogeography of the genus must take this lack of knowledge into account.

Conclusion. – Numerous existing names and species delimitations of Cordyceps were considered for possible application to C. cardinalis. Descriptions and examination of specimens of C. washingtonensis did not match morphologically (TABLE I), and sequence data are not available. Descriptions of C. rosea are similar to both C. pseudomilitaris and C. cardinalis but differed in size of perithecia, asci, ascospores and its 8–10 septated ascospores (Kobayasi and Shimizu 1982, TABLE I). Cordyceps rosea is known only from the type collection, whose whereabouts are unknown and thus its utility in Cordyceps taxonomy is limited. Finally, C. roseostromata is a morphologically similar species that grouped strongly among C. militaris sequences. This finding is consistent with its original morphological description, especially in the size of partspores, but differ in the size of the perithecium (Kobayasi and Shimizu 1983, TABLE I). This suggests that either it is potentially conspecific with C. militaris and indicates its variability in the size of perithecium or the cultures that we used are not indicative of C. roseostromata sensu Kobayasi. Additional research is required before we are able to address the taxonomic status of C. roseostromata.

Taxonomy and systematics of Cordyceps are complicated by both the large number of species and synonyms (Mains 1957, Kobayasi 1982) and the inability of us and others (Hywel-Jones, pers comm) to locate numerous type specimens. This latter point is especially problematic with respect to the collection of Y. Kobayasi, who described more than 100 species of Cordyceps (Kobayasi 1982). In discovering and describing C. cardinalis we attempted to apply all known names to this species. However, none of them proved to be suitable in comparison of morphological and/or molecular data.

	ACKNOWLEDGMENTS

 
We would like to acknowledge K. Cook for assistance in preparing and sectioning of stromata, Drs R.A. Humber and W. Gams for providing ARSEF and CBS cultures, Dr J. Trappe for providing a Latin diagnosis, Dr D. Murray for assistance in identifying the lepidopteran host of C. cardinalis, H. Manabe for providing collections of Japanese specimens and photographs of C. cardinalis, and the National Science Foundation for its financial support (DEB-9806936 and DEB-0129212) to J. W. Spatafora.

	FOOTNOTES

 
Accepted for publication September 17, 2003.

¹ Corresponding author. E-mail: Spatafoj{at}science.oregonstate.edu

	LITERATURE CITED

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Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791.

Gams W, O’Donnell K, Schroers H-J, Christensen M. 1998. Generic classification of some more hyphomycetes with solitary conidia borne on phialides. Can J Bot 76:1570–1583.

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Kobayasi Y. 1941. The genus Cordyceps and its allies. Science Report of the Tokyo Bunrika Daigaku (Section B, No. 84) 5:53–260.

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