Phylogenetic Evidence for an Animal Pathogen Origin of Ergot and The
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Molecular Ecology (2007) 16, 1701–1711 doi: 10.1111/j.1365-294X.2007.03225.x PBlackwell Puhblishing Ltdylogenetic evidence for an animal pathogen origin of ergot and the grass endophytes J. W. SPATAFORA,* G.-H. SUNG,* J.-M. SUNG,† N. L. HYWEL-JONES‡ and J. F. WHITE, JR§ *Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA, †Department of Applied Biology, Kangwon National University, Chuncheon 200-701, Korea, ‡Mycology Laboratory, National Center for Genetic Engineering and Biotechnology, Science Park, Pathum Thani 12120, Thailand, §Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, USA Abstract Grass-associated fungi (grass symbionts) in the family Clavicipitaceae (Ascomycota, Hypo- creales) are species whose host range is restricted to the plant family Poaceae and rarely Cyperaceae. The best-characterized species include Claviceps purpurea (ergot of rye) and Neotyphodium coenophialum (endophyte of tall fescue). They have been the focus of con- siderable research due to their importance in agricultural and grassland ecosystems and the diversity of their bioactive secondary metabolites. Here we show through multigene phylogenetic analyses and ancestral character state reconstruction that the grass symbionts in Clavicipitaceae are a derived group that originated from an animal pathogen through a dynamic process of interkingdom host jumping. The closest relatives of the grass symbi- onts include the genera Hypocrella, a pathogen of scale insects and white flies, and Metarhizium, a generalist arthropod pathogen. These data do not support the monophyly of Clavicipitaceae, but place it as part of a larger clade that includes Hypocreaceae, a family that contains mainly parasites of other fungi. A minimum of 5–8 independent and unidi- rectional interkingdom host jumps has occurred among clavicipitaceous fungi, including 3–5 to fungi, 1–2 to animals, and 1 to plants. These findings provide a new evolutionary context for studying the biology of the grass symbionts, their role in plant ecology, and the evolution of host affiliation in fungal symbioses. Keywords: Clavicipitaceae, evolution, fungi, grass symbionts, host affiliation, interkingdom host- jumping, symbioses Received 30 July 2006; revision received 9 October 2006; accepted 6 November 2006 hypothesized that psychoactive side effects from ingesting Introduction ergot alkaloids were connected with early biblical events, Grass-associated fungi (grass symbionts) of the family played an important ceremonial role in ancient Greece, Clavicipitaceae have had a significant impact on human and were partially responsible for the 16th and 17th civilization for thousands of years (White et al. 2003). The century witch trials in both Europe and colonial America best-documented case is that of Claviceps purpurea (ergot), (Matossian 1989; Hudler 1998; White et al. 2003). It was not the causal agent of ergotism, which is expressed in the until the mid-1700s, however, that Cl. purpurea was identi- form of numerous human maladies including gangrene, fied as the cause of ergotism (Tissot 1765). The biological convulsions and seizures, hallucinations and hysteria, and activity of Cl. purpurea and related fungi has been used susceptibility to other diseases (Matossian 1989; Hudler in numerous cultures and traditional and modern medi- 1998). The physiological and psychoactive effects of cine to facilitate childbirth and abortions (Riddle 1997), Cl. purpurea are due to its ability to produce potent to treat migraines (Landy 2004), and in recreational drug biologically active alkaloids that have detrimental effects use, that is, LSD25 (Hofmann 1980). on the central nervous system (Tudzynski et al. 2001). It is More recently, mycologists, plant ecologists, and plant pathologists have documented the prevalence and importance Correspondence: Joseph W. Spatafora, Fax: 1-541-737-3573; of fungal endophytes of plants, especially those of grasses E-mail: [email protected] (Schardl 1996; Saikkonen et al. 1998; Clay & Schardl 2002; © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 1702 J. W. S PAT AFO RA E T A L. Fig. 1 Exemplar species of Clavicipitaceae. (a) Cordyceps militaris on lepidopteran pupa. (b) Cordyceps ophioglossoides on Elaphomyces sp. (truffle, Ascomycota). (c) Metarhizium on Graptopsaltria nigrofuscata (cicada, Hemiptera). (d) Hypocrella on scale insects (Hemiptera). (e) Claviceps purpurea on Spartina alterniflora (Poaceae). (f) hyphae of Neotyphodium lolii growing endophytically in Lolium perenne (Poaceae). Schardl et al. 2004). The primary fungi that comprise In addition to the grass symbionts, Clavicipitaceae the grass endophytes are Clavicipitaceae species of comprises a diverse assemblage of fungi characterized by the sexually reproductive genus Epichloë and its asexual symbioses and associations with other eukaryotes, includ- counterpart Neotyphodium. These organisms grow as true ing pathogens of animals and parasites of other fungi endophytes and typically colonize the aerial portion of the (Kobayasi 1941, 1982; Mains 1957, 1958, 1959; Fig. 1). Sig- plant. Epichloë species produce sexually reproductive stro- nificantly, most Clavicipitaceae species are pathogens of mata that physically encase the florets, preventing seed arthropods and are classified in the sexual genera Cordyceps, development, a syndrome known as choke (White et al. Hypocrella, and Torrubiella (Kobayasi 1941, 1982; Mains 1991). Neotyphodium species produce no reproductive 1957, 1958, 1959). As a group, these fungi attack hosts from structures on the plant, but grow as a filamentous stage at least 10 orders of Arthropoda, although any one fungus throughout the plant and are vertically transmitted with species has a narrow host range of a single species or the host’s seeds. The interaction of Epichloë species with the closely related species and attacks a particular stage of the host plant is generally considered antagonistic, while arthropod life cycle. Fungal parasites in Clavicipitaceae Neotyphodium species are usually considered mutualistic. comprise a group of approximately 20 species of Cordyceps, Infected host plants benefit from increased drought which exhibit host specificity to the truffle genus Elapho- tolerance, enhanced growth and competitive ability, and myces (Ascomycota) (Mains 1957). Additional pathogens decreased herbivory (Clay 1988, 1990; Clay & Schardl and parasites of the animal and fungal kingdoms include 2002). Much of the beneficial effect of the interaction is numerous asexual genera that attack microinvertebrates attributed to the secondary metabolites produced by the (Gams & Zare 2003) and other fungi, including rust fungi fungus, although the mode of action is only known in the and the commercially cultivated button mushroom (Gams case of herbivory; many of the metabolites have antagonistic & van Zaayen 1982). Like the grass symbionts, animal effects on insects and significantly deter insect feeding pathogens and fungal parasites in Clavicipitaceae produce (Clay 1988; Bush et al. 1997; Siegel & Bush 1997; Wilkinson a myriad of secondary metabolites (Isaka et al. 2003) with et al. 2000; Clay & Schardl 2002; Tanaka et al. 2005). several extracts, purified compounds, and species used in © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd E V OL U TI ONAR Y OR IGI N OF GR ASS-A SSOCI AT E D F UNGI 1703 traditional (Zhu et al. 1998) and modern (Dunn et al. 2001) related families in Hypocreales. Emphasis was placed on medicine, research (Naula et al. 2003), and in agriculture as broad representation of host affiliation and morphological biological control agents (Shah & Pell 2003). and taxonomic diversity. Two species, Glomerella cingulata Clavicipitaceae is in the order Hypocreales (Ascomycota) and Verticillium dahliae, from the closely related order and its broad host range and diverse ecology is unique for the Phyllachorales were included as outgroup taxa. Information order. Other major families and ecologies of Hypocreales on source of fungal isolates is provided in Table 1. include Hypocreaceae, with most species being parasites of fungi, and Nectriaceae and Bionectriaceae, which com- Molecular techniques prise numerous plant pathogens and saprobes of woody and herbaceous plants (Rossman et al. 1999). As such, the Six loci including those that encode for the nuclear small evolutionary history of Hypocreales and Clavicipitaceae is (SSU rRNA) and large (LSU rRNA) ribosomal RNAs, characterized by interkingdom host jumping, with the best β-tubulin (β-tub), translation elongation factor 1-α (tef1) studied example being among closely related species of the largest (rpb1) and second largest (rpb2) subunits of RNA Cordyceps that attack cicadas nymphs and Elaphomyces, polymerase II genes were sampled. DNA was isolated and which share a common subterranean habitat (Nikoh & rRNA loci were amplified, sequenced and aligned as pre- Fukatsu 2000). To better understand the evolutionary rela- viously described (Sung et al. 2001). Protein-coding genes tionships among the animal, fungal, and plant-associated were amplified and sequenced with primers listed in Table 2. fungi in Clavicipitaceae and to provide a phylogenetic Sequencing was performed with BigDye version 3 (Applied foundation for future studies, we conducted a multigene Biosystems Inc.) and run on either an ABI 3100 or ABI 3700 phylogenetic study of the family. Using this phylogeny, we automated sequencer in the Central