Bioactive Alkaloids in Vertically Transmitted Fungal Endophytes

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Functional Ecology 2014, 28, 299–314 doi: 10.1111/1365-2435.12076

DEFENSIVE SYMBIOSIS Bioactive alkaloids in vertically transmitted fungal endophytes

Daniel G. Panaccione*,1, Wesley T. Beaulieu2 and Daniel Cook3

1 Division of Plant & Soil Sciences, West Virginia University, 1090 Agricultural Sciences Building, Morgantown, WV 26506-6108 USA; 2Department of Biology, Indiana University, Bloomington, IN, USA; and 3USDA ARS Poisonous Plant Research Laboratory, Logan, UT, USA

Summary 1. Plants form mutualistic symbioses with a variety of microorganisms including endophytic fungi that live inside the plant and cause no overt symptoms of infection. Some endophytic fungi form defensive mutualisms based on the production of bioactive metabolites that protect the plant from herbivores in exchange for a protected niche and nutrition from the host plant. Key elements of these symbioses are vertical transmission of the fungus through seed of the host plant, a narrow host range, and production of bioactive metabolites by the fungus. 2. Grasses frequently form symbioses with endophytic fungi belonging to the family Clavicipit- aceae. These symbioses have been studied extensively because of their significant impacts on insect and mammalian herbivores. Many of the impacts are likely due to the production of four classes of bioactive alkaloids – ergot alkaloids, lolines, indole-diterpenes and peramine – that are distributed in different combinations among endophyte taxa. 3. Several legumes, including locoweeds, are associated with a toxic syndrome called locoism as a result of their accumulation of swainsonine. Species in two genera were recently found to contain previously undescribed endophytic fungi (Undifilum spp., family Pleosporaceae) that are the source of that toxin. The fungi are strictly vertically transmitted and have narrow host ranges. 4. Some plant species in the morning glory family (Convolvulaceae) also form symbioses with endophytic fungi of the Clavicipitaceae that produce ergot alkaloids and, perhaps in at least one case, lolines. Other species in this plant family form symbioses with undescribed fungi that produce swainsonine. The swainsonine-producing endophytes associated with the Convolvula- ceae are distinct from the Undifilum spp. associated with locoweeds and the Clavicipitaceous fungi associated with Convolvulaceae. 5. In the establishment of vertically transmitted symbioses, fungi must have entered the symbiosis with traits that were immediately useful to the plant. Bioactive metabolites are likely can- didates for such pre-adapted traits which were likely useful to the free-living fungi as well. With future research, vertically transmitted fungi from diverse clades with narrow host ranges and that produce bioactive compounds are likely to be found as important mutualists in additional plants.

Key-words: defensive mutualism, ergot alkaloids, indole-diterpenes, lolines, peramine, plant-herbivore interactions, swainsonine, symbiosis

including energetic (e.g. photosynthetic Chlorella in Anth- Introduction ozoans), nutritional (e.g. mycorrhizae), transport (e.g. pol- Several classes of mutualisms have been recognized based lination) and defensive (Boucher, James & Keeler 1982; upon the beneﬁts exchanged between the partners Janzen 1985; Douglas 1994). In defensive mutualisms, one partner provides protection from or resistance to one or *Correspondence author. E-mail: [email protected] more of its partner’s natural enemies. A classic example is

Published 2013. This article is a U.S. Government work and is in the public domain in the USA. Functional Ecology 2013 © British Ecological Society 300 D. G. Panaccione et al. that of the Ant-Acacia system in which voracious ants pro- pletely dependent upon the host’s reproductive success tect Acacia trees from megaherbivores (e.g. elephants) in (Ferdy & Godelle 2005). exchange for shelter in plant structures called domatia The natural host range of a symbiont (as opposed to the (Janzen 1966; Palmer et al. 2008). Defensive mutualisms range of species it could infect in a laboratory setting) is involving microbial symbionts that produce protective another defining characteristic of endophytic fungi associ- chemistry have repeatedly evolved in diverse taxa and can ated with defensive symbioses. Clavicipitaceous endo- have resounding effects on host success as well as commu- phytes capable of vertical transmission are associated with nity structure and dynamics (White & Torres 2009). For select taxa of the Poaceae and Convolvulaceae and have example, marine bryozoans harbour bacterial symbionts narrow host ranges (Clay & Schardl 2002; Steiner et al. that produce polyketide bryostatins, without which hosts 2011). In most instances, a given fungal taxon is associated are vulnerable to fish predation (Lopanik, Lindquist & with a specific plant host species (Schardl 2010). In con- Targett 2004; Lopanik, this issue), leaf cutter ants utilize trast, many horizontally transmitted endophytes represent an actinomycete that produces antibiotics to ward off fungal genera that are ubiquitous in the environment and yeasts that would otherwise degrade the ants’ fungal gar- are often associated with multiple host taxa (Rodriguez den (Currie, Mueller & Malloch 1999), and aphids host et al. 2009). facultative bacterial symbionts that can provide resistance The defensive mutualism hypothesis does not exclude to both abiotic (e.g. heat) and biotic (e.g. parasitoids) the possibility of negative effects to the host under certain stressors (Oliver et al. 2010; Oliver, this issue). Defensive environmental conditions, especially since the host must mutualism was proposed by Clay (1988) to describe the incur some due to endophyte infection. For example, relationship between certain fungi and their grass hosts, in Cheplick, Clay & Marks (1989) observed reduced growth which the fungi are afforded a habitat and carbohydrates in endophyte-infected tall fescue under low nutrient condi- by their host plant and provide their host with protection tions. Turf varieties of tall fescue infected with a particular from biotic stress (e.g. herbivory). Neotyphodium endophyte were more susceptible to root Fungi that participate in defensive mutualisms typically disease caused by Pythium graminicola (Rodriguez et al. are referred to as endophytes, even though some produce 2009). Wali€ et al. (2006) showed that red fescue (Festuca epiphytic structures. The term ‘endophyte’, however, also rubra) infected with Epichloe€ festucae in subarctic regions is used more broadly for any fungus, bacterium or other suffered more damage from the snow mould, Typhula microorganism that colonizes living plants without caus- ishikariensis than did non-infected red fescue growing in ing overt detrimental symptoms and typically has no obvi- these same areas. Despite the occasional example that ous external signs of infection. Rodriguez et al. (2009) indicates a detriment of endophyte infection to the host, proposed four classes of endophytic fungi that were complete dependence on the host plant for transmission of grouped according to criteria such as host range, tissues the endophyte provides a means for selecting fungi that colonized, in planta colonization, in planta biodiversity, are beneficial to their host plants. Fungal endophytes that mode of transmission and the nature of the benefits affor- are strictly vertically transmitted must be beneficial in ded to the host. Two criteria we feel are diagnostic in order for the host plants to retain them (Ewald 1987). In defining fungal endophytes that participate in defensive cases where negative effects have been seen, it does not mutualisms are the capacity for vertical transmission of preclude the existence of some positive benefit that has not the endophyte via seed and a narrow host range, both of been measured that results in maintenance of the mutual- which are characteristic of class 1 endophytes as described ism, such as reduced survival but increased regeneration by Rodriguez et al. (2009), although those authors limited resulting in net positive population growth (Rudgers et al. class 1 endophytes to species in the fungal family Clavici- 2012). pitaceae. In addition to vertical transmission and a narrow host Hereditary symbionts that are strictly vertically trans- range, a third and striking feature of many plant-endo- mitted are completely dependent on host reproduction for phytic fungus associations – particularly those that fit well their own propagation and reproductive fitness. When a with the definition of defensive mutualism – is the produc- vertically transmitted symbiont benefits the host, it has an tion of bioactive secondary metabolites by the fungal sym- indirect positive effect on its own fitness. Any symbiont bionts. Whereas fungi in general produce a wide array of that is strictly vertically transmitted and puts its host at a secondary metabolites, we propose that bioactive metabo- disadvantage would go extinct because its host would be lites are particularly important to vertically transmitted outcompeted by non-infected conspecifics (Ewald 1987). fungi, which in the establishment of their fungus–plant Vertically transmitted symbionts are essentially a trait of symbioses must have provided some immediate benefit to the host and if one is ‘in the least degree injurious’, first the host plant. We describe here selected examples of such principles would suggest it would be ‘rigidly destroyed’ by vertically transmitted endophytic fungi, the chemicals they natural selection (Darwin 1859). Horizontally transmitted produce and the ways their chemicals contribute to the symbionts, on the other hand, exploit the host’s ability to symbioses. We also include examples of vertically transmit- survive and contact non-infected individuals and are often ted symbiotic fungi that produce noteworthy bioactive more virulent as the symbiont’s reproduction is not com- chemicals in their symbiotic state but for which roles of

Published 2013. This article is a U.S. Government work and is in the public domain in the USA. Functional Ecology 2013 © British Ecological Society, Functional Ecology, 28, 299–314 Bioactive alkaloids of fungal endophytes 301 the chemicals in the symbioses have not been experimen- amine. Although each of these four classes of alkaloids is tally established. In each case, the metabolites observed derived in some way from amino acid precursors, the four originally appeared to be components of the plant or pathways are completely independent of one another. No would have appeared as such if the symbiosis had not individual fungal isolate is known to produce representa- already been discovered. tives of all four classes; most epichloae produce metabolites belonging to one to three of the chemical classes (Schardl et al. 2011). Clavicipitaceous endophytes of grasses Fungi of the genera Epichloe€ and Neotyphodium grow sym- ERGOT ALKALOIDS biotically with many cool season grasses. Neotyphodium species are exclusively asexual and grow within the inter- Ergot alkaloids are a diverse family of secondary metabo- cellular spaces of their grass hosts (Fig. 1a). Epichloe€ spe- lites produced by certain epichloae, ergot fungi (Claviceps cies represent the teleomorphic states of several spp.) and related species of Balansia and Periglandula (dis- Neotyphodium species. In addition to the symbiotic phase cussed later), and in the opportunistic human pathogen typical of Neotyphodium species, Epichloe€ species are capa- Aspergillus fumigatus. The biosynthetic pathway has been ble of exiting their plant hosts via the formation of sexual studied extensively and recently reviewed (Lorenz et al. reproductive stroma on plant inflorescences. Those sexu- 2009; Panaccione 2010; Wallwey & Li 2011). The diverse ally reproducing Epichloe€ species are thus capable of verti- metabolites in the ergot alkaloid family can be grouped as cal transmission through their asexual stage and clavines, simple amides of lysergic acid, or ergopeptines occasional horizontal transmission when they reproduce based on their complexity and relative position in the sexually. Schardl (2010) referred to representatives of the pathway (Fig. 2). Various ergot alkaloids interact as two genera collectively as epichloae. agonists or antagonists at receptors for the monoamine In addition to direct effects on herbivores, epichloid en- neurotransmitters serotonin, dopamine, adrenaline and dophytes of grasses can significantly affect ecological noradrenaline. Resulting activities include vasoconstric- communities. Neotyphodium coenophialum-infected tall tion, uncontrolled muscle contraction and disturbance in fescue (Lolium arundinaceum) suppresses both plant (Clay the central nervous system and reproductive systems & Holah 1999) and arthropod diversity (Finkes et al. (reviewed in Lorenz et al. 2009; Panaccione 2010; Wallwey 2006), affects the outcome of competitive interactions & Li 2011). Numerous feeding studies with a variety of (Clay, Marks & Cheplick 1993), alters plant soil feedbacks mammals indicate that ergot alkaloids, at concentrations (Matthews & Clay 2001), slows succession to forest at which they are found in endophyte-infected grasses, communities (Rudgers et al. 2007), and disrupts relation- have significant detrimental effects on mammalian health ships between diversity and ecosystem properties such as and reproduction (e.g. Hill et al. 1994; Filipov et al. 1998; productivity (Rudgers, Koslow & Clay 2004). Gadberry et al. 2003; Parish et al. 2003a,b). Ergot alka- The epichloae collectively produce four classes of bioac- loids also affect insects and nematodes, which contain tive metabolites in their symbiotic associations with plants: homologous neurotransmitters. Activities in insects include ergot alkaloids, indole-diterpenes, loline alkaloids and per- feeding deterrence, delayed development and increased

(a) (b)

Fig. 1. Class 1 endophytic fungi. (a) Ani- line blue-stained hypha of Neotyphodium (c) (d) (e) coenophialum growing intercellularly in a peeled leaf sheath of tall fescue (Lolium arundinaceum); (b) Hyphae of GFP- expressing Undiﬁlum oxytropis in locoweed vascular tissue; (c) Numerous, small colonies of Periglandula ipomoeae growing epiphytically on the adaxial leaf surface of Ipomoea asarifolia; (d) Fungicide-treated I. asarifolia leaf lacking fungal colonies; (e) Aniline blue-stained colonies of P. ipomoeae from the adaxial leaf surface of I. asarifolia. (Photos: the authors)

Fig. 2. Diversification of ergot alkaloids associated with endophyte–plant symbioses. Double arrows indicate one or more omitted intermediates. Dashed arrows indicate uncharacterized steps. Relevant enzymes associated with catalysis at branch points are indicated. At the first branch point, alternative forms of EasA form festuclavine (not pictured) and agroclavine (Coyle et al. 2010); additional alternative forms are hypothesized to produce cycloclavine and lysergol in Periglandula-infected Ipomoea spp. At the second branch point, combinations of peptide synthetases Lps1, Lps2 and Lps3 are required to produce ergopeptines or simple amides of lysergic acid (Lorenz et al. 2009; Ortel & Keller 2009). Lysergic acid is bracketed to indicate that it is not typically considered a clavine. mortality (Clay & Cheplick 1989; Ball, Miles & Prestidge Notably, the knockout strain accumulated the same molar 1997; Potter et al. 2008). quantity of ergot alkaloids as the wild type, but the alka- The ergot alkaloid pathway is notable for its accumula- loids were restricted to earlier pathway intermediates and tion of intermediates and spur products to concentrations spur products. Thus, the accumulation of both intermedi- that approach or exceed the amounts of the pathway end ates and end products is beneficial to the fungus and product (Panaccione et al. 2003; Panaccione & Coyle its grass host in resisting vertebrate and invertebrate 2005). Panaccione (2005) hypothesized that this ineffi- herbivore pressures. ciency in turning over intermediates has been selected for The general significance of ergot alkaloids to because those accumulating intermediates or spur products Lp1-infected perennial ryegrass was apparent from the provide some benefit to the producing fungi (or its grass observation that perennial ryegrass containing a different host, in the case of endophytes) that differs from the bene- knockout mutant, which was completely devoid of ergot fit(s) provided by the pathway end product. Differences in alkaloids but still colonized by the fungus (Wang et al. activities of clavine intermediates and spur products com- 2004), was strongly preferred by rabbits, even over the pared to ergopeptines or the simple amides are apparent endophyte-free perennial ryegrass (Panaccione et al. from direct exposure of bacteria and nematodes to these 2006b). Thus, without any ergot alkaloids this grass would alkaloids in vitro (Panaccione 2005; Panaccione et al. be subject to increased herbivory and likely at competitive 2006a; Bacetty et al. 2009a,b). In a more natural setting, disadvantage compared to grasses containing ergot studies with perennial ryegrass (Lolium perenne) and gene alkaloid-producing endophytes. knockout mutants of the endophyte Epichloe€ typhin- a 9 Neotyphodium lolii isolate Lp1 (hereafter simply Lp1) INDOLE-DITERPENES provide more support for this hypothesis. A knockout mutant that accumulated certain clavines but not ergopep- The indole-diterpenes represent another important class of tines or simple amides of lysergic acid deterred rabbit feed- diverse alkaloids produced by some epichloae as well as by ing on infected grasses as well as or better than the wild certain Claviceps spp. and some members of the Tricho- type of the fungus (Panaccione et al. 2003, 2006b). In con- comaceae (e.g. Aspergillus and Penicillium spp.) (Saikia trast, perennial ryegrass containing the same knockout et al. 2008). Indole-diterpenes have been studied inten- endophyte had reduced insecticidal and insect feeding sively because certain members of this class of metabolites deterrent properties compared to wild-type endophyte, have strong tremorgenic activity in mammals. For exam- indicating a role for ergopeptines and simple amides of ple, the lolitrems produced by Neotyphodium lolii in peren- lysergic acid in these anti-insect traits (Potter et al. 2008). nial ryegrass cause ryegrass staggers (Gallagher, White &

Mortimer 1981; Gallagher et al. 1982, 1984), which can indole-diterpenes could be beneficial to their host by acting result in significant economic losses. against insects, as has been demonstrated for the biogeni- Similar to the ergot alkaloids, the indole-diterpenes of cally related yet structurally distinct compound nodulisp- endophytes are very diverse. A simplistic view of the diver- oric acid. Nodulosporic acid is produced in culture by sification of indole-diterpenes can be based on the oxida- Nodulisporium sp. (an anamorphic fungus in the Xylaria- tion and prenylation of intermediate terpendole I and its ceae that was isolated from an unidentified woody plant) subsequent metabolites independently, resulting in differ- and has good insecticidal activity against a range of insects ent end products including terpendoles, lolitrems and (Byrne, Smith & Ondeyka 2002). The less commonly janthitrems (Fig. 3). encountered but biogenically related janthitrems also may Much of the early analysis of grass endophytes for be associated with insecticidal activity. Janthitrems accu- indole-diterpenes focused intensively on lolitrem B. Evi- mulate in plants with N. lolii isolate AR37, an endophyte dence for lolitrem B as the key tremorgenic toxin in strain that is included in some commercial varieties of N. lolii-infected perennial ryegrass has come from animal perennial ryegrass because of its low tremorgenic activity. feeding studies (e.g. Gallagher, White & Mortimer 1981; AR37-infected perennial ryegrass varieties are notably Gallagher et al. 1982) as well as from comparisons of natu- resistant to the insect pest Wiseana cervinata (porina) rally occurring isolates that vary in indole-diterpene pro- (Jensen & Popay 2004); however, a direct linkage of the files (e.g. Bluett et al. 2005a,b). More recent genetic anti-insect activities of AR37 with the janthitrems has not screening, facilitated by a more thorough understanding of been established. Several other indole-diterpenes have been indole-diterpene biosynthesis, indicated that some epichloid isolated from the sclerotia of various Aspergillus spp. endophytes that do not produce lolitrem B still produce less (Trichocomaceae, Eurotiales), and these indole-diterpenes complicated indole-diterpenes such as terpendoles (Gate- have been demonstrated to have anti-insect activities nby et al. 1999; Young et al. 2009; Schardl et al. 2011). through feeding and topical assays (Gloer 1995). Interestingly, fungal endophytes producing terpendoles but The production of indole-diterpenes and ergot alkaloids lacking lolitrem B have been successfully marketed in for- by certain representatives of two phylogenetically disjunct age varieties of perennial ryegrass in New Zealand as less families, the Clavicipitaceae and the Trichocomaceae (and toxic alternatives to traditional perennial ryegrass varieties very rarely by fungi outside these families), is remarkable. (Bluett et al. 2005a,b). Lolitrem B-deficient varieties may Whereas the known alkaloid-producing Clavicipitaceae still induce minor tremoring in mammals, presumably due (order Hypocreales) all are associated with living plants, to the presence of janthitrems or other indole-diterpenes, the alkaloid-producing Trichocomaceae (order Eurotiales) but the effects are minimal (Bluett et al. 2005a,b). are primarily saprotrophs on plant matter. Although ergot The observation that some non-tremorgenic epichloae alkaloids and indole-diterpenes are assembled from some retain the ability to produce intermediates in the indole- common precursors, the biosynthetic pathways for these diterpene pathway is interesting, considering their negligi- alkaloids are completely independent. The polyphyletic ble anti-mammalian activity compared to the lolitrems. distribution of the two independent pathways among such Young et al. (2009) speculated that the less tremorgenic diverse fungi cannot be explained at present.

Fig. 3. Diversiﬁcation of indole-diterpenes associated with endophyte-plant symbioses. Double arrows indicate one or more omitted intermediates. Dashed arrows indicate uncharacterized steps. LtmE/LtmJ and LtmF/LtmK represent separate prenyl transferase/monooxygen- ase (respectively) combinations that work on opposite ends of members of the indole-diterpene family (Young et al. 2006, 2009). Each combination can act on multiple substrates.

LOLINES Schardl et al. (2007) carefully reviewed studies on vertebrate toxicity of lolines and concluded that anti-vertebrate The loline alkaloids are a family of aminopyrrolozidine effects of lolines were likely to be negligible, because studies alkaloids, derived from homoserine and proline joined in a indicating such effects were either confounded by the non-peptidic manner. Lolines have been most intensively presence of ergot alkaloids in the same plant tissues or con- studied in endophytic Neotyphodium spp. but also have ducted with exceptionally high concentrations of lolines. been reported in the plants Adenocarpus decorticans (Faba- ceae) (reviewed by Schardl et al. 2007) and Argyreia mollis PERAMINE (Convolvulaceae) (Tofern et al. 1999). Whether the lolines in the Adenocarpus and Argyreia species are of plant origin Peramine is the most widely distributed of the four classes or derived from endophytic fungi has not yet been deter- of epichloae-derived secondary metabolites (Schardl et al. mined. The biosynthesis and activities of lolines has been 2011), but its production is not known outside of the epi- reviewed in detail by Schardl et al. (2007). Lolines occur in chloae (Clay & Schardl 2002; Tanaka et al. 2005). Its ori- exceptionally high concentration in Neotyphodium coeno- gin as a fungal metabolite was shown by its production by phialum-infected tall fescue (Lolium arundinaceum) and isolated fungi in vitro (Rowan 1993), and more convinc- also are found abundantly and in variable forms in several ingly by its disappearance from grass–endophyte symbiota other epichloae-infected grasses (Schardl et al. 2007, 2011). upon mutation of the relevant gene in the fungus (Tanaka Variability among lolines is mainly generated by the pres- et al. 2005). Peramine is unique among the four major ence or absence of methyl, formyl or acetyl groups on the classes of epichloae-produced alkaloids, in that it is a sin- homoserine-derived amine group (Fig. 4a). gle chemical as opposed to a family of chemicals, and it The insecticidal and insect feeding deterrent activities of appears to be the product of a single multifunctional lolines have been shown in a series of feeding experiments enzyme as opposed to a complex pathway (Tanaka et al. with either endophyte-infected plants (e.g. Yates, Fenster 2005). Peramine is derived from a dipeptide possibly made & Bartelt 1989; Siegel et al. 1990; Jensen, Popay & Tapper up of arginine and a precursor to proline (Fig. 4b). 2009) or with purified lolines (e.g. Riedell et al. 1991). Rie- Peramine is a strong feeding deterrent for Argentine dell et al. (1991) also applied lolines topically to aphids stem weevil, an important pest of perennial ryegrass in and noted that toxicity of lolines was comparable to that New Zealand, and several other insects (Clay, Hardy & of nicotine. A convincing demonstration of the significance Hammond 1985; Johnson et al. 1985; Rowan, Hunt & of lolines to insect resistance in an endophyte-infected Gaynor 1986; Rowan, Dymock & Brimble 1990; Rowan grass came from an elegant genetic study conducted by 1993). The anti-feeding effects of peramine are not univer- Wilkinson et al. (2000) who observed co-segregation of sal, however, as the aphid Rhopalosiphum padi appears not activity against two different aphid species and loline pro- to be deterred by its presence (Johnson et al. 1985; Gaynor duction in a genetic cross among Epichloe€ festucae isolates & Rowan 1986). The significance of peramine to defending in meadow fescue (Lolium pratense). Moreover, aphid plant material against herbivory by Argentine stem weevil mortality increased with increasing concentrations of was convincingly demonstrated in a study by Tanaka et al. lolines in plants containing loline-positive progeny. (2005) in which the gene encoding the multifunctional In addition to the well-documented effect on insects, enzyme responsible for peramine biosynthesis was inacti- lolines also are nematicidal (Bacetty et al. 2009a). The vated by knockout. Resulting peramine-deficient mutants effects of lolines appear to be restricted to invertebrates. were as susceptible to feeding by the Argentine stem weevil as endophyte-free plants of the same variety. Unlike the ergot alkaloids and indole-diterpenes, which (a) are found mainly in tissues that are colonized by the endophyte (pseudostem or seeds), peramine is water soluble and dispersed throughout the plant (Ball et al. 1997a,b; Spiering et al. 2002, 2005; Koulman et al. 2007). Peramine is found in fluids exuded from cut leaves of all tested endophyte-infected varieties of perennial ryegrass, tall fescue, and Elymus sp. and in the guttation fluid of endophyte- infected perennial ryegrass and Elymus sp. (Koulman et al. (b) 2007). This localization pattern would allow peramine to protect tissues remote from the fungus, and its presence in guttation fluid would conceivably allow it to deter feeding by sensitive insects without the insects breaching the cuti- cle. The activity of peramine against many phloem feeders Fig. 4. Structures of (a) lolines and (b) peramine. Variation and its presence in roots (a tissue not well colonized by among lolines derives from substituents on the indicated nitrogen peramine-producing fungi) indicates the presence of (Schardl et al. 2007). peramine in phloem.

DISTRIBUTION OF BIOACTIVE ALKALOID CLASSES and only four of those 15 produce both ergot alkaloids AMONG EPICHLOAE and indole-diterpenes (Table 1). The data show that anti-insect alkaloid classes are more likely to be present The capacity to produce the four classes of bioactive alka- in plants containing epichloae endophytes than are anti- loids varies among epichloae taxa. Two classes of epi- vertebrate alkaloids. chloae-produced alkaloids – the ergot alkaloids and the The distribution of lolines, ergopeptines (but not other indole-diterpenes – have anti-vertebrate activities, whereas ergot alkaloids) and peramine among sexual stroma-pro- three classes – the ergot alkaloids, lolines and peramine – ducing Epichloe€ spp. (those capable of horizontal transmis- have anti-invertebrate properties (with the anti-insect sion) compared to strictly asexual, vertically transmitted activities of epichloae-derived indole-diterpenes still uncer- Neotyphodium spp. was investigated by Leuchtmann, tain). In the list of grass–epichloae symbiota compiled by Schmidt & Bush (2000) who observed greater production Schardl et al. (2011), there are 29 symbiota for which the of lolines and ergopeptines in the vertically transmitted en- presence of all four classes of endophyte alkaloids has been dophytes. The reduced level of anti-insect alkaloids in tested. Among these 29 symbiota, 86% produce at least grasses hosting sexually reproducing epichloae is consistent one of the three established anti-insect classes of alkaloids, with the dependence of the sexual Epichloe€ spp. on insects and 48% have at least two classes of anti-insect alkaloids for spermatization, or transfer of gametes, among fungi of (Table 1). The common toxic endophyte isolate of N. coe- opposite mating types. nophialum is the only endophyte known to produce all three classes of anti-insect alkaloids. The anti-vertebrate alkaloids are less common than the anti-insect compounds Clavicipitaceous endophytes of Convolvulaceae among this same set of 29 symbiota for which data are The genus Periglandula consists of clavicipitaceous epibiot- available. Approximately one-half (15 of 29) of the symbi- ic fungal symbionts of the Convolvulaceae (morning ota contain at least one class of anti-vertebrate compound, glories) that produce ergot alkaloids in the seeds and, in

Table 1. Distribution of anti-vertebrate and anti-insect alkaloids among epichloae–grass symbiota in which all four classes of bioactive alkaloids have been assayed*

Fungus Host plant Anti-vertebrate alkaloids† Anti-insect alkaloids‡

Epichloe€ elymi Elymus canadensis ERG ERG, PER Epichloe€ festucae Festuca longifolia ERG, IDT ERG, PER E. festucae Festuca ovina ERG ERG, PER E. festucae Festuca rubra subsp. commutata ERG ERG, PER E. festucae F. rubra subsp. commutata – PER E. festucae F. rubra subsp. rubra IDT – E. festucae F. rubra subs. rubra ERG ERG E. festucae Lolium giganteum ERG ERG, LOL Epichloe€ typhina Lolium perenne – PER Neotyphodium aotearoae Echinopogon ovatus – LOL Neotyphodium coenophialum Lolium arundinaceum ERG ERG, LOL, PER N. coenophialum L. arundinaceum – LOL, PER Neotyphodium huerfanum Festuca arizonica – PER Neotyphodium gansuense Achnatherum inebrians ERG ERG Neotyphodium lolii Lolium perenne ERG, IDT ERG, PER N. lolii L. perenne IDT PER N. lolii L. perenne IDT – N. lolii 9 E. typhina isolate Lp1 L. perenne ERG, IDT ERG, PER Neotyphodium siegelii Lolium pratense – LOL, PER Neotyphodium starrii Bromus anomalus ERG ERG, PER Neotyphodium sp. E55 Poa autumnalis – LOL, PER Neotyphodium sp. E4074 Lolium sp. – LOL, PER Neotyphodium sp. E4078 Lolium sp. ERG, IDT ERG, PER Neotyphodium sp. Festuca paradoxa – PER Neotyphodium sp. Festuca subverticillata –– Neotyphodium sp. Hordelymus europaeus –– Neotyphodium tembladerae F. arizonica – PER Neotyphodium typhinum Poa ampla – PER Neotyphodium uncinatum L. pratense – LOL

*Refer to Schardl et al. (2011) for details on symbiota. †ERG, ergot alkaloids; IDT, indole-diterpenes. ‡ERG, ergot alkaloids; LOL, loines; PER, permine.

Published 2013. This article is a U.S. Government work and is in the public domain in the USA. Functional Ecology 2013 © British Ecological Society, Functional Ecology, 28, 299–314 306 D. G. Panaccione et al. some cases, the foliage of infected plants (Markert et al. amides and ergopeptines) have each been reported from 2008; Steiner et al. 2011). The two species described of the Convolvulaceae (Eich 2008), including the ergopeptine Periglandula are fungi that form systemic infections in the ergobalansine (Jenett-Siems, Kaloga & Eich 1994), origi- above-ground parts of their host plants and are vertically nally discovered in the clavicipitaceous fungi Balansia ob- transmitted (Steiner et al. 2006). Epiphytic mycelia are vis- tecta which form epiphytic infections of Cenchrus echinatus ible to the naked eye on young leaves (Fig. 1c). Steiner (Sandbur Grass) (Cyperaceae) (Powell et al. 1990). In et al. (2011) have stated that there is no evidence that the addition to ergot alkaloids known from other Clavicipita- fungi ever penetrate the host plant; however, the seed ceae, unique ergot alkaloids have been discovered in the transmissibility of the fungi indicates that there must be Convolvulaceae, notably cycloclavine from Ipomoea hilde- some internal growth of the fungus. Workers demon- brandtii, an African shrub (Stauffacher et al. 1969). All strated that this fungus was responsible for ergot alkaloid reports of ergot alkaloids from the Convolvulaceae have production when they observed that treatment of Ipomoea come from the speciose tribe Ipomoeeae (ca. 900 species) asarifolia with fungicides resulted in the loss of epiphytic and show no clear phylogenetic pattern (Eich 2008); how- mycelia concomitantly with loss of detectable ergot alka- ever, the phylogeny of this large family is still not clearly loids in the foliage (Kucht et al. 2004). Unlike other occur- resolved. Both major clades within the Ipomoeeae (Stefa- rences of plant-associated Clavicipitaceae, Periglandula novic, Krueger & Olmstead 2002) have ergot alkaloid-posi- species are the only clavicipitaceous fungi known to associ- tive representatives, and there is variation within sections ate with a dicotyledonous host (Steiner et al. 2006). These with respect to the presence of ergot alkaloids (Eich 2008). ‘endophytes’ also appear to have a narrow host range: the Expanded sampling of the Convolvulaceae for ergot alka- two described Periglandula species are chemically dissimi- loids will surely reveal more species infected by Periglandu- lar and occur on different host plants (Steiner et al. 2011). la. Extrapolation from available data would suggest A third, undescribed, Clavicipitaceae from Ipomoea tri- upwards of 250 species of ergot alkaloid-positive color, which does not form epiphytic mycelia, was detect- Ipomoeeae. able via PCR and groups with described Periglandula Interestingly, loline and ergot alkaloids have been species, yet it is also phylogenetically distinguishable detected in the seeds and foliage of one species, Argyreia (Ahimsa-Muller et al. 2007). mollis (Tofern et al. 1999), suggesting Periglandula may Prior to the discovery of Periglandula species, the occur- also produce other classes of secondary metabolites found rence of ergot alkaloids in the Convolvulaceae was thought in other Clavicipitaceae. There have been no published to be a case of convergent evolution or horizontal gene reports from the Convolvulaceae of the other two major transfer (Steiner, Hellwig & Leistner 2008). Although pub- classes of clavicipitaceous secondary metabolites – indole- lished evidence for Periglandula species colonization has diterpenes and peramine – although we do not know of been provided for only three species of Convolvulaceae, any studies that explicitly tested for them. There are, how- ergot alkaloids are known to occur in many more species ever, reports of tremorgenic symptoms in livestock caused in this diverse plant family (Eich 2008), each of which by grazing of foliage from ergot alkaloid-positive Convol- likely harbours a species of Periglandula. Whereas ergot vulaceae, specifically caused by sheep feeding on Ipomoea alkaloids were discovered in grasses due to their influence muelleri in Australia (Gardiner, Royce & Oldroyd 1965) on agriculture (Lyons, Plattner & Bacon 1986), their dis- and sheep and cattle feeding on I. asarifolia in Brazil covery in the Convolvulaceae followed from the work of (Araújo et al. 2008). Livestock grazing on grasses contain- ethnobotanist Richard Schultes in Central America in the ing indole-diterpenes also suffer from tremorgenic symp- late 1930s (Schultes 1941; Schultes 1969). He reported that toms (Belesky & Bacon 2009). Because the tremoring the seeds of Turbina corymbosa (host to P. turbinae), called symptoms were caused by grazing on these two ergot ‘ololiuqui’ by the Aztecs, and the seeds of I. tricolor, called alkaloid-positive species (one of which is host to ‘badoh negro’ by the Zapotecs, were consumed ritualisti- P. ipomoeae), the possibility that some Periglandula species cally for divination. Two decades later, Albert Hofmann produce indole-diterpenes should be investigated. isolated the lysergic acid amides ergine and lysergic acid The ecological effects of Periglandula infection or ergot a-hydroxyethylamide from T. corymbosa (Hofmann & alkaloid presence in the Convolvulaceae has yet to be stud- Tscherter 1960; Hofmann 1961), which were likely respon- ied. As Periglandula species are closely related to Neoty- sible for the hallucinogenic effects of these plants. phodium species endophytes and share some important The discovery of ergot alkaloids in a dicotyledonous characteristics (e.g. vertical transmission and production of plant spurred several studies on the occurrence of ergot ergot alkaloids), their presence also may confer similar alkaloids in the Convolvulaceae; Eich (2008) has critically benefits to their convolvulaceous hosts such as resistance reviewed this work. In the genus Ipomoea (ca. 500 spp.), to herbivory (Clay & Schardl 2002). Unlike the cool sea- 79 species have been screened for ergot alkaloids and 23 son pooid grasses which host epichloae, there is striking (29%) are unambiguously positive (Eich 2008). The genera variation in life history and habitat even among the limited Argyreia, Stictocardia and Turbina also have ergot alka- number of known ergot alkaloid-positive Convolvulaceae. loid-positive representatives (Eich 2008). Moreover, the They include herbaceous twining vines (e.g. I. tricolor), three major types of ergot alkaloids (clavines, lysergic acid woody lianas (e.g. Argyreia nervosa), sprawling vines (e.g.

I. pes-caprae) and shrubs (e.g. Ipomoea leptophylla and I. hildebrandtii) which can be found worldwide in deserts, sand dunes, forests and grasslands in North and South America, Africa, Asia and Australia (Verdcourt 1978; Devall 1992; Austin, Jarret & Johnson 1993; Austin & Huaman 1996). Many ergot alkaloid-positive Convolvula- ceae are restricted to a single continent, but one species, Ipomoea pes-caprae, is found on tropical and subtropical beaches worldwide where it grows as a pioneer species just above the high tide line (Devall 1992). Fig. 5. Proposed pathway for swainsonine and slaframine biosyn- While many studies in epichloae-grass systems demon- thesis. Pathway is based on studies conducted in Rhizoctonia legu- strate increased resistance to foliar herbivory, resistance to minicola (Harris et al. 1988b). seed predation may be an important dimension of the Peri- glandula-Convolvulaceae symbioses. Some studies have indicated that alkaloids can be present in the seeds but not lysosomal storage disease and altered glycoprotein synthe- the foliage (Chao & Der Marderosian 1973; Jirawongse, sis (Hartley 1971; Dorling, Huxtable & Vogel 1978). Pharadai & Tantivatana 1977). Convolvulaceae seeds can Recently, a fungal endophyte, Undifilum oxytropis be large, often >5 mm in diameter (Verdcourt 1978), and (Pryor et al. 2009), previously described as an Embellesia presumably represent a large investment of plant species (Wang et al. 2006), was reported to produce resources. Additionally, many species are highly parasit- swainsonine in locoweeds (Braun et al. 2003). The Undifi- ized by bruchine beetles (Coleoptera: Bruchinae) (Reyes, lum genus (Pleosporaceae) is closely related to the genera Canto & Rodriguez 2009). The larvae of these beetles can Alternaria, Embellesia and Ulocladium (Pryor et al. 2009). bore into the seed, consume the cotyledons or embryo and Undifilum species are only associated with swainsonine- leave a characteristic circular exit hole. Reports of bru- containing Astragalus and Oxytropis species with one chine parasitism rates range from 4 to 85% in I. pes-caprae exception, Undifilum bormuelleri, a pathogen of the legume (Wilson 1977; Devall & Thien 1989), and 34 to 100% in Securigera varia that does not contain swainsonine. Undifi- I. leptophylla, a shrub found in the short grass prairies of lum species have been found to be associated with swainso- the Central United States (Keeler 1980, 1991). Whereas nine-containing Astragalus and Oxytropis species in North several factors may contribute to this variation, especially America and China (Pryor et al. 2009; Yu et al. 2010; visitation by ants to extra floral nectaries (Keeler 1980), Baucom et al. 2012). Like many epichloae and the known population differences in ergot alkaloid content may play a Periglandula species, Undifilum species associated with role. Other possibilities are that the specialized bruchines locoweeds are vertically transmitted and have no apparent associated with ergot alkaloid-positive Convolvulaceae sexual stage (Oldrup et al. 2010; Ralphs et al. 2011). have overcome the plant’s acquired defence from Periglan- Undifilum species also appear to have a narrow host range dula or that there is ongoing co-evolution between the as different plant species are associated with unique Undifi- plant–fungal symbiota and the beetles. lum species (Pryor et al. 2009; Baucom et al. 2012). In addition to the legumes, swainsonine occurs sporadi- cally in two other plant families, the Convolvulaceae and Endophytes of locoweeds and related taxa the Malvaceae. In the Convolvulaceae, some Ipomoea and Several species in the legume genera Astragalus, Oxytropis Turbina species are reported to contain swainsonine, for and Swainsona have been found to be toxic to grazing live- example, I. carnea and T. cordata (de Balogh et al. 1999; stock in the Americas, Asia and Australia (Marsh 1909; Dantas et al. 2007), while in the Malvaceae, Sida carpino- Marsh & Clawson 1936; Gardiner, Linto & Aplin 1969; folia is reported to contain swainsonine (Colodel et al. Huang, Zhang & Pan 2003). Locoism, a neurologic dis- 2002). Like the legumes, swainsonine was identified in the ease, was first noted by the Spanish conquistadors, and plant species associated with these families due to livestock again during the settlement of Western North America by poisoning and subsequent economic impact. pioneers (Marsh 1909; Marsh & Clawson 1936; Jones, Swainsonine is also reported to be produced by two Hunt & King 1997). Clinical signs and pathology of loco- phylogenetically disjunct fungi, Rhizoctonia leguminicola ism are similar in animals intoxicated by locoweed species (Ceratobasidiaceae) and Metarhizium anisopliae (Clavici- and Swainsona species (James, Van-Kampen & Hartley pitaceae) (Schneider et al. 1983; Patrick, Adlard & Kesha- 1970; Panter et al. 1999). Swainsonine (Fig. 5), a trihydr- varz 1993). Rhizoctonia leguminicola is a fungal pathogen oxyindolizidine alkaloid, was first identified as the active of red clover (Trifolium pratense) that causes black patch principle in Swainsona canescens, a legume native to Aus- disease in the plant. Metarhizium anispoliae is an entomo- tralia (Colegate, Dorling & Huxtable 1979), and subse- pathogen that attaches to the outside of an insect, grows quently identified as the active principle in locoweeds internally and causes death. The roles swainsonine plays in (Molyneux & James 1982). Swainsonine inhibits the either of these biological systems have not been elucidated. enzymes a-mannosidase and mannosidase II resulting in Like the ergot alkaloids, swainsonine appears to be more

Published 2013. This article is a U.S. Government work and is in the public domain in the USA. Functional Ecology 2013 © British Ecological Society, Functional Ecology, 28, 299–314 308 D. G. Panaccione et al. widely distributed in fungi other than seed-transmitted matures (Cook et al. 2012). Finally, swainsonine concen- endophytes. trations are greatest in floral parts and seeds (Grum et al. The biosynthesis of swainsonine has been investigated in 2012), consistent with the optimal defence theory. the fungus R. leguminicola (Harris et al. 1988b). Swainso- There are few studies regarding the ecological role of nine is derived from lysine which is converted into pip- swainsonine and how it responds to environmental ecolic acid. Two precursors of swainsonine in the fungal changes. Swainsonine concentrations do not change in biosynthetic pathway were detected in the shoots of Diablo respond to clipping used to simulate herbivory, nor does it locoweed (Astragalus oxyphysus) (Harris et al. 1988a,b); as deter grazing as animals become progressively more intoxi- a result, Harris et al. (1988a) proposed that the biosynthet- cated (Ralphs et al. 2002; Pfister et al. 2003). In fact ani- ic pathway of swainsonine in R. leguminicola is similar to mals take 2–3 weeks to show clinical signs and continue the pathway in locoweeds, where swainsonine was later grazing locoweeds after becoming intoxicated (Pfister et al. found to be produced by Undifilum species. 2003). Activity of swainsonine against insects, fungi or Support for the fact that swainsonine is a fungal-derived bacteria has not been definitively tested in published stud- secondary metabolite in locoweeds is based on the follow- ies, although preliminary results show that swainsonine ing observations: (i) locoweed plants infected with Undifi- has no effect on some insect species (Parker 2008). lum species contain swainsonine; (ii) plants derived from Legumes are known for forming symbioses with N-fixing Astragalus and Oxytropis embryos in which the seed coat, bacteria and investigators found that swainsonine concen- the primary location of Undifilum, was removed have no trations were greater in plants inoculated with one strain detectable swainsonine, or have concentrations less than of Rhizobium but not others (Valdez Barillas et al. 2007), 0Á001% (Oldrup et al. 2010; Grum et al. 2012); (iii) plants suggesting an interaction between the two classes of symbi- derived from fungicide-treated Astragalus and Oxytropis onts. An alternative interpretation is that the improved seeds have no detectable swainsonine or have concentra- nitrogen status of the host may have increased substrate tions less than 0Á001% (Grum et al. 2012); (iv) Undifilum availability for swainsonine production; however, no con- species isolated from locoweeds produce swainsonine in sistent differences in swainsonine concentrations were pure culture (Braun et al. 2003); (v) plants derived from observed in locoweed plants, whether nitrogen deficient or embryos that were inoculated with Undifilum have swains- adequate, when nitrogen was supplied through fertilizer onine concentrations greater than 0Á01% (Grum et al. (Delaney et al. 2011). Lastly, swainsonine concentrations 2012); and (vi) rats fed U. oxytropis developed lesions and were shown to increase slightly in response to water stress clinical signs similar to those fed swainsonine-containing in some locoweed species but not others (Vallotton et al. Oxytropis lambertii (McLain-Romero et al. 2004). 2012). Swainsonine concentrations vary greatly among species, It has not been determined whether swainsonine is varieties and populations. For example, Astragalus species plant- or fungal-derived in the legume Swainsona canes- generally have greater swainsonine concentrations than do cens, in the Convolvulaceous genera of Ipomoea and Turbi- Oxytropis species in North America (Ralphs et al. 2008) na,orinSida carpinfolia, a species of the Malvaceae while different varieties of O. lambertii vary greatly in their family. The presence of swainsonine in these species may swainsonine concentrations (Gardner, Molyneux & Ralphs be a case of convergent evolution or horizontal gene 2001). Additionally, in toxic populations of locoweeds, transfer; however, due to the sporadic occurrence of two chemotypes of plants have been identified, namely swainsonine in these genera, it seems probable that a chemotype one plants, which contain swainsonine concen- swainsonine-producing fungal endophyte is associated with trations >0Á01%, and chemotype two plants, which have these taxa that contain swainsonine. In fact, recent concentrations <0Á01% (generally near 0Á001% or not research suggests that a fungal endophyte is present in the detected) (Cook et al. 2009, 2011). These two chemotypes swainsonine-positive taxa, S. canescens and Ipomoea car- differ significantly in the amount of endophyte they nea (Cook and Beaulieu, unpublished data). The fungal contain which may explain the difference in swainsonine endophyte associated with S. canescens appears to be a concentrations (Cook et al. 2009, 2011). novel Undifilum species, while the endophyte associated Swainsonine and endophyte amounts have been investi- with I. carnea appears to belong to an Ascomycete family gated in different plant parts at different phenological not related to the Pleosporaceae family that contains stages (Cook et al. 2012). Swainsonine is found in all plant Undifilum. Preliminary data suggest that these endophytes parts although concentrations are greater in above-ground produce swainsonine in vitro, are vertically transmitted, parts than in below-ground parts (Cook et al. 2009, 2011). and have a narrow host range. Endophytic Undifilum species also are found in all plant A comparison of the locoweed/swainsonine-producing parts with only small quantities found in the root (Cook endophyte system(s) to those involving clavicipitaceous et al. 2009, 2011). The root crown appears to be a major endophytes reveals some similarities but also significant reservoir for the endophyte during the following year’s differences (Table 2). (Note that the Convolvulaceae are growth as many locoweeds are perennial plants (Cook interesting in that individual species have symbioses with et al. 2009, 2011), and swainsonine and endophyte Periglandula species, presently undescribed swainsonine amounts increase in above-ground parts as the plant producers, or in some cases no endophytes.) First, in the

Table 2. Important characteristics of plant–fungal symbiota considered in this review

Grasses Morning Glories Locoweeds

Associated Fungal Clavicipitaceae Clavicipitaceae Unknown Pleosporaceae Family Bioactive Chemicals Ergots, lolines, peramine, Ergots, lolines2, indole-diterpenes3 Swainsonine Swainsonine Produced indole-diterpenes Major Clade Monocots Eudicots Eudicots Growth Form Herbaceous Mostly woody vines (also shrubs, Herbaceous trees, herbaceous vines) Distribution Mostly temperate Mostly tropical & subtropical Semiarid to temperate Economic Forage, crops Some crops1, agricultural pests Agricultural pests Importance Pollination System Wind Pollinated Insect pollinated Insect pollinated Mode(s) of Strictly vertical, mixed Strictly vertical (?) Strictly Strictly vertical (?) Transmission (vertical and horizontal), vertical (?) and strictly horizontal

1Ipomoea batatas (sweet potato) and Ipomoea aquatica (water spinach) are crop species but do not contain ergot alkaloids (Eich 2008) and have never been reported to produce swainsonine. 2Lolines were reported from A. mollis (Tofern et al. 1999), but it has not been demonstrated that they are produced by Periglandula. 3Indole diterpenes have not been reported from morning glories, but there have been reports of tremorgenic symptoms, characterisitc of indole diterpene poisoning, in livestock feeding on species infected by Periglanudla (Araújo et al. 2008). grass-epichloae and Convolvulaceae-Periglandula species which they engage; however, toxins are noteworthy endophyte symbioses, plants completely free of the endo- because they have a clear impact on humans, grazing phyte are occasionally encountered (Schardl et al. 2009; animals and/or insect pests. For fungi to persist as verti- Schardl 2010; Beaulieu, Clay & Panaccione, unpublished), cally transmissible endophytes, they need to be advanta- whereas in the locoweed system, an endophyte-free plant geous for their host plants, otherwise they would be has not yet been detected in a natural population (Cook outcompeted by non-infected conspecifics, which typically et al. 2009, 2011). Instead, locoweeds occur in two chemo- exist among endophyte-infected populations because endo- types in native plant populations that differ greatly in the phyte transmission is rarely 100% (Afkhami & Rudgers amount of swainsonine and extent of endophyte coloniza- 2008). The advantage provided to the host by the endo- tion. Second, a small amount of Undifilum species myce- phyte may be sporadic in nature, resulting in the infected lium is detected in below-ground plant parts, whereas and non-infected hosts within a population. Bioactive epichloae typically are not reported to be in below-ground metabolites are a pre-adaption that fungi can bring with parts (Schardl, Leuchtmann & Spiering 2004). Third, them in the establishment of a symbiosis. swainsonine is found in all tissues in symbiotic plants Pathways for many of the metabolites discussed here (Cook et al. 209; Cook et al. 2011) as is the clavicipita- may have evolved in fungi prior to their lineage becoming ceous water-soluble alkaloid peramine, while the ergot and associated with plants as endophytes and have been indole-diterpene alkaloids produced by the clavicipitaceous retained because the toxins provided the free-living fungus endophytes are found in above-ground parts only (Ball protection from insects or other animals. The clavicipita- et al. 1997a,b; Spiering et al. 2002, 2005). Lastly, all verti- ceous endophytes of grasses and morning glories may pro- cally transmitted endophytic fungi that produce ergot alka- vide an illustration of this point. Spatafora et al. (2007) loids, indole-diterpenes, lolines or peramine are derived demonstrated through phylogenetic analyses that the from Clavicipitaceae, regardless of the plant family with plant-infecting Clavicipitaceae (which includes the epi- which they are associated, whereas strictly vertically trans- chloae and the Periglandula spp.) likely evolved from mitted endophytes that produce swainsonine are derived insect-infecting Clavicipitaceae. Insect-pathogenic Meta- from different fungal families that form relationships with rhizium spp. share a recent common ancestor with the specific plant families. In regard to this observation, the clade that contains the epichloae and related plant-infect- evolutionary history of the swainsonine biosynthetic path- ing Clavicipitaceae; these fungi are part of a monophyletic way in these diverse fungi is particularly intriguing. group (Clavicipitaceae clade A) whose most recent common ancestor was likely an insect pathogen (Spatafora et al. 2007). The genomes of M. anisopliae and Metarhizi- Concluding remarks um acridum (Gao et al. 2011) contain gene clusters very In this study, we have focused on the bioactive metabolites similar to the ergot alkaloid gene clusters of other ergot of vertically transmitted endophytes and the effects of alkaloid-producing fungi, although ergot alkaloid produc- these metabolites on herbivores. Certainly, there are other tion has not been demonstrated experimentally in these ways that endophytes can contribute to the symbioses in species. Considering the distribution of ergot alkaloid

Published 2013. This article is a U.S. Government work and is in the public domain in the USA. Functional Ecology 2013 © British Ecological Society, Functional Ecology, 28, 299–314 310 D. G. Panaccione et al. biosynthetic capacity among the epichloae, Periglandula et al. (2009) expanded upon this work and showed verti- spp., and Metarhizium spp., and the toxicity of certain cally transmitted Neotyphodium spp. reach infection fre- ergot alkaloids to insects, ergot alkaloid biosynthetic quencies 40–130% greater than Epichloe€ species that have capacity may have been present in the insect-pathogenic mixed horizontal and vertical transmission. In aphid–bac- ancestor that evolved into the plant-infecting Clavicipita- terial interactions, the vertically transmitted Buchnera spe- ceae. The biosynthetic capacity may have then diversified cies is fixed in most populations and considered obligate, to include other forms that are more effective against ver- whereas there are many horizontally transmitted symbio- tebrate herbivores. So long as there is some initial benefit nts that exhibit variability in their presence (Oliver et al. to harbouring the fungal endophyte, the fungus may be 2010). Finally, the rapidly expanding cache of genomic selected for and then evolve other bioactive metabolites or data available to researchers may further facilitate the beneficial mechanisms apart from secondary compounds. search for endophytes involved in defensive mutualisms. The ability of fungi in this lineage to colonize both plants Genomic studies of plants may provide an opportunity to and insects is demonstrated by Metarhizium robertsii, search in silico for evidence of these symbionts by looking which can grow endophytically in switch grass (Panicum for gene clusters of fungal metabolites or conserved fungal virgatum) and bean (Phaseolus vulgaris) roots, in addition genes. to parasitizing insects (Sasan & Bidochka 2012). It also is In summary, plant–fungal associations with the charac- interesting to note that M. anisopliae produces swainso- teristics of vertical transmission, narrow host range, and nine despite being phylogenetically disjunct from the other the production of bioactive secondary metabolites resulting swainsonine-producing fungi. Thus, it is possible that in a generally mutualistic association extend beyond the swainsonine may have first arisen in an insect pathogen as well-studied grass–epichloae systems. As other major clas- well. ses of plant–microbial symbioses involve diverse taxa – for Considerable effort has gone into understanding ecologi- example, mycorrhizae are formed by Glomeromycota and cal impacts of endophytes (reviewed in Clay & Schardl Basidiomycota while nitrogen-fixing associations are 2002; Schardl et al. 2009). Similarly, molecular and bio- formed by Rhizobium (Proteobacteria) and Frankia chemical approaches have improved our understanding of (Actinobacteria) – class 1 endophytes as defined by Rodri- endophyte-produced bioactive metabolites and their bio- guez et al. (2009) are not limited to the Clavicipitaceae. synthetic pathways (reviewed in Lorenz et al. 2009; Panac- Considering advances in knowledge from work in the cione 2010; Schardl et al. 2011). Future research should grass–epichloae symbiota, studying additional endophyte connect these two lines of investigation to molecularly dis- symbioses may provide key insights into the organization sect the roles of various chemicals in fungus–grass–herbi- of ecological communities and provide excellent case vore interactions and assess the impact of eliminating or studies on the evolution and diversification of mutualisms. adding specific chemicals via genetic modification of the endophyte. Moreover, these approaches need to be applied Acknowledgements to a broader range of vertically transmitted endophytes, including the clavicipitaceous endophytes of the Funding from the U.S. Department of Agriculture National Institute of Ipomoeeae and the swainsonine-producing endophytes of Food and Agriculture (2012-67013-19384) to D.G.P. is gratefully acknowl- edged. We thank Keith Clay for helpful guidance on the content of this locoweeds and related legumes. review, Christopher Schardl and an anonymous reviewer for constructive Finally, the possibility that similar endophyte associa- comments on a previous version of this article, and Sarah Robinson for tions are more widely present among plant taxa should be assistance with the bibliography. This article is published with permission of the West Virginia Agriculture and Forestry Experiment Station as considered. The examples described herein were evident to scientific article number 3155. us because of their impact on animal agriculture or because of the effects of the bioactive metabolites on humans. Other such symbioses may occur in plants that References are not food for humans or livestock and, thus, we have Afkhami, M.E. & Rudgers, J.A. (2008) Symbiosis lost: imperfect vertical not been confronted with them. While searching for other transmission of fungal endophytes in grasses. 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Published 2013. This article is a U.S. Government work and is in the public domain in the USA. Functional Ecology 2013 © British Ecological Society, Functional Ecology, 28, 299–314