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Differential Allocation of Seed-Borne Ergot Alkaloids During Early Ontogeny of Morning Glories (Convolvulaceae) Wesley T. Beaulieu & Daniel G. Panaccione & Corey S. Hazekamp & Michelle C. Mckee & Katy L. Ryan & Keith Clay Received: 26 April 2013 / Revised: 11 June 2013 / Accepted: 14 June 2013 / Published online: 9 July 2013 # Springer Science+Business Media New York 2013 Abstract Ergot alkaloids are mycotoxins that can increase host plant resistance to above- and below-ground herbivores. Some morning glories (Convolvulaceae) are infected by clavicipitaceous fungi (Periglandula spp.) that produce high concentrations of ergot alkaloids in seedsup to 1000-fold greater than endophyte-infected grasses. Here, we evaluated the diversity and distribution of alkaloids in seeds and seed- lings and variation in alkaloid distribution among species. We treated half the plants with fungicide to differentiate seed-borne alkaloids from alkaloids produced de novo post- germination and sampled seedling tissues at the cotyledon and first-leaf stages. Seed-borne alkaloids in Ipomoea amnicola, I. argillicola, and I. hildebrandtii remained primarily in the cotyledons, whereas I. tricolor allocated lysergic acid amides to the roots while retaining clavines in the cotyledons. In I. hildebrandtii, almost all festuclavine was found in the cotyledons. These observations suggest differential allocation of individual alkaloids. Intraspecific patterns of alkaloid distri- bution did not vary between fungicide-treated and control seedlings. Each species contained four to six unique ergot alkaloids and two species had the ergopeptine ergobalansine. De novo production of alkaloids did not begin immediately, as total alkaloids in fungicide-treated and control seedlings did not differ through the first-leaf stage, except in I. argillicola. In an extended time-course experiment with I. tricolor, de novo production was detected after the first-leaf stage. Our results demonstrate that allocation of seed-borne ergot alkaloids varies among species and tissues but is not altered by fungicide treatment. This variation may reflect a response to selection for defense against natural enemies. Keywords Ergoline alkaloids . Fungal endophyte . Ipomoea . Symbiosis . Periglandula . Defensive mutualism Introduction Defensive mutualisms are a widespread class of symbioses that often involve the production of bioactive secondary metabolites by microbes and the protection of hosts from natural enemies (White and Torres 2009). These relationships have been documented in diverse taxa involving invertebrate and plant hosts associated with bacterial and fungal symbionts (Clay and Schardl 2002; White and Torres 2009). One well known example is cool-season grasses (Poaceae) symbiotic with fungal endophytes in the family Clavicipitaceae, specifi- cally Neotyphodium and Epichloë spp. (Schardl 2010). In these interactions, the plant provides a habitat and resources for the fungus and in turn receives protective secondary metabolites, which may include indole-diterpenes, peramine, loline alkaloids, and ergot alkaloids, depending upon the fungal species and isolate (Panaccione et al. 2013; Schardl et al. 2012). While endophyte infection per se can have other effects such as increased drought tolerance and competitive ability (Clay and Schardl 2002), the only known function of these alkaloids is protection. For example, the production of alkaloid compounds by these fungi in planta increases resis- tance to vertebrate and invertebrate herbivores in many grass- fungal symbiota (Clay et al. 2005; Koh and Hik 2007; Schardl et al. 2009) with cascading effects on communities (Clay et al. 2005; Finkes et al. 2006; Rudgers et al. 2004, 2007). Ergot alkaloids are a particularly noteworthy class of secondary metabolites given their phylogenetic distribution W. T. Beaulieu (*) : M. C. Mckee : K. Clay Department of Biology, Indiana University, Bloomington, IN, USA e-mail: [email protected] D. G. Panaccione : C. S. Hazekamp : K. L. Ryan Division of Plant & Soil Sciences, West Virginia University, Morgantown, WV, USA J Chem Ecol (2013) 39:919930 DOI 10.1007/s10886-013-0314-z

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Page 1: Differential Allocation of Seed-Borne Ergot Alkaloids During Early … · 2013-10-01 · in the cotyledons, whereas I. tricolor allocated lysergic acid amides to the roots while retaining

Differential Allocation of Seed-Borne Ergot AlkaloidsDuring Early Ontogeny of Morning Glories (Convolvulaceae)

Wesley T. Beaulieu & Daniel G. Panaccione &

Corey S. Hazekamp &Michelle C. Mckee &Katy L. Ryan &

Keith Clay

Received: 26 April 2013 /Revised: 11 June 2013 /Accepted: 14 June 2013 /Published online: 9 July 2013# Springer Science+Business Media New York 2013

Abstract Ergot alkaloids are mycotoxins that can increasehost plant resistance to above- and below-ground herbivores.Some morning glories (Convolvulaceae) are infected byclavicipitaceous fungi (Periglandula spp.) that produce highconcentrations of ergot alkaloids in seeds—up to 1000-foldgreater than endophyte-infected grasses. Here, we evaluatedthe diversity and distribution of alkaloids in seeds and seed-lings and variation in alkaloid distribution among species.We treated half the plants with fungicide to differentiateseed-borne alkaloids from alkaloids produced de novo post-germination and sampled seedling tissues at the cotyledonand first-leaf stages. Seed-borne alkaloids in Ipomoeaamnicola, I. argillicola, and I. hildebrandtii remained primarilyin the cotyledons, whereas I. tricolor allocated lysergic acidamides to the roots while retaining clavines in the cotyledons.In I. hildebrandtii, almost all festuclavine was found in thecotyledons. These observations suggest differential allocationof individual alkaloids. Intraspecific patterns of alkaloid distri-bution did not vary between fungicide-treated and controlseedlings. Each species contained four to six unique ergotalkaloids and two species had the ergopeptine ergobalansine.De novo production of alkaloids did not begin immediately, astotal alkaloids in fungicide-treated and control seedlings did notdiffer through the first-leaf stage, except in I. argillicola. In anextended time-course experiment with I. tricolor, de novoproduction was detected after the first-leaf stage. Our resultsdemonstrate that allocation of seed-borne ergot alkaloids variesamong species and tissues but is not altered by fungicide

treatment. This variation may reflect a response to selectionfor defense against natural enemies.

Keywords Ergoline alkaloids . Fungal endophyte . Ipomoea .

Symbiosis .Periglandula . Defensivemutualism

Introduction

Defensive mutualisms are a widespread class of symbiosesthat often involve the production of bioactive secondarymetabolites by microbes and the protection of hosts fromnatural enemies (White and Torres 2009). These relationshipshave been documented in diverse taxa involving invertebrateand plant hosts associated with bacterial and fungal symbionts(Clay and Schardl 2002; White and Torres 2009). One wellknown example is cool-season grasses (Poaceae) symbioticwith fungal endophytes in the family Clavicipitaceae, specifi-cally Neotyphodium and Epichloë spp. (Schardl 2010). Inthese interactions, the plant provides a habitat and resourcesfor the fungus and in turn receives protective secondarymetabolites, which may include indole-diterpenes, peramine,loline alkaloids, and ergot alkaloids, depending upon thefungal species and isolate (Panaccione et al. 2013; Schardlet al. 2012). While endophyte infection per se can have othereffects such as increased drought tolerance and competitiveability (Clay and Schardl 2002), the only known function ofthese alkaloids is protection. For example, the production ofalkaloid compounds by these fungi in planta increases resis-tance to vertebrate and invertebrate herbivores in many grass-fungal symbiota (Clay et al. 2005; Koh and Hik 2007; Schardlet al. 2009) with cascading effects on communities (Clay et al.2005; Finkes et al. 2006; Rudgers et al. 2004, 2007).

Ergot alkaloids are a particularly noteworthy class ofsecondary metabolites given their phylogenetic distribution

W. T. Beaulieu (*) :M. C. Mckee :K. ClayDepartment of Biology, Indiana University, Bloomington, IN, USAe-mail: [email protected]

D. G. Panaccione : C. S. Hazekamp :K. L. RyanDivision of Plant & Soil Sciences, West Virginia University,Morgantown, WV, USA

J Chem Ecol (2013) 39:919–930DOI 10.1007/s10886-013-0314-z

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and physiological effects. They are psychoactive chemicalsthat mimic neurotransmitters including dopamine, serotonin,and adrenaline that cause vasoconstriction, smooth musclecontraction, bewilderment and hallucinations in vertebrates.Chronic exposure to ergot alkaloids can result in a syndromeknown as ergotism with physical effects such as weight lossand gangrene of the extremities, as in “St. Anthony’s Fire”(White et al. 2003) caused by contaminated grain stocksduring the Middle Ages. In agricultural systems, there canbe significant losses in grazing livestock caused by con-sumption of infected forage (Vanheeswijck andMcDonald 1992). Wild vertebrates such as voles (Microtusspp.) and pika (Ochotona spp.) also are deterred from plantsby infection with fungal endophytes known to produce ergotalkaloids and potentially other compounds (Fortier et al. 2000;Koh and Hik 2007). Ergot alkaloids also have activitiesagainst insects (Clay and Cheplick 1989; Potter et al. 2008),nematodes (Bacetty et al. 2009a, b) and bacteria (Eich et al.1984; Panaccione 2005). By contrast, New World plantscontaining ergot alkaloids are used to treat migraines and postnatal bleeding (Plowman et al. 1990), just as preparations ofClaviceps spp. historically were used during labor and deliv-ery in the Old World (White et al. 2003). Purified ergotalkaloids are used to treat these same symptoms in modernmedicine (Henrich et al. 2008; Lorenz et al. 2009). Ergotalkaloid-containing seeds of the morning glories Ipomoeatricolor and Turbina corymbosawere ritualistically consumedas entheogens by the Zapotec and Aztec peoples, respectively(Schultes 1969) and LSD, a semi-synthetic ergot alkaloid, hasbeen consumed intentionally by humans for its psychoactiveeffects (Hofmann 2009).

The genetic capacity to produce ergot alkaloids is knownonly from ascomycetous fungi, primarily species in theClavicipitaceae but also species in the Trichocomaceae,e.g., Aspergillus fumigatus (Panaccione 2010; Wallwey andLi 2011). Within the Clavicipitaceae, ergot alkaloids areproduced by systemic fungal endophytes in planta such asNeotyphodium spp. and Epichloë spp. infecting cool-seasongrasses, Balansia spp. infecting warm-season grasses andsedges (Cyperaceae), as well as Claviceps spp., ovarianparasites of grasses, sedges and rushes (Panaccione 2010).Recently, ergot alkaloids in morning glories (Convolvulaceae)were shown to be produced by clavicipitaceous fungi classifiedin the genus Periglandula (Ahimsa-Muller et al. 2007; Kuchtet al. 2004; Steiner et al. 2011). While two species ofPeriglandula have been formally described (Steiner et al.2011), ergot alkaloids have been reported in at least 40morningglory species (Eich 2008), with concentrations in seeds up to1000 times greater than seeds of grasses infected byNeotyphodium and Epichloë spp. (Ahimsa-Muller et al. 2007;Kucht et al. 2004; Panaccione et al. 2003). This high concen-tration of ergot alkaloids, along with the metabolic demands ofthe fungal symbiont, presumably comes at a cost to the plant.

Given that Periglandula spp. infecting morning gloriesappear to be vertically transmitted through seeds, theoreticaland empirical work from a variety of systems would predictthat the infection has adaptive value for the host since anotherwise purely maternally-transmitted symbiont would notpersist (Bull et al. 1991; Ewald 1987; Herre 1993). Onepossible benefit to both plant and fungus from high ergotalkaloid concentrations in the seed could be increased resis-tance to seed predation (Zhang et al. 2012). Another poten-tial benefit that may result from ergot alkaloids that areredistributed during germination and seedling growth isgreater resistance to natural enemies compared to uninfectedseedlings lacking seed-borne ergot alkaloids. Seedlings maybe especially vulnerable to predators and pathogens, as theyhave limited resources to divert from growth to defensivechemistry. Since ergot alkaloids are known to be effectiveagainst many types of natural enemies in grasses (Panaccioneet al. 2013; Schardl et al. 2012), optimal defense theory predictsthat the seed-borne ergot alkaloids already present in the seedupon germination will be preferentially allocated to the mostvaluable tissues (Rhoades 1979). As plant and fungal repro-duction are linked by vertical transmission through seeds, thefungal symbiont would increase its own fitness if it increasedhost plant fitness.

Previous research has demonstrated that fungal alkaloidscan be mobilized in planta. For example, Koulman et al.(2007) found that ergot alkaloids, as well as peramine andloline alkaloids, were detected in the guttation (leaf exudate)and cut-leaf fluid of tall fescue (Festuca arundinacea) infectedby N. coenophialum, in contrast to perennial ryegrass (Loliumperenne) infected by N. lolii or E. festucae where ergot alka-loids were detected in plant tissues but not the guttation or cut-leaf fluids. Similarly, Spiering et al. (2005) showed in peren-nial ryegrass infected byN. lolii that endophyte concentration,as measured by molecular markers and hyphal counts,explained only 20% of the variance of in planta ergot alkaloiddistribution, with a much larger effect of plant genotype.Research on the Convolvulaceae-Clavicipitaceae symbiosisis in a nascent stage with many basic questions unanswered,such as the distribution of the fungus or ergot alkaloids inconvolvulaceous plants. One previous study conducted beforethe discovery of ergot alkaloid-producing Periglandula spp.demonstrated that alkaloids synthesized in the leaves of I.tricolor (referred to as I. violacea by the authors) can betranslocated (Mockaitis et al. 1973). When the authors graftedleaves of I. tricolor onto I. nil (a species known to not containergot alkaloids), ergot alkaloids were detected in the stem androots of I. nil. One alternative but less likely explanation totranslocation of alkaloids is that the fungal symbiont in thegrafted I. tricolor leaves colonized the stem and roots of the I.nil plant onto which it was grafted.

In this study, we investigated the in planta distribution ofseed-borne ergot alkaloids in four Ipomoea species during

920 J Chem Ecol (2013) 39:919–930

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early ontogeny. Specifically, we addressed four questions:(1) Are ergot alkaloids synthesized de novo during earlyseedling ontogeny? (2) What is the in planta distribution ofergot alkaloids during ontogeny? (3) Are individual ergotalkaloids differentially allocated among tissues within hostseedlings? (4) Does the in planta distribution of ergot alkaloidsvary among species? To address these questions, we conductedtwo sequential harvest experiments where plants were destruc-tively harvested at distinct life stages, and the identity andamount of ergot alkaloids were determined in separate tissues.In each experiment, we included a fungicide treatment todifferentiate between ergot alkaloids that were seed-borne vs.those produced by de novo synthesis during ontogeny. Oneexperiment focused on I. tricolor, and addressed questions 1–3.A second experiment included I. tricolor and three additionalIpomoea spp. to address questions 1–4.

Methods and Materials

Multispecies Experiment To address questions 1–4, weconducted a sequential harvest experiment with four speciesof Ipomoea. Three species were obtained from the UnitedStates Department of Agriculture Agricultural ResearchService Genetic Resource Initiative Network (USDA-ARSGRIN): Ipomoea amnicola (PI 553010, collected in Texas,USA), a twining vine found in South America, CentralAmerica, and Texas; I. argillicola (PI 538265, collected inQueensland, Australia), a sprawling vine found in grasslandsin Western Australia; and I. hildebrandtii (PI 643125, col-lected in Sipi Falls, Uganda), a shrub found in semi-aridgrasslands of East Africa. Ipomoea tricolor var. “FlyingSaucers” was purchased commercially from W. Atlee Burpee& Co (Warminster, PA, USA). In nature, this species is atwining vine native to Central America. Adult plants of I.amnicola, I, argillicola, and I. hildebrandtii derived from thesame seed stocks used in this experiment exhibited epiphyticmycelia characteristic of Periglandula spp. on the upper leafsurface of adult leaves, thus suggesting that the plants usedin our experiment did indeed have a viable endosymbiont.The undescribed endosymbiont of I. tricolor was previouslyreported not to produce epiphytic mycelia (Ahimsa-Mulleret al. 2007). Furthermore, adult plants of all four speciesderived from our seed stocks produce seed with detectableergot alkaloids (Beaulieu et al. data unpublished).

Seeds of each species were surface sterilized by soakingfor 5 min in 10 % commercial bleach, 5 min in 95 % ethanol,and 5 min in distilled water. Following surface sterilization,seeds were scarified and soaked overnight in either distilledwater (control) or a fungicide solution (1.3 % pyraclostrobin,BASF). The purpose of the fungicide treatment was to killPeriglandula-like fungal endosymbionts that could produceergot alkaloids de novo after the seedling germinated. In a

separate study, treatment with this fungicide yielded adult I.amnicola plants lacking surface mycelia characteristic ofPeriglandula spp., as well as detectable ergot alkaloids inthe foliage (Beaulieu et al., data unpublished). After soakingovernight, seeds were rinsed with distilled water and plantedin the Indiana University greenhouse into 7.6-cm diameterpots with a 4:1:1 mix of soil, vermiculite, and perlite topromote drainage. Eight individual plants (4 fungicide-treated and 4 controls) from each species were harvested ateach of two stages: first, when the cotyledons had fullyexpanded, and second, when the first true leaf had fullyexpanded. On average, the first and second harvests weremade 11 and 22 days post-planting but varied by species.Roots, hypocotyl, and cotyledons were sampled at the firstharvest, while the same three tissues along with the first trueleaf were sampled at the second harvest. The experiment wasa full factorial design with four species, two fungicide treat-ments (treated or not), two harvests and four replicates pertreatment level per harvest. The only exception was I.hildebrandtii control plants at the first leaf stage for whichwe had three, not four, replicates.

Independently, four seeds from each species were surfacesterilized as described above, soaked overnight in distilledwater to imbibe and then dissected to separate the cotyle-dons, embryonic axis, endosperm, and seed coat. Alkaloidconcentrations in these seed tissues then were measured toprovide a baseline for determining whether ergot alkaloidsare mobilized and redistributed post germination.

Extended Harvest Experiment To address questions 1–3, weconducted an extended sequential harvest experiment whereseedlings of I. tricolor were destructively harvested weeklyfor five consecutive weeks, which extended beyond the firstleaf stage. Seeds of I. tricolor var. “Heavenly Blue” wereobtained from Hirt’s Garden (Medina, OH, USA) and ger-minated on filter paper saturated with water (control) or a2 mg/ml aqueous solution of the systemic fungicidetebuconazole (Chem Service, West Chester, PA, USA) to killany Periglandula-like endosymbionts that could produceergot alkaloids de novo after germination. This fungicidewas demonstrated to be effective in killing Periglandulaipomoeae, which infects I. asarifolia (Kucht et al. 2004).Seedlings in the fungicide treatment group additionally weresprayed to run off with the same fungicide solution in weeks1 and 2 to increase the efficacy of the treatment. In a previousstudy, 18 out of 18 I. tricolor plants undergoing a similarfungicide treatment produced seeds lacking detectable ergotalkaloids (Beaulieu et al., data unpublished). Between threeand six plants in the control and fungicide-treated groupswere harvested each week. The roots, hypocotyl, and coty-ledons were sampled for each harvest, while the first true leafwas sampled beginning at week 2 and the fourth true leaf wassampled beginning at week 4.

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Ergot Alkaloid Analysis To determine ergot alkaloid content,all plant tissues were dried for 3 days at 40 °C, and total dryweights were recorded (multispecies experiment only).Tissues were pulverized with 3-mm diam silica beads for30 s at 6 m/s in a FastPrep-24 homogenizer (MP Bio) ina cold room. The resulting fine powder was soaked inmethanol (99.93 % A.C.S. HPLC Grade, Sigma-Aldrich)for 3 days at 4 °C with daily vortexing to extract ergotalkaloids. Ergot alkaloids in the methanol extract wereanalyzed by reverse-phase HPLC on a C18 column(Prodigy 5-μm ODS3 [150 mm by 4.6 mm]; Phenomenex,Torrance, CA, USA) with dual fluorescence detectors set atexcitation and emission wavelengths of 272 nm/372 nm and310 nm/410 nm as described by Panaccione et al. (2012).Identity of individual peaks was confirmed by LC-MS withelectrospray ionization in positive mode as described by Ryanet al. (2013). Ergot alkaloids used as standards were obtainedfrom the following sources: ergobalansine was isolated fromsclerotia of Balansia obtecta provided by James F. White, Jr.(Rutgers University); chanoclavine was provided by Alfarma(Prague, Czech Republic) while lysergol and ergonovinewere purchased from Sigma (St. Louis, MO, USA). Peakscorresponding to cycloclavine, festuclavine, ergine, andlysergic acid α-hydroxyethylamide (LAH) were identifiedbased on their fluorescence properties (Panaccione et al.2012), molecular masses, and their disappearance from produc-ing plants following treatment with fungicide. Chanoclavine,festuclavine, and cycloclavine (which fluoresce maximallyat 272 nm excitation/372 nm emission wavelengths) werequantified by comparison of peak areas relative to a stan-dard curve prepared from agroclavine (Sigma) as an exter-nal standard. All other alkaloids (which fluoresce maximallyat 310 nm excitation/410 nm emission wavelengths) werequantified based on peak areas relative to a standard curveprepared from ergonovine as an external standard.

Statistical Analyses For the multispecies experiment, totalamounts of ergot alkaloids were analyzed by ANOVA foreach species at each life stage: seed, first harvest (cotyledonstage), and second harvest (first true leaf stage). The onlyfactor for the seed stage was tissue, while factors for the firstand second harvests included tissue, fungicide, and theirinteraction to test for effects of the fungicide. Models werefit with the aov function in R (R Development Core Team2012) and type-III sums of squares and F tests were calcu-lated with the Anova function in the car package (Fox andWeisberg 2011). The response variable was natural log-transformed prior to analysis to approximate a normal distri-bution, and plots of fitted values vs. residuals were inspectedto verify constant variance and independence of error. Post-hoc t-tests were calculated with the TukeyHSD function. Forthe extended harvest experiment with I. tricolor, ergot alka-loid concentrations in tissues of fungicide-treated vs. control

seedlings in five consecutive weekly harvests were com-pared by Student’s t-test. Data from the extended harvestexperiment are expressed as ergot alkaloid concentration, asopposed to total ergot alkaloids, because total tissue masseswere not available for this experiment.

Results

Ergot Alkaloid Diversity Each species contained betweenfour and six unique ergot alkaloids with a total of eightunique ergot alkaloids across all species (Table 1, Fig. 1).Two ergot alkaloids, ergine and chanoclavine, were found inall four species, while three ergot alkaloids were each foundin only one species—lysergol in I. amnicola and cycloclavineand festuclavine in I. hildebrandtii. Considering the threemain classes of ergot alkaloids, we detected four clavines,three lysergic acid amides, and one ergopeptine. Clavinesand lysergic acid amides were detected in all species, whilethe one ergopeptine (ergobalansine) was detected only in I.amnicola and I. argillicola. We observed the greatest ergotalkaloid diversity, as measured by the Shannon Index, in I.argillicola. Despite having the greatest number of alkaloids(six), I. hildebrandtii had the lowest diversity, as its profilewas dominated by cycloclavine. We note that ergine, a simpleamide of lysergic acid, is not thought to be a biosynthetic endpoint but rather a breakdown product from the more complexlysergic acid derivatives such as LAH or ergopeptines (Fig. 1)(Flieger et al. 1982; Panaccione et al. 2003).

De novo Production of Ergot Alkaloids In the multispeciesexperiment, we did not detect a difference between totalergot alkaloids in tissues of fungicide-treated and controlplants for any species at the cotyledon stage, indicating thatde novo production of ergot alkaloids does not occur atdetectable levels from seed germination through cotyledonstage in any of the four Ipomoea species. At the first leafstage, total amounts of ergot alkaloids were greater in controltissues only in I. argillicola. While we did not find a signif-icant treatment by tissue interaction for I. argillicola, the dataindicated that the difference between control and fungicide-treated plants was greatest in the first leaf where the controlplants had, on average, 2.16 times the amount of ergotalkaloids, suggesting de novo synthesis. In contrast, the ratioof total ergot alkaloids in control vs. fungicide-treated seed-lings of I. argillicola among other tissues ranged from 0.95to 1.13. Average days to harvest, post germination, at the firstleaf stage were as follows: 10 (I. tricolor), 20 (I. amnicola),26 (I. argillicola), and 30 (I. hildebrandtii).

In the extended harvest experiment with I. tricolor seed-lings, we first detected increased ergot alkaloid concentra-tions in control plants relative to fungicide-treated plants inweek 3 in the hypocotyl and first true leaf, indicating de novo

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production (Fig. 2), suggesting the fungicide-treatment ei-ther killed the endosymbiont or inhibited ergot alkaloidproduction. Ergot alkaloid concentrations also were signifi-cantly higher in the cotyledons, first true leaf, and fourth trueleaf of controls plants sampled in weeks 4 and 5 and thehypocotyl again in week 4 (but not week 5). The greatestdifferences in ergot alkaloid concentrations were seen in thetrue leaves where concentrations in control tissues ranged

from ca. 5 to 20 times greater than in fungicide-treatedtissues. Ergot alkaloid concentrations in roots did not differsignificantly between treatments in any week, indicating thatnewly synthesized alkaloids were not transported to, or pro-duced in, the roots. As in the multispecies experiment, weobserved no differences between control and fungicide-treated seedlings at the cotyledon stage, and the largestdifferences were seen among the true leaves, as was the case

Table 1 Ergot alkaloid concentrations in seeds of four species ofConvolvulaceae; values are mean μg alkaloid per g seed±standarderror; empty cells indicate a specific ergot alkaloid was not detected in

that species; N=4 for Impomoea amnicola, I. argillicola and I.hildebrandtii and N=3 for I. tricolor

Class Ergot Alkaloid Species

I. amnicola I. argillicola I. hildebrandtii I. tricolor

Clavines Chanoclavine 625±66 151±9 31±11 208±30

Lysergol 42±4

Cycloclavine 4755±337

Festuclavine 306±49

Simple Amides of Lysergic Acid Ergine 20±4 271±59 27±3 1267±63

LAH 332±28 27±6 105±12

Ergonovine 514±119 9±2 93±9

Ergopeptines Ergobalansine 1456±254 573±34

Total 2143±291 1841±188 5155±401 1673±109

Ergot alkaloid richness 4 5 6 4

Shannon diversity 0.74±0.03 1.50±0.01 0.33±0.03 0.80±0.02

LAH lysergic acid alpha-hydroxyethylamide

Fig. 1 Ergot alkaloidsaccumulating in morning glorysymbioses. This abbreviatedpathway, composited frommultiple species (and notrepresenting the pathway of anyindividual fungus), indicates therelationship among ergotalkaloids observed in thepresent study. Double arrowsindicate one or more omittedintermediates. Dashed arrowsindicate hydrolysis to formergine. Additional details onergot alkaloid pathways can befound in recent reviews (Lorenzet al. 2009; Panaccione 2010;Panaccione et al. 2012;Wallwey and Li 2011)

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with I. argillicola, indicating a developmental lag in ergotalkaloid production.

Distribution of Seed-Borne Ergot Alkaloids At the seedstage, there was a substantial tissue effect on total ergot alka-loids for each species (Table 2, P<0.001). Among all species,total ergot alkaloids were greatest in the cotyledons followed bythe embryonic axis (Fig. 3). Ergot alkaloids were more highlyconcentrated in the endosperm than the seed coat in I.amnicola and I. argillicola, whereas concentrations in thesetissues were similar in I. hildebrandtii and I. tricolor.

At the cotyledon stage in the multispecies experiment,there was a significant tissue effect on total ergot alkaloidsfor each species (Table 2, P<0.001) indicating differentialallocation to the roots, hypocotyls, and cotyledons amongthe four species (Fig. 4a). In I. amnicola, I. argillicola, and I.hildebrandtii, ergot alkaloids were most abundant in thecotyledons, followed by the hypocotyl, and least abundantin the roots (P<0.05 for all comparisons). In contrast, totalamount of ergot alkaloids did not differ among tissues of I.tricolor (P>0.05 for all comparisons). However, the distri-bution of individual alkaloids did vary as described below.

At the first leaf stage in the multispecies experiment, againthere was a significant tissue effect on total ergot alkaloids foreach species (Table 2, P<0.001). The greatest amount of alka-loids in I. amnicola, I. argillicola, and I. hildebrandtiiwas foundin the cotyledons, while the greatest amount in I. tricolor wasfound in the roots (Fig. 4b). Ergot alkaloids were least abundantin the first true leaf for all species compared to other tissues.

Differential Allocation of Individual Alkaloids Two speciesexhibited differential allocation of individual alkaloids inplanta. In I. tricolor, lysergic acid amides were redistributedbetween the seed and cotyledon stages from the cotyledons tothe roots, whereas the only clavine detected, chanoclavine, didnot move, given that the quantity of chanoclavine in the coty-ledon did not change significantly from the seed stage throughthe first leaf stage (Fig. 5). The quantity of chanoclavine in theroots, hypocotyl, and first true leaf of these seedlings could beaccounted for by the quantity of chanoclavine in the embryonicaxis at the time of germination. Thus, the quantities ofchanoclavinemeasured among seedling tissues through the firstleaf stage were consistent with a lack of mobility of thisalkaloid. Conversely, the quantity of lysergic acid amides inthe roots and hypocotyl at the cotyledon expansion stage great-ly exceeded what could have been supplied by the embryonicaxis, indicating redistribution of alkaloids from the cotyledonsto the hypocotyl and roots. The lysergic acid amides of I.

23

45

6

Week 1

L4 L1 C H R

ControlFungicide

01

23

4

Week 2

L4 L1 C H R

01

23

4

Week 3

L4 L1 C H R

**

01

23

4

Week 4

L4 L1 C H R

* **

*

01

23

4

Week 5

L4 L1 C H R

*

**

Log(

µg/g

Erg

ot A

lkal

oids

+1)

�Fig. 2 Concentration of ergot alkaloids (log-transformed) in controland fungicide-treated tissues from seedlings of Ipomoea tricolor over afive-week time course (extended harvest experiment). Asterisks indicatea significant difference (P<0.05) in t-tests. Abbreviations: L4, leaf 4;L1, leaf 1; C, cotyledons; H, hypocotyl; and, R, roots

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tricolor—LAH, ergonovine, and ergine—were consideredtogether since ergine is not a biosynthetic end point and itmay be derived from more than one parent ergot alkaloid(Fig. 1). We detected no difference between control andfungicide-treated plants, indicating no de novo synthesis atthis stage. At the time of first leaf expansion, most of thelysergic acid amides in I. tricolor were located in the roots,with significantly smaller quantities found in the hypocotyl,cotyledons and first leaf. In I. hildebrandtii, almost allfestuclavine remained in the cotyledons, after germination(1.7±0.1 μg, mean ± s.e.), with only a small amount foundin the hypocotyl (0.08±0.01 μg) and roots (0.01±0.008 μg).At the first leaf stage, festuclavine was detected in the first leafof only one of the plants and at a very low level (0.01 μg).

Discussion

Our results demonstrate that the diversity and distributionof ergot alkaloids produced by fungal endosymbionts that

infect morning glories is highly regulated among species.We observed differential allocation of seed-borne ergotalkaloids among seedling tissues of four morning gloryspecies (Fig. 4). In addition, de novo production of ergotalkaloids was not detectable until true leaves have developed(Fig. 2, Table 2), suggesting that the high concentrations anddistribution of ergot alkaloids in seeds may have adaptive

Table 2 Results from the multi-species experiment analyzed withANOVA for total ergot alkaloids(μg alkaloids, log transformed) inseed and seedling tissues fromfour species of Ipomoea(Convolvulaceae); factors includ-ed the tissue sampled and thefungicide treatment

Source Harvest Species

I. amnicola I. argillicola I. hildebrandtii I. tricolor

Tissue Seed Stage <0.001 <0.001 <0.001 <0.001

Treatment Cotyledon Stage N.S. N.S. N.S. N.S.

Tissue <0.001 <0.001 <0.001 N.S.

Trt*Tissue N.S. N.S. N.S. N.S.

Treatment First Leaf Stage N.S. 0.026 N.S. N.S.

Tissue <0.001 <0.001 <0.001 <0.001

Trt*Tissue N.S. N.S. N.S. N.S.

−2

−1

01

2

Log(

Tota

l µg)

I. amnicola I. argillicola I. hildebrandtii I. tricolor

A

B

C

D

A

B

C

D

A

B

CC

A

B

BC

C

Tissue

CotyledonEmbyronic axis

EndospermSeed coat

Fig. 3 Total amount of ergot alkaloids (log transformed) in seed tissuesfrom four species of Ipomoea (multispecies experiment). Differentletters indicate significant differences among tissues within a species(Tukey’s Honest Significant Difference t-test, P<0.05)

−1

01

2

Log(

Tota

l µg)

I. amnicola I. argillicola I. hildebrandtii I. tricolor

a

Tissue

1st LeafCotyledons

HypocotylRoots

A

BC

A

BC

A

B

CA

A A

−1

01

2

Log(

Tota

l µg)

I. amnicola I. argillicola I. hildebrandtii I. tricolor

b

C

A

B

C

C

A

B

C B

A

B

B

C

B B

A

Fig. 4 Total amount of ergot alkaloids (log transformed) in seedlingtissues from four species of Ipomoea at the cotyledon stage (a) and firstleaf stage (b) (multispecies experiment). Control and fungicide-treatedseedlings are grouped together as we detected no fungicide by tissueinteractions. Different letters indicate significant differences among tissueswithin a species (Tukey’s Honest Significant Difference t-test, P<0.05)

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value for the seedling and its fungal endosymbiont whosefitness is dependent upon seedling survival and reproduction.Experimental work is needed to confirm that ergot alkaloid-containing seedlings have increased resistance to natural ene-mies, but we expect this would be the case given that ergotalkaloids have been shown to be effective against a variety ofabove and below-ground enemies including vertebrates,invertebrates, nematodes, and bacteria (Bacetty et al. 2009a, b;Clay and Schardl 2002; Panaccione 2005; Panaccione et al.2006b; Potter et al. 2008). In addition to interspecific differencesin total ergot alkaloid allocation, individual alkaloids were dif-ferentially allocated in planta, as observed with lysergic acidamides mobilized to the roots of I. tricolor (Fig. 5) andfestuclavine in I. hildebrandtii that was primarily found inthe cotyledons. Differential allocation of specific alkaloids, asopposed to total alkaloids, may represent another mechanismby which species can respond to selection by plant pests.

We found no evidence for de novo production of ergotalkaloids through the cotyledon stage in any of the fourspecies. At the first leaf stage, only I. argillicola showed anincrease in total alkaloids relative to fungicide-treated seed-lings (Table 2), and then largely in the first leaf only.Treatment with systemic fungicide, therefore, prevented denovo synthesis of ergot alkaloids. Furthermore, we found no

difference in the pattern of allocation among tissues due tothe fungicide treatment. Comparison of alkaloid concentra-tions over a longer time course in I. tricolor showed thatalkaloid synthesis was detectable as a difference in concen-tration between control and fungicide-treated seedlings 3-weeks after germination (Fig. 2), approximately 10 dayslater than the first leaf stage in the multispecies experiment.We did not detect synthesis of ergot alkaloids in the roots,consistent with grass-epichloae symbiota (Panaccione et al.2006b). In I. tricolor, the concentration of alkaloids found inthe leaves 5 weeks post-germination was much lower thanthe concentrations in seeds and seedling tissues, but stillgreater than concentrations in grass leaves. We do not havedata on concentrations in leaves of older plants from theother species since the multispecies experiment did not sam-ple past the first true leaf stage. However, we anticipate thatwe would observe the same pattern of reduced amounts ofalkaloids in adult leaves compared to seedling tissues, as theamount of ergot alkaloids found in the first leaf generallywas the lowest of all tissues sampled (Fig. 4). Eventually, theseed-borne alkaloids must be diluted as plants grow so thatthe alkaloids present in adult tissues must be primarily pro-duced de novo. Maintaining high concentrations of ergotalkaloids in adult tissues, similar to concentrations observed

Fig. 5 Total amount of lysergicacid amides (top) andchanoclavine (bottom) alkaloidsin tissues of Ipomoea tricolorcollected after overnightimbibition (seed) at completeexpansion of cotyledons(cotyledon stage; 5 days) and atexpansion of first true leaf (firstleaf stage; 10 days)(multispecies experiment).Error bars represent standarderror. Different letters indicatesignificant differences amongtissues within a species(Tukey’s Honest SignificantDifference t-test, P<0.05)

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in seedling tissues, would represent a high cost to the plant-fungal symbiotum.

In addition to differential allocation of ergot alkaloids toseedling tissues among species, we found evidence for dif-ferential allocation and translocation of individual alkaloidswithin species. For example, lysergic acid amides (LAH,ergonovine, and ergine) in I. tricolor exhibited mobility, asthey were most abundant in the cotyledons at the seed stage,but they were most abundant in the roots at the seedlingstages (Fig. 5). By contrast, chanoclavine exhibited restrictedmobility as it remained primarily in the cotyledons post-germination. Mobility is not simply a matter of polarity ofthe individual molecules since these alkaloids all have similarpolarity as assessed by their retention times in reverse-phaseHPLC on a C18 column (Coyle et al. 2010; Panaccione et al.2003). Further, these same individual alkaloids were distributeddifferently among morning glory species, demonstrating thattheir distribution may be a labile trait. For example, lysergicacid amides (LAH, ergonovine, and ergine), which wereredistributed from the cotyledons to the roots in I. tricolor, alsowere found in high concentrations in the cotyledons of I.argillicola (and lesser concentrations in I. hildebrandtii), butthe majority of the alkaloids remained in cotyledons in thesespecies post-germination (Figs. 2 and 3). This suggests that thedifference in distribution is caused by the plant-fungalsymbiotum and is not an intrinsic property of the alkaloid.Furthermore, these patterns did not differ between fungicide-treated and control plants, suggesting that the re-allocation ofalkaloids in I. tricolor is not caused by a living fungus. We arenot aware of any studies that explicitly test the effects of LAHand ergine on below-ground enemies, but ergonovine, one ofthe alkaloids that accumulates in roots of I. tricolor, has beendemonstrated to be repellent and nematistatic to the lesionnematode Pratylenchus scribneri in vitro (Bacetty et al.2009a, b). Whether I. tricolor faces higher pressures from rootparasites or pathogens compared to the other Ipomoea species,however, is unknown. We also found evidence for differentialallocation of individual alkaloids in I. hildebrandtii, wherefestuclavine primarily remained in the cotyledons and did notmobilize to other tissues post-germination. Thus, species candifferentially allocate the total amount of ergot alkaloids(Fig. 4) as well as individual alkaloids (Fig. 5).

Because unique ergot alkaloids have different biologicalactivities, the ability to differentially allocate specific alka-loids may allow a more precise response to selective pres-sures from pests. For example, Potter et al. (2008) found thatlysergic acid derivatives (simple amides and ergopeptines)that accumulate in perennial ryegrass symbiotic with thefungus Neotyphodium lolii x Epichloë typhina contributedto repellent and insecticidal activity against the crown-feeding cutworm Agrotis ipsilon, whereas simpler clavineswere significantly less effective, as evidenced by experi-ments with knockout mutants. Similarly, by using knockout

mutants, Panaccione et al. (2006a) demonstrated thatclavines in endophyte-infected perennial ryegrass (Loliumperenne) caused rabbits to prefer endophyte-free plants, butthe presence of an ergopeptine (ergovaline) also reducedtheir appetite. Finally, Clay and Cheplick (1989) found thatthe fall armyworm (Spodoptera frugiperda) had differentialsurvivorship and growth in response to the inclusion ofdifferent ergot alkaloids in their diet. Thus, previous studiessuggest that the specific types and allocation of ergot alka-loids will have consequences for biological interactions withIpomoea spp. infected by Periglandula spp..

Although there has been no formal description of fungalsymbionts in the Ipomoea species investigated here, thedescription of Periglandula spp. that infects two other speciesof Convolvulaceae (Steiner et al. 2011) provides proof ofprinciple that morning glories that contain ergot alkaloids areinfected by a fungal endosymbiont responsible for their pro-duction, as has been documented in grasses infected by otherclavicipitaceous fungi. The endosymbiont of I. tricolor hasbeen detected by polymerase chain reaction, and sequence datasuggest that the fungus is closely related to the two describedPeriglandula species (Ahimsa-Muller et al. 2007). However,unlike P. ipomoeae and P. turbinae, the fungal symbiont of I.tricolor does not produce visible epiphytic mycelia and was notformally described by Steiner et al. (2011). Fungal endosymbi-onts of I. argillicola, I. amnicola, and I. hildebrandtii have notbeen reported previously, but their presence is evidenced by theaccumulation of ergot alkaloids and epiphytic growth of hy-phae on adaxial leaf surfaces. Eich (2008) reported over 40species of morning glories that contain ergot alkaloids out ofapproximately 100 species tested. Given that there are ca. 1650species of Convolvulaceae (Mabberley 1987), there are likelymany more species infected by Periglandula-like endosymbi-onts that produce ergot alkaloids. Curiously, Ipomoea carnea,is infected by a vertically-transmitted fungal endosymbiont inthe fungal order Chaetothryiales that produces the indolizidinealkaloid swainsonine (Cook et al. 2013). There may be othermorning glory species also infected by swainsonine-producingfungi, and it would be interesting to determine if swainsonineexhibits differential allocation among species as described herefor ergot alkaloids.

The only ergopeptine alkaloid detected, ergobalansine,was found in the two most closely related species in thisstudy, I. amnicola and I. argillicola (Miller et al. 1999),suggesting that the fungi infecting the plants also may beclosely related, even though the former is native to the NewWorld and the latter to Australia. If all ergot alkaloid-producing symbionts of morning glories are vertically trans-mitted, as is the case for the two described species ofPeriglandula, they should co-diverge with their hosts, giventhat host and symbiont reproduction are linked. Co-divergence between hosts and vertically-transmitted symbi-onts has been demonstrated for grass-epichloae associations

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(Schardl et al. 1997, 2008), as well as for several insect-bacterial systems (Hosokawa et al. 2006; Jousselin et al.2009). If this is the case for morning glories infected byPeriglandula, then closely related plant species should beinfected by closely related fungal endosymbionts that mayproduce similar alkaloid profiles. In addition to ergotalkaloids, clavicipitaceous fungi are also known to produceadditional compounds including indole-diterpenes, peramineand loline alkaloids (Panaccione et al. 2013; Schardl et al.2012). While we did not screen for these compounds here, P.ipomoeae infecting I. asarifolia also produces indole-diterpenes (Schardl et al. 2013). Additionally, loline alkaloidshave been reported in the morning glory species Argyreiamollis, though whether this is a fungal product is unclear(Tofern et al. 1999). Thus, Periglandula-like symbiontsinfecting morning glories may be capable of producing someor all of the other alkaloids produced by grass-associatedNeotyphodium.

Differing selective pressures may cause the asymmetricaccumulation and allocation of different ergot alkaloids withinseedling tissues among Ipomoea species. The concentrations ofergot alkaloids found in seeds of several species of morningglories (including the four species studied here) are up to 1000-fold greater than those previously reported to deter herbivoresin ergot alkaloid-containing grasses (Clay and Schardl 2002;Panaccione et al. 2006b; Potter et al. 2008). For example, tallfescue and perennial ryegrass infected by Neotyphodium endo-phytes typically have ergot alkaloid concentrations in the 0.5 to10 μg/g range (Panaccione et al. 2003), whereas the morningglory species in this study exhibited ergot alkaloid concentra-tions from approximately 1,600 to 5,100 μg/g. Several speciesof Convolvulaceae are highly parasitized by specializedbruchine beetles in the genus Megacerus (Coleoptera:Bruchinae) whose larvae bore into the seed and consume theembryo, rendering it inviable (Reyes et al. 2009). Beetle para-sitism rates as high as 85 % and 100 % have been reported inpopulations of I. pes-caprae (Devall and Thien 1989; Wilson1977) and I. leptophylla (Keeler 1980, 1991), respectively.However, as Megacerus spp. are highly-specialized seed para-sites, we should expect them to have some level of adaptationto ergot alkaloids (Ehrlich and Raven 1964; Levin 1976).Alternatively, to protect from seed parasites and predators, theseeds may provide a source of ergot alkaloids that can beredistributed to protect the seedlings from more generalistenemies. Because the endosymbiont is vertically transmittedthrough seeds, benefits of protection accrue to both partners.Interspecific differences in allocation patterns to seedling tis-sues (Fig. 4) suggest that the pattern of allocation is, thus, labileand potentially under selection. In environments where seed-lings face greater pressure from above-ground pests, we wouldpredict greater allocation to the cotyledons and hypocotyl (as inI. amnicola, I. argillicola and I. hildebrandtii), whereas wewould expect greater allocation to roots (as in I. tricolor) if

the species faces greater pressure from below-ground pestssuch as nematodes or soil-borne pathogens. Whether the pat-terns of allocation observed here correlate with environmentalpest pressure requires field-based studies.

Our results indicate that there is differential allocation oftotal and individual ergot alkaloids within and amongIpomoea species, and that this pattern is not affected byfungicide treatment. These patterns suggest that diversity,concentrations, and the distribution of ergot alkaloids duringmorning glory ontogeny are highly regulated and may reflectselection for defense against pests and pathogens, given thatergot alkaloids have well-known protective effects and thatindividual ergot alkaloids can have differing effects on dif-ferent enemies. As many species of Convolvulaceae havebeen reported to contain ergot alkaloids, with most speciesstill unexamined, and given the worldwide distribution andecological diversity of morning glories, future researchefforts should evaluate the distribution and dynamics of thesecompounds in relation to environmental and life history traitsand their effectiveness against natural enemies.

Acknowledgments W.T.B. acknowledges funding from an Anne S.Chatham Fellowship in Medicinal Botany from the Garden Club ofAmerica and useful discussions with Chunfeng Huang (Indiana UniversityDepartment of Statistics) as well as Stephanie Dickinson and Xuefu Wang(Indiana Statistical Consulting Center). C.S.H. was supported by NationalScience Foundation grant DBI-0849917. Additional funding was providedby United States Department of Agriculture National Institute of Food andAgriculture grant 2012-67013-19384 to D.G.P. We thank the greenhousestaffs of Indiana University and West Virginia University as well asChristopher T. Moore for technical support. This article is published withpermission of the West Virginia Agriculture and Forestry ExperimentStation as scientific article number (3173).

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