Oecologia (2012) 168:471–481

DOI 10.1007/s00442-011-2114-x

PLANT-ANIMAL INTERACTIONS - ORIGINAL PAPER

Changes in plant chemical defenses and nutritional quality as a function of ontogeny in Plantago lanceolata (Plantaginaceae)

Carolina Quintero · M. Deane Bowers

Received: 10 July 2010 / Accepted: 26 July 2011 / Published online: 13 September 2011© Springer-Verlag 2011

Abstract Numerous empirical studies have examinedontogenetic trajectories in plant defenses but only a fewhave explored the potential mechanisms underlying thosepatterns. Furthermore, most documented ontogenetic tra-jectories in plant defenses have generally concentrated onaboveground tissues; thus, our knowledge regarding wholeplant trends in plant defenses throughout development orpotential allocation constraints between growth anddefenses is limited. Here, we document changes in plantbiomass, nutritional quality and chemical defenses forbelow- and aboveground tissues across seven age classes ofPlantago lanceolata (Plantaginaceae) to evaluate: (1) par-tial and whole plant ontogenetic trajectories in constitutivechemical defenses and nutritional quality, and (2) the roleof resource allocation constraints, namely root:shoot (R:S)ratios, in explaining whole plant investment in chemicaldefenses over time. Overall investment in iridoid glyco-sides (IGs) signiWcantly increased, while water and nitro-gen concentrations in shoot tissues decreased with plantage. SigniWcant variation in IG content between shoot androot tissues across development was observed: allocation ofIGs into root tissues linearly increased from younger toolder plants, while non-linear shifts in allocation of IGsduring ontogeny were observed for shoot tissues. Finally,R:S ratios only weakly explained overall allocation ofresources into defenses, with young stages showing a

positive relationship, while older stages showed a negativerelationship between R:S ratios and IG concentrations.Ontogenetic trajectories in plant quality and defenseswithin and among plant tissues can strongly inXuenceinsect herbivores’ performance and/or predation risk; thus,they are likely to play a signiWcant role in mediating speciesinteractions.

Keywords Iridoid glycosides · Constitutive defenses · Root:shoot ratios · Resource allocation · Root defenses

Introduction

Plant susceptibility to herbivory, as well as the impact thatherbivore damage has on plant Wtness, vary markedly overa plant’s lifetime. For example, variation in herbivore dam-age from the seedling stage to senescence can change byseveral orders of magnitude for the same plant–herbivoreinteraction (e.g., Pires and Price 2000; Fonseca et al. 2006;Loney et al. 2006), and may even involve considerablechanges in the composition of the herbivore communityinXicting damage throughout plant ontogeny (e.g., Waltzand Whitham 1997; Boege 2005; Thomas et al. 2010). Sim-ilarly, the impact of herbivore damage on plant Wtness alsodepends on the ontogenetic stage being attacked, rangingfrom complete compensation by the host plant to long-last-ing reductions in Xower production and fruit set (Warnerand Cushman 2002; Boege et al. 2007; Hanley and Fegan2007). As a result, the selective pressure that herbivoresexert on plant traits that confer resistance should vary withplant ontogeny, leading to qualitative and/or quantitativechanges in plant resistance traits during plant development.Despite the relevance that assessing developmental changesin plant defenses may have for the improvement of plant

Communicated by Jacqui ShykoV.

C. Quintero (&) · M. D. BowersDepartment of Ecology and Evolutionary Biology and University of Colorado Museum, University of Colorado, Campus Box 334, Boulder, CO 80309-0260, USAe-mail: carolina.quintero@colorado.edu

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defense theories, as well as our understanding of ecologicalinteractions, relatively little is known about how allocationof resources to plant defenses varies throughout plantdevelopment (Boege and Marquis 2005; Hanley et al.2007).

Numerous models have been proposed to explain pat-terns of variation in plant constitutive chemical defensesas a function of plant development. With the exception ofboreal woody plants, where the ontogenetic trajectories inplant defenses have been proposed to strongly reXect theselective pressure exerted by mammalian herbivores(Bryant et al. 1992), most arguments propose that intrin-sic forces, namely resource acquisition and allocationconstraints, are responsible for ontogenetic variability inplant defenses (Bryant et al. 1991; Herms and Mattson1992; Boege and Marquis 2005). Among these, the theo-retical model proposed by Boege and Marquis (2005) isthe more comprehensive and detailed model, encompass-ing all stages from cotyledon to senescent plants at thewhole plant level. This model predicts that multipleresource allocation and architectural constraints through-out plant development will lead to a non-linear relation-ship between plant age and defenses. SpeciWcally, themodel predicts relatively higher levels of defenses inyounger over older seedlings until resources stored incotyledons and seeds are depleted, followed by a decreasein defenses in response to large root:shoot (R:S) ratios anda disproportional allocation of resources to the productionof photosynthetic area. Once R:S ratios start to decrease,growth rate might be more limited than photosyntheticrate allowing for a signiWcant increase in defensesthroughout juvenile stages. Finally, when plants reachreproductive maturity, constraints due to reproductionmay again result in decreased levels of defenses or theymay remain constant depending on the plant’s reproduc-tive strategy (Boege and Marquis 2005). A recent meta-analysis conducted by Barton and Koricheva (2010),including 116 studies and 153 plant species, concludedthat ontogenetic trajectories in plant defenses tend toshow non-linear patterns. However, while non-linearityappears to be the norm, the shape of the relationshipseems to depend on plant life form (i.e. woody plants,herbs or grasses) and defensive traits (i.e. physical traits,chemical traits, tolerance) (Barton and Koricheva 2010).

The absence of a generalized pattern describing theontogeny of plant defenses suggests that allocation con-straints and costs of defenses through plant developmentare not consistent across all plant life forms and defensivetraits. Nevertheless, despite the large number of studiesdocumenting ontogenetic trajectories in plant defenses,only a few have tested the potential mechanisms drivingthese patterns, such as those looking at age-speciWc alloca-tion costs and trade-oVs between growth and defenses

(e.g., Goodger et al. 2006; Barton 2007; Boege et al. 2007;Orians et al. 2010). Hence, the potential of diVerences inallocation constraints among grasses, herbs and woodyplants, as well as among diVerent kinds of defensive traits,as the source of discrepancies in ontogenetic patterns ofdefenses remains unclear. Furthermore, inconsistencyamong results from investigations of ontogenetic patternsof plant defense might reXect the lack of studies assessingontogenetic trajectories at the whole plant level. Most stud-ies to date concentrate on aboveground tissues, especiallyleaves, while ontogenetic trajectories in root defenses havebeen largely ignored. However, the few studies that haveexplored ontogenetic trajectories in below- and above-ground tissues have consistently documented dissimilarontogenetic patterns of defenses among tissues (Hartmannand Zimmer 1986; Williams and Ellis 1989; Bellostas et al.2007; Beninger et al. 2009). Therefore, without a completeassessment of defensive traits at the whole plant level,meaningful variability within and among tissues duringplant development may obscure overall patterns of plantinvestment in defenses over time, as well as potential trade-oVs between growth and defenses.

In sum, in the last two decades, numerous attempts topredict theoretical patterns and drivers of ontogeneticvariation in constitutive plant defenses have been made;yet few studies to date have empirically tested the rela-tionship between plant development and defenses acrossmultiple developmental stages and plant tissues, and/ortested the mechanisms driving these patterns. In thisstudy, we used seven pre-reproductive age classes ofPlantago lanceolata L. (Plantaginaceae) to investigateontogenetic variation in below- and aboveground consti-tutive plant chemical defenses (iridoid glycosides), andthe role of resource allocation constraints as the poten-tial mechanism driving these ontogenetic trajectories. Inaddition, we assessed ontogenetic variation in otherplant traits relevant to herbivores, including leaf waterand nitrogen concentration, as well as below- and above-ground biomass. Because our interest was to assess over-all variation in constitutive trajectories at the wholeplant level, we did not assess here within-tissue varia-tion (e.g., young, intermediate and old leaf ages) whichmay add considerable variation in levels of defenses andnutritional quality for herbivores (e.g., Bowers andStamp 1992, 1993). In particular, we addressed the fol-lowing three questions: (1) how do plant traits relevantto herbivores, including constitutive chemical defensesand nutritional quality, change throughout plant devel-opment?; (2) do plant chemical defenses in below- andaboveground tissues follow similar ontogenetic trajecto-ries?; and (3) can R:S ratios help to predict the alloca-tion of resources to constitutive chemical defenses at thewhole plant level?

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Methods

Study system

Plantago lanceolata is a cosmopolitan, short-lived peren-nial herb with a rosette growth form introduced to NorthAmerica from Eurasia more than 200 years ago (Caverset al. 1980). This plant species produces, in addition to irid-oid glycosides (Ronsted et al. 2000), a number of bioactivephenolic compounds such as Xavonoids and phenyletha-noid glycosides (Tamura 2001; Galvez et al. 2005). How-ever, iridoid glycosides are the primary allelochemicalsfound in large concentrations in both below- and above-ground tissues and inXuencing feeding preferences of gen-eralist and specialist rhizophagous and folivorousherbivores (e.g., Bowers and Puttick 1989; Biere et al.2004; De Deyn et al. 2009); thus, here we speciWcallyfocused on the ontogenetic trajectories of these compounds.

Iridoid glycosides (IGs) are a group of cyclopentanoidmonoterpene-derived compounds that occur in over 50families of plants (Jensen 1991). Plantago lanceolata pro-duces primarily two iridoid glycosides: aucubin and catal-pol, and amounts of these compounds may be as high as10–12% dry weight (Bowers and Stamp 1992, 1993). Highconcentrations of IGs, especially of catalpol, indicate notonly a greater biosynthetic investment by the plant but mayalso result in a higher unpalatability and/or toxicity to gen-eralist herbivores (Bowers and Stamp 1993). Although welack speciWc knowledge regarding IG production sites (i.e.shoots vs. roots), these carbon-based defenses are synthe-sized through the isoprenoid biosynthetic pathway and areusually stored in cell vacuoles (Croteau 1987). They arealso likely phloem mobile (Gowan et al. 1995; Beningeret al. 2007), and concentrations in leaves and roots inP. lanceolata were found to be positively correlated (DeDeyn et al. 2009). Previous studies have identiWed plant ageas an important intrinsic factor aVecting P. lanceolata chem-istry (e.g., Bowers and Stamp 1993; Fuchs and Bowers 2004;Barton 2007); and these ontogenetic trajectories in IG pro-duction signiWcantly vary among both maternal families andpopulations (Bowers and Stamp 1993; Barton 2007).

In addition, P. lanceolata has been incorporated into thediet of many North American insect herbivores, and thusmultiple specialist and generalist herbivores can aVect andbe aVected by IG content in this species. For example, highIG concentrations usually deter generalist herbivores feed-ing on shoot or root tissues (e.g., Bowers and Puttick 1989;Biere et al. 2004; De Deyn et al. 2004; Wurst and van derPutten 2007). Yet several specialist herbivores use IGs asfeeding and/or oviposition stimulants (e.g., Prudic et al.2005; Talsma et al. 2008), and some have the ability tosequester these compounds from leaves and roots and usethem for their own protection against natural enemies (e.g.,

Strohmeyer et al. 1998; Baden and Dobler 2009; Opitzet al. 2010). Furthermore, although most herbivores associ-ated with this species are either rhizophagous or folivorous,there is at least one example of a species of Longitarsus(Chrysomelidae) Xea beetle that feeds on and sequestersIGs from both tissues, as larvae and adults (Willinger andDobler 2001; Baden and Dobler 2009). As a result, ontoge-netic trajectories in constitutive variation of shoot and rootIGs in this species can have profound consequences on theidentity, abundance and performance of the herbivore com-munity associated with it throughout its lifetime.

Ontogenetic variation in plant traits

In order to evaluate the eVect of plant ontogeny on plantquality and chemical defenses, seven distinct age classes ofPlantago lanceolata (3, 5, 8, 10, 12, 15, and 18 weeks oldsince germination) were grown at the University of Colo-rado greenhouse during the summer of 2008. The Wrst twostages (3 and 5 weeks old) correspond to two distinctiveseedling stages, the next three stages (8, 10 and 12 weeksold) correspond to juvenile stages, and the last two ageclasses (15 and 18 weeks old) to pre-reproductive stages.All age classes were harvested simultaneously in order tocontrol for confounding environmental factors that couldinXuence plant performance such as photoperiod and tem-perature. Synchronization among ontogenetic stagesoccurred by germinating seeds at intervals of 15–30 daysfrom March to July. In this way, although average environ-mental conditions from sowing to harvest varied among ageclasses, we ensured that all individuals were exposed to thesame conditions at harvest time. Since IG concentrationsin this as well as in other IG-containing plant species canvary substantially over days and even hours (Hogedal andMolgaard 2000; Fuchs and Bowers 2004), or be inXuencedby temperature (Tamura 2001), simultaneous harvest acrossall age classes should minimize variables other than plantage that could inXuence IG content.

Seeds were collected from >20 maternal plants from apopulation in Boulder County, Colorado, mixed, and ran-domly assigned to treatments. Seeds were germinated inFafard mix and transplanted to a growth medium of equalparts Metro Mix 350, sterilized sand, and turface after15 days. Plants were grown in extra-deep 13 l pots in orderto minimize root-binding eVects. We grew 20 plants in eachage class, for a total of 140 plants. Replicates were ran-domly placed on a single 1.2 £ 8 m greenhouse bench,exposed to natural daylight, and watered daily. Fertilizerwas supplied to all plants every 3–4 days throughout thelength of the experiment. The fertilizer solution was thestandard solution used in the University of Colorado green-houses, which was Scotts Peter’s Excel (Scotts-SierraHorticultural Products, Marysville, OH, USA) mixed at a

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ratio of 15–5–15 N–P–K which also provides trace amountsof micronutrients. Temperatures Xuctuated over the courseof the season but greenhouse temperatures did not exceed32°C or fall below 15°C at any time. Extra-deep pots wereused in order to ensure natural growth rate of root as well asshoot tissues and to prevent pot-binding, which can aVectplant secondary metabolite production (Baldwin 1988).However, we believe that the combination of abundantresources and nutrient availability together with moderategreenhouse temperatures delayed the onset of Xowering(usually recorded at 15 weeks after sowing under green-house conditions; personal observation) during this Wrstgrowing season.

At harvest, plants were separated into roots and shoots.Shoot tissues represented mostly leaves but in a few of theoldest plants (n = 14) these also contained immature inXo-rescences accounting for less than 1% of the total shoot bio-mass. Statistical analyses with and without these 14individual plants yielded similar results; thus, we presentthe results for the complete dataset. All plant tissues wereweighed fresh, oven-dried at 50°C for 48 h to a constantmass, and weighed again to the nearest 0.01 g. The ratiobetween root and shoot dry biomass was calculated for eachindividual plant.

Analyses of plant water, nitrogen and iridoid glycoside content

To assess variation in plant quality throughout develop-ment, we measured plant water content and nitrogen con-tent. Percent water was calculated as [(wet weight¡dryweight)/wet weight] £ 100. Nutritional quality was mea-sured as total leaf nitrogen, and quantiWed by Micro-Dumascombustion on a NA1500 C/H/N analyzer, using a samplesize of ten plants per age class. Due to the low biomass ofplants at the 3-week stage, we were unable to measure leafnitrogen content for those plants.

To assess concentrations of iridoid glycosides, rootsand shoots were separately ground into a Wne powder, and2–50 mg subsamples (entire above- and/or belowgroundtissue for some seedlings) were processed for IG extractionand analyzed by gas chromatography following previouslydescribed methods (Bowers and Stamp 1993; Barton 2007).BrieXy, samples were extracted overnight in 95% metha-nol, the methanol extract was Wltered to remove the plantmaterial and the residue evaporated to dryness. Phenyl-�-D-glucose (PBG) was added as an internal standard and thesamples were then partitioned between water and ether toremove hydrophobic compounds. An aliquot of the remain-ing solution was removed, evaporated, and derivatized withTri-Sil-Z™ (Pierce Chemical) and injected into a HP7890A gas chromatograph (Agilent Technology) using anAgilent DB-1 column (30 m, 0.320 mm, 0.25 �m particle

size). Amounts of aucubin and catalpol were quantiWedusing ChemStation B-03-01 software and they are pre-sented as percent dry weight for comparative purposes(e.g., Fuchs and Bowers 2004; Barton 2007; Wurst and vander Putten 2007).

Statistical analyses

Developmental variation in root and shoot biomass, R:Sratios and percentage of water and nitrogen in abovegroundtissues were analyzed using one-way ANOVAs, followedby Bonferroni post hoc tests to distinguish mean diVerencesamong age classes if overall signiWcant diVerences werefound. Variation in production of IGs across plant ontogenywas Wrst assessed simultaneously for both plant tissues,shoots and roots, and both iridoids, aucubin and catalpol.Because aucubin and catalpol are correlated and their con-centrations are also potentially correlated between shootand root tissues, we used multivariate analysis of variance(MANOVA) to examine variation in these four variablesdue to plant age. When signiWcant eVects were detected byMANOVA, we followed up with univariate ANOVAs foreach of the four variables. Bonferroni post hoc tests wereused to assess signiWcant diVerences among the seven ageclasses. Lastly, we did a repeated measures ANOVA inorder to test overall variation in total IGs (aucubin + catal-pol) among age classes, between plant tissues (shoots vs.roots; the repeated measure), and their interaction. Water,nitrogen, and IG concentrations as well as R:S ratios werearcsine square-root transformed to meet assumptions ofnormality. Biomass measures did not require transforma-tion.

To assess whether investment in chemical defenses atthe whole plant level as well as for below- and above-ground tissues showed a linear or non-linear increase withplant age, we regressed the concentration of total IGs(aucubin + catalpol) at the whole plant level (shoots +roots), as well as at the shoot and root tissue level sepa-rately, on plant age and tested whether the linear and qua-dratic slopes associated with age diVer signiWcantly fromzero. Because age is treated as a continuous variable, forthese analyses we Wrst needed to center this predictor vari-able (Judd et al. 2009). Then, to evaluate whether the shapeof the curve of total IG content as a function of plant agediVers between shoot and root tissues, we Wrst computed anew dependent variable that describes the diVerence inchemical defenses between tissues (i.e. within-subjectdiVerence variable IGdiV = IGs shoots ¡ IGs roots) andregressed it on plant age. When a within-subject diVerenceis regressed on a between-subject predictor, the resultingslopes will be testing for an interaction between the within-subject independent variable (tissue) and the between-sub-ject one (age). Thus, the linear and quadratic term in this

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case will assess whether the linear or quadratic eVect of ageon plant defenses interacts with plant tissue (i.e. whetherthe linear and quadratic slopes for shoot and root tissuesdiVer from each other) (Judd et al. 2009).

Finally, in order to assess whether R:S ratios can help toexplain total IG production across developmental stages,we performed a linear regression to test the prediction of anegative relationship between R:S ratios and plant invest-ment in defenses as proposed by Boege and Marquis(2005). In addition, in order to test whether the relationshipbetween R:S ratios and IG concentrations shifts with plantage, we performed an analysis of covariance with totalplant IG levels as a function of age using R:S ratios as acovariate and testing for an age by R:S ratio interaction. Forthe Wrst regression we used all seven plant stages (n = 140),then after we conWrmed a signiWcant interaction betweenage and R:S ratios (see “Results”), we performed two sepa-rated linear regressions for younger life stages (age classes3, 5, and 8 week-old, n = 60) and older life stages (age clas-ses 10, 12, 15, and 18 week-old, n = 80).

Results

There were pronounced diVerences in plant biomass, nutri-tional quality and chemical defenses among the seven Plan-tago lanceolata age classes. Plant age signiWcantlyinXuenced total plant biomass (F6,132 = 103.4, p < 0.0001)and R:S ratios (F6,132 = 8.9, p < 0.0001) (Fig. 1a, b). Asexpected, overall biomass of both shoot and root tissuesincreased with plant age; however, some exceptions can beobserved such as lower biomass in shoots of the 15-week-old class and in roots of the 18-week-old class than previ-ous age classes (Fig. 1a). Given our experimental design,this unexpected variation is unlikely to be the result ofpotential genetic biases or limited resources altering growthrates. Furthermore, because we did not follow individualplants during a growing season but rather harvested all ageclasses simultaneously from cohorts germinated at diVerenttimes from March to July, these changes in biomass cannotbe interpreted as loss of biomass. Instead, they may haveemerged as a result of diVerential average environmentalconditions (i.e. day length and temperature) shaping devel-opmental trajectories in biomass partitioning, as seen inother systems (McConnaughay and Coleman 1999; Moriu-chi and Winn 2005). Nevertheless, after performing all sta-tistical analyses with and without these age classes, becauseoverall patterns remained the same (except where indi-cated), we present here the results of the complete data set.

Plant age signiWcantly inXuenced shoot nutritional qual-ity measured as percentage of water (F6,133 = 85.5,p < 0.0001) (Fig. 1c), and nitrogen (F5,54 = 34.2,p < 0.0001) (Fig. 1d), both of which declined as plants

aged. Mean water (Fig. 1c) and nitrogen (Fig. 1d) concen-trations were signiWcantly higher for seedling and youngjuvenile stages (i.e. 3–8 weeks old) than older juvenileand pre-reproductive plants (i.e. 10–18 weeks old). In con-trast, constitutive production of total iridoid glycosides(aucubin + catalpol) increased over time, ranging from aslow as 0.003 to as high as 8.86% dry weight from seedlingto pre-reproductive plants, respectively. Aucubin and catal-pol in shoot and root tissues varied considerably throughoutplant development as shown by the MANOVA (Wilks’� = 0.229, F4,126 = 9.67, p < 0.0001) (Fig. 2). UnivariateANOVAs also demonstrated a signiWcant eVect of plant ageon shoot aucubin and catalpol concentrations (F6,129 =43.31, p < 0.0001 and F6,129 = 11.63, p < 0.0001, respectively)as well as on root aucubin and catalpol concentrations(F6,129 = 16.44, p < 0.0001 and F6,129 = 5.26, p < 0.0001,respectively).

In turn, repeated measures ANOVA demonstrated thattotal IG concentration was signiWcantly inXuenced by plantage (F6,129 = 38.6, p < 0.0001) and plant tissue (F1,129 =104.3, p < 0.0001), with higher IG concentrations in shootcompared to root tissues and overall increase in IGs asplants aged, although in a non-linear fashion (Fig. 2). Fur-thermore, there was a signiWcant interaction between plantage and tissue (F6,129 = 26.02, p < 0.0001), indicating thatdiVerences in IG content between shoot and root tissueschanged throughout plant development. Thus, three maintrends emerged from our data. First, concentrations of IGsin shoot tissues were, on average, three times higher thanwere concentrations in root tissues. Second, mean groupdiVerences in aucubin and catalpol among all seven ageclasses followed similar trends for both shoot (Fig. 2a) androot tissues (Fig. 2b). Third, aucubin concentrations inshoot tissues drive most of the changes in total IGs through-out plant ontogeny.

Regression models demonstrated that constitutive varia-tion in total IGs increased non-linearly with plant age forshoots and linearly for roots, indicating that ontogeneticpatterns in defenses between tissues were dissimilar(Fig. 3). At the whole plant level, ontogenetic trajectories inconstitutive IG production were also non-linear (Table 1;Fig. 3a). This non-linear trend was mostly driven by shootssince partial investment in defenses for shoot tissues alsoshowed a signiWcant linear and quadratic term (Table 1;Fig. 3b), but roots, with signiWcantly less variation acrossage classes than shoots, showed a signiWcant linear increasein IGs with plant age but not a signiWcant quadratic trend(Table 1; Fig. 3c). Furthermore, the shape of the curvesdescribing IG content as plants age diVered between tis-sues, demonstrated by a signiWcant interaction betweenplant tissue and the linear or quadratic eVect of age on plantdefenses (for IGdiV linear t = 7.72, p < 0.0001; IGdiV qua-dratic t = 6.37, p < 0.0001). SpeciWcally, we found that the

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linear and quadratic eVect is signiWcantly stronger in shootsthan in roots, indicating that, as plants age, more abruptdiVerences in IG content can be observed for shoots thanfor roots (see Fig. 3).

Finally, variation in R:S ratios across all age classes didnot follow the negative association predicted by Boege andMarquis (2005) to explain plant allocation of resources tochemical defenses (slope = ¡0.09, R2 = 0.001, p = 0.29).Similarly, the ANCOVA showed that total IG concentra-tions at the whole plant level were not explained by R:Sratios once age was included in the model (R:S ratio: F1,120

0.03, p = 0.85; plant age: F6,120 28.4, p < 0.0001), but a sig-niWcant age by R:S ratio interaction was observed (F1,20

5.61, p < 0.0001), indicating that the relationship betweenR:S ratios and IGs varied among age classes. Because seed-ling R:S ratios were not higher than R:S ratios of older ageclasses (Fig. 1b) and a signiWcant age by R:S ratio interac-tion was detected, two separate linear regressions were runon the 3- to 8-week-old individuals and the older 10- to 18-week-old individuals. In this case, although much of thevariation in IGs across plant age classes is still poorlyexplained by diVerences in R:S ratios, an interesting patternarose. For seedling and young juvenile stages, contrary topredictions by the non-linear model, larger R:S ratios had aweak but signiWcant positive relationship with proportion

of total IGs (slope = 0.31, R2 = 0.08, p = 0.02) (Fig. 4a). Incontrast, for older juvenile and pre-reproductive stages, thedecrease in R:S ratios as plants grow showed a signiWcantnegative relationship with plant investment in defenses(slope = ¡0.47, R2 = 0.22, p < 0.0001) as predicted by themodel (Fig. 4b). However, this negative relationship ismostly driven by the older pre-reproductive age class(18 weeks old), as removal of this stage leads to a non sig-niWcant relationship between R:S ratios and IG content inolder stages (slope = 0.04, R2 = 0.002, p = 0.74).

Discussion

Plant defense theories predict that defenses should be allo-cated to plant tissues as a function of the value of those tis-sues for plant Wtness and their susceptibility to damage bynatural enemies (Feeny 1976; McKey 1979). Although boththe value and the susceptibility to damage of a tissuechange as plants develop, few studies to date have exploredchanges in chemical defenses of diVerent plant tissuesacross multiple developmental stages. Here, we showedthat the defensive chemistry of shoot and root tissues sig-niWcantly varies during plant development and that this var-iation is mostly non-linear, as predicted by Boege and

Fig. 1 Plantago lanceolata ontogenetic variation (mean § 1SE) ina shoot and root biomass, b root:shoot ratios, and leaf nutritional qual-ity measured as c percentage water and d percentage nitrogen acrossseven age classes. Except for biomass, original data were arcsine-square root transformed for statistical analyses, actual values are

shown for illustrative purposes only. Letters indicate mean groupdiVerences as tested by a Bonferroni post-hoc test (p < 0.05). In(a) capital letters represent group mean diVerences for shoot biomassand lower-case letters represent group mean diVerences for rootbiomass

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Marquis (2005). Thus, our data add to the growing numberof empirical studies supporting non-linear trends in ontoge-netic trajectories of plant defenses (Barton and Koricheva2010 and references therein). These ontogenetic trajectoriesin plant defenses indicate that population level demograph-ics may contribute to the observed spatiotemporal variationin plant defenses, and may help to explain ecological inter-

actions among plants and herbivores (Lawrence et al. 2003;Boege 2005; Fonseca et al. 2006; Thomas et al. 2010).

In this study, the youngest seedling stage (3-week-oldplants), consisting of the cotyledons and up to two trueleaves, had twice as much total concentration of IGs thanolder seedling and young juvenile plants with 6–10 leaves(5 and 8 weeks old). Similar to our results and supportingthe proposed model by Boege and Marquis (2005), a fewother empirical studies have reported decreases in plantallelochemicals during early seedling development (Wal-lace and Eigenbrode 2002; Orians et al. 2010). However,this trend does not seem to apply across taxa (see Bartonand Koricheva 2010), and is slightly diVerent from a previ-ous study showing positive ontogenetic trajectories in IGsin P. lanceolata seedlings (Barton 2007). Following thisinitial decrease, our study showed that allocation ofresources to defenses during the juvenile stages (from 8 to18 weeks old) increased almost exponentially, from anaverage of less than 0.4 to over 4% dry weight (Fig. 2).This result agrees with the well-documented trend ofincreases in plant defenses during immature plant stages(Barton and Koricheva 2010), also supporting the Boegeand Marquis (2005) model.

Given that plant secondary compounds are diVerentiallysynthesized and stored among plant tissues, whole plantassessment of defenses is needed for understanding plantpartial and total investment of defenses over time. In thissystem, concentrations of IGs were three times larger inshoot as compared to root tissues across all developmentalstages, suggesting that resource allocation to defenses inaboveground tissues may prevail over belowground tissuesduring P. lanceolata development. Furthermore, althoughnot as pronounced as in other systems, the pattern of varia-tion in total IGs in leaves and roots diVered, indicating thatIG content changed diVerently in shoot and root tissuesthroughout plant development. SpeciWcally, allocation ofIGs into root tissues constantly increased from younger toolder plants while non-linear shifts in allocation of IGs

Fig. 2 Plantago lanceolata ontogenetic variation (mean § 1SE) inconstitutive defenses measured as proportion of aucubin and catalpolin a shoot and b root tissues. Original data were arcsine square-roottransformed for statistical analyses, percentage data are shown forillustrative purposes only. Letters indicate mean group diVerencesamong age classes as tested by Bonferroni post-hoc tests (p < 0.05).Capital letters represent group mean diVerences for aucubin and low-er-case letters represent group mean diVerences for catalpol

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IGs

(% d

ry w

eigh

t)

0.0

1.0

2.0

3.0

4.0

5.0

AucubinCatalpol

(a) Shoots

(b) Roots

Aab

Ba

Ba

ABab

Cb

Cb

Dc

Bb

Aa A

a

Aa

Aab

ABab

Aab

Age class (wks-old)

IGs

(% d

ry w

eigh

t)

Fig. 3 Proportion of total IGs (arcsine square-root transformed dryweight) for each individual plant measured (n = 140, 20 per age class)at the a whole plant level as well as separated for b shoot and c root

tissues regressed across seven pre-reproductive age classes of Plan-tago lanceolata. Lines of best Wt for linear (solid) and quadratic(dashed) models are shown for illustrative purposes

0 3 5 8 10 12 15 18 20

0.0

0.1

0.2

0.3

0.4 (a) Whole plant

Plant age class (wks-old)

0 3 5 8 10 12 15 18 20

(c) Roots

0 3 5 8 10 12 15 18 20

Tot

al IG

s (t

rans

f dry

wt) (b) Shoots

123

478 Oecologia (2012) 168:471–481

during ontogeny were observed for shoot tissues (Table 1;Fig. 3). DiVerential ontogenetic trajectories between below-and aboveground tissues have also been observed in othersystems (Hartmann and Zimmer 1986; Williams and Ellis1989; Bellostas et al. 2007; Beninger et al. 2009), but noconsistent trends can be described. For example, in two IGcontaining plants, Linaria vulgaris and Antirrhinum majus,Beninger et al. (2009) reported a linear decrease as well asa lack of variation in root iridoids from seedling to repro-ductive plants, respectively, while defenses in abovegroundtissues of both species seemed to vary in a non-linear fash-ion. In contrast, total alkaloid concentrations in Papaversomniferun reported by Williams and Ellis (1989) showedsigniWcant non-linear trends in both shoot and root tissues,although maximum and minimum peaks did not necessarilymatch (see also Hartmann and Zimmer 1986). This dispar-ity in ontogenetic trajectories between shoot and root tis-sues highlights the need to consider all plant tissuesavailable at any stage to accurately test for overall plantinvestment in defenses throughout development.

Changes in resource allocation among plant tissues dur-ing ontogeny reXect variation in growth rate, physiologicaland anatomical constraints, as well as changing priorities(e.g., growth vs. reproduction) of an organism during thecourse of its development. Thus, researchers have used

these allocation patterns, such as R:S ratios, to predictresource allocation to plant defenses (e.g., Boege andMarquis 2005). Here, we demonstrate that R:S ratios canpartially help to predict ontogenetic trajectories in plantdefenses. While juvenile to pre-reproductive plants followthe predicted trend of a negative relationship between R:Sratios and constitutive defenses (Fig. 4b), allocation ofdefenses in early ontogenetic stages showed the oppositetrend (Fig. 4a). The early decrease in IGs as well as R:Sratios from young seedling to young juvenile stages mayreXect a trade-oV between investment in photosyntheticarea versus defenses or simply a dilution eVect due tohigher growth rate compared to biosynthetic synthesis ofchemical defenses. Following this initial positive relation-ship, the decrease in R:S ratios was associated with anincrease in IGs, which is consistent with an increase insynthesis of defenses as growth rate becomes more limitingthan photosynthetic rate (Herms and Mattson 1992).Furthermore, this shift in the relationship between R:Sratios and IG concentrations may suggest potentially tran-sient costs of defenses, as recently demonstrated by Orianset al. (2010). Nevertheless, these trends need to beinterpreted with caution since the amount of variation inconstitutive levels of IGs explained by R:S ratios was verylow and some trends may be driven by a single age class,

Table 1 Total IG concentrations at the whole plant level, as well as separately for shoots and roots, regressed as a function of plant age inP. lanceolata, from 3 to 18 weeks-old plants

Parameter estimates (�) for the constant and the linear and quadratic slopes

Slopes signiWcantly diVerent from zero are highlighted in bold type

Whole plant Shoots Roots

� t p � t p � t p

Constant 0.060 12.4 0.0001 0.063 11.1 0.0001 0.048 14.1 0.0001

Age

Linear 0.008 12.6 0.0001 0.010 12.1 0.0001 0.003 6.7 0.0001

Quadratic 0.001 7.8 0.0001 0.001 7.2 0.0001 0.000 0.9 0.33

Fig. 4 Linear regression between R:S ratios and proportion of totalIGs production across a young life stages (3-, 5- and 8-week-oldplants), and b older life stages (10-, 12-, 15- and 18-week-old plants).

Proportion of total IGs at the whole plant level were arcsine square-root transformed

(a)

R:S ratio

0.0 0.2 0.4 0.6 0.8 1.0

Tot

al IG

s (t

rans

f dry

wt)

0.00

0.05

0.10

0.15

0.20

3 wk5 wk8 wk

(b)

R:S ratio

0.0 0.2 0.4 0.6 0.8 1.00.00

0.10

0.20

0.30

0.40

10 wk 12 wk15 wk18 wk

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Oecologia (2012) 168:471–481 479

indicating that production of IGs depends on several otherfactors besides the allocation of biomass to diVerent tissues.Alternatively, lack of strong correlations between R:Sratios and IG concentrations may be due to partial assess-ment of P. lanceolata overall investment in defenses.Because P. lanceolata also invest in phenolic compounds(Galvez et al. 2005), as well as in physical defenses such asleaf toughness and trichomes (Schipper and OlV 2000; de laFuente 2002), we cannot rule out the potential predictedvalue of R:S ratios in overall defenses.

Whatever the mechanism, the ontogenetic variation inIGs described here, encompassing four- to Wvefoldincreases in shoot and root tissues, can have important con-sequences for plant–herbivore interactions. Laboratory andWeld experiments have shown that specialist butterXies pre-fer to oviposit on P. lanceolata tissues with higher IG con-tent (Prudic et al. 2005; Talsma et al. 2008), and that larvaeof these herbivores grew better on diets with higheramounts of IGs (e.g., Bowers and Puttick 1989; Harveyet al. 2005; Saastamoinen et al. 2007). In contrast, general-ist herbivores and pathogenic fungi damaging below- oraboveground tissues are usually deterred by high IG con-tent (Strohmeyer et al. 1998; Biere et al. 2004; De Deynet al. 2004; Harvey et al. 2005; Wurst and van der Putten2007). As a consequence, the relative palatability ofP. lanceolata to below- and aboveground herbivores andpathogens will signiWcantly vary through the season, andmay determine the impact of these antagonists on plantWtness. Lastly, for sequestering specialist herbivores, onto-genetic trajectories in plant defenses can, in turn, modifyherbivore unpalatability and thus can indirectly mediate tri-trophic interactions (e.g., Strohmeyer et al. 1998; Harveyet al. 2005; Baden and Dobler 2009; Smilanich et al. 2009).Thus, mainly for specialist herbivores that consume andsequester IGs from single or multiple tissues, these ontoge-netic trajectories in plant constitutive defenses may impactherbivore population dynamics and community structurethrough seasonal changes in host selection, herbivore per-formance, and levels of resistance to natural enemies.

Ontogenetic trajectories in leaf nutritional quality werealso evident, but the trend was opposite to the onedescribed for chemical defenses, showing a decrease in leafwater and nitrogen concentration as plants aged. Similardecreases in nutritional quality have been observed innumerous plant taxa (e.g., Gaudet et al. 2001; Bowers andStamp 1993; Goodger et al. 2006; Merilo et al. 2009),directly inXuenced by plant age or indirectly mediatedthrough their correlation with other ontogenetic shifts inleaf anatomy and physiology (Apple et al. 2002; Ishidaet al. 2005). In our study, ontogenetic variation in nutri-tional quality encompassed an average decrease of 10% inpercentage water and 30% in nitrogen concentration from3- to 18-week-old plants (Fig. 1c, d). This variation can

also have important implications for plant-insect interac-tions since diets with low water and nitrogen concentrationstypically have negative impacts on herbivore host selection,growth rates, and Wtness (Mattson 1980; Awmack andLeather 2002). SpeciWcally for this system, similar varia-tion in leaf nitrogen content (»30%) among individuals ofP. lanceolata resulted in a signiWcant eVect on ovipositionpreferences of the specialist Buckeye butterXy (Junoniacoenia, Nymphalidae), with females showing preferencefor high-nitrogen plants (Prudic et al. 2005). Thus, theontogenetic trajectories in nutritional quality showed heremay also help to explain temporal variation in species inter-actions as seen in other systems (e.g., Gaudet et al. 2001;Barrett and Agrawal 2004).

In summary, developmental variation in chemicaldefenses across Plantago lanceolata ontogeny appears tobe similar, and sometimes greater, in magnitude thangenetic or environmental variation in a single age class(Bowers et al. 1992; Bowers and Stamp 1993; Barton2007). Recent empirical studies have shown similar pat-terns in other systems (e.g., Donaldson et al. 2006; Holeskiet al. 2009; Lawrence et al. 2003). Thus, these studies high-light the importance of separating the proportion of varia-tion in plant defenses that has a developmental basis fromstrict genetic or environmentally dependent variation, inorder to improve our understanding of the evolution ofplant defenses. Furthermore, these ontogenetic trajectoriesin plant defenses and nutritional quality are likely to inXu-ence below- and aboveground herbivores alike, providing apotential mechanism to explain temporal variation in herbi-vore community composition and species interactions overtime.

Acknowledgments We thank K. Barton, K. Boege, Y. Linhart, theBowers lab, and three anonymous reviewers for valuable commentsand suggestions on this manuscript. In addition, we gratefullyacknowledge A. Hill, L. Mulder, and T. J. Lemieux for greenhouse andlaboratory assistance; and N. Robinson and C. M. Judd for their assis-tance with data analyses. Funding for this project was provided byBeverly Sears Graduate Student Grant, the Undergraduate ResearchOpportunity Program and the Department of Ecology and Evolution-ary Biology at the University of Colorado, and a National ScienceFoundation grant DEB 0909717.

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