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American Journalof Botany 8 1(3): 278-286. 1994.

CARBON INTEGRATION IN ARISTATA (): THE REPRODUCTIVE EFFECTS OF DEFOLIATION1

MARGARET HORTON AND ELIZABETH P. LACEY2 Departmentof Biology, 312 EberhartBuilding, University of North Carolina, Greensboro,North Carolina 27412

Patternsof carbon integration in aclonalspecies are poorlyunderstood in spiteof their potential to influenceindividual fitness.To providemore information about these patterns, we performeda defoliation experiment with P. aristata.We examined,at themetameric level, the reproductive responses to theremoval of the major carbon sources within metamers. Bractson markedreproductive spikes and leaves subtendingthese spikes were removed at threestages of reproductive maturity:spike elongation, flowering, and fruiting. Spike dry weight and length, capsule number, seeds per capsule, and seed weightwere measured. We testedthe hypothesis that seed weight would respond least to defoliation.We also performeda complementary14C translocationexperiment to measurethe amount of radioactive carbon moving into the marked spikes fromoutside the metamer. Defoliation depressed all componentsof reproduction within marked spikes, and little14C was translocatedfrom outside the metamer into the reproductive spikes, even those that were defoliated. Both results support theview that reproductive metamers in thisspecies are largely autonomous with respect to theircarbon budget. Defoliation duringspike elongation most depressed reproduction, and removal depressed reproduction more than did leaf removal. The data suggestthat compensate for leaf removal by increasingtheir photosynthetic rate; however, the ability to compensatediffers among populations. Of all thereproductive components, seed weight was least affected by defoliation. The data show,however, that the time of defoliationrelative to reproductivedevelopment influences which reproductive componentsare affected.

Ecological and evolutionaryinterest in thephysiological 1988; Thomas and Watson, 1988; Garrishand Lee, 1989; integrationof has blossomed in thelast few decades. Tuomi et al., 1989; Kemball, Palmer,and Marshall, 1992; This interesthas been motivatedby the desire to under- Lacey and Marshall, 1992). Translocationhas been mea- stand how such integrationaffects a genotype'sability to sured both in stressedand unstressedplants and at dif- persist in a particularenvironment. Interest in physio- ferentdevelopmental stages. These studies provide in- logical integrationprompted Watson and Casper (1984) formationabout the degree of integrationof different to coin the term integratedphysiological unit (IPU) to morphologicalunits, i.e., the IPU boundaries,in a variety emphasize thefact that the functional unit of organization of environmentalconditions. in a plant conceptuallydiffers from the morphological Fewerempirical studies have examinedthe relationship units of organization.Within an IPU the assimilation, betweenthe IPU and fitness.Patterns of carbon integra- distribution,and utilizationof a resource,like carbon, is tion have the potentialto influencelifetime reproductive regulated.The IPU boundaries withina plant may co- output,which contributesstrongly to fitness.A few bi- incide loosely with any one of the morphologicalunits ologistshave performeddefoliation experiments in aclon- constitutinga plant, e.g., metamer,branch, ramet, and al species to examine the reproductiveresponses of re- usuallychange as a plantdevelops (see reviewsby Watson movingcarbon sources or sinks(Janzen, 1976; Stephenson, and Casper, 1984; Watson, 1986). 1980; Marquis, 1988; Thomas and Watson, 1988; Garrish Much of the empirical researchon IPUs has focused and Lee, 1989; Marshall, 1989; Shea and Watson, 1989; on identifyingthe boundaries of the IPU for carbon in Tuomi et al., 1989). However, because most of the de- differentplant species. Biologists have measured carbon foliationexperiments have been used to measure the de- translocationamong ramets withinclonal plant species gree of integrationamong morphologicalunits, i.e., used (e.g.,see reviewsby Pitelka and Ashmun,1985; Hutchings to identifyIPU boundaries, informationabout the rela- and Bradbury,1986; Marshall, 1990; Schmid, 1990; more tionshipbetween the IPU and fitnessis limited. Ideally, recently:Chapman, Robson, and Snaydon, 1992; Landa one would like a prioriknowledge of the IPU boundaries. et al., 1992). A few have also examined carbon translo- Then afterdefoliation, one could observethe reproductive cation among branchesand metamersin aclonal species response,not just at the whole plant level but also at the (Janzen,1976; Stephenson,1980; Bertin,1982; Marquis, level of the IPU. We conducted such a defoliation experimentusing ' Receivedfor publication 28 June1993; revision accepted 12 October Plantago aristata,bracted plantain. P. aristata is an an- 1993. nual rosette species that produces reproductivespikes The authorsthank K. Landa,C. Marshall,and M. Watsonfor helpful fromthe axils of mature leaves. Subtendingeach flower commentson a previousdraft of thispaper; M. Watsonfor use of on a spike is a greenbract. In an unstressedplant of this laboratoryand greenhouse facilities to gather the 14C translocationdata; species, verylittle carbon is translocatedbetween repro- K. Landa and T. Preuningerfor help collecting data; and D. Herrfor statisticaladvice. This studywas partiallysupported by a SigmaXi ductivemetamers, a metamerbeing operationally defined grant-in-aidto MH. as a spike and its subtendingleaf (Lacey and Marshall, 2 Authorfor correspondence. 1992). Based on this observation,we made the a priori 278 March 1994] HORTON AND LACEY-CARBON INTEGRATIONIN PLANTAGO 279 assumption that a reproductivemetamer is an IPU for few seedlingsthat died duringthe firstwk in the growth carbon in P. aristata. Then we asked 1) what would be a chamber were replaced with seedlingsfrom the original metamer's reproductiveresponse to removal of its two germinationpots. Of the 360 plants transplanted,ex- major carbon sources,leaf and bracts,separately and to- cludingreplacements, 341 survived to harvest. getherand 2) how would the response change with de- During germination,the plants grewunder 12-hrd at velopmentalmaturity of the spike?Because the degreeof approximately22 C. For the first7 wk aftertransplanting, reintegrationof the defoliated metamer with carbon the plants experienced"winter" conditions: 8 hr lightat sources in othermetamers could influencethe response, 19 C/16 hr dark at 13 C. They were wateredthree times we also conducteda "4C translocationexperiment to mea- per wk and fertilizedonce per wk with ?/4-strengthHoag- sure the amount of reintegration.Because neitherauthor land's solution. At the end of 7 wk, we reprogrammed has observed much herbivoryof P. aristata in the field, thegrowth chamber for "summer" conditions: 14 hrlight our experimentwas performednot to mimic herbivory at 26 C/10 hr dark at 20 C. We watered the plants four but ratherto learn more about carbon allocation patterns times and fertilizedthem twice in an 8-d cycle until se- in the species. nescence began, which was approximately 17 wk after Reproduction can be divided into a number of com- transplanting.At that time we reduced the wateringto ponents,e.g., inflorescencenumber, fruit number per in- threetimes and fertilizationto once in the 8-d cycle. florescence,seed numberper fruit,and seed size (Adams, At the time of transplanting,we randomly assigned 1967; Primack, 1978). All or some of these components threeseedlings from each of six familiesper population can theoreticallybe alteredby defoliation.Harper, Lovell, to one of ten treatments:nine experimentaltreatments and Moore (1970) proposed that seed size is the most and one controlgroup. Each experimentaltreatment was conservativereproductive component, responding least characterizedby the removal of some combination of to stress,because seed size stronglyinfluences individual photosyntheticsource organsfrom a markedmetamer at fitness. Others (e.g., Lloyd, 1980; Stephenson, 1981; a particulardevelopmental stage of the metamer'srepro- Bookman, 1983) proposed that fruitnumber responds ductive spike. We performedthree different types of re- greatlyto stress. Previous studies (e.g., Harper, Lovell, movals. With scissorswe clipped eitherthe bractson the and Moore, 1970; Stephenson, 1984; Marshall, Levin, reproductivespike, or the leaf subtendingthe spike, or and Fowler, 1986; Agren, 1989; Marshall, 1989) have both the bracts and leaf. The cuts were made as close to tested the hypothesisthat stress negativelyaffects fruit the bottom of the targetorgans as possible. We clipped number more than it does seed size at the whole-plant the experimentalplants at one of three developmental level, and most provide supportingevidence forthe hy- stages: duringspike elongation,when the spike was 1-3 pothesis. In the experimentdescribed below, we tested cm long; duringflowering, just afterthe firstflower of the thishypothesis at thelevel ofthe metamer. Lee and Bazzaz inflorescencehad opened; and during fruiting,after all (1980) proposed that the time of the stress relative to the flowerson the spike had opened. During spike elon- reproductivedevelopment should influencewhich repro- gation,the inflorescencedevelops, and bracts on the in- ductive componentsare affectedby the stress.Therefore, florescencecontinue to grow. Therefore,we retrimmed we also tested the hypothesisthat the negative effectof the bracts an additional zero to fivetimes, as necessary. defoliationon seed size would be manifestedlater in spike In contrastto the bracts, subtendingleaves were fully developmentthan would thenegative effect on fruitnum- expanded at thetime of initial clipping. No leaves or bracts ber. were removed fromthe plants in the controlgroup. Reproductive spikes firstappeared 12 wk aftertrans- MATERIALS AND METHODS planting.The firstmetamer to produce a spike on each plant was tagged and used as eitheran experimentalor Study species-Plantago aristata Michx. (Plantagina- controlmetamer. When the spike in the taggedmetamer ceae), bracted plaintain,is an acaulescent winterannual turned brown, we harvested the plant. We dried and that is native to the southeasternUnited States. Unlike weighed each marked spike, withoutits bracts and sub- many annuals that grow vegetativelyas a rosette,P. ar- tendingleaf. We also weighedthe remainingportions of istata retainsits rosetteform throughout its life.The re- the shoot, which we collected and dried separately.All productivespikes arise fromthe axils of fullyexpanded plant parts were oven dried at approximately65 C for leaves. Each spike bears numeroussessile cleistogamous 2 d. flowers,and each floweris subtended by a conspicuous Data forseveral componentsof reproductionwere col- green bract. Flowers develop into two-seeded capsules lectedfrom each markedmetamer: spike dry weight, spike (Johnson,1981). length,and capsule number.We measuredspike biomass, because, despite its shortcomings,biomass is most com- Defoliationexperiment-We collectedseeds forour ex- monlyused to measure the resourcesa plant allocates to perimentsfrom two populations approximately 4 km apart reproduction(e.g., Bazzaz and Reekie, 1985; Marshall in Guilford County, North Carolina. Seeds were stored and Watson, 1992). We measured spike lengthbecause separatelyby maternalfamily in paper bags in the lab- spike elongationin P. aristatacontinues during flowering oratoryfor approximately6 mo. Then we sowed seeds and fruiting.If carbon resourcesavailable to themetamer froma haphazardlyselected sample offamilies, by family, are limited,a reductionin spike lengthmight occur, es- into Terralite pottingsoil in small pots. Afterthe- first peciallyif carbon was limitedearly in spike development leaves appeared, we transplantedseedlings individually when elongationis most rapid. into pots ( approximately7 cm diam) and randomlyas- For a subset of treatments(i.e., all defoliationtreat- signed each plant a position in a growthchamber. The ments during spike elongation,bract and leaf removal 280 AMERICAN JOURNAL OF BOTANY [Vol. 81

TABLE 1. Mean square (MS) values foranalyses of variance and ANOVA contrastsfor reproductive components in Plantago aristata.Main sources of variationwere treatments(T), which correspondedto the defoliationby developmentstage combinations,populations (P), and families(F) nestedwithin population. Shoot dryweight (S) was included as a covariate. The defoliationtreatments included bractand leaf removal (B&L), bract removal (B), and leaf removal (L); the development stages were spike elongation (E), flowering(F), and fruiting(Fr). C = control. Contrastswere averaged over families.a

Spike dryweight Spike length Capsule no. Total plant spike dry weight Source df MS MS MS MS Full model 120 16,047**** 2,269**** 378**** 1.04**** T 9 101,174**** 15,621**** 2,593**** 0.056ns P 1 419,284**** 39,063**** 5,184**** 3.361**** T*P 9 7,882* 1,132ns 281** 0.060ns F(P) 10 15,088**** 2,458*** 386**** 0.577*** T*F(P) 90 3,666ns 5,894ns 114ns 0.050ns S 1 1,58Ins 871ns 98ns 40.113**** Error 217 3,183 698 95 0.043 Contrasts E vs. Fl 1 260,412**** 53,208**** 6,923**** Fl vs. Fr 1 18,990* 458ns 217ns Fr vs. C 1 7,779ns 567ns 1 Ins B&L vs. C 1 183,976**** 15,973**** 3,055**** B vs. C 1 93,219**** 11,875**** 1,073** L vs. C 1 9,441ns 1,189ns' 1 Ins B&L vs. B 1 30,555** 583ns 1,002** a **** = P < 0.0001; P < 0.001;**= P < 0.01; * = P < 0.05; ns = P 2 0.10. duringflowering and fruiting,and the controlgroup), we We then establishedfour treatment groups, each con- also determinedthe mean number of seeds per capsule tainingthree plants. Firstwe placed plants into two cat- and mean seed weightper spike. Mean seed numberper egories based upon the presence of a spike in eitherthe capsule was determinedby countingthe matureseeds on elongation or floweringdevelopmental stage. The elon- each spike and dividingby capsule numberfor that spike. gatingor floweringspike was marked.Then withinthese To determinemean seed weightper spike, we divided two developmental categories,we randomlydesignated total seed weightby seed numberfor each spike. Finally, individuals as eitherexperimentals or controls.Prior to to examinethe effect of the treatments on thereproductive 14CO2 exposure, we removed the bracts and subtending output of the restof the plant, we weighedall the repro- leaf on the marked metamer of the experimentals.No ductive spikes on the plant,excluding the marked spike. removals were made on the controls. The data wereexamined with analyses of variance (SAS Twenty-threehours afterthe removal of source organs, Institute,1985) to evaluate the effectsof treatment,pop- we placed all of the shoot except the markedmetamer in ulation, familynested within population, and their in- an acetate chamber(one plant per chamber)and exposed teractionson each reproductivetrait. We included total the shoot to 5 ml of 2 mCi of 14CO2.The chamberswere shoot biomass as a covariate in each model to adjust for removed after30 min, and the plants were left in the individualdifferences in plantsize. We used theContrasts greenhousefor 26 hr,at which time theywere harvested. option to examine the specificeffects of treatmentwith We separatedthe exposed portionof the shoot,the roots, regardto both the developmental stage at removal and and the unexposed spike and dried them at 65 C for the source organs removed. Three plants were excluded approximately72 hr. For the controlplants, we removed fromthe analyses because the value of one of theirre- the bracts and subtendingleaf fromthe spike and dried productivetraits differed from the treatmentgroup mean each part separately. bymore than three standard deviations. Because we found Then we measured the amount of radioactive carbon no evidence priorto the analyses that the data were not in each part of threereplicate plants per treatment.Plant normallydistributed, we did not transformthe data. parts were weighed and ground prior to combustion in oxygen.We oxidized two 20-mgsamples fromeach shoot Translocation experiment-We germinateda second and fromeach root system,and one 20-mg sample from sample of seeds fromthe same populations,but different each unexposed spike. For the control plants, we also families, under conditions described in the defoliation oxidized a 20-mg sample fromthe bracts and fromthe experiment.After 4 wk we transplantedseedlings indi- subtendingleaf on the unexposed metamer. When the vidually into pots and randomlyassigned the pots to the floweringspike, bracts,or leaf weighed less than 20 mg, remainingspaces in the growthchamber, where they ex- we oxidized the entirepart. Each sample was combusted perienced "summer" conditions. We wateredthe plants at 900 C using a RJ Harvey InstrumentCorporation Bi- fourtimes and fertilizedthem twice every 8 d forthe next ological Oxidizer 0X400. The liberated 14CO2 was col- 6 wk. At thattime we reduced the wateringand fertiliza- lected in separatescintillation vials containingC1 4 cock- tion to threetimes and once, respectively,per 8-d cycle. tail (Harvey InstrumentCorp., Hillsdale, NJ) and counted Three months after transplanting,we transportedthe witha BeckmanLS-230 liquid scintillationcounter. These floweringplants to Bloomington,Indiana for the tracer methodsclosely followedthose describedby Landa et al. study.The plantswere placed in a greenhouse(14 hrlight, (1992). We used the counts, along with the mass of the 24-40 C) and allowed to acclimate for48 hr. sample combusted and the total mass of the plant part, March 1994] HORTON AND LACEY- CARBON INTEGRATION IN PLANTAGO 281 to compute the percentageof the total 14C02 uptake of TABLE 2. Mean square (MS) values foranalyses of variance and ANO- the plantimported by each unexposed spike. To compare VA contrastsfor reproductive components in P. aristata.a the experimentaland controlspikes, we used thebiomass Seeds per capsule Mean seed weight and cpm of the control spike minus its bracts. Analysis of variance was used to determineif developmental stage Source df MS df MS and defoliationinfluenced assimilate movementinto the Full model 72 0.160**** 71 0.09 1**** marked spike. T 5 0.638**** 5 0.185**** P 1 1.107**** 1 0.362*** T*P 5 0.659**** 5 0.021ns F(P) 10 0.145** 10 0.183**** RESULTS T*F(P) 50 0.075* 49 0.033ns S 1 0.028ns 1 0.316*** Defoliation experiment-Treatment significantlyaf- Error 122 0.047 121 0.0256 fectedall aspects of reproductionin the markedmetamer Contrasts per plant: spike lengthand dry weight,capsule number, E vs. Fl 1 1.79**** 1 0.0003ns seeds per capsule, and seed weight(Tables 1, 2). However, Fl vs. Fr 1 0.001ns 1 0.171* it did not affectthe dryweight of all otherreproductive Fr vs. C 1 0.OOlns 1 0.102ns structureson the experimentalplants. B&L vs. C 1 1.770**** 1 0.487*** B vs. C 1 0.144ns 1 0.490*** Defoliation reduced reproductiveoutput of the meta- L vs. C 1 0.0005ns 1 0.103ns mer,but the negativeeffect was restrictedalmost entirely a = 1). Only seed **** p < 0.0001; * = P < 0.001; ** = P < 0.01; * = P < 0.05; to the earliest developmental stage (Fig. ns = p > 0.10. weight was affectedby defoliation during floweringor fruiting.All reproductivecomponents were negatively af- fectedby defoliationduring spike elongation.Spike dry 50% when bracts and leaf were removed during spike weight and capsule number were reduced by approxi- elongation. mately 30% fromthose of the controls when averaged The two populations and the familieswithin popula- over all combinations of bract and leaf removal. The tionssignificantly differed in all reproductivecomponents contrasts(Tables 1, 2) show significantdifferences in re- measured (Tables 1, 2). Additionally,the treatmentby productive responses to defoliation made during spike population interactionswere oftensignificant. The effects elongationand those made duringflowering, even when of removingdifferent combinations of carbon sources as one takes into account the numberof multiplecontrasts manifestedin spikedry weight, capsule number,and seeds performed.By floweringtime the defoliationeffects dis- per capsule differedfor the two populations (Fig. 2). In appeared. one population,removing the bractsand subtendingleaf The differentcomponents of reproductionshowed two substantiallydepressed spike dry weight, capsule number, major patternsof response to defoliation(Fig. 1). First, and seeds percapsule fromthat observed when only bracts the componentsquantitatively differed in theirresponse. were removed. In the other population, bract and leaf Spike dry weightand capsule number were reduced the removal and bract removal, alone, produced similar re- mostby defoliationduring spike elongation (3/3% and 277% sponses. reduction,respectively, when averaged over all combi- nations of bract and leaf removal). Spike lengthwas re- Translocation experiment-The 14C measurements duced by 22%. Seeds per capsule and seed weightwere showed that the marked spikes did receive carbon from reduced the least, by only 7% and 5%, respectively.Sec- sourcesoutside the spike's metamer. However, the amount ond, the reproductivecomponents differed in the persis- totaledless than 1.5%, even forthose plants that had their tence of theirresponse. Mean seed weightper spike was carbon sources removed. The percentimported into de- reducedby defoliationduring flowering as much as it was foliated and control spikes duringspike elongationwas by defoliationduring spike elongation(see contrasts,Ta- 1.189 ? 0.082 and 1.276 ? 0.815 (mean + S.E.), re- ble 1). Only afterflowering had begun did the defoliation spectively.The percentimported into defoliatedand con- effectbegin to disappear. In contrast,none of the other trolspikes duringflowering was 1.247 ? 0.455 and 0.349 reproductivecomponents showed any negative effectof + 0. 166, respectively. defoliationbeyond the spike elongationphase. Spike size increased dramaticallyfrom time of elon- Removing differentcombinations of carbon sources gationto flowering.Mean spike dryweights for defoliated within a metamer stronglyinfluenced the reproductive and controlspikes at elongationwere 20.7 and 22.6 mg, response (Fig. 1). Removing the subtendingleaf, alone, respectively;spike weightsat floweringwere 119.4 and did not affectany reproductivecomponent when com- 93.9 mg, respectively.When the percent 14C imported pared to the control group. In contrast,bract and leaf into the spike was standardizedfor spike size, less than removal,as well as removal of bractsalone, depressedall 0.1% per mg was detectedin the spike. of the reproductivecomponents. These reductionswere The analysis of variance detectedno significanteffects highlysignificant when compared to reproductionin the of defoliationor developmental stage on the percentof controlgroup (contrasts,Tables 1, 2). Bract and leaf re- 14Cimported into the markedspikes (Table 3). On a per- moval produced a significantlygreater negative response mg spike basis, however, developmental stage did sig- in spikedry weight, capsule number,and seeds percapsule nificantlyinfluence the amount imported. There was a than did bractremoval alone (see contrasts,Tables 1;,2). six- to tenfolddrop in percent imported per mg spike We observedthe largest reduction in spike dryweight and fromspike elongationto flowering.Thus, it appears that capsule number,which were reduced by approximately while the total amount of translocatedcarbon into the 282 AMERICAN JOURNAL OF BOTANY [Vol. 81

4Q0- __220- _ PARTS REMOVED

350 E A 2 BRACT&LEAF ~~~~~300 O~~~~~~~~~~~~~~~~~~LEAF - t140 250 12 ~~~~~~~~~~~~~~~~~~~NONE

200 ~i100' 80' l0 60 100 40 E FL FR C E FL FR C

70 2.2

60 2C

50-

40 1.6-

30- A

20

101

E FL FR c E FL FR C

1.6 3- E ~~~~2.8 F 2.67 1.4

1.3 27

1.2' z 1.1~~~~~~~~~~~~~~~~~~.

E FL FR C E FL FR C

DIEVELOPMENTAL STAGE Fig. 1. Reproductiveresponses to defoliationat differentdevelopmental stages in Plantagoaristata expressed in termsof mean ? 2 SE forboth populationscombined. Graphs A-E referto theresponse of themarked metamer in termsof: A) dryweight of thereproductive spike, B) spike length,C) capsulenumber, D) seedsper capsule, E) seedweight. Graph F showsthe dry weight of all reproductivespikes per plant, not including thespike on themarked metamer. Developmental stages at timeof removal: spike elongation (E), flowering(Fl), fruiting(Fr). No removalswere madeon thecontrols (C). N = 33-36. spike remains fairlyconstant, the translocatedcarbon is location to various structuresin a plant reflectpast se- being spread over a greatervolume as spike biomass in- lection forpatterns that maximize fitness.These two as- creases. sumptionssuggest that if carbon sourcesin a reproductive metamerare removed, the plant will reallocate more of DISCUSSION its remainingresources to reproductivecomponents that contributethe most to fitnessand less to componentsthat There are two assumptionsthat underliemost studies contributeleast to fitness.There is, at best,weak evidence of plant growthand development:first that resources are to support this predictionin P. aristata. In P. aristata, limited,and second that extant patternsof resource al- traitslike capsule number, seeds per capsule, and seed March 1994] HORTON AND LACEY-CARBON INTEGRATIONIN PLANTAGO 283

400 TABLE 3. ANOVA meansquare (MS) valuesfor percent of totalas- similatedradioactive carbon that moved into the marked spike.a -~350 730- Percentimported into spike Percentimported (per mg spike 250 into spike dryweight) Source df MS MS 200 1 1 0.4926ns 0.0004ns 04 RemovalTreatment (T) 150. Developmental Stage (D) 1 0.5669ns 0.0071*** 100' T-D 1 0.7285ns 0.00008ns Error 8 0.6799 0.0002 50 a **** = p < 0.0001; * = P < 0.001; ** = P < 0.01; * = P < 0.05; 0 ns = p > 0. 10. E FL FR C

70- The above assumptions about limited resources and 60- allocation patternsalso suggestthat defoliation should 50' reduce capsule number more than it should seed weight z (e.g., Adams, 1967; Harper, Lovell, and Moore, 1970; 40- Stephenson, 1981). Fruit abortion has been considered the most importantstage of reproductiveregulation be- 30 cause it is energeticallyefficient to abort young fruits (Bookman, 1983). Seed weightis thoughtto change little 20' in response to stressbecause of its large contributionto offspringfitness. (However, Marshall, Levin, and Fowler E FL FR C [1986] have correctlynoted that differenthabitats may select for differentpatterns of plasticityin reproductive components.)In P. aristata,defoliation did reducecapsule 2.2- numbermore than it did seed weight.When we defoliated 2- the metamersduring spike elongation,capsule number was reduced by 27% and seed weightby only 5%. Thus, 1.8- seed weightwas themore conservative reproductive char- U1.6- acter.The proportionalreduction in numberof seeds per capsule was also low (7%). We did not include seeds per 1.4 capsule in our a prioripredictions about the differences 1.2- in reproductiveresponses because P. aristata produces a maximum of only two seeds per capsule. Thereforewe suspected that the proportionalreduction in seeds per 0.8- capsule would be low forthis reason alone. We were, in fact,surprised that defoliationproduced any significant E FL FR C reductionin seeds per capsule. Primack (1978) foundno DEVELOPMENTAL STAGE deviation fromtwo seeds per capsule in the herbarium Fig. 2. Reproductiveresponses to defoliationat differentdevelop- specimens that he examined. mental stages in Plantago aristata expressedin termsof mean ? 2 SE Lee and Bazzaz (1980) notedthat decline in seed weight shown by population. The two populations are identifiedby different relativeto otherreproductive components should depend amounts of shading. Graphs A-C referto the response of the marked on thetime ofdefoliation. Our data providegood evidence metamerin termsof: A) dryweight of thereproductive spike, B) capsule forthis. The negativeeffects of defoliationpersisted into number,C) seeds per capsule. Developmental stagesat timeof removal: the floweringand fruitingphases only for seed weight. spike elongation(E), flowering(F1), fruiting(Fr). Removal treatments: I = bract and leaf removal, 2 = bract removal, 3 = leaf removal. No Therefore,if we had chosen to defoliateour experimental removals were made on the controls(C). plants only at onset of flowering,or later,we would have erroneouslyconcluded that seed size showed a greater response to defoliation. Agriculturalstudies have also weightstrongly influence fitness, while a traitlike spike shown that delayingthe time of stressrelative to floral lengthprobably does not. Because the species is cleistog- and fruitdevelopment decreases the negative effecton amous, it "need not" display its flowersto pollinatorsto fruitand seed number but increasinglydepresses seed effectseed set. Also, seeds typicallyfall close to theparent weight(e.g., Sharrow,1990; Rajewski and Francis, 1991). plant regardlessof spike length.While defoliationduring Reproductivestructures have been shownto contribute spike elongation reduced capsule number more than it an importantpercentage of the carbon needed forrepro- did spike length,it reduced spike lengthmore than it did duction in many trees,shrubs, and herbaceousplant spe- seeds per capsule and seed weight.Spike dryweight was cies (e.g.,Flinn and Pate, 1970; Bazzaz and Carlson, 1979; also greatlyreduced, although seed weightand the weight Bazzaz, Carlson, and Harper, 1979; Watson and Casper, of accessory reproductivestructures are confounded in 1984; Heilmeier and Whale, 1987). In P. aristata, these this measurement. reproductivestructures are the bracts. In fact,the bracts 284 AMERICANJOURNAL OF BOTANY [Vol. 81 play a largerrole than does the subtendingleaf in main- (Welker et al., 1985; Wallace, 1990; Dyer et al., 1991) taininga local carbonbalance withina reproductivespike. have detected interpopulationaldifferences in the phys- Bract removal alone reduced all components of repro- iological responsesto herbivoryand artificialdefoliation. duction, while removal of the subtendingleaf alone re- Our defoliationexperiment shows thatthe removal of duced none. We do not believe thatthese resultssuggest carbon sources within P. aristata produces a localized that the leaf does not provide carbon for the spike. In response.Defoliation reduced reproductive output within another study of P. aristata, more than 70% of the ra- the defoliated metamerbut not the total dry weightof dioactive carbon exportedfrom a subtendingleaf moved otherspikes on the plant. These data are consistentwith into its associated spike duringspike elongation (Lacey Lacey and Marshall's (1992) study,which showed little and Marshall, 1992). Therefore,subtending leaves do carbon translocationamong reproductivemetamers in translocatecarbon to theirassociated spike. Rather, we intact plants. The data are also consistentwith the '4C hypothesize that bracts may be compensatingfor leaf measurementsmade in thisstudy, which showed thatthe removal by increasing their photosyntheticrate in re- amount of carbon moving fromthe shoot into marked sponse to changesin the source-sinkbalance of the meta- metamerswas small even when the metamer's leaf and mer. bracts were removed. Thus, our initial assumption that Our hypothesisis supportedby the nonadditiveeffects a reproductivemetamer is an IPU forcarbon in P. aristata ofremoving both bracts and subtendingleaf. The removal is supported. of both bractsand leafdepressed reproduction more than Two other studies of carbon integrationat the meta- we would have predictedfrom the independenteffects of meric level in naturallygrowing species permita com- removingthe bracts alone and theleafalone (Fig. 1). When parison with our data. Marquis (1988) observed a local- the bracts were missing,neither the rest of the metamer ized effectof defoliation in Acerpennsylvanicum. Removal nor the rest of the shoot compensated forthe loss of the of leaves directlysubtending fruits reduced seed produc- leaf. Other studies have shown that afterpartial defoli- tion in those fruits,while removal of neighboringleaves ation or shading,remaining leaves increase theirphoto- produced no effect.In contrast,Garrish and Lee (1989) syntheticrates per unitarea (e.g., Sticklerand Pauli, 1961; observed that the number of fruitsand seeds produced Thorne and Koller, 1974; Satoh, Kriedemann,and Lov- by defoliatedvs. intact metamersdid not differsignifi- eys, 1977; Thomas and Stoddart, 1980; McNaughton, cantlyin Cassiafasciculata. Thus, at the metamericlevel, 1983; Yamashita and Fujino, 1986; Mendoza, Pinero, patternsof carbon integration,as measured in termsof and Sarukhan,1987; Gold and Caldwell, 1990). Our data its reproductiveconsequences, differ among species. Fur- suggestthat the bracts,modified leaves, compensate for thermore,the data show thatintegration among metamers leaf removal by respondingsimilarly. is not correlatedwith the compactnessof a species,as has It appears, however,that the importanceof a subtend- been hypothesizedby Schmid (1990) forclonal plants. ing leaf as a directcarbon source forspike development Our defoliationexperiment was not performedto mim- varies among individual plants. In one population, the ic herbivoryin P. aristata. Rather it was performedto leaf contributedlittle to reproductiveoutput, evidenced help learn more about carbon allocation patternsin the both by the observationthat its removal, alone, did not species. P. aristatais one of threeannual species of Plan- depress output,when compared to the controland by the tago indigenous to the southeasternUnited States. All observationthat its removal withbracts did not depress threespecies can be foundgrowing in frequentlydisturbed output below that observed when only bracts were re- fields and roadsides; sometimes they have been found moved (Fig. 2). In contrast,in the otherpopulation, the growingtogether (Lacey, personal observation). All in- leaf did appear to contributeto reproductiveoutput. Re- digenous species of Plantago germinateat approximately moval ofboth bractsand leafdepressed output below that the same time; however,P. aristata survives longerand observed when only bracts were removed. Thus, in the reproduceslater in the growingseason. In some summers second population,bracts appeared to compensatefor loss it can persistthrough the summerto flowera second time of a leaf,whereas in the first,they did not. Eitherthe leaf (Primack, 1979). In contrastto P. virginica,one of the contributedlittle to spike developmentor the restof the othertwo indigenousannuals, P. aristata shows little'4C metameror shoot compensated forthe loss. translocation among reproductive metamers in intact There are two possible explanations for these inter- plants (Lacey and Marshall, 1992). Our data show thata populational differencesin responseto defoliation.First, metamerof P. aristataremains largely autonomous even the differencesmight reflect environmentally induced pa- afterdefoliation within the metamer.It is possible that rentaleffects. The effectsof the maternalor paternalen- this differencein patternof carbon integrationmay con- vironmentmight have been carriedover into the gener- tributeto the species' increased longevity.Carbon re- ation used forour experiment.Although environmentally sources may not be drainedaway fromother shoot meta- induced parentaleffects have usually been detectedonly mers and the root to make up for lost resourcesin the in seed traits,they sometimes persist into the adult, e,g., damaged metamer. reproductive,phase of a life cycle (e.g., see reviews by Future defoliationstudies should consider the effects Roach and Wulff,1987; Lacey, 1990). Source-sinkdy- of defoliationboth at the whole plant level and at the namics mightbe involved in theselatter effects, but pres- level of the IPU. As Harper (1977) has compellinglyar- entlyno studiesaddress thisissue. Alternatively,the pop- gued,a plantis an assemblageof reiterated morphological ulations may geneticallydiffer in source-sinkdynamics. units,each of which may have its own physiologicalpro- If this latterexplanation is correct,then it indicates the gram. Understandinghow an individual plant interacts existenceof the potentialfor evolutionary change in car- withits environment requires understanding not only how bon integrationpatterns. Recent studies of grass species a whole plant respondsto the environmentbut also how March 1994] HORTON AND LACEY-CARBON INTEGRATIONIN PLANTAGO 285

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