HERBIVORE RESISTANCE IN BETULA PENDULA : EFFECT OF FERTILIZATION, DEFOLIATION, AND PLANT GENOTYPE

49

Ecology, 81(1), 2000, pp. 49–65q 2000 by the Ecological Society of America

HERBIVORE RESISTANCE IN BETULA PENDULA: EFFECT OFFERTILIZATION, DEFOLIATION, AND PLANT GENOTYPE

PIA MUTIKAINEN,1,2,5 MARI WALLS,1 JARI OVASKA,1,3 MARKKU KEINANEN,4

RIITTA JULKUNEN-TIITTO,4 AND ELINA VAPAAVUORI3

1Department of Biology, University of Turku, FIN-20014 Turku, Finland2ETH-Zurich, Experimental Ecology, ETH-Zentrum NW, CH-8092 Zurich, Switzerland

3The Finnish Forest Research Institute, Suonenjoki Research Station, FIN-77600 Suonenjoki, Finland4University of Joensuu, Department of Biology, P.O. Box 111, FIN-80101 Joensuu, Finland

Abstract. Plant resistance to herbivores is affected both by genetic and environmentalfactors. The carbon–nutrient balance hypothesis (CNB) explains environmentally induced vari-ation in both constitutive and delayed herbivore-induced resistance (DIR) in terms of variationin soil fertility and light regime. The CNB hypothesis predicts that an increase in the availabilityof nutrients (e.g., fertilization) decreases both constitutive and induced resistance against her-bivores. We tested the relative roles of plant genotype, defoliation, and soil fertility in deter-mining herbivore resistance of cloned silver birch Betula pendula Roth saplings. As indicatorsof insect and mammalian resistance we conducted bioassays with a geometrid moth, Epirritaautumnata (Borkhausen), and counted the resin droplets on the shoot of the saplings, respec-tively. In addition, we measured rapid induced resistance (RIR) against the insect herbivore.Finally, we analyzed leaf secondary chemistry to investigate the correlations of secondarychemicals with the level of resistance measured using the performance of E. autumnata.

With respect to the constitutive resistance against an insect herbivore, our results supportthe CNB hypothesis; the larvae of E. autumnata had a higher relative growth rate and pupalmass on fertilized saplings compared to nonfertilized saplings, i.e., the fertilized saplings hada lower resistance level. However, the relative growth rate of E. autumnata was significantlydecreased by defoliation only when the larvae were grown on fertilized saplings. The numberof resin droplets increased due to fertilization and, in fertilized saplings, following defoliation,but these responses were highly determined by the genotype of the sapling. Altogether, theresults on resin droplets are not in accordance with the CNB hypothesis.

The concentration of condensed tannins correlated negatively with E. autumnata growthrate and pupal mass in both fertilization levels, whereas the concentration of total nontanninphenolics correlated positively with the E. autumnata growth rate in nonfertilized saplings. Inaddition, the concentration of myricetin glycosides correlated negatively with the pupal massof E. autumnata, whereas the correlations between E. autumnata performance indices and othergroups of flavonol glycosides were either significantly positive (kaempferol glycosides) ornonsignificant (quercetin glycosides). Further, the concentration of 3,49-dihydroxypropiophen-one 3-glucoside (DHPPG) correlated positively with the magnitude of induction in E. autumnatagrowth rate and pupal mass in fertilized saplings, where the significant induction in resistanceoccurred. The correlations of secondary chemistry and E. autumnata performance indicessuggest that the constitutive level of resistance of B. pendula against E. autumnata is mainlydetermined by the concentration of condensed tannins, whereas the induced resistance is de-termined by the concentration of nontannin phenolics, such as flavonol glycosides and DHPPG.

We observed significant differences among the clones in their insect and mammalianresistance (i.e., genetic basis for the resistance), which indicates that resistance can evolveas a response to herbivory. However, fertilization explained a higher proportion of variancein insect performance indices than the genotype of the plant, whereas the opposite was truefor the amount of resin droplets, which we used as an indicator of mammalian resistance.

Key words: Betula pendula; carbon/nutrient balance; clonal variation; defoliation; Epirrita autumnata;fertilization; herbivore resistance; plant–herbivore interactions; resin droplets; secondary chemicals.

INTRODUCTION

Phenotypic variation in plant resistance to herbivoryis caused by genetic and/or environmentally induced

Manuscript received 9 December 1997; revised 20 October1998; accepted 8 December 1998; final version received 14January 1999.

5 E-mail: [email protected]

differences among plant individuals (for a review, seeMarquis [1992]). The maintenance of genetic variationin resistance is a dilemma because if resistance is ben-eficial in terms of plant fitness, plant populationsshould evolve towards the highest level of resistanceand lose additive genetic variation in resistance (Fisher1930). However, genetic variation in resistance traits

50 Ecology, Vol. 81, No. 1PIA MUTIKAINEN ET AL.

within populations has been observed in numerousstudies (e.g., Hanover 1966, Berenbaum et al. 1986,Fritz 1990, Kennedy and Barbour 1992, Zangerl andBerenbaum 1997). The relative contributions of geneticand environmental factors, and their interaction, to thevariation in resistance have not been extensively stud-ied until recently (Maddox and Cappuccino 1986, Mad-dox and Root 1987, Fritz 1990, Karban 1992, Kennedyand Barbour 1992). In his review, Karban (1992) con-cluded that plant genotype has a stronger effect onresistance to herbivores (i.e., on herbivore populations)than the interaction between genotype and environ-mental factors.

Environmentally induced variation in plant resis-tance may be due to both biotic (e.g., previous herbiv-ory) and abiotic factors. The growth–differentiationbalance (GDB, Loomis 1932, Lorio 1986, Herms andMattson 1992) and carbon–nutrient balance (CNB,Bryant et al. 1983, 1988, 1993, Tuomi et al. 1984)hypotheses explain environmentally induced variationin both constitutive and herbivore-induced resistancewith variation in growth-limiting environmental fac-tors, such as soil fertility and light regime. Accordingto the CNB hypothesis, the balance between carbonand nutrients is changed in nutrient-limited plants,whose growth is limited more than photosynthesis,leading to the accumulation of carbon-based secondarymetabolites. These metabolites (e.g., phenolics) mayreduce plant quality as food for herbivores and thuscontribute to the resistance against herbivores (e.g.,Scriber and Slansky 1981). In plants not suffering fromnutrient limitation, the carbon is used for growth andresistance is decreased. The CNB hypothesis also pro-vides an explanation for DIR in deciduous plants. Thewithin-plant balance between carbon and nutrients isdisturbed by defoliation which removes more nutrientsthan carbon, again followed by the accumulation ofcarbon-based secondary metabolites and the expressionof DIR. Consequently, the CNB hypothesis predictsthat an increase in the availability of nutrients (e.g.,fertilization), coinciding with the defoliation mitigatesthe induction of resistance and, consequently, the her-bivores perform better. Shading, by decreasing theamount of carbon available for secondary metabolites,also decreases the level of induced resistance. Studiesthat have tested the CNB hypothesis in deciduous treesin relation to DIR have obtained contrasting results.For example, Bryant et al. (1993) found strong supportfor the CNB hypothesis as an explanation for DIR inBetula resinifera–Rheumaptera hastata system in Alas-ka, whereas the results of Ruohomaki et al. (1996) fromBetula pubescens ssp. tortuosa–Epirrita autumnatasystem do not seem to support the CNB hypothesis. Inaddition to the DIR observable in leaf chemistry andperformance of insect herbivores, the CNB hypothesishas been tested in relation to the resinous substanceson the bark of young B. pendula which have beenshown to determine the resistance against mammalian

herbivores (Rousi et al. 1991, Rousi et al. 1993). Thesestudies have also given contrasting results on the effectof fertilization on resistance: resistance against hareswas not decreased (Rousi et al. 1991) whereas resis-tance against voles was decreased due to fertilization(Rousi et al. 1993). Taken together, the explanatorypower of CNB hypothesis seems to depend on both theplant and herbivore species and group of secondarychemicals in question (Reichardt et al. 1991, Gershen-zon 1994, Hartley et al. 1995, Kyto et al. 1996). Further,most studies on CNB hypothesis in relation to inducedresistance have concentrated on the across-year induc-tion of resistance, i.e., on DIR (e.g., Bryant et al. 1993,Ruohomaki et al. 1996). However, Hunter and Schultz(1995) found out that fertilization prevented inductionin oak within one season, and suggested that the effectsof fertilization on induction of resistance may occur atmore than one temporal scale.

To test the relative role of genotype, defoliation andsoil fertility in determination of induced and consti-tutive plant resistance against herbivores we studiedthe effects of fertilization on resistance of cloned silverbirch (Betula pendula Roth) saplings against both in-sect and mammalian herbivores. As indicators of insectand mammalian resistance we conducted bioassays tomeasure the performance of a geometrid moth, Epirritaautumnata, and counted the amount of resin dropletson the shoot of the saplings, respectively (Rousi et al.1991). According to the CNB hypothesis, the resistanceof fertilized saplings is lower than that of unfertilizedsaplings and thus the herbivores should perform betteron fertilized saplings, all this providing that low nu-trient availability limits growth. Consequently, to con-firm that nutrients limited growth in our experimentalfield, we also measured the effect of fertilization onheight growth and total biomass of B. pendula. In thesame experimental design, we measured rapid inducedresistance (RIR) against an insect herbivore. Finally,we analyzed leaf secondary chemistry to investigatewhether the concentrations of secondary chemicals cor-related with the level of resistance measured using theperformance of E. autumnata.

METHODS

Plant material

We used commercially available Betula pendula sap-lings (Hortus-Puutarha Ltd., Kaarina, Finland) whichwere cloned using a tissue culture technique. We used15 clones originating from mother trees and crossesbetween trees growing in southern and central Finland(Table 1). The microcuttings were propagated duringthe winter of 1992. In the beginning of June 1992, themicrocuttings were individually planted into plastictrays filled with fertilized peat. For the following grow-ing season, the saplings were grown in a greenhouse.After overwintering in cold storage at 248C, the stillleafless saplings were transplanted to the experimental

January 2000 51HERBIVORE RESISTANCE IN BETULA PENDULA

TABLE 1. Origins of the Betula pendula clones used in the experiment.

CloneCommercial

code Origin CoordinatesSecondarychemistry

47

262840

728-4028-7828-26828-28828-40

ValkeakoskiPunkaharju 3 PunkaharjuLoppi 3 Rautalampi(Loppi 3 Rautalampi) 3 E/KKangasala 3 Nummi-Pusula

618089 N, 288499 E618439 N, 298259 E608399 N, 248259 E608309 N, 248429 E618239 N, 278009 E–608259 N, 238519 E

noyesyesyesyes

5560707980

928-55928-60928-70928-79928-80

KeuruuVarkausKeuruuPielavesiEno

618489 N, 298179 E618489 N, 298179 E618489 N, 298179 E638189 N, 268479 E628489 N, 308059 E

noyesnonoyes

84869093

124

928-84928-86928-90028-93028-124

PielavesiPielavesiEnoKangasalaKihnio

638189 N, 268479 E638189 N, 268479 E628489 N, 308059 E618239 N, 278009 E628089 N, 238189 E

yesyesnoyesyes

Notes: Clones with only one place of origin mentioned were derived from selected trees fromthese sites, and clones with two or three places of origin mentioned were crosses between trees.Coordinates show the growing site of the trees when they were selected for micropropagation.

field in Suonenjoki Research Station of the FinnishForest Research Institute on 15 May 1993.

Design of the experimental field

The saplings were planted to the experimental area(54 3 140 m2) according to a fully randomized blockdesign. The experimental field was divided into twoseparate subareas, each consisting of ten blocks (9 324 m2). One of the subareas was used for measurementsof height growth and biomass accumulation, and theother one was used in the bioassays with Epirrita au-tumnata and in the analyses of leaf chemistry.

The two subareas were divided into ten blocks witheach block constituting one replicate. To each of theten blocks, we planted 240 saplings, i.e., 16 saplingsfrom each of the 15 clones. The 16 saplings from eachclone were randomly divided to four subgroups. Thesaplings belonging to a subgroup were planted together,each sapling approximately 1 m apart from other sap-lings. Within each block, there were 60 marked loca-tions for the subgroups. The location for each subgroupwithin a block was randomized. Within each block,each subgroup consisting of the four saplings from thesame clone was randomly assigned to one of the fol-lowing treatments: (1) control (no fertilization, no de-foliation); (2) no fertilization 1 defoliation; (3) fertil-ization, no defoliation; (4) fertilization 1 defoliation.Within each block, the experiment was conducted as atwo-way factorial design, having fertilization and de-foliation as factors. Within each subgroup of the foursaplings, one of the saplings was randomly assignedfor measurements conducted in 1993 and one was ran-domly assigned for measurements to be conducted in1994. The two unassigned saplings were used for otherpurposes. Altogether, the experimental field included4800 saplings.

Fertilization, defoliation, and plant growth andbiomass

For the subgroups of four saplings assigned to thefertilization treatment, we applied NPK fertilizer (18:5:10, Typpirikas Y-lannos, Kemira Ltd., Helsinki, Fin-land) twice during both growing seasons. The fertilizerwas distributed around each sapling in an area 15 cmin diameter. By the end of 1993 and 1994 the fertilizedsaplings had received 6 and 12 g of fertilizer, respec-tively. The amount of fertilizer applied corresponds to12 kg N·ha21·yr21, 3.3 kg P·ha21·yr21, and 6.7 kgK·ha21·yr21.

Defoliation of the saplings was performed with scis-sors at the end of June in both study years. Thus thesaplings used in 1994 were defoliated only once. Weremoved 50% of the leaves. The defoliation was con-ducted in two phases: first we cut off half of the leaflamina from every second leaf and in the next morningremoved the rest of the leaf, including the petioles.

In the blocks assigned to the measurements of growthand biomass accumulation, the height of the saplingswas measured in the beginning and at the end of thegrowing season in both study years. The relative in-crease in height during the growing season was cal-culated as (L2 2 L1)/L1, where L1 5 length in the be-ginning and L2 5 length at the end of growing season.At the end of both growing seasons, we collected allabove- and belowground biomass of saplings assignedto the year in question. The collected plant parts wereoven dried (808C, 48 h) and weighed.

Bioassays and resin droplets

The saplings in the blocks assigned to the measure-ments of resistance and leaf chemistry were fertilizedand defoliated as described above. For both control anddefoliated saplings, we measured the resistance levels


of previously undamaged leaves in terms of the relativegrowth rate of the fourth instar larvae of a generalistgeometrid moth, Epirrita autumnata. It is known fromprevious studies that slow relative growth rate indicatespoor performance of E. autumnata (e.g., Ayres et al.1987, Haukioja et al. 1988). In both silver birch (Mu-tikainen et al. 1996) and mountain birch, Betula pu-bescens ssp. tortuosa (Haukioja and Niemela 1977,Haukioja 1980, Haukioja and Hanhimaki 1985, Han-himaki and Senn 1992), defoliation has been shown torapidly increase foliage resistance, measured as the rel-ative growth rate of E. autumnata.

Prior to the experiment, E. autumnata larvae werereared solitarily in plastic tubes (48 ml) on leaves ofmountain birch (Betula pubescens ssp. tortuosa). Thelarvae were randomly assigned to different treatments.For the bioassays, we collected two undamaged leavesfrom each sapling (i.e., from both control and defoli-ated ones) 24 h after conducting the defoliation. In thestatistical analyses, the mean of these two leaves wasused as a replicate. Each leaf was placed in a rearingtube with one weighed larva. An individual larva wasused only once. After 24 h at 138C, the larvae wereweighed again. We calculated the relative growth rate(RGR) of the larva as (mass gained [mg])·(initial bio-mass [mg])21·d21. The fresh mass of the larva was trans-formed to dry mass using the equation: dry mass 5exp(1.113 3 ln[fresh mass] 2 2.079) (Neuvonen andHaukioja 1984).

The relative growth rate of E. autumnata was mea-sured in both 1993 and 1994 using 1200 larvae eachyear. In 1993, the seedlings were sprayed with syper-metrine (0.05% solution, trade name Ripcord, KemiraLtd., Helsinki, Finland) to protect the saplings fromleaf-eating beetles attacking the experimental field. Thepesticide was applied 3 wk before the bioassays and,according to the information provided by the manu-facturer, we conducted the bioassays with E. autumnataclearly after the safety time for the pesticide. However,633 out of the 1200 larvae died during the 24 h growthexperiment. Since the larvae died in similar percent-ages from each treatment the results from 1993 mayunderestimate the responses due to the lower level ofreplication. In 1994, after finishing the 24 h bioassay,we continued rearing the larvae on the leaves of thesame saplings until they quit feeding and began to pu-pate. Two days after pupation, the pupas were weighed.

The number of resin droplets on the top of stems ofbirch has been shown to correlate with resistance tohares (Rousi et al. 1991). We calculated the number ofresin droplets from a 1 cm wide band around the stembetween the third and fourth lateral bud from the plantapex with a stereomicroscope using 2- to 4-fold mag-nification.

Analysis of leaf chemistry

Leaf secondary chemicals were analyzed in 1994using two leaves collected from the top of the saplings

in the blocks used for the bioassays. The leaves werecollected 2 d after the defoliation was conducted andair dried. For the analyses of leaf chemistry we usedten clones from eight experimental blocks (Table 1)and thus the number of replicates is eight. We analyzedaltogether 34 different secondary chemicals. In this pa-per, we only present the correlations of condensed tan-nins; total nontannin phenolics; flavonol glycosides;flavone aglycones; and 3,49-dihydroxypropiophenone3-glucoside (DHPPG) with the performance indices ofEpirrita autumnata. Condensed tannins and total non-tannin phenolics were chosen because they are oftenconsidered to reduce herbivore performance (e.g.,Feeny 1976, Scriber and Slansky 1981, Coley 1983,1986, Ayres et al. 1997). Flavonol glycosides are clas-sified into three groups based on their aglycone moi-eties: kaempferol, quercetin and myricetin glycosideshave one, two, or three adjacent hydroxyls, respec-tively, in the B-ring of the flavonoid basic structure.The number of vicinal hydroxyl groups corresponds tothe chemical and biological activity of the compound;thus kaempferol is often less active than quercetin andmyricetin (Elliger et al. 1980, Laks 1989). Based onthis knowledge on the biological activity of these com-pounds, we expected them to differ in their correlationswith the E. autumnata performance indices. DHPPGand flavone aglycones were chosen for the correlationsbecause their concentration increased due to defoliationin this experiment (Keinanen et al. 1999). The leaveswere extracted according to Keinanen and Julkunen-Tiitto (1996), and the concentrations of nontannin phe-nolics and DHPPG were analyzed by HPLC as in Kein-anen and Julkunen-Tiitto (1998). Condensed tanninswere analyzed by vanillin-HCl assay (Julkunen-Tiitto1985).

Statistical analysis

The data were analyzed as a mixed-model three-wayrandomized block analysis of variance having fertil-ization and defoliation as fixed effects and clone as arandom effect. The error terms were determined ac-cording to Zar (1984). The experiment was conductedaccording to a factorial design having 2 (fertilization)3 2 (defoliation) 3 15 (clones) 3 10 (blocks) 5 600saplings for both study years. In the blocks that wereused for the growth measurements, 23 saplings diedduring the first season. In the bioassay blocks, 7 sap-lings died during the first season. Due to the deaths ofE. autumnata larvae in the 1993 bioassay, the relativegrowth rate data of 1993 was analyzed without usingthe block as factor. The data were checked for normalityand homogeneity of variances. The data on the totalbiomass of the saplings for 1993 and 1994 were logtransformed to meet the assumptions of ANOVA. Wecalculated correlations of E. autumnata performancewith the concentrations of secondary chemicals usingPearson’s correlation coefficients. The correlationswere based on mean values for each clone. For the


TABLE 2. Results of analysis of variance for (A) height growth and (B) total plant biomass (log-transformed) at the end ofthe first (1993) and second (1994) growing season.

Effect df

1993

MS F P

1994

MS F P

A) Height growthBlockCloneDefoliation†Fertilization‡Defol. 3 Fertil.§Defol. 3 CloneFertil. 3 CloneDefol. 3 Fertil. 3 Clone

Error

914

111

141414

525

0.290.070.48

17.900.530.040.030.020.03

11.562.82

13.49531.29

24.201.391.320.86

0.0000.0000.0030.0000.0000.1510.1900.599

0.070.040.270.530.000.000.010.000.00

16.429.18

77.8898.44

0.000.831.310.55

0.0000.0000.0000.0000.9690.6390.1990.900

B) Total biomassBlockCloneDefoliation†Fertilization‡Defol. 3 Fertil.§Defol. 3 CloneFertil. 3 CloneDefol. 3 Fertil. 3 Clone

Error

914

111

141414

527

1.500.764.41

168.070.240.050.070.030.07

22.0411.1895.68

2365.397.490.681.050.48

0.0000.0000.0000.0000.0160.7950.4050.943

0.510.571.70

266.910.010.070.070.060.09

5.786.54

25.204055.20

0.110.770.750.64

0.0000.0000.0000.0000.7410.7060.7240.836

† Defoliation 3 Clone as error term.‡ Fertilization 3 Clone as error term.§ Defoliation 3 Fertilization 3 Clone as error term.

correlations, we calculated the magnitude of inductionas the difference between nondefoliated and defoliatedsaplings in RGR and pupal mass of E. autumnata, andin the amount of secondary chemicals.

RESULTS

Height growth and biomass of Betula pendula

In both 1993 and 1994, height growth and total bio-mass of Betula pendula increased due to fertilization(Table 2, Figs. 1 and 2), suggesting that nutrient avail-ability limited growth in our experimental field. Judg-ing from the mean squares and F values of the ANOVA,fertilization explained the highest amount of variancefor these traits in both study years (Table 2). In 1993,fertilized saplings had a total biomass of 28.96 6 0.64g (mean 6 1 SE) and their relative height growth was0.49 6 0.01. Nonfertilized saplings weighed 8.96 60.15 g and their relative height growth was 0.14 60.01. In 1994, the fertilized saplings weighed 111.656 1.99 g and their relative height growth was 0.28 60.00. Nonfertilized saplings weighed 28.50 6 0.53 gand their relative height growth was 0.22 6 0.00.

Contrary to the effects of fertilization, height growthand total biomass of Betula pendula were decreaseddue to defoliation (Table 2, Figs. 1 and 2). On average,in 1993, the relative height growth of control plantswas 0.35 6 0.02 and that of defoliated plants was 0.296 0.01. Undamaged control plants weighed 20.81 60.81 g whereas the defoliated plants weighed 17.11 60.66 g. In 1994, the relative height growth of controland defoliated saplings was 0.27 6 0.00 and 0.23 60.00, respectively. Undamaged control plants weighed

74.04 6 2.95 g and defoliated plants weighed 66.11 62.66 g.

In both study years, the differences in total biomassand relative height growth among the clones were sta-tistically significant (Table 2, Figs. 1 and 2). However,there were no significant interactions between cloneand fertilization or between clone and defoliation, sug-gesting that all clones responded similarly to both fer-tilization and defoliation in terms of biomass and rel-ative height growth (Table 2). In 1993, there was asignificant interaction between fertilization and defo-liation for both relative height growth and total biomass(Table 2, Fig. 3). Therefore, we tested the differencesbetween the defoliation treatments separately in fertil-ized and nonfertilized saplings. When fertilized, thedefoliated saplings grew slower than the control sap-lings (F1 5 22.92, P , 0.0001; Fig. 3). In the nonfer-tilized saplings, there was no significant difference inheight growth between control and defoliated saplings(F1 5 0.05, P 5 0.820; Fig. 3). The defoliated saplingsweighed less than the control saplings (nonfertilized:F1 5 18.99, P , 0.0001; fertilized: F1 5 24.16, P ,0.0001). These results suggest that the effects of de-foliation were relatively stronger in fertilized saplingscompared to nonfertilized saplings. In 1994, there wereno significant interactions for height growth or totalbiomass (Table 2).

Bioassays with Epirrita autumnata

For both the RGR and pupal mass of Epirrita au-tumnata fertilization explained the highest proportionof variance (Table 3, Figs. 4 and 5). RGR of E. au-


FIG. 1. Mean (61 SE) relative height growth of nonfertilized and fertilized Betula pendula saplings representing 15 clonesduring (A) the second growing season (1993) and (B) the third growing season (1994).

FIG. 2. Mean (61 SE) total biomass (g) of nonfertilized and fertilized Betula pendula saplings representing 15 clonesduring (A) the second growing season (1993) and (B) the third growing season (1994).


FIG. 3. Relative height growth and total biomass (mean6 1 SE) of nonfertilized and fertilized Betula pendula saplingsin the second growing season (1993).

tumnata larvae was significantly increased by fertil-ization in both growing seasons (Table 3, Fig. 4). In1993, RGR of larvae fed on leaves of fertilized saplingswas 0.21 6 0.02 and that of nonfertilized saplings was0.01 6 0.02. In 1994, the corresponding values were0.35 6 0.01 and 0.15 6 0.01, respectively. In addition,the pupal mass of E. autumnata was increased by fer-tilization (Table 3, Fig. 5); the pupae of larvae fed onleaves from fertilized saplings weighed on average75.58 6 0.66 mg whereas those fed on leaves fromnonfertilized saplings weighed 59.00 6 0.63 mg. Thusresistance against an insect herbivore decreased due tofertilization, i.e., the larvae grew better, in accordancewith the CNB hypothesis.

There were also significant differences among theclones in RGR and pupal mass of E. autumnata (Table3, Figs. 4 and 5). In terms of RGR and pupal mass, allclones responded similarly to fertilization and defoli-ation, indicated by the nonsignificant interaction termsin the ANOVA models (Table 3). However, there wasan almost significant (P 5 0.057) interaction betweendefoliation and fertilization for RGR in 1993 (Table 3,Fig. 6). There was no significant difference in the RGRbetween control and defoliation in nonfertilized sap-lings (F1 5 0.10, P 5 0.748; Fig. 6). In fertilized sap-lings, the larvae that fed on leaves from defoliatedsaplings tended to have slower RGR than larvae that

were fed on leaves from control saplings (F1 5 3.48,P 5 0.063; Fig. 6). These results suggest that in 1993,fertilization decreased the constitutive resistanceagainst an insect herbivore, but fertilized saplings wereable to respond to defoliation with rapid induced re-sistance. In 1994, the results are qualitatively similar,i.e., a decrease in RGR of larvae that were fed onfertilized saplings following defoliation (Fig. 6). How-ever, in 1994, the interaction between defoliation andfertilization for RGR of E. autumnata was not statis-tically significant (Table 3). Further, in 1994, the in-teraction between defoliation and fertilization for pupalmass was significant (Table 3, Fig. 6). However, theinteraction was not similar to that observed for RGRin 1993 (Fig. 6). In this case, in nonfertilized saplings,the pupal mass of larvae in the defoliation treatmentwas higher than the pupal mass of larvae in the controltreatment (F1 5 12.86, P , 0.0001). In fertilized sap-lings, there was no significant difference in pupal massbetween defoliated and control saplings (F1 5 0.25, P5 0.614).

Correlations of leaf chemistry with the performanceof E. autumnata

In order to determine the relationship between sec-ondary chemicals and E. autumnata performance, wecalculated Pearson’s correlation coefficients betweenRGR and pupal mass of E. autumnata, and the con-centrations of secondary chemicals. Since we used themean values of undefoliated saplings of each clone (n5 10), the correlations can be considered as genetic(e.g., Simms 1992). Furthermore, we calculated thecorrelations separately for nonfertilized and fertilizedsaplings.

In both nonfertilized and fertilized saplings, the con-centration of condensed tannins correlated negativelywith both the RGR and pupal mass of E. autumnata(Table 4, Figs. 7A, B). In nonfertilized saplings, theconcentration of total nontannin phenolics was posi-tively correlated with the RGR of E. autumnata (P ,0.10, Table 4, Fig. 7C). In fertilized saplings, the con-centration of kaempferol glycosides correlated signif-icantly positively with RGR whereas the correlation ofmyricetin glycosides with pupal mass was negative (P, 0.10, Table 4, Figs. 7D, E).

In fertilized saplings, the magnitude of induction inboth E. autumnata RGR and pupal mass were signif-icantly positively correlated with the concentration ofDHPPG (Table 4, Figs. 8A, B). Further, in nonfertilizedsaplings, the induction in RGR correlated positivelywith the induction in DHPPG (Table 4, Fig. 8C). Innonfertilized saplings, induction in pupal mass corre-lated significantly positively with the concentration oftotal nontannin phenolics (Table 4, Fig. 8D), suggestingthat nontannin phenolics promoted the induction of re-sistance measured in terms of difference in E. autum-nata pupal mass between defoliated and control sap-lings. Further, in fertilized saplings, the concentration


TABLE 3. Results of analysis of variance for (A) the relative growth rate (RGR) of Epirrita autumnata in the first (1993)and second (1994) growing season and for (B) the pupal mass in the second growing season (1994) and the number ofresin droplets at the end of the first growing season (1993).

A) RGR

Effect df

1993

MS FVar. expl.

(%)

1994

MS FVar. expl.

(%)

RGRBlockCloneDefoliation‡Fertilization§Defol. 3 Fertil.\Defol. 3 CloneFertil. 3 CloneDefol. 3 Fertil. 3 Clone

Error

914111

141414

315/527¶

0.170.092.980.130.670.570.280.11

1.58†1.24

49.29***4.30†0.670.570.28

6.68.1

77.923.5

2.92.51.2

0.030.110.086.130.050.020.030.020.02

1.615.62***3.37†

217.82***2.281.211.481.23

2.713.019.494.014.0

3.13.83.2

B) Resin droplets and pupal mass

Effect df

Resin droplets

MS FVar. expl.

(%)

Pupal mass

MS FVar. expl.

(%)

BlockCloneDefoliation‡Fertilization§Defol. 3 Fertil.\Defol. 3 CloneFertil. 3 CloneDefol. 3 Fertil. 3 Clone

Error

914111

141414

527/498#

1426.924672.041497.655957.775435.33705.28950.46562.75261.92

5.45***17.84***

2.126.27*9.66**2.69**3.63***2.15**

8.532.213.230.940.8

6.78.85.4

1035.48373.93579.94

36 766.911083.51130.67152.03167.50101.28

10.22***3.69***4.44†

241.84***6.47*1.291.501.65†

15.69.4

24.194.531.6

3.54.04.4

Note: The percentage of total variance explained (Var. expl.) was calculated as sseffect/(sseffect 1 sserror) and thus does not add upto 100%.

† P , 0.10, *P , 0.05, ** P , 0.01, *** P , 0.001.‡ Defoliation 3 Clone as error term.§ Fertilization 3 Clone as error term.\ Defoliation 3 Fertilization 3 Clone as error term.¶ The first number is the df for 1993; the second is the df for 1994.# The first number is the error df for resin droplets; the second is the error df for pupal mass.

of kaempferol glycosides correlated positively with in-duction in pupal mass, although this correlation wasonly marginally significant (P , 0.10, Table 4, Fig.8E). These results suggest that the concentration oftannins and myricetin glycosides may be significant forthe level of constitutive resistance whereas nontanninphenolics and DHPPG may be more important in theinduction of resistance.

Resistance against mammalian herbivores:resin droplets

The number of resin droplets differed significantlyamong clones and between fertilized and nonfertilizedsaplings whereas there was no significant differencebetween defoliated and control saplings (Table 3, Fig.9). The number of resin droplets was higher in fertilizedsaplings (58.28 6 1.09) than in nonfertilized saplings(51.84 6 1.30), contrary to the predictions of CNBhypothesis. However, there was a significant interactionbetween defoliation and fertilization for the number ofresin droplets (Table 3, Fig. 10). This interaction wasdue to significantly higher number of resin droplets in

fertilized and defoliated saplings than in fertilized con-trol saplings (F1 5 18.40, P , 0.0001) whereas innonfertilized saplings, the difference between controland defoliation was not significant (F1 5 1.29, P 50.257; Fig. 10). This interaction suggests that defoli-ation induced an increase in mammalian resistance butonly in fertilized saplings, which also had a higher levelof constitutive resistance against mammalian herbi-vores.

There were also significant interactions between de-foliation and clone, and fertilization and clone for thenumber of resin droplets (Table 3). In addition, thethree-way interaction among clone, defoliation and fer-tilization was significant (Table 3), suggesting that dif-ferent clones responded differently to defoliation andthat the responses also depended on the availability ofnutrients. Due to the significant three-way interactionwe tested the difference in number of resin dropletsbetween control and defoliated saplings separately foreach clone in both fertilization treatments. In nonfer-tilized saplings, defoliation did not significantly in-crease the number of resin droplets in any of the clones


FIG. 4. Mean (61 SE) relative growth rate of Epirrita autumnata larvae fed on leaves of nonfertilized or fertilized Betulapendula saplings representing 15 clones. Growth rate trials were conducted during (A) the second growing season of thesaplings (1993) and (B) the third growing season (1994).

FIG. 5. Mean (61 SE) pupal mass (mg) of Epirrita au-tumnata larvae reared on leaves of nonfertilized or fertilizedBetula pendula saplings representing 15 clones.

(P . 0.05 for all clones; Fig. 9). Clones 4, 7, 26, 60,90, and 124 responded differently to defoliation in thetwo fertilization treatments. In nonfertilized saplingsof clones 4 and 124, defoliation decreased the numberof resin droplets (P , 0.05 for both clones) whereasthere were no significant differences between defoli-ation and control in fertilized saplings (P . 0.05 forboth clones; Fig. 9). In clones 7, 26, 60 and 90, de-foliation significantly increased number of resin drop-lets in fertilized saplings (P , 0.05 for all clones),whereas there were no significant differences betweenthe defoliation and control in non-fertilized saplings ofthese four clones (P . 0.05 for all clones). Thus theseresults indicate that, on the average, the number of resindroplets increased due to fertilization and, in fertilizedsaplings, following defoliation, but that these responseswere highly determined by the genotype of the sapling.

DISCUSSION

Variation in plant quality as food for herbivores hasbeen explained by the growth-differentiation balance(GDB) hypothesis which predicts ‘‘that any environ-mental factor that limits growth more than photosyn-thesis can increase the resource pool available for al-location to secondary metabolism’’ (Herms and Matt-son 1992). In particular, the CNB hypothesis more ex-plicitly predicts that nutrient deficiency limits growthmore than photosynthesis, which leads to the accu-mulation of carbon-based secondary chemicals (e.g.,


FIG. 6. Mean (61 SE) relative growth rate (1993, 1994)and pupal mass (1994) of Epirrita autumnata larvae rearedon nonfertilized or fertilized Betula pendula saplings.

Bryant et al. 1983, 1988, 1993, Tuomi et al. 1984,1990). These secondary chemicals are known to affectplant resistance to herbivores (e.g., Scriber and Slansky1981). In our experimental field, height growth andbiomass accumulation of nonfertilized 2 year old Bet-ula pendula saplings were 30.9% and 28.6% of thoseof fertilized saplings, respectively. These results clearlyindicate that nutrient availability limited growth, andthus our experimental design fulfills the assumption ofnutrients as growth limiting factor inherent in the CNBhypothesis. However, our results on both insect andmammalian resistance are somewhat contradictory tothe CNB hypothesis.

Performance of an insect herbivore

Our results on insect resistance partly support theCNB hypothesis. The performance of Epirrita autum-nata larvae fed on leaves from fertilized saplings wasbetter compared to those fed on leaves from nonfer-tilized saplings. This suggests that the constitutive levelof resistance changed due to fertilization, in accordancewith the CNB hypothesis. In a previous experimentwith the E. autumnata–mountain birch system, the pu-pal mass was higher and developmental time was short-er in larvae which were fed on leaves of nitrogen fer-tilized trees (Ruohomaki et al. 1996). Thus, as in ourstudy, the constitutive resistance in mountain birchseemed to function according to the CNB hypothesis.However, in the study of Ruohomaki et al. (1996), theperformance of E. autumnata did not follow the pre-dictions of CNB hypothesis on DIR. It is interestingto notice that the effects of fertilization on DIR inmountain birch were similar to those we observed inRIR in silver birch in this experiment.

An alternative, though not mutually exclusive (Kar-ban and Baldwin 1997) hypothesis for the CNB hy-pothesis suggests that herbivore induced changes areactive defensive responses (Rhoades 1979, 1985, Hau-kioja 1980, Haukioja and Neuvonen 1985, Neuvonenand Haukioja 1991). This hypothesis produces two pre-dictions. First, if the induced responses are defensivethey should be buffered against changes in the avail-ability of nutrients. Second, increasing the availabilityof nutrients (or other limiting resource) should increasethe vigor of the plant and its ability to produce defen-sive compounds. This is also a prediction of the plantvigor hypothesis presented by Price (1991). In thisstudy, we observed rapid induced resistance only in thefertilized saplings, measured in terms of growth rateof E. autumnata. However, we observed no defoliation-induced decreases in the pupal mass, which is a directindicator of the effects of food quality on herbivoreperformance (Karban and Myers 1989, Haukioja 1990).Clearly these two measures indicate changes in leafquality in two different time scales: the bioassay tomeasure growth of E. autumnata was started 24 h afterthe defoliation whereas the pupal mass indicates leafquality for larvae that were reared on the leaves forabout 3 wk. Furthermore, if defoliation induced RIR,it is most likely that induction of control saplings alsooccured due to the collection of leaves both for thebioassay and for rearing the larvae for pupation. Con-sequently, we did not observe induction in pupal mass.Thus, in studies on rapid induced resistance, short termgrowth rate bioassays may be better indicators of leafquality.

To conclude, with respect to the constitutive resis-tance against an insect herbivore, our results supportthe CNB and GDB hypotheses. However, the resultson rapid induced resistance against an insect herbivoreare more consistent with the active defense hypothesis.


TABLE 4. Pearson correlation coefficients for correlations of Epirrita autumnata performancewith secondary chemicals separately for (A) nonfertilized and (B) fertilized saplings.

Chemical RGRPupalmass

Induction inRGR

Induction inpupal mass

A) NonfertilizedCondensed tanninsTotal nontannin phenolics

20.450.60†

20.80**0.49

0.220.39

20.500.65*

Flavonol glycosidesKaempferol glycosidesQuercetin glycosidesMyricetin glycosides

0.3820.18

0.4820.14

0.0520.13

0.2620.23

0.500.180.090.41

0.1020.08

0.3120.24

Flavone aglyconesInduction in flavone aglycones

0.10···

0.13···

20.320.27

0.0820.08

DHPPGInduction in DHPPG

20.20···

20.23···

20.030.69*

0.020.18

B) FertilizedCondensed tanninsTotal nontannin phenolics

20.61†0.24

20.520.05

0.2520.07

20.4420.17

Flavonol glycosidesKaempferol glycosidesQuercetin glycosidesMyricetin glycosides

20.490.66*

20.0320.42

20.4620.02

0.2120.58†

20.360.33

20.1420.18

20.480.57†

20.2520.19

Flavone aglyconesInduction in flavone aglycones

DHPPGInduction in DHPPG

20.17···

20.05···

0.42···

20.05···

20.070.450.80**0.36

0.310.300.75*0.24

Notes: Induction in RGR and pupal mass is the difference in RGR and pupal mass, respec-tively, between undefoliated and defoliated saplings. Induction in flavone aglycones andDHPPG is the difference in flavone aglycones and DHPPG, respectively, between defoliatedand undefoliated saplings. The correlations are based on mean values of each clone (n 5 10).

† P # 0.10, * P # 0.05, ** P # 0.01.

The significant differences among the clones in theirinsect resistance suggest genetic basis for the resistanceindicating that resistance can evolve as a response toinsect herbivory.

Correlations of leaf chemistry with E. autumnataperformance

The quality of leaves as food for herbivores may bedetermined by their concentration of secondary chem-icals as well as by their nutrient content. Fertilizationincreases nutritional quality of the host plant and thusbenefits herbivores (Ohmart et al. 1985, Bjorkman etal. 1991, Joseph et al. 1992, Mopper and Whitham1992, Potter 1992). Thus the relative contributions ofnutrients and secondary chemicals may be difficult todistinguish. In this experiment, the performance of E.autumnata larvae increased significantly if they werefed on foliage of fertilized saplings compared to larvaefed on foliage of unfertilized saplings (Fig. 3). In thissame experiment, the concentration of condensed tan-nins decreased due to fertilization whereas that of totalnontannin phenolics did not show any significantchanges due to the fertilization (Keinanen et al. 1999;Figs. 7 and 8). Thus the higher RGR and pupal massof E. autumnata may reflect either the decrease in con-densed tannins or the putative increase in leaf nitrogen.Unfortunately we did not measure the nitrogen con-centration of the foliage in this experiment. However,

the strong negative correlations between the concen-tration of condensed tannins and E. autumnata perfor-mance indices observed in both fertilization levels sug-gest that condensed tannins may play a significant rolein determining the resistance of B. pendula against aninsect herbivore. This result is in accordance with sev-eral previous studies (e.g., Rossiter et al. 1988, Hermsand Mattsson 1992, Bryant et al. 1993, Sagers andColey 1995, Ayres et al. 1997).

In our experiment, the concentration of condensedtannins was the only major group of secondary chem-icals analyzed showing a negative correlation with E.autumnata performance. Another generalized group ofsecondary metabolites, flavonol glycosides, showed nosignificant correlations with growth rate or pupal massof E. autumnata. However, when individual flavonolglycosides were analyzed, there was a significant pos-itive correlation between kaempferol glycosides andgrowth rate of E. autumnata larvae, whereas the cor-relation of pupal mass with myricetin glycosides wasnegative (Table 4). Similarly, in the study of Ruoho-maki et al. (1996), the negative relationship betweenherbivore performance and condensed tannins and totalphenolics was not perfect. This and other recent studiesthus clearly suggest that these generalized classes ofsecondary metabolites may be poor predictors of fo-liage quality for herbivores (Clausen et al. 1991, Neu-vonen and Haukioja 1991, Suomela et al. 1995, Ayres


FIG. 7. Correlations between secondary chemicals and Epirrita autumnata performance indices (RGR, pupal mass). Eachpoint represents one clone (n 5 10). Concentrations of secondary chemicals are in terms of dry mass.

et al. 1997). This may be due to the fact that the eco-logical consequences of a specific chemical compoundfor herbivores may be determined by its additive orsynergistic effects with other compounds (McKey1979, Berenbaum 1985). Further, instead of the abso-lute amounts, the relative proportions of different com-pounds may be of substantial ecological importance(Langenheim 1994). Finally, we analysed just a smallproportion of the myriad of secondary chemicals, eventhough the chemical assays we conducted were morecomprehensive than in many previous studies. Of thegroups of secondary chemicals not described here, forexample, terpenoids (Pokhilo and Uvarova 1988, Har-

borne 1997), papyriferic acid (e.g. Reichardt et al.1984, Bryant et al. 1987), and proteinase inhibitors(Niemela et al. 1979, Seldal et al. 1994) may affectfood choice and growth of herbivores feeding on Betulaspecies.

In this study, the constitutive level of insect resis-tance seems to be mainly determined by the concen-tration of condensed tannins whereas the induced re-sistance is determined by the constitutive level of small-molecular weight compounds, especially DHPPG. In astudy of Watanabe et al. (1990, as cited in Mori et al.1992), attack of Betula platyphylla var. japonica bygypsy moth (Lymantria dispar) increased the amount


FIG. 8. Correlations between secondary chemicals and Epirrita autumnata performance indices (induction in RGR andinduction in pupal mass). Induction was calculated as the difference in RGR and pupal mass, respectively, between defoliatedand control saplings within each treatment combination (clone–fertilization). Each point represents one clone (n 5 10).Concentrations of secondary chemicals are in terms of dry mass.

of DHPPG in leaves. Later, Mori et al. (1992) testedthe antifeedant properties of DHPPG against gypsymoth and found DHPPG to be a weak antifeedant. How-ever, since the concentration of DHPPG in B. platy-phylla is generally much lower than that in B. pendula(Keinanen, unpublished data), it is also likely that thetotal antifeedant effect of DHPPG is stronger in B.pendula. DHPPG is a vacuolar compound, whereas theother defoliation-induced group of secondary chemi-cals, flavonoid aglycones, is found on the surfaces ofbirch leaves (Keinanen and Julkunen-Tiitto 1998). Wealso calculated the correlations of the defoliation-in-duced chemicals (DHPPG and flavonoid aglycones)

with insect performance indices (RGR, pupal mass)using the defoliated saplings. Interestingly, none of thecorrelations were significant (P . 0.05 for all corre-lations). This suggests that the constitutive concentra-tions of these chemicals are more important predictorsof the induced response in insect performance than thedefoliation-induced increase in the concentrations ofthese chemicals per se.

Indicators of mammalian resistance: resin droplets

The resin droplets of some birch species (e.g., Betularesinifera, B. pendula) contain large amounts of pa-pyriferic acid which functions as a deterrent against


FIG. 9. Mean (61 SE) number of resin droplets at the tipof nonfertilized and fertilized Betula pendula saplings rep-resenting 15 clones. Resin droplets were calculated from a 1cm wide band around the stem between the third and fourthlateral bud from the plant apex.

FIG. 10. Mean (61 SE) number of resin droplets at thetip of nonfertilized and fertilized Betula pendula saplings.Resin droplets were calculated from a 1 cm wide band aroundthe stem between the third and fourth lateral bud from theplant apex.

mammalian herbivores (Reichardt et al. 1984, Bryantet al. 1987, Julkunen-Tiitto et al. 1996). Consequently,the number of resin droplets on the bark of birch seed-lings has been found to correlate negatively with thefeeding preference of hares (Rousi et al. 1991, Tah-vanainen et al. 1991). According to our results, thenumber of resin droplets increases due to fertilizationand, in fertilized saplings, following defoliation. Theeffect of fertilization is in accordance with the resultsof Rousi et al. (1991, 1993) who found that fertilizationincreased the number of resin droplets near the tips ofthe seedlings but did not have significant effect at thebase of the seedlings. We also checked the number ofresin droplets at the base of the saplings, and in ac-cordance with the results of Rousi et al. (1993), foundno differences between fertilized and nonfertilized sap-lings (P. Mutikainen, unpublished data).

The increase in the number of resin droplets due tofertilization (Rousi et al. 1991, 1993) is contrary to theCNB hypothesis. However, Bryant et al. (1987) foundthat fertilization decreased the concentration of papyr-iferic acid and increased the palatability of Alaskanpaper birch to hares consistent with the CNB hypoth-esis. Rousi et al. (1991, 1993) suggest that these con-troversial results are mainly caused by an age differ-ence between seedlings tested; the seedlings used inthe studies of Rousi et al. were 1 year old whereas theones Bryant et al. used were older (,3 m tall, ,3 cmin diameter at 1 m above the ground). To our knowl-

edge, the prediction that the effect of fertilization onthe amount of resin droplets depends on the age of theseedlings has not been tested explicitly. Alternatively,the differences in resin production within the saplingsmay be consequences of growth processes. If resin pro-duction is linked to primary growth, the number ofdroplets at the base of the seedling will be renderedwider apart, and they may also wither off during thegrowing season (Lapinjoki et al. 1991). Further, theepidermal cells that produce secondary metabolites inthe bark are often active only in the first year of in-ternode growth, because subsequent radial growth re-sults in their destruction (Esau 1965). Since the di-ameter growth occurs mainly at the base, the numberof resin droplets at the base of the sapling is likely tobe less affected, or even reduced, by fertilization thanthat at the top of the saplings.

Defoliation induced an increase in the number ofresin droplets in the fertilized saplings (Fig. 10). Thisresponse may be considered as delayed induced resis-tance since the resin droplets differentiated in one sea-son determine the resistance level during the followingwinter and thus describe across-year induction. Ourresults on resin droplets as an indicator of mammalianresistance thus contrast with the CNB hypothesis whichpredicts a weakened induction in fertilized saplings.These results seem to indirectly support the active de-fense hypothesis, parallel to the results on the inductionof resistance against an insect herbivore (Fig. 6). How-ever, the increase in resin droplets in defoliated saplingsmight have been caused by the corresponding decreasesin height growth, biomass accumulation, (Figs. 1A and2A) and especially in diameter growth (data not shown)which occurred only in fertilized saplings. Further, de-foliation mimics insect herbivory and thus an inductionin mammalian resistance seems to be poorly targeted.

Finally, the benefit of the increase in resin droplets


due to fertilization (12% increase) or defoliation (17%increase) to the seedling is equivocal. In a study ofRousi et al. (1993), hares did not differentiate betweenthe fertilization treatments even if the number of resindroplets at the tips of the fertilized seedlings was al-most 50% higher than that of unfertilized seedlings.

Effect of genotype vs. effect of fertilization

This experimental data shows that there are signif-icant differences among the clones of Betula pendulain resistance traits. However, for the insect performanceindices, fertilization explained a higher proportion ofvariance than genotype (Table 3). The substantial roleof fertilization in shaping resistance levels in this studymay partly be explained by the artificial nature of thefertilization treatment. The fertilization level chosenfor this experiment does not necessarily correspond tothe among-site differences that exist in nature and thusthe explanatory power of fertilization might not havebeen as substantial as observed if we had used a lowerfertilization level. The explanatory power of fertiliza-tion was substantial also for plant growth traits (Table2), indicating that fertilization clearly affected the per-formance of the experimental plants. The variationcaused by the defoliation and plant genotype may betterlie within the limits of natural variation and thus theireffects may be masked by the artificially strong effectof fertilization on variation in the resistance traits.

For resin droplets, used here as an index of mammalianresistance, the genotype explained a higher proportion ofvariance than fertilization (Table 3). Correspondingly,Rousi et al. (1991, 1993) found significant differences inthe number of resin droplets among families. We alsofound significant differences among the clones in theirmammalian resistance responses to fertilization and de-foliation. Our results indicate that the genetic variationin resistance against mammalian herbivores is substantial,even among clones originating from a restricted area insouthern and central Finland. Furthermore, even if thebackground level of mammalian resistance in B. pendulaseems to be genetically determined, environmental factorsmay contribute significantly to the phenotypic variationin resistance.

It is possible to separate the clones from each otherusing their chemical profile based on 34 secondary chem-icals analyzed (Keinanen et al. 1999). Further, in non-fertilized saplings, the most resistant clone was 34% moreresistant than the least resistant clone in terms of pupalmass of E. autumnata. For resin droplets, the correspond-ing value was almost 70%. Although these indices defi-nitely do not correspond one by one to the differences inresistance against herbivores, they indicate high variationin resistance among the genotypes. Coleman and Jones(1991) list aspects of plant genotype that determinewhether induction in resistance occurs. First, some ge-notypes simply do not posses the capacity for induction.Further, the threshold level of damage before inductionoccurs may differ among genotypes and may be affected

by environmental conditions. Constitutive resistance mayalso mask induced resistance. In relation to the resistanceagainst an insect herbivore, our results show that allclones showed similar responses to defoliation, i.e., theoccurrence of induction was not dependent on fertilizationlevel, whereas fertilization affected the overall level ofresistance. In terms of resin droplets, some clones wereinduced only when fertilized and some when not fertil-ized, suggesting that in addition to the genotype, the oc-currence of induction was modified by environmental fac-tors.

If herbivory is detrimental, and resistance to herbivoryis beneficial in terms of plant fitness, plant populationsshould evolve towards the highest level of resistance andloose genetic variation in resistance (Fisher 1930). How-ever, genetic variation in resistance traits within popu-lations has been observed in numerous studies (this study,Hanover 1966, Berenbaum et al. 1986, Zangerl and Ber-enbaum 1990, 1997, Han and Lincoln 1994, Sagers andColey 1995). One possible factor that constrains the evo-lution of high level of resistance are the negative geneticcorrelations between resistance traits and other plant func-tions, i.e., the costs of resistance (Feeny 1975, 1976, Ber-enbaum et al. 1986, Simms 1992, Han and Lincoln 1994,Sagers and Coley 1995, Zangerl and Berenbaum 1997).In this study, we have shown that simulated herbivory iscostly to the plants in terms of biomass accumulation orheight growth. Consequently, the next step is to inves-tigate whether costs limit the increase in resistance levelsin B. pendula.

We studied the resistance responses of B. pendulausing indicators of both insect and mammalian resis-tance. In conclusion, these and previous results suggestthat a single hypothesis on environmental effects is notcomprehensive enough to explain the phenotypic vari-ation in plant resistance traits even within a single plantspecies. On the other hand, a more comprehensive ex-planation for the phenotypic variation in resistance mayalso require that the role of natural enemies (such asherbivores) and other ecological factors (such as re-source availability) as factors imposing selection onresistance traits will be better understood.

ACKNOWLEDGMENTS

We are extremely grateful to Kaisa Siik, Maija Piitulainen,Suvi Vesanto, and Susanna Haapala for help in the field andin conducting the bioassays. Jukka Jokela, Rick Karban andan anonymous reviewer gave valuable comments on themanuscript. This study was funded by the Academy of Fin-land, Emil Aaltonen Foundation, and Ministry of Agricultureand Forestry.

LITERATURE CITED

Ayres, M. J., T. P. Clausen, S. F. MacLean, A. M. Redman,and P. B. Reichardt. 1997. Diversity of structure and an-tiherbivore activity in condensed tannins. Ecology 78:1696–1712.

Ayres, M. J., J. Suomela, and S. MacLean. 1987. Growthperformance of Epirrita autumnata (Lepidoptera: Geomet-ridae) on mountain birch: trees, broods, and tree 3 broodinteractions. Oecologia 74:450–457.


Berenbaum, M. 1985. Interactions among allelochemicals inplants. Pages 139–169 in G. Cooper-Driver, T. Swain, andE. Conn, editors. Chemically mediated interactions be-tween plants and other organisms. Plenum Press, NewYork, New York, USA.

Berenbaum, M. R., A. R. Zangerl, and J. K. Nitao. 1986.Constraints on chemical coevolution: wild parsnips and theparsnip webworm. Evolution 40:1215–1228.

Bjorkman, C., S. Larsson, and R. Gref. 1991. Effects ofnitrogen fertilization on pine needle chemistry and sawflyperformance. Oecologia 86:202–209.

Bryant, J. P., F. S. Chapin, III, and D. R. Klein. 1983. Carbon/nutrient balance of boreal plants in relation to vertebrateherbivory. Oikos 40:357–368.

Bryant, J. P., F. S. Chapin, III, P. B. Reichardt, and T. P.Clausen. 1987. Response of winter chemical defense inAlaska paper birch and green alder to manipulation of plantcarbon/nutrient balance. Oecologia 72:510–514.

Bryant, J. P., P. B. Reichardt, T. P. Clausen, and R. A. Werner.1993. Effects of mineral nutrition on delayed inducibleresistance in Alaska paper birch. Ecology 74:2072–2084.

Bryant, J., J. Tuomi, and P. Niemela. 1988. Environmentalconstraints of constitutive and long-term inducible defensesin woody plants. Pages 367–389 in K. C. Spencer, editor.Chemical mediation of coevolution. Academic Press, SanDiego, California, USA.

Clausen, T., P. Reichardt, J. Bryant, and R. Werner. 1991.Long-term and short-term induction in quaking aspen: re-lated phenomena? Pages 71–83 in D. W. Tallamy and M.J. Raupp, editors. Phytochemical induction by herbivores.John Wiley, New York, New York, USA.

Coleman, J. S., and C. G. Jones. 1991. A phytocentric per-spective of phytochemical induction by herbivores. Pages3–45 in D. W. Tallamy and M. J. Raupp, editors. Phyto-chemical induction by herbivores. John Wiley, New York,New York, USA.

Coley, P. D. 1983. Herbivory and defensive characteristicsof tree species in a lowland tropical forest. EcologicalMonographs 53:209–233.

Coley, P. D. 1986. Cost and benefits of defense by tanninsin a neotropical tree. Oecologia 70:238–241.

Elliger, C., B. Chan, and A. Waiss Jr. 1980. Flavonoids aslarval growth inhibitors. Structural factors governing tox-icity. Naturwissenschaften 67:358–360.

Esau, K. 1965. Plant anatomy. John Wiley, New York, NewYork, USA.

Feeny, P. 1975. Biochemical coevolution between plants andtheir insect herbivores. Pages 3–19 in L. H. Gilbert and P.H. Raven, editors. Coevolution of animals and plants. Uni-versity of Texas Press, Austin, Texas, USA.

Feeny, P. 1976. Plant apparency and chemical defense. Pages1–40 in J. W. Wallace and R. L. Mansell, editors. Bio-chemical interactions between plants and insects. PlenumPress, New York, New York, USA.

Fisher, R. 1930. The genetical theory of natural selection.Oxford University Press, Oxford, UK.

Fritz, R. S. 1990. Effects of genetic and environmental vari-ation on resistance of willow to sawflies. Oecologia 82:325–332.

Gershenzon, J. 1994. The cost of plant chemical defenseagainst herbivory: a biochemical perspective. Pages 105–173 in E. A. Bernays, editor. Insect plant interactions. CRCPress, Boca Raton, Florida, USA.

Han, K., and D. E. Lincoln. 1994. The evolution of carbonallocation to plant secondary metabolites: a genetic analysisof cost in Diplacus aurantiacus. Evolution 48:1550–1563.

Hanhimaki, S., and J. Senn. 1992. Sources of variation inrapidly inducible responses to leaf damage in the mountainbirch-insect herbivore system. Oecologia 91:318–331.

Hanover, J. 1966. Genetics of terpenes. I. Gene control of

monoterpene levels in Pinus monticola Dougl.. Heredity21:73–84.

Harborne, J. B. 1997. Plant secondary metabolism. Pages132–155 in M. J. Crawley, editor. Plant ecology. BlackwellScience, Oxford, UK.

Hartley, S. E., K. Nelson, and M. Gorman. 1995. The effectsof fertilizer and shading on plant chemical composition andpalatability to Orkney voles, Microtus arvalis orcadensis.Oikos 72:79–87.

Haukioja, E. 1980. On the role of plant defences in the fluc-tuation of herbivore populations. Oikos 35:202–213.

Haukioja, E. 1990. Induction of defenses in trees. AnnualReview of Entomology 36:25–42.

Haukioja, E., and S. Hanhimaki. 1985. Rapid wound-inducedresistance in white birch (Betula pubescens) foliage to thegeometrid Epirrita autumnata: a comparison of trees andmoths within and outside the outbreak range of the moth.Oecologia (Berlin) 65:223–228.

Haukioja, E., and S. Neuvonen. 1985. Induced long-termresistance of birch foliage against defoliators: defensive orincidental? Ecology 66:1303–1308.

Haukioja, E., S. Neuvonen, S. Hanhimaki, and P. Niemela.1988. The autumnal moth in Fennoscandia. Pages 163–178in A. Berryman, editor. Dynamics of forest insect popu-lations. Patterns, causes, and implications. Plenum Press,New York, New York, USA.

Haukioja, E., and P. Niemela. 1977. Retarded growth of ageometrid larva after mechanical damage to leaves of itshost tree. Annales Zoologici Fennici 14:48–52.

Herms, D. A., and W. J. Mattson. 1992. The dilemma ofplants: to grow or defend. Quarterly Review of Biology67:283–335.

Hunter, M. D., and J. C. Schultz. 1995. Fertilization miti-gates chemical induction and herbivore responses withindamaged oak trees. Ecology 76:1226–1232.

Joseph, G., R. Kelsey, A. Moldenke, J. Miller, R. Berry, andJ. Wernz. 1992. Effects of nitrogen and Douglas-fir alle-lochemicals on development of the Gypsy moth, Lymantriadispar. Journal of Chemical Ecology 19:1245–1263.

Julkunen-Tiitto, R. 1985. Phenolic constituents in the leavesof northern willows: methods for the analysis of certainphenolics. Journal of Agricultural and Food Chemistry 33:213–217.

Julkunen-Tiitto, R., M. Rousi, J. Bryant, S. Sorsa, M. Keina-nen, and H. Sikanen. 1996. Chemical diversity of severalBetulaceae species: comparison of phenolics and terpen-oids in northern birch stems. Trees 11:16–22.

Karban, R. 1992. Plant variation: its effects on populationsof herbivorous insects. Pages 195–215 in R. S. Fritz andE. L. Simms, editors. Plant resistance to herbivores andpathogens. Ecology, evolution, and genetics. The Univer-sity of Chicago Press, Chicago, Illinois, USA.

Karban, R. and I. T. Baldwin. 1997. Induced responses toherbivory. The University of Chicago Press. Chicago, Il-linois, USA.

Karban, R., and J. H. Myers. 1989. Induced plant responsesto herbivory. Annual Review of Ecology and Systematics20:331–348.

Keinanen, M., and R. Julkunen-Tiitto. 1996. Effect of samplepreparation method on birch (Betula pendula Roth) leafphenolics. Journal of Agricultural and Food Chemistry 44:2724–2727.

Keinanen, M., and R. Julkunen-Tiitto. 1998. High-perfor-mance liquid chromatographic determination of flavonoidsin Betula pendula and Betula pubescens leaves. Journal ofChromatography A 793:370–377.

Keinanen, M., R. Julkunen-Tiitto, P. Mutikainen, M. Walls,J. Ovaska, and E. Vapaavuori. 1999. Trade-offs in phenolicmetabolism of silver birch: effects of fertilization, defoli-ation, and genotype. Ecology 80:1970–1986.


Kennedy, G., and J. Barbour. 1992. Resistance variation innatural and managed systems. Pages 13–41 in R. S. Fritzand E. L. Simms, editors. Plant resistance to herbivoresand pathogens. Ecology, evolution, and genetics. The Uni-versity of Chicago Press, Chicago, Illinois, USA.

Kyto, M., P. Niemela, and S. Larsson. 1996. Insects on trees:population and individual response to fertilization. Oikos75:148–159.

Laks, P. E. 1989. Chemistry of the condensed tannin B-ring.Pages 249–263 in R. W. Hemingway and J. J. Karchesy,editors. Chemistry and significance of condensed tannins.Plenum Press, New York, New York, USA.

Langenheim, J. 1994. Higher plant terpenoids: a phytocentricoverview of their ecological roles. Journal of ChemicalEcology 20:1223–1280.

Lapinjoki, S. P., H. A. Elo, and H. T. Taipale. 1991. Devel-opment and structure of resin glands on tissues of Betulapendula Roth during growth. New Phytologist 117:219–223.

Loomis, W. E. 1932. Growth-differentation balance vs. car-bonhydrate-nitrogen ratio. Proceedings of the AmericanSociety of Horticultural Science 29:240–245.

Lorio, P. L., Jr. 1986. Growth-differentation balance: a basisfor understanding southern pine beetle-tree interactions.Forest Ecology and Management 14:259–273.

Maddox, G., and N. Cappuccino. 1986. Genetic determi-nation of plant susceptibility to an herbivorous insect de-pends on environmental context. Evolution 40:863–866.

Maddox, G., and R. Root. 1987. Resistance to 16 diversespecies of herbivorous insects within a population of gold-enrod, Solidago altissima: genetic variation and heritability.Oecologia 72:8–14.

Marquis, R. 1992. Selective impact of herbivores. Pages301–325 in R. S. Fritz and E. L. Simms, editors. Plantresistance to herbivores and pathogens. Ecology, evolution,and genetics. The University of Chicago Press, Chicago,Illinois, USA.

McKey, D. 1979. The distribution of secondary compoundswithin plants. Pages 55–133 in G. A. Rosenthal and M. R.Berenbaum, editors. Herbivores: their interaction with sec-ondary plant metabolites. Volume II. Ecological and evo-lutionary processes. Academic Press, Orlando, Florida, USA.

Mopper, S., and T. G. Whitham. 1992. The plant stress par-adox: Effects in pinyon sawfly sex ratios and fecundity.Ecology 73:515–525.

Mori, K., Z.-H. Qian, and S. Watanabe. 1992. Synthesis of 3,49-dihydroxypropiohenone 3-b-D-glucopyranoside, a constituentof Betula platyphylla var. japonica, by enzymatic transglu-cosylation. Liebigs Annalen der Chemie485–487.

Mutikainen, P., M. Walls, and J. Ovaska. 1996. Herbivore-induced resistance in Betula pendula: the role of plant vas-cular architecture. Oecologia 108:723–727.

Neuvonen, S., and E. Haukioja. 1984. Low nutritive qualityas defence against herbivores: induced responses in birch.Oecologia 63:71–74.

Neuvonen, S., and E. Haukioja. 1991. The effects of induc-ible resistance in host foliage on birch-feeding herbivores.Pages 277–291 in D. W. Tallamy and M. J. Raupp, editors.Phytochemical induction by herbivores. John Wiley, NewYork, New York, USA.

Niemela, P., E. A. Aro, and E. Haukioja. 1979. Birch leavesas a resource for herbivores. Damage-induced increase inleaf phenols with trypsin-inhibiting effects. Reports fromthe Kevo Subarctic Research Station 15:37–40.

Ohmart, C., L. Stewart, and J. Thomas. 1985. Effects of foodquality, particularly nitrogen concentrations, of Eucalyptusblakelyi foliage on the growth of Paropsis atomaria larvae(Coleoptera: Chrysomelidae). Oecologia 65:543–549.

Pokhilo, N. D., and N. I. Uvarova. 1988. Isoprenoids ofvarious species of the genus Betula. Chemistry of NaturalCompounds 24:273–285.

Potter, D. 1992. Abundance and mortality of a specialistleafminer in response to experimental shading and fertil-ization of American holly. Oecologia 91:14–22.

Price, P. W. 1991. The plant vigor hypothesis and herbivoreattack. Oikos 62:244–251.

Reichardt, P. B., J. P. Bryant, T. P. Clausen, and G. D. Wieland.1984. Defence of winter-dormant Alaska paper birchagainst snowshoe hares. Oecologia 65:58–69.

Reichardt, P. B., F. S. Chapin, III, J. P. Bryant, B. R. Mattes,and T. P. Clausen. 1991. Carbon/nutrient balance as a pre-dictor of plant defense in Alaskan balsam poplar: Potentialimportance of metabolite turnover. Oecologia 88:401–406.

Rhoades, D. F. 1979. Evolution of plant chemical defenseagainst herbivores. Pages 3–54 in G. A. Rosenthal and M. R.Berenbaum, editors. Herbivores: their interaction with sec-ondary plant metabolites. Volume II. Ecological and evolu-tionary processes. Academic Press, Orlando, Florida, USA.

Rhoades, D. 1985. Offensive-defensive interactions betweenherbivores and plants: their relevance in herbivore popu-lation dynamics and ecological theory. American Naturalist125:205–238.

Rossiter, M. C., J. C. Schultz, and I. T. Baldwin. 1988. Re-lationships among defoliation, red oak phenolics and gypsymoth growth and reproduction. Ecology 69:267–277.

Rousi, M., J. Tahvanainen, H. Henttonen, and I. Uotila. 1993.Effects of shading and fertilization on resistance of winter-dormant birch (Betula pendula) to voles and hares. Ecology74:30–38.

Rousi, M., J. Tahvanainen, and I. Uotila. 1991. A mechanismof resistance to hare browsing in winter-dormant Europeanwhite birch (Betula pendula). American Naturalist 137:64–82.

Ruohomaki, K., F. S. Chapin, III, E. Haukioja, S. Neuvonen,and J. Suomela. 1996. Delayed inducible resistance inmountain birch in response to fertilization and shade. Ecol-ogy 77:2302–2311.

Sagers, C., and P. Coley. 1995. Benefits and costs of defensein a neotropical shrub. Ecology 76:1835–1843.

Scriber, J., and F. J. Slansky. 1981. The nutritional ecologyof immature insects. Annual Review of Entomology 26:183–211.

Seldal, T., E. Dybwad, K.-J. Andersen, and G. Hogstedt.1994. Wound-induced proteinase inhibitors in grey alder(Alnus incana): a defence mechanism against attacking in-sects. Oikos 71:239–245.

Simms, E. 1992. Costs of plant resistance to herbivory. Pages392–425 in R. S. Fritz and E. L. Simms, editors. Plantresistance to herbivores and pathogens. Ecology, evolution,and genetics. The University of Chicago Press, Chicago,Illinois, USA.

Suomela, J., V. Ossipov, and E. Haukioja. 1995. Variationin foliage phenols, carbohydrates, and amino acids, andgrowth of Epirrita autumnata larvae is distributed differ-ently among and within mountain birch trees. Journal ofChemical Ecology 21:1421–1446.

Tahvanainen, J., R. Julkunen-Tiitto, M. Rousi, and P. Reichardt.1991. Chemical determinants of resistance in winter-dormantseedlings of European white birch (Betula pendula) to brows-ing by the mountain hare. Chemoecology 2:49–54.

Tuomi, J., P. Niemela, E. Haukioja, S. Siren, and S. Neuvonen.1984. Nutrient stress: an explanation for anti-herbivoreresponses to defoliation. Oecologia 61:208–210.

Tuomi, J., P. Niemela and S. Siren. 1990. The Panglossianparadigm and delayed inducible accumulation of foliar phe-nolics in mountain birch. Oikos 59:399–410.

Zangerl, A., and M. Berenbaum. 1997. Cost of chemicallydefending seeds: furanocoumarins and Pastinaca sativa.American Naturalist 150:491–504.

Zangerl, A. R., and M. R. Berenbaum. 1990. Furanocou-marin induction in wild parsnip: genetics and populationalvariation. Ecology 71:1933–1940.

Zar, J. H. 1984. Biostatistical analysis. Prentice-Hall, En-glewood Cliffs, New Jersey, USA.

HERBIVORE RESISTANCE IN BETULA PENDULA : EFFECT OF FERTILIZATION, DEFOLIATION, AND PLANT GENOTYPE

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