OIKOS 98: 437–446, 2002

Developmental plasticity in birch leaves: defoliation causes a shiftfrom glandular to nonglandular trichomes

Pasi Rautio, Annamari Markkola, Jocelyn Martel, Juha Tuomi, Esa Harma, Karita Kuikka, Annika Siitonen,Iola Leal Riesco and Marja Roitto

Rautio, P., Markkola, A., Martel, J., Tuomi, J., Harma, E., Kuikka, K., Siitonen, A.,Riesco, I. L. and Roitto, M. 2002. Developmental plasticity in birch leaves: defolia-tion causes a shift from glandular to nonglandular trichomes. – Oikos 98: 437–446.

The structures on leaf surfaces, e.g. trichomes, can act as effective antiherbivorymechanisms as chemical repellents. Structural defences usually represent constitutiveresistance, but there are also a few cases of inducible morphological defences. Wetested whether defoliation may induce changes in trichome production in white birch(Betula pubescens). The studied birches were either 0, 50 or 100% defoliated duringthe previous or current summer, and we measured the alterations in the productionof glandular vs. nonglandular leaf trichomes, developmental instability (fluctuatingasymmetry, FA) and leaf and shoot growth. We detected a clear shift from glandularto nonglandular leaf trichomes following previous-year defoliation but not aftercurrent-year defoliation. Furthermore, the density of nonglandular trichomes aroundthe mid-vein of leaves increased following previous-year defoliation but decreasedafter current-year defoliation. While leaf and shoot growth showed a distinct decreasein response to defoliation, FA turned out to be less sensitive. Consequently, previous-year defoliation can induce the production of nonglandular trichomes in birch leaves.Because this response was accompanied by a reduction in glandular trichomes, thepresent results may suggest a trade-off between the different trichome types of birchleaves.

P. Rautio, A. Markkola, J. Tuomi, E. Harma, K. Kuikka, A. Siitonen, M. Roitto,Uni�. of Oulu, Dept of Biology, P.O. Box 3000, FIN-90014, Oulu, Finland(pasi.rautio@oulu.fi). – J. Martel, Uni�. of Turku, Dept of Biology, FIN-20014, Turku,Finland. – I. L. Riesco, Uni�. of Barcelona, Dept of Biology, A�. Diagonal 645,ES-08028, Barcelona, Catalonia, Spain.

Plants have diverse strategies for coping with herbivory.Foraging herbivores must first deal with the defencemechanisms associated with the leaf surface. For in-stance, leaf pubescence might be an equally effectiverepellent as chemical defences for small insect herbi-vores. Leaf pubescence provides a mechanical barrierthat hinders insects from effectively moving and feedingdue to the presence of hairs (i.e. trichomes) on thesurface (Levin 1973, Stipanovic 1983, Southwood 1986,Baur et al. 1991). Furthermore, some plant species haveglandular trichomes with excretory cells that may pro-duce repellent volatile compounds (Duffey 1986) orsticky substances (Stipanovic 1983). Some species, such

as Arabidopsis thaliana, possess both mechanical andchemical defences, containing both trichomes and toxicglucosinolates (Mauricio and Rausher 1997). In addi-tion to providing a mechanical barrier against herbi-vores, leaf pubescence protects against drought stress(Korner 1999) and heat load by decreasing light ab-sorbance (Ehleringer et al. 1976).

The plant allocation theory assumes trade-offs be-tween alternative defence mechanisms (Berenbaum etal. 1986, Tuomi et al. 1989a, Bjorkman and Anderson1990, Mauricio and Rausher 1997) and between defenceand life-history traits (i.e. defence costs, Rhoades 1979,Herms and Mattson 1992, Mutikainen and Walls 1995).

Accepted 28 February 2002

Copyright © OIKOS 2002ISSN 0030-1299

OIKOS 98:3 (2002) 437

Because the allocation theory assumes a constantamount of resources to be available for distributionbetween the alternative demands for the limited re-sources (Tuomi et al. 1982), no trade-offs betweendefence mechanisms (Steward and Keeler 1988, Mauri-cio et al. 1997) or costs (Simms and Rausher 1987,A� gren and Schemske 1993, Karban 1993) may be de-tected when this assumption is violated. For instance,A� gren and Schemske (1993) noticed that high-trichomeindividuals of Brassica rapa produced more flowers,although their flowering was delayed compared to low-trichome plants. Agrawal (2000) noticed that, in Lepid-ium �irginicum, induced plant defence (trichome densityand chemical compounds) caused an allocation shiftfrom root growth to aboveground shoot growth, butthere was no total biomass loss due to defence invest-ments. On the other hand, Elle et al. (1999) suggestedthat the fitness cost of glandular trichomes in Daturawrightii is due to pleiotropic effects of the gene codingfor glandular trichome production. In some cases, hairi-ness has been reported to increase susceptibility toherbivores (Webster 1975, Southwood 1986), and herbi-vores may have evolved counter-adaptations to copewith plant trichomes (Rathcke and Poole 1975, see alsoSiebert 1975, Hulley 1988).

In birches, chemical resistance against insect andmammalian herbivores has been widely studied (Tah-vanainen et al. 1991, Ossipov et al. 1997, Kause et al.1999), but less attention has been paid to the mechani-cal and structural defence of birch leaves. Birch specieshave two main types of trichomes – multicellular glan-dular trichomes and uni- or multicellular m onopodialnonglandular trichomes (cf. Esau 1953). On the stemsof young shoots of silver birch (Betula pendula), resinglands originating from multicellular glandular tri-chomes contribute to resistance against browsing mam-mals, e.g. the hare (Tahvanainen et al. 1991, Rousi etal. 1991). The functional importance of shoot and leafhairs in white birch (Betula pubescens Ehrh.) is un-known, but it can be assumed that if they participate inresistance, their targets would be rather insect herbi-vores than mammals. For instance, leaf margin hairi-ness may adversely affect leaf-chewing insects, such asmoth caterpillars, which usually start feeding from theleaf margin (cf. Hulley 1988). Furthermore, there aregood reasons to expect a trade-off between the differenttypes of trichomes in birch leaves. Because leaf epider-mal cells can differentiate into either glandular or nong-landular trichomes (Glover 2000), both genetic andenvironmental variation enhancing the development ofone leaf hair type may simultaneously reduce the devel-opment of the other type.

In the present study, we tested whether differentdefoliation histories and levels of leaf removal mayinduce developmental and morphological changes inthe leaves of white birch seedlings grown in a commongarden experiment. For that purpose, we studied leaf

hair production and fluctuating asymmetry, both dur-ing the same growing season and one year after thedefoliation treatment. We consider leaf hairs as poten-tial defensive traits of birch leaves and hence expecteddefoliation to induce an increase in leaf morphologicaldefences. Most morphological defences usually repre-sent constitutive resistance, but there is also some evi-dence of inducible morphological defences (Myers 1987,Baur et al. 1991, Karban and Baldwin 1997, Agrawal2000). We also tested whether defoliation affects glan-dular and nonglandular trichomes differently, as itcould be expected, provided that there is a trade-offbetween the different trichome types. In addition to leafhair production, previous defoliation may affect birchgrowth, leaf size (e.g. Tuomi et al. 1988), phenology(Tuomi et al. 1989b) and developmental stability. Inorder to quantify developmental instability, we mea-sured leaf fluctuating asymmetry (FA), i.e. the magni-tude of random deviations from the symmetricalbilateral shape of the leaf, which has been shown toincrease as a consequence of herbivory (Martel et al.1999) and plant stress (Kozlov et al. 1996, Wilsey et al.1998, Lappalainen et al. 2000). We therefore expectedthat defoliation may adversely affect leaf developmentand hence increase deviations from the bilateral symme-try of birch leaves.

Material and methods

Experimental design

Nursery-grown, one-year-old white birch (Betulapubescens) seedlings were planted in an experimentalfield of the Botanical Gardens of the University of Oulu(65°00� N, 25°30� E) in mid-June 1997. They weregrown in a mixture of nutrient-poor peat and mineralsoil, limed and fertilised with NPK (7 years before theexperiment). The seedlings were transplanted at inter-vals of 0.5 m in rows distanced 1 m apart. In thefollowing summer (1998), the seedlings were randomlydistributed into the following five treatments, with 20seedlings per treatment: (1) control (no leaf removal),(2) 50% of leaves per seedling removed (cut from thepetiole with scissors) during the summer 1998, (3) 100%of leaves removed in 1998, (4) 50% of leaves removedduring the summer 1999, and (5) 100% leaves removedin 1999. Defoliation was carried out at the end of Junein both years, when the early leaves (short-shoot leavesand basal leaves of long shoots) were mature (seeMacdonald and Mothersill 1983, Macdonald et al.1984). The seedlings produced new leaves on longshoots during the growing season. According to thetreatments, 50% or 100% of these late leaves wereremoved after 2 weeks (mid-July) and 7 weeks (lateAugust) after the first defoliation in June (Fig. 1).

438 OIKOS 98:3 (2002)

Leaf hair measurements

Leaf hairs (glandular and nonglandular trichomes) werecounted from long-shoot leaves sampled twice. The firstsampling was carried out before the first defoliation inthe summer 1999 (late June, Fig. 1) from seedlings thathad been 50% and 100% defoliated in 1998 and fromseedlings that had not yet been defoliated. We used theseedlings of treatment 5 (100% defoliation in 1999) as acontrol in this comparison, to avoid removing leavesfrom the control seedlings of treatment 1. The sampledleaves in the first sampling were the lowest leaves in thelong shoots (i.e. early leaves). The second sampling wascarried out before the last defoliation in 1999 (lateAugust) simultaneously with the leaf collection for theFA measurements described below (see Fig. 1). Thesampled leaves in the second sampling were matureleaves at the tips of long shoots (i.e. late leaves formedafter the defoliations in 1999). For the leaf hair mea-surements, 5 leaves from 6 seedlings per treatment werecollected in the first sampling, and 5 leaves from 7seedlings per treatment were collected in the secondsampling. The sampled leaves were kept frozen (−20°C) until the measurement.

Leaf hairs were counted under a light microscope at40× magnification. Leaf margin hairs (nonglandulartrichomes) were counted at the widest section of theleaf along a distance of 1.5 mm (1500 �m) and fromboth the left and the right margins. Leaf margin hairi-ness was calculated as the mean of the left and rightmargins. Leaf trichomes and glandular hairs on theupper side of each leaf were counted at the widestsection of the leaf from an area of 4.5 mm2 (4.5×106

�m2), half of which was situated on either the left or theright side of the mid-vein. The mid-vein thus ran acrossthe middle of the examined field. Trichomes and glan-dular hairs on the lower side of the leaves were countedin a similar way. The mid-vein on the lower side of theleaves occasionally had a ‘‘woolly’’ appearance due tosmall hairs. These hairs could not be counted at theabove magnification if they were abundant. Hence,these small ‘‘mid-vein hairs’’ were counted from asmaller area (40×103 �m2) at larger magnification ifnecessary. Furthermore, for every studied leaf, thelength (from the leaf tip to the borderline of the lamina/petiole) and the width (widest section of the lamina) ofthe leaf were recorded.

FA measurements

Before the third defoliation in 1999 (late August, cf.Fig. 1), 10 long-shoot leaves and 10 short-shoot leavesper seedling were collected for FA measurements from20 control seedlings, 20 seedlings 50% defoliated and 20seedlings 100% defoliated in 1999 (treatments 1, 4 and5). Two seedlings from the control treatment wereexcluded from further analyses because most of thecollected leaves were twisted during the drying process(see the procedure below). The sampled long-shootleaves had grown after the first and second defoliationsduring 1999 (Fig. 1). We sampled the largest leaf in themost distal third of the current-year long shoot, whichwas usually the third or fourth leaf from the apex of thelong shoot. The sampled short-shoot leaves, on theother hand, had a different developmental history foreach treatment. In the 100% defoliated seedlings, short-shoot leaves had grown after the defoliations, whereasin the control seedlings and the 50% defoliatedseedlings, the short-shoot leaves were early leavesformed at the beginning of the growing season. Fluctu-ating asymmetry (FA) was measured from pressed anddried leaves. All the studied leaves were measured byone of us (A.S.) by recording the width of the right (R)and left (L) leaf halves (from the mid-vein to the leafmargin) to the nearest mm at the mid-point of the leafmid-vein.

The absolute FA was computed as the absolute rightminus left value �R−L�, and the size-corrected FA as�R−L�/[(R+L)/2] (Palmer and Strobeck 1986). Tofurther study the effect of leaf size on FA, we computedestimated marginal means where absolute FA was ad-justed for covariate (leaf width=R+L). The presenceof antisymmetry or directional asymmetry can impedethe detection of fluctuating asymmetry. Hence, weanalysed the presence of directional asymmetry by us-ing one-sample t-test (seedling-specific R−L meansagainst zero) and the presence of antisymmetry bytesting the normality of the R−L values (Palmer andStrobeck 1986). The presence of antisymmetry anddirectional asymmetry was analysed separately for eachtreatment and for both long and short shoots. TheShapiro-Wilk test revealed no significant deviation ofthe R−L values from normality (p=0.119–0.658), i.e.we did not detect any signs of antisymmetry. In all butone treatment-shoot type combination (p=0.042), nocases of directional asymmetry were found (p=0.087–1.0). Re-measurement of 122 leaves verified the preci-sion of the FA measurements: the intraclass correlationbetween the two measurements was 0.76 (Zar 1996).

Growth parameters

The growth of all seedlings was measured as final stemheight and diameter at the end of the experiment

Fig. 1. Procedures for defoliation (below the lines) and samplecollection (above the lines, in italics).

OIKOS 98:3 (2002) 439

Table 1. Results of the analysis of variance of the glandular trichomes (=glands) and non-glandular trichomes (= trichomes)on lower and upper sides of leaves and hairs on leaf margins and on midvein. ANOVA was performed separately withinprevious and current summer. Symbols after the F-values indicate statistical significance: ***=p�0.001, **=p�0.01,*=p�0.05, °=0.05�p�0.1 and ns=p�0.1.

Current summerPrevious summer

SS df F FSS df

Lower glandsDefoliation 336.65 0.088ns2 8.57** 1.52 2Linear 334.96 1 0.09ns17.05*** 1.511 1Quadratic 1.96 1 0.1ns 0.001ns0.009 1Error 294.73 15 154.74 18

Lower trichomesDefoliation 13875.2 1.32ns2 20.38*** 2276.1 2Linear 12701.01 1 1.94ns37.31*** 1673.28 1Quadratic 1174.19 1 3.45ns 0.7ns602.82 1Error 5106.1 15 15522.1 18

Upper glandsDefoliation 1.37 0.32ns2 2.34ns 4.85 2Linear 1.33 1 0.02ns4.54° 0.183 1Quadratic 0.04 1 0.14ns 0.62ns4.67 1Error 4.39 15 136.18 18

Upper trichomesDefoliation 2216.59 2 17.83*** 467.42 2 1.14nsLinear 2197.81 1 1.84ns35.35*** 376.48 1Quadratic 18.78 1 0.3ns 0.44ns90.94 1Error 932.6 15 3688.3 18

Margin hairsDefoliation 448.04 4.21*2 18.06*** 140.23 2Linear 447.74 1 6.06*36.09*** 100.98 1Quadratic 0.3 1 0.02ns 2.36ns39.25 1Error 186.08 15 299.74 18

Midvein hairsDefoliation 51.47 2 25.16*** 95.52 2 19.44***Linear 48.8 1 47.72*** 90.27 1 36.74***Quadratic 2.67 1 2.14ns2.61ns 5.25 1Error 15.34 15 44.22 18

(mid-September 1999). Stem diameter is an average oftwo measurements at a 90° angle and at about 1 cmfrom the ground. These parameters were also recordedat the beginning of the experiment before the firstdefoliation (late June 1998) and used as a covariate inthe statistical analyses (see below). From the growthmeasurements, we discarded six of the 20 seedlings ofthe 100% defoliation treatment of 1998 because themain stem apex died and the seedlings produced newsprouts from the stem base. These seedlings were notused in the leaf trichome or FA measurements either.

Statistical analyses

Concerning the leaf hair data, we tested a single hy-pothesis (changes in structural defence in response todefoliation) with multiple parameters (number of glan-dular and nonglandular trichomes on the upper andlower sides, trichomes in leaf margins and small hairson the mid-vein) and thus performed a protectedANOVA for the data. In this approach, which com-bines MANOVA and ANOVA, univariate analyses are

performed if a multivariate analysis yields a significantresult. This procedure is less conservative than thesequential Bonferroni correction (i.e. Dunn-Sidakmethod) used to correct the p values of multiple tests ofa single hypothesis (Scheiner 1993). Since MANOVA(Pillai’s trace statistics) indicated a significant interac-tion between the degree and timing of defoliation(F12,58=4.3, p�0.001), and because we used seedlingsdefoliated 100% in 1999 (treatment 5) as controls forthe 1998 defoliated seedlings (not yet defoliated at thetime of leaf collection, cf. Fig. 1), we performed uni-variate tests (one-way ANOVA with the degree ofdefoliation as a grouping factor) separately within thestudy years (Table 1).

The effect of defoliation on abosolute and size-cor-rected FA was analysed with one-way ANOVA withinlong-shoot leaves and short-shoot leaves separately(Table 2). Because the classifying factor (defoliation) inall of the above tests was quantitative, we studied thedifferences between the treatment means with polyno-mial contrasts after ANOVA (Steel et al. 1997). Sincewe had three levels of defoliation, up to second-degreepolynomial contrast (linear and quadratic) could be

440 OIKOS 98:3 (2002)

fitted to the data. Absolute FA values were adjusted forcovariate under one-way ANCOVA with defoliation asfixed factor and leaf width as covariate (Fig. 4c).

The possible statistical differences in the final heightand diameter of the seedlings between the treatmentswere studied by means of 1-way ANCOVA, with theextent of defoliation as a fixed factor and the initialheight or diameter as a covariate. The specific compari-sons between the different treatments following AN-COVA were studied by comparing estimated marginalmeans (Searle et al. 1980) with simple contrasts (controlvs other treatments). The estimated marginal means arepresented in Fig. 5a, b, where the treatments signifi-cantly different from the control are indicated by aster-isks. The possible statistical differences in leaf widthand length in the different defoliation treatments weretested with 1-way ANOVA for each year separately.

Results

Leaf hairs

In seedlings defoliated during the previous summer(1998), a clear shift from glandular to nonglandular

trichomes was observed on both the upper and thelower sides of the leaves. While glandular hairs showeda significant linear decrease in response to the extent ofdefoliation, a completely opposite trend was seen innonglandular trichomes (Fig. 2a vs b, 2c vs 2d, Table1). This trend was not, however, present in the seedlingsdefoliated earlier in the same (1999) summer (Fig.2a–d, Table 1). On the other hand, leaf margin tri-chomes in the seedlings defoliated in the previous sum-mer showed a significant linear increase in response todefoliation (Fig. 3a, Table 1). The number of marginhairs also increased after complete defoliation duringthe current summer. Defoliations in the previous andcurrent years had significant but opposite effects onmid-vein hairs on the lower leaf surface. Mid-vein hairsincreased in seedlings defoliated in the previous summerbut decreased in seedlings defoliated in the same sum-mer (Fig. 3b, Table 1).

Fluctuating asymmetry

Fluctuating asymmetry did not show any clear or con-sistent response to defoliation. In both short- and long-shoot leaves, 100% defoliation seemed to decrease the

Table 2. Results of the analysis of variance of the absolute and size-corrected fluctuating assymmetry (FA) in short-shoot andlong-shoot leaves. Symbols after F-values: see Table 1.

Size-corrected FAAbsolute FA

Long-shoot leavesShort-shoot leavesLong-shoot leavesShort-shoot leaves

Source FSS df F SS df F SS df F SS df

2 1.81ns0.52 2 2.15ns 1.145 2 4.26* 0.016 2 6.9** 0.0023Defoliation3.14°10.00213.82***10.0166.2*10.8331.49ns10.18Linear

1 0.47ns0.34 1 2.82° 0.312 1 2.32ns 0.000 1Quadratic 0.0ns 0.0003576.88Error 550.035570.066557.39

Fig. 2. Number (mean�1S.E.) of (a) glandular and (b)nonglandular trichomes(hairs) on the under side ofthe leaves and (c) glandularand (d) nonglandulartrichomes on the upper sidein the examined area of theleaves in birches defoliated (0,50 or 100%) in either theprevious or the currentsummer.

OIKOS 98:3 (2002) 441

Fig. 3. Number (mean�1S.E.) of (nonglandular) hairson leaf margin (a) and onmid-vein on underside of theleaves (b) in the examinedarea in birches defoliated (0,50 or 100%) in either theprevious or the currentsummer.

Fig. 4. Absolute (in mm) (a),size-corrected (b) andcovariate (leaf width) adjusted(c) fluctuating asymmetry(mean�1 S.E.) in short-shootand long-shoot leaves inbirches defoliated (0, 50 or100%) earlier in the samesummer.

Fig. 5. Height (a) and diameter (b)(estimated marginal means�1 S.E.,in cm) of control (no defoliation)seedlings and seedlings defoliated50% or 100% in the either previous(Prev) or the current summer (Cur).The asterisks indicate the treatmentssignificantly (p�0.05) different fromthe control treatment.

absolute deviation from symmetrical development (ab-solute FA: Fig. 4a), though this decrease was statisti-cally significant only in long-shoot leaves (Table 2).This trend, however, disappeared when the measure ofasymmetry was corrected with leaf size, despite the factthat absolute asymmetry was not related to leaf size(within-treatment correlations between absolute FAand leaf size: r= −0.02–0.2). The size-corrected FAincreased linearly with the degree of defoliation inshort-shoot leaves (Fig. 4b, Table 2), but in long-shootleaves this trend was not statistically significant (Table2). When absolute FA was adjusted for leaf size (Fig.4c), defoliation had no effect on short-shoot leaves(F2,56=0.928, p=0.401) or long-shoot leaves (F2,54=0.184, p=0.833).

Shoot and leaf growth

The final height and diameter of the defoliatedseedlings significantly decreased compared to the con-trol seedlings (Fig. 5a, b, Table 3). Growth retardationin the seedlings defoliated earlier in the same growingseason was not equally pronounced as in those defoli-ated during the previous growing season. This differ-ence between the previous and current summerdefoliations was most evident in the 100% defoliatedseedlings. While the height of the seedlings defoliated100% during the previous summer was less than half ofthat of the control seedlings, the decrease in growth inthe other defoliation treatments was below 20%. Instem diameter, a similar trend was seen (Fig. 5a, b).

442 OIKOS 98:3 (2002)

Current-year 100% defoliation affected leaf dimen-sions much more than it affected seedling height ordiameter, whereas 50% defoliation caused no detectablechanges in leaf size. Defoliation in the previous summer(1998) reduced the size of the leaves that formed earlyin the following summer (1999), and this reduction inleaf size was proportional to the degree of defoliation(Fig. 6a, b, Table 4).

Discussion

Defoliation performed in the previous growing seasonclearly decreased seedling growth and leaf size. Thedecrease in leaf size was not, however, in proportion tothe trends seen in the number of leaf trichomes. Whilethe leaves in the control seedlings were roughly 1.5times wider and longer than the leaves in the 100%defoliated seedlings, the latter had over 10 times moretrichomes on their upper leaf surface than the controlseedlings. This clearly indicates that the increase in the

number of trichomes was not merely due to increaseddensity as a function of decreased leaf size. Further-more, previous and current-year defoliations tended toaffect leaf size similarly, but their effects on leaf hairswere different.

The primordia of early leaves (short-shoot leaves andbasal leaves on long shoots) that expand in the springand early summer have already been formed in theprevious summer during the development of axillarybuds (Macdonald and Mothersill 1983, Macdonald etal. 1984). Hence, the effect of the leaf loss during theprevious summer (1998) on the dimensions and hairi-ness of the following summer’s early leaves (collected inJune 1999, Fig. 1) was likely to be mediated via thesedevelopmental processes. These results thus suggest thatthe induction of epidermal cell differentiation for vari-ous kinds of trichomes may already occur during theprevious growing season, thus resembling a delayedinduced response. This process may, however, dependon the type and location of trichomes: defoliation eitherin the previous or the current summer increased nong-landular trichomes on the leaf margins, but defoliation

Table 3. Results of the analysis of covariance of the final height and diameter of the studied birches. Covariate is initial heightor diameter (measured before the first defoliation, see Fig. 1). Symbols after F-values: see Table 1.

Height Diameter

FdfSS dfSS F

79.4***1762.97Covariate 61.0***138251.41246.19Defoliation 4 32.44***60264.6 4 24.0***

845.18 88Error 55178.1 88

Fig. 6. Average (�1 S.E.)leaf width (a) and length (b)(in cm) in birches defoliated(0, 50 or 100%) in either theprevious or the currentsummer.

Table 4. Results of the analysis of variance of leaf length and width. ANOVA was performed separately within previous andcurrent summer. Symbols after F-values: see Table 1.

Previous summer Current summer

dfSS FdfSSF

Leaf lengthDefoliation 4.544 2 11.06** 11.06** 2 32.5***

22.11***Linear 6.231 1 44.86***4.539 1Quadratic 0.005 1 0.02ns 2.81 1 20.23***Error 3.08 15 2.5 18

Leaf widthDefoliation 9.08**21.937 42.7***210.54

11.936Linear 18.15*** 7.82 1 63.4***0.001Quadratic 22.1***12.720.01ns1

182.22151.6Error

OIKOS 98:3 (2002) 443

in the previous summer increased and that in the cur-rent summer decreased mid-vein trichome density.

Our results strongly support the idea that potentiallydefensive morphological traits, such as hairiness, aredevelopmentally plastic traits and that both previousand current-year defoliation may induce changes inthese morphological traits. If these changes really weredefensive responses to simulated herbivory, we wouldhave expected the morphological defences to have in-creased as a response to a previous attack. This was thecase in leaf trichomes and mid-vein hairs, but reversechanges took place in glandular hairs. This may indi-cate that trichomes and glandular hairs have differentfunctions or, alternatively, that there is a trade-offbetween these traits. The striking difference in theresponses of the different types of hairs to defoliation,i.e. glandular hairs showing a linear decrease in re-sponse to the degree of defoliation, and the preciselyopposite trend seen in nonglandular leaf trichomes maybe explained by the different costs of producing the twotypes of hairs. The cost of building glandular hairs isprobably higher due to the complex terpenoid com-pounds produced by excretory cells in glands comparedto the cost of building the simple cellulose walls oftrichomes. The glucose requirement for the construc-tion of volatile terpenoids (limonene) has been calcu-lated to be 18.8 mmol g−1 and that for cellulose andhemicellulose well below half of that, i.e. 6.5 and 7.1mmol g−1 respectively (Lambers and Poorter 1992).Further, the shift from glandular to nonglandular tri-chomes is also partly physiologically or genetically con-strained. Glover (2000) has shown with Arabidopsis thattrichome development in the leaf epidermis is not ran-dom but likely to be caused by interactions between thedeveloping epidermal cells that determine which cellsare committed to trichome production. Further, asGlover (2000) suggests, even when the molecular mech-anisms behind the developmental steps of various tri-chome types are different, these differential pathwaysinteract with each other, i.e. if a cell is differentiatedinto a trichome, it may inhibit the neighbouring cellsfrom adopting the trichome role. Hence, our resultssuggest that when birches faced an intensive and repet-itive loss of photosynthetic capacity, they adopted a lesscostly structural defence mechanism.

Even though defoliation altered leaf size and inducedchanges in the production of leaf hairs, we did not findany significant increase in deviations from the bilateralsymmetry of the leaves, despite the potential effects onthe diverse developmental processes involved. For in-stance, in trees with all leaves removed earlier in thesummer, the new (short- and long-shoot) leaves pro-duced after defoliation retained the symmetrical bilat-eral shape. In fact, the most extensive defoliationtended to reduce deviations from the bilateral form.When absolute deviations were corrected with leaf sizethe size-corrected FA showed a different trend, even

though leaf size and asymmetry seemed to lack anyclear relation within treatment groups. This inconsis-tency between absolute and size-corrected FA is pre-sumably a mere consequence of the fact that leaf sizedecreases more due to defoliation than deviation fromsymmetrical leaf form. When, for instance, absolute FAof short shoot leaves in 100% defoliated trees was about86% of that in control (0% defoliated) trees, the leafwidth in 100% defoliated trees was 54% of that incontrol trees. Thus the increasing trend that can be seenin size corrected FA (Fig. 4b) raises from the fact thanthe denominator in the formula �R−L�/[(R+L)/2] de-creases more than the numerator. Consequently, whenthe defoliation-induced decrease in the leaf size wasremoved (covariate adjusted FA, Fig. 4c), there was noclear trends in leaf asymmetry in relation to defoliation.Hence, these results seem to suggest that, in the case ofartificial defoliation, FA may be a poorly sensitive toolto detect induced responses in plants (cf. Lappalainenet al. 2000, see also Wilsey and Saloniemi 1999, Bjork-sten et al. 2000). There is some correlative evidence thatFA may be associated with a higher risk of herbivory:larvae of the geometrid Epirrita autumnata consumedmore from the birch leaves of high than of low FA trees(Lempa et al. 2000). This relationship may not, how-ever, be causal because larval consumption rate proba-bly relates to high concentrations of hydrolysabletannins in high FA leaves, leading to compensatoryfeeding by larvae (Kause et al. 1999).

In conclusion, our results are consistent with thehypothesis that trichomes might operate as morpholog-ical defences. However, the potential defensive functionof trichomes should be separately tested since leafpubescence has other functions, such as reducing heatload (Ehleringer et al. 1976) and preventing water loss(Korner 1999). Hairiness can, on the other hand, pro-tect leaves from evapotranspiration and herbivoressimultaneously (Woodman and Fernandes 1991). It is,nevertheless, plausible that an increased trichome den-sity might prevent insect larvae from feeding on leaves(Levin 1973, Baur et al. 1991), though some indicationsof increased herbivore success on hairy leaves have alsobeen reported (Webster 1975). According to the gener-ally acclaimed definitions given by Karban and Myers(1989), we can say with certainty that the changes intrichome density and type observed in our study aredefoliation-induced responses. Whether these responsesreally represent induced resistance or defence againstherbivores requires further studies evaluating the effectsof increased trichome density on herbivore preferenceand, further, on plant fitness in the presence ofherbivores.

Acknowledgements – The personnel at the Botanical Gardensof the University of Oulu provided facilities and help duringthe work. Dr. Anneli Kauppi (Univ. of Oulu, Dept of Biol.)presented useful facts regarding the morphology and physiol-ogy of birches. Dr. Julia Koricheva (Univ. of Turku, Dept of

444 OIKOS 98:3 (2002)

Biol.) made useful comments on the manuscript. Sirkka-LiisaLeinonen (Lic. Phil.) revised the English of the manuscript.The study was funded by the Foundation of Finnish NaturalResources (Suomen Luonnonvarain Tutkimussaatio, project‘‘Effects of defoliation on mycorrhizal symbiosis and defencein forest trees’’) and the Academy of Finland (project no.c40951).

ReferencesAgrawal, A. A. 2000. Benefits and costs of induced plant

defense for Lepidium �irginicum (Brassicaceae). – Ecology81: 1804–1813.

A� gren, J. and Schemske, D. W. 1993. The cost of defenseagainst herbivores: an experimental study of trichome pro-duction in Brassica rapa. – Am. Nat. 141: 338–350.

Baur, R., Binder, S. and Benz, G. 1991. Nonglandular leaftrichomes as short-term inducible defense of the grey alder,Alnus incana (L.), against the chrysomelid beetle, Agelas-tica alni L. – Oecologia 87: 219–226.

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

Bjorkman, C. and Anderson, D. B. 1990. Trade-off amongantiherbivore defences in a South American blackberry(Rubus bogotensis). – Oecologia 85: 247–249.

Bjorksten, T. A., Fowler, K. and Pomiankowski, A. 2000.What does sexual trait FA tell us about stress? – TREE 15:163–166.

Duffey, S. S. 1986. Plant glandular trichomes: their partial rolein defence against insects. – In: Juniper, B. and South-wood, R. (eds), Insects and the plant surface. EdwardArnold Publ., London, pp. 151–172.

Ehleringer, J., Bjorkman, O. and Mooney, H. A. 1976. Leafpubescence: effects on abosorptance and photosynthesis ina desert shrub. – Science 192: 376–377.

Elle, E., van Dam, N. M. and Hare, J. D. 1999. Cost ofglandular trichomes, a ‘‘resistance’’ character in Daturawrightii Regel (Solanaceae). – Evolution 53: 22–35.

Esau, K. 1953. Plant anatomy. – John Wiley & Sons, Inc.Glover, B.J. 2000. Differentiation in plant epidermal cells. – J.

Exp. Bot. 51: 497–505.Herms, D. A. and Mattson, W. J. 1992. The dilemma of

plants: to grow or defend. – Q. Rev. Biol. 67: 283–335.Hulley, P. E. 1988. Caterpillar attacks plant mechanical de-

fence by mowing trichomes before feeding. – Ecol. Ento-mol. 13: 239–241.

Karban, R. 1993. Induced resistance and plant density of anative shrub, Gossypium thurberi, affect its herbivores. –Ecology 74: 1–8.

Karban, R. and Baldwin, I. T. 1997. Induced responses toherbivory. – Univ. of Chicago Press.

Karban, R. and Myers, J. H. 1989. Induced plant responses toherbivory. – Annu. Rev. Ecol. Syst. 20: 331–348.

Kause, A., Ossipov, V., Haukioja, E. et al. 1999. Multiplicityof biochemical factors determining the quality of growingbirch leaves. – Oecologia 120: 102–112.

Korner, C. 1999. Alpine plant life: functional plant ecology ofhigh mountain ecosystems. – Springer.

Kozlov, M. V., Wilsey, B. J., Koricheva, J. and Haukioja, E.1996. Fluctuating asymmetry of birch leaves increases un-der pollution impact. – J. Appl. Ecol. 33: 489–495.

Lambers, H. and Poorter, H. 1992. Inherent variation ingrowth rate between higher plants: a search for physiologi-cal causes and ecological consequences. – Adv. Ecol. Res.23: 187–261.

Lappalainen, J. H., Martel, J., Lempa, K. et al. 2000. Effectsof resource availability on carbon allocation and develop-mental instability in cloned birch seedlings. – Int. J. Plant.Sci. 161: 119–125.

Lempa, K., Martel, J., Koricheva, J. et al. 2000. Covariationof fluctuating asymmetry, herbivory and chemistry duringbirch leaf expansion. – Oecologia 122: 354–360.

Levin, D. A. 1973. The role of trichomes in plant defense. –Q. Rev. Biol. 48: 3–15.

Macdonald, A. D. and Mothersill 1983. Shoot development inBetula papyrifera. I. Short-shoot organogenesis. – Can. J.Bot. 61: 3049–3065.

Macdonald, A. D., Mothersill, D. H. and Caesar, J. C. 1984.Shoot development in Betula papyrifera. III. Long-shootorganogenesis. – Can. J. Bot. 62: 437–445.

Martel, J., Lempa, K. and Haukioja, E. 1999. Effects of stressand rapid growth on fluctuating asymmetry and insectdamage in birch leaves. – Oikos 86: 208–216.

Mauricio, R. and Rausher, M. D. 1997. Experimental manipu-lation of putative selective agents provides evidence for therole of natural enemies in the evolution of plant defence. –Evolution 51: 1435–1444.

Mauricio, R., Rausher., M. D. and Burdick, D. S. 1997.Variation in the defense strategies of plants: are resistanceand tolerance mutually exclusive. – Ecology 78: 1301–1311.

Mutikainen, P. and Walls, M. 1995. Growth, reproductionand defence in nettles: responses to herbivory modified bycompetition and fertilization. – Oecologia 104: 487–495.

Myers, J. H. 1987. Nutrient availability and the deployment ofmechanical defenses in grazed plants: a new experimentalapproach to the optimal defense theory. – Oikos 49:350–351.

Ossipov, V., Loponen, J., Ossipova, S. et al. 1997. Gallotan-nins of birch pubescens leaves: HPLC separation andquantification. – Biochem. Syst. Ecol. 25: 493–504.

Palmer, A. R. and Strobeck, C. 1986. Fluctuating asymmetry:measurement, analysis, patterns. – Annu. Rev. Ecol. Syst.17: 391–421.

Rathcke, B. J. and Poole, R. W. 1975. Coevolutionary racecontinues: butterfly larval adaptation to plant trichomes. –Science 187: 175–176.

Rhoades, D. F. 1979. Evolution of plant chemical defenseagainst herbivores. – In: Rosenthal, G. A. and Janzen, D.H. (eds), Herbivores: their interaction with secondary plantmetabolites. Academic Press, pp. 4–54.

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

Scheiner, S. M. 1993. MANOVA: multiple response variablesand multispecies interactions. – In: Scheiner, S. M. andGurevitch, J. (eds), Design and analysis of ecological ex-periments. Chapman and Hall, pp. 94–112.

Searle, S. R., Speed, F. M. and Milliken, G. A. 1980. Popula-tion marginal means in the linear model: an alternative toleast squares means. – Am. Stat. 34: 216–221.

Siebert, M. W. 1975. Candidates for the biological control ofSolanum eleagnifolium Cav. (Solanaceae) in South Africa.1. Laboratory studies on the biology of Gratiana lutescens(Boh.) and Gratiana pallidula (Boh.) (Coleoptera: Cassi-didae). – J. Entomol. Soc. S. Africa 38: 297–304.

Simms, E. L. and Rausher, M. D. 1987. Costs and benefits ofplant resistance to herbivory. – Am. Nat. 130: 570–581.

Southwood, R. 1986. Plant surfaces and insects – an overview.– In: Juniper, B. and Southwood, R. (eds), Insects and theplant surface. Arnold, London, pp. 1–22.

Steel, R. G. D., Torrie, J. H. and Dickey, D. A. 1997.Principles and procedures of statistics: a biometrical ap-proach, 3rd ed. – McGraw-Hill.

Steward, J. L. and Keeler, K. H. 1988. Are there trade-offsamong antiherbivore defenses in Ipomoea (Convolvu-laceae)? – Oikos 53: 79–86.

Stipanovic, R. D. 1983. Function and chemistry of planttrichomes and glands in insect resistance. – In: Hedin, P.

OIKOS 98:3 (2002) 445

A. (ed.), Plant resistance to insects. Am. Chem. Soc. Symp.Ser. 208. Am. Chem. Soc, Washington, pp. 69–100.

Tahvanainen, J., Julkunen-Tiitto, R., Rousi, M. and Reichardt,P. B. 1991. Chemical determinants of resistance in winter-dormant seedlings of European white birch (Betula pen-dula) to browsing by mountain hare. – Chemoecology 2:49–54.

Tuomi, J., Niemela, P. and Mannila, R. 1982. Resource alloca-tion of dwarf shoots of birch (Betula pendula): reproductionand leaf growth. – New Phytol. 91: 483–487.

Tuomi, J., Niemela, P., Rousi, M. et al. 1988. Induced accumu-lation of foliage phenols in mountain birch: branch responseto defoliation? – Am. Nat. 132: 602–608.

Tuomi, J., Ilvessalo, H., Niemela, P. et al. 1989a. Within-plantvariation in phenolic content and toughness of the brownalga Fucus �esiculosus L. – Bot. Mar. 32: 505–509.

Tuomi, J., Niemela, P., Jussila, I. et al. 1989b. Delayedbudbreak: a defensive response of mountain birch to early-season defoliation? – Oikos 54: 87–91.

Webster, J. A. 1975. Association of plant hairs and insectresistance. – Agric. Res. Service. USDA Misc. Publ. No.1297.

Wilsey, B. J. and Saloniemi, I. 1999. Leaf fluctuating asymmetryin tree-line mountain birches, Betula pubescens ssp. tortuosa :genetic or environmentally influenced. – Oikos 87: 341–345.

Wilsey, B., Haukioja, E., Koricheva, J. and Sulkinoja, M. 1998.Leaf fluctuating asymmetry increases with hybridization andelevation in tree-line birches. – Ecology 79: 2092–2099.

Woodman, R. I. and Fernandes, G. W. 1991. Differentialmechanical defense: herbivory, evapotranspiration and leaf-hairs. – Oikos 60: 11–19.

Zar, J. H. 1996. Biostatistical analysis, 3rd ed. – Prentice Hall.

446 OIKOS 98:3 (2002)