Interspecific defoliation responses of trees depend on sites of winter nitrogen storage

Functional Ecology

2001

15

, 535–543

© 2001 British Ecological Society

535

Blackwell Science, Ltd

Interspecific defoliation responses of trees depend on sites of winter nitrogen storage

P. MILLARD,† A. HESTER, R. WENDLER and G. BAILLIE

Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen AB16 8QH, UK

Summary

1.

To determine if the response of trees to herbivory is related to their ability to storenitrogen, saplings of

Pinus sylvestris

L.,

Betula pendula

Roth. and

Sorbus aucuparia

L.were clipped, either when dormant or at spring bud burst, to remove half the previousyear’s shoot growth. The impact of clipping on N remobilization and uptake was quanti-fied in relation to their growth responses.

2.

Pinus sylvestris

stored N during the winter in needles grown the previous summer,whereas

B. pendula

and

S. aucuparia

used their woody roots and older stems. Therefore,N remobilization was unaffected by clipping the deciduous species, but was reduced byhalf in the evergreen.

3.

For both

P. sylvestris

and

B. pendula

, root uptake contributed N for leaf growthimmediately after bud burst, concurrently with remobilization.

Sorbus aucuparia

hadremobilized half the N from storage before any N was taken up by the roots.

4.

Pinus sylvestris

, which has a fixed pattern of growth, produced a lower total needlemass when clipped, but individual needles were heavier. Nitrogen uptake during sum-mer was reduced by 26 and 44% for winter- and spring-clipped saplings, respectively.Both deciduous species showed compensatory leaf growth such that, by the end of thesummer, leaf mass (and

B. pendula

leaf area) were unaffected by clipping. Nitrogenuptake by both deciduous species was unaffected by clipping.

5.

We conclude that the site of N storage during winter is a crucial factor in determin-ing the response of a sapling to herbivory, as interactions with the growth patterns ofthe three species can lead to different responses to damage. We suggest that for ever-green trees, the production of antiherbivory compounds serves primarily to protect Nstored in their foliage, rather than the leaves themselves.

Key-words

: Evergreen and deciduous trees, herbivory, internal cycling

Functional Ecology

(2001)

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, 535–543

Introduction

Pinus sylvestris

L.,

Betula pendula

Roth. and

Sorbusaucuparia

L. are common species in native, temperatewoodlands (Rodwell 1991), and their natural regenera-tion in hill and upland areas is often limited due todamage caused by browsing herbivores (Gill 1992c;Humphrey

et al

. 1998). From the few published studiesto date, browsing damage appears to be more detri-mental to the growth of

P. sylvestris

, and saplings ofthis species appear more likely to be killed by browsingdamage than either

B. pendula

or

S. aucuparia

(Miller

et al

. 1982). However, the reasons for these differencesare not fully understood.

The physiological responses of

P. sylvestris

tobrowsing damage are variable, and have been shown to

depend on factors such as the extent of biomassremoved and the timing of the damage. Honkanen,Haukioja & Suomala (1994), for example, found thatbud damage resulted in positive growth responses,whereas needle damage could result in stimulated orinhibited growth depending on the relative position ofthe shoot and the timing of defoliation. Furthermore,defoliation of 1-year-old needles (Honkanen, Haukioja& Kitunen 1999) reduced the mean length of needles innew shoots, whereas removal of 2-year-old needles hadno effect upon current needle growth.

Betula

spp. alsorespond to herbivory in a variety of ways according totiming, severity and location of damage. For example,moderate browsing of

Betula pubescens

and

B. pendula

was reported by Danell & Huss-Danell (1985) to pro-duce trees with fewer leaves, but which were larger,heavier and contained more nitrogen than leaves fromcontrol trees. Removal of apical buds in the winterfrom

B. pubescens

also resulted in the production of

†Author to whom correspondence should be addressed. E-mail:[email protected]

FEC541.fm Page 535 Monday, July 2, 2001 1:45 PM

536

P. Millard

et al.

© 2001 British Ecological Society,

Functional Ecology

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larger leaves (Senn & Haukioja 1994), but bud removalat bud burst in spring produced little or no such response.Hjaltén, Daniel & Ericson (1993) also found thatremoval of apical dominance by browsing can leadto overcompensation in growth.

Sorbus aucuparia

iswidely quoted as being a highly preferred browsespecies (Bergström & Danell 1987; Gill 1992a, Gill1992b; Miller

et al

. 1982), but the physiological responsesof this species to browsing damage have been littlestudied (Gill 1992c).

Several authors have suggested that the response oftrees to herbivory-induced damage is dependent ontheir ability to grow using resources from the previousyears (Haukioja

et al

. 1990; Honkanen

et al

. 1999).Much of the N used by trees for leaf growth in springcomes from the remobilization of stored reserves(Millard 1996). The site of storage of N during the winterdepends on the growth habit of the tree. Coniferous,evergreen species such as

P. sylvestris

store N in theirfoliage (Miller

et al

. 1979; Nambiar & Fife 1991; Proe,Midwood & Craig 2000), often in the youngest ageclass of needles which grew the previous summer(Millard & Proe 1992; Millard & Proe 1993). Nitrogendoes not appear to be stored in the roots of eitheryoung (Millard & Proe 1993) or older (Nambiar 1987)conifers. In contrast, deciduous trees store N in theirwoody tissues, often in specific wood and bark storageproteins that are degraded during spring shoot growth(Coleman

et al

. 1991; Nsimba-Lumaki & Peumans1986; Sauter & van Cleve 1992). Roots of young decidu-ous trees have also been reported to store N duringthe winter (Millard & Proe 1991; Tromp 1983), including

B. pendula

saplings (Millard

et al

. 1998). The site of Nstorage in a tree thus might be important in determiningresponses to herbivory, given that browsing damagein winter is more likely to lead to losses of N stored infoliage or above-ground woody tissues than in roots.However, such relationships have been little studied.

In the experiment reported here, we examined theimpact of simulated mammalian browsing damage onthe internal cycling of N in saplings of

P. sylvestris

,

B.pendula

and

S. aucuparia

. Saplings below 1 m high wereselected for study because at this stage all the above-ground biomass is vulnerable to damage by large mam-mals. Saplings were grown in sand culture for 2 years,during the first of which they were supplied with

15

N.In the second year the trees received N at natural abund-ance and were clipped (to remove half of the previousyear’s above-ground growth) either in January (whenthe trees were still dormant), or at bud break in Marchor April, or were left intact. During the spring andsummer a series of destructive harvests were taken inorder to: (i) determine the impact of clipping on thecontribution of both N remobilization and N uptakefor leaf growth; (ii) relate the growth responses of thethree species following clipping to their sites of N stor-age; and (iii) determine the impact of the timing ofdamage in relation to bud burst on the amount of Nremobilization.

Materials and methods

Two-year-old seedlings of

Pinus sylvestris

L.,

Betulapendula

Roth. and

Sorbus aucuparia

L. (105 of eachspecies) were lifted from a nursery while dormant, inMarch 1996, and planted in pots (30 cm diameter

×

26cm deep). The trees had previously received a moder-ate N supply. The pots were arranged randomly in aLatin square design in a greenhouse, and all trees werewatered with 300 cm

3

of a nutrient solution containing3·0 mol N m

–3

as

15

NH

4

15

NO

3

enriched with

15

N to 2·8atom percentage excess. Other nutrients were suppliedas described by Millard & Proe (1991). Trees werewatered every 2 days throughout the spring, summerand autumn of 1996. A natural photoperiod was used,and the greenhouse ventilated to provide temperaturesclose to ambient. Throughout the winter of 1996/97the trees were kept frost-free (

≥

2

°

C) and moist. Inearly January 1997 trees were carefully removed fromthe pots and all sand washed off the roots before beingplanted in fresh sand in a new, larger pot (40 cmdiameter

×

35 cm deep).Morphological measurements to characterize sap-

ling pre-clipping treatment variability were taken inJanuary 1997, before application of the experimentalclipping treatments, by measuring their height, thediameter of the main stem at the sand surface, thenumber of branches, the number of stems with lateralbranches, the number of buds, and the number ofshoots grown during 1996. Out of that list, a subset ofmeasurements which, together, explained the highestproportion of between-sapling variability were thenused as covariates in all subsequent data analyses, asdescribed in the data analysis section below.

Thirty-five trees of each species were clipped on 14January 1997 to remove half of the previous year’s(1996) shoot growth. This was designated the winter(dormant) clipping treatment. Clipping treatmentswere designed to mimic as closely as possible browsingdamage by Red deer: removal of 1996 shoots (includ-ing buds/leaves) up to the maximum stem diametersnormally eaten by Red deer for each of the three spe-cies (L.A. Shipley, unpublished data; Shipley

et al

.1999). As the basal diameters of all 1996 shoots werewithin the ranges taken by Red deer, whole shoots wereclipped on all species. If more than one 1996 shoot waspresent on a sapling, every second shoot was clipped tojust above the point where 1996 growth was initiated,starting with the leader/top shoot. If only one 1996shoot was present (some individuals of

S. aucuparia

),only the top 50% of that one shoot was removed. Thedry weights and total number of buds removed in clip-pings from each tree were recorded. As the morpho-logy of the three species is very different, the totalnumber of shoots per sapling and the number and pro-portion of buds on old and new shoots differed betweenspecies. Thus, although the dry weight removed was


537

Remobilization of N in browsed trees


Functional Ecology

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similar for all species, the total numbers of shoots andbuds removed and, importantly, the proportions ofbuds removed, differed between species.

From the January 1997 repotting onwards, treeswere watered with 300 cm

3

of nutrient solution, con-taining unlabelled N at natural abundance, once aweek until the beginning of March, when the volumeapplied was increased to 500 cm

3

and the frequency ofapplication to every 2 days. A second clipping treat-ment was applied to a further 30 trees of each specieson the date of their bud burst. This was designated asthe spring (bud burst) clipping treatment. The timingwas assessed by observing buds daily; for each indi-vidual tree, the day the first bud opened was noted anddesignated the date of bud burst. These observationswere made on all trees in the experiment. The springclipping treatment was applied in exactly the same wayas the winter clip. Unlike the two deciduous species, inpine considerable bud elongation can occur beforeactual bud burst, which is likely to have physiologicalimplications for this species, but it is not known howthe response to damage might differ if this species wereclipped at the time of first shoot elongation rather thanbud burst. Therefore, for consistency between the threespecies we chose to clip all species at the time of emer-gence of the first needles/leaves.

At each of seven destructive harvests, five replicatesaplings of each species were randomly selected. Sap-lings were removed from their pots and all remainingsand was gently washed from the roots. The

P. sylvestris

trees were then separated into 1995 needles; 1996 needles;current year’s (1997) needles; 1995 (and older) stem;1996 stem; current year’s (1997) stem; woody roots; andfine roots. Both

B. pendula

and

S. aucuparia

were separ-ated into leaves (1997); 1995 (and older) stem; 1996stem; 1997 stem; woody roots (including tap roots);and fine roots. All plant samples were then freeze-dried, weighed and milled prior to

15

N analysis. ATracer MAT continuous flow mass spectrometerwas used for determinations of both

15

N enrichmentand the total N concentrations in samples. The

15

Nenrichment was used to calculate the uptake of labelledN (Millard & Neilsen 1989). Recovery of labelled N inleaves grown during 1997 gave a direct measure of Nthat had been taken up during 1996 and remobilizedfor leaf growth during spring 1997.

Data for N uptake and remobilization were related tothe stage of leaf development by using the number ofdays from bud burst as a measure of time. Rates ofuptake and remobilization with time were analysedusing exponential curve-fitting, with treatment as agrouping factor (

5; Genstat 5 Committee1993). Comparisons of proportional

15

N data per sapling

were made using paired

t

-tests (see Table 3). In viewof the large variation in sapling sizes and morpho-logy, all analyses of actual values (see Tables 4 and 5),rather than proportions, used residual maximumlikelihood (REML) (

5) with: (i) treatment(timing of clip) and harvest date as fixed factors;(ii) sapling number as a random factor; and (iii) threeor four pretreatment morphological measurements(described below) as covariates. Wald tests were usedto test for significance of main effects and interactions,and least significance difference tests were used to testdifferences between tables of means (

5). Prin-cipal component analyses (

5) were used toselect the covariates by determining which pretreat-ment (January 1996) morphological measurements foreach species made the most substantial contribution tothe first two principal components and were also relat-ively uncorrelated. The following four measurementswere identified for

B. pendula

and

P. sylvestris

: height;diameter of main stem; number of 1996 shoots; numberof overwintering 1996/97 buds; for

S. aucuparia

meas-urements were the same except for height. The use ofcovariates in the REML analyses allowed explicitexamination of the variation between individuals notcaused by the treatments, together with analyses oftreatment effects on the residual variation allowing forthe covariates. This removed the risk of false ‘treat-ment differences’ caused by groups of larger or smallersaplings randomly occurring within any one harvest.

Results

The total biomass of 1996 shoots removed was approx-imately 50% for all species, but the number and pro-portion of buds removed per sapling differed betweenthe three species, reflecting their different morphology(Table 1). Because of the architecture of the

S. aucuparia

trees, with many buds on older stems, removing halfthe previous year’s growth removed only about a quar-ter of the buds, as compared to about half the budsremoved from other two species (Table 1). In addition toremoving just over half the buds from the

P. sylvestris

trees, clipping removed 55% of the needle pairs fromthe previous year’s growth.

To determine which tissues of each species were usedto store N during the winter, the

15

N content of peren-nial tissues of control trees of each species was meas-ured before bud break when the trees were dormant(14 January), and again after remobilization had fin-ished (66

±

1·4 days from bud burst for

P. sylvestris

;56

±

1·1 days from bud burst for

B. pendula

; 59

±

1·5days from bud burst for

S. aucuparia

), and expressedas a proportion of the total

15

N content of the wholetree (Table 2).

Pinus sylvestris

stored N predominantly


538

P. Millard

et al.


Functional Ecology

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, 535–543

in the 1996 needles, although there were also smallerproportional decreases in the

15

N content of 1995 needlesand stem (formed before

15

N addition) following remobil-ization. In contrast,

B. pendula

stored N predomin-antly in the woody roots and 1995 stem, and

S. aucuparia

in these tissues plus the 1996 stem (Table 2).

The provision of labelled N to trees during 1996allowed the recovery of labelled N in the needles orleaves which grew in 1997 to be used as a direct meas-ure of remobilization (Fig. 1). Remobilization startedstraight after bud burst in all three species, but con-tinued for significantly longer in

P. sylvestris

than in the

deciduous trees. Clipping significantly reduced theamount of remobilization by

P. sylvestris

(

P

< 0·001),with the spring-clipped trees being more affected thanthose clipped in the winter. The reduced remobiliza-tion following clipping presumably reflected the factthat the clipping treatments removed over half the 1996needles, which were the main site of N storage duringthe winter (Table 2). Neither the amount nor the durationof N remobilization were affected by clipping of

B. pendula

or

S. aucuparia

(Fig. 1), because the N had been storedin older, woody tissues unaffected by the clipping.

The recovery of unlabelled N in the needles andleaves grown during 1997 represented N that had beentaken up by the roots after bud burst in 1997. For both

P. sylvestris

and

B. pendula

, root uptake contributed Nfor leaf growth immediately following bud burst, con-currently with N remobilization (Fig. 1). In contrast,no unlabelled N was recovered in the leaves of

S. aucuparia

until 22 days from bud burst (Fig. 1), by which timesome 53% of the N remobilization had alreadyoccurred. The rates and total amounts of uptake ofunlabelled N by

S. aucuparia

differed slightly betweentreatments (

P

< 0·01), with winter-clipped saplingshaving the highest total uptake of unlabelled N(Fig. 1). The parameters from the curves in Fig. 1 wereused to calculate when the amounts of N in leavesfrom remobilization and root uptake were equal. For

P. sylvestris

needles this was 65 days from bud burstin the control trees, and 36 and 24 days for the winter-and spring-clipped trees. For

B. pendula

the compar-able value was after 61 days’ leaf growth irrespectiveof clipping treatment. Leaf growth by

S. aucuparia

was supported predominantly by remobilization ofN for longer than in the other two species, up to 103days for the control trees, and 91 and 108 days for thewinter- and spring-clipped trees, respectively.

The effects of clipping treatments on individual leafgrowth differed among the three species. Removal ofhalf the previous year’s growth (including half thebuds) from

P. sylvestris

resulted in a significantly lowermass of 1997 needles at the end of the growing season(

P

< 0·001; Table 3), reflecting the lower number ofneedle pairs produced (

P

< 0·001; Table 3) and thereduced amount of N remobilized for needle growth(Fig. 1). However, both clipping treatments signific-antly increased the mass per needle pair (

P

< 0·001;Table 3)

.

The impacts on total 1997 needle mass andnumber of needle pairs were more severe for treesclipped at bud burst than trees clipped in winter (both

P

< 0·05; Table 3), also reflecting the reduced amountsof N remobilized for needle growth (Fig. 1). However,the clipping treatments had no significant effect on thenumber of needle pairs that grew per bud remaining onthe tree after the spring clipping treatment was appliedat bud burst (shown as the needle/bud ratio in Table 3).In contrast, clipping the

B. pendula

trees resulted in

Table 1.

Number and proportion of buds removed in shoot clippings from each spe-cies studied

Species

Number of buds

Total per sapling

Removed by clipping

Percentage removed by clipping

Pinus sylvestris

31·9

±

2·35 18·5

±

1·49 58·5

±

1·98

Betula pendula

288·5

±

13·56 125·0

±

6·11 43·4

±

0·92

Sorbus aucuparia

17·1

±

1·05 5·1

±

0·98 28·7

±

1·92

Values are mean

±

SE of 30 replicates of spring-clipped trees.

Table 2.

Storage of nitrogen in perennial tissues of

Pinus sylvestris

,

Betula pendula

and

Sorbus aucuparia

trees during winter

Species Tissue

Proportional

15

N content

Dormant sapling†

After remobilization‡ Significance

P. sylvestris

Buds/1997 growth 1

±

0·2 45

±

5·4 ***1996 needles 47

±

2·0 21

±

3·3 ***1996 stem 7

±

0·6 6

±

0·5 ns1995 needles 1

±

0·3 0·1

±

0·1 ***1995 stem 9 ± 1·1 3 ± 0·7 ***Woody roots 8 ± 3·7 5 ± 0·7 nsFine roots 27 ± 4·1 21 ± 1·2 ns

B. pendula Buds/1997 growth 0 ± 0·0 36 ± 2·4 ***1996 stem 3 ± 0·4 2 ± 0·2 *1995 stem 36 ± 4·1 14 ± 1·5 ***Woody roots 27 ± 1·5 8 ± 0·9 ***Fine roots 34 ± 6·0 40 ± 3·9 ns

S. aucuparia Buds/1997 growth 2 ± 0·1 49 ± 1·4 ***1996 stem 15 ± 1·4 7 ± 1·2 **1995 stem 34 ± 5·4 18 ± 1·0 *Woody roots 27 ± 2·2 11 ± 1·1 ***Fine roots 23 ± 4·7 15 ± 2·3 ns

†SED value for loge transformed means. ‡SED value for square root transformed means. Values are given for the proportional 15N content (± SEM) of tissues of control trees from the first harvest when the trees were dormant and in trees harvested after the majority of remobilization had finished (30 June for P. sylvestris; 12 May for B. pendula and S. aucuparia). Data are means ± SEM of five replicates. Significance values (t-test): ns, not significant, *, P < 0·05; **, P < 0·01; ***, P < 0·001.


539Remobilization of N in browsed trees

© 2001 British Ecological Society, Functional Ecology, 15, 535–543

P. sylvestris B. pendula S.aucuparia

0

100

200

300

0 50 100 150 200 0 50 100 150 200 0 50 100 150 200

0 50 100 150 200 0 50 100 150 200 0 50 100 150 200

Labe

lled

N (

mg

tree

–1)

Unl

abel

led

N (

mg

tree

–1)

0

100

200

300

400

500

600

0

100

200

300

0

100

200

300

400

500

600

Days from bud burst

0

100

200

300

0

100

200

300

400

500

600

(a) (c) (e)

(b) (d) (f)

Fig. 1. Effect of the timing of clipping on recovery of labelled 15N (top) and unlabelled N (bottom) in new needles of Pinussylvestris and leaves of Betula pendula and Sorbus aucuparia in relation to the days from bud burst at harvest for each tree. Datafor individual control trees (h); those clipped when dormant (d); and those clipped at bud burst (s) are fitted with logisticcurves. Pinus, (a) control, y = 181 – 151(0·9795)x; dormant clip, y = 142 – 151(0·9795)x; bud burst clip, y = 112 – 151(0·9795)x (totalvariance, r 2 = 0·70, P < 0·001, difference P < 0·001); (b) y = 761 – 790(0·9963)x (r 2 = 0·67, P < 0·001). Betula, (c) y = 142 –153(0·9663)x (r 2 = 0·78, P < 0·001); (d) y = 246–251(0·9886)x (r 2 = 0·86, P < 0·001). Sorbus, (e) y = 125–135(0·9625)x (r 2 = 0·67,P < 0·001); (f ) control, y = 49(1·0133)x – 68; dormant clip, y = 57(1·0133)x – 67; bud burst clip, y = 45(1·0133)x– 64 (totalvariance, r 2 = 0·93, P < 0·001, difference P < 0·01).

Table 3. Effect of winter or spring clipping on leaf growth characteristics at final harvest§

Species Control Winter clipped Spring clipped SED df Significance

Pinus sylvestrisTotal 1997 needle mass 53·8c 36·0b 24·2a 0·29‡ 28 ***Mass per 97 needle pair 0·027a 0·051b 0·057b 0·167† 28 ***Number of needle pairs 1837c 723b 428a 2·63‡ 28 ***Needle/bud ratio 43·7 41·2 49·6 8·15 28 ns

Betula pendulaTotal leaf mass 15·2 13·2 15·1 0·17‡ 40 nsMass per leaf 0·020a 0·027b 0·028b 0·003 45 *Leaf area 2148 1819 1790 2·94‡ 40 nsArea per leaf 2·6a 3·6b 3·2ab 0·41‡ 40 *Leaf number 737b 474a 484a 51·0 59 ***Leaf/bud ratio 2·4a 2·8a 3·7b 0·29 59 **

Sorbus aucupariaTotal leaf mass 18·6 23·1 20·6 1·43 42 nsMass per leaf 0·21 0·28 0·26 0·057‡ 44 nsLeaf area 1610b 1675b 1155a 3·04‡ 41 *Area per leaf 37·8 49·4 35·9 0·57‡ 40 nsLeaf number 74b 47a 51a 0·61‡ 49 **Leaf/bud ratio 2·7 3·6 3·4 0·77 49 ns

†SED value for loge-transformed means. ‡SED value for square root-transformed means. §Final harvest 8 September for P. sylvestris; 3 September for B. pendula and S. aucuparia. All leaf /needle masses in g; leaf areas in cm2. Data are REML-adjusted means of five replicates. Where data were transformed for analysis (see superscript on SED), means have been back-transformed for clarity. Within each row means with the same superscript letter are not significantly different at P < 0·05. Significance values (least significant difference tests): ns, not significant; *, P < 0·05; **, P < 0·01; ***, P < 0·001.


540P. Millard et al.


fewer leaves being produced (P < 0·001), but the massper leaf and area per leaf were significantly greater(both P < 0·05). Thus there were no significant differ-ences in total leaf mass per tree (Table 3) or total Nuptake for leaf growth (Fig. 1) by the time of the lastharvest in September. Betula pendula also respondedto clipping by altering the leaf/bud ratio, with thespring-clipped trees producing significantly moreleaves per bud than the control or winter-clipped trees(P < 0·01; Table 3). The clipped S. aucuparia trees pro-duced significantly fewer leaves (P < 0·01), but areaper leaf and mass per leaf were highly variable and didnot differ significantly between treatments. Total leafarea per sapling was thus also highly variable, and wassignificantly smaller only in spring-clipped trees(P < 0·05). Total leaf mass did not differ significantlybetween treatments by the time of last harvest in Sep-tember, as per the few significant differences in total Nuptake for leaf growth (Fig. 1). Leaf/bud ratios did notdiffer significantly between treatments for this species,

although values for the clipped trees were greater thanfor the controls.

The amounts of unlabelled N recovered in the varioustissues of the trees by final harvest are shown inTable 4. Clipping had significantly (P < 0·05; Table 4)reduced the amount of unlabelled N in P. sylvestristrees and, as a consequence, reduced recovery in bothneedles (P < 0·05) and stem (P < 0·01) which had grownduring the year of clipping (1997), and in woody andfine roots (P < 0·05; P < 0·01). However, the propor-tion of total unlabelled N recovered in the 1997 needleswas not greatly altered by clipping, being 40% for thecontrol and 45% for both the clipped treatments. Incontrast, clipping had no effect on total N uptake byB. pendula, but very slightly increased the amountof unlabelled N allocated to the growth of leaves (P <0·05). Total N uptake by S. aucuparia was not sig-nificantly affected by clipping, but clipping in wintersignificantly reduced the amount of unlabelled N allo-cated to 1996 stem (P < 0·01) and new buds (P < 0·05).

Discussion

Many studies have considered the consequences ofherbivory on the carbon physiology of trees (Heichel &Turner 1983; Reich et al. 1993; Honkanen et al. 1999;Vanderklein & Reich 1999). Fewer studies have con-sidered the impacts of herbivory on their N physiology(Holopainen et al. 1995; Lovett & Tobiessen 1993).Our data suggest that the different growth response toherbivory of the evergreen species compared to thedeciduous trees was due to two main contrastingaspects of their physiology. First, storage of N inneedles that are susceptible to browsing resulted inP. sylvestris trees having less N available for remobil-ization after they had been clipped, whereas thedeciduous species were unaffected. Second, P. sylvestrishas a fixed pattern of growth whereas both B. pendulaand S. aucuparia had an indeterminate growth form,allowing compensatory growth.

In P. sylvestris, remobilization and root uptake wereconcurrent. By the time remobilization had finished,labelled N accounted for nearly half the N in the needlesof control trees. The fact that P. sylvestris relies to suchan extent on remobilization to provide N for needlegrowth might explain why Honkanen et al. (1999)found that defoliation of 1-year-old but not 2-year-oldneedles reduced the subsequent mass and length ofneedles growing on new shoots. These authors inter-preted this response as being due to an alteration ofshoot sink strength for carbon as a consequence ofdefoliation (Honkanen et al. 1999). Our data suggestthat the removal of N stored in the 1-year-old needles

Table 4. Uptake and partitioning of nitrogen by Pinus sylvestris, Betula pendula andSorbus acuparia following winter or spring clipping

Species Tissue

Unlabelled N content

Control clipped

Winter clipped Spring SED df Significance

P. sylvestris Buds 22b 14ab 8a 0·45† 15 *1997 needles 353b 295ab 221a 1·42‡ 28 *1996 needles 45b 30b 22a 0·22† 60 *1995 needles 3 6 24 1·28† 27 ns1997 stem 89c 61b 29a 0·74‡ 40 **1996 stem 51b 29a 24a 0·23† 59 *1995 stem 38 39 29 0·24† 59 nsWoody roots 57b 36a 30a 0·54‡ 60 *Fine roots 224a 141ab 110a 0·27† 58 **Total 882b 651a 497a 0·18† 58 *

B. pendula Buds 15 16 18 3·01 8 nsLeaves 154a 182ab 196b 18·2 45 *1997 stem 31 31 25 0·6‡ 28 ns1996 stem 36 26 35 0·7‡ 60 ns1995 stem 35 36 26 0·25‡ 60 nsWoody roots 248 189 172 0·27† 60 nsFine roots 330 388 393 0·29† 59 nsTotal 849 868 865 1·5‡ 60 ns

S. aucuparia Buds 5b 3a 6b 0·29† 9 *Leaves 380 407 318 0·38† 44 ns1997 stem 111 126 142 0·27† 42 ns1996 stem 27b 14a 27b 0·46‡ 66 **1995 stem 161 122 144 0·32† 66 nsWoody roots 133 82 90 0·34† 66 nsFine roots 145 121 177 0·29† 66 nsTotal 962 872 904 0·3† 66 ns

†SED value for loge-transformed means. ‡SED value for square root-transformed means. Values are given for unlabelled N content (mg per tree) of tissues from final harvest of the experiment (8 September for P. sylvestris; 3 September for B. pendula and S. aucuparia). Data presented are REML-adjusted means of five replicates. Where data were transformed for analysis (see superscripts on SED), means have been back-transformed for clarity. Within each row, means with the same superscript letter are not significantly different at P < 0·05. Significance values (least significant difference tests): ns, not significant; *, P < 0·05; **, P < 0·01; ***, P < 0·001.




could also be an important factor in determining thesubsequent growth response of new foliage.

Clipping also reduced N uptake by P. sylvestris dur-ing the summer, while allocation patterns within thetree were unaltered. This effect on N uptake might befurther exacerbated under field conditions, as above-ground herbivory can reduce both fine root produc-tions (Ruess, Hendrick & Bryant 1998) and the extent ofroot colonization by mycorrhizal fungi (Rossow, Bryant& Kielland 1997). As a consequence, less N was recoveredin the current year’s needles at the end of the summer,which in turn would probably mean that less N wouldbe available for remobilization the following year.

Remobilization of N is unaffected by the currentN supply to a range of tree species, being dependentonly on the amount of N in store (Millard 1996). Thissuggests that the process of remobilization is source-rather than sink-driven. The present study providedfurther evidence for remobilization by B. pendula beingsource-driven. Betula pendula is heterophyllous, andwinter buds contain only a limited number of leafprimordia which form the first population of ‘early’ leaves(Maillette 1982). We found that this initial populationof early leaves remained constant until after the harv-ests taken on 12 May, 99 ± 2·3 days from bud burst.This corresponded with the time that remobilizationfinished. The control trees had produced 606 ± 3·2leaves per tree, and the winter- and spring-clippedtrees 329 ± 2·4 and 348 ± 4·1, respectively (control versusclipped, P < 0·001). The amount of N remobilized perleaf was 0·23 mg per leaf in the control trees, and 0·43and 0·41 mg per leaf for the winter- and spring-clippedtrees, respectively (control versus clipped; P < 0·01).No such relationship between bud and initial leafnumbers and the amount of N remobilized was foundfor S. aucuparia. However, the fact that leaf growthproceeded for some 22 days before N from root uptakewas recovered in the leaves, and remobilization wasunaltered by clipping, suggests that remobilization islikely to be source-driven in this species as well.

Nitrogen was stored by the deciduous trees inorgans which were unaffected by clipping. In addition,as a source-driven process, remobilization providedthe same amount of N in total for the buds remainingafter clipping, allowing the fewer leaves that grew tocontain more N and to have a greater area per leafcompared to the control trees (except for the spring-clipped S. aucuparia). The compensatory growth whichboth species were able to produce also meant that clip-ping had no effect on N uptake during the summer,with the amount of N allocated to storage also beingunaffected. It is likely therefore that the amount of Navailable for remobilization the following year afterdamage would have been unaffected by the clipping.

Pinus sylvestris produces only preformed buds, and sohas a fixed pattern of growth (Rook 1985). However,

P. sylvestris is capable of compensatory growth in responseto herbivory, although these responses depend on thetiming and severity of the defoliation. For example,Honkanen et al. (1994) found that branch defoliationretarded the growth of foliage in whorls above thedefoliated branches, but produced a positive effect ongrowth of needles below. Debudding resulted in noshoot extension and, as a consequence, existing needlesincreased in both length and mass (Honkanen et al.1994). Edenius, Danell & Bergström (1993) reportedthat P. sylvestris was able to compensate for needle loss,but that regrowth was delayed in the year followingintense clipping, which they suggested was a mechanismby which trees were temporarily released from furtherherbivory. We found that in the growing season follow-ing clipping P. sylvestris was unable to increase thenumber of needle pairs produced per bud, but thatthere was an increase in the mass of needles growingfrom the remaining buds.

Betula pendula saplings responded to removal ofnearly half their buds by increasing both the area ofindividual leaves and the number of leaves producedper bud. However, the timing of our clipping treatments(at dormancy and bud burst) was important in deter-mining these responses. Ovaska, Walls & Vapaavuori(1993a) found that saplings of B. pendula partiallydefoliated after some 4 weeks’ leaf growth were incap-able of compensatory growth, which they ascribed toa decreased total carbon gain, despite upregulationof photosynthesis in the remaining leaf area (Ovaskaet al. 1993b). In contrast Danell & Bergström (1989)applied clipping treatments to B. pendula before budburst and, as in the present study, found no effect onshoot biomass.

A proportion of the buds produced by B. pendularemain dormant during spring and summer, and con-tribute to the population of buds for the next year(Maillette 1982). Our clipping treatments removedapical buds and so will have reduced apical dominance(Senn & Haukioja 1994), potentially affecting the sub-sequent growth of axillary buds, as found by Collinet al. (2000) for Fraxinus excelsior. Clipping to removeeither buds or whole shoots of Fraxinus pennsylvanicawas shown by Davidson & Remphrey (1994) to increaseneoformed leaf production relative to control trees,thereby allowing them to re-establish their leaf areaquickly after injury. Our data do not show if the com-pensatory growth we found in B. pendula was due to:(i) neoformed growth within existing buds; (ii) a decreasein the proportion of buds remaining dormant; or(iii) the production of new buds which, in turn, grewnew leaves. However, given that compensatory growthby B. pendula is apparently not possible if defoliationoccurs after the initial phase of leaf growth (Ovaska et al.1993a), this suggests that the response we measuredwas unlikely to be regulated by the carbon balance of theplants. The same was probably true for S. aucupariasaplings, which also exhibited compensatory growth inresponse to clipping.


542P. Millard et al.


It is likely that the consequences of large mammal her-bivory on P. sylvestris will last for several years, because:(i) clipping reduced growth that year and resulted infewer buds being set in late summer (Rook 1985) toprovide for growth next year; and (ii) the slower rate ofN uptake after clipping resulted in there being less Navailable for remobilization the following year. In con-trast, the two deciduous species showed considerableplasticity in their growth response to clipping, suchthat neither bud numbers nor N allocated to storagethe following winter was much affected. Both specieshad almost recovered from the effects of simulated her-bivory by the end of the summer. The site of N storageduring the winter therefore appears to be an importantfactor in determining the response of a tree species toherbivory.

The carbon/nutrient balance hypothesis (Bryantet al. 1983; Bryant et al. 1991) suggests that speciesadapted to fertile sites will respond to herbivory byutilizing stored resources for compensatory growth,whereas slower-growing species adapted to nutrient-poor sites will instead protect their leaves by investinga greater proportion of their carbon in antiherbivorydefence compounds. Trees whose growth is limited bynutrient availability rely on the internal cycling ofnutrients for growth to a greater extent than those wellsupplied with nutrients (Millard 1996). Long-lived, slow-growing evergreen species are commonly found in themost nutrient-deficient sites, and because they store Nin their foliage it is susceptible to loss by herbivory. Inaddition to protecting the leaves per se, we suggest thatantiherbivory compounds may serve primarily to pro-tect the nutrients stored within them.

Acknowledgements

We thank A. Midwood for the 15N analyses and theScottish Executive Rural Affairs Department for fund-ing the research.

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Received 30 October 2000; accepted 29 March 2001


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Interspecific defoliation responses of trees depend on sites of winter nitrogen storage