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Environmental and Experimental Botany 81 (2012) 72– 78

Contents lists available at SciVerse ScienceDirect

Environmental and Experimental Botany

journa l h o me pa g e: www.elsev ier .com/ locate /envexpbot

atent soil effects of grazing and ammonium deposition on Deschampsia flexuosaillers inserted and grown in heather moorland soil

osalind Jonesa, David Caustona, Bridget Emmettb, Luis Mura, Dylan Gwynn-Jonesa,∗

Institute of Biological, Environmental and Rural Science, Aberystwyth University, Ceredigion SY23 3DA, UKCentre for Ecology and Hydrology (CEH) Bangor, Environment Centre Wales, Deiniol Road, Bangor LL57 2UW, UK

r t i c l e i n f o

rticle history:eceived 6 July 2011eceived in revised form 7 February 2012ccepted 15 February 2012

eywords:itrogen depositionrazingeschampsia flexuosaioindicatoretabolic fingerprinting

TIR

a b s t r a c t

Upland heather moorlands support a range of increasingly rare and threatened biota, making them apriority habitat for conservation and restoration. Over-grazing and nitrogen deposition are two of themost important threats to maintaining these heather moorlands, yet there remains a paucity of researchinto their combined long-term effects. During the summer of 2008, we established an experiment withinan existing research site at Pwllpeiran, mid-Wales. The original site, established in 1996, investigatedlong-term grazing and N deposition treatments (ammonium and nitrate) on heather moorland. Previousfindings from the site, following a decade of treatment, suggested no significant, detectable impacts oftreatments on soil C:N ratio and the foliar nitrogen of vegetation.

The aim of our study was to investigate short- (2008) and long-term (1996–2008) N deposition treat-ment impacts, in combination with past grazing (1990–2007), on soil nutrient bioavailability. Soil coreswere harvested and aboveground vegetation removed. Tillers of the grass Deschampsia flexuosa wereplanted into these cores which were then reciprocally transplanted back into the field experiment. TheN deposition treatment was continued but grazing was excluded. D. flexuosa biomass changes were nextassessed and leaf chemistry investigated using the metabolic fingerprinting method Fourier-transforminfrared spectroscopy (FTIR) following three months of growth in the field (May–August 2008).

Grazing treatment (on its own) had significant negative impacts on aboveground biomass and signifi-cant changes in plant chemistry were also revealed through the metabolic fingerprinting method Fouriertransform infrared spectroscopy (FTIR). Short-term N deposition treatments during 2008 had no impacts

on D. flexuosa growth or chemistry. There were also no detectable latent effects of long-term nitrate treat-ments on either growth or chemistry of D. flexuosa. However, plants grown in plots that had receivedlong-term treatments of ammonium (NH4

+) had significantly lower poly-phenolic contents (revealed byFTIR) than plants grown in either nitrate (NO3

−) or control plots, suggesting detectable latent effects of Napplication in its reduced form. Further work needs to be undertaken to assess the relevance of residualsoil nitrogen pools post N deposition and grazing.

. Introduction

Heather moorland is characterised as a mosaic of semi-naturalwarf-shrub heaths, bogs and grassland (Bardgett et al., 1995).s a result these are diverse habitats which support a range ofpecies, and some of the rarer habitat classes are protected underC ‘Habitats Directive’ 92/43/EEC (Thompson et al., 1995). Grazingas historically been used as a management tool for maintain-

ng heather moorland habitats (Thompson et al., 1995; Worrall

nd Adamson, 2008; Clay et al., 2009). Since the Second Worldar, changes in management practices (focussed on increasingoorland productivity) have resulted in degradation and loss of

∗ Corresponding author.E-mail address: dyj@aber.ac.uk (D. Gwynn-Jones).

098-8472/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2012.02.006

© 2012 Elsevier B.V. All rights reserved.

such habitats (Bardgett et al., 1995; Ross et al., 2003). Governmentsubsidies for cutting drainage ditches, and European Common Agri-cultural Policy (CAP) funding for increasing stocking densities wereamongst key drivers for this change (Holden et al., 2004, 2007).

Declines in the heather species Calluna vulgaris have been asso-ciated with over-grazing (Alonso et al., 2001) and increased grasscover (Thompson et al., 1995). Causes include extraction of plantmaterial, high stocking densities impacting soil structure and sitehydrology (Archer, 2007; O’Connell et al., 2007; Marshall et al.,2009). Futhermore, compaction and reduced porosity increase sur-face run-off and compound flooding during heavy rainfall events(Langlands and Bennett, 1973; Meyles et al., 2006; Marshall et al.,

2009).

The effects of heavy grazing, in combination with increasednitrogen deposition (primarily from anthropogenic sources), canprovide suitable conditions for nitrophilous grasses, such as Molinia

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aerulea, to invade and dominate large areas (Alonso et al., 2001).he resultant loss of biodiversity and habitat change can, in someases, occur at a rate that is too fast or at a scale that is too largeor mitigation measures to be implemented (Heil and Diemont,983). Heather moorland, for example, may be converted to acidrassland when over-grazed and subjected to nitrogen deposition.vidence to support this has been shown experimentally (Hartleynd Mitchell, 2005) and at a national level (Smart et al., 2005;askell et al., 2010; RoTAP, 2010).Previous studies have shown that grasses are more tolerant

f grazing than dwarf shrubs (Alonso and Hartley, 1998). Shrubpecies such as C. vulgaris are sensitive to grazing but when grazings prevented they can dominate as their canopies limit light avail-bility for the germination and development of competing grasseedlings (Alonso et al., 2001).

Nitrogen deposition in excess of Critical Loads (CLs) is anothermportant driver of degradation in such heather moorland commu-ities. Many studies, both at the experimental and wider landscapecales, have shown that N deposition has resulted in damage andabitat change (e.g. Arróniz-Crespo et al., 2008; Härdtle et al.,009; Maskell et al., 2010; RoTAP, 2010), and the forms of nitro-en deposited will dictate effects. Biologically, ammonium (NH4

+)s more readily accumulated in heather moorland communitieshan nitrate (NO3

−). It has therefore been suggested that the pol-uting effects of ammonium could prove longer lasting and have areater impact than nitrate (RoTAP, 2010). The effects of the dif-erent forms of deposited nitrogen may be further complicated bydaphic and climatic factors. For example, nitrate is more readilyeached from the soil, particularly in areas of high rainfall, suchs those where upland heather moorland is found (Evans et al.,008). However, ammonium can be retained in the soil (particu-

arly if acidic) gradually promoting nitrophilous grasses to occupyormerly low-nutrient habitats such as heather moorlands. Theseffects are exacerbated in the presence of grazing which opens andisrupts the dwarf shrub canopy (Alonso and Hartley, 1998).

An experiment was established in mid Wales (Pwllpeiran) in996 to explore the interactive effects of long-term grazing andimulated N deposition on soil and community (Emmett et al.,007; RoTAP, 2010). Following a decade of exposure to the treat-ents, Emmett et al. (2007) carried out a suite of measurements

ncluding vegetation composition, soil C:N ratio or foliar N of veg-tation. Their results suggested that biomass was lower whererazing had been more intense but there were no detectablempacts of N on plant growth and foliar nitrogen concentrationsf existing vegetation. They also found no effects of N depositionn soil C:N ratio (Emmett et al., 2007). However, gaps remain inur understanding of plant community level responses to N deposi-ion, particularly our understanding of potential nutrient retentionn the soil and implications to plants.

New metabolic fingerprinting methods that examine plant andoil samples have shown promise as tools to help understand theesponses of these systems to N deposition (Kalaitzidis et al., 2008;idman et al., 2005). Such methods require small tissue samplesnd can be used to assess metabolite changes in plants across largepatial scales via analysis of multivariate data sets using power-ul multivariate statistical methods (Fiehn, 2002). For example,iological samples can be analysed by Fourier transform infraredpectroscopy (FTIR), which exploits variability in the vibrations ofifferent bonds when different wavelengths in the infrared spec-rum are simultaneously directed onto a sample (see Gidman et al.,005). Comparison of the collected spectra can be used to screenignificant differences between plants subjected to different treat-

ents, in terms of functional groups, thus providing insight into

he metabolite responses.To specifically assess the short- and longer-term relevance of

deposition (ammonium and nitrate) and grazing via the soil,

rimental Botany 81 (2012) 72– 78 73

we exploited and manipulated the extant experimental design atthe Pwllpeiran site (see Emmett et al., 2007). From this experi-ment, we extracted soil/community cores from each treatment andexisting aboveground biomass was harvested, dried and weighed.Deschampsia flexuosa tillers were next planted into the soil coreswhich were reciprocally transplanted across the experiment. Thecores had previously been part of a long-term experimentalset up in which treatment plots received regular N additions(1996–2007, ammonium or nitrate) and grazing (1990–2007),some also received a single dose of phosphorus (2000). Coresincluding tillers of D. flexuosa were maintained in the field exper-imental treatments for three months. Ammonium and nitratetreatments were continued but grazing was prevented during the2008 field study.

Our primary objective was to assess the long-term effects of pastgrazing (1990–2007) and nitrogen treatments (1996–2008) on theexisting aboveground biomass in the plots. Our first hypothesis,therefore, was that N addition would have a positive effect on exist-ing vegetation and grazing would have a negative impact (due to theeffects of defoliation and of soil compaction by grazing herbivores).By extracting vegetation and inserting D. flexuosa tillers into the soilcores we would test for residual treatment effects within the soil.We hypothesised that previous grazing would have negative effectswhilst past N additions would have positive effects on the growthof D. flexuosa. Past treatments would also affect foliage metabolites.Overall, we expected that the source of nitrogen applied would dic-tate metabolic and growth responses of the plants, with ammoniumhaving stronger effects as it is more likely to be retained in the soilthan nitrate at a low pH in such moorland systems.

To test these hypotheses we measured aboveground biomassof D. flexuosa plants after three months of exposure and analysedleaf tissue via the metabolic fingerprinting method Fourier trans-form infrared spectroscopy (FTIR; as described by Gidman et al.,2005). This method was specifically employed due to its capacity toscreen for differences in D. flexuosa across the experimental designand, where differences occurred, to allow identification of potentialmetabolites responsible for this variation. By using this method weaimed to explore the significance of residual and recent N depositson D. flexousa growth and metabolites.

2. Materials and methods

2.1. Experimental set up

The original long-term experiment employed for this studywas at Pen y Garn, Pwllpeiran on moorland habitats (GR: SN 798772), near Cwmystwyth in mid-Wales and consisted of two pad-docks, one with high grazing pressure of 1.5 sheep ha−1 (equatingto 1095 sheep days) and the other with low grazing pressure of1.0 sheep ha−1 (730 sheep days) established in 1990 (see Emmettet al., 2007). Nitrogen treatment plots were added to both grazingregimes in 1996 to gain an understanding of the combined effects ofnitrogen and grazing on a heather moorland community. The siteis located 300 m a.s.l. and receives annual rainfall of ∼2000 mm.Within each paddock three replicate blocks were set up, each con-taining four nitrogen treatment regimes.

Treatment plots within each block were fully randomised: therewere four replicates of four treatments consisting of a control(CONT, rain water addition alone), ammonium sulphate applied at10 kg N ha−1 yr−1 (AS10), ammonium sulphate at 20 kg N ha−1 yr−1

(AS20) and sodium nitrate at 20 kg N ha−1 yr−1 (SN20). In June 2000,AS10 plots received a single phosphorous treatment of 20 kg P ha−1

in the form of sodium dihydrogen orthophosphate. This providedthe opportunity to further assess the effects of N deposition (evenat the relatively low rate of 10 kg N ha−1) in relation to P addition(see Emmett et al., 2007, for full details).

7 d Experimental Botany 81 (2012) 72– 78

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Table 1ANOVA of biomass of D. flexuosa plants exposed to seasonal (3 months) nitrogentreatments (‘Nitrogen’) and grown in soil previously subjected to different nutrient(‘Core’) and grazing (‘Grazing’) regimes at Pwllpeiran field site.

Factor DoF SS MS F p

Main plotGrazing 1 0.515 0.515 17.942 0.001***

Block in Grazing 4 0.023 0.012 0.400 0.805Nitrogen 3 0.010 0.033 1.157 0.366Grazing × Nitrogen 3 0.009 0.003 0.098 0.960Block × Nitrogen in Grazing 12 0.172 0.029 – –

Sub-plotCore 3 0.082 0.023 1.087 0.356Grazing × Core 3 0.161 0.036 1.410 0.241Block × Core in Grazing 12 0.128 0.021 0.847 0.602Nitrogen × Core 9 0.197 0.022 0.868 0.555Grazing × Nitrogen × Core 9 0.064 0.007 0.280 0.980Residual 212 2.689 0.025 – –Total 271 4.085 – – –

4 R. Jones et al. / Environmental an

For the current study, in May 2008, four soil cores were removedrom each treatment plot within the above experiment and recip-ocal transplants made within the same block so there was one soilore for each of the four different treatments in each plot. Eachore was 20 cm deep with a diameter of 10 cm, enclosed withineavy-duty PVC tubes that were free draining. Before placing thexperimental cores back into the field site the existing abovegroundegetation biomass was harvested and the number of speciesecorded (according to whether they were monocotyledonous,icotyledonous or bryophytes). Dry weights were measured fol-

owing a 48-h period of drying at 50 ◦C.Three mini soil cores (2.5 cm in diameter) were removed from

ach of the extracted main cores for planting of the test species. flexuosa L. Tillers were taken from potted plants growing in alasshouse and inserted in Levingtons John Innes seed compost (aow nutrient medium containing 100 mg N l−1) in 2.5 cm diametereed plugs for two weeks in a misting glasshouse to allow rootso establish. These were next placed out of doors for two weekso harden. By using only small plugs to establish plants we min-mised the amount of compost transferred into the experimentalite and at the same time ensured optimal root growth. At the timef transplanting, plants were chosen so that they were similar inize (2–4 cm tall) and had uniform tiller numbers (three tillers). Tis-ue nitrogen was not measured throughout the study due to smallmounts of available tissue and our focus on D. flexuosa metabolitend growth responses.

Three plants were transplanted into each core (with the excep-ion of one block where only two plants were available due to plant

ortality). Once all of the grasses were planted, wire hanging bas-ets (30 cm in diameter) were upended over the cores and held inlace using metal pegs to prevent direct grazing of the plants byheep. The mesh size of the baskets was small enough to preventisturbance by sheep but caused minimal shading. The plants werehen left to grow under natural conditions for a period of three

onths, until the 28th August, at which point D. flexuosa above-round biomass was harvested and dried for 48 h at 50 ◦C theneighed.

.2. FT-IR analyses of D. flexuosa leaf tissue

Following drying, a sub-sample of D. flexuosa leaf tissue fromach core (200 mg ± 10%) was ground to a fine powder in a ball millnd prepared for FTIR analysis. Water was added to each sampleo form slurry of 90 �g−1 �l−1. A 5 �l aliquot of each sample wasdded, in a randomised order, to a pre-designated well on a siliconample carrier plate. Once all samples had been added the plateas dried at 50 ◦C for 15 min until all samples were fully dry.

Fully automated collection of FTIR reflectance/absorbance spec-ra was carried out following recognised protocols as per Johnsont al. (2007) and updated for the use of silicon carrier plates by Rinut al. (2007).

.3. Statistical analyses

The main experiment consisted of four factors: grazing (at twoevels), block (at three levels), nitrogen applied (at four levels) andistorical nitrogen in soil cores (at four levels). The biomass couldherefore be assessed by analysis of variance (ANOVA) and the FTIRata by multiple analysis of variance (MANOVA) using MiniTabRelease 12.23) and MatLab (Version 7.0.4.) as described by Johnsont al. (2007).

Once all of the values had been calculated and a summary of theANOVA produced, the factors whose interactions were significantere submitted to canonical variate analysis in CANVAR (FORTRANrogram written by Dr. D.R. Causton, Aberystwyth University). The

Significance shown as *p ≤ 0.05, **p ≤ 0.01.*** p ≤ 0.001.

outputs of these analyses were then plotted in MatLab, with 95%confidence circles, so that differences could be visualised.

3. Results

3.1. Pre-insert vegetation: species counts and abovegroundbiomass

We counted the number of original monocotyledonous anddicotyledonous species from the four harvested cores for eachplot to assess any detectable effects of nitrogen, grazing or bothon higher plant species. A Bartlett’s homogeneity of variance testcarried out for block against the responses revealed homogeneitywithin the data.

There were no significant differences (p > 0.05) for dicotyle-donous species number in relation to past grazing, nitrogentreatment or their interactions (data not shown for brevity). How-ever, the number of monocotyledonous species was significantlylower in the ammonium plus phosphorus treatment, but only inthe less intensely grazed paddock (F(3,11) = 5.23, p = 0.027, Fig. 1a).

We also analysed total plant biomass from the initial core vege-tation and ANOVA revealed a significant and unsurprising negativeeffect of grazing (F(1,84) = 3.986, p = 0.049) and positive nitro-gen treatment effect (F(3,84) = 3.423, p = 0.021) but there was nosignificant interaction (F(3,84) = 1.868, p = 0.141). Data presentedshow the nitrogen plus phosphorus response (Fig. 1b) and high-light increased biomass resulting from the higher proportion andgrowth-rate of bryophytes.

3.2. D. flexuosa biomass following growth in cores and exposureto nitrogen applications for three months

The final aboveground biomass data of D. flexuosa plants in eachcore was analysed via ANOVA. The results showed that only grazinghad a significant impact on the biomass of D. flexuosa (Table 1).However, this was a residual effect of previous grazing as exclosureshad been installed around the grasses to prevent herbivores eatingthem during the three-month duration of the experiment.

3.3. Metabolite fingerprinting of D. flexuosa leaves followingexposure to nitrogen and grazing treatments

The FTIR data were reduced to eight principal components (PCs)which explained 99% of total explained metabolomic variation.These were then used to carry out a MANOVA that indicated a signif-icant grazing effect plus a residual (historical) effect of nitrogen on

R. Jones et al. / Environmental and Experimental Botany 81 (2012) 72– 78 75

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. flexuosa metabolites via the soil cores (‘Core’, Table 2). The short-erm (3 months) addition of nitrogen treatments had no significantffects (either individually or interactively with other treatmentactors). Overall, results showed that application of relatively lowitrogen doses over a growing season were not detectable in theiomass (Table 1) or metabolic fingerprint (Table 2) of D. flexuosa.

A canonical variate analysis (CVA) was carried out using theC scores to better ascertain the differences in the metabolomicesponses of D. flexuosa to the soils that had received 12 years of

revious nitrogen treatment (Fig. 2). The CVA showed that the twoifferent ammonium treatments were distinct from the control anditrate treatments but not from each other. Although the CVA did

able 2ANOVA of FTIR metabolic fingerprints showing responses of D. flexuosa exposed to

easonal (3 months) nitrogen treatments (‘Nitrogen’) and grown in soil previouslyubjected to different nutrient (‘Core’) and grazing (‘Grazing’) regimes at Pwllpeiraneld site.

Factor DoF Wilk’s � F p

Main plotGrazing (8,5) 0.0992 5.677 0.036*

Block in Grazing (32,20) 0.0147 1.341 0.248Nitrogen (24,15) 0.0256 1.597 0.175Grazing × Nitrogen (24,15) 0.0289 1.507 0.207

Sub-plotCore (24,85) 0.2189 2.429 0.002**

Grazing × Core (24,85) 0.4358 1.170 0.293Block × Core in Grazing (96,206) 0.0472 1.228 0.114Nitrogen × Core (72,184) 0.1285 1.025 0.439Grazing × Nitrogen × Core (72,184) 0.1161 1.085 0.328

* p ≤ 0.05.** p ≤ 0.01.**p ≤ 0.001.

Fig. 2. Canonical variate analysis based on principal component scores derivedfrom outputs of FTIR analysis of D. flexuosa plants exposed to four differentsoil N treatments. CONT (blue) = control, AS10 + P (red) = ammonium sulphate at10 kg N ha−1 yr−1 plus a one-off application of phosphorus at 20 kg P ha−1 yr−1, AS20(brown) = ammonium sulphate at 20 kg N ha−1 yr−1, and SN20 (green) = sodiumnitrate at 20 kg N ha−1 yr−1. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)

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6 R. Jones et al. / Environmental an

ot show significant differences between the treatments it wouldot be expected to exactly reflect the MANOVA (CVA is equivalento a multiple range test used in association with an ANOVA, seeohnson et al., 2007). However, the implications were clear thatontrol and nitrate were different from the ammonium treatment.

In detecting responses to ammonium we next identified chem-cal bonds and/or functional groups by examining the primaryransformation matrix for the CVAs of grazing and nitrogen. PCs 3nd 5 contributed most variation to the significant ‘Grazing’ result.Cs 5 and 6 (on CV1) and 3 (on CV2) contributed most to the vari-tion for ‘Core’.

Means and standard deviations were calculated from the load-ngs of these PCs. Any wavenumber that had a loading more thanwo standard deviations either side of the mean was then chosenor further analysis, which was carried out in PyChem (Jarvis et al.,006). Peaks in the fingerprint region (∼900 nm−1) were excluded.elected peaks were compared with a table of characteristic bondbsorbances to putatively identify them. The peaks identified were:henolic (C O); d-glucopyranose; C S (bond); polymeric; (O H)henolic; (O H) phenols/alcohols; carbonyl group (CO); polysac-harides. The analysis was carried out again using only these peaks.indings from previous N deposition studies (e.g. Gidman et al.,005, 2006) indicated that amide regions should have contributedost significantly towards the observed results. However, a PC-CVA

arried out on the amide region (Fig. 3a) in the current experimentid not support this.

A second PC-CVA was completed based on the main peaks iden-ified (above) to assess how they related to the different treatmentsFig. 3b). Plant material from control plots showed some separa-ion from the nitrogen treatments but assessment of the loadingsor each of the seven sub-peaks on the PC-CVA (data not shown)evealed effects of treatment on the phenolic (C O) bond. Thisndicated that the phenolic compounds in D. flexuosa tissue weretrongly modified by the residual effects of nitrogen accumulatedn these soils.

. Discussion

Heather moorlands are important international habitats, hav-ng been awarded protection under European law (Thompson et al.,995), but still face the threat of degradation from anthropogenicources of nitrogen (Heil and Diemont, 1983; Maskell et al., 2010),ver-grazing (Thompson et al., 1995; Marshall et al., 2009) and theirombined effects (Alonso et al., 2001). Detecting and understand-ng how these perturbations impact on heathlands over long timecales is essential, particularly in relation to the future managementf such habitats.

Previous research at our study site suggested that followingong-term exposure to N, only subtle interactive effects (Graz-ng × N) were detectable on the cover of existing shrub and sedgeegetation, Vaccinium myrtillus and Carex pilulifera (Emmett et al.,007). From these observations it was suggested that the absence of

effects could either be due to leaching of the additional nitrogen,r because the species present had become insensitive or adaptedo the addition of nitrogen.

Before planting tillers of the grass D. flexuosa into the cores,xisting vegetation was harvested and weighed. Species numbernd plant biomass were determined and revealed that the long-erm (12 years) N treatments had little or no effect on the number oficotyledon species (Fig. 1a). However, in the low-grazed paddock,he plots where P had been added resulted in a significant reduction

n monocotyledonous species numbers, probably due to increasedrowth of bryophytes. This was also reflected by higher biomass inhese plots (Fig. 1b), mostly a result of increased bryophyte growth.his indicated that bryophytes, at least, were P limited in the

rimental Botany 81 (2012) 72– 78

presence of the nitrogen additions, but following the addition of P inthese paddocks were able to respond to the increase in both nutri-ents. Even though P was only added once to the system (in 2000),the bryophytes were able to retain and recycle nutrients efficientlyto maintain dominance within the plots where P was previouslyadded. There was also a significant negative effect of grazing onbiomass, which was lower in the paddock with the more intensivegrazing regime.

In the current study we further assessed residual effects of graz-ing and nitrogen via the soil using a typical acid grassland speciesD. flexuosa. We hypothesised that there would be a long-term neg-ative residual effect of grazing on grass growth and alterations inplant chemistry that would arise from past defoliation and soil com-paction effects (Lipiec and Stepniewski, 1995). Results suggestedthat the aboveground biomass of D. flexuosa plants was significantlylower in the high-grazed paddock compared to the low-grazedpaddock (Table 1). This result is supported by earlier findings thatbiomass was lower where grazing had been more intense (Emmettet al., 2007). However, in this instance the grasses themselves werenot directly grazed, indicating a residual effect of grazing on the soil,which in turn affected the growth of the vegetation. It is possiblethat the removal of nutrients from the system by grazers, and theassociated trampling, resulted in less favourable growth conditionsfor these plants compared to those in the lower grazed paddock.

We further hypothesised that D. flexuosa tillers would respondto short- and long-term effects of nitrogen but that in the longerterm this would be dictated by the source of nitrogen used. Weexpected residual effects of ammonium but no long term effects ofthe nitrate treatment as it is more likely leached from such systems.Previous residual nitrogen application for 12 years (1996–2008)and seasonal N application (2008) to the soil cores did not cause achange in the biomass of D. flexuosa (Table 1). This suggested thatany nutrients remaining in the soil from past treatments, or thoseapplied during the three months of the experiment, did not elicit agrowth response in this species.

D. flexuosa has an inherently slow growth rate (Van de Vijver etal., 1993) and may not have the capacity to respond to additionalnutrient availability during the three-month period of the studyin 2008. However, plants in the control plots were visibly redder(likely to be a result of higher anthocyanin levels) than those inthe nutrient treatment plots. Hence plants were responding to thetreatments. A carryover nutrient effect (via nutrients from the pre-grown tillers and their associated soil prior to insertion into the soilcores) overriding treatment effects on growth is therefore unlikely.Anthocyanin accumulation is often observed when plant nutrientsare limited (Hilbert et al., 2003; Diaz et al., 2005) and this maysuggest that control plants were limited by soil nutrient availabilityin the original soil cores. This was in contrast to the ammoniumtreatment plants which did not show these visible signs.

The study employed FTIR to interrogate any metaboliteresponses to the various treatment combinations on D. flex-uosa. Results showed that grazing effects were detectable inthe metabolic fingerprints analysed (Table 2). This supports ourhypothesis that there would be altered chemistry in response to ahigher intensity of grazing, even though the plants were not directlygrazed.

FTIR analyses also revealed significant metabolite differencesaccording to nitrogen treatment. However, these results only par-tially confirmed our experimental hypothesis, that all nitrogentreatments would be detected in the metabolic fingerprints of theplants: CVA of metabolite data revealed that both of the ammo-nium treatments were different from the nitrate and control (Fig. 2).

Although the nitrate was applied long-term at a rate of 20 kg N ha−1

it was not possible to detect an effect relative to the control. Thiswas probably the result of soil leaching of nitrate as this has beenobserved and measured experimentally at this site in the past

R. Jones et al. / Environmental and Experimental Botany 81 (2012) 72– 78 77

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ig. 3. PC-CVA showing effects of different nitrogen treatments in D. flexuosa on:wo standard deviations from the mean. Nitrogen treatments were CONT (+) = conthosphorus at 20 kg P ha−1 yr−1, AS20 (�) = ammonium sulphate at 20 kg N ha−1 yr−

Emmett et al., 2004), and elsewhere in other studies (e.g. Evans etl., 2008). In contrast, ammonium is more typically found in suchcidic heather moorland soil (Emmett et al., 1998) and thereforeotentially accessible to plants in the longer term.

To gain a more complete understanding of the long-term effectsf the ammonium treatment, further investigation of the FTIR dataevealed which metabolites in the grasses were most affected byhe ammonium treatments. Previously Gidman et al. (2005, 2006)ndicated that compounds possessing amide bonds were amongsthe most significantly affected metabolites in ammonium treatedeath plants. However, our results did not agree with this pre-ious work (Fig. 3a). Seven other chemical bonds were found toespond to the treatments and responsible for control and nitro-en treatment differences (Fig. 3b). Peaks identified in the spectra,hich were most strongly associated with the nitrogen treat-ents, were related to bonds within secondary metabolites such

s phenolics and the most strongly affected was the C O bondhenolic compounds. Our study supports previous observationshere phenolic compound concentrations have been found toecrease when nutrients are applied (Balsberg Påhlsson, 1992).ur field observations of visible reddening of leaves of D. flex-osa plants in the control plots relative to those treated withmmonium nitrogen adds some observational support to this con-lusion. We believe that such reddening was due to anthocyanin,

phenolic pigment that has been shown to be lowered in plantshen exposed to N fertilisation (Hilbert et al., 2003; Diaz et al.,

005).This study evaluated D. flexuosa for detecting short- and long-

erm deposition of nitrogen in combination with varying historicalrazing intensities on an upland degraded heather moorland using

mides, and (b) bonds identified from wavenumbers that had loadings more than10 + P (©) = ammonium sulphate at 10 kg N ha−1 yr−1 plus a one-off application of

SN20 (�) = sodium nitrate at 20 kg N ha−1 yr−1.

a series of reciprocal soil core transplants. Results suggestedresidual effects of the higher grazing regime existed, causing sig-nificant reduction in biomass of D. flexuosa compared to the lowergrazed regime. There was no detectable effect of either the longor short-term nitrogen treatments on the biomass of D. flexuosaduring a three-month experimental period, contrary to expecta-tions. However, metabolic fingerprinting results did show an effecton the metabolic signatures of the grasses with lower leaf poly-phenolic content in ammonium-treated plants versus control andnitrate treatments. These data highlight the importance of histor-ical residual soil ammonium at such heather moorland sites plusscope for plant metabolomic approaches to provide insight intovegetation response to past N deposition.

Acknowledgements

The authors would like to thank ADAS Pwllpeiran for assistancewith maintaining the treatments at Pwllpeiran field site and JulieHirst for assistance with fieldwork. RJ was supported by a NaturalEnvironment Research Council Ph.D. CASE studentship in associ-ation with the Centre for Ecology and Hydrology. We thank AlanJones and John Scullion for useful comments on the manuscript.

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