changes in plant community structure and soil biota along soil nitrate gradients in two deciduous...

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ARTICLE IN PRESSG ModelEDOBI 50395 1–7

Pedobiologia xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Pedobiologia - Journal of Soil Ecology

j ourna l homepage: www.elsev ier .de /pedobi

hanges in plant community structure and soil biota along soil nitrateradients in two deciduous forests

atja Steinauera,∗, Sharon Zytynskab, Wolfgang W. Weisserb, Nico Eisenhauera

Friedrich Schiller University Jena, Institute of Ecology, Dornburger Str. 159, 07743 Jena, GermanyTechnische Universität München, Terrestrial Ecology, Department of Ecology and Ecosystem Management, Center for Life and Food Scienceseihenstephan, Hans-Carl-von-Carlowitz-Platz 2, 85354 Freising, Germany

r t i c l e i n f o

rticle history:eceived 2 July 2013eceived in revised form 20 January 2014ccepted 26 January 2014

eywords:arthwormsertilizationicrobial growth

lant community compositionoil microorganisms

a b s t r a c t

Anthropogenic nitrogen (N) deposition is a serious threat to biodiversity and the functioning of manyecosystems, particularly so in N-limited systems, such as many forests. Here we evaluate the associationsbetween soil nitrate and changes in plant community structure and soil biota along nitrate gradientsfrom croplands into closed forests. Specifically, we studied the composition of the understory plant andearthworm communities as well as soil microbial properties in two deciduous forests (Echinger Lohe (EL)and Wippenhauser Forst (WF)) near Munich, Germany, which directly border on fertilized agriculturalfields. Environmental variables, like photosynthetically active radiation, distance to the edge and soil pHwere also determined and used as co-variates. In both forests we found a decrease in understory plantcoverage with increasing soil nitrate concentrations. Moreover, earthworm biomass increased with soilnitrate concentration, but this increase was more pronounced in EL than in WF. Soil microbial growth afteraddition of a nitrogen source increased significantly with soil nitrate concentrations in WF, indicatingchanges in the composition of the soil microbial community, although there was no significant effect in
EL. In addition, we found changes in earthworm community composition along the soil nitrate gradientin WF. Taken together, the composition and functioning of forest soil communities and understory plantcover changed significantly along soil nitrate gradients leading away from fertilized agricultural fields.Inconsistent patterns between the two forests however suggest that predicting the consequences of Ndeposition may be complicated due to context-dependent responses of soil organisms.
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ntroduction

Forest edges have received much attention in ecology andcosystem management, since they represent a transition zoneetween the forest habitat and adjacent ecosystems. Surround-

ng areas such as pasture and cropland can trigger changes in thepecies composition and nutrient cycles across edges (Murcia 1995;ies et al. 2004; Harper et al. 2005). Additionally, differences inicroclimate occur between the two sides of the edge, which can

lso lead to changes in the rate of decomposition and nutrientobilization (Murcia 1995). Edges are also subjected to the influx

f chemical compounds from the atmosphere or via drift fromurrounding land (Thimonier et al. 1992; Wuyts et al. 2008). There-

Please cite this article in press as: Steinauer, K., et al., Changes in planin two deciduous forests. Pedobiologia - J. Soil Ecol. (2014), http://dx.d

ore, increased deposition of potentially acidifying and eutrophyingmmonium, nitrate and sulphate depositions at the edge has beeneported (Wuyts et al. 2008). Such influences result in differences in

∗ Corresponding author. Tel.: +49 3641 949419; fax: +49 3641 949402.E-mail address: [email protected] (K. Steinauer).

ttp://dx.doi.org/10.1016/j.pedobi.2014.01.007031-4056/© 2014 Published by Elsevier GmbH.

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© 2014 Published by Elsevier GmbH.

top soil properties and understory plant species composition withincreasing distance to the edge (Matlack 1994; Wuyts et al. 2011).

Nitrogen (N) is one of the key elements affecting the diversity,dynamics and functioning of terrestrial, freshwater and marineecosystems. Many organisms have adapted to low levels of N(Vitousek et al. 1997); moreover, the availability of N stronglyinfluences the growth and abundance of organisms (Vitousek andHowarth 1991). Anthropogenic N deposition sources, like agricul-ture or combustion of fossil fuels, thus exert strong effects onthe biodiversity and functioning of many ecosystems (Sala et al.2000). In N-limited ecosystems, such as forests, changes in herba-ceous layer biodiversity and composition are of special interestdue to their functional relevance and fast response to changes in Navailability (Tamm 1991; Gilliam 2006; Gilliam 2007; Bernhardt-Römermann et al. 2010).

Elevated N availability has often been reported to increase

t community structure and soil biota along soil nitrate gradientsoi.org/10.1016/j.pedobi.2014.01.007

plant productivity, but to decrease plant diversity by favoring thefew species that are most efficient in N uptake (Gilliam 2006;Harpole and Tilman 2007; Clark and Tilman 2008). For instance,comprehensive long-term studies within the BioCON experiment

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dx.doi.org/10.1016/j.pedobi.2014.01.007


http://www.sciencedirect.com/science/journal/00314056

http://www.elsevier.de/pedobi

mailto:[email protected]


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ARTICLEEDOBI 50395 1–7

K. Steinauer et al. / Ped

Reich et al. 2001) showed an increase in plant productivity inesponse to N addition, but a decrease in soil microbial biomassDijkstra et al. 2005; Eisenhauer et al. 2012), plant (Reich 2009)nd soil animal diversity (Eisenhauer et al. 2012). In contrast tohe above-mentioned studies, Bernhardt-Römermann et al. (2010)ound increasing functional diversity of plants with increasing soil

levels in the deciduous forest investigated in the present paper.owever, as typical for studies in terrestrial ecology, belowground

esponses to global change agents, such as fertilization effects, aretill less well explored. Moreover, it is unclear if fertilization ofgricultural fields influences the composition and functioning ofdjacent ecosystems.

Soil organisms play major roles in several ecosystem func-ions; for example, promoting plant productivity, regulatingutrient mineralization and promoting decomposition of organicatter (Neher 1999; Wardle et al. 2004). Thus, shifts in soil

ood web composition in response to fertilization may causeronounced alterations in ecosystem functioning. Given that dif-erent groups of soil microorganisms use different resourcesith respect to complexity and quality (Griffiths et al. 1999;ramer and Gleixner 2006), fertilization may cause compositionalhifts in communities of soil microbes and animals (Frey et al.004).

Although previous studies showed inconsistent responses ofoil organisms to N inputs (e.g., Niklaus and Körner 1996; Zakt al. 2000; Dijkstra et al. 2005), some general patterns tend tomerge. For instance, based on a meta-analysis, Treseder (2008)ypothesized that N input through fertilization negatively affectsoil microbial biomass due to one or more mechanisms includinglterations in soil pH, carbon and N availability, and below- andboveground productivity of plants. Further, N input may decreasehizodeposition fueling rhizosphere communities (Högberg et al.010; Eisenhauer et al. 2012). Treseder (2008) concluded that Nddition has overall negative effects on microbial biomass and thatong fertilization periods and large amounts of N even intensify theeduction of microbial biomass (Treseder 2008). Likewise, DeForestt al. (2004) reported decreased microbial enzyme activity after Nertilization, indicating shifts in the functioning of soil microbialommunities. Moreover, root colonization and species richness ofycorrhizal fungi have been reported to decrease due to higher

availability (Egerton-Warburton and Allen 2000; Lilleskov et al.002a,b).

Responses of different groups of soil organisms to fertilizationay however differ. Several experiments have shown that both

rganic and inorganic N fertilizers can have beneficial effects onarthworm populations (Edwards and Lofty 1982; Timmermant al. 2006). For example, higher nutrient availability throughrganic N has been show to increase earthworm biomass andumbers (Edwards and Lofty 1982; Hansen and Engelstad 1999),lthough extremely high levels of N input may inhibit their prolifer-tion (Haynes and Naidu 1998). The effects of inorganic N fertilizersan be explained by an increasing amount of plant material andhe subsequent higher amount of decomposing organic matterEdwards and Lofty 1982). Plant litter decomposition has long beenecognized as an essential process for organic matter turnover andutrient fluxes in most ecosystems. The subsequent release of car-on and nutrients represents the primary source of nutrients forlants and microbes (Berg and McClaugherty 2008). Rates of plant

itter decomposition and nutrient mineralization are, in turn, influ-nced by litter quality; higher nitrogen contents mostly enhanceecomposition.

Despite all this previous work, there is still a lack of under-


tanding of the responses of ecosystems to N addition effects (Westt al. 2006; Bardgett and Wardle 2010; Decaëns 2010). Most stud-es on high soil N concentrations are either based on permanentlot observations or on studies along environmental gradients

PRESSgia xxx (2014) xxx–xxx

(Brunet et al. 1998). The present study focuses exclusively onsoil nitrate gradients in forest ecosystems. We investigated theassociations between soil nitrate concentrations and plant com-munity properties, soil biota and functioning in forests that areadjacent to fertilized agricultural fields. To achieve this, we studiedunderstory plant community composition, earthworm communi-ties and soil microbial properties in two deciduous forests nearMunich, Germany. We used an observational approach to inves-tigate N gradients as was also done by Bernhardt-Römermannet al. (2007, 2010) in one of the forests investigated in thepresent study. Such gradients in soil nitrate concentrations couldarise from the drift and/or lateral flow of mineral N applied viafertilizers. Given inconsistent results in previous studies, we inves-tigated two forests differing in tree, understory plant and soilcommunity composition. We expected soil nitrate gradients lead-ing away from fertilized agricultural fields to be associated withsignificant changes in plant community composition and coveras well as in the diversity of soil organisms of adjacent foreststands (Bernhardt-Römermann et al. 2007). More specifically, wehypothesized an increase in diversity and productivity of plantcommunities (Bernhardt-Römermann et al. 2010), a decrease insoil microbial biomass (Treseder 2008) and an increase in earth-worm biomass (Edwards and Lofty 1982) with increasing nitrateconcentrations accompanied by significant alterations in soil pro-cesses.

Materials and methods

Study sites and sampling design

We used two study sites located in two different deciduousforests close to the city of Munich, Germany. The first one isthe ‘Echinger Lohe’ (EL), a nature reserve located 20 km north-east of Munich on the ‘Münchner Schotterebene’, a plain whichwas formed at the end of the last ice age. The texture of this Lep-tosol changes from carbonate-rich sandy soil toward humus-richsandy – loamy gravel with increasing distance to the edge. Theforest patch covers an area of about 24 ha and is surrounded byintensively used agricultural land. The dominating tree species areCarpinus betulus L., Quercus robur L. and Acer pseudoplatanus L., andthe understory vegetation is dominated by Colchicum autumnale L.,Anemone nemorosa L. and Carex alba Scop. The surrounding fieldsrepresent a diverse mixture of conventionally fertilized barley, ryeand potato fields.

The second forest site is the ‘Wippenhauser Forst’ (WF), whichis located north of Freising, approximately 40 km northeast ofMunich. The texture of this Cambisol is dominated by loess loamand sandy gravel belonging to the upper fresh water molasse (Ter-tiary). Similar to the EL, the WF has a sharp border between theforest and agricultural field at its southern edge. In 2012 rye wasgrown on the conventionally fertilized agricultural field. The forestoverstory is dominated by Fagus sylvatica L. and A. pseudoplatanusL., while the understory is dominated by Carex brizoides L. and Rubusfructiosus L.

Twenty monitoring plots were set up per forest, and all mea-surements were done in both forests in May 2012. In EL, onetransect was set up with 20 plots (1 m × 1 m) spaced at 20 m inter-vals along a soil nitrate gradient (Fig. S1) reported in a previousstudy (Bernhardt-Römermann et al. 2007). In WF, two transects(Fig. S2, 10 plots each, 1 m × 1 m, spaced at 20 m intervals) were set


up leading from the edge to the center of the forest.Supplementary material related to this article can be

found, in the online version, at http://dx.doi.org/10.1016/j.pedobi.2014.01.007.

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lant community and explanatory variables

Plant species within the monitoring plots were identified using botanical key (Schmeil 2000), and plant species-specific cov-rage was estimated (5% intervals). Similarly, the coverage ofhe tree canopy was estimated at each plot. Coverage data werercsin-transformed. Photosynthetically active radiation (PAR; LI-90 Quantum Sensor from LI-COR Biosciences) was measured inach plot. PAR was measured twice on sunny days between 11 amnd 2 pm, and the mean PAR was used for statistical analyses. Tenoil samples were taken in October 2013 to measure soil pH alonghe transects in each forest. Soil pH and PAR were measured tonvestigate if potential relationships between soil nitrate concen-ration and plant and soil community variables were confoundedy other environmental factors.

arthworms

Earthworm density and diversity were determined using theustard extraction method (Eisenhauer et al. 2008) whereby 0.4%ustard solutions were prepared by shaking 400 g of standardustard with 5 l of water 24 h before extraction. An additional

l of water were added to each canister and the solution wasixed thoroughly right before application. Briefly, a metal frameas pushed into the soil to keep the mustard solution in a defined

rea of 0.5 m × 0.5 m (0.25 m2). Litter material within the frame wasand-sorted for earthworms. Then, 5 l of mustard solution wereoured into the metal frame and emerging earthworms were sam-led for 15 min. Afterwards, another 5 l of mustard solution waspplied and earthworms were sampled again for another 15 min.arthworms were preserved in 70% ethanol until identification topecies level (after Schaefer (2000) in the laboratory.

oil microbial and nitrate measurements

Ten soil samples were taken to a depth of 10 cm after removinghe litter layer per monitoring plot using a metal corer (diam-ter 2 cm) and pooled in a plastic bag. Afterwards, the samplesere sieved (2 mm) to remove large stones and roots. One part

f the soil, approximately 4.5 g soil (fresh weight), was used toeasure soil microbial respiration and biomass. Basal respirationas determined (without addition of substrate) and measured

s the mean of the O2 consumption rates of hours 14–24 afterhe start of the measurements using an automated respirometerased on electrolytic O2 microcompensation (Scheu 1992). Soilicrobial biomass was calculated from the maximum initial res-

iratory response (MIRR) using the substrate-induced respirationethod (SIR; Anderson and Domsch 1978). Following previous

tudies, SIR was calculated from the respiratory response to D-lucose-monohydrate. Catabolic enzymes of soil microorganismsere saturated by adding 40 mg glucose/g soil dry weight as an

queous solution. The soil dry weight was determined by puttinghe soil in a drying oven overnight at 60 ◦C and calculating theifference in weight between fresh and dried soil. The specific res-iratory quotient (qO2) was calculated by dividing basal respirationy microbial biomass, and served as an indicator of disturbance and-use efficiency of soil microorganisms (Eisenhauer et al. 2010).

Additionally, soil microbial growth was measured after N addi-ion ((NH4)2SO4) at a C:N ratio of 10:2 (Anderson and Domsch978). 500 �l of an aqueous solution of glucose and (NH4)2SO4 wasdded to each sample. Microbial growth was determined within therst 10 h (MIRR) (Eisenhauer et al. 2010). The respiration rates were


atural log-transformed assuming an exponential growth of theoil microorganisms after nutrient addition, which was followedy linear regression to determine the slope of soil microbial growthEisenhauer et al. 2010).

PRESSgia xxx (2014) xxx–xxx 3

The second part of the soil sample was used to determinesoil nitrate concentrations. 200 ml of extraction solution (0.005 MCaCl2) was mixed with 100 g (fresh weight) of the soil sample in abottle. It was shaken by hand for 5–7 min (depending on the soiltype) to break up the different soil aggregates (Schmidhalter 2005).Soil nitrate (NO3

−) concentration was measured after Schmidhalter(2005) using nitrate-test strips (Reflectoquant Cat. No. 1.1671.0001,5–225 mg/l NO3

−, E. Merck, Darmstadt, Germany) and a reflec-tometer (RQflex® Cat. No. 116955, E. Merck, Darmstadt, Germany).The reactive zone of the strip was moistened with the extractionsolution for approximately 2 s. Within the reactive zone of thetest strip, chemical reactions form a red-violet dye, which can bedetermined using a reflectometer (Schmidhalter 2005). Again, thegravimetric water content of the soil was determined by calculat-ing the difference in weight between fresh and dried (60 ◦C for 20 h)soil.

Statistical analysis

General linear models (GLMs, type I sum of squares) were usedto analyze the effects of forest (EL and WF; categorical variable) andsoil nitrate concentrations (continuous variable), and the interac-tion between forest and soil nitrate concentrations on understoryvegetation properties (plant species richness, Shannon diversityindex and plant community coverage), earthworms (density andbiomass), and soil microbial properties (basal respiration, micro-bial biomass, specific respiratory quotient and microbial growthafter N addition). We chose to employ a sequential GLM approachsince the forests differed considerably in soil nitrate concentrations(Table 1; F1,37 = 111.49; P < 0.001) to avoid the general differencebetween forests influencing potential effects of soil nitrate gradi-ents. GLMs were performed using SPSS (IBM SPSS Statistics 20, NewYork, USA).

In addition to the GLM approach, we used path analysis to inves-tigate if soil nitrate effects were partly due to changes in pH and PARalong the transects. Path analysis allows testing of the strength ofdirect and indirect relationships between variables in a multivari-ate approach (Grace 2006). Path analyses were done with AMOS 5(Amos Development Corporation, Crawfordville, FL, USA).

For the community analysis, the Bray–Curtis index was usedto calculate community dissimilarity between the communities ofearthworms and plants in each plot. This produces a distance matrixcontaining all pair-wise dissimilarity values. A distance matrix wasalso created for soil nitrate concentration. The association betweenthese matrices was analyzed using Mantel and partial Mantel tests(when controlling for variation in the third variable); variablestested were plant community, earthworm community and nitrateconcentration. Community analyses were performed in R (ver-sion 2.14.0) using the Vegan Community Ecology Package (Version2.0-2; Oksanen et al. 2011), and PaSSAGE (Pattern Analysis, SpatialStatistics and Geographic Exegesis; Rosenberg and Anderson 2011).

Results

Explanatory variables

The mean soil pH (Table 1, Figs. S3 and S4) differed significantlybetween forests (F1,11 = 225.95, P < 0.0001), but not along the tran-sects of either forests (F1,13 = 2.74, P = 0.12). PAR was significantly


higher in WF than in EL (F1,35 = 5.27; P = 0.03), but did not changelinearly along the transect (Table 1; edge: EL: 44.87 W/m2, WF:40.76 W/m2; middle of the forest: EL: 7.74 W/m2, R2 = 0.02, P = 0.60;WF: 19.28 W/m2, R2 = 0.04, P = 0.39).

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Table 1Mean values and standard deviation of explanatory variables, earthworm biomass and soil microorganisms in Echinger Lohe and Wippenhauser Forst.

Variables Echinger Lohe Wippenhauser Forst

Mean| SD Mean SD

Soil nitrate concentration [kg NO3−-N/ha] 228.09 53.67 57.20 31.46

Soil pH 6.81 0.4 3.90 0.2PAR [W/m2] 10.43 10.15 35.98 28.51Cover understory plants [%] 61.70 39.09 41.90 28.86Cover trees [%] 92.55 21.38 75.50 25.07Earthworm biomass [g/0.25 m2] 1.72 1.10 0.22 0.18Basal respiration [�g O2 h−1 g soil dw−1] 16.13 2.03 4.81 1.44

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Microbial biomass [�g Cmic g soil dw ] 2309.20

Specific respiratory quotient [�L mg Cmic−1 h−1] 7.05Microbial growth 0.013

Supplementary material related to this article can beound, in the online version, at http://dx.doi.org/10.1016/.pedobi.2014.01.007.

Soil nitrate concentrations differed considerably betweenorests (Table 1; WF +300%) and decreased with increasing distanceo the edge in EL (EL: R2 = 0.41, P = 0.003). WF showed changing soilitrate concentrations along the transect, although this pattern wasot as clear as in EL (R2 = 0.28, P = 0.116). EL is located on flat ter-ain, which is probably why we found a linear decline in soil nitrateoncentrations from the forest edge toward the forest center. Byontrast, WF is located on uneven terrain, which is why varyingoil nitrate concentrations can be expected. Consequently, we onlyound a trend of decreasing soil nitrate concentrations toward theenter of the forest, and we thus used soil nitrate concentrations

not distance to forest edge – as the explanatory variable in alltatistical analyses.

lant community

The coverage of the understory community decreased signifi-antly with increasing soil nitrate concentrations (Tables 1 and 2,ig. 1). Although this pattern was true for both forests, the rela-ionship was somewhat stronger in EL (resulting in a marginallyignificant interaction between forest and soil nitrate concentra-


ions; Table 2). Nitrophilic species such as Urtica dioica L. andegopodium podagraria L. were mainly growing at the edge of the

orests supporting the results of our soil nitrate measurements. In

ig. 1. Coverage of the understory plant community ([%]; arcsin-transformed) asffected by soil nitrate concentrations [kg NO3

−N/ha] and forest (Echinger Lohe (EL)nd Wippenhauser Forst (WF)).

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324.02 376.07 194.780.92 14.66 2.5480.004 0.056 0.016

contrast to plant cover, soil nitrate concentrations did not affectthe Shannon diversity index and species richness of the understoryvegetation, and there were no significant interactions betweenforest and soil nitrate concentrations (Table 2). Moreover, treecoverage (Table 1) increased significantly with soil nitrate con-centrations in EL (R2 = 0.26, P = 0.02), but remained unaffected inWF (R2 < 0.01, P = 0.94). Detailed information about plant speciespresent in each forest and their mean coverage within definedintervals can be found in the Supplementary Material (Tables S1and S2). Information on tree species-specific coverage-weightednitrogen Zeigerwert of Ellenberg along the soil nitrate gradients isgiven in Figs. S5 and S6); no clear patterns were observed.

Supplementary material related to this article can befound, in the online version, at http://dx.doi.org/10.1016/j.pedobi.2014.01.007.

Earthworms

While earthworm densities (Table 1) were not significantlyaffected by forest and soil nitrate concentrations, earthwormbiomass was significantly higher in EL than in WF (+682%; Table 1).Moreover, earthworm biomass increased with increasing soilnitrate concentrations, but the increase in earthworm biomass wasmore pronounced in EL than in WF resulting in a significant interac-tion between forest and soil nitrate concentrations (Table 2, Fig. 2).


Community analyses revealed that plots with more similarplant communities also had more similar communities of earth-worms (EL: Mantel test: r = 0.259, P = 0.002; WF: Partial Mantel test:r = 0.227, P = 0.058). Thus, changes in the plant community were

Fig. 2. Earthworm biomass [g/0.25 m2] plotted as affected by soil nitrate concentra-tions [kg NO3

−N/ha] and forest (Echinger Lohe (EL) and Wippenhauser Forst (WF)).

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K. Steinauer et al. / Pedobiologia xxx (2014) xxx–xxx 5

Table 2Effects of forest (Echinger Lohe and Wippenhauser Forst; categorical variable), soil nitrate concentrations (continuous variable) and the interaction between forest andsoil nitrate concentrations on understory vegetation properties (plant species richness, Shannon diversity index and plant community coverage), earthworms (density andbiomass), and soil microbial properties (basal respiration, microbial biomass, specific respiratory quotient and microbial growth after N addition) were generated usinggeneral linear models (GLM, type I sum of squares).

Variable Forest Nitrate Forest × nitrate

F-value P-value F-value P-value F-value P-value

Understory vegetationShannon diversity index 19.75 0.0001 1.04 0.3148 0.01 0.9254Species richness 20.35 0.0000 0.13 0.7210 0.10 0.7550Arcsin coverage 3.16 0.0836 0.60 0.4456 2.96 0.0940

MacrofaunaEarthworm density 0.02 0.887 0.961 0.3340 1.948 0.1710Earthworm biomass 43.45 <0.0001 3.20 0.0820 6.13 0.0180

Soil microorganismsBasal respiration 360.39 <0.0001 0.61 0.4390 0.75 0.3940Microbial biomass 505.75 <0.0001 0.74 0.3950 0.01 0.9110Specific respiratory quotient 156.26 <0.0001 1.54 0.2230 0.28 0.5990

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Microbial growth 189.67 <0.0001

ignificant results (P < 0.05) are highlighted in bold, with marginally significant resu

ssociated with changes in the earthworm community. In WF, weound Dendrodrilus rubidus Savigny and Dendrobaena octaedra Sav-gny (both epigeic species), and in EL, we found Octolasion lacteumerley and Allolobophora sp. Savigny (endogeic species), Lumbri-us rubellus Hoffmeister an epi-endogeic species and Lumbricusastaneus Savigny (an epigeic earthworm species). There were noignificant associations between soil nitrate levels and plant com-unity structure in WF (Mantel test: r = −0.092, P = 0.389). In EL,

o association was found between the nitrate concentrations andither the plant community (Mantel test: r = 0.012, P = 0.435) or thearthworm communities (Mantel test: r = 0.042, P = 0.254).

oil microorganisms

Soil basal respiration, microbial biomass and specific respiratoryuotient (Table 1) differed significantly between forests, but wereot significantly affected by soil nitrate concentrations or the inter-ction between forests and soil nitrate concentrations (Table 2).y contrast, soil microbial growth after N addition differed signifi-antly between forests and along the soil nitrate gradient (Table 1),


ith the latter being due to an increase in soil microbial growthith increasing soil nitrate concentrations in WF, but not in EL

Table 2, Fig. 3).

ig. 3. Soil microbial growth as affected by soil nitrate concentrationskg NO3

−N/ha] and forest (Echinger Lohe (EL) and Wippenhauser Forst (WF)).

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11.38 0.0018 5.59 0.0238

bold and italics (P < 0.10).

Because a number of abiotic conditions are likely to change alongthe transect from the forest edge to the interior, we carried out apath analysis to test for the effect of distance to the forest edge, aswell as the covariates pH, PAR, leaf litter biomass and herb coverage.Soil nitrate always had stronger associations with the earthwormbiomass and microbial growth than distance to forest edge did,and the nitrate effect remained significant or a strong explana-tory variable in the analyses. The complete dataset is given in theSupplementary material (Table S3).

Supplementary material related to this article can befound, in the online version, at http://dx.doi.org/10.1016/j.pedobi.2014.01.007.

Discussion

According to our expectation, soil nitrate gradients leadingaway from fertilized agricultural fields were significantly asso-ciated with the composition of plant communities as well asthe compositions and functioning of soil organisms of adjacentforest stands. In contrast to our hypothesis and former studiesin EL (Bernhardt-Römermann et al. 2007, 2010), increasing soilnitrate concentrations did not correlate with the diversity of theunderstory plant community, however the coverage of the vegeta-tion decreased with increasing soil nitrate concentrations in bothforests. Although we could not confirm the meta-analysis results byTreseder (2008) of decreasing soil microbial biomass with increas-ing nitrate concentrations, we found pronounced changes in soilmicrobial growth after the addition of a N source in WF, indicatingalterations in the composition and functioning of the soil microbialcommunity (Eisenhauer et al. 2010). In line with our expectations,earthworm biomass increased with soil nitrate concentrations inboth forests resulting in altered earthworm communities in WF.However, due to the monitoring approach taken in the presentstudy, we cannot rule out feedback effects of earthworms on soilnitrate concentrations.

Forest soils generally act as a sink for N input when highamounts of N accumulate in the humus layer as organically boundN (Melin et al. 1983; Melin and Nömmik 1988). Therefore, N avail-ability for plants is often low. Falkengren-Grerup (1993) foundthat experimental N addition in a F. sylvatica forest in south-ern Sweden resulted in a significant reduction in the biomassof some of the common understory plant species. These results


are in line with our findings as increasing soil nitrate concentra-tions were associated with low understory plant coverage in bothforests. Thus, understory plants may not represent a nitrate sinkin the investigated forest stands (Mäkipää 1994). In EL, and in line

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2009a. Plant community impacts on the structure of earthworm communitiesdepend on season and change with time. Soil Biol. Biochem. 41, 2430–2443.

Eisenhauer, N., Milcu, A., Nitschke, N., Sabais, A.C.W., Scherber, C., Scheu, S., 2009b.

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ith the study by Diekmann and Falkengren-Grerup (2002), treeoverage increased significantly with increasing soil nitrate con-entrations. This increased canopy likely shaded the understorylants, potentially overriding beneficial effects of nitrate availabil-

ty on understory plant growth. Such indirect effects of higheroil nitrate concentrations most likely also depend on the sea-on, due to differential growth of herbaceous plants and trees,nd this may explain the inconsistent findings of previous stud-es (Bernhardt-Römermann et al. 2007, 2010) and the present onen EL.

In both forests, plant and earthworm communities were asso-iated across the monitoring plots, meaning that those plots withore similar plant communities also had more similar earthworm

ommunities. It may be that the earthworms move to areas with areferred plant community (e.g., Milcu et al. 2008; Eisenhauer et al.009a), although the reverse may also be true with earthwormsltering the plant community (Partsch et al. 2006; Eisenhauer et al.009b). However, the earthworm communities in the present studyonsisted of only a few species and thus, the power to detect anyifferences was low.

The lack of evidence for an association between soil nitrateevels and earthworm community structure in EL is likely due tohe small number of species found in the area. Nonetheless, thereas a marginally significant association between these variables

n WF, with soil nitrate concentration explaining up to 5.4% ofhe variation in the earthworm community. We found increas-ng earthworm biomass with increasing soil nitrate concentrationsn EL. Those findings are confirmed by several studies (Edwardsnd Lofty 1982; Makeschin 1997; Curry 2004; Jordan et al. 2004),hich reported that fertilizers enhance earthworm biomass linkedith increasing pH and organic matter content, although very high

evels of fertilization may inhibit their proliferation (Haynes andaidu 1998). Considering the pronounced effects of earthworms on

oil processes and plant growth (Scheu 2003; Edwards 2004), it isery likely that anthropogenic nitrate inputs like fertilization mayhange the functioning of ecosystems through changes in earth-orm biomass (EL) and community composition (WF).

Previous studies found both positive (Roberge and Knowles967; Roberge 1976) and negative (Dijkstra et al. 2005; Treseder008; Eisenhauer et al. 2012) effects of fertilization on micro-ial biomass and microbial growth. In WF, microbial growth afterddition of a N source increased with soil nitrate concentrations,hile understory plant coverage decreased. Indeed, plants and

oil microbes have been reported to often compete for soil nutri-nts (Månsson et al. 2009; Kuzyakov and Xu 2013). Furthermore,everely N-limited soil heterotrophic microflora may inhibit plant-uptake in forest soils (Hart and Stark 1997; Kaye and Hart 1997).any studies have focused on such competition under both field

nd controlled conditions (Bardgett et al. 2003; Cheng and Bledsoe004; Xu et al. 2008). In WF, the increase in microbial growthith increasing soil nitrate concentration, recorded after addition

f (NH4)2SO4, indicates a shift in the community composition of soilicroorganisms and/or potential synergistic effects of soil nitrate

nd the added ammonium sulfate on the existing soil microbialommunity. Probably, soil microbial communities at the forest edgexperiencing frequent inputs of mineral N forms were dominatedy opportunistic microbes, such as many bacterial species. Theseicrobes would then be adapted to easily accessible N forms and

hus likely responded with rapid growth to addition of an N source.y contrast, more autochthonous microbial communities in theiddle of the forest, likely dominated by soil fungi, may be used

o degrade complex organic compounds and thus have respondedith lower growth rates. Although the proposed changes in the

oil microbial community remain to be tested, the altered micro-


ial growth rates indicate changes in soil functions along soil nitrateradients.

PRESSgia xxx (2014) xxx–xxx

Conclusion

The results reported here show that gradients in soil nitrateconcentrations leading away from fertilized agricultural fields andinto forests are associated with significant changes in ecologicalcommunity compositions below-ground and in plant communitycoverage, both indicating significant shifts in ecosystem function-ing. However, such compositional and functional changes differedbetween the forests studied, suggesting that the consequences ofanthropogenic N inputs like fertilization are context-dependentand deserve further attention.

Acknowledgements

We thank Klaus Neugebauer from the District Government ofUpper Bavaria for logistics help and Michael Miesl (TU München)for technical support. Particular thanks go to the many forestry stu-dents of the TU München that helped establish the experimentalfield sites and collected data in the field.

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