Changes in plant community structure and soil biota along soil nitrate gradients in two deciduous forests

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Pedobiologia - Journal of Soil Ecology journal homepage: www.elsevier.de/pedobi

Changes in plant community structure and soil biota along soil nitrate gradients in two deciduous forests

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Katja Steinauer a,∗ , Sharon Zytynska b , Wolfgang W. Weisser b , Nico Eisenhauer a a

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

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Article history: Received 2 July 2013 Received in revised form 20 January 2014 Accepted 26 January 2014

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Keywords: Earthworms Fertilization Microbial growth Plant community composition Soil microorganisms

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Introduction

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Anthropogenic nitrogen (N) deposition is a serious threat to biodiversity and the functioning of many ecosystems, particularly so in N-limited systems, such as many forests. Here we evaluate the associations between soil nitrate and changes in plant community structure and soil biota along nitrate gradients from croplands into closed forests. Specifically, we studied the composition of the understory plant and earthworm 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 agricultural fields. Environmental variables, like photosynthetically active radiation, distance to the edge and soil pH were also determined and used as co-variates. In both forests we found a decrease in understory plant coverage with increasing soil nitrate concentrations. Moreover, earthworm biomass increased with soil nitrate concentration, but this increase was more pronounced in EL than in WF. Soil microbial growth after addition of a nitrogen source increased significantly with soil nitrate concentrations in WF, indicating changes 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 gradient in WF. Taken together, the composition and functioning of forest soil communities and understory plant cover 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 N deposition may be complicated due to context-dependent responses of soil organisms. © 2014 Published by Elsevier GmbH.

Forest edges have received much attention in ecology and ecosystem management, since they represent a transition zone between the forest habitat and adjacent ecosystems. Surrounding areas such as pasture and cropland can trigger changes in the species composition and nutrient cycles across edges (Murcia 1995; Ries et al. 2004; Harper et al. 2005). Additionally, differences in microclimate occur between the two sides of the edge, which can also lead to changes in the rate of decomposition and nutrient mobilization (Murcia 1995). Edges are also subjected to the influx of chemical compounds from the atmosphere or via drift from surrounding land (Thimonier et al. 1992; Wuyts et al. 2008). Therefore, increased deposition of potentially acidifying and eutrophying ammonium, nitrate and sulphate depositions at the edge has been reported (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).

top soil properties and understory plant species composition with increasing 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 marine ecosystems. Many organisms have adapted to low levels of N (Vitousek et al. 1997); moreover, the availability of N strongly influences the growth and abundance of organisms (Vitousek and Howarth 1991). Anthropogenic N deposition sources, like agriculture or combustion of fossil fuels, thus exert strong effects on the biodiversity and functioning of many ecosystems (Sala et al. 2000). In N-limited ecosystems, such as forests, changes in herbaceous layer biodiversity and composition are of special interest due to their functional relevance and fast response to changes in N availability (Tamm 1991; Gilliam 2006; Gilliam 2007; BernhardtRömermann et al. 2010). Elevated N availability has often been reported to increase plant productivity, but to decrease plant diversity by favoring the few 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

http://dx.doi.org/10.1016/j.pedobi.2014.01.007 0031-4056/© 2014 Published by Elsevier GmbH.

Please cite this article in press as: Steinauer, K., et al., Changes in plant community structure and soil biota along soil nitrate gradients in two deciduous forests. Pedobiologia - J. Soil Ecol. (2014), http://dx.doi.org/10.1016/j.pedobi.2014.01.007

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(Reich et al. 2001) showed an increase in plant productivity in response to N addition, but a decrease in soil microbial biomass (Dijkstra et al. 2005; Eisenhauer et al. 2012), plant (Reich 2009) and soil animal diversity (Eisenhauer et al. 2012). In contrast to the above-mentioned studies, Bernhardt-Römermann et al. (2010) found increasing functional diversity of plants with increasing soil N levels in the deciduous forest investigated in the present paper. However, as typical for studies in terrestrial ecology, belowground responses to global change agents, such as fertilization effects, are still less well explored. Moreover, it is unclear if fertilization of agricultural fields influences the composition and functioning of adjacent ecosystems. Soil organisms play major roles in several ecosystem functions; for example, promoting plant productivity, regulating nutrient mineralization and promoting decomposition of organic matter (Neher 1999; Wardle et al. 2004). Thus, shifts in soil food web composition in response to fertilization may cause pronounced alterations in ecosystem functioning. Given that different groups of soil microorganisms use different resources with respect to complexity and quality (Griffiths et al. 1999; Kramer and Gleixner 2006), fertilization may cause compositional shifts in communities of soil microbes and animals (Frey et al. 2004). Although previous studies showed inconsistent responses of soil organisms to N inputs (e.g., Niklaus and Körner 1996; Zak et al. 2000; Dijkstra et al. 2005), some general patterns tend to emerge. For instance, based on a meta-analysis, Treseder (2008) hypothesized that N input through fertilization negatively affects soil microbial biomass due to one or more mechanisms including alterations in soil pH, carbon and N availability, and below- and aboveground productivity of plants. Further, N input may decrease rhizodeposition fueling rhizosphere communities (Högberg et al. 2010; Eisenhauer et al. 2012). Treseder (2008) concluded that N addition has overall negative effects on microbial biomass and that long fertilization periods and large amounts of N even intensify the reduction of microbial biomass (Treseder 2008). Likewise, DeForest et al. (2004) reported decreased microbial enzyme activity after N fertilization, indicating shifts in the functioning of soil microbial communities. Moreover, root colonization and species richness of mycorrhizal fungi have been reported to decrease due to higher N availability (Egerton-Warburton and Allen 2000; Lilleskov et al. 2002a,b). Responses of different groups of soil organisms to fertilization may however differ. Several experiments have shown that both organic and inorganic N fertilizers can have beneficial effects on earthworm populations (Edwards and Lofty 1982; Timmerman et al. 2006). For example, higher nutrient availability through organic N has been show to increase earthworm biomass and numbers (Edwards and Lofty 1982; Hansen and Engelstad 1999), although extremely high levels of N input may inhibit their proliferation (Haynes and Naidu 1998). The effects of inorganic N fertilizers can be explained by an increasing amount of plant material and the subsequent higher amount of decomposing organic matter (Edwards and Lofty 1982). Plant litter decomposition has long been recognized as an essential process for organic matter turnover and nutrient fluxes in most ecosystems. The subsequent release of carbon and nutrients represents the primary source of nutrients for plants and microbes (Berg and McClaugherty 2008). Rates of plant litter decomposition and nutrient mineralization are, in turn, influenced by litter quality; higher nitrogen contents mostly enhance decomposition. Despite all this previous work, there is still a lack of understanding of the responses of ecosystems to N addition effects (West et al. 2006; Bardgett and Wardle 2010; Decaëns 2010). Most studies on high soil N concentrations are either based on permanent plot observations or on studies along environmental gradients

(Brunet et al. 1998). The present study focuses exclusively on soil nitrate gradients in forest ecosystems. We investigated the associations between soil nitrate concentrations and plant community properties, soil biota and functioning in forests that are adjacent to fertilized agricultural fields. To achieve this, we studied understory plant community composition, earthworm communities and soil microbial properties in two deciduous forests near Munich, Germany. We used an observational approach to investigate N gradients as was also done by Bernhardt-Römermann et al. (2007, 2010) in one of the forests investigated in the present study. Such gradients in soil nitrate concentrations could arise from the drift and/or lateral flow of mineral N applied via fertilizers. Given inconsistent results in previous studies, we investigated two forests differing in tree, understory plant and soil community composition. We expected soil nitrate gradients leading away from fertilized agricultural fields to be associated with significant changes in plant community composition and cover as well as in the diversity of soil organisms of adjacent forest stands (Bernhardt-Römermann et al. 2007). More specifically, we hypothesized an increase in diversity and productivity of plant communities (Bernhardt-Römermann et al. 2010), a decrease in soil microbial biomass (Treseder 2008) and an increase in earthworm biomass (Edwards and Lofty 1982) with increasing nitrate concentrations accompanied by significant alterations in soil processes.

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Materials and methods

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Study sites and sampling design

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We used two study sites located in two different deciduous forests close to the city of Munich, Germany. The first one is the ‘Echinger Lohe’ (EL), a nature reserve located 20 km northeast of Munich on the ‘Münchner Schotterebene’, a plain which was formed at the end of the last ice age. The texture of this Leptosol changes from carbonate-rich sandy soil toward humus-rich sandy – loamy gravel with increasing distance to the edge. The forest patch covers an area of about 24 ha and is surrounded by intensively used agricultural land. The dominating tree species are Carpinus betulus L., Quercus robur L. and Acer pseudoplatanus L., and the understory vegetation is dominated by Colchicum autumnale L., Anemone nemorosa L. and Carex alba Scop. The surrounding fields represent a diverse mixture of conventionally fertilized barley, rye and potato fields. The second forest site is the ‘Wippenhauser Forst’ (WF), which is located north of Freising, approximately 40 km northeast of Munich. The texture of this Cambisol is dominated by loess loam and sandy gravel belonging to the upper fresh water molasse (Tertiary). Similar to the EL, the WF has a sharp border between the forest and agricultural field at its southern edge. In 2012 rye was grown on the conventionally fertilized agricultural field. The forest overstory is dominated by Fagus sylvatica L. and A. pseudoplatanus L., while the understory is dominated by Carex brizoides L. and Rubus fructiosus L. Twenty monitoring plots were set up per forest, and all measurements were done in both forests in May 2012. In EL, one transect was set up with 20 plots (1 m × 1 m) spaced at 20 m intervals along a soil nitrate gradient (Fig. S1) reported in a previous study (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.

Please cite this article in press as: Steinauer, K., et al., Changes in plant community structure and soil biota along soil nitrate gradients in two deciduous forests. Pedobiologia - J. Soil Ecol. (2014), http://dx.doi.org/10.1016/j.pedobi.2014.01.007

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Plant community and explanatory variables

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Plant species within the monitoring plots were identified using a botanical key (Schmeil 2000), and plant species-specific coverage was estimated (5% intervals). Similarly, the coverage of the tree canopy was estimated at each plot. Coverage data were arcsin-transformed. Photosynthetically active radiation (PAR; LI190 Quantum Sensor from LI-COR Biosciences) was measured in each plot. PAR was measured twice on sunny days between 11 am and 2 pm, and the mean PAR was used for statistical analyses. Ten soil samples were taken in October 2013 to measure soil pH along the transects in each forest. Soil pH and PAR were measured to investigate if potential relationships between soil nitrate concentration and plant and soil community variables were confounded by other environmental factors.

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Earthworms

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Earthworm density and diversity were determined using the mustard extraction method (Eisenhauer et al. 2008) whereby 0.4% mustard solutions were prepared by shaking 400 g of standard mustard with 5 l of water 24 h before extraction. An additional 5 l of water were added to each canister and the solution was mixed thoroughly right before application. Briefly, a metal frame was pushed into the soil to keep the mustard solution in a defined area of 0.5 m × 0.5 m (0.25 m2 ). Litter material within the frame was hand-sorted for earthworms. Then, 5 l of mustard solution were poured into the metal frame and emerging earthworms were sampled for 15 min. Afterwards, another 5 l of mustard solution was applied and earthworms were sampled again for another 15 min. Earthworms were preserved in 70% ethanol until identification to species level (after Schaefer (2000) in the laboratory. Soil microbial and nitrate measurements Ten soil samples were taken to a depth of 10 cm after removing the litter layer per monitoring plot using a metal corer (diameter 2 cm) and pooled in a plastic bag. Afterwards, the samples were sieved (2 mm) to remove large stones and roots. One part of the soil, approximately 4.5 g soil (fresh weight), was used to measure soil microbial respiration and biomass. Basal respiration was determined (without addition of substrate) and measured as the mean of the O2 consumption rates of hours 14–24 after the start of the measurements using an automated respirometer based on electrolytic O2 microcompensation (Scheu 1992). Soil microbial biomass was calculated from the maximum initial respiratory response (MIRR) using the substrate-induced respiration method (SIR; Anderson and Domsch 1978). Following previous studies, SIR was calculated from the respiratory response to Dglucose-monohydrate. Catabolic enzymes of soil microorganisms were saturated by adding 40 mg glucose/g soil dry weight as an aqueous solution. The soil dry weight was determined by putting the soil in a drying oven overnight at 60 ◦ C and calculating the difference in weight between fresh and dried soil. The specific respiratory quotient (qO2 ) was calculated by dividing basal respiration by microbial biomass, and served as an indicator of disturbance and C-use efficiency of soil microorganisms (Eisenhauer et al. 2010). Additionally, soil microbial growth was measured after N addition ((NH4 )2 SO4 ) at a C:N ratio of 10:2 (Anderson and Domsch 1978). 500 ␮l of an aqueous solution of glucose and (NH4 )2 SO4 was added to each sample. Microbial growth was determined within the first 10 h (MIRR) (Eisenhauer et al. 2010). The respiration rates were natural log-transformed assuming an exponential growth of the soil microorganisms after nutrient addition, which was followed by linear regression to determine the slope of soil microbial growth (Eisenhauer et al. 2010).

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The second part of the soil sample was used to determine soil nitrate concentrations. 200 ml of extraction solution (0.005 M CaCl2 ) was mixed with 100 g (fresh weight) of the soil sample in a bottle. It was shaken by hand for 5–7 min (depending on the soil type) 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 reflectometer (RQflex® Cat. No. 116955, E. Merck, Darmstadt, Germany). The reactive zone of the strip was moistened with the extraction solution for approximately 2 s. Within the reactive zone of the test strip, chemical reactions form a red-violet dye, which can be determined using a reflectometer (Schmidhalter 2005). Again, the gravimetric water content of the soil was determined by calculating 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 used to analyze the effects of forest (EL and WF; categorical variable) and soil nitrate concentrations (continuous variable), and the interaction between forest and soil nitrate concentrations on understory vegetation properties (plant species richness, Shannon diversity index and plant community coverage), earthworms (density and biomass), and soil microbial properties (basal respiration, microbial biomass, specific respiratory quotient and microbial growth after N addition). We chose to employ a sequential GLM approach since the forests differed considerably in soil nitrate concentrations (Table 1; F1,37 = 111.49; P < 0.001) to avoid the general difference between forests influencing potential effects of soil nitrate gradients. GLMs were performed using SPSS (IBM SPSS Statistics 20, New York, USA). In addition to the GLM approach, we used path analysis to investigate if soil nitrate effects were partly due to changes in pH and PAR along the transects. Path analysis allows testing of the strength of direct and indirect relationships between variables in a multivariate 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 used to calculate community dissimilarity between the communities of earthworms and plants in each plot. This produces a distance matrix containing all pair-wise dissimilarity values. A distance matrix was also created for soil nitrate concentration. The association between these matrices was analyzed using Mantel and partial Mantel tests (when controlling for variation in the third variable); variables tested were plant community, earthworm community and nitrate concentration. Community analyses were performed in R (version 2.14.0) using the Vegan Community Ecology Package (Version 2.0-2; Oksanen et al. 2011), and PaSSAGE (Pattern Analysis, Spatial Statistics and Geographic Exegesis; Rosenberg and Anderson 2011).

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Results

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Explanatory variables

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The mean soil pH (Table 1, Figs. S3 and S4) differed significantly between forests (F1,11 = 225.95, P < 0.0001), but not along the transects 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 change linearly 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).

Please cite this article in press as: Steinauer, K., et al., Changes in plant community structure and soil biota along soil nitrate gradients in two deciduous forests. Pedobiologia - J. Soil Ecol. (2014), http://dx.doi.org/10.1016/j.pedobi.2014.01.007

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

Soil nitrate concentration [kg NO3 − -N/ha] Soil pH PAR [W/m2 ] Cover understory plants [%] Cover trees [%] Earthworm biomass [g/0.25 m2 ] Basal respiration [␮g O2 h−1 g soil dw−1 ] Microbial biomass [␮g Cmic g soil dw−1 ] Specific respiratory quotient [␮L mg Cmic−1 h−1 ] Microbial growth

Echinger Lohe SD

Mean

SD

228.09 6.81 10.43 61.70 92.55 1.72 16.13 2309.20 7.05 0.013

53.67 0.4 10.15 39.09 21.38 1.10 2.03 324.02 0.92 0.004

57.20 3.90 35.98 41.90 75.50 0.22 4.81 376.07 14.66 0.056

31.46 0.2 28.51 28.86 25.07 0.18 1.44 194.78 2.548 0.016

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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. Soil nitrate concentrations differed considerably between forests (Table 1; WF +300%) and decreased with increasing distance to the edge in EL (EL: R2 = 0.41, P = 0.003). WF showed changing soil nitrate concentrations along the transect, although this pattern was not as clear as in EL (R2 = 0.28, P = 0.116). EL is located on flat terrain, which is probably why we found a linear decline in soil nitrate concentrations from the forest edge toward the forest center. By contrast, WF is located on uneven terrain, which is why varying soil nitrate concentrations can be expected. Consequently, we only found a trend of decreasing soil nitrate concentrations toward the center of the forest, and we thus used soil nitrate concentrations – not distance to forest edge – as the explanatory variable in all statistical analyses.

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Plant community

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Wippenhauser Forst

Mean|

contrast to plant cover, soil nitrate concentrations did not affect the Shannon diversity index and species richness of the understory vegetation, and there were no significant interactions between forest and soil nitrate concentrations (Table 2). Moreover, tree coverage (Table 1) increased significantly with soil nitrate concentrations in EL (R2 = 0.26, P = 0.02), but remained unaffected in WF (R2 < 0.01, P = 0.94). Detailed information about plant species present in each forest and their mean coverage within defined intervals can be found in the Supplementary Material (Tables S1 and S2). Information on tree species-specific coverage-weighted nitrogen Zeigerwert of Ellenberg along the soil nitrate gradients is given in Figs. S5 and S6); no clear patterns were observed. 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. Earthworms

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The coverage of the understory community decreased significantly with increasing soil nitrate concentrations (Tables 1 and 2, Fig. 1). Although this pattern was true for both forests, the relationship was somewhat stronger in EL (resulting in a marginally significant interaction between forest and soil nitrate concentrations; Table 2). Nitrophilic species such as Urtica dioica L. and Aegopodium podagraria L. were mainly growing at the edge of the forests supporting the results of our soil nitrate measurements. In

Fig. 1. Coverage of the understory plant community ([%]; arcsin-transformed) as affected by soil nitrate concentrations [kg NO3 − N/ha] and forest (Echinger Lohe (EL) and Wippenhauser Forst (WF)).

While earthworm densities (Table 1) were not significantly affected by forest and soil nitrate concentrations, earthworm biomass was significantly higher in EL than in WF (+682%; Table 1). Moreover, earthworm biomass increased with increasing soil nitrate concentrations, but the increase in earthworm biomass was more pronounced in EL than in WF resulting in a significant interaction between forest and soil nitrate concentrations (Table 2, Fig. 2). Community analyses revealed that plots with more similar plant communities also had more similar communities of earthworms (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 concentrations [kg NO3 − N/ha] and forest (Echinger Lohe (EL) and Wippenhauser Forst (WF)).

Please cite this article in press as: Steinauer, K., et al., Changes in plant community structure and soil biota along soil nitrate gradients in two deciduous forests. Pedobiologia - J. Soil Ecol. (2014), http://dx.doi.org/10.1016/j.pedobi.2014.01.007

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

Forest F-value

Understory vegetation Shannon diversity index Species richness Arcsin coverage Macrofauna Earthworm density Earthworm biomass Soil microorganisms Basal respiration Microbial biomass Specific respiratory quotient Microbial growth

Forest × nitrate

Nitrate P-value

F-value

P-value

F-value

P-value

19.75 20.35 3.16

0.0001 0.0000 0.0836

1.04 0.13 0.60

0.3148 0.7210 0.4456

0.01 0.10 2.96

0.9254 0.7550 0.0940

0.02 43.45

0.887 <0.0001

0.961 3.20

0.3340 0.0820

1.948 6.13

0.1710 0.0180

360.39 505.75 156.26 189.67

<0.0001 <0.0001 <0.0001 <0.0001

0.4390 0.3950 0.2230 0.0018

0.75 0.01 0.28 5.59

0.3940 0.9110 0.5990 0.0238

0.61 0.74 1.54 11.38

Significant results (P < 0.05) are highlighted in bold, with marginally significant results in bold and italics (P < 0.10).

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associated with changes in the earthworm community. In WF, we found Dendrodrilus rubidus Savigny and Dendrobaena octaedra Savigny (both epigeic species), and in EL, we found Octolasion lacteum Öerley and Allolobophora sp. Savigny (endogeic species), Lumbricus rubellus Hoffmeister an epi-endogeic species and Lumbricus castaneus Savigny (an epigeic earthworm species). There were no significant associations between soil nitrate levels and plant community structure in WF (Mantel test: r = −0.092, P = 0.389). In EL, no association was found between the nitrate concentrations and either the plant community (Mantel test: r = 0.012, P = 0.435) or the earthworm communities (Mantel test: r = 0.042, P = 0.254).

Soil microorganisms Soil basal respiration, microbial biomass and specific respiratory quotient (Table 1) differed significantly between forests, but were not significantly affected by soil nitrate concentrations or the interaction between forests and soil nitrate concentrations (Table 2). By contrast, soil microbial growth after N addition differed significantly between forests and along the soil nitrate gradient (Table 1), with the latter being due to an increase in soil microbial growth with increasing soil nitrate concentrations in WF, but not in EL (Table 2, Fig. 3).

Fig. 3. Soil microbial growth as affected by soil nitrate concentrations [kg NO3 − N/ha] and forest (Echinger Lohe (EL) and Wippenhauser Forst (WF)).

Because a number of abiotic conditions are likely to change along the transect from the forest edge to the interior, we carried out a path analysis to test for the effect of distance to the forest edge, as well as the covariates pH, PAR, leaf litter biomass and herb coverage. Soil nitrate always had stronger associations with the earthworm biomass and microbial growth than distance to forest edge did, and the nitrate effect remained significant or a strong explanatory variable in the analyses. The complete dataset is given in the Supplementary material (Table S3). 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. Discussion According to our expectation, soil nitrate gradients leading away from fertilized agricultural fields were significantly associated with the composition of plant communities as well as the compositions and functioning of soil organisms of adjacent forest stands. In contrast to our hypothesis and former studies in EL (Bernhardt-Römermann et al. 2007, 2010), increasing soil nitrate concentrations did not correlate with the diversity of the understory plant community, however the coverage of the vegetation decreased with increasing soil nitrate concentrations in both forests. Although we could not confirm the meta-analysis results by Treseder (2008) of decreasing soil microbial biomass with increasing nitrate concentrations, we found pronounced changes in soil microbial growth after the addition of a N source in WF, indicating alterations in the composition and functioning of the soil microbial community (Eisenhauer et al. 2010). In line with our expectations, earthworm biomass increased with soil nitrate concentrations in both forests resulting in altered earthworm communities in WF. However, due to the monitoring approach taken in the present study, we cannot rule out feedback effects of earthworms on soil nitrate concentrations. Forest soils generally act as a sink for N input when high amounts of N accumulate in the humus layer as organically bound N (Melin et al. 1983; Melin and Nömmik 1988). Therefore, N availability for plants is often low. Falkengren-Grerup (1993) found that experimental N addition in a F. sylvatica forest in southern Sweden resulted in a significant reduction in the biomass of some of the common understory plant species. These results are in line with our findings as increasing soil nitrate concentrations were associated with low understory plant coverage in both forests. Thus, understory plants may not represent a nitrate sink in the investigated forest stands (Mäkipää 1994). In EL, and in line

Please cite this article in press as: Steinauer, K., et al., Changes in plant community structure and soil biota along soil nitrate gradients in two deciduous forests. Pedobiologia - J. Soil Ecol. (2014), http://dx.doi.org/10.1016/j.pedobi.2014.01.007

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with the study by Diekmann and Falkengren-Grerup (2002), tree coverage increased significantly with increasing soil nitrate concentrations. This increased canopy likely shaded the understory plants, potentially overriding beneficial effects of nitrate availability on understory plant growth. Such indirect effects of higher soil nitrate concentrations most likely also depend on the season, due to differential growth of herbaceous plants and trees, and this may explain the inconsistent findings of previous studies (Bernhardt-Römermann et al. 2007, 2010) and the present one in EL. In both forests, plant and earthworm communities were associated across the monitoring plots, meaning that those plots with more similar plant communities also had more similar earthworm communities. It may be that the earthworms move to areas with a preferred plant community (e.g., Milcu et al. 2008; Eisenhauer et al. 2009a), although the reverse may also be true with earthworms altering the plant community (Partsch et al. 2006; Eisenhauer et al. 2009b). However, the earthworm communities in the present study consisted of only a few species and thus, the power to detect any differences was low. The lack of evidence for an association between soil nitrate levels and earthworm community structure in EL is likely due to the small number of species found in the area. Nonetheless, there was a marginally significant association between these variables in WF, with soil nitrate concentration explaining up to 5.4% of the variation in the earthworm community. We found increasing earthworm biomass with increasing soil nitrate concentrations in EL. Those findings are confirmed by several studies (Edwards and Lofty 1982; Makeschin 1997; Curry 2004; Jordan et al. 2004), which reported that fertilizers enhance earthworm biomass linked with increasing pH and organic matter content, although very high levels of fertilization may inhibit their proliferation (Haynes and Naidu 1998). Considering the pronounced effects of earthworms on soil processes and plant growth (Scheu 2003; Edwards 2004), it is very likely that anthropogenic nitrate inputs like fertilization may change the functioning of ecosystems through changes in earthworm biomass (EL) and community composition (WF). Previous studies found both positive (Roberge and Knowles 1967; Roberge 1976) and negative (Dijkstra et al. 2005; Treseder 2008; Eisenhauer et al. 2012) effects of fertilization on microbial biomass and microbial growth. In WF, microbial growth after addition of a N source increased with soil nitrate concentrations, while understory plant coverage decreased. Indeed, plants and soil microbes have been reported to often compete for soil nutrients (Månsson et al. 2009; Kuzyakov and Xu 2013). Furthermore, severely N-limited soil heterotrophic microflora may inhibit plant N-uptake in forest soils (Hart and Stark 1997; Kaye and Hart 1997). Many studies have focused on such competition under both field and controlled conditions (Bardgett et al. 2003; Cheng and Bledsoe 2004; Xu et al. 2008). In WF, the increase in microbial growth with increasing soil nitrate concentration, recorded after addition of (NH4 )2 SO4 , indicates a shift in the community composition of soil microorganisms and/or potential synergistic effects of soil nitrate and the added ammonium sulfate on the existing soil microbial community. Probably, soil microbial communities at the forest edge experiencing frequent inputs of mineral N forms were dominated by opportunistic microbes, such as many bacterial species. These microbes would then be adapted to easily accessible N forms and thus likely responded with rapid growth to addition of an N source. By contrast, more autochthonous microbial communities in the middle of the forest, likely dominated by soil fungi, may be used to degrade complex organic compounds and thus have responded with lower growth rates. Although the proposed changes in the soil microbial community remain to be tested, the altered microbial growth rates indicate changes in soil functions along soil nitrate gradients.

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