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Keywords:

  • 15N tracer experiment;
  • biodiversity and ecosystem functioning;
  • complementarity;
  • native species;
  • niche;
  • species combination;
  • stable isotopes

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  1. Afforestation is globally increasing to produce timber and pulp wood, but also to enhance ecosystem services such as carbon sequestration, nutrient retention or groundwater recharge. In China, large areas have been and will be afforested in order to compensate for the negative impacts of former clear-cuttings and to make use of the ecosystem services associated with afforestation. In order to further optimize these services with regard to balanced nutrient (particularly nitrogen) cycles, it is important to know whether the use of mixtures of native tree species in afforestation projects promotes the acquisition and retention of nitrogen compared with the currently established large-scale monocultures.

  2. To test the effect of species richness on system N retention and tree sapling N uptake, we conducted a 15N tracer experiment in a young tree plantation. To this end, saplings of four abundant early successional tree species were planted in monocultures, in two- and four-species mixtures and as single trees.

  3. Nitrogen retention increased with higher species richness due to enhanced N pools in sapling biomass. These species richness effects strengthened over time.

  4. Species-specific differences in 15N recoveries over time revealed below-ground niche differentiation with regard to N uptake, which is likely to result in complementary resource use among coexisting species.

  5. Synthesis and applications. This study provides evidence that mixed afforestation promotes N retention from the sapling stage. To further improve ecosystem services associated with afforestation, we strongly suggest the use of mixtures of native tree species instead of monocultures. Mixtures of four species may reduce system N losses and thus may lessen groundwater contamination due to N leaching. We encourage further investigations to find optimal species combinations that promote a wide range of ecosystem services related to more closed nutrient cycles and minimized soil erosion. In our study, the plantations' capability to retain N could be optimized by means of both increasing tree species richness and by choosing the optimal species combinations.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Forests provide important ecosystem goods such as production of timber, fuel and pulp wood, but also support ecosystem functions and services such as element cycling, carbon sequestration or erosion control. Furthermore, forests are key systems to stabilize climate conditions at the regional and global scales (Dixon & Wisniewski 1995). However, changes in land use and deforestation caused a world-wide and extensive decline of forest ecosystems in recent decades. As a consequence, a range of policies have been designed to support forest management strategies aiming at the prevention of deforestation and the establishment of new forests, mainly on former agricultural or degraded land (Smith et al. 2000; Madsen 2002; Bauhus & Schmerbeck 2010). One important political instrument in the framework of the Kyoto Protocol is that carbon emissions are allowed to be offset by demonstrable removals of carbon from the atmosphere (Smith et al. 2000). Using this instrument, many countries of Asia, Europe and America made huge efforts to increase the area covered by forests to reduce atmospheric CO2 levels in recent decades (Houghton 2003; Willson 2006). The main aim of these large afforestation (i.e. forest planting in areas that previously were not classified as forests) or reforestations is to enhance carbon sequestration. However, afforestation is often established as easily manageable monocultures, although there is accumulating evidence of a positive relation between tree diversity and productivity (Paquette & Messier 2011; Cardinale et al. 2012). In addition, Gamfeldt et al. (2013) found that mixed stands in contrast to monocultures may not only offer the potential for high carbon sequestration, but meanwhile improve further ecosystem services such as game production potential, bilberry production or soil carbon storage. In addition, diverse stands have been found to enhance ecosystem functions and services such as ecosystem resilience over space and time (Thompson et al. 2009), or nutrient cycling and decomposition rates (Nadrowski, 2010; Scherer-Lorenzen 2013). It is also debated whether societal benefits provided by diverse stands are accompanied by economical profits as well (such as harvest of timber; e.g. Knoke et al. 2008). Although tree plantations are currently established throughout many countries in Asia, Europe and America, information about species richness–ecosystem service relationships in such afforestation is limited. Furthermore, it is not known which time frame is needed for mixture effects to occur.

The maintenance of high groundwater quality is considered one of the most important ecosystem services of forest ecosystems. Watershed protection is especially important in areas with intensive agriculture, where N leaching through the soil contaminates groundwater resources (Schläpfer & Erickson 2001). Nitrogen is an essential plant nutrient but can also be a potential pollutant; therefore, it is interesting to reveal the fate of N within a system. The afforestation of former agricultural fields with saplings provides the unique opportunity to retain N in the soil plant system and thereby prevent N from contaminating ground water.

One potential mechanism leading to increased system N retention of mixed afforestation in comparison with monocultures is complementarity of soil N use. Plants may minimize interspecific competition for N by means of niche differentiation due to differences in temporal and spatial uptake patterns or preferred N forms (Kahmen et al. 2006; von Felten et al. 2009). Thus, niche differentiation is likely to cause complementarity in N uptake, which in turn might cause a higher N use efficiency in mixtures compared with monocultures (for grasslands e.g. Kahmen et al. 2006; Roscher et al. 2008; Nyfeler et al. 2009; Ashton et al. 2010). In an experimental plantation in Panama, increased N pools in above-ground biomass were found in mixtures (Oelmann et al. 2010; Zeugin et al. 2010) due to species-specific N use efficiency. Some species performed well in particular species combinations but not in others, which points to the importance of species identity (Zeugin et al. 2010). While the increased exploitation of soil N by complementary species mixtures results in higher N pools in plant biomass, soil N pools and N leaching were found to decrease in grassland experiments (Tilman, Wedin & Knops 1996; Niklaus et al. 2001; Scherer-Lorenzen et al. 2003). This could result in an overall increased N retention of the system. Furthermore, the occupation of niches due to interspecific below- (root segregation) and above-ground interaction (canopy space division), that is, niche consolidation, may result in increased complementarity over time. Oelmann et al. (2011) found that plant diversity–N cycling relationships in newly established grasslands changed with time because of the accumulation of organic matter in the soils. The positive relationship between plant diversity and soil N storage that evolved after the establishment phase improved soil fertility, reduced the need for fertilizer application and promoted closed N cycles, for example due to decreasing leaching losses to ground water (Oelmann et al. 2011). Furthermore, van Ruijven & Berendse (2005) found species richness effects on productivity to increase with time due to enhanced resource use efficiency.

China is one of the largest and most heavily populated countries in the world. Its long history of agriculture in combination with large-scale food production has resulted in an overexploitation of forest resources. However, a new juridical framework was established, and there is a strong political will to enhance and enlarge the existing forest areas. Namely, the National Forest Protection Plan, established in 1998, comprises a logging ban on state-owned forests and the plan to afforest 30·8 million hectares of degraded land (Jintao, Katsigris & White 2002). Afforestation is the primary factor increasing forest cover in China (Zhang & Song 2006). From 2005 to 2010, more than 45 million ha of plantation forests was planted which represent unprecedented large-scale afforestation world-wide (Cao et al. 2011). Afforestation is mainly established as monospecific stands, and the use of coniferous species such as Platycladus orientalis, Pinus massoniana and Cunninghamia lanceolata is most common. However, monospecific afforestation is susceptible to insect infestations and disease (Wenhua 2004). Moreover, the planting of only few commercially available and economically important species causes further ecological problems such as a decrease in soil water and nutrient content in areas which are not suited for these species. Therefore, species selection for afforestation should be location specific and not a ‘one size fits all’ approach (Cao et al. 2011). To develop a more natural approach, a mix of native pioneer species from the species pool of the potential natural vegetation at any site should be chosen (Cao, Chen & Yu 2009; Cao et al. 2011; Fang, Yang & Zhang 2012). Moreover, the establishment of mixed forest stands of native species might have the potential to promote various ecosystem services at the same time leading to increased economical, ecological and social value (Bauhus & Schmerbeck 2010). To be able to estimate the value of native mixed forest plantations, knowledge about the ecology and physiology of native species, which might be planted, needs to be established. Most of the native broad-leaved species in subtropical China, where our study took place, are not commercially used so far, and knowledge of their ecological requirements, growth behaviour and interactions as well as raising conditions of seedlings is scarce.

To address these questions and practical considerations, we designed a mixed species afforestation experiment to test the effects of species identity and species richness on ecosystem N retention. Furthermore, we wanted to test whether species richness effects (and thus related ecosystem services) may occur already at the sapling stage. In our afforestation experiment, we used tree saplings of four abundant native broad-leaved tree species (two evergreen and two deciduous) as model system to address our research questions. Since we established our experiment on a former field, we chose early successional species, which are as well suited for open space afforestation. To analyse species identity and richness effects on system N retention and N allocation patterns, we planted saplings in monocultures, in two- and four-species mixtures and as individual trees. Sapling N uptake and system N retention was quantified by applying a 15N tracer. More precisely, we wanted to test the following hypotheses:

  • (H1) Nitrogen acquisition and retention increases with species richness due to complementary effects in species mixtures.
  • (H2) Species richness effects strengthen over time.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Study area and experimental design

The experiment was set up in the north-east part of Jiangxi Province, P.R. China (N 29° 06·29 E 117° 55·28). The study area is characterized by a subtropical climate with a mean annual precipitation of approximately 2000 mm and a mean temperature of 15·1 °C. The soils of the region are mainly Cambisols, and in the lower parts of the landscape, Acrisols and Ferralsols are prevailing. The potential natural vegetation is a subtropical evergreen broad-leaved forest with a dominance in abundance of evergreen species (Bruelheide et al. 2011), but with a balanced number of co-occurring deciduous and evergreen species (Lou & Jin 2000).

The experimental area was a former agricultural field, which was ploughed, harrowed and divided into four blocks prior to the set-up of the experiment in March 2009. Weeding took place throughout the experimental time. Four highly abundant, early successional species were chosen for the experiment: Schima superba Gardn. et Champ. and Elaeocarpus decipiens Hemsley (evergreen), Quercus serrata Murray and Castanea henryi (Skan) Rehd. et Wils. (deciduous; Yu et al. 2001; Table 1).The following planting schemes were established on 1-m² plots.

Table 1. Information on the four species used in the experimental planting
Species C. henryi E. decipiens Q. serrata S. superba
  1. Indication of mycorrhizal associations is based on personal observation and literature. Planting height and planting ground basal diameter are given as means with standard deviation.

FamilyFagaceaeElaeocarpaceaeFagaceaeTheaceae
Leaf habitDeciduousEvergreenDeciduousEvergreen
Inferred mycorrhizal associationEctomycorrhizaArbuscular mycorrhizaEctomycorrhizaArbuscular mycorrhiza
Planting height [cm]46·4 ± 14·128·7 ± 6·922·9 ± 8·934·0 ± 8·3
Planting ground basal diameter [mm]5·2 ± 1·57·7 ± 3·33·3 ± 1·54·5 ± 1·5

To test for the effects of competitor presence, one-year-old saplings were planted either as individual trees or 16 saplings in an array of four by four. The 16-sapling array was used to establish monocultures, two-species combinations and four-species combinations. The four monocultures of each species, all six possible two-species combinations and one-four-species combination gave a total of eleven species combinations. The planting distance between saplings was 15 cm, producing a planting density of 44 000 saplings per ha. Planting density was chosen to mimic an early successional stand of high competition between saplings. In the nearby Gutianshan National Nature Reserve (N 29°8·18-29°17·29, E 118°2·14–118°11·12), densities of 16,000 individuals per ha were found in stands of about 20 years age (i.e. after self-thinning took place; Bruelheide et al. 2011). In the species combinations, each species was represented by the same number of individuals in both, the peripheral rows (i.e. 12 individuals) as well as in the centre (i.e. four individuals). To avoid edge effects, all analyses were performed using the four central individuals. All treatments were replicated four times, once in each of the four blocks. The total number of plots was 88 (11 species combinations x four blocks + 11 single saplings x four species – spread over four blocks). All treatments were randomly assigned to plots within blocks. The experiment was run from March 2009 until September 2010 when sapling harvest took place.

15N tracer application

Pulse labelling with 15NH415NO3 (98% 15N) was performed in August and in September 2009. Tracer application at 10 cm soil depth took place at eight points in a grid of 7 cm spacings surrounding single trees and each of the four central individuals. Each sapling received an amount of 0·01314 g of 15NH415NO3 per application dissolved in water. 15N addition was carried out by means of an Eppendorf Repeater (4780, Eppendorf AG, Hamburg, Germany) extended by an injection needle with four openings.

Sampling and analyses of leaf, fine root and soil N and 15N contents

Young, fully developed leaves were sampled 6 days before and after each application as well as in April and September 2010. Leaves were dried in a drying oven (24 h, 70 °C). The species-specific allocation of biomass to foliage was estimated using allometric equations on the basis of the ground basal diameter [measured at the time of leaf sampling, R² of allometries ≥0·76; (Trogisch 2012)]. The species mean leaf biomass per individual and plot has been used for the estimation of the N pool.

Fine roots (diameter <2 mm) were excavated in September 2010, washed with tap water, dried (48 h, 60 °C) and weighed. Species mean root biomass per individual and plot has been calculated for the estimation of the N pool. Fine root samples were pooled per species and plot for 15N analyses. We restricted biomass sampling to plant organs of high N content.

Five soil samples were taken by means of a soil corer (10 cm depth) before and after the first 15N application, after the second application, as well as in April and September 2010. Soil samples were pooled per plot and dried (24 h, 70 °C). The soil pool was calculated as the volume of soil below the four central individuals (30 × 30 × 20 cm soil depth) and multiplied with bulk density (1·09 and 1·64 g cm−3 for 0–10 and 10–20 cm depth, respectively).

Plant and soil samples were sheared with a mixer mill (MM 400; Retsch, Haan, Germany) and redried at 70 °C before weighing. Total N and 15N were determined using a continuous flow elemental analyser-isotopic ratio mass spectrometer (vario El cube, Elementar, Hanau, Germany, coupled to an Isoprime IRMS, Isoprime Ltd., Cheadle Hulme, UK).

Calculation of 15N content, 15N enrichment and 15N tracer recovery

15N contents from references and samples were calculated as

  • display math(eqn1)

where Rsample and Rstandard are the ratios of 15N to 14N of the sample and the standard, respectively. The standard is conventionally set as atmospheric N2 (Rstandard = 0·0036765; Coplen, Krouse & Böhlke 1992).

15N enrichment is the per mille isotope enrichment of a sample (δ15Nsample) versus a reference (δ15Nref; for leaves and soil: samples taken before the first application, for roots: samples from untreated saplings; (Fry 2006):

  • display math(eqn2)

15N tracer recovery (at.%) in leaves and fine roots of saplings and of soil was calculated as

  • display math(eqn 3)

where mpool is the amount of N in the biomass of leaves/fine roots per sapling (g), at.%15Npool, at.%15Nref and at.%15Ntracer is the at.%15N of labelled leaves/fine roots/soil samples, the unlabelled reference and the added tracer, respectively (Fry 2006). 15N tracer recoveries (% 15Nrec) represent masses of 15N tracer recovered as the percentage of total 15N tracer masses applied per sapling.

In addition to the recovery of leaves, fine roots and soil, N retention was calculated as the total recovery (sum of plot soil, leaf and fine root recovery as these components take up most of N) for the plots with competitors. To calculate the plot recovery of leaves and fine roots, we used the biomass of leaves and roots of all four central individuals and the respective species-specific enrichment values. The relative leaf, root and soil recovery in N retention was calculated as percentages of total recovery.

Statistical analyses

All statistical analyses were carried out using R (version 2.12.1 R Development Core Team 2010). The data were analysed by linear mixed effects models using the R package ‘nlme’ (Pinheiro et al. 2010). All significant categorical variables were further examined by a post hoc Tukey's test [package ‘multcomp’ (Hothorn, Bretz & Westfall 2008)]. To adjust for the unbalanced experimental design, ANOVA Type II [package ‘car’ (Fox & Weisberg 2011)] was used to test for main effects. Model simplification was performed by stepwise backward selection of fixed factors, removing the least significant variables until only significant predictor variables remained (< 0·05). All recovery data were square root–transformed prior to analyses to meet the criteria of normal distribution.

In a first step, the whole data set was used to analyse the effect of competitor presence on leaf and root recovery at the end of the experiment. Fixed effects were fit as ‘initial diameter’ + ‘presence of competitors’ + ‘species’. We included the initial diameter to adjust for differences in the mean diameter size of saplings at the time of planting. Random effects were plot nested in block to account for spatial nestedness.

In a second step, we tested for the effects of neighbour identity on leaf and root recovery at the end of the experiment. The data set was divided by species and analysed by linear mixed effects models, using the initial dbh and neighbour identity (i.e. no neighbour, each of the four species or all species) as fixed factors and plot nested in block as random intercept.

In a third step, the effects of species richness and species combination on N retention and the relative recovery of leaf/root/soil were analysed by simple linear mixed effects models, using either species richness or species combination as fixed effects (for N retention the model accounted for initial mean basal diameter of saplings) and block as random intercept. For this analysis, only data from plots with competitors have been used.

In a last step, a repeated measures linear mixed effects model was used to analyse leaf recovery over time, with time fitted as random intercept (c. time|block/plot). Fixed effects were fitted in the order ‘initial diameter’, ‘time’, ‘species’, ‘species richness’/'species combination', ‘time*species’, ‘time*species richness’. Two separate models were fitted, one including species richness and one species combination due to restricted degrees of freedom.

Model residuals of all models did not show violation of modelling assumptions (normality and homogeneity of variances).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Species identity and competition effects on 15N recovery

Species identity of saplings was a significant predictor of leaf and root 15N recovery. As revealed by post hoc Tukey's test, C. henryi had significantly lower leaf recovery than the other species as well as significantly lower root recovery than E. decipiens and S. superba.

Surprisingly, the recovery of leaves and roots was significantly enhanced in individuals with competitors compared with individual saplings (Chisq: 15·0, 9·0; < 0·001, = 0·0027). Furthermore, the ratio of leaf to fine root recovery did not differ between individual saplings and those with competitors.

Neighbourhood effects on leaf N recovery (i.e. no neighbour, monospecific neighbours, neighbours in the two- or four-species mixtures) were not significant for any of the species (> 0·05, Fig. 1). However, the root recovery of C. henryi and Q. serrata showed a significant effect of neighbour identity. For C. henryi, both the individual saplings as well as the saplings in monocultures had significantly reduced root recovery compared with saplings with neighbours of E. decipiens or the four-species mixture (post hoc Tukey's test < 0·05, Fig. 1). Furthermore, individual saplings of Q. serrata had a lower root recovery than Q. serrata saplings in monocultures or in mixtures with E. decipiens (post hoc Tukey's test < 0·05, Fig. 1).

image

Figure 1. 15N recovery (square root–transformed) of leaves and fine roots without neighbours (no), specific species combinations (i.e. target species in combinations with: Ch = C. henryi, Ed = E. decipiens, Qs = Q. serrata, Ss = S. superba) and four-species mixture (all). Significant differences as revealed by post hoc Tukey's test (< 0·05) are indicated by different letters.

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15N retention and relative 15N recovery in leaves, roots and soil

System 15N retention (overall plot recovery) was positively affected by species richness at the level of = 0·0576 (Chisq: 5·7; Fig. 1a) and was between 3·5 and 31·9% of applied 15N. The analysis of the different system compartments (leaves, fine roots and soil) revealed that fine root recovery was lower than leaf recovery, and biomass recovery (leaves and fine roots) was lower than soil recovery. Whereas the relative leaf and root recovery were significantly higher in species mixtures compared with monocultures (Table 2; Figs. 2b and 1c), the relative soil recovery was significantly reduced (Table 2; Fig. 2d). Besides species richness, species combination significantly affected the relative leaf, root and soil recovery (Table 2). Most remarkably, combining S. superba with C. henryi resulted in a significantly higher 15N accumulation in roots and leaves compared with monocultures of C. henryi, Q. serrata or E. decipiens (post hoc Tukey's test: < 0·05, Fig. 3a,b), but not compared with the monoculture of S. superba. The relative root recovery in the four-species combination was also higher than in the C. henryi monoculture (Fig. 3b).

Table 2. The effect of species richness and species combination on the relative nitrogen (N) recovery of leaves, roots and soil (as percentages from overall N retention)
Response variableChisq.P-value
  1. Given are the model equations and results of mixed effects models.

Response ~ richness, random = 1|block
Relative leaf recovery13·10·0014
Relative root recovery14·30·0008
Relative soil recovery8·00·0187
Response ~ combination, random = 1|block
Relative leaf recovery43·0<0·0001
Relative root recovery35·8<0·0001
Relative soil recovery22·50·0129
image

Figure 2. Nitrogen (N) retention affected by species richness. N retention summed as the recovery of soil, roots and leaves (a), relative leaf recovery (b), relative root recovery (c) and relative soil recovery (d). Significant differences as revealed by post hoc Tukey's test (< 0·05) are indicated by different letters.

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image

Figure 3. The effects of species combination on relative leaf (a) and root (b) recovery. Significant differences are indicated by different letters as revealed by post hoc Tukey's test (< 0·05).

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Leaf recovery as a function of time

15N leaf recovery of saplings was significantly affected by time (Chisq: 75·5; < 0·0001). The effect of time was different for each species as indicated by the significant interaction of species and time (Chisq: 38·1; < 0·0001; Fig. 4a). However, for all sampling dates, the evergreen species had higher leaf recoveries in comparison with the deciduous species. As shown in Fig. 4a, evergreen species had higher uptake rates immediately after the first application. The higher 15N uptake rates of evergreens were approximately maintained throughout the whole experiment. However, deciduous species took up more 15N after the second application and 15N recovery even increased till spring. From spring to the end of the experiment in late summer, 15N recovery of deciduous species decreased. We did not find a significant effect of species richness on leaf recovery; however, the interaction of species richness and time was significant (Chisq: 8·9, = 0·0118, Fig. 4b). Although differences in recovery were minimal shortly after the two applications, they became more pronounced with ongoing experimental time. By the end of the experiment, individuals had elevated leaf recovery in mixtures compared with monocultures. Interestingly, in the repeated measures model, no significant effect of species combination on recovery was found.

image

Figure 4. 15N leaf recovery over time. Black arrows indicate the two application dates of 15N Ammonium nitrate. (a) Interaction between species and time. Symbols: triangles – evergreen (grey – E. decipiens; black – S. superba); points – deciduous (grey – C. henyri; black – Q. serrata). (b) Interaction between species richness and time. Symbols: black – monocultures; dark grey – two-species mixtures; light grey – four-species mixtures. Symbols are staggered within sampling time to improve visibility of standard error bars.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Species richness and identity effects on N retention

Our results demonstrate that species richness of mixtures increases system N retention in young subtropical tree plantations. To find out more about the underlying mechanisms of this pattern, we compared the relative recoveries of soil, leaves and roots. Although relative soil recovery was highest compared with relative leaf and root recovery, soil recovery decreased with species richness (Fig. 2). Thus, the observed positive relationship between species richness and system N retention is caused by an increase in relative N recovery of sapling biomass (fine roots and leaves) with higher species numbers in mixtures. Increasing above-ground N pools in three-species mixtures compared with monocultures were also found in a tropical tree plantation in Panama and attributable to complementary resource uptake (Oelmann et al. 2010; Zeugin et al. 2010). For the same species as examined in the present study, Trogisch (2012) revealed short-term (6 days) spatial, temporal and chemical N form complementary effects with regard to sapling N sequestration. Such complementarity effects might be due to a decrease in niche overlap between individuals in species mixtures versus monocultures (von Felten et al. 2009). However, niche overlap and overall niche breadth (calculated as the overall niche of all four species) remained unaffected by tree species richness when measured at the four distinct seasons (Trogisch 2012). The present study, in contrast, acquired recovery data from longer-term (1 year) N sequestration spanning all four seasons. Our findings suggest that complementary space use may have evolved in the course of the experiment. This may be due to both, an increased niche differentiation by root growth and segregation (i.e. complementary use of below-ground space) and enhanced canopy space division as a result of above-ground species interactions. Increasing complementarity over time is considered a main mechanism that explains positive species diversity–productivity relationships (van Ruijven & Berendse 2005; Cardinale et al. 2007).

In mixtures, N recovery of leaves and roots was higher compared with the individual trees (independent of species identity). This is in accordance with Ashton et al. (2010) and we attribute this to enhanced rooting, invoked by the higher sapling density in mixtures and thus increased below-ground competition. If mixtures are compared, we also found a significant species identity effect, likely attributable to species-specific N uptake rates or preferences in N forms (Table 2, Figs 1 and 3; Kahmen et al. 2006; von Felten et al. 2009; Ashton et al. 2010). From an application point of view, it is important to note that the relative leaf and root recovery of combinations of the evergreen species S. superba with the deciduous species C. henryi was distinctly higher than in all monocultures (with the exception of S. superba; Fig. 3a,b). This finding supports the hypothesis that increasing N uptake (and retention) was attributable to complementarity effects among co-occurring species. Complementarity of S. superba and C. henryi was also mirrored by the above-ground biomass values found for the saplings (Lang et al. 2012). The biomass in the mixture was higher than in monoculture of C. henryi, which was attributed to niche separation in canopy space use (Lang et al. 2012). However, whereas C. castanea exhibited strong intraspecific competition (as indicated by the low root recovery of the individual in monoculture; Fig. 1), intraspecific competition for Q. serrata was not stronger than interspecific competition. For both species, below-ground competition for N was low in mixtures with E. decipiens. Furthermore, species differed in their leaf recovery over time (significant interaction), which suggests a temporal differentiation with regard to N use. These results indicate that in practice, the N retention of afforestation could be optimized by means of both increasing the number of planted tree species (i.e. the tree species richness level) and by choosing the optimal species combinations.

Individuals of the deciduous species Q. serrata and C. henryi frequently showed root tip mycelia in our experiment. Both species belong to the plant family Fagaceae which is known to form ectomycorrhiza. In contrast, the two evergreen species S. superba (Theaceae) and E. decipiens (Elaeocarpaceae) are likely to form arbuscular mycorrhiza. Interestingly, species of the genus Elaeocarpus are able to form both arbuscular and ectomycorrhiza (Haug et al. 1994). Since ectomycorrhizal plants are depleted in foliar 15N compared with arbuscular mycorrhizal plants (Craine et al. 2009), it is conceivable that the trees' mycorrhizal associations might have affected the tissue 15N values found for the four tree species (i.e. lower values found for C. henryi which is associated with ecotomycorrhiza). However, we assume this effect to be low, since findings of Craine et al. (2009) refer to natural 15N (background) concentrations, whereas in our experiment, soil 15N concentrations have been manipulated by tracer additions.

Species richness effects strengthened over time

Our results indicate that species richness effects on N acquisition and retention may already establish within a one-year time span. The general pattern of leaf recovery of all species richness levels was similar. However, the changes in leaf recovery over time differed between species richness treatments. More interestingly, differences between species richness treatments strengthened, while the variation within treatments remained at a similar range. Therefore, the effect of species richness on leaf recovery was enhanced with time. Since we found that the relative leaf recovery was one of the crucial factors determining the positive relationship between species richness and system N retention, we expect this relationship to strengthen with time alike. For grassland studies, it was found that diversity effects increased with time due to increased complementarity (Cardinale et al. 2007; Oelmann et al. 2011). However, whereas these studies considered timeframes of several years, the present study may give evidence for seasonal niche differentiation or niche consolidation to cause complementarity effects within 1 year.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Our findings suggest positive species diversity effects for an important ecosystem service, which is highly relevant for afforestation programmes as currently applied in China on a large scale. The positive relationship between species richness and system N retention together with the increasing effect of species richness on leaf N recovery over time suggests that mixed plantings even at the sapling stage of a restricted species pool may considerably increase N retention in subtropical forest systems even after a few years. This in turn has the potential to significantly reduce N losses and thus N accumulation in the leachate or groundwater. Since species identity also has strong effects on ecosystem functions and services such as N retention (Zeugin et al. 2010; Oelmann et al. 2011), our findings also stress the importance of the respective species combinations to be planted in afforestation projects. From our experiment, we expect that a combination of evergreen and deciduous species more precisely of S. superba and C. henryi will hold the most promise for enhancing N retention of afforestation.

Following the Kyoto Protocol, current policy focuses primarily on carbon sequestration of afforestation. However, we want to stress that by establishing mixed afforestation of native species, society could profit from a portfolio of other ecosystem services such as prevention from erosion, aesthetic forest landscape or diverse fruit harvest. Further research is needed to evaluate the species mixtures with the most pronounced effects on overall ecosystem multifunctionality (Hillebrand & Matthiessen 2009; Isbell et al. 2011). Moreover, the use of a wide range of native species in afforestation projects could contribute to both an improvement of ecosystem services and the preservation of diverse forest ecosystems as they are typical in the study area (Myers et al. 2000).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

This study was funded by the German Research Foundation (DFG FOR 891/1). We are grateful for this support. We would like to thank the whole research group BEF China and especially the group of PhD students for the successful and cooperative teamwork. Furthermore, we appreciate very much the knowledge and help of Susanne Wedi-Pumpe and Ingelore Strube during the stable isotope analyses and laboratory work. Thanks to the helpful comments of two anonymous reviewers, this manuscript could be further improved.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
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