2.3.1 Study sites
The experiments were conducted on three permanent grassland sites typical for Northern Germany. The whole region is characterized by a temperate, sub-continental climate.
The south-eastern lowland site (SEL) is located near Göttingen in the Leine valley, the north-western lowland site (NWL) is located near Oldenburg in the Northern German Plain and the sub-mountainous site (SMS) is located in Silberborn near Uslar in the Solling mountain range.
Site details are presented in Table 8-1.
2.3.2 Experimental design
In a three-year experiment (2011 – 2013) on all three sites, we investigated the effects of drought (with and without rain-out shelters), sward composition (with and without reduction of dicot species cover), and nitrogen fertilization (with 180 kg N ha-1 a-1 or without) in a completely randomized block design with four replicates. Treated plots had a size of 1.8 m by 1.8 m (3.24 m²). All measurements and samples were taken from a core area of 0.4 x 0.4 m (0.16 m²) in the center of each plot.
Spring and summer droughts of, on average, 36 days were induced by installing rain-out shelters with an inclined roof allowing rain to run off that covered the whole plot with UV-permeable greenhouse film (GeKaHo GbR, Gewächshausfolie SPR 5, 200my) at 1.5 m mean height. Measurements of photosynthetically active radiation (PAR) with the SunScan Canopy Analysis System on a sunny day in May 2012 around noon showed a significant difference (chi-squared = 44.9032, p-value <0.0001) of the ambient mean PAR between plots with and without shelters (n = 32; without shelter: 1757 ± 48 W m-², with shelter 1275 ± 86 W m²).
Due to the lower radiation intensity under the shelters we expect photosynthetic rates and thus assimilation to be smaller, leading to a similar effect as the drought stress treatment, although we cannot distinguish between the effects of water supply and PAR on plants.
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Spring drought stress periods started at the end of April or the beginning of May, about seven to eight weeks after the start of the year’s growing season. The start of the growing season was determined by five consecutive days with an average temperature > 5°C (Jones et al., 2002). After the end of the spring drought stress period, the greenhouse films were removed for three weeks to allow rewetting of stress-treated plots by natural precipitation.
Summer drought stress periods started around the end of June. Afterwards, natural precipitation was again allowed on all plots until the following spring. No irrigation was applied during the three years of experiments.
We follow the definition of a functional group as a group of species that share morphological, and perhaps physiological, traits (Lauenroth et al. 1978) and thus divided the species found in our swards into the functional groups grasses, forbs, and legumes. The functional group composition of swards was manipulated by the application of herbicides against dicotyledonous species (forbs and legumes). Thus, on half of the plots, Starane Ranger (100 g l-1 Fluroxypyr and 100 g l-1 Triclopyr, 2 l 1) and Duplosan KV (600 g l-1Mecoprop-P, 2 l ha-1) were applied one year before the start (2010) and in the course of the experiment (2012).
Herbicide treatment resulted in two sward types: diverse swards with the original species composition and grass-dominated swards (Table 8-2).
On all plots, 200 kg ha-1 potassium chloride (in the form of 40 % K2O) and 30 kg ha-1 triple superphosphate (in the form of 46 % P2O5) were applied at the beginning of the growing season to ensure plant nutrient supply. Additionally, to half of the plots 180 kg nitrogen (N) ha-1 were applied. N application was split into 90 kg N ha-1 at the beginning of the growing season and 45 kg N ha-1 after the each cut-. K application was split into 100 kg ha-1 at the beginning of the growing season and 100 kg N ha-1 after the first cut at the end of the spring drought stress period.
Whole plots were cut directly after each stress period and once again in the beginning of October at 7 cm stubble height. Biomass samples were taken only from the core area.
Samples were sorted by functional group (grass, forbs, and legumes), dried at 60 °C for 48 hours and weighed. Aboveground production was determined as accumulated dry weight per year.
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We cumulated the sample weights of the first two as well as of the third annual cuts over all three years and analyzed both datasets for the influence of sward type and fertilization level on sward biomass production during drought stress events.
2.3.3 Water and climate relations
The microclimate under the rain-out shelters and above control plots was surveyed by data loggers (CiK Solutions GmbH, Haxo-8 LogTag), which recorded values every 30 minutes from April until the last cut during all three years. Temperature and relative humidity showed no significant difference (Wilcoxon signed rank test with continuity correction, P = 0.1763) between treatments with and without shelters..
We did not apply barriers to prevent water run-off into the plots, to minimize disturbance to the swards’ root system. Soil water content under rain-out shelters was monitored by gravimetric sampling. Even after major rainfall events, the core area from which all samples were taken proved to remain dry. During the induced droughts, which lasted on average 36 days, stressed plots received no rainfall. The control plots received natural precipitation. The rain-out shelters held back 73 ± 5% precipitation (SEL), 52 ± 8% (NWL), and 60 ± 5% (SMS), respectively, during the experimental period. On average over all locations and years, rain-out shelters held back 148 mm of the 240 mm precipitation (62 ± 8%) that fell from the beginning of the growing season until the end of the second drought stress treatment.
2.3.4 Statistical analysis
Statistical analyses were performed with R version 3.0.2 (R Core Team 2013) using a significance level of α ≤ 0.05 throughout. Two datasets each (spring and summer harvest weights, autumn harvest weights) of total plot biomass as well as grass functional group biomass were analyzed by applying linear mixed models using the additional software package nlme by Pinheiro et al. (2011). All models were tested for normal distribution
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(quantile-quantile-plots and Shapiro-Wilk test) and homoscedasticity (residual plots and Levene Test). If these criteria were not met, models were corrected by including a variance function or transformation. Fixed effects in the analyses of whole swards were sward type, fertilization, drought stress, year, and season. Site and block were included as nested random effects. In the fixed effects of the analyses of grass biomass sward type was substituted by the cumulative biomass share (%) of forbs and legumes, because forb and legume share as separate variables resulted in model overload.
All full models (initial models before optimization) included all possible interactions and were subsequently optimized according to the methods suggested by Pinheiro & Bates (2000) and Zuur et al. (2009) to obtain a model that optimally fit the data and had an AIC as low as possible. Fixed effects and their interactions that appear as not significant in the results tables were excluded from the model during optimization, which resulted in a better model fit to the data.
After all models were fit, we calculated and grouped post-hoc pairwise contrasts of the most influential variables and their interactions using the Tukey method for comparing families found in the packages lsmeans (Lenth 2016) and multcompView (Graves et al. 2015). Most influential variables were selected by the magnitude of the F-value.