Study sites
The study was conducted in six mature European beech forests on different bedrock in Lower Saxony, Germany, covering the whole spectrum of soil types colonized by beech in this region.
Five study sites (HM, RU, EG, DR, GW) are located in the central German uplands on Mesozoic or Tertiary bedrock in the surroundings of Goettingen, one site (GR) is situated 33 km northwest of Hannover in the Pleistocene lowlands on glacial moraine deposits (Saalian). The climate of the study region is cool-temperate with mean annual temperature ranging between 7.1 and 8.7 °C, and mean annual precipitation between 709 and 902 mm (World Clim data base). The stands are either pure beech stands or dominated by F. sylvatica with admixture of single trees of other species and sufficiently comparable in terms of forest structure (26–36 m in height, 111–300 stems ha-1) and age (95-166 years) (Table 3.1). Three sites are characterized by deep soil profiles (> 2 m), which developed from Pleistocene fluvial and aeolian sandy deposits (GR), Tertiary sand (HM), or Quaternary loess (RU). The soils of the other three sites developed from Triassic sandstone (EG), Tertiary basalt (DR) or Triassic limestone (GW), and are comparatively shallow with a maximum profile depth of 60-80 cm. Due to the largely different bedrock types, the soils differ widely in their chemical and physical properties, in particularly in terms of soil texture, cation exchange capacity and base saturation (Table 3.2).
Soil sampling and analyses
Soil sampling was conducted in June 2013 (GR) and May 2014 (HM, RU, EG, DR, GW) along three randomly distributed transects per site with a minimal distance of 10 m from each other, each aligned towards a main tree. The transects of 330 cm length were excavated to a maximum depth of 200 cm (or to a maximum depth of 80 cm at the shallow sites EG, DR and GW).
Samples were taken in a regular grid with vertical and horizontal dimensions of 185 and 315 cm, respectively (Angst et al., 2016). The regular grid started close (10–50 cm) to a main tree (Fagus sylvatica L.) and extended horizontally in steps of 45 cm and downwards in 25 cm-steps. Soil samples were taken at each grid intersection with a circular steel core sampler (diameter: 8.5 cm, height: 6 cm) at 0, 45, 90, 135, 180, 225, 270, 315 cm horizontal distance and 10, 35, and 60 cm
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depth at all sites, and additionally at 85, 110, 135, 160, and 185 cm depth at the sites GR, HM and RU. Thus, a total of 24 (EG, DR, GW) or 64 (GR, HM, RU) soil samples were taken in each transect, resulting in a total of 72 or 192 samples per site. Immediately after sampling, the fresh soil samples were sieved (< 2 mm) to remove stones and roots. The soil samples were stored after sieving in polyethylene bags at 4 °C.
For the analysis of soil organic carbon and total nitrogen, the soil samples were prepared through drying for 3 d at 50 °C and grinding by a planet micromill (Fritsch Pulverisette 7). Total C and N contents were determined by gas chromatography (Vario EL elemental analyzer, Elementar, Hanau, Germany).
The pH of the fresh soil samples was measured in a 0.01 M CaCl2 solution (ratio soil to liquid:
1:2.5). Since all samples were free of carbonate, all C referred to organic C. For the analysis of soil texture, soil samples with a SOC content >1.2 % were pretreated with H2O2 to oxidize the organic matter. Soil texture was analyzed with a particle sizer (Analysette 22, Fritsch, Idar-Oberstein, Germany); the samples were separated into two size fractions (>0.2 and < 0.2 mm) with a 0.2 mm sieve, and each fraction was measured separately to increase the accuracy of the measurement. The base saturation and cation exchange capacity (CEC) of the samples were determined through ICP analysis after BaCl2 percolation.
Root sampling and fine root system analysis
We used the soil coring method to study beech fine root density (in g d.m. m-3 soil volume) and abundance (in g m-2 ground area per soil layer) in the top- and subsoil of profiles dug to 2 m depth, or to the surface of the bedrock. In each forest, a plot of ca. 30 x 30 m was demarcated, and each three subplots of ca. 25 m² size were placed by random in the direct neighborhood of the stems of a mature beech tree. Each six samples from the forest floor and the upper mineral soil layer were extracted at random locations using a soil corer of 3.5 cm in diameter; the extracted soil cores were sliced into the organic layer, and the 0-10 cm, 10-20 cm and 20-40 cm layers of the mineral soil. For every sample, the thickness of the organic layer (consisting of the L, Of, Oh layers) was measured at the undisturbed wall left after the extraction of the soil core.
Samples of the lower profile (≥ 40 cm depth) were taken in each one soil pit of 1.35 m length per
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subplot (i.e. three per study site) that was dug with an excavator to 2 m depth (or to bedrock depth at the sites EG, DR and GW). Using a steel cylinder of 12.3 cm in diameter, each three soil samples were taken from the three walls of a pit at depth intervals of 20 cm, corresponding to a sample volume of ~ 2.4 L. Each sample was taken right under the sample of the next higher depth. In total, nine samples (three per soil pit, three pits) were extracted per soil depth and site, resulting in a total soil volume of ca. 22 L that was analyzed per depth layer. All samples were transferred to plastic bags and stored at 4 °C until being processed in the laboratory.
In the laboratory, the samples were gently washed over sieves of 0.25 mm mesh size to separate the roots from adhering soil particles. We soaked the sample remains in demineralized water and extracted all roots greater 10 mm length with tweezers for further examination; smaller root fractions were neglected in two out of three samples to keep the workload reasonable. Under the stereo-microscope, the larger rootlets >10 mm length were separated into live (biomass) and dead (necromass) roots, and subsequently into fine (≤2 mm in diameter) and coarse roots (> 2 mm in diameter). The distinction of live and dead roots was done based on the criteria root and periderm color, tissue elasticity, and cohesion of cortex, periderm and stele (Hertel et al. 2013). Even though the small root fragments < 10 mm in length were neglected, the procedure has been found to be suitable to collect the largest part of fine root biomass (> 95%) (Bauhus and Bartsch 1996;
Leuschner et al. 2001). Fine root necromass, however, is considerably underestimated by the neglect of root fragments < 10 mm length. We used soil depth-specific regression equations to correct this error by extrapolating the necromass from precisely analyzed samples to those samples that were only partly analyzed. The regressions relate the mass of dead fine roots < 10 mm in length to the necromass particles ≥ 10 mm in length, established separately for every third sample. In these precisely investigated samples, the mass of small dead roots was quantified with a method introduced by van Praag et al. (1988) and modified by Hertel (1999). After the extraction of the large rootlets, the sample residue was evenly spread on a filter paper (730 cm²), which was divided into 36 even-sized small quadrats. Six of the quadrats were randomly selected and even the finest necromass particles collected under the microscope. All live and dead root samples were dried at 70°C for 48 h and weighed.
42 Analysis of fine root morphology
For determining specific root length (SRL, in cm g d.m.-1), specific root surface area (SRA, in cm² g d.m.-1), and the mean root diameter (MD, in mm) of the live fine root fraction < 2 mm, the fine roots of each sample were scanned (EPSON expression 1680, EPSON America Inc.) and analyzed using the WinRHIZO 2005c image analysis software (Régent Instruments Inc., Quebec, Canada). Fine root length index (RLI, in m root length m-2 ground area) and fine root area index (RAI, in m2 root area m-2 ground area) were calculated by multiplying SRL or SRA with the fine root biomass of the respective depth layers and integrating fine root length or surface area over the whole soil profile. Root tip frequency (RTF, in n mg-1 root d.m.) was determined by counting all turgescent tips of two live fine root strands per sample and relating the number to the respective root dry mass; root tip abundance (RTA, n m-2 ground area) was calculated by multiplying RTA with the fine root biomass of the respective soil layer, root tip density (RTD, n L-1) by relating to soil volume.
Statistical analyses
Means and standard errors were calculated by averaging over the each three soil pits of a study site, while the each three samples taken in the same depth of a pit were treated as pseudo-replicates by averaging over them.
We used the SAS package, version 9.3 (Statistical Analyses System, SAS Institute Inc., Cary, NC, USA) for statistical analysis. A non-parametric Mann-Whitney (Wilcoxon) two-sample test was used to determine significant differences between the different soil layers at a site, and difference between the six sites for a given soil layer. Significance was determined at P ≤0.05 throughout. Multivariate regression analyses were performed to analyze the influence of soil characteristics on fine root biomass, and of fine root biomass and soil characteristics on fine root morphological traits. We used the backward elimination procedure at the significance level P
≤0.05. Vertical root mass distribution was modeled as y = 1-βd, where y is the cumulative root fraction from the soil surface to depth d and β is the extinction coefficient (Gale and Grigal 1987;
Jackson et al. 1996). High β values correspond to a larger proportion of root mass in greater soil depth. We applied a Pearson correlation analysis to search for relationships between root
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morphological traits and measures of fine root system size and structure, and edaphic and stand structural parameters. We conducted a Principal Components Analysis (PCA) to analyze relationships between fine root biomass, root morphological traits and soil properties (for a list of matrix factors see Table 3.7). The PCA was conducted with the package CANOCO, version 4.5 (Biometris, Wageningen, The Netherlands).