Ecological Archives E096-017-A2
Martine Kos, Tibor Bukovinszky, Patrick P. J. Mulder, and T. Martin Bezemer. 2015. Disentangling above- and belowground neighbor effects on the growth, chemistry, and arthropod community on a focal plant. Ecology 96:164–175. http://dx.doi.org/10.1890/14-0563.1
Appendix B. Description of the methods of the chemical and molecular analyses and SEM.
B.1 Chemical analysis of monospecific and control soil
To determine the abiotic soil characteristics of the five replicates of the monospecific and control soils, a sub-sample of 100 g of each soil samples was sieved (4 mm mesh size) and dried (5 days at 40 °C). In this sub-sample, phosphorous (P) was extracted by shaking 5 g dry soil with 50 mL 0.01 M CaCl2 for 2 h and concentrations of P were determined colorimetrically using a BioTek Synergy HT multi-mode microplate reader (Beun- de Ronde, Abcoude, the Netherlands). Mineral nitrogen (N) was extracted by shaking 10 g dry soil with 50 mL 1 M KCl for 2 h and concentrations of ammonium (NH4-N) and nitrate (NO3--N) were determined colorimetrically using a SEAL QuAAtro SFA system (Beun- de Ronde). Total plant-available amounts of phosphorous (Olsen-P) were determined using the method of Olsen et al. (1954) and absorbance was measured at 720 nm. The % OM was determined as weight loss of a soil sample after ignition at 430 °C for 24 h. Total % carbon (C) and % N was measured on a FlashEA 1112 Elemental Analyzer (Interscience, Breda, the Netherlands). pH was measured in 2:5 dry soil:water suspensions.
B.2 Molecular analysis of fungal and bacterial communities in monospecific and control soil
The composition of the soil fungal and bacterial communities in the five replicates of the monospecific and control soils was determined by Terminal restriction fragment length polymorphisms analyses (T-RFLP). Total DNA was extracted from 0.25 g frozen soil (-20 °C) with a Power Soil DNA isolation kit (MOBIO laboratories, Carlsbad, CA, USA) using a bead beating system. DNA concentration was measured using the NanoDrop 2000. For the samples with a DNA concentration below 10ng/µL a new DNA-extraction was performed.
For analysis of the fungal communities, the ITS region of the fungal rDNA was amplified by PCR using the primers ITS1F (White et al. 1990) and ITS4 (Gardes and Bruns 1993), dual-end labeled with 6FAM and NED respectively. The PCR reaction contained 13.8 μL milli-Q water, 2.5 μL 10x Fast Start High Fidelity Reaction Buffer (Roche Diagnostics, Almere, The Netherlands), 2.5 μL dNTP mix (2 mM of each), 2.5 µL ITS1Ff-6FAM primer (10 µM), 2.5 µL ITS4r-NED primer (0.2 μM), 0.2 μL Fast Start High Fidelity Enzyme Blend (5 U μL-1) (Roche Diagnostics) and 1 μL template DNA. PCR cycle conditions were 5 min at 95 °C, 34 cycles of 30 s at 95 °C, 40 s at 55 °C and 1 min at 72 °C, followed by 10 min at 72 °C before cooling. PCR product presence and quality were verified on 1.5% agarose gels prior to restriction digestion. Two restriction enzymes, HhaI and TaqαI (New England Biolabs, Ipswich, MA, USA), were used to digest the dual end-labelled DNA amplicons in a mixture containing 3.5 μL ddH2O, 1 μL buffer, 0.1 μL Bovine Serum Albumin, 5 μL PCR product and 0.4 μL restriction enzyme, incubated at 37 °C (HhaI) or at 65 °C (TaqαI) for 3 h, followed by inactivation at 80 °C for 20 min. Restriction products were purified using ethanol precipitation.
For analysis of the bacterial communities, DNA was amplified by PCR using the fluorescently-labelled forward primer FAM-27f and the unlabeled reverse primer 1492r, which target the bacterial 16S rRNA gene (Moeseneder et al. 1999). The PCR reaction contained 17.8 µL milli-Q water, 2.5 µL 10x Fast Start High Fidelity Reaction Buffer (Roche Diagnostics), 2.5 µL dNTP Mix (2 mM of each), 0.5 µL of each primer 27f-FAM and 1492r (10 µM), 0.2 µL Fast Start High Fidelity Enzyme Blend (5 U µL-1, Roche Diagnostics) and 1 µL template DNA. PCR amplification conditions were: 5 min at 95 °C, 34 cycles of 30 s at 95 °C, 40 s at 55 °C and 1 min at 72 °C, followed by 10 min at 72 °C before cooling. PCR product presence and quality were verified on 1.5% agarose gels prior to restriction digestion. The restriction enzymes HhaI and MspI (New England Biolabs, Ipswich, MA, USA) were used to digest the amplified DNA in a mixture containing 3.5 µL ddH2O, 1 µL buffer, 0.1 µL Bovine Serum Albumin, 5 µL PCR product and 0.4 µL of each restriction enzyme, held at 37 °C for 3 h, followed by enzyme inactivation at 80 °C for 20 min. Restriction products were purified using ethanol precipitation.
Fragment length polymorphism analysis was performed on an automated 3130 Genetic Analyser sequencer, using GeneScan-500 LIZ (Applied Biosystems, Carlsbad, CA, USA) as a size standard. Samples which were over- (highest peak > 80 000 relative fluorescence units, rfu) or under-loaded (highest peak < 1000 rfu) were re-run with an adjusted concentration. Peaks at sizes < 50 base pairs (bp) and > 500 bp were removed. Peaks were aligned to TRFs among the samples by applying a clustering threshold of 0.5 bp. All peaks higher than 0.3% of the sum of all peaks in a sample were included in the analysis (Bezemer et al. 2013). In soil conditioned by the five neighboring plant species and the control soil in total 620 fungal and 198 bacterial TRFs were detected.
B.3 Chemical analysis of focal plants
C and N content of J. vulgaris leaves were determined using a FlashEA 1112 Elemental Analyzer (Interscience, Breda, the Netherlands). After destruction of the organic matter by ignition at 550 °C, the total amount of P was released by digestion in an autoclave for 30 min at 121 °C with 2.5% potassium persulfate and converted into orthophosphate. The liberated orthophosphate was measured by the colorimetric technique of Murphy and Riley (1962), with a SEAL QuAAtro SFA system (Beun- de Ronde).
PA analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following the procedure outlined by Cheng et al. (2011). In short, 10 mg of freeze-dried ground plant material was extracted with 1.0 mL 2% formic acid solution containing heliotrine (1 µg mL-1) as internal standard. Following centrifugation and filtration, 25 µL of the extracted filtrate was diluted and neutralized with 975 µL of 10 mM ammonium hydroxide solution. Ten µL of the dilution was injected in a Waters Acquity ultra-performance chromatographic system coupled to a Waters Quattro Premier tandem mass spectrometer (Waters, Milford, MA, USA), run in multiple reaction monitoring mode (MRM). For the chromatographic separation of PAs a Waters UPLC BEH C18 (150 × 2.1 mm, 1.7 µm particles) analytical column was used. The column was kept at 50°C and run at 0.4 mL/min using an acetonitrile/water gradient containing 6.5 mM ammonium hydroxide. The gradient started at 100% water and was changed linearly to 50% acetonitrile in 12 min. For each compound two product ions were selected, based on their fragmentation spectra, to be included in the MRM. See Table C1 for the mass spectrometric settings used for each compound. PAs were quantified against a set of calibration samples of PA reference standards (corresponding to a concentration range of 0 to 500 ng/mL in the diluted plant extracts). Data were processed using Masslynx 4.1 software. The mean concentrations obtained in the J vulgaris focal plants (215 individuals) for 45 PAs as well as the sum concentration for PA tertiary amines and PA N-oxides are shown in Table C1. Data were processed using Masslynx 4.1 software.
B.4 Structural equation modeling
Structural equation modeling (SEM) was used to explore the possible mechanisms by which neighboring plants affected arthropod abundance on the focal plant. SEM was performed with the 'sem' package in R (version 3.0.1, R Development Core Team 2013). We performed two different SEM analyses. In the first SEM analysis, we aimed to disentangle AG and BG neighbor effects. The conceptual model for this SEM analysis considered both direct and indirect neighbor effects, AG or BG, on the abundance of herbivores and carnivores on J. vulgaris. All possible indirect neighbor effects, either via changes in biomass or via changes in chemistry of the focal plant, were included in the model. Indirect effects were calculated by computing the product of the path coefficients along the indirect path. This SEM analysis was performed for all neighboring species combined, but also separately per neighboring species. Treatment 4 with AG and BG effects in experimental units with connected (open) pots was kept out of the SEM analysis. Prior to the SEM analysis, variables were transformed as described for the univariate analyses. For plant chemistry, the sample scores on the first axis of a Principle Component Analysis, including foliar % P, C:N ratio, total PA concentration and the proportion of PA tertiary amines, was used. We removed non-significant paths from our model to select the model that best fitted our data. In SEM, the goodness-of-fit of the model is assessed by comparing the observed and model-predicted covariances with a Χ² test. If the Χ² values have an associated P value of > 0.05, the model is acceptable (there is reasonable fit between model and the data) (Grace 2006).
In the second SEM analysis, we studied which AG traits of the neighboring plants affected arthropod abundance on the focal plant. We included foliar and reproductive biomass and herbivore and carnivore abundance on the neighboring plants as continuous predictor variables. Furthermore, we modelled all other unmeasured plant traits in a single predictor variable: the factor 'Neighbor identity'. Foliar and reproductive biomass were included as separate predictor variables because the effect of flowers and leaves on arthropod attraction may differ. For this SEM, only the data for experimental units in which an AG neighbor was present were included in the model (AG and AG+BG treatments, although treatment 4 with AG and BG effects in experimental units with connected pots was kept out of the analysis). This second SEM analysis was performed for all neighboring species combined. The conceptual model considered both direct and indirect effects of neighbor identity and biomass on the abundance of herbivores and carnivores on J. vulgaris. All possible indirect effects of neighbor identity and biomass, either via changes in biomass or chemistry of the focal plant, or via changes in arthropod abundance on the neighboring plants themselves, were included in the model.
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