Materials and Methods ESM1

Materials and methods ESM1

Field site and experimental set-up

At the field site, the organic horizon is about 3 m (from 2-4 m) thick, with a mean soil organic matter (SOM) content of 29.2% (range=20.1-55.4%, Walkley-Black method), a clay content of 25%, a pH of 4.9, and a bulk density of 0.5 g cm-3 in the topsoil layer (0-30 cm deep).

The climate is Mediterranean, with dry and hot summers and rainfall mainly concentrated in autumn and spring (mean annual rainfall = ca. 945 mm year-1) and mean monthly air temperature ranging from 7°C in February to 30°C in August (mean = 14.8°C year-1). Further details on climate conditions are found in Pellegrino et al. (2014).

Soil analytical methods

Soil pH and EC were measured in deionised water (1:2.5 and 1:2, w/v, respectively). Kexch was determined by atomic absorption. Pavail was determined by colorimetry using an extraction with sodium bicarbonate (Olsen and Sommers, 1982). Ntot was determined by the macro Kjeldahl digestion procedure, while NO3- and NH4+ were determined by the Kjeldahl method after KCl 2 N extraction and, in the case of NO3-, after reduction with Devarda's alloy. SOM was measured using the modified Walkley-Black wet combustion method (Nelson and Sommers, 1982). Exchangeable cations were determined by the BaCl2 method (pH 8.1) (Soil Survey Staff, 1992), and then the extracted solution was analysed by atomic absorption (Thomas, 1982). CEC was determined from soil using the BaCl2-triethanolamine method (pH 8.1). Base saturation (Bas Sat) was calculated by dividing the sum of the exchangeable cations by the CEC, and the soil C:N ratio was calculated by dividing soil organic carbon ((SOM / 1.7) × 10) by Ntot.

DNA extraction, PCR amplification, cloning and sequencing

The quality of the extracted DNA and amplicons was checked on a ND-1000 spectrophotometer (NanoDrop Technology, Wilmington, DE, USA). PCR total soil fungal amplicons were generated from 10 ng μL-1 genomic DNA in volumes of 20 µL with 0.5 U of GoTaq® Hot Start Polymerase (Promega Corporation, Madison, WI, USA), 0.5 μM of each primer, 0.2 mM dNTPs, 1.25 mM of MgCl2 and 1× reaction buffer.

PCR amplicons of arbuscular mycorrhizal fungi (AMF) were generated from 10 ng μL-1 genomic DNA in volumes of 20 µL with 0.5 U of GoTaq® Hot Start Polymerase (Promega Corporation, Madison, WI, USA), 0.2 μM of each primer (NS31/AM1), 0.2 mM of each dNTP, 1.25 mM of MgCl2 and 1× reaction buffer. All PCR reactions were run on a S1000 Thermal Cycler™ (Bio-Rad, Hercules, CA, USA).

An ABI Prism® 3730XL automated sequencer (Applied Biosystems, Carlsbad, CA, USA) was used for sequencing (High-Throughput Genomics Unit, Seattle, WA, USA).

Data and sequence analysis

The updated AMF reference dataset is composed of 59 NS31/AM1 public sequences created using only AMF morphotypes. The reference dataset provides the correspondence of the sequences with the closest virtual taxon (VT) after BLAST search against the MaarjAM database (Öpik et al., 2010). The alignment is in an open-access database available at https://sites.google.com/site/restomedpeatland/microbiology. Individual-based rarefaction curves (Gotelli and Colwell, 2001) were calculated using clones/sequences as units of replication.

Statistical analyses

To infer that the effects we detected were unlikely to have been caused by a purely spatial phenomenon, we accounted for plot positions by applying analysis of covariance (ANCOVA). In this way, we factored out the distance variable (using the spatial coordinates as covariables), which may have affected the dependent variables studied. This allowed a proper comparison of the treatments. To use the spatial coordinates in the ANCOVAs, the variables were transformed into radiants and then standardised.

Regarding multivariate analysis, we used the partial redundancy analysis (pRDA) linear method (Lepš and Šmilauer, 2003) when the length of the gradient of the detrended correspondence analysis (DCA) was lower than four, otherwise the partial canonical correspondence analysis (pCCA) unimodal method was used. Variation partitioning (VarPart) was achieved by applying a model that tested the unique effects of predictors (VarPart-2-groups-Conditional effects-tested) (ter Braak and Šmilauer, 2012). All data were log-transformed, centred and standardised by the response variables. Monte-Carlo permutation tests were performed using 499 random permutations (unrestricted permutation) to determine the statistical significance of the relations between land-use type and response variables.

References

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Lepš J,Šmilauer P(2003)Multivariate Analysis of Ecological Data Using canoco. Cambridge University Press, Cambridge, UK

Nelson DW. Sommers, LE, (1982) Total carbon, organic carbon and organic matter Page AL, Miller RH, Keeney DR (Eds.) Methods of Soil Analysis, Part 2. Chemical and microbiological properties, 2nd edn. American Society of Agronomy, Madison, WI, USA, pp. 539-579

Olsen SR, Sommers LE (1982) Phosphorus. In: Page AL, Miller RH, Keeney DR (Eds.) Methods of Soil Analysis, Part 2. Chemical and microbiological properties, 2nd edn. American Society of Agronomy, Madison, WI, USA, pp 403-430

Öpik M, Vanatoa A, Vanatoa E, Moora M, Davison J, Kalwij JM, Reier U, Zobel M (2010) The online database MaarjAM reveals global and ecosystemic distribution patterns in arbuscular mycorrhizal fungi (Glomeromycota) New Phytol 188: 223-241

Pellegrino E, Bosco S, Ciccolini V, Pistocchi C, Sabbatini T, Silvestri N, Bonari E (2014) Agricultural abandonment in Mediterranean reclaimed peaty soils: long-term effects on soil chemical properties, arbuscular mycorrhizas and CO2 flux.Agr Ecosyst Environ 199: 164-175

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Thomas GW (1982) Exchangeable cations. In: Page AL, Miller RH, Keeney DR (Eds.) Methods of soil analysis. Part 2. Chemical and microbiological properties, 2nd edn. American Society of Agronomy, Madison, WI, USA, pp 159-165

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