Soil Parameter and Molecular Fungal Community Analysis

In the laboratory, gravimetric soil moisture content, pH in CaCl₂, total carbon (C_tot), inorganic C, and total nitrogen (N_tot) contents were determined according to standard procedures as outlined in Online Resource 1, Supplementary Materials and Methods. Organic carbon (C_org) was calculated as the difference between C_tot and inorganic C. In early June 2015, detailed physio-chemical analyses were conducted on soil samples derived from additional soil pits (n = 4) to characterize the habitat. The following parameters were determined: fine and coarse root biomass, stone fraction, bulk density, pH, C_org, N_tot, soil nutrients, and exchangeable ions (Online Resource 1, Table S1).

For DNA isolation from soil samples, 800 μl of the soil suspension in LifeGuard Soil Preservation Solution (see above) was transferred to the wells of a Bead Plate from the PowerSoil-htp 96-Well Soil DNA Isolation Kit (MO BIO, Carlsbad, CA, USA). After centrifugation (4000g, 15 min) and removal of the supernatant, the protocol of the manufacturer was followed with modifications (see Online Resource 1, Supplementary Materials and Methods). The fungal ITS2-region, which has been suggested as preferred barcoding region for fungal community analyses [44], was amplified with primer pair ITS3Mix/ITS4Mix (adapted from [37], for details see Online Resource 1, Supplementary Materials and Methods). Library preparation and Illumina MiSeq sequencing of fungal amplicons were conducted as described [45]. Illumina MiSeq PE250 sequencing was performed at the NGS Unit of the Vienna Biocenter Core Facility GmbH (Vienna, Austria). For details, see Online Resource 1.