Sampling
The MFD sample collection includes samples collected as part of the MFD sampling campaign, as well as samples contributed by members of the MFD consortium. The samples taken as part of the MFD sampling campaign were registered and associated with the appropriate metadata using codeREADr (https://www.codereadr.com) using a linear barcode attached to sterile 100 ml sample containers. After collection, the samples were stored between 4 °C and 10 °C for up to 48 h before being deposited at −20 °C for later processing.
As we wanted to cover as much of the Danish environmental landscape as possible, we requested expert collaborators send existing samples from interesting environments or environments that are not easily sampled. These include samples from existing publications, samples collected as part of governmental monitoring, but also samples from collaborators with no current publication. If not otherwise stated, these samples were acquired as frozen sample material. We divided each set of samples into projects, in which samples of the same type (soil, sediment, water) were subjected to the same treatment. Based on this, we constructed summary tables over the different protocols used for sampling and DNA extraction methodology (Supplementary Data 5). Most samples (across the biggest sample groups) were treated similarly, but we acknowledge that the different treatments might affect the results; consequently we applied the appropriate filtering where needed. The number of subsamples and other related sampling metadata are provided at GitHub and in Supplementary Data 6.
Soil samples
Topsoil samples from the MFD sampling campaign were collected as up to five subsamples (0–20 cm), taken within a ∼80 m2 (5 m radius) sampling area using a weed extractor, which was cleaned with 70% ethanol between sampling sites. As DNA from microorganisms could potentially be overwhelmed by the DNA from whole specimens in the sample material, we visually inspected each subsample with the naked eye and avoided including complete specimens (grass, leaves, sticks or larger animals) in the samples. After specimen removal on site, the subsamples were combined in a sterile plastic bag, the bag closed and the collective sample homogenized by hand before up to 100 ml was transferred to the barcoded sample container (P04_2, P04_4, P04_6, P04_7, P08_1, P08_2, P08_3, P08_5, P08_6, P08_7, P08_8 and P17_1). The samples from projects P19_1, P20_1, P21_1 (ref. 64) and P25_1 were collected as single subsamples. The subset of topsoil samples from the Land Use and Coverage Area Frame Survey (P04_8)25 were collected by collaborators from Aarhus University as described in previously65, in a manner very similar to the MFD sampling campaign.
A subset of the topsoil samples (P01_1)31 from both natural and agricultural habitats were provided by collaborators from Aarhus University and Copenhagen University. These were collected as described previously31. In brief, 81 subsamples, spanning a 9 × 9 grid covering a 40 × 40 m plot, were mixed into a representative sample from which we acquired a subsample. New sample projects were added to extend the existing project with wet terrestrial habitats (P01_2), agricultural and semi-agricultural habitats (P02_1), sites with different agricultural practices (P02_2 (ref. 66)) and urban habitats (P03_1 (ref. 67)). These were all collected as 81 subsamples except in the case of P03_1 which was mixed from 9 subsamples.
Samples from subterranean soils were collected as single samples from different depths using a soil drill (P06_1, P06_2, P06_3). Subsurface soil (P06_1) was collected with PVC liners by percussion hammering using a Geoprobe (NIRAS) drill rig. Soil samples were then collected around the oxic-anoxic interface with 5 ml cut-off syringes through openings cut into the core liners. We acquired the samples from P06_3 as DNA, which had previously been extracted using the DNeasy PowerLyzer PowerSoil Kit (QIAGEN) according to the manufacturer’s protocol. In the case of agricultural soils from croplands, six out of every seventh sample was provided by SEGES. As part of the collection, the individual samples were frozen, crushed to particles below 1 cm in size and dried at 37 °C (P04_3, P04_5), the effect of which was investigated (Supplementary Note 3).
Sediment samples
Surface sediment samples (0–10 cm) from the MFD sampling campaign were collected as up to five sediment subsamples from across the sampling area using a gravity corer, which was cleaned with 70% ethanol between sampling sites. The subsamples were combined in a sterile plastic bag, the bag was closed and the collective sample was homogenized by hand after careful removal of larger debris and any collected water. Up to 100 ml of the homogenized sample was transferred to the barcoded sample container. For the sediment from standing water sources (P05_1, P05_2, P08_5, P09_1, P09_2, P11_1 and P11_3) the top 10 cm was collected, while only the top 5 cm was collected from streams (P10_1, P10_2 and P10_3). Pond sediment (P09_2) was collected from the deepest point of each pond. Stream samples were collected as three subsamples across a 20 m transect of the stream, two at 25% distance from each brim and one in the middle of the stream.
Lake sediments provided by University of Southern Denmark were from either lakes selected for investigation of biotic phosphorus dynamics (P09_3) or a lake restauration initiative (P09_4). For P09_3, the cores were taken from the deepest part of the lake. For P09_4, the cores were taken at five different sampling stations. In both cases, a gravity corer was used for the sampling68. Sediment samples from coastal areas (P11_2) were collected at a single point using a HAPS bottom corer, as described previously69. Each sample was mixed from 10 subsamples of the sediment core (0–2 cm and 5–7 cm). We acquired the samples as DNA, which had previously been extracted with the DNeasy PowerMax Soil Kit (QIAGEN) according to the manufacturer’s protocol.
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