BIODIVERSITY GENOMICS EUROPE WP4
Ecological Restoration - Soil sampling
SOP
First version: 23 June 2023
Last version: 10 November 2024
Laura Nájera-Cortazar [BIOPOLIS-CIBIO]
Albano Beja-Pereira [BIOPOLIS-CIBIO]
Pedro Beja [BIOPOLIS-CIBIO]
Associação Biopolis - CIBIO – Centro de Investigação em Biodiversidade e Recursos
Genéticos [BIOPOLIS - CIBIO]
BIODIVERSITY GENOMICS EUROPE
receives funding from the European Union's Horizon Europe Research and Innovation Action.
https://biodiversitygenomics.eu/
2 | Ecological Restoration-Soil Sampling | PlutoF
Table of contents
Introduction 2
Sampling Design 3
Before starting 6
During sampling 7
After sampling 9
Registering samples in PlutoF Go app 10
Acknowledgements 13
References / Bibliography 13
Useful contact 13
Introduction
The Biodiversity Genomics Europe (BGE) Consortium has the overriding aim of accelerating the
use of genomic science to enhance understanding of biodiversity, monitor biodiversity change,
and guide interventions to address its decline. The objective is to establish functioning
biodiversity genomics networks, data generation and pipelines to characterize biodiversity, and
to improve management intervention and biomonitoring programs by practical application of
genomic tools.
Soils are one of the main global reservoirs of biodiversity, and a key determinant of ecosystems
functioning and performance (Wagg et al., 2014). Despite soil’s biodiversity exceeding that of
other terrestrial systems, it remains highly understudied (FAO et al. 2020), particularly regarding
microorganisms community diversity and interactions in wild vs cultivated lands. In collaboration
with the wIldE project, that searches for climate-smart rewilding as tools against the threats of
climate change and biodiversity loss, the Ecological Restoration - Soil case study is designed to
characterize soil biodiversity in Europe using genomic tools, and to evaluate how soil’s
biodiversity changes across ecological gradients of land abandonment and/or post-disturbance
vegetation stages. The case study will inform if changes in diversity, species composition and
functional composition of soil communities share similarities across Europe, or in alternative, if
they are largely idiosyncratic and site-specific.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
2
3 | Ecological Restoration-Soil Sampling | PlutoF
Sampling Design
To evaluate changes in soil biodiversity during the process of ecological succession following
land abandonment (or a major disturbance), metabarcoding techniques will be used to assess
soil biodiversity, targeting fungi, arthropods, and microbiome. For each local case study, the
sampling design involves a chronosequence of replicated plots at different stages of recovery
since land abandonment or the last major disturbance (Figure 1). The chronosequence should
start with plots representative of early successional stages, mostly old fields (i.e., at some time
after land abandonment) and end in late successional old-growth or mature forests, including
also the intermediate recovery stages. Other chronosequences representing ecological
restoration/rewilding are also considered.
For each case study, four restoration stages will be sampled (early successional, two
intermediate stages, and late successional, with no fixed distance between each other), with six
independent replicates per restoration stage (i.e., six different plots per each stage, at a
minimum of 1 km from each other); this can be adjusted depending on the particular conditions
of each case study.
Figure 1. Example of a chronosequence (space for time substitution) representing the stages of
vegetation recovery after land abandonment.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
3
4 | Ecological Restoration-Soil Sampling | PlutoF
At each replicate and sampling occasion, five sample cores should be sampled within each
replicate, collecting two soils samples/depths per core, one at a 10-20 cm depth, and another
one at a 20-30 cm depth (Figure 2). As a reference, cores within each replicate can be taken in
a square-like manner, with no fixed distance between them (100 mt distance is suggested when
possible, but there is no fixed distance required for this study). In total, there will be 240
samples (60 samples per stage, per four stages). Individual labeled tubes will be sent to
BIOPOLIS-CIBIO for pooling and downstream analyses.
Figure 2. Example of the sampling scheme per successional stage, per replicate. The soil probe
(right end in the image) samples one core that can include two depths (below the red lines), if the
terrain is deep enough.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
4
5 | Ecological Restoration-Soil Sampling | PlutoF
Materials
List of materials needed for soil sampling (Figure 3)
A. Soil probe (a length of more than 1m is recommended to facilitate manipulation)
B. Scaled shovel (e.g. gardening kit)
C. Bulb planter
D. Soil tester (temperature, pH)
1
E. Gloves, (e.g. latex, nitrile)
F. Sterile 50ml conical tubes
G. Spatulas/spoons
H. 10% bleach solution
I. Dry ice
2
(important), or exceptionally in normal ice (min. Temp -2ºC) in sufficient quantity to cover
the expected tubes and some extras
J. Cool boxes or hermetic containers for dry ice (calculate space for 240 tubes plus dry ice)
K. Labels and/or predefined QR codes for samples
3
L. Tape
M. Hermetic plastic bags (e.g. zip bags -preferable with hard lock)
Figure 3. Materials needed for soil sampling. Letters refer to the materials list.
3
Be sure that the labels support negative storing temperatures. Use tape for extra help to fix it to the tube.
2
Once collected, the soil samples must be promptly stored at negative temperatures. Dry or carbonic ice
is the quickest and stablest way of reducing temperature. The quick reduction of temperature inactivates
microorganisms and stops enzymatic degradation of its DNA. Once frozen, the samples cannot be
thawed but for the genomic DNA extraction process.
1
Optional
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
5
6 | Ecological Restoration-Soil Sampling | PlutoF
Before starting
Make sure you have all sampling materials (Figure 3) organized and cleaned (10% bleach,
NaOCl), to have sorted fieldwork logistics (e.g., vehicle, budget, personnel, etc.), to consider soil
characteristics (type, depth, size of grain, etc.), sampling location, storing equipment, permits (if
needed), area accessibility, etc.
Taking the soil sample is a demanding physical activity. It is strongly advised to use protective
gloves when manipulating the soil probe, and if possible, a team of three or four persons for the
sampling would be the best option for a faster and more organized sampling (e.g. one person
taking the samples, one recording metadata in PlutoF, two persons rotating to dig the soil).
Bring enough cool boxes to store all the material collected (approximately four boxes of 40 x 40
cm), calculate how much space is needed for the tubes and the dry ice in each box. Take
precautions about dry ice supply if the sampling locations are isolated and with no possibility to
acquire more when needed. It is advisable to consider at least 30 kg of dry ice for a sampling
mission of four days, in properly isolated boxes. The recommendation is to open the boxes the
least possible, and to tape the edges to insure isolation, if needed.
Particular attention shall be given to the sampling spot. Avoid sampling at the very border of big
rocks, near water streams or within flooding areas, within the tree root perimeter (avoid big
trees), and take a photo from the landscape caring to have the sampling point at the center of
the photo. If possible, do some previous research on the sampling area looking for any recent
events that might have caused a significant perturbation of the soil (e.g., fire, flood, construction,
animal parking, clear cut forest, historical site, etc.). Although that sort of event does not
preclude the sampling, it would be good to have that information further downstream when
interpreting the data.
For recording sample data, the PlutoF plattform (Kessy et al. 2010) will be used as a workbench
for soil records. It includes a mobile app, PlutoF Go, that will record sample data during
fieldwork. A set of stickers with unique QR codes will be supplied for adding them to each
sample. To optimize time of sampling and storing, the QR codes stickers should be placed on
the sterile tubes and reinforced over with tape prior to sampling. A collector must be registered
in the PlutoF platform (Register → fill in details) prior to sampling in order to appear as collector
in the PlutoF Go app. Alternatively, project leaders can add “persons” into the platform (Menu
“Persons” → Add → fill in details). Make sure you have downloaded the app and enter your
personal data correctly. Further information on PlutoF is given on page 10. An on-line training
session on using PlutoF is available upon request, and support will be provided wherever
needed before, during and after the field work. QR codes will be read by the PlutoF Go app
when entering the data. Taking time to open, explore, and practice taking metadata with the app
is strongly recommended, as is meant to standardize and optimize time while doing fieldwork.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
6
7 | Ecological Restoration-Soil Sampling | PlutoF
During sampling
During all the process, it is important to be careful of not contaminating samples at any stage.
The use of gloves when manipulating samples is mandatory, taking particular care when placing
the soil sample in each tube, ensuring that whatever touches the sampled soil has not been
used before/cleaned to prevent any cross-contamination. For more efficient work, at least two
persons should be taking the soil sample: one using the sampling materials, and other one
using new sterile gloves for collecting the soil to the sterile tubes. Store the tubes to belong to
one particular replicate in separated labeled plastic bags (e.g. zip bags); this will help further
arrangement of samples (see Figure 4). Is recommended to have the zip bag labeled with the
collection date, collector name(s) and stage/replicate number, you can put it inside the cool box
and directly store the tubes with samples inside their correspondent zip bag.
In most types of soil:
1. Before taking the samples, make sure QR codes are well placed in the collection tubes
2. Scan both QR codes tubes prior to sampling the soil in the PlutoF Go app
4
(Add material
sample → Choose form → Soil → Sample ID code icon). This will optimize time of
storage
3. Remove the organic layer in the selected area, and then introduce the soil probe, going
at 30 cm in depth
5
3.1. If there is not enough soil depth, use the bulb planter to get the sample
4. Carefully remove the probe/bulb planter to prevent the sampled soil to fall
5. Introduce the probe tip inside the collection tube, be careful of not touching the collected
soil and the inside part of the tube without gloves
5.1. When using the bulb planter, just place the collection tube in the center of the soil
sampled an carefully release it, untouched soil will fall into the tube
6. Using gloves and a sterilized spatule, slide the soil sample out of the probe to the 50 ml
sterile collection tube (the probe might have already incorporated its own device for
depositing the soil). If the soil is rather thin or shallow, be sure to remove the top 4-5 cm
layer and collect the soil beneath at its maximum depth. Whenever the soil is deep
enough (>30 cm), the core samples can be collected from two distinct layers [20-30 cm
and 10-20 cm]
6.1. Fill up 2⁄3 of the tube with soil collected at a depth between 20-30 cm
6.2. Take another tube and do the same but this time for a depth between 10-20 cm
6.3. In case of shallow soils use a single 50ml tube and register the depth interval
5
For shallow soils (<20cm depth) the sample shall be collected at its maximum depth and the depth
interval information should be registered.
4
The PlutoF Go app can be found for Android, and for iOS systems. Instructions and other information
can be found in the link provided. A quick guide for PlutoF Go is provided in this document.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
7
8 | Ecological Restoration-Soil Sampling | PlutoF
7. Close the tube and immediately store it in its correspondent zip bag in dry ice or,
exceptionally, on ice and keep it (at least) at -2ºC during transportation
6
8. Measure the humidity, PH and temperature by inserting the soil tester probe in the hole
left by the collection of soil (>5 minuter).This is needed only for one core per replicate.
9. Record the rest of the material collection information in PlutoF Go app (Add material
sample → Choose form → Soil → fill in the correspondent details)
10. Store samples properly labeled at -20ºC at its final destination
In sandy or unstructured soils:
1. Scratch the top layer of the sand with the shovel, digging approximately 4-5 cm
2. Scan both QR codes tubes prior to sampling, and record the material collection
information in PlutoF Go app (Add material sample → Choose form → Soil → fill in the
correspondent details)
3. From the cleared surface, take a sample with a scaled gardening spoon or with the bulb
planter
4. Place the sample in a 50 ml sterile collection tube with the QR labels (prevent
contamination of sample using gloves and/or a sterilized spatule)
5. Immediately store the tube in dry ice or, exceptionally, on ice and keep it (at least) at
-2ºC during transportation. Do not forget to store all the collected core samples in
separate labeled hermetic bags.
6. Measure the humidity, PH and temperature by inserting the soil tester probe in the hole
left by the collection of soil (>5 minuter).This is needed only for one core per replicate.
7. Store samples properly labeled at -20ºC at its final destination
IMPORTANT: Clean all the collecting materials before and after sampling using a solution of
10% bleach, (i.e., between different sampling sites), and replace the hand gloves when
manipulating a new sample
6
For the purpose of this study, each depth per core sampled was kept in the original 50 mL tube, closed
and directly put in dry ice. Another alternative, for more experienced workers and to save space when
transporting, would be to pool the same five depths per core, per stage replicate, on field. In this way,
each stage will result with two pools, one for depth a (10-20 cm), and another one for depth b (20-30 cm).
For more information about this sampling scheme, please contact L. Nájera-Cortazar (Useful contact).
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
8
9 | Ecological Restoration-Soil Sampling | PlutoF
After sampling
Remember: immediately after collecting each sample in the tube, make sure it is properly
closed, scanned, and temporarily store it in dry ice (or regular ice if not possible otherwise), to
prevent contamination and overgrowth of any microorganism contained in the sample. When
returning from fieldwork, organize samples in plastic zip bags (if you have not done it during
sampling) placing the 10 cores of each replicate within a zip bag, correctly labeled (i.e.
stage number, replicate number, collector, site, date, etc.), and store the samples at -20ºC or
more as soon as possible (Figure 4).
Before shipping out the samples to the lab, make sure that the lab confirms the availability to
receive the samples and ask the carrier for the time the parcel will spend in transit. Make sure to
add sufficient dry ice to last for the entire journey. And lastly, add any permit/document that
could be needed to depart from your country.
Figure 4. Space occupation of 240 tubes in -20ºC freezer drawers. Total core’s samples of each
replicate are contained in separated zip hermetic bags (10 tubes per bag).
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
9
10 | Ecological Restoration-Soil Sampling | PlutoF
Registering samples in PlutoF Go app
PlutoF is an online data management and computing service provider for biological data. PlutoF
Go is an app that will be used to record samples directly from fieldwork. Before using the app,
the data collector should be registered on the PlutoF website. This can be done by the user in
the option “Become a user”, or to be added by the BGE project manager directly on her/his
workbench
7
. You can fill all the available information within the soil option, but it is required at
least to have the data detailed below. Images below the following list will correspond with the
numbers in this list:
1. Open the PlutoF Go app
2. Go to Add material sample box (If this option is not visible in the main page, go to
Settings, scroll down to Material sample, activate Enable material sample gathering and
make sure the Soil form is highlighted in green as well)
3. The Location button will show your position in the map. You should record all the
necessary information at the moment of the sample collection, therefore it should
capture the coordinates detected by your device’s GPS. If there is no internet signal you
can still get the coordinates by pressing the compass icon (top right inside the map box).
To georeference your site with an external GPS, go to the search icon on the right
(magnifying glass icon) and place the complete coordinates in the top box that says
7
The PlutoF project manager will be the only one authorized to add any person to the working project.
Any team member will be automatically notified by email when added to any project.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
10
11 | Ecological Restoration-Soil Sampling | PlutoF
Location, address or… and click search. Once that is set, click the back option on the top
left corner and continue with the data entry.
4. Start date: the date of the sample collection
5. Project: choose the “Ecological Restoration Soil -
Institution” option. Choose the already created project
according to your institution acronym
6. Choose form: select “Soil”, highlights
7. Sample ID: click on the code icon and point your
device camera to the corresponding QR code provided for
the soil sampling. QR codes are unique.
8. Subcode: Country initials (separator point) + Stage
& number + Replicate & number + depth option (a = 10-20
cm, b = 20-30 cm) XX.S1R1a - XX.S4R6a
9. Core ID
8
: Add the core number (C1-5) and depth (a:
10-20 cm; b: 20-30 cm) that you are sampling (i.e. C1a). The
first sample taken will be the depth b (20-30 cm), as is the
one in the bottom part of the probe.
10. Sample type: select “Soil”
8
In the previous versions, Core ID was requested to be added in Description, before added to the app.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
11
12 | Ecological Restoration-Soil Sampling | PlutoF
11. Collectors: Any person has to be previously
registered/added in the PlutoF platform
12. Habitat description: Fill in details of the dominant
vegetation of the area, or edit in platform. Complement this
information by adding a picture (see point 18).
13. Soil texture: this optional, but is strongly
recommended. Using the app +camera icon, take a picture
of one core per replica (if there is time, a photo of each core
would be better), to further assess other soil characteristics
for future individual projects.
14. PH: This optional, recommended for future individual
projects.
15. Temperature: This optional, recommended for future
individual projects.
16. Horizon description: When there is not enough soil or
structured soil it can be specified here.
17. Photo: Add a picture of the sampled habitat using the
panel that is in the top of the Material sample menu. Use the
+camera icon (top left) to generate a picture. One set of habitat
pictures per replica is enough. Additionally (optional), you can
add any extra information/metadata you think convenient.
18. Review all the information submitted is accurate and click “Save” (bottom of the screen).
19. In the main screen, your entries will be waiting in the
queue to be synchronized. Click on the cloud icon on
the top right to do it.
In the image, there is one good sample entry (dark
letters, intense color) that can be synchronized, and
another entry with missing information (red letters, faded
color) that will not be able to sync until the missing
information is filled. This can be done by clicking in the
entry and revising the info submitted. Make sure you
have a good internet connection to sync your entries, and
try to sync often to ensure the data is saved. You are
ready for the next sampling collection.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
12
13 | Ecological Restoration-Soil Sampling | PlutoF
Acknowledgements
Biodiversity Genomics Europe (Grant no.101059492) is funded by Horizon Europe under the
Biodiversity, Circular Economy and Environment call (REA.B.3); co-funded by the Swiss State
Secretariat for Education, Research and Innovation (SERI) under contract numbers 22.00173
and 24.00054; and by the UK Research and Innovation (UKRI) under the Department for
Business, Energy and Industrial Strategy’s Horizon Europe Guarantee Scheme.
References / Bibliography
Abarenkov, K.; Tedersoo, L; Nilsson, R. H.; Vellak, K.; Saar, I.; Veldre, V.; Parmasto, E.; Prous,
M.; Aan, A.; Ots, M.; Kurina, O.; Ostonen, I.; Jõgeva, J.; Halapuu, S.; Põldmaa, K.; Toots, M.;
Truu, J.; Larsson, K-H.; and Kõljalg, U. 2010. PlutoF - a Web Based Workbench for Ecological
and Taxonomic Research, with an Online Implementation for Fungal ITS Sequences.
Evolutionary Bioinformatics, 6, 189 - 196.
FAO, ITPS, GSBI, CBD and EC. 2020. State of knowledge of soil biodiversity - Status,
challenges and potentialities, Report 2020. Rome, FAO. https://doi.org/10.4060/cb1928en
Wagg, C.; Bender, S. F.; Widmer, F.; and van der Heijden, M. G. A. 2014. Soil biodiversity and
soil community composition determine ecosystem multifunctionality. Proc. Natl. Acad. Sci. USA.
111 (14) 5266-5270 https://doi.org/10.1073/pnas.1320054111
Useful contact
Main: Laura Nájera Cortazar | Associação Biopolis - CIBIO – Centro de Investigação em
Additional: Pedro Beja | Associação Biopolis - CIBIO – Centro de Investigação em
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
13
