Creamer 2015

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APSOIL 2259 No. of Pages 13
Applied Soil Ecology xxx (2015) xxxxxx
Contents lists available at ScienceDirect
Applied Soil Ecology

journal homepage: www.elsevier.com/locate/apsoil
Ecological network analysis reveals the inter-connection between soil

biodiversity and ecosystem function as affected by land use across
Europe
R.E. Creamera,* , S.E. Hannulab , J.P.Van Leeuwenc , D. Stonea,d , M. Rutgerse , R.M. Schmelze ,
P.C.de Ruiterg , N.Bohse Hendriksenh , T. Bolgeri , M.L. Bouffaudj, M. Bueek , F. Carvalhol ,
D. Costal , T. Dirilgeni , R. Franciscom , B.S. Grifthsn , R. Grifthso , F. Martink ,
P.Martins da Silval , S. Mendesl, P.V. Moraism , C. Pereiral , L. Philippotj , P. Plassartj,
D. Redeckerp , J. Rmbkef , J.P. Sousal, M. Woutersee, P. Lemanceauj
a
Teagasc, Johnstown Castle Research Centre, Ireland

Netherlands Institute of Ecology, The Netherlands
Wageningen University and Research Centre, The Netherlands
d
Leeds University, UK
e
National Institute for Public Health and the Environment, The Netherlands
f
ECT Oekotoxikologie GmbH, Germany
g
University of Amsterdam, The Netherlands
h
Aarhus University, Denmark
i
University College Dublin, Ireland
j
INRA, UMR 1347 Agrocologie, Dijon, France
k
INRA, Laboratory of Excellence Advanced Research on the Biology of Tree and Forest Ecosystems (ARBRE), UMR 1136, Champenoux, France University of
Lorraine, UMR 1136, Champenoux, France
l
Centre for Functional Ecology, University of Coimbra, Portugal
m
CEMUC and Department of Life Sciences, University of Coimbra, Portugal
n
Crop and Soils Systems Research Group, SRUC, UK
o
Centre for Ecology and Hydrology, UK
p
Universit de Bourgogne, UMR1347 Agrocologie, Dijon, France
b
c
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 15 April 2015
Received in revised form 5 August 2015
Accepted 11 August 2015
Available online xxx
Soil organisms are considered drivers of soil ecosystem services (primary productivity, nutrient cycling,
carbon cycling, water regulation) associated with sustainable agricultural production. Soil biodiversity
was highlighted in the soil thematic strategy as a key component of soil quality. The lack of quantitative
standardised data at a large scale has resulted in poor understanding of how soil biodiversity could be
incorporated into legislation for the protection of soil quality. In 2011, the EcoFINDERS (FP7) project
sampled 76 sites across 11 European countries, covering ve biogeographical zones (Alpine, Atlantic,
Boreal, Continental and Mediterranean) and three land-uses (arable, grass, forestry). Samples collected
from across these sites ranged in soil properties; soil organic carbon (SOC), pH and texture. To assess the
range in biodiversity and ecosystem function across the sites, fourteen biological methods were applied
as proxy indicators for these functions. These methods measured the following: microbial diversity: DNA
yields (molecular biomass), archaea, bacteria, total fungi and arbuscular mycorrhizal fungi; micro fauna
diversity: nematode trophic groups; meso fauna diversity: enchytraeids and Collembola species;
microbial function: nitrication, extracellular enzymes, multiple substrate induced respiration,
community level physiological proling and ammonia oxidiser/nitrication functional genes. Network
analysis was used to identify the key connections between organisms under the different land use
scenarios. Highest network density was found in forest soils and lowest density occurred in arable soils.
Key taxomonic units (TUs) were identied in each land-use type and in relation to SOC and pH
categorisations. Top-connected taxonomic units (i.e. displaying the most co-occurrence to other TUs)
were identied for each land use type. In arable sites this was dominated by bacteria and fungi, while in
Keywords:
Soil biodiversity
Ecosystem function
Carbon cycling and storage
Nitrogen
Phosphorus
Nutrient cycling
Network analysis
* Corresponding author.
E-mail address: rachel.creamer@teagasc.ie (R.E. Creamer).
http://dx.doi.org/10.1016/j.apsoil.2015.08.006
0929-1393/ 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: R.E. Creamer, et al., Ecological network analysis reveals the inter-connection between soil biodiversity and
ecosystem function as affected by land use across Europe, Appl. Soil Ecol. (2015), http://dx.doi.org/10.1016/j.apsoil.2015.08.006
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R.E. Creamer et al. / Applied Soil Ecology xxx (2015) xxxxxx
grassland sites bacteria and fungi were most connected. In forest soils archaeal, enchytraeid and fungal
TUs displayed the largest number of neighbours, reecting the greatest connectivity. Multiple regression
models were applied to assess the potential contribution of soil organisms to carbon cycling and storage
and nutrient cycling of specically nitrogen and phosphorus. Key drivers of carbon cycling were microbial
biomass, basal respiration and fungal richness; these three measures have often been associated with
carbon cycling in soils. Regression models of nutrient cycling were dependent on the model applied,
showing variation in biological indicators.
2015 Elsevier B.V. All rights reserved.
1. Introduction
Soil organisms are considered as drivers of ecosystem services,
in particular those soil ecosystem services associated with
sustainable agricultural production. These include primary production of food, bre and fuel, nutrient cycling, carbon cycling and
storage, and water inltration and purication (Hooper et al.,
2005). As such, soil biodiversity is therefore highlighted in the Soil
Thematic Strategy (EU (European Union), 2002) as a key
component of soil quality. Soil quality is dened as the capacity
of soil to function, within natural or managed ecosystem
boundaries, to sustain plant and animal production, maintain or
enhance water and air quality, and support human health and
habitation (Karlen et al., 1997). Many of these functions depend on
the diversity and activities of soil organism communities.
Increasingly we require a multi-faceted approach to land
management, with an increasing need for greater food production,
while simultaneously delivering other ecosystem services or soil
functions, such as carbon (Tardy et al., 2015) and nutrient cycling
(Fierer et al., 2012). Land management can lead to the degradation
of carbon stocks in soils, and therefore understanding the role of
soil biota in carbon cycling and storage is vital. The soil carbon pool
is 3.3 and 4.5 times the size of the atmospheric (760 Gt) and the
biotic pool (560 Gt), respectively (Lal, 2004). It is essential from a
climate change perspective that we protect carbon storage
potential in our soils, furthermore, active cycling of carbon,
combined with large amounts of organic carbon temporarily
stored in soils, increases primary productivity, stabilises soil
structure, increases nutrient retention and water ltration (Turb
et al., 2010 De Vries et al., 2013). Land management also has a
signicant impact on the capacity of the system to cycle nutrients,
providing a constant supply to crops as needed to ensure optimum
productivity. This has traditionally been a high input system, with
the addition of synthetic fertilisers to promote availability of
essential nutrient for plant growth (especially nitrogen (N) and
phosphorus (P)), however it is becoming increasingly apparent
that soil organisms have a strong role to play in the cycling of
nutrients due to their involvement in the geochemical cycles
(Lemanceau et al., 2015).
In 2012, the European Commission acknowledged the importance of soil biodiversity in the role of ecosystem functioning, stating
that these functions are worthy of protection because of their socioeconomic as well as environmental importance (Jones et al., 2012).
However, the lack of quantitative standardised data on soil
biodiversity at the European scale has resulted in poor understanding of both the role that soil organisms play in soil ecosystem services
and the need to protect soil biodiversity to ensure the future
provision of such functions. This was also highlighted in the EUs 6th
Framework programme nanced project: environmental assessment of soil for monitoring (ENVASSO) established in 2005, that
recommended pan-European indicators to assess the potential loss
of soil biodiversity (Bispo et al., 2009). This work has been followed
up by the Ecological Function and Biodiversity Indicators in
European Soils (EcoFINDERS) project, nanced under the EUs 7th
Framework programme and established in 2009, to support the
European Union soil policy making by providing the necessary tools

to design and implement strategies for sustainable use of soils, with a
specic focus on soil biodiversity and associated ecosystem
functioning.
There have been many studies which have quantied the impact
of land management and land use on the diversity and functioning of
soil biota (a few examples include; Trasar-Cepeda et al., 2008; Lohaus
et al., 2013; Mills and Adl, 2011; Bartz et al., 2014). Tsiafouli et al.
(2015) highlights the lack of integrative approach, with many of
these studies focussing on one aspect of soil biodiversity (e.g. species
richness, abundance, food webs, community structure), promoting
the need for more multi-factorial approaches. Tsiafouli et al. (2015)
analysed the effect of agricultural intensication across Europe on
the structure, diversity, food web assembly and community
dynamics of soil biota, summarising that agriculture intensication
reduces soil biodiversity, resulting in fewer functional groups and
reduce diversity.
Traditional methods such as diversity estimates and multivariate statistical techniques describe beta-diversity and can reveal the
role of biotic and abiotic factors in shaping the communities.
However, they do not take into account the interactions among
organisms, a very important factor shaping any natural community
(Bohan et al., 2013; Mulder et al., 2011). Much of the focus in nature
conservation has been on protection of individual species while
biotic interactions are increasingly at risk from local and global
extinction as a consequence of (anthropogenic) environmental
disturbances (Pocock et al., 2012). Using a network based
approach, the relationship between organisms within and across
taxonomic units/trophic levels can be analysed even from very
large datasets. In ecology, networks have been long used for
macro-organisms (Bascompte et al., 2003) but recently the
approach of analysing large datasets using summarizing network
analysis based on ecological theories has become popular in the
eld of soil microbial ecology (see for example Barbern et al.,
2012).
The aim of this study was to investigate the biological diversity
(soil microbial and faunal communities) associated with major
land use management types found across Europe and to examine
how these various ecological networks relate to two key ecosystem
services in soil; (1) carbon cycling and storage potential and (2)
nutrient cycling, specically nitrogen (N) and phosphorus (P). To
achieve this, a pan-European transect was sampled in 2011 at
81 sites, across 11 European countries, covering ve biogeographical zones (Alpine, Atlantic, Boreal, Continental and Mediterranean)
and three land use types (arable, grass, forestry) (Stone et al., 2015,
this issue). These sites represent a wide range of soil properties,
specically chosen to provide a wide spectrum of measurements
for SOC, pH and texture (sand, silt and clay content). Fourteen soil
biological properties were measured: (i) microbial diversity; DNA
yields (molecular biomass), archaea, bacteria, fungi, arbuscular
mycorrhizal fungi (AMF), (ii) micro fauna diversity; nematodes
trophic groups, (iii) meso fauna diversity; enchytraeid, and
Collembola species, (iv) functional indicators; nitrication, extracellular enzyme assays (EEA), multiple substrate induced respiration (MSIR) and community level physiological proling (CLPP),
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and the abundance of key functional genes involved in ammonia

oxidation and denitrication. Network analysis was used to
identify the key connections between organisms/trophic groups
under the different land use management types and multiple
regression analyses were then employed to examine the relationship between the various organisms/trophic groups and soil
functions.
2. Methods
2.1. Site sampling and sample processing
81 sites were sampled across Europe, as part of the EcoFINDERS
project; this study is known as the European transect (Fig. 1).
Sites were selected from within European Union countries using a
spatial random sampling model, weighed to derive a spectrum of
sites representative of the range of soil properties; (SOC, textural
class (representing by% clay) and pH), land-use and biogreographical zones across Europe (EEA, 2012). Data used to spatially
derive potential locations for sampling were based on the
European Food Safety Authority (EFSA) database, the Corine
landcover map and soils database (Gardi et al., 2011) and the
European Environment Agency map of biogeographical zones
(EEA, 2012). Full details of the development of the site selection
model and sampling can be found in Stone et al. (2015). In brief, soil
was sampled from each site following a pre-agreed standard
operating procedures (SOPs) within EcoFINDERS, guaranteeing
that all sites were sampled in a consistent manner. Soil was taken
from the top 5 cm of the prole using plastic cores. All cores were
packed in pre-labelled bags and posted (24 h delivery) in cooled
boxes to Teagasc Research Institute, in Ireland. On receipt, soils
were sieved to <2 mm fraction and sub-sampled following a
specied coning and quartering technique to attain homogenous
sub-samples (except the mesofaunal samples which were kept
untouched). All sub-samples for molecular analyses were frozen to
40 C and DNA extracts at 80 C. All sub-samples for microbial
functional analyses and cores for faunal analyses were kept at 4 C.
Sub-samples were posted onto the nal laboratory for analysis
within 2 weeks of receipt, using freezer packs to keep the sample
cooled. Sub-samples for the measurement of soil physical/
chemical properties were dried at 40 C (air dried).
2.2. Measurement of soil properties
Soil texture was measured using the particle size pipette
method (ISO (International Organization for Standardization),
1998), providing information on total sand (%), silt (%) and clay (%)
fractions. Total carbon (C) and nitrogen (N) were analysed
Fig. 1. Map of transect sites, based upon the biogeographical zones of Europe (EEA, 2012).
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following (ISO (International Organization for Standardization),

1995) and organic carbon (OC) was determined by LECO elemental
analysis, this was conducted on 0.25 mm milled dry soil subsamples (Massey et al., 2014). Cation exchange capacity (CEC) was
measured using BaCl2 extraction method (ISO (International
Organization for Standardization), 1994). pH was measured in a
1:2.5 soil in water suspension using a glass electrode (van
Reeuwijk, 2002). N mineralisation was analysed using the Illinois
soil nitrogen test for amino sugar-N (McDonald et al., 2014). This
Illinois soil nitrogen test (ISNT) method was developed by Khan
et al. (2001) and modied by Klapwyk and Ketterings (2005) to
estimate the amount of amino-sugars plus NH4-N in the soil. The
concentration of ISNT-N liberated by NaOH and captured as NH4-N
by the boric acid was quantied by colorimetric analysis with an
Aquakem 600A (Aquakem 600A, 1621, Vantaa, Finland). Phosphorus was measured using the Mehlich 3 methodology (Mehlich,
1984) and analysed on a Varian Vista MPX ICP-OES.
2.3. Measurements of soil biodiversity
2.3.1. Microbial diversity
The methodology used for phospholipid fatty acids (PLFA)
extraction, separation, transmethylation and GC analysis was the
MIDI PLFA hybrid method described by Francisco et al. (2015, this
issue). Briey, soils were lyophilized and lipids extracted using the
Bligh and Dyer (1959) extraction procedure. Lipid extracts were
separated by solid-phase extraction (SPE) using an SI-column and
organic solvents as eluents. Phospholipids were eluted with
methanol. Phospholipids were derivatised and transmethylated
using the MIDI FAME protocol (MIDI, Inc., Newark, DE, United
States). Fatty acid methyl esters (FAME) were measured by Gas
Chromatography (GC) (Agilent Technologies, Wilmington, DE,
USA), identied and quantied using standards (internal FAME
19:0 and calibration mixtures) and Sherlock MIS data base, based
on the calculated equivalent chain lengths (ECL). The biomarkers
were dened according to Francisco et al. (2015, this issue).
DNA for all molecular work was extracted using the method
described in Plassart et al. (2012). Crude DNA extracts were
resolved by electrophoresis in gel, stained with ethidium bromide
and a standard curve of DNA was used to estimate the nal DNA
concentration in the extracts allowing the assessment of so-called
molecular microbial biomass (Dequiedt et al., 2011). In this paper
this will be referred to as molecular microbial biomass. After
microbial DNA extraction, Terminal restriction fragment length
polymorphism (T-RFLP) was applied to measure the three
microbial domains (bacteria, archaea, fungi), based on the length
and abundance of unique restriction fragments found in each
sample. Bacterial and archaeal T-RFLP community proles were
generated by amplifying specic 16S rRNA gene sequences, while
the fungal T-RFLP community proles were generated by
amplifying the ITS1-ITS4 region as described by Grifths et al.
(2011) and Plassart et al. (2012).
To determine fungal richness and relative frequency, fungal
ITS2 region was amplied from these metagenomic DNA samples
(Ihrmark et al., 2012). Amplicon libraries were pyrosequenced and
fungal community diversity was generated from the analysed
sequences as described by Coince et al. (2014).
Fungal copy numbers were determined using the same primers
as for 454-pyrosequencing using real-time PCR mix from RotorGene SYBR Green PCR Kit (Qiagen). T4 Gene 32 protein (Roche) was
used to enhance the reaction and ensure similar amplication from
all soils. The samples were analysed on a Rotor-Gene 3000 machine
(Gorbett Research, Sydney, Australia). The reaction mixtures were
processed using a pipetting robot (Gorbett Research, Sydney,
Australia) in 20 ml volume and contained 0.3 mM each primer,
0.25 ml T4 and 1.010.0 ng template DNA. The cycling conditions
were: 40 sec at 95 C, 1 min at 58 C and 1 min at 72 C. Plasmids

extracted from pure fungal cultures were serial diluted and used as
a reference for the copy numbers. As ITS2 region can vary in length,
three different plasmids extracted from three different species
were used as standards. All samples were analysed in at least two
different runs and in two different concentrations to conrm the
reproducibility of the quantication and lack of inhibition due to
i.e. humic acids.
To analyse AMF diversity, nested PCRs were performed on three
replicates from all samples. The rst PCR was performed using
0.4 U of Phusion High Fidelity DNA polymerase (Thermo Fisher
Scientic, Courtaboeuf, France), 1x Phusion HF buffer, 0.5 mM of
the primers SSUmCf and LSUmBr (Krger et al., 2009), 0.2 mM of
each dNTPs and 1 ml of genomic DNA, in a nal volume of 20 ml.
The PCR conditions used were 5 min at 99 C, 35 cycles of 10 s at
99 C, 30 s at 63 C and 1 min at 72 C, followed by 10 min at 72 C,
using an Eppendorf Mastercycler epgradient S (Vaudaux-Eppendorf, Schnenbuch, Switzerland). The nested PCR was done using
1 U of Phusion High Fidelity polymerase, 1 HF buffer, 0.5 mM of
the primers ITS3m (Zhong et al., 2010) and ITS4 (White et al., 1990)
with barcodes, 0.2 mM of each dNTPs and 2 ml of PCR product
diluted at 1:50, in a total volume of 50 ml. PCR conditions were 30 s
at 98 C, 30 cycles of 10 s at 98 C, 30 s at 64 C and 20 s at 72 C,
followed by 10 min at 72 C, in an Eppendorf Mastercycler
epgradient S. The three PCR replicates of each sample were pooled
and puried using the High Pure PCR Product Purication Kit
(Roche Applied Science, Meylan, France) following the manufacturers instructions. After quantication with Picogreen, the
puried PCR products were mixed equimolarly to prepare
sequencing libraries. The libraries were sent to Beckman Coulter
Genomics (Grenoble, France) for sequencing using 454 GS FLX
technology.
2.3.2. Micro- and meso-fauna
Enchytraeids were extracted from three replicate soil cores
(5.0 cm depth 5.0 cm width) per site with OConnors hot/wet
funnel method (OConnor, 1962) following ISO standards (ISO
(International Organization for Standardization), 2006). Specimens were identied to species using light-microscopically in vivo,
applying the keys and techniques in Schmelz and Collado (2010,
2012), together with primary literature.
Collembola were extracted from three replicate soil cores
(5.0 cm depth 5.0 cm width) per site. The samples were
transferred by courier mail to IMAR, University of Coimbra,
Portugal, where they were extracted using a modied Macfadyen
High Gradient Extractor (Macfadyen, 1961) for seven days.
Collembola were mounted on slides and identied to species
level, in most cases, using primary literature on European
Collembolan identication.
Nematode trophic groups were determined by morphological
analysis, using a Doncaster counting plate (Doncaster, 1962), and
identied to trophic level (plant-parasites, bacterial-feeders,
fungal-feeders, omnivores and predators) by observing the
head/mouth structures under an inverted microscope (100 and
200 magnication).
2.4. Measurements of soil biological functioning
To determine multiple substrate induced respiration (MSIR)
proles, the MicroResp methodology adapted from Campbell et al.
(2003) and reported by Creamer et al. (2009) was applied in this
study. A spectrum of seven substrates was selected: D-(+)-galactose, L-malic acid, gamma amino butyric acid, n-acetyl glucosamine, D-(+)-glucose, alpha ketogluterate, citric acid and water for
basal respiration measurements. Details of the methodology are
described in Creamer et al. (2015, this issue).
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Community level physiological proles (CLPP) using Biolog

ECO-plates were analysed in the European transect and the
Netherlands Soil Monitoring Network (Rutgers et al., this issue).
This method is considered to determine multiple functional
endpoints represented by a sample of the heterotrophic soil
bacterial community (Winding et al., 2005).
Extracellular enzyme activities (EEA) in the soils were determined on 8 different uorogenic model substrates related to the
hydrolysis of O-glycosyl linkages of ve di- and poly-saccharides
including starch, cellulose, hemicellulose and chitin, ester linkages
of organic phosphates and sulfates and peptide linkages of
proteins. The assay was performed in microtiter plates as described
by Johansen et al. (2005).
Potential nitrication was measured using the method described by Kandeler (1996) but adapted to the microplate (Ng et al.,
2014). Soil samples (2 g moist soil) were incubated for 5 h on
rotatory shaker at room temperature in 20 ml (NH4)2SO4 (10 mM)
and 0.1 ml NaClO (1.5 M). Control was kept at 20 C during
incubation and thawed at room temperature after incubation
period. After incubation, 6 ml KCl (2 M) solution was added to
samples and controls and shaken (30 min) followed by centrifugation (4 min, 3000 rpm). 5 ml of ltrate was mixed with 3 ml NH4Cl
and 2 ml colour reagent (2 g sulphanilamide and 0.1 g N-(1naphthyl)-ethylenediamine hydrochloride in 150 ml distilled
water and 20 ml concentrated phosphoric acid) and allowed to
stand (15 min, room temperature). NO2 was measured spectrophotometrically (OD 540 nm) on a microplate reader. The NO2 -N
concentration was calculated using a calibration curve made with a
standard solution of NaNO2 (10 mg NO2 -N ml 1).
Quantication of the bacterial and archaeal ammonia-oxidizers
(AOA and AOB) and of the nitrous oxide reducers (nosZ1 and
nosZ2) was performed according to Tourna et al. (2008), Leininger
et al. (2006) and Jones et al. (2013), respectively. The real-time PCR
assays were carried out in a ViiA7 (Life Technologies, USA) with a
15 ml reaction volume containing the SYBR green PCR Master Mix
(Absolute Blue QPCR SYBR Green Low Rox Mix, Thermo, France),
1 mM of each primer, 250 ng of T4 gene 32 (QBiogene, France) and
0.5 ng of DNA. Standard curves were obtained with serial plasmid
dilutions of a known amount of a plasmid DNA containing
fragment of the amoA, nosZ1 and nosZ2 genes.
2.5. Statistical procedures
2.5.1. Network analysis for soil biodiversity linkages
To construct networks, TRFLP data were used for fungi, archaea
and bacteria, species numbers for Enchytraeids and Collembola,
trophic groups for nematodes, and amplicon sequence-data
grouped into family level for AMF (Table 1biodiversity). All of
the levels are further considered as taxonomic unit. Taxonomic
units (TU) that were present in only one sample per category (land
Table 1
Biological Indicators applied at 81 sites across Europe, to assess soil biodiversity or functions; C-cycling and nutrient (N&P) cycling.
Main Indicator
C storage and cycling
Extracellular enzyme activity
(EEA)
Multiple substrate induced
respiration (MicroResp)
Biolog
Measures
Paper recommending indicator
Beta-glucosidase; sum of enzyme activity
Kivlin and Treseder, (2014);

Sinsabaugh et al. (2008)
Campbell et al. (2003),
Creamer et al. (2015)
Rutgers et al. (2015), Rutgers
and Breure, (1999)
Francisco et al. (2015);
Herman et al. (2012);
Fernandes et al. (2013)
Dequiedt et al. (2011)
Graefe and Beylich, (2003);
Cole et al. (2000)
Grifths et al. (2007)
Van Der Heijden et al. (2008)
Basal respiration, L-malic acid, D-(+)-glucose, alpha ketogluterate, PCA1, PCA2

1/GG50
Phospholipid fatty acids (PLFA)
Fungal: bacterial, ergosterol (18:2w6,9), AMF (16:1w5c and 18:1w9c)
DNA yields
Enchytraeids
(ng microbial DNA g soil 1)

Relative abundance of Enchytraeid acidity indicators
Nematodes
AMF families
Feeding guild richness (plant-feeders, fungal-feeders, omnivores, bacterial-feeders, predators)

Acaulosporaceae, Ambisporaceae, Archaeosporaceae, Claroideoglomeraceae, Diversisporaceae,
Gigasporaceae, Glomeraceae,Pascisporaceae, Paraglomeraceae
Fungal abundance and richness Fungal copy numbers, fungal richness
Nutrient cycling of N and P
Enchytraieds
Species richness and Abundance per m2
Nematode
Plant-feeders, Fungal-feeders, Omnivores, Bacterial-feeders, Predators and total abundance
Extracellular Enzyme Activity

(EEA)
Biolog
Arylsulfatase, phosphomonoesterase, Leucin aminopeptidase
Nitrication potential
Functional Gene A
L_Arginine, L-asparagine, L-phenylalanine, L-serine, N-acetyle-D-glucosamine, L-threonine, Dglucosaminic acid, glycyl-L-glutamic acid, phenyl-ethylamine, putrescine
Amount of NO2-N released (ng/g soil dm/h)
nosZ1 (denitrier) gene, nosZ2 (denitrier) gene
Functional Gene B
AOB (Bacteria: ammonia oxidizers) gene; AOA (Archea: ammonia oxidizers) gene
Molecular microbial biomass
(ng microbial DNA g soil-1)
Fungal abundance and richness Fungal copy numbers, fungal richness
Biodiversity
Enchytraeid
Nematode
Collembola
AMF families
archaea, bacteria and fungi
Species diversity
Feeding guild richness
Species richness
Acaulosporaceae, Ambisporaceae, Archaeosporaceae, Claroideoglomeraceae, Diversisporaceae,
Gigasporaceae, Glomeraceae, Pascisporaceae, Paraglomeraceae
T-RFLP copies per ng DNA (abundance); Dikarya Richness;
Coince et al. (2014)
Cole et al. (2000) (N

mineralisation only)
Xiao et al. (2010)
Grifths and Bardgett (1997)
Sinsabaugh et al. (2014)
Van Eekeren et al. (2008)
Schloter et al. (2003)
Jones et al. (2013); Philippot
et al. (2013)
Leininger et al. (2006)
Dequiedt et al. (2011)
Coince et al. (2014)
Rmbke et al. (2013)

Donn et al. (2012)
Yeates et al. (1993)
Bispo, et al. (2009)
Hart and Reader (2002)
Plassart et al. (2012)
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use, pH or SOC) were removed prior to analysis. The remaining

number of TUs after singleton removal per category is presented in
Supplementary Table A and the numbers were used to scale the
sizes of the nodes in the network. Spearman-rank correlation
matrixes were calculated in R (R Core Development Team, R
Foundation for Statistical Computing, Vienna, Austria) using
abundance data. Only signicant positive correlations were used
in further analysis. The percentage of signicant correlations from
total possible correlations was used as a measure of interaction
strength between TUs, and edge size and darkness were scaled to
this. Within taxa correlations (groups/species or trophic group)
were calculated but not displayed. The data were transferred to
Cytoscape (Shannon et al., 2003) for further analysis and
visualization. Network density, clustering coefcient and average
number of neighbours was calculated using network analysis tools
within Cytoscape.
2.5.2. Potential C storage and cycling
Using total C content and land-management type (arable,
grassland and forest), sites have been ranked according to C cycling
and storage potential. For this ranking, coefcients for land use
type were calculated using linear regression with total C as
dependent, and land use type as categorical independent variables.
The coefcients thus found were: 0.414 for arable elds, 0.917 for
grasslands and 2.040 for forests. These coefcients were multiplied
by the amount of normalised SOC content in the topsoil (sample
divided by the average) to get a quantication of C storage potential
corrected for land use.
Carbon storage potential was subsequently used as a dependent
variable in a forward stepwise multiple linear regression. All suitable
biological measurements (Table 1carbon cycling) were included in
the analysis, and all non-standardized parameters were log-transformed before inclusion in the model. Based on amount of explained
variance, and the Akaike information criterion (AIC), we selected the
most parsimonious model for predicting C storage potential.
2.5.3. Nutrient cycling of N and P
Three models have been applied to assess the cycling of
nutrients at these sites: (i) normalised N mineralisation alone has
been modelled to address the contribution of soil organisms to the
nitrogen mineralisation in soils. (ii) a P availability model using
inversely normalised P availability (average divided by sample)
only to assess the role of soil biology to P cycling in soils. (iii) a
combined model to assess overall nutrient cycling. This uses the
product of the normalised N mineralisation (sample divided by
average) multiplied by the inversely normalised P availability
(average divided by sample) in the topsoil to get a quantication of
combined N and P cycling. For all three models forward stepwise
multiple linear regression was applied.
All suitable biological measurements (Table 1nutrient cycling) were included in the analysis, and all non-standardized
parameters were log-transformed before inclusion in the model.
Based on amount of explained variance, and the Akaike information criterium (AIC), we selected the most parsimonious model for
predicting nutrient cycling. This procedure was also executed with
normalised N mineralisation and inversely normalised P availability as dependent variables separately, to disentangle the respective
nutrient cycles. Statistical analyses were carried out using SPSS
(20.0.0) and R (R Core Development Team, 2012).
3. Results
3.1. Review of soil properties and measurements
In total, 76 sites were analysed from the 81 sites sampled (Stone
et al., this issue), as these sites had a complete set of parameters.
Table 2 shows the range of soil properties for the 76 sites; SOC
ranged from 0.45% to 51.1% (Fig. 2a), lowest mean SOC was found in
arable sites, while the highest SOC concentrations were found in
forest sites. pH varied considerably across sites, ranging from 3.7 to
8.2, with the lowest mean pH found in forest sites and highest
mean pH in arable sites (4.99 and 7.07, respectively). Soil texture
varied across all sites and is represented in this paper by clay
content. Clay content varied from <1% to 70%.
3.2. Co-occurrence of soil organisms
To investigate the biological diversity associated with the main
drivers of biological activity described across Europe, co-occurrence patterns of TUs were created using network inference for
each land use type, SOC category and pH group (Figs. 3, 4 and 6). It
was not feasible to create co-occurrence patterns for biogeographical zones, due to the low number of sites within the Boreal and
Mediterranean zones. The size of the node represents the diversity
of a given TU. However, this information is dependent upon the
method applied to derive the diversity data. Within this study,
different methodologies were applied to derive community
assemblage information, for example the TRFLP method provides
a large number of peaks described as ngerprints providing
information on the genetic structure of the community targeted,
whereas nematode and Collembola identication are only given to
trophic groups and species, respectively. Therefore the size of the
node cannot be used as an indication of diversity of a specic TU.
The thickness of the line connecting different TUs, represents the
percentage of possible connections that are signicantly correlated
for a specic combination of TU connections. Using the percentage
of signicant correlations from possible connections instead of
absolute numbers corrects for the difference in number of TUs per
Table 2
Summary of soil properties.
Land use within
biogeographical zone
No. of
sites
SOC mean
(%)
SOC St.
dev
P mean
(mg/l)
P Std
dev
N min (mg N kg
dry soil)
Grass_Continental
Grass_Atlantic
Grass_Mediterranean
Grass_Alpine
Arable_Continental
Arable_Atlantic
Arable_Mediterranean
Arable_Alpine
Forest_Continental
Forest_Atlantic
Forest_Alpine
Forest_Boreal
11
13
2
8
10
13
2
1
5
5
2
4
6.18
4.48
1.65
6.18
1.76
2.06
1.28
3.17
9.07
4.27
12.68
32.08
4.98
2.19
0.11
2.89
0.43
1.34
0.63
0.00
6.24
1.79
4.55
15.28
64.21
68.58
69.86
105.20
156.12
137.09
17.54
27.33
46.43
39.40
54.67
121.72
49.89
52.50
30.30
101.19
130.88
109.85
19.66
0.00
51.97
50.46
51.04
62.18
435.28
361.41
133.77
488.25
168.61
159.53
97.58
207.43
383.62
288.61
637.88
540.27
N min Std
dev
pH
mean
pH Std
dev
Clay mean
(%)
Clay Std
dev
284.24
146.24
4.08
114.29
33.66
77.48
14.94
0.00
180.83
92.76
361.26
228.30
6.36
6.24
7.41
6.16
6.89
7.13
7.17
7.78
5.20
5.32
5.56
4.02
1.06
0.96
0.20
0.92
0.74
0.86
1.51
0.00
1.59
0.85
2.16
0.13
30.7
27.2
20.0
28.8
25.5
23.3
21.5
57.0
22.8
20.6
36.0
14.0
14.0
15.6
4.2
10.0
13.3
18.7
16.3
0.0
16.1
13.3
19.8
0.0
G Model
Fig. 2. (a) Soil organic carbon content by land use within biogeographical zone. The
x-axis description is provided by (b) and (c). N mineralisation potential by land use
within biogeographical zone. The x-axis description is provided by (c). P availability
summarised by land use within biogeographical zone.
group of organisms and between categories, and reveals the

strongly connected TUs.
The key connectivity of TUs occurring across the different land use
categories were illustrated in Fig. 3a which shows the strength of the
connection between key TUs. The highest density networks were
found in the forest soils (density of 0.041) followed by grasslands
(density 0.027) and arable lands (0.025) (Supplementary Table A).
The higher the density the larger the number of signicant
connections found. In the arable sites the AMF families and
nematode trophic groups showed the strongest association. This
is due to strong positive correlation between plant-feeding
nematodes and AMF (Fig. 3a). This connection is completely absent
in grassland soils and only weak in forest soils. A similar cooccurrence can be seen for AMF families and archaea, where a
connection is visible in arable systems, very weak in grassland
systems and missing in forest soils. There was a strong connection
between enchytraeid species and Collembola species in arable and
grassland sites but is much weaker in forest soils. In the grassland
sites the dominant connections were found between the bacteria
and archaea TUs, with signicant correlations also found between
enchytraeids and nematodes with archaea. In forest systems, there is
a strong connection between enchytraeids and AMF families. The
connection between bacteria and fungi, the two largest TUs of soil
organisms (Francisco et al., 2015, this issue), gets stronger when land
use intensity diminishes (arable < grasslands < forest). Furthermore, enchytraeids, nematodes and archaea all display weaker
correlations with bacteria in arable sites compared to grassland and
forestry sites.
Arable sites displayed a lower average number of neighbours

(i.e. the number of connections for a specic TU (with >30 neighbours)) compared to the other land use classes (Supplementary
Table A). The top twenty connected TUs present in each land-use
class are displayed in Fig. 3b, with the number of neighbours (other
TUs which show a signicant correlation with this TU) represented
on the y-axis, the higher the number of neighbours the more stable
a network. Archaea (peak IDs; trf_246, and trf_359 and bacteria
(peak IDs; trf_470)) (Fig. 3b), were found to be the top three
connected TUs in arable sites and overall archaea and bacteria TUs
were the most connected in these soils. Only one species of
Collembola (Isotoma viridis) was found in the top twenty connected
TUs and two fungi TUs (peak IDs; trf_205, trf_266). AMF families,
nematode functional groups and enchytraeid species were not
found among the top twenty-connected TUs in arable soils.
In the grassland sites, the top-connected TUs were bacteria
(Peak IDs; trf_412, trf_88, trf_413, and trf_226 (Acidobacteria)) and
fungi (Peak IDs; trf_205 and trf_230) which were found to have
more than 50 neighbours. Some enchytraeid species (Fridericia
cylindrical (CYL), Enchytronia parva (PAR),) were present in the toptwenty connected species in grassland sites. Fridericia cylindrica
was the most connected enchytraeid species, this species was
observed only in grassland sites. This species was found to have
strong connectivity in sites that were categorized by SOC 215%
and pH 57 (Supplementary graphs A and B), suggesting it is not
commonly connected in more extreme environments, but rather
prefers slightly acidic to neutral pH and none peaty conditions (i.e.
SOC < 15%). While Enchytronia parva was found in both grassland
and forest sites and appeared to be strongly connected across all
pH categories and in sites that had SOC ranges from <2% up to 15%.
Archaea TUs (Peak IDs; trf55, trf151) were found to have more than
40 neighbours. One collembolan (Isotomorus palustris) was among
top-twenty connected TUs. Isotomorus palustris was the most
connected collembolan species in grassland sites, also well
connected in sites categorised by SOC content of 215% and pH
57 (Supplementary Graphs A and B). Similar to arable sites, AMF
families and nematode trophic groups were not present in the top
twenty connected TUs in grassland soils.
In forest soils, TUs of archaea (Peak IDs; trf_288, trf_359;
trf_277; trf_343) had the most number of neighbours. Enchytraeid
species (Enchytraeus bulbosus (BUS), Fridericia benti (BEN), Enchytraeus buchholzi (PALE)), fungal (Peak IDs; trf492, trf122, trf338,
trf270 and trf141) and bacterial (trf153, trf121, trf91, trf162) TUs
had more than 50 neighbours in forest soils. These enchytraeid
species are not necessarily the most commonly occurring, but
rather display the most connections in forest soils. Enchytraeus
bulbosus was the most connected enchytraeid species in forest
sites, but the co-occurrence of these species was not strongly
connected to other TUs in any SOC or pH category (Supplementary
Graphs A and B). AMF families, Collembola species and nematode
trophic groups did not appear in the top twenty connected TUs.
3.3. Carbon cycling and storage potential
Sites were ranked on their potential for C cycling and storage
potential. To assess which biological measures play an important
role in these functions, nine soil biological indicators of carbon
cycling and potential storage were selected by an expert group and
through literature review (Table 1). The two sites with the lowest
SOC ranking were FRA_01 and FRA_04, both representing Atlantic
arable sites. The lowest overall mean SOC for land use within a
biogeographical zone was found in the Mediterranean arable sites
(Fig. 2a).
The regression model accounted for 82% of the variance in the
data described by the following indicators; basal respiration,
G Model
Fig. 3. (a) Network of biotic interaction based on signicant positive Spearman correlations in each land use type. The nodes are sized to the number of species included in the
analyses. The size and darkness of the connecting edges is sized to the proportion of signicant positive correlations from all possible correlations between taxonomic units in
the land-use type. For arable soils the threshold for positive interaction was Spearman correlation >0.40, for grasslands >0.35 and for forests >0.45, respectively. Land use
categories were classied on the basis of Stone et al., 2015 (this issue). (b) Top 20 connected species in the three land-use categories (i) arable, (ii) grassland and (iii) forests.
Colour legend indicates the following; fungi (pink), archeae (purple), bacteria (blue), enchytraeids (green), collembolan (orange), nematodes (light blue-turqouise) and AMF
(dark red).
Fig. 4. Network of biotic interaction based on signicant positive Spearman correlations in each organic matter (SOC%) category. The nodes are sized to the number of species
included in the analyses and their darkness is relational to the connectedness of the node to other nodes. The size and darkness of the connecting edges is sized to the
proportion of signicant positive correlations from all possible correlations between taxonomic units in each organic matter% category. For soils with organic matter% <2 the
treshold for positive interaction was Spearman correlation >0.44, for soil with org matter 215% >0.27 and for soils with organic matter >15% >0.77, respectively. Carbon
categories were classied on the basis of Stone et al., 2015 (this issue).
molecular microbial biomass and fungal richness, with a Std. Error

of the estimate of 0.591.
Model: Ln(Rank-C) = 1.818 + 0.849 Ln(MicroResp_Water) +
0.983 Ln(Molecular Microbial Biomass) 1.920 Ln(Fungal
Richness).
In the network analysis used to determine the key connections
of soil TUs in relation to carbon cycling and storage, sites were
analysed according to the SOC categories dened in Stone et al.

(2015, this issue) which represents sites with low carbon content
<2%, medium carbon content (215%) and high carbon content
(>15%).
The highest network densities were detected in sites of medium
SOC content (215%) (density of 0.040). In sites, where soil SOC
exceeded 15% and in sites with less than 2% SOC, density was very
G Model
low (0.023 and 0.024, respectively) (Supplementary Table B).

Interactions were more evenly distributed at sites with 215% SOC
content, compared to low and high SOC categories where some
connections were missing. For example, the connection between
collembolan species and enchytraeid species was much stronger in
<2% SOC (11.5% possible connections signicant) and >15% SOC
(12.5% connections signicant) categories compared to 215%
category where only 5.9% of the signicant connections were
evident between these two groups.
As the regression model highlighted, the fungal richness was
key in explaining the variance accounted for in the carbon cycling
and storage model. Using the network analysis we can assess the
composition and connectivity of the fungal community in the
different SOC categories. The highest density of fungal rst degree
networks was found in the category 215% (0.027) while in both
low SOC (<2%) and high SOC (>15%) the fungi formed much looser
networks (densities of 0.019) (Fig. 5).
3.4. Nutrient cycling of N and P
Nitrogen mineralisation availability varied across the 76 sites
from 39 to 1092 (mg N kg 1 dry soil). N mineralisation was lowest
on average in arable sites in the Mediterranean region and greatest
in the Alpine forest sites (Fig. 2b). Phosphorus availability was
equally diverse, ranging from 3.64 to 451 (mg/l 1) between sites.
The lowest available P was quantied in arable Mediterranean
sites, while the greatest availability of P was measured in arable
Continental sites (Fig. 2c).
Three models were applied to quantify nutrient cycling across
the sites. The rst model used normalised N mineralisation data as
the dependent variable and was regressed against nine biological
indicators chosen to represent nutrient cycling (Table 1). This

model described a large proportion of the variation across sites
with an adjusted R2 of 0.734 and a Std. Error of the estimate of
0.332. The model included the following signicant parameters:
Model 1: (normalised N min) = 7.71 + 0.75 ln(Mol.Microbial.
Biomass) 0.11 Biolog L_Threonine.
P availability (Model 2) only accounted for a small amount of
the variability using the same biological indicators as model 1, with
an adjusted R2 of 0.196 and a Std. Error of the estimate of 2.904. The
model included the following signicant parameters:
Model 2: (normalized P) = 12.97 1.93 ln(EEA_phosphomonoesterase) 1.03 ln(potential nitrication) + 1.35 ln(EEA_Leucin.aminopeptidase) 1.01 ln(Enchy_SpRichness).
A combined model of N P was applied to assess the
contribution of soil organisms to nutrient cycling in general. This
model resulted in an adjusted R2 of 0.482 and a Std. Error of the
estimate of 1.978, including the following signicant parameters:
Model 3 (N P) = 12.72 + 3.28 Ln(Molecular microbial biomass) 1.16 Ln(Potential nitrication) + 0.55 Ln(AOA) 3.72
Ln(Fungal Richness).
Accounting for 48% of the variation found across sites, this
model suggests a microbial driven system for cycling of nutrients.
4. Discussion
This study sought to determine the covariation in TUs in soils
across three broad European land uses. This has been achieved
identifying the major connections between TUs for the different
land uses and the stability (number of connections) associated
with the land use types. In addition, this paper has summarised key
Fig. 5. Fungal rst degree networks in different organic matter categories. All signicant connections between fungi (red) and the other organisms are depicted here. The
darkness of the edges is scaled to the interaction strength and the size of the nodes to the average abundance of the TRFs in each organic matter category. Interactions between
fungal TRFs are not drawn. Colour legend indicates the following; fungi (pink), archeae (purple), bacteria (blue), enchytraeids (green), collembolan (orange), nematodes (light
blue-turqouise) and AMF (dark red).
G Model
10
Fig. 6. Network of biotic interaction based on signicant positive Spearman correlations in each pH category. The nodes are sized to the number of species included in the
analyses and their darkness is relational to the connectedness of the node to other nodes. The size and darkness of the connecting edges is sized to the proportion of signicant
positive correlations from all possible correlations between taxonomic units in the land-use type. For pH<5 the treshold for positive interaction was Spearman correlation
>0.57, for pH 57 correlation >0.31 and for pH >7 correlation >0.38, respectively. pH categories were classied on the basis of Stone et al., 2015 (this issue).
indicators for large scale monitoring or measurement of two

ecosystem services (cycling of carbon, nitrogen and phosphorus).
the large number of TUs extractable from these kingdoms, it would

be expected to have a large number of connections found in all
sites.
4.1. Co-occurrence of soil biota in different land use types

4.2. Carbon cycling and storage
The density of network connections provide a useful insight
into the potential food web dynamics taking place in soils across
Europe and how these change with land use. Assessment of the
three land-use types (arable, grass and forest) clearly showed that
land use intensication resulted in lower density networks, a
reduction in the strength of the connections between bacteria and
most other TUs (with the exception of collembolan and AMF) and
an overall reduction in the average number of neighbours. Forest
soils displayed the greatest density of network connections of the
three land use categories, suggesting a more stable system with a
strongly developed food web in place (Digel et al., 2014). In
comparison the arable sites revealed relatively poor density, with a
dominance of a few taxonomic groups, suggesting a partial food
web driven by AMF and plant feeding nematodes. These two TUs
are well known to co-exist on plant roots (Hol and Cook, 2005),
competing for root space and potential feeding sites (Francl, 1993).
These ndings corresponds with the work of Tsiafouli et al.
(2015) who found that increasing land-use intensity resulted in a
decrease in soil faunal taxonomic groups, diversity among
functional groups and a reduction in the average trophic level in
the soil food web. De Vries et al. (2013) also found that land use
intensication reduced the abundance of most functional groups of
soil organisms in four climatically different regions in Europe.
The difference in community connectivity with land use type
also reected the trend to have more connections in forest soils
than in arable with more groups connected (i.e. co-occurred at
more than 1 site, with other TUs). Archaea and bacterial TUs were
the most connected in arable sites, while the interconnection
dominance shifted to bacteria and fungi in grassland sites. In forest
soils archaeal, enchytraeid and fungal TUs displayed the largest
number of neighbours, reecting the greatest connectivity.
Changes in community composition may reect the substrate
availability and disturbance associated with the different land use
systems. For example, the absence of enchytraeids from arable
systems in the top 5 cm can be related to physical or chemical
disturbance (ploughing, soil compaction or contamination), or
moisture conditions near the surface (Didden, 1993; Rhrig et al.,
1998; Didden and Rmbke, 2001). In forest systems the acidic
nature of the soils, reduces the presence of competing earthworms,
resulting in the occurrence of certain enchytraeid species, notably
Cognettia sphagnetorum (CON) (Huhta et al., 1986; Graefe and
Beylich, 2003; Rty, 2004). Archaeal, bacterial and fungal TUs were
found across all land uses and are considered the rst order
primary consumers of all food web systems (Powell, 2007). Due to
This paper assessed whether it was possible at a large scale to

relate the ecosystem service; carbon cycling and storage to key
components of the soil biota. Sites were ranked by a combination
of SOC (%) and disturbance intensity of the soil, for example arable
sites are ploughed on a regular basis, creating high disturbance
intensity over a short time period, oxidising the more labile soil
organic carbon fractions and releasing carbon to the atmosphere as
CO2 (Chan et al., 2002). At the other end of the spectrum of
disturbance intensity, forest sites are left relatively undisturbed for
the growth period of the trees, this can range from 15 years to
100 years.
In this study basal respiration, molecular microbial biomass and
fungal richness were strong indicators associated with the
functional capacity of a system to cycle and store soil organic
carbon over time. Unsurprisingly, the rst two indicators describe
the capacity of the system to turnover carbon (Vance et al., 1987;
Meidute et al., 2008). Microbes are the primary decomposers of
plant material due to the diversity of the enzymes produced and
their unique ability to produce enzymes to break down both simple
molecules such as cellulose and more complex plant derived
compounds such as lignin (Romani et al., 2006). While the
microbial (bacterial and fungal) community are commonly
associated with transformations of SOC in soils (Tardy et al.,
2015) it has also been shown that the interaction between
microbes and soil fauna (including mites, earthworms, collembolans, enchytraeids and nematodes) aid this process and typically
simulate decomposition thus affecting carbon cycling (Nielsen
et al., 2011). In this study the highest network density was found in
sites with SOC between 2 and 15%, showing a hump-back model,
describing the response of the biotic community to extreme
conditions of SOC. The connectivity of the fungal TUs was greatest
in the forest sites, suggesting their importance in the food webs of
forest systems and in terms of cycling SOC. Francisco et al. (2015,
this issue) supported this nding showing that fungal abundance
was greatest in the forest sites and lowest in arable sites. However
the fungal richness was low in forest sites compared to the arable
and grassland sites (Stone et al., 2014), this could be due to a
predominance of key soil fungi in forest systems (Bouffaud, in
press). Authors have dened soil microbial biomass and fungi as
principally responsible for carbon sequestration in soil (Giller et al.,
1997; Clemmensen et al., 2013). These last authors showed that
5070% of stored carbon in a chronosequence of boreal forested
islands derives from roots and root-associated microorganisms.
G Model
Therefore the inclusion of soil microbial biomass, respiration and

fungal richness have been found to be key indicators of carbon
cycling and potential storage and should be considered in further
soil monitoring frameworks assessing this ecosystem service.
4.3. Nutrient cycling of nitrogen and phosphorus
The assessment of nutrient cycling at 76 sites across Europe was
achieved by comparing N mineralisation and P uptake by plants at
these sites. Three initial models were derived, the models were
statistical, and often do not necessarily reect true causal
relationships (Mac Nally, 2002). However, they are very useful
to quantitatively describe complex soil systems. A model using N
cycling only, accounted for 73% of the variation in N mineralisation
between sites and was described by the measures of molecular
biomass and L-threonine substrate utilisation in the Biolog assay. Lthreonine contains nitrogen (C:N ratio of 4:1), suggesting its
relevance as an indicator for N-cycling. This suggests a reliance on
the SOC availability to support N mineralisation. Fierer et al. (2012)
suggest that phylogenetic or physiological reponses of the
microbial community to N concentrations may be the result of
the amount or type of organic carbon substrate present in soils.
However, in both the European and Dutch datasets (Rutgers et al.,
2015 this issue), L-threonine demonstrates higher variation than
average within samples.
While the N model only adequately described the biological
contribution to N cycling across these sites it did not account for
the contribution of soil biota to P cycling in soils. The second model
addressed P cycling only, but this resulted in a very poor estimate
of variance. Soil enzymes were most signicant in describing the
variation of P availability across the sites, specically phosphomonoesterase and Leucin aminopeptidase. Phosphomonoesterase
is responsible for the mineralisation of organic P to the inorganic P
form, which is utilised by plants and microbes (Nannipieri et al.,
2011). Hendriksen et al. (2015, this issue) found that climatic and
land-use had no signicant impact on the behaviour of soil enzyme
activity but that soil organic carbon content was a strong regulator
of enzyme activity and pH had a signicant effect on specic
enzymes such as phosphomonoesterase.
Enchytraeid species richness also contributed signicantly to
the model; however, this may be a statistical rather than an
ecological phenomenon (Mac Nally, 2002) as no previous research
has identied the role of enchytraeid species richness in
contributing to P availability and variation between sites was low.
Finally, a combined model including molecular microbial
biomass, potential nitrication, abundance of archaeal ammonia-oxidizers and the structure of the fungal community provided
a better description of variance for P availability, but less robust for
N mineralisation potential across sites. This model, while it
accounts for less variation across sites, compared to the model
using N only, provides a reasonable assessment of biological
indicators for both N and P cycling in soils across Europe. The
positive relationship between nutrient cycling (N P) and
microbial biomass can be attributed to the important role that
micro-organisms play in nutrient mobilisation. The microbial
biomass is essentially a labile pool of P, which is resistant to
xation by abiotic conditions (clay content, Fe, Al, Ca) and loss by
leaching (Brookes, 2001). Enhanced P availability for plants is often
attributed to arbuscular mycorrhizae, for example Van der Heijden
et al. (1998) showed that increasing arbuscular mycorrhizal
diversity and hyphal length were signicant for increasing plant
P concentrations. Interestingly N nitrication was identied in the
combined model, but not considered signicant in the model
considering N cycling only. Van der Heijden et al. (2008) reported
that mycorrhizal fungi and nitrogen-xing bacteria were
11
responsible for up to 80% of all nitrogen, and up to 75% of

phosphorus, that is acquired by plants annually.
5. Conclusion
This is the rst pan-European study to measure such a range of
soil biological parameters across Europe. The network analysis has
demonstrated the variation in co-occurrence of TUs across the
three different land use classes. It has shown the impact of land use
intensity on the density of network connections, highlighting that
arable systems display much lower network density compared to
grass and forest systems. There were also changes associated with
pH and SOC. Key biological indicators were identied in relation to
the cycling of carbon and nutrients (N and P). Most of the indicators
identied were comparable to those identied in more mechanistic studies, showing the applicability of these indicators for larger
scale studies or monitoring networks. In some cases, statistical
relationships were acknowledged where no prior research was
available explain the underlying mechanisms and therefore using
such large scale sampling campaigns must be interpreted with
some caution. This collation and analysis of soil biodiversity data
shows the importance of large datasets to understand the
community dynamics and the quantitative patterns for prediction
of soil ecosystem services at a continental scale.
Acknowledgements
This work was supported by the European Union within the
projects EcoFINDERS (FP7-264465) and by the Rural and Environment Science and Analytical Services Division of the Scottish
Government. Many thanks to the laboratory staff at Teagasc,
Johnstown Castle Research Centre for their support in abiotic
laboratory analyses, in particular Pat Sills, Paul Massey, Carmel
OConnor and Olivia Fagan. Thanks to Rogier Schulte, Teagasc,
Johnstown Castle Research Centre for advice in modelling design
for nutrient cycling and carbon cycling statistical models.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.apsoil.
2015.08.006.
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APSOIL 2259 No. of Pages 13

Applied Soil Ecology xxx (2015) xxxxxx

Contents lists available at ScienceDirect

Applied Soil Ecology

Ecological network analysis reveals the inter-connection between soil

Teagasc, Johnstown Castle Research Centre, Ireland

R.E. Creamer et al. / Applied Soil Ecology xxx (2015) xxxxxx

European Union soil policy making by providing the necessary tools

R.E. Creamer et al. / Applied Soil Ecology xxx (2015) xxxxxx

and the abundance of key functional genes involved in ammonia

R.E. Creamer et al. / Applied Soil Ecology xxx (2015) xxxxxx

following (ISO (International Organization for Standardization),

were: 40 sec at 95  C, 1 min at 58  C and 1 min at 72  C. Plasmids

R.E. Creamer et al. / Applied Soil Ecology xxx (2015) xxxxxx

Community level physiological proles (CLPP) using Biolog

Paper recommending indicator

Beta-glucosidase; sum of enzyme activity

Kivlin and Treseder, (2014);

Basal respiration, L-malic acid, D-(+)-glucose, alpha ketogluterate, PCA1, PCA2

Phospholipid fatty acids (PLFA)

Fungal: bacterial, ergosterol (18:2w6,9), AMF (16:1w5c and 18:1w9c)

(ng microbial DNA g soil 1)

Feeding guild richness (plant-feeders, fungal-feeders, omnivores, bacterial-feeders, predators)

Species richness and Abundance per m2

Plant-feeders, Fungal-feeders, Omnivores, Bacterial-feeders, Predators and total abundance

Extracellular Enzyme Activity

Arylsulfatase, phosphomonoesterase, Leucin aminopeptidase

Coince et al. (2014)

Cole et al. (2000) (N

Rmbke et al. (2013)

R.E. Creamer et al. / Applied Soil Ecology xxx (2015) xxxxxx

use, pH or SOC) were removed prior to analysis. The remaining

R.E. Creamer et al. / Applied Soil Ecology xxx (2015) xxxxxx

group of organisms and between categories, and reveals the

Arable sites displayed a lower average number of neighbours

R.E. Creamer et al. / Applied Soil Ecology xxx (2015) xxxxxx

molecular microbial biomass and fungal richness, with a Std. Error

analysed according to the SOC categories dened in Stone et al.

R.E. Creamer et al. / Applied Soil Ecology xxx (2015) xxxxxx

low (0.023 and 0.024, respectively) (Supplementary Table B).

indicators chosen to represent nutrient cycling (Table 1). This

R.E. Creamer et al. / Applied Soil Ecology xxx (2015) xxxxxx

indicators for large scale monitoring or measurement of two

the large number of TUs extractable from these kingdoms, it would

4.1. Co-occurrence of soil biota in different land use types

This paper assessed whether it was possible at a large scale to

R.E. Creamer et al. / Applied Soil Ecology xxx (2015) xxxxxx

Therefore the inclusion of soil microbial biomass, respiration and

responsible for up to 80% of all nitrogen, and up to 75% of

R.E. Creamer et al. / Applied Soil Ecology xxx (2015) xxxxxx

microbial communities by using whole soil. Appl. Environ. Microbiol. 69,

R.E. Creamer et al. / Applied Soil Ecology xxx (2015) xxxxxx

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were: 40 sec at 95 C, 1 min at 58 C and 1 min at 72 C. Plasmids