Journal of Ecology 2010, 98, 50–61 doi: 10.1111/j.1365-2745.2009.01601.

The ecological engineering impact of a single tree

species on the soil microbial community
Ruth J. Mitchell1,2*, Colin D. Campbell1,3, Stephen J. Chapman1 and Clare M. Cameron1
1
The Macaulay Institute, Craigiebuckler, Aberdeen AB15 8QH, UK; 2Centre for Ecology and Hydrology Banchory,
Hill of Brathens, Banchory, Aberdeenshire AB31 4BW, UK; and 3Department of Soil and Environment, Swedish
University of Agricultural Sciences, Box 7014, SE-750 07 Uppsala, Sweden

Summary
1. Ecosystem engineering by a single species can have a cascading effect on many ecosystem
processes. While the impact of above-ground ecological engineers on soil chemical properties has
been studied, few studies have assessed their impact on the soil microbial community, which is lar-
gely responsible for many ecosystem functions.
2. Utilizing a long-term experiment where birch was planted on heather moorland 20 years ago,
the engineering impact of a single tree species (Betula pubescens) on the soil microbial community
was assessed using phospholipid fatty acid (PLFA) analysis and fungal polymerase chain reaction–
denaturing gradient gel electrophoresis (PCR-DGGE). Changes in the soil microbial community
were then related to soil chemical and physical variables and tree performance variables.
3. Under birch, total microbial biomass (total PLFA) declined, species richness increased and the
ratio of fungal : bacterial PLFA declined. The fungal PLFA marker increased with increasing
organic matter and depth of the LFH and O soil horizons, characteristics associated with moorland
soils. Bacterial PLFAs increased with increasing birch canopy cover. The fungal community of the
birch plots was different from that in the heather plots and changes in the fungal community com-
position were related to the size of the birch trees in the plots.
4. Changes in the soil microbial community were also related to changes in mineralizable N.
Mineralizable N was correlated with both decreasing total soil microbial biomass and decreasing
fungal : bacterial ratio.
5. The durability of the engineering effects of birch was studied in a second experiment. Plots were
established in first-generation birch woodland that had developed on Calluna-dominated
moorland. The plots were cleared of birch and planted with heather. After 20 years, there was no
difference in the community composition of the PLFAs. In contrast, there were significant differ-
ences in the fungal community composition as judged by DGGE analysis, with the fungal commu-
nity in the felled birch plots being similar to the heather moorland.
6. Synthesis. This work demonstrates that addition of a single tree species to heather moorland
results in changes in below-ground soil microbial communities and in nutrient cycling.
Key-words: bacteria, Betula, Calluna, ecosystem engineer, fungi, mineralizable nitrogen,
moorland, PCR-DGGE, PLFA, succession

result in the modification and ⁄ or creation of habitats are

Introduction
Species may change their environment by transforming living
or non-living materials from one physical state to another.
Such processes that are not directly trophic or competitive but
*Correspondence author. Natural Research Projects, Brathens
Business Park, Hill of Brathens, Glassel, Banchory, Aberdeen-
shire AB31 4BY, UK. E-mail: r.mitchell@abdn.ac.uk
collectively termed ecosystem engineering (Jones, Lawton &
Shachak 1994). Ecosystem engineering occurs through activi-
ties such as digging burrows (Bancroft, Hill & Roberts 2004;
Casas-Criville & Valera 2005); building dams (Wright, Jones &
Flecker 2002; Wright, Flecker & Jones 2003; Bailey et al.
2004), building shelters (Cardinale, Gelmann & Palmer 2004;
Kagata & Ohgushi 2004) or simply vegetation growth (e.g. tree
growth, Jones, Lawton & Shachak 1994). These activities have

 2009 The Authors. Journal compilation  2009 British Ecological Society


been shown to result in changes in the physical properties of
the soil (Lavelle et al. 1997), in plant communities and soil
chemical properties (Folgarait et al. 2002), the community
structure of ground-nesting ants (Reyes-Lopez, Ruiz & Fer-
nandez-Haeger 2003) and even the weather (Holling 1992,
quoted in Jones, Lawton & Shachak 1994). Such ecosystem
engineering by a single species can have a cascading effect on
many other ecosystem processes. While the impact of above-
ground engineers on soil chemical proprieties has been studied,
few studies have followed the cascading effects of an above-
ground engineer on the soil microbial community (SMC). The
drivers of change in the SMC composition are often not fully
known, yet knowledge of such processes may offer opportuni-
ties for a better understanding of how ecosystems function and
the ecological consequences of global change phenomena
(Wardle 2005).
Where changes in vegetation have been related to the SMC
it has been done in the context of habitat changes such as the
abandonment of arable land (Van der Wal et al. 2006), the res-
toration of habitats (Zhang et al. 2006) or long-term (28 000-
year) succession (Williamson, Wardle & Yeates 2005). Such
work is often carried out within the context of developing a
better understanding of the relationship between above-
ground and below-ground diversity (Wardle 2005). The impact
of the addition of a single species to the above-ground commu-
nity on the below-ground community is rarely studied and
most of the available studies have dealt with grassland species
(Bardgett et al. 1999; Wardle et al. 1999; Innes, Hobbs &
Bardgett 2004).
Soil microbial communities are hard to study due to the
scale of the extant diversity and the inability to culture and
identify most species, which all leads to difficulties in assessing
changes in composition. One method that overcomes some of
these problems is phospholipid fatty acid (PLFA) analysis.
Fatty acids are essential membrane components of all living
cells and generally constitute a relatively constant proportion
of the biomass of an organism. Under comparable environ-
mental conditions, changes in fatty acid patterns are thought
to reflect changes within microbial communities and therefore
act as indicators of microbial community composition. Some
fatty acids are considered as ‘key signatures’ (Zelles 1999) and
can be used to distinguish between different bacterial taxa or
between bacteria and fungi. Other fatty acids are ubiquitous
and therefore have poor taxonomic potential but can be used
to help estimate microbial biomass. A second method that also
overcomes traditional limitations to study microbial diversity
is denaturing gradient gel electrophoresis (DGGE), a type of
finger printing of polymerase chain reaction (PCR)-amplified
DNA. Different sequences of DNA will denature at different
concentrations of the denaturant as the sample migrates
through the electrophoresis gel. This results in a pattern of
bands where each band may represent a different microbial
population present within the community. Here, we use both
PLFAs and fungal PCR-DGGE to assess the impact of a sin-
gle above-ground species on the SMC.
It is not known whether a single above-ground engineering
species can drive changes in the SMC through changes in soil
 2009 The Authors. Journal compilation  2009 British Ecological Society, Journal of Ecology, 98, 50–61
Tree impacts on the soil microbial community 51
properties. However, we know that changes in soil chemical
and physical properties, such as changes in pH, the quality of
the organic matter and soil moisture, drive changes in the
SMC (Bååth & Anderson 2003; Hackl et al. 2005; Williamson,
Wardle & Yeates 2005; Högberg, Högbeg & Myrold 2007),
and that birch (Betula pubescens) has been shown to change
these soil properties (Mitchell et al. 2007). Acidic soils are
more often dominated by fungi while bacteria dominate less
acidic soil (Wardle 2005), and birch has been shown to increase
the pH of the soil. Thus, the first hypothesis we tested is that
the composition of the SMC and the total soil microbial bio-
mass changes when birch is planted on moorland; in particular
we expected a change towards a more bacteria-dominated
SMC on the birch plots. The SMC has been shown to be differ-
ent for different grass species and even under different clones
of spruce (Korkama et al. 2007). Consequently, our second
aim was to test the hypothesis that different Betula species will
have differing effects on the SMC. The durability (sensu Jones,
Lawton & Shachak 1997) of a SMC once the ecosystem engi-
neer has been removed is also untested and thus the third aim
was to test the hypothesis, that with the removal of the ecosys-
tem engineer (Betula) and the re-establishment of moorland
plant species, that the SMC would revert to one typical of a
moorland. Soil microorganisms play an important role in eco-
system processes such as organic matter decomposition and
nutrient cycling. If birch does cause the SMC to change
towards a more bacteria-dominated system (Hypothesis 1),
rates of nutrient cycling would be expected to increase (Wardle
et al. 2004; Van der Heijden, Bardgett & van Straalen 2008),
resulting in an increase in mineralizable nitrogen (N). There-
fore, our fourth hypothesis was that if the SMC changes to a
more bacteria-dominated community, this will result in a fur-
ther cascading effect on ecosystem process with an increase in
mineralizable N, which we aimed to test.
Materials and methods
EXPERIMENT 1: PLANTED BIRCH PLOTS ON HEATHER
MOORLAND
Three experimental sites, Delnalyne (Banffshire, north-eastern Scot-
land, 5714¢27.8¢¢ N, 320¢38.71¢¢ W), Craggan A (Banffshire, north-
eastern Scotland, 5722¢29.35¢¢ N, 320¢17.36¢¢ W) and Kerrow
(Inverness-shire, West Scotland, 5719¢48.15¢¢ N, 445¢55.41¢¢ W),
were established on open Calluna-dominated moorland in the 1980s.
The distance between the sites ranged from 12 to 75 km. The soil
type at all sites was a humus-iron podzol. Twelve plots were
established at Delnalyne and Craggan A in a paired-plot design.
In each pair, the two treatments, heather control and planted
with Betula pubescens, were randomly assigned to the plots. At
Kerrow, there were 18 plots in groups of three; the three treat-
ments, heather control, planted with Betula pendula and planted
with Betula pubescens, were randomly assigned to plots within
each group. The plots were 16 · 16 m, except at Delnalyne where
they were 12 · 12 m. There was a gap of about 6 m between
each plot. The experimental area at each site was fenced to
exclude grazing by large herbivores (deer, sheep and rabbits) from
the experiment. For further details of the plot layout, see Mitchell
52 R. J. Mitchell et al.

et al. (2007). The heather control and planted Betula pubescens tography (GC) conditions were as described by Certini, Campbell &
plots at Delnalyne, Craggan A and Kerrow formed Experiment Edwards (2004). Briefly, 500 mg milled, freeze-dried soil was
1a: the impact of Betula pubescens on moorland systems. The 12 extracted with a chloroform–methanol–citrate butter mixture
planted Betula sp. plots at Kerrow formed Experiment 1b: a (1:2:0.8), and the phospholipids were separated from other lipids on
comparison of Betula pubescens and Betula pendula. a silicic acid column. The phospholipids were subjected to a mild-
alkali methanolysis, and the resulting fatty acid methyl esters
(FAMES) were separated and identified by GC. PLFA identities were
EXPERIMENT 2: THE DURABILITY OF CHANGES
assigned based on retention time and comparison with known
CAUSED BY BETULA
standards and verified by GC mass spectrometry. Saturated and
A second experiment, to test the durability of changes caused by unsaturated FAMES were identified by making silver adducts, and
Betula on moorland, was established at Craggan B (Banffshire, the position of unsaturated bonds was determined using disulphide
north-eastern Scotland, 5720¢20.8¢¢ N, 320¢38.71¢¢ W) in 1978. bridging.
Twelve plots arranged in a paired-plot design were established in The PLFAs were classified according to Frostegård, Tunlid &
mature Betula pubescens woodland that was first-generation wood- Bååth (1996) for fungi and actinobacteria and according Frostegård,
land on previous Calluna-dominated moorland (Mitchell et al. 2007). Tunlid & Bååth (1996) and Zogg et al. (1997) for bacteria (Table 1).
The Betula pubescens trees in one of each of the paired plots were Bacterial PLFAs were classified according to Zogg et al. (1997) into
felled in June 1978 and the wood and brash removed. The vegetation Gram-positive and Gram-negative bacteria (Table 1). Those PLFAs
in the felled plots was sprayed once with Asulox to kill Pteridium listed by Frostegård, Tunlid & Bååth (1996) as actinobacteria were
aquilinum and NaCl to kill all Betula seedlings that had established; added to Zogg et al.’s (1997) list of Gram-positive bacteria. The
spot treatments were then carried out to kill any P. aquilinum or PLFA 18:2x6,9 is found in both plants and fungi, but as most roots
Betula sp. that had re-established. Calluna vulgaris was planted in the were removed from the soil prior to analysis this marker was taken to
felled plots and the plots were weeded occasionally over the next indicate the presence of fungi (Frostegård, Tunlid & Bååth 1993). By
20 years to remove any Betula seedlings that had established. The summing PLFAs known to be of bacterial or fungal origin, it is also
felled plots were 24 · 24 m, within which a 16 · 16 m plot was estab-
lished. The birch control plots, mature Betula pubescens, were also
16 · 16 m. The felled plots were fenced to exclude large grazing Table 1. Classification of phospholipid fatty acids (PLFAs) as
herbivores, but the birch control plots were not fenced. The treat- fungal, bacterial or unclassified*. The list is limited to PLFAs
ments, felled birch or birch control, were randomly assigned to plots extractable by the modified Bligh and Dyer method
within each pair of plots (see Mitchell et al. (2007) for further details
of plot treatments). Non-specific
Fungal eucaryotes† Bacterial Unclassified*
SOIL SAMPLING
18:2x6,9 20:4x6,9,12,15c 15:0ia 13:0
Tree growth within individual planted birch plots was very variable 20:5x3 15:0aia 14:0i
with some parts of many plots containing only a few small trees and 20:4x2,6,10,14 15:0 14:0
being similar to the heather control plots. In order to more accurately 20:4x3,6,9,12 16:0ia 14:1x9c
20:1x9 16:1x7cb 14:1x9t
assess the impact of the trees on the microbiological community, the
20:1 16:1x7tb 16:0
plots were divided into quarters and the tree growth was assessed
20:0 16:1x5cb 16:0br
(Mitchell et al. 2007). The microbiological community was sampled 16:0(10Me)a,d 16:1i
from the quarter plot of each plot with the greatest tree growth and 17:0(10Me)a,d 16:1x11c
the corresponding quarter plot of the heather control plot. Three 17:0ia 16:1x11t
cores were taken from the chosen quarter of each plot using a box 17:0aia 16:0(12 Me)
corer (width 5 cm) to a depth of 15 cm in September 2003. For Exper- 17:0cyb 17:0br
iment 2 (Craggan-felled birch site), three soil cores were taken from 17:0 17:1x8t
one random quarter of each plot. All cores were collected at any one 18:1x7b 17:1x8c
site in the same day in order to minimize variation between the cores. 18:0(10Me)a,d 17:1x7
19:0cyb 17:0(12Me)
The soil cores were stored at 4 C and processed within 5 days of field
18:0
sampling. The depth of the humified (LFH) layer and O horizon of
18:3x6,8,13
each core was recorded, and the O horizon samples were bulked for 18:2x8,12
each quarter plot. The soil samples were sieved (< 2 mm) and roots, 18:1x9
stones, etc. removed prior to subsampling and storage. Samples for 18:1x13
molecular analysis were stored at )80 C, samples for PLFA analysis 18:1x10 or 11
were stored at )20 C and samples for chemical analysis were 19:1x6
air-dried at 30 C. 19:1x8

The PLFAs were classified according to Frostegård, Tunlid &

MICROBIOLOGICAL ANALYSIS
Phospholipid fatty acid extraction (PLFA)
Lipid extraction and PLFA analyses were performed as described by
Frostegård, Tunlid & Bååth (1993) using the modified Bligh and Dyer
method (Bligh & Dyer 1958). Specific modifications and gas chroma-
Bååth (1996) and Zogg et al. (1997).
a
Gram-positive bacteria, bGram-negative bacteria, cprotozoa,
d
actinobacteria.
*Unclassified PLFAs occur in both prokaryotic and eukaryotic
organisms and can not be classified as being either specifically
fungal or bacterial.
†PLFAs found in fungi and other eucaryotes.

 2009 The Authors. Journal compilation  2009 British Ecological Society, Journal of Ecology, 98, 50–61

possible to quantify bacterial or fungal biomass separately (e.g.
Pennanen et al. 1998). The ratio of fungal to bacterial PLFA was cal-
culated by taking the fungal marker 18:2x6,9 and dividing it by the
sum of the predominant bacterial PLFAs (Table 1) (Frostegård &
Bååth 1996).
DNA Extraction and PCR amplification of fungal ITS
regions
The DNA from 250 mg soil, previously stored at )80 C, was
extracted using a modified FastDNA Spin Kit for Soil (QBiogene,
Carlsabad, CA, USA) where the sodium phosphate buffer, MT
buffer and PPS reagent were replaced with sodium phosphate buffer
(100 mm, pH8), lysing buffer (100 mm NaCl, 500 mm (pH8) Tris–
HCl, 10% SDS) and 0.4 V Ammonium acetate (7.5 m) as described
by Berthelet, Whyte & Greer (1996). Fungal ITS regions were PCR-
amplified using ITS1F and ITS4 (White, Lee & Taylor 1990). Cycling
parameters were 95 C for 5 min, followed by 35 cycles of 94 C for
30 s, 55 C for 1 min and 72 C for 2 min with a final extension at
72 C for 10 min. These products were then used as template in a
nested PCR using 1 lL in 50 lL reaction volumes with primers
ITS1F (with a GC clamp) and ITS2 (White, Lee & Taylor 1990), and
separated using DGGE.
Denaturing grade gel electrophoresis
Polyacrylamide gels (8% Acrylogel 2.6 solution; BDH, Poole,
UK) were prepared with a vertical gradient of 25% (1.7 m Urea –
10% (vol ⁄ vol) Formamide) – 55% (3.8 m Urea – 22% (vol ⁄ vol)
Formamide) as described by Anderson, Parkin & Campbell
(2008). Two lL of each nested PCR product was loaded onto the
gels and electrophoresis was performed in 1· TAE buffer (40 mm
Tris–acetate, 1 mm EDTA) at 75 V and 60 C for 16 h. Gels were
silver-stained as previously described by McCaig, Glover & Pros-
ser (2001) and scanned using an Epson Expression 1680 Pro scan-
ner at a resolution of 600 dpi. DGGE gels were analysed using
Gel Compar II software v 4.5 (Applied Maths, Saint-Martens-
Latem, Belgium). The presence of bands was determined using a
minimum profiling of 10% and binary matrices were produced by
performing band matching using an optimization tolerance setting
of 0.1%.
COLLECTION OF EXPLANATORY VARIABLES
Data on soil chemistry and tree performance collected from these
plots were used as explanatory variables for changes in the micro-
bial community. Full details of these explanatory variables are
available in Mitchell et al. (2007); however, a brief description of
the methods used to collect the data is provided. For the quarter
plot that was sampled the canopy cover was recorded (using a
hemispherical densiometer), the number of trees counted and the
height and basal diameter measured on 10 randomly selected trees.
Tree growth varied greatly between the plots (Mitchell et al. 2007),
and hence the inclusion of tree variables within the analysis was
seen as important explanatory variable. Soil chemical analysis was
carried out on the air-dried, 2-mm sieved subsamples previously
mentioned. Available Na, K, Ca, Mg, Fe and Al (extracted in
ammonium acetate pH 7) were analysed following Thomas (1982).
Total P was measured using the method in Smith & Bain (1982)
and total C and N were measured using the methods of Pella &
Colombo (1973). Available P (extracted in Truog’s reagent) was
analysed following the methods in Allen (1989), and pH was mea-
 2009 The Authors. Journal compilation  2009 British Ecological Society, Journal of Ecology, 98, 50–61
Tree impacts on the soil microbial community 53
sured in water (McLean 1982). Loss-on-ignition (LOI) and soil
moisture content were measured following Gardner (1965). Miner-
alizable N was analysed following the aerobic method in Allen
(1989). Briefly, 15 g of sand and 6 mL of water were added to each
of two 5 g of air-dried and sieved soil to bring the moisture to an
optimal level for aerobic mineralization. One sample was extracted
immediately in 6% KCl and the second extracted after it had been
incubated for 14 days at 30 C. The total inorganic nitrogen in
both extracts was measured and the mineralizable N calculated as
the difference in total inorganic nitrogen between the second and
first samples.
DATA ANALYSIS
Three groups of data were analysed. First, the impact of B. pubescens
was assessed by analysing the data from the heather control plots and
the planted B. pubescens plots at Delnalyne, Craggan A and Kerrow
(Experiment 1a). Secondly, the effect of different birch species was
assessed by analysing the data from Kerrow (Experiment 1b).
Thirdly, the durability of the birch engineering was assessed by analy-
sing the data from the control birch and felled birch plots at Craggan
B (Experiment 2).
Univariate data were analysed by fitting linear mixed models to
plot-level data using the Proc mixed procedure in sasv9.1 (SAS
2005). Treatment and site were included as fixed effects. Variation
across the site was accounted for by the paired-plot design of the
experiment. Categorical variables for plot pair were included as ran-
dom effects in all models. Tukey’s pairwise comparisons were used
to determine differences between treatments and adjusted using the
Tukey–Kramer correction for multiple tests. PLFA data were analy-
sed as nmol g)1. The relationship between mineralizeable nitrogen
and PLFA content (nmol g)1) was also assessed using the Proc
mixed procedure with PLFA content and site included as fixed
effects and plot pair included as a random effect. Shannon’s diver-
sity index was calculated for the % mol PLFA data. Differences
between treatments in Shannon’s diversity index were analysed as
above using Proc mixed.
Multivariate analysis was carried out on the % mol PLFA data
and the binary fungal DGGE data. In all multivariate analyses, site
was included as a covariable and the analysis was carried out using
canoco 4.5 (Ter Braak & Smilauer 2002). The PLFA data from Exper-
iment 1a were analysed using redundancy analysis (RDA) as initial
data analysis showed that the gradients on the axes were short, indi-
cating that a linear response model, such as RDA, was more appro-
priate than a unimodal model such as canonical correspondence
analysis (Ter Braak & Smilauer 2002). The PLFA data were log-
transformed prior to analysis. Environmental variables included for
possible selection within the analysis were: number of trees, tree
cover, total basal area of trees, average height of trees, depth of the
LFH horizon, depth of the O horizon, moisture, total C, total N, total
P, C : N ratio, LOI, pH, moisture and available Na, K, Ca, Mg, P,
Al, Fe and Mn. Forward selection of environmental variables was
used with each variable being tested using a Monte Carlo permuta-
tion test (9999 permutations). Selection of variables stopped when the
addition of variables did not significantly explain more of the varia-
tion. The PLFA data from Experiment 2 were analysed using detr-
ended correspondence analysis (DCA). As the DGGE data were
presence ⁄ absence data, it was not possible to analyse the data by
RDA. Instead DCA was used for both Experiments 1a and 2. The
axes scores for the plots from the DCA were analysed using Proc
mixed (as above) to assess if plots with different treatments had differ-
ent scores.
54 R. J. Mitchell et al.

ve : Gram-negative bacteria. There were significant differences

Results
between sites in all the above analyses.
EXPERIMENT 1A: THE IMPACT OF BETULA PUBESCENS
ON THE SOIL MICROBIAL COMMUNITY Changes in PLFA composition and diversity.
Phospholipid fatty acid composition differed between heather
Changes in PLFA content.
control and planted birch plots. In an RDA analysis of the
Total PLFA was significantly lower on planted birch plots PLFA composition, the environmental variables tree cover,
than on the heather control plots (F1,15 = 11.5, P = 0.0041) LOI, depth of the LFH horizon, depth of the O horizon, K,
(Fig. 1a). When the PLFA was split into different groups, the %N, Ca and Fe were all significant (P < 0.05) in explaining
fungal PLFA was significantly lower in the planted birch plots the variation in PLFAs, and in total explained 31% of the vari-
than on the heather control plots (F1,15 = 12.9, P = 0.0027) ation. After the fitting of covariables (sites), which explained
(Fig. 1c). In addition, the ratio of fungal : bacterial PLFA was 49% of the variation, the first axis of the ordination explained
lower in the planted birch plots than on the heather control 31% of the remaining variation in PLFAs and the second axis
plots (F1,15 = 7.8, P = 0.01) (Fig. 1d). There was no effect of 13%. The first axis was positively correlated with tree cover
B. pubsecens on the PLFA markers for 16:1x5c, Gram- and to a lesser extent Fe, and samples from the planted birch
positive or Gram-negative bacteria or the ratio of Gram-positi- plots, with good birch growth, were generally found along the

(a) 3000
(b) 1400

2500 1200
Bacterial PLFA nmolg–1
Total PLFA nmolg–1

1000
2000
800
1500
600
1000
400

500
200

0 0
Delnalyne Craggan A Kerrow Craggan B Delnalyne Craggan A Kerrow Craggan B
Site Site

(c) 600 (d) 1

0.9
500
0.8
Fungal PLFA nmolg–1

Fungal:bacterial ratio

0.7
400
0.6
300 0.5

0.4
200
0.3

0.2
100
0.1
0 0
Delnalyne Craggan A Kerrow Craggan B Delnalyne Craggan A Kerrow Craggan B
Site Site

(e) 3.1

3
Shannon's diversity index

Heather control
2.9 Planted birch

2.8
Felled birch
Birch control Fig. 1. Changes in phospholipid fatty acids
2.7
(PLFA) content and diversity with the
2.6 addition (Delnalyne, Craggan A and Ker-
row) and removal (Craggan B) of Betula
2.5
pubescens. (a) Total PLFA; (b) bacterial; (c)
2.4 fungal; (d) fungal : bacterial ratio; (e) Shan-
Delnalyne Craggan A Kerrow Craggan B non’s diversity index for PLFAs. Mean
Site values (n = 6)± 1 SE are shown.

 2009 The Authors. Journal compilation  2009 British Ecological Society, Journal of Ecology, 98, 50–61
 Tree impacts on the soil microbial community 55

1.0
Cover
K
N

Axis 2 (13%)
ENV. Variables
LOI
Fig. 2. Biplot of samples and environmental
variables from redundancy analysis (RA) of Fe
Ca Samples
phospholipid fatty acids (PLFA) data from LFH
OHor Delnalyne heather
three sites where Betula pubescens was
Delnalyne birch
planted on moorland (birch) and open moor-
land control plots (heather). Environmental Craggan A heather
variables are: Birch canopy cover (cover) Craggan A birch
and soil chemistry of the organic layer for Kerrow heather
potassium (K), nitrogen (N), iron (Fe), loss- Kerrow birch
on-ignition (LOI), depth of LFH layer –1.0
(LFH), depth of organic layer (OHor). –1.0 Axis 1 (31%) 1.0
0.8

18:0(10Me)

16:0i
17:0(10Me) 15:0i
15:0
16:1w7c
14:0i
17:0
Cover
16:1w11t 17:0ai
Axis 2 (13%)

K 17:0br
N
16:1w7t
Fig. 3. Biplot of species and environmental 17:1w8c
17:0(12Me) 14:0 15:0ai
20:4 w6,9,12,15 19:0cy
17:0cy
variables from redundancy analysis (RA) of LOI 16:1w11c
16:1i 20:4 w3,6,9,12 16:1w5c
18:1w9
phospholipid fatty acids (PLFA) data from 14:1w9c 19:1w6
16:0(12Me) 18:1w10or11
18:1w7 17:0i
heather control and planted birch plots. Fe
17:1w7
19:1w8 14:1w9t
Ca 18:2w8,12
Environmental variables are: Birch canopy LFH 20:1 20:1w9 17:1w8t
13:0 18:1w13 18:3w6,8,13
cover (cover) and soil chemistry of the 20:4w2,6,10,14
OHor
16:0(10Me)
20:0
organic layer for available potassium (K), 18:2w6,9 16:0br
20:5w3
total nitrogen (N), available iron (Fe), loss- 18:0
on-ignition (LOI), depth of LFH layer 16:0
–0.6

(LFH) and depth of organic layer (OHor)

(see Table 1 for classification of PLFA
markers). –1.0 Axis 1 (31%) 1.0

positive half of this axis (Fig. 2). LOI and K, and to a lesser
extent Ca, and the depth of the LFH and O horizons were neg-
atively correlated with the first axis, and samples from the
heather control plots were generally found along the negative
half of this axis. The second axis was positively correlated with
total nitrogen as well as tree cover, K and LOI and negatively
correlated with Ca, and the depth of the LFH and O horizons.
The samples did not split into clear groups along the second
axis.
The majority of the PLFA markers increased with increas-
ing birch cover indicating an increase in their biomass with
birch cover (Fig. 3). In particular, most of the bacterial PLFA
markers, especially 15:0i, 17:0ai, 15:0ai, 17:0cy, 16:1x7t,
16:1x5c, 16:0(10Me), 17:0i, 19:0cy and 18:1x7, increased with
increasing birch cover (Fig. 3). The fungal PLFA marker
18:2x6,9 declined with increasing birch cover indicating a
decline in fungi with birch establishment. Twenty-four PLFAs
were also recorded (Table 1) for which it is not possible to be
specific about which organisms they come from as they occur
in both prokaryotic and eukaryotic organisms in the soil.
These unclassified PLFAs largely come from soil microorgan-
isms as most visible roots and animals, if present, were
removed. A small fraction could, however, have come from
another source within the soil (e.g. plant fine roots, algae,
micro ⁄ mesofauna). Shannon’s diversity index for the PLFAs
was significantly greater under the planted birch plots than
under the heather control plots (F1,17 = 21.2, P = 0.0003)
(Fig. 1e) suggesting an overall increase in diversity with birch
cover, which concurs with the results from the RDA.
Relationship between PLFA and mineralizable nitrogen
Mineralizable nitrogen increased as total PLFA declined
(F1,29 = 20.5, P < 0.0001) (Fig. 4a). When the sites were
analysed separately, the pattern was still significant for Delna-
lyne (F1,6 = 43.9, P < 0.001) and Kerrow (F1,6 = 20.8,
P < 0.01) but not for Craggan. Mineralizable N was also
related to the change in species composition with an increase in
mineralizable N as the proportion of bacteria increased in the
community relative to fungi (F1,21 = 15.5, P = 0.007)

 2009 The Authors. Journal compilation  2009 British Ecological Society, Journal of Ecology, 98, 50–61
56 R. J. Mitchell et al.
(a) 4000
Total PLFA nmolg–1
3000
2000
1000
2 0.2
R = 0.32
0 R = 0.66
0 20 40
Mineralizable N mg 100 g–1
Delnalyne
Fig. 4. Relationship between mineralizable nitrogen (N) and (a) total phospholipid fatty acids (PLFA) and (b) changes in the soil microbial com-
munity (PLFA fungal : bacterial ratio). Data points are from both planted birch plots and heather plots. Trend lines for each site are shown
together with R2-values.
(Fig. 4b). This pattern was also significant when the sites were
analysed separately for Delnalyne (F1,6 = 11.1, P < 0.05)
and Kerrow (F1,6 = 19.9, P < 0.01) but not for Craggan. As
increased mineralizable N had been showed to be closely corre-
lated with PLFA and is likely to be a result of changes in the
microbial community rather than a driver of changes in com-
munity composition, mineralizable N was not included as an
environmental variable in the multi-variate analysis.
Changes in fungal DGGE bands
There were significant differences in fungal composition. A
DCA of the fungal DGGE bands separated out the samples
along the first axis; birch plots with good birch growth (as
measured by the total basal area of the trees within the
plot) had low scores on the first axis and those plots with
low birch growth and the heather control plots had higher
scores on the first axis (see Fig. S1 in Supporting Informa-
tion). After the fitting of the covariables (sites), which
explained 12% of the variation, the first axis explained 15%
of the variation in the presence ⁄ absence of fungal DGGE
bands and the second axis a further 6%. The heather con-
trol plots had significantly higher scores than the planted
birch plots on the first axis (F1,15 = 37.1, P < 0.0001).
There was no difference between the treatments in the
scores on the other axes (2–4). The first axis scores did not
correlate with changes in soil total carbon or nitrogen or
organic content (LOI).
EXPERIMENT 1B: COMPARISON OF BETULA
PUBESCENS AND B. PENDULA
There was no difference in total PLFA content between the
plots with the two birch species (B. pubescens and B. pendula)
at Kerrow. When the PLFAs were classified into groups as
markers for bacteria, fungi, 16:1x5c, Gram-positive bacteria,
Gram-negative bacteria, there were no differences between the
birch species for any of the groups. The Shannon’s diversity
 2009 The Authors. Journal compilation  2009 British Ecological Society, Journal of Ecology, 98, 50–61
(b) 1.0
Fungal PLFA: Bacterial PLFA ratio
0.8
0.6
R 2 = 0.03
R 2 = 0.62
0.4
R 2 = 0.30
R 2 = 0.47
2
0.0
60 80 0 20 40 60 80
Mineralizable N mg 100 g–1
Craggan A Kerrow
index for the PLFAs was also not significantly different
between the two birch species.
EXPERIMENT 2: THE DURABILITY OF BETULA IMPACTS
Total PLFA content was lower on the felled birch plots than
the birch control plots (F1,5 = 16.5, P = 0.0098) (Fig. 1a).
Bacterial PLFA followed the same pattern (F1,5 = 9.8,
P = 0.025) (Fig. 1b) as did fungal PLFA but this was only sig-
nificant at the 10% level (F1,5 = 5.9, P = 0.059) (Fig. 1c).
The ratio of fungal : bacterial PLFA was not significantly
different between the birch control and felled birch plots.
Gram-positive bacteria content was lower in the felled birch
than the birch control (F1,5 = 6.79, P = 0.047); Gram-nega-
tive bacteria content followed the same pattern but this was
only significant at the 10% level (F1,5 = 6.3, P = 0.053). The
ratio between Gram-positive and Gram-negative bacteria was
not different between the two treatments. The PLFA 16:1x5c
content was lower in the felled birch plots than in the birch
control (F1,5 = 7.0, P = 0.045). There was no difference in
the Shannon’s diversity index for the PLFAs between the felled
birch plots and the birch control plots.
There was no significant difference in the DCA axes scores
for the birch control plots and the felled birch plots when the
PLFA data were analysed by DCA, indicating that there was
no difference in the microbial composition of these plots as
assessed by PLFA analysis. In contrast, the composition of the
fungal DGGE from the felled birch plots was significantly dif-
ferent from the birch control plots. There was a significant dif-
ference in the scores on the first axis of a DCA analysis
between the two types of plots (F1,5 = 40.4, P = 0.0014) with
the birch control plots at the positive end of the first axis and
the felled birch plots at the negative end (Fig. 5a). To test if the
fungal community was similar to that occurring under heather
moorland, a second analysis was carried out with data from
the heather control and planted birch plots from Craggan A
and data from the birch control and felled birch plots from
Craggan B. This second analysis showed the planted birch and
 Tree impacts on the soil microbial community 57

(a) 3.0 dicted in our first hypothesis. Although one cannot directly
Birch control
translate changes in the PLFA pattern into changes in species
Felled birch
composition, these results indicate that similar microbial com-
munities were induced at the three sites when birch was
planted on moorland soils. The SMC changed to a more bac-
teria-dominated one; this pattern was shown in both the
results of the ratio of fungal : bacterial PLFA and in the
RDA. This shift towards a bacteria-dominated SMC is con-
Axis 2 (14%)

sistent with the theory that birch turns mor soils into mull
soils (Miles 1981b). Mor soils are characterized by deep acidic
organic surface horizons where the SMC is dominated by
fungi; mull soils are relatively more fertile and have more bac-
teria-dominated food webs (Wardle 2005). Keith et al. (2006)
found that the relative increase in bacteria-feeding nematodes
was greater than that of fungal feeders when birch colonized
moorland and suggested that this indicated a shift from a
fungi-dominated SMC towards a bacteria-dominated SMC.
Our work here has shown this to be the case. The lower total
–0.5

biomass of the SMC under birch is consistent with a change

–0.5 Axis 1 (19%) 0.5 towards a bacteria-dominated SMC and bacteria-based
energy channels. As woodland develops on moorland there is
2.0

(b) an increase in predators, such as nematodes (Keith et al.

Heather control
2006). In bacteria-based energy channels, predation is more
Planted birch
important than resource regulation in regulating different tro-
Birch control
phic levels compared to a fungi-based energy system (Wardle
Felled birch
2005). In a bacteria-based energy system, a higher proportion
of bacterial productivity and biomass is predated than is the
Axis 2 (8%)

case for fungi in a fungal-based energy system; the net result

is a more rapid cycling of nutrients in a bacteria-based energy
system than a fungi-based energy system (Wardle et al. 2004;
Wardle 2005; Van der Heijden, Bardgett & van Straalen
2008). This leads to a lower total microbial biomass in the
soil, a faster rate of carbon turnover and higher rates of
decomposition (Rinnan et al. 2007). This concurs with Mitch-
–0.5

ell et al. (2007) who found that decomposition was greater

–0.5 Axis 1 (14%) 2.5
Fig. 5. Ordination diagrams of plots from detrended correspondence
analysis (DCA) of fungal denaturing gradient gel electrophoresis
(DGGE) data using data from (a) birch control and felled birch plots
(Craggan B) and (b) birch control, felled birch, heather control and
planted birch plots (Craggan A & B).
birch control plots towards the positive end of the first axis and
the heather control and felled birch towards the origin of
the first axis (Fig. 5b). Sample scores on the first axis were
significantly different between treatments (F3,20 = 6.1, P =
0.0039). Felled birch plots had significantly lower scores than
both the birch control plots (P = 0.01) and the planted birch
plots (P = 0.028) but were not significantly different from the
heather control plots.
under birch than heather. The diversity of PLFAs provides
an indication of the diversity of the SMC (Zelles 1999; Frost-
egård, Tunlid & Bååth 1993, Frostegård & Bååth 1996) and
Shannon’s diversity indices show that, while the total biomass
of the SMC declines when birch colonizes moorland, the
diversity of PLFAs increases.
The comparison of presence ⁄ absence of bands on the
DGGE profiles showed that the species composition of the
fungal community changed. This change was directly related
to the total basal area of the trees in the plot, a measure of birch
growth. Plots with small trees and a largely moorland-domi-
nated ground flora had a fungal community similar to the
moorland, while plots with large trees had a different fungal
community.

THE IMPACT OF DIFFERENT BIRCH SPECIES

Discussion (HYPOTHESIS 2)

Whereas these results show that birch is a major ecosystem

THE IMPACT OF BIRCH (HYPOTHESIS 1)
engineer of the SMC, different Betula species were not
Birch drove changes in both the composition of the SMC and shown to have different effects on the SMC, and we thus
the total soil microbial biomass (total PLFA content) as pre- have to reject our second hypothesis. This might have been
 2009 The Authors. Journal compilation  2009 British Ecological Society, Journal of Ecology, 98, 50–61
58 R. J. Mitchell et al.
expected as Mitchell et al. (2007) showed no difference in
plant species richness, soil properties or decomposition
between the different tree species, and other drivers of
change, such as shade, water uptake and litter input, are
unlikely to vary greatly between the two birch species. If
conditions at the site had favoured the growth of one species
or the other (Gimingham 1984), then differences between the
SMC may have been expected, but this was not the case
here.
DRIVERS OF CHANGE
This work suggests that changes in soil chemistry (Ca, pH,
total N and K) and soil physical properties (organic matter
content, depth of LFH and O horizons) caused by birch are
driving changes in the SMC. Understanding the drivers of
changes in the SMC is a contemporary question for soil ecolo-
gists (Bardgett, Yeates & Anderson 2005). Soil communities
are constantly changing, with changes ranging from seasonal
ones in abundance to those in diversity over successional time.
The degree to which soil communities’ change over time is
highly context-dependent, varying with a range of factors such
as the nature and frequency of disturbance regimes, vegetation
change, and changes in soil and climatic conditions (Bardgett,
Yeates & Anderson 2005). SMCs have been shown to respond
at different rates to the same change of management (cessation
of fertilizer application) in different grasslands (Bardgett,
Hobbs & Frostegård 1996; Smith et al. 2003) and changes in
the SMC have been demonstrated to vary depending on plant
species, plant taxonomic (or functional) groups and site-spe-
cific differences in abiotic and biotic soil properties (Bezemer
et al. 2006). Thus, trying to understand the mechanisms by
which birch is driving the changes in the SMC may not be
straightforward.
C : N ratios have often been cited as key in explaining
changes in SMC (Williamson, Wardle & Yeates 2005; Hög-
berg, Högbeg & Myrold 2007) but the C : N ratio was not sig-
nificant in explaining any of the variation in the PLFA
community in this data set; however, total N was significant in
explaining the variation in the PLFA community. Changes in
the nutrient status of the soil have also been shown to drive
changes in the SMC. High nutrient concentrations, low C : N
ratio and high quality of organic matter have been shown to
favour bacterial instead of fungal communities (Williamson,
Wardle & Yeates 2005). Birch has long been thought of as a
soil improver (Dimbleby 1952) and the planted birch plots
have been demonstrated to have lower total C and higher
available P than the heather control plots (Mitchell et al.
2007), indicating a change in nutrient concentrations within
the soil. In addition, the quality of the litter also increases as
birch colonizes moorland (Iason & Hester 1993; De Kovel
et al. 2000). Thus, changes in litter quality and nutrient content
of the soil driven by birch may be responsible for driving
changes in the SMC.
The soil PLFAs of beech-oak forests have been shown to
be related to soil pH (Bååth & Anderson 2003) and other
studies have linked changes in soil pH to changes in the
 2009 The Authors. Journal compilation  2009 British Ecological Society, Journal of Ecology, 98, 50–61
SMC (Hackl et al. 2005; Högberg, Högbeg & Myrold 2007).
In this study, the PLFAs 14:0i, 16:1x5c and 18:1x7 all
increased with increasing birch cover (Fig. 3) and have all
previously been shown to increase with increasing pH
(Bååth & Anderson 2003). While pH was not shown to sig-
nificantly increase in the planted birch plots (Mitchell et al.
2007), other work has shown that birch does increase soil
pH (Miles & Young 1980; Miles 1981a; Mitchell et al. 1997).
Small, localized and possibly insignificant changes in soil pH
may have a significant effect on PLFAs; an example from
Bååth & Anderson (2003) showed that a pH difference of
only 0.5 units would induce a change of more than 1 mol%
in some PLFAs, a difference that would usually be highly
significant.
Birch causes changes, either directly or indirectly, in the
structure and physical properties of the soil, in particular a
reduction in the organic and LFH horizons (Mitchell et al.
2007), and these variables were shown to be related to changes
in the SMC. The mechanism through which changes in these
variables drive changes in the SMC is possibly via microsite
availability. Soil is not homogenous but composed of different
types of microsites related to the structure of the soil. Changes
in the organic content of the soil, moisture and soil horizons
will result in a change in the types of microsites available within
the soil. Different types of microsites within the soil support
different species of microorganisms (Schimel, Bennett & Fierer
2005; Nielsen et al. 2008).
Birch colonization of moorland also drives changes in plant
species richness (Miles 1981a; Mitchell et al. 2007). There has
been a growing interest in determining whether plant species
richness is an ecological driver of SMC in its own right (Wardle
2005). Evidence for this is mixed with Chung et al. (2007) and
Högberg, Högbeg & Myrold (2007) showing that increasing
plant species richness increased microbial diversity while War-
dle et al. (1999, 2001) found that the alteration of plant diver-
sity did not cause predictable unidirectional changes in the
diversity of the SMC. Due to the density of birch on the
planted birch plots, plant species richness was lower in
the planted birch plots than the heather control plots (Mitchell
et al. 2007), yet the diversity of the SMC as measured by
PLFAs increased. Thus, in this study, it was not an increase in
plant diversity, which was driving the increase in diversity of
the SMC.
DURABILITY (HYPOTHESIS 3)
The SMC, as shown by the PLFA analysis, showed little
change in community composition even 20 years after the
removal of the engineering species, birch. However, the fungal
community composition (as shown by DGGE) in the felled
birch plots was different from the birch control plots and simi-
lar to nearby heather control plots. Long time-lags in the
response of SMC have been found in arctic systems (Rinnan
et al. 2007) and abandoned arable land reverting to heathland
(Van der Wal et al. 2006). The slow response in our experiment
(PLFA data) cannot be attributed to a slow response of the
plant species composition driving the change, as was the case

in the arctic (Rinnan et al. 2007). In Experiment 2, not only
was the engineering species (birch) removed but also the domi-
nant heathland species (Calluna vulgaris) planted. This speeded
up the return of the plant community to a community more
similar to heather moorland, yet the SMC was still similar to
birch woodland. Mitchell et al. (2007) showed that soil chemi-
cal properties had not changed on the felled birch plots after
20 years; we therefore propose that further changes in physical
and chemical soil properties may be a prerequisite for a change
in the SMC to occur. A similar reason was given by Van der
Wal et al. (2006) for the slow rate of increase in the fungal
community following agricultural abandonment and reversion
to heathland.
While the PLFA composition did not change, the biomass
of PLFAs did change with a lower total, bacterial and fungal
PLFA in the felled birch plots than the birch control plots. This
is contrary to what was expected from the results from the
planted birch plots. If the SMC were changing towards a com-
munity more typical of moorland, then it was expected that the
PLFA results from the felled birch plots would be higher than
the birch control plots, not lower. This may in part be
explained by the vegetation composition of the site. The birch
control plots were located in degenerating birch woodland that
contained 60- to 80-year-old trees, the trees were widely spaced
and the ground flora was dominated by grasses (Mitchell et al.
2007). It may be that the SMC was more influenced by the
grass-dominated ground vegetation than the birch trees and
the comparison was really between grassland plots and felled
birch plots (moorland). The total PLFA content was a lot
lower at Craggan B than at the other sites, particularly Crag-
gan A (c. 1000 vs. 2000 nmol g)1) and the soil at Craggan B
had a lower organic content than Craggan A; this may explain,
in part, why the patterns in PLFAs were not as predicted.
BIRCH AS AN ECOSYSTEM ENGINEER AND ITS
CASCADING EFFECTS (HYPOTHESIS 4)
Whether a species is an ecosystem engineer can be assessed
according to (i) the extent of the impact of the engineering on
the ecosystem and (ii) the durability of the engineered impacts
once the engineering species has been removed. The impacts of
an engineering species are greatest when the resources that are
modified are utilized by many other species or when the engi-
neer affects abiotic forces that then affect many other species
(Jones, Lawton & Shachak 1994). This work has provided fur-
ther evidence of the cascading effects of birch as an ecosystem
engineer altering the not only the community composition but
also the ecosystem processes as shown by the negative correla-
tion between total PLFA and fungal : bacterial PLFA with
mineralizable N. Birch causes a shift towards a more bacterial
dominated SMC, which results in faster rates of nutrient
cycling as bacteria-dominated food webs are generally associ-
ated with faster rates of nutrient cycling than fungi-dominated
food webs (Wardle et al. 2004; Van der Heijden, Bardgett &
van Straalen 2008); this in turn results in an increase in miner-
alizable N. Until recently, N mineralization was thought to be
carried out in a similar way across a wide range of organisms
 2009 The Authors. Journal compilation  2009 British Ecological Society, Journal of Ecology, 98, 50–61
Tree impacts on the soil microbial community 59
so that it was not affected by changes in community composi-
tion. However, recent work suggests that N mineralization is
governed by a number of different factors, which are more
sensitive to changes in community composition such as the
production of extracellular enzymes by specific groups of
microorganisms (Schimel & Bennett 2004) and the type of
microsites available within the soil regulating which micro-
organisms live and function within the soil (Chen & Stark
2000; Schimel & Bennett 2004). This work has only shown that
N mineralization is correlated with a change in the microbial
community. However, N mineralization is not just driven by
the SMC, but also by substrate quality and quantity both of
which have been shown to change during succession (Iason &
Hester 1993; De Kovel et al. 2000). Birch may thus be driving
changes in N mineralization in two ways; first by causing a
change in substrate quality and quantity, and secondly by
driving changes in soil moisture, bulk density, organic matter
and pH (Miles & Young 1980; Miles 1981a; Mitchell et al.
1997, 2007). Both, which in turn, lead to changes in the micro-
bial community, which then result in an increase in the rate of
N mineralization. We have therefore added to the work of
Mitchell et al. (2007) showing that changes in soil chemical
and physical properties driven by birch have cascading effects
on key below-ground communities (the microbial community)
and processes (N mineralization), which are important for a
range of other ecosystem components.
The durability of the changes in the SMC caused by birch
varied depending on what was measured. While the quantity
of PLFAs had changed, their composition had not changed
20 years after the removal of the engineering species. The fun-
gal community (as shown by DGGE) was shown to be less
durable, with significant changes occurring within the
20 years.
This work has followed the cascading effects of single species
through changes in soil nutrients to changes in the SMC and
changes in nutrient cycling. While species from late-succes-
sional forests may be expected to have a greater engineering
effect than early successional forests (Jones, Lawton & Sha-
chak 1997), we have shown a single early successional wood-
land species to have significant engineering impacts on the
microbial community.
Acknowledgements
We are grateful to the late John Miles whose long-term vision established this
experiment. William Young planted the trees and fenced the plots and Roger
Cummins maintained the plots and provided useful background information
on the experimental design. This work was initiated by a British Ecological
Early Career grant to R.J.M. (ref: 03 ⁄ 18), while C.D.C., C.M.C., S.J.C. were
funded by the Scottish Government’s Rural and Environment Research and
Analysis Directorate.
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Received 11 June 2009; accepted 14 October 2009
Handling Editor: Marcel van der Heijden
Supporting Information
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sion of this article:
Figure S1. Ordination diagram of plots from DCA analysis of fungal
DGGE from planted birch and heather plots.
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