Plant Soil (2013) 367:521534

DOI 10.1007/s11104-012-1484-0

REGULAR ARTICLE

Bracken fern (Pteridium aquilinum L. kuhn) promotes an open

nitrogen cycle in heathland soils
T. H. DeLuca & S. A. Zewdie & O. Zackrisson &
J. R. Healey & D. L. Jones

Received: 7 June 2012 / Accepted: 1 October 2012 / Published online: 14 October 2012
# Springer Science+Business Media Dordrecht 2012

Abstract
Background and Aims In spite of the broad array of
studies conducted on the ecology of bracken fern
(Pteridium aquilinum (L.) kuhn), there is currently
only a limited understanding of how P. aquilinum
alters the soil environment in which it succeeds. P.
aquilinum is one of the worlds most aggressive invasive species and is known to effectively invade conservation priority habitats such as Calluna vulgaris
(L.) heathland. The aim of this study was to evaluate
differences in soil properties between intact stands of
C. vulgaris and neighboring P. aquilinum to assess
Responsible Editor: Stefano Manzoni.
Electronic supplementary material The online version of this
article (doi:10.1007/s11104-012-1484-0) contains
supplementary material, which is available to authorized users.
T. H. DeLuca (*)
School of Environmental and Forest Sciences,
University of Washington,
107 Anderson Hall,
Seattle, WA 98195-2100, USA
e-mail: deluca@uw.edu
T. H. DeLuca : S. A. Zewdie : J. R. Healey : D. L. Jones
School of the Environment,
Natural Resources and Geography, Bangor University,
Bangor, Gwynedd LL57 2UW, UK
T. H. DeLuca : O. Zackrisson
Institute for Subarctic Landscape Research,
Silvermuseet,
Arjeplog S930 90, Sweden

how P. aquilinum alters soil N transformations in a

manner that might promote its success.
Methods Replicate plots in five independently paired
stands of P. aquilinum and C. vulgaris were established
on land in which P. aquilinum is actively invading. Soils
under the two plant types were evaluated for total N,
mineralisable N, net nitrification, nitrifier activity, denitrification enzyme activity, polyphenol N complexing
capacity, and resin sorption of inorganic N.
Results Soils under P. aquilinum were consistently
higher in NO3- and NH4+ concentrations compared to
C. vulgaris. Extractable organic and inorganic N concentrations for soil under P. aquilinum were respectively 65 %, 77 % and 358 % greater in amino N
NH4+-N and NO3--N compared to that under C. vulgaris. In-situ net nitrification (NO3- sorption to ionic
resins) was found to be nearly 300 times greater under
P. aquilinum than under C. vulgaris.
Conclusions P. aquilinum alters the soil environment
as to create an inorganic N-rich environment that is
favorable to its growth and development.
Keywords Below-ground competition . Invasive
species . Nutrient cycling . Nitrogen
transformations . Shrubland

Introduction
The influence of invasive and ruderal plant species on
nitrogen (N) availability is known to influence competitive

522

outcomes between plant communities (Rout and Callaway

2009), however, we currently posses an incomplete picture of the direct and indirect influence of plant species on
soil N cycling. Bracken fern (Pteridium aquilinum (L.)
kuhn) is a native to the British Isles, but arguably one of
the most aggressive ruderal species in the region as well as
many parts of the world (Marrs and Watt 2006). P. aquilinum invasion is important in economic terms due to the
loss of productive agricultural land and interference with
forest regeneration but also in conservation terms for the
loss of biodiversity in priority habitats (e.g. heathlands).
The role of P. aquilinum in the transformation of soil
ecosystems is well recognized, but also remains poorly
understood.
Nitrogen is commonly a limiting nutrient in most
temperate and maritime ecosystems (Vitousek and
Howarth 1991) and mineral N availability is known
to be scarce under ericaceous heaths even in locations
with relatively high rates of N deposition (Emmett and
Reynolds 1996). Interestingly, P. aquilinum response
to increased N has been found to be minimal (Gordon
et al. 1999b; Werkman and Callaghan 2002; Werkman
et al. 1996) suggesting that it can meet its N needs,
thereby reducing its response to applied inorganic N,
either through mechanisms that greatly increase availability of inorganic N in soil or by sequestering N in
rhizomes.
P. aquilinum success on Atlantic heathlands and its
ability to invade existing stands of dwarf shrub heather
(Calluna vulgaris L. Hull) is often attributed to its
ability to overtop C. vulgaris. Although P. aquilinum
has the capacity to compete well for water (Roberts et
al. 1980), C. vulgaris has proven to be a superior
competitor for water in moorland settings (Gordon et
al. 1999a; Gordon et al. 1999b) suggesting that differences in competition for other resources, and perhaps
specifically N-uptake mechanisms, may be important
below-ground drivers of P. aquilinums success in
plant community dynamics.
The interaction between P. aquilinum and heathland
dwarf shrubs is related to their large functional differences and is underscored by the difference in temporal
and spatial components of their niches. C. vulgaris is a
slow-growing evergreen shrub, a stress-tolerant competitor (Grime et al. 2007) whilst P. aquilinum is a fastgrowing, competitive deciduous herb with persistent
and copious rhizomes (Gordon et al. 1999b). Although
both species overlap in habitat, the success of one is
dependent in part on the developmental stage of the

Plant Soil (2013) 367:521534

other. A degenerate stand of C. vulgaris is thus more

susceptible to P. aquilinum invasion than a vigorous
stand at an early stage of its growth cycle (Marrs and
Pakeman 1995; Watt 1955). To date, there has been no
distinct attempt to assess niche separation based on
differences in how these species access and cycle N.
There is no single consistent picture of N dynamics
in the P. aquilinum rhizosphere. Some previous studies
suggest that P. aquilinum may stimulate N turnover
and the accumulation of mineralizable N and NH4+ in
rooting zone and deep soils compared to that under
ericaceous shrubs (Marrs et al. 1992; Mitchell et al.
1997). Soulsby and Reynolds (1994) conducted investigations on the relative contribution of deciduous
trees, woody shrubs and P. aquilinum to throughfall
nutrient deposition in North Wales. They reported
significantly higher rates of N in throughfall under P.
aquilinum than under the other two vegetation types
(Soulsby and Reynolds 1994). In contrast, other studies have found reduced inorganic N and specifically
reduced levels of NO3- in the soils invaded by P.
aquilinum (Griffiths and Filan 2007; Smart et al.
2007). A reduced presence of NO3- could be a function of rapid uptake of NO3- during the growing season; however, in NH 4 + amendment experiments,
Smart et al. (2007) found reduced nitrification rates
under P. aquilinum compared with soils under grass.
Such findings have led to the hypothesis that P. aquilinum influences community dynamics by maintaining
a low inorganic-N concentration within the rhizosphere (Griffiths and Filan 2007). The lack of consistency in the evidence of inorganic-N dynamics in the
rooting zone of P. aquilinum is likely to be due partially to differences in seasonal effects (mineralization
versus uptake) and in part to the fact that most nutrient
studies were carried out using inappropriate, incomplete, or outdated methods of assessing N turnover
(Schimel and Bennett 2004). By applying multiple
methods of assessing N turnover and methods that
integrate across seasonal influences, we hope to overcome this historical lack of consistency.
Plants directly and indirectly influence soil N cycling through their influence on the soil biotic community (Rout and Callaway 2009; Van Der Heijden et
al. 2008). However, the influence of plant invasion on
soil nutrient cycling must be viewed in the context of
the pre-invasion conditions and the nature of the species involved (Dassonville et al. 2008). Dassonville et
al. (2008) reported that nutrient poor soils were often

Plant Soil (2013) 367:521534

523

enriched following invasion whereas nutrient rich soils

experienced an opposite effect. P. aquilinum is a remarkable plant in terms of the proportion of biomass C allocated below-ground. Rhizome bud density often exceeds
500 m2 and total rhizome biomass in upper soil layers
ranges between 1 and 5 kgm2 dry weight (Marrs and
Watt 2006). The rapid development of this great mass of
rhizomes on the advancing front of P. aquilinum encroachment (Whitehead and Digby 1997) has the capacity to greatly alter the physical morphology of the soil
(Miles 1985) thereby creating mull type humus, reversal
of podzolization, and reduction of soil acidity, all of
which would greatly alter soil microbial processes.
The aeration of soils often increases N mineralization rates (Marrs et al. 1988) and disturbance induced
increases in aeration can be facilitated by anthropogenic activities such as plowing (Voroney et al. 1981),
faunal activities (Baxter and Hole 1966), or tree tipping (Schliemann and Bockheim 2011), but few
papers have documented rhizo-pedoturbation as a
mechanism for increased N availability. While roots
and rhizomes are known to enhance the aeration of
soils and to mechanically induce aeration and therefore oxidative processes in the rooting zone of wetland
soils (Armstrong et al. 1992), the role of rhizome
aeration on soil N cycling in upland soils has received
limited attention. This mechanism may be a differentiating feature between P. aquilinum and other species
in terms of N acquisition. Conversely, dwarf shrubs,
including C. vulgaris have been found to produce
phenol-rich litter that has the potential to complex
protein N, thereby reducing the formation of inorganic
N (Hofland-Zijlstra and Berendse 2010). These same
plants retain ericoid mycorrhizas which can access
organic N via hyphal networks (Nsholm and
Table 1 Summary of estimated
elevation, aspects, slope and
slope description of study sites
of P. acquilinum and C. vulgaris
communities on the uplands of
North Wales

Site
Abergwyn
Moel y Ci
Holyhead
Capelulo
Penmaenmawr

Plant Grid

Persson 2001) creating a tight internal N cycle in

which inorganic N is sparingly accumulated.
We suggest that differences in studies are partially a
result of inconsistent analyses between studies and
hypothesize that P. aquilinum stimulates the mineralization of organic N and net nitrification in contrast to
neighboring stands of C. vulgaris. The specific objectives the present study were to: (1) Determine N mineralization and nitrification dynamics in soils under P.
aquilinum and C. vulgaris using a variety of assessment approaches; (2) Evaluate possible physical and
biochemical mechanisms that drive differences in N
turnover in soils associated with the two plant species.

Methods and materials

Sites
We investigated N mineralization and nitrification dynamics on replicated paired stands of P. aquilinum and
C. vulgaris in North Wales (Table 1). Five individual
sites were identified on lands protected for high conservation value (by the National Trust and Royal Society
for the Protection Birds) where P. aquilinum and C.
vulgaris were in close proximity. It must be emphasized
that each pair of stands, although located as close as
possible together, was often separated by a small (0
30 m) difference in elevation and no assumptions can be
made about the history of vegetation cover at any of the
sites. The sampled stands were each of an area of
approximately 5050 m. Soils at each site were poorly
developed acidic brown podzolics (Dystric Eutrudepts).
Soil physical and chemical properties are presented in
Table S1. Vegetative cover was characterized by a dense
Ref

Elevation (m)

Aspect

Slope (%)

C. vulgaris

SH65514

460

Northwest

3045

P. aquilinum

SH65698

417

Northwest

3045

C. vulgaris

SH58774

316

Northwest

3045

P. aquilinum

SH58733

274

Northwest

1530
915

C. vulgaris

SH 21120

120

Southwest

P. aquilinum

SH21181

107

Southwest

915

C. vulgaris

SH74967

470

Southwest

1530

P. aquilinum

SH74938

455

Southwest

1530

C. vulgaris

SH73388

450

west

3545

P. aquilinum

SH73408

427

west

3545

524

monotypic stand of P. aquilinum or of C. vulgaris with

varied densities of grasses (Deschampsia flexuosa L.,
Festuca ovina L., and F. rubra rubra) and of gorse (Ulex
europeaus L, and U. galii L.) which was found almost
exclusively in the C. vulgaris stands.
Sample plots 11 m were established at each site on
a set of three transects run in a northsouth orientation
and separated by 10 m. Four plots (three at
Penmaenmawr and Capelulo) were located randomly
along each transect with a minimum distance between
plots of 2 m. Four separate samples of the O horizon
(depth 217 cm per stand (Table S1)) and surface mineral soil (010 cm depth immediately below the O
horizon) were collected from random positions in each
plot. The four samples were then bulked to create a
single sample for each horizon per plot giving a total
of 12 (9 at Penmaenmawr and Capelulo) samples for
each horizon per site or 24 samples per pair. Organic
horizon samples were collected with a 15 cm diameter
PVC ring, with all of the contents of the O horizon
placed into a bag to allow for estimation of bulk density.
Mineral soil samples were collected with a 2.5 cm diameter soil core where possible, and with a trowel when
soils were excessively rocky. Mineral soil bulk density
was determined by collecting three 5 cm diameter by
5 cm deep (100 cm3) cores in each vegetation type
creating six bulk density samples per site. Soil pH was
determined on all mineral soil samples by suspending
soil in 0.01 M CaCl2 in a 2:1 solution to soil ratio.
Soil samples intended for fresh soil analyses were
collected from Abergwyngregyn, Moel-y-Ci, and
Holyhead in October 2010 and in May 2011, two additional sites were sampled, Penmaenmawr and Capelulo,
to expand the breadth of the study. Soil samples were
returned to the laboratory in sealed bags, sieved to pass
4 mm to remove large stones and roots and analyzed for
moisture content, total C and N, 1.0 M KCl-extractable
N (inorganic and free amino acids), mineralizable N, net
nitrification, and nitrifier activity. In each plot in each of
the ten stands one polyester mesh ionic resin capsule
(Unibest, Walla Walla, WA, USA) was buried 10 cm
deep in the mineral horizon of both species for approximately 4 months (June to October) to monitor in-situ N
mineralization and nitrification rates.
Soil N cycling
Soil moisture content was calculated by moisture loss
after drying for 24 h at 80 C. To account for N

Plant Soil (2013) 367:521534

transformations, we performed in-vivo net nitrification and mineralization using a 28-d aerobic
incubation assay. In brief, a soil sub-sample was
immediately extracted with 1.0 M KCl and analyzed by the microplate-colorimetric technique using the salicylate-nitroprusside method for NH4+
(Mulvaney 1996) and the vanadium method for
NO3- (Miranda et al. 2001). A second soil sample
was incubated at 25 C for 28 d; followed by
extraction and analyses of NH4+ and NO3- as described
above. Net nitrification was then calculated as NO3- at
day 28 minus NO3- at time zero; net ammonification
was calculated as NH4+ at 28 d minus NH4+ at time zero,
and net mineralization was calculated as total inorganic
N (NH4+ plus NO3-) at time 28 d minus total inorganic N
at time zero. Potentially mineralizable N was determined by placing field-moist soil (5 g) and distilled
water (12.5 ml) into 50 cm3 polypropylene tubes. The
tubes headspace was then flushed with N2(g), the tubes
sealed and incubated at 30 C for 14 d (Hart et al. 1994).
A 12.5 ml aliquot of 2 M KCl was then added to each
tube (to create 25 ml of 1 M KCl) and the soils shaken
(30 min, 200 rev min1), centrifuged (18,000 g, 5 min)
and NH4+ determined as described above. Total potentially mineralizable N (PMN) was determined as the
difference between the extractable NH4+ concentration
at 0 d and 14 d. Total dissolved N (TDN) and total
dissolved carbon (TDC) in K2SO4 extracts of soil samples were analyzed using a Shimadzu TCV-TNM1 analyzer (Shimadzu Corp., Kyoto, Japan). The K2SO4
extracts were 10-fold diluted with deionized water prior
to analyses. Dissolved organic N (DON) was calculated
as TDN (NH4+ + NO3-).
Denitrification enzyme activity (DEA) was determined using a modified version of the method described by Tiedje (1994). Briefly, 5 g of soil was
weighed into 50 cm 3 polypropylene tubes, and
1.25 ml of distilled water was added along with 5 ml
of KH2PO4 buffer. The tubes were capped with a
septum, and flushed with N2(g). Using a syringe,
10 % of the headspace was removed and replaced with
acetylene and the tubes gently shaken for 4 h. Using a
jumper needle, the headspace was transferred to an
evacuated vial and analyzed for total N2O on a Varian
450 gas chromatograph (Santa Clara California, USA)
equipped with an electron capture detector. Dissolved
N2O was accounted for using Bunson adsorption coefficients (Tiedje 1994) and the values reported as mg
N2O kg soil1 d1.

Plant Soil (2013) 367:521534

Plant foliage phenolics and protein complexation

potential
Senescent leaf litter was collected in all 10 stands in
September 2011 as summer months are known to be a
period of high phenolic production in C. vulgaris
(Jalala et al. 1982). Total C and total N were determined in samples that were dried at 80 C for 48 h and
ground to pass a 100 mesh sieve (150 m) using a ball
mill. The samples were then analyzed for C and N by
dry combustion using a Leco True Spec CN analyzer
(Leco Corp., St Joseph, MI, USA). Ground litter samples were analyzed for total soluble phenolics by placing 0.5 g of dried material into 20 ml of distilled water,
shaken for 30 min, filtered through Whatman 42 filter
paper and analyzed for total soluble phenolics using
the Folin and Ciocalteus reagent (Swain and Hillis
1959). Litter from both plant communities was then
assessed for its capacity to complex proteins using the
method described by Gundale et al. (2010). Briefly,
plant tissue samples were placed in a buffered solution
and then treated with either water or a 1000 mgl1
protein (bovine serum albumin) solution. Protein concentrations in litter without protein added were then
subtracted from protein concentrations in the litter+
protein samples. Protein complexation potential was
estimated as the difference between the original solution protein concentration and that after addition to the
litter samples (Gundale et al. 2010).
As a follow up to the protein-complexationpotential assay and to more carefully evaluate the
influence of plant community type on N mineralization, an arginine mineralization assay (Kemmitt et al.
2006) was conducted using fresh replicate soil samples from the P. aquilinum and C. vulgaris stands at
Moel-y-Ci. The litter layer was moved aside and mineral soil samples (010 cm) were collected in January
2012 along the previously used transects at Moel-y-Ci
using a 2.5 cm dia soil probe, passed through a coarse
(4 mm) sieve to remove roots and stones, and stored at
5 C until initiating the assay. Soils were weighed (1 g)
into 50 cm3 centrifuge tubes and incubated with
100 1 of 14C-labeled arginine (25 mM; 1.85 kBq
ml1) for 348 h at 10 C, with 14CO2 evolution measured with 1 M NaOH traps (Kemmitt et al. 2006).
Radioactivity in the traps was determined by liquid
scintillation counting using a Wallac 1404 liquid scintillation counter and Wallac Optiphase 3 scintillation
fluid (Wallac EG& G Ltd, Milton Keynes, UK). At the

525

end of the assay, the soil was extracted with 0.1 M

NaCl, and NH4+ and NO3- concentrations measured as
described above.
Statistical analyses
Data were analyzed for differences between the two
vegetation (stand) types using a one-way analysis of
variance separately in each of the five sites using each
plot as a replicate (n0212). All data were analyzed to
determine whether they met the assumptions of parametric statistical analyses. Data not conforming to these
assumptions were transformed using a log +1 transformation (NO3- and net nitrification data). The data from
the 12 plot samples within each stand were then combined to give an average value for each stand and used to
determine the significance of differences between P.
aquilinum and C. vulgaris across all sites using a two
way ANOVA (two plants by five sites). All data were
analyzed using SPSS 16 for Windows.
For the time-dependent 14C-labelled arginine mineralization experiment, a double exponential decay model
(Boddy et al. 2007) was fitted to the experimental data




t
f a1  expk 1 a2  expk 2 t
1
where a1 describes the amount of 14C allocated to the
first mineralizable pool and k1 is the rate constant for a1.
The proportion of C partitioned to the second slower
pool a2 is described by the rate constant k2 and t is time.
Microbial C-use efficiency (CUE) of the 14C-arginine
substrate was described as follows
CUE a2 =a1 a2

Results
Soil C and N pool sizes
Total C concentration in surface mineral soils of the
two vegetation types was significantly greater under
C. vulgaris than under P. aquilinum when averaged
across the five sites (P<0.01, Table 2); however, this
trend varied among sites. This general trend was surprising given the greater depth of O horizon associated
with P. aquilinum compared to that associated with C.
vulgaris (mean 7.9 cm and 5.4 cm respectively, P<
0.01, Table 2). There was no significant difference in

526

Plant Soil (2013) 367:521534

Table 2 Total C, total N, and C:N in 010 cm surface mineral

soils under P. aquilinum and under C. vulgaris at five paired
field sites in North Wales. Mean and (in parentheses) one
standard error of the mean, and statistical test results for the
difference between P. aquilinum and C. vulgaris stands are

shown, within each site for the 12 individual plot values (9 for
Penmaenmawr and Capelulo) per stand type, and across the
sites for the 5 individual site values per stand type; *, P<0.05;
**, P<0.01

Site

Plant

Total C (gkg1)

Total N (gkg1)

C:N

Abergwyngregyn

C. vulgaris

168 (39.7)

6.4 (1.4)

26 (0.7)

Abergwyngregyn

P. aquilinum

109** (4.1)

8.0* (0.3)

14** (0.2)

Moel-y-Ci

C. vulgaris

177 (7.4)

8.0 (0.2)

22 (0.4)

Moel-y-Ci

P. aquilinum

162 (10.1)

9.5* (0.6)

17* (0.2)

Holyhead

C. vulgaris

312 (2.4)

12.5 (0.4)

25 (0.7)

Holyhead

P. aquilinum

191** (34.8)

10.6 (2.6)

19* (2.1)

Penmaenmawr

C. vulgaris

134 (12.8)

6.0 (0.7)

23 (0.5)

Penmaenmawr

P. aquilinum

168* (13.1)

7.9 (0.4)

21* (0.4)

Capelulo

C. vulgaris

306 (48.5)

10.9 (1.7)

28 (1.1)

Capelulo

P. aquilinum

123** (9.4)

6.5** (0.4)

19** (1.2)

Mean

C. vulgaris

219 (41.1)

8.7 (1.4)

25 (1.2)

Mean

P. aquilinum

151** (19.8)

8.5 (1.2)

18** (1.4)

surface mineral soil total N concentration between the

two vegetation types across the five sites. However,
the trend was highly variable amongst the sites. Total
N concentration of the O horizon was significantly
higher under P. aquilinum than under C. vulgaris in
two of the five sites (P<0.05), but was significantly
lower under P. aquilinum at a third site (P<0.01 data
not shown). The C:N ratio in surface mineral soil was
consistently lower under P. aquilinum than under C.

vulgaris (across all sites P <0.01, Table 2), which

would potentially result in greater mineralization rates
under P. aquilinum. There was no significant effect (P
>0.05) of vegetation type on mineral soil bulk density
across the sites, though it was significantly higher
under P. aquilinum than under C. vulgaris in two of
the sites (Table 3). In contrast O horizon bulk density
was significantly lower under P. aquilinum than C.
vulgaris across the sites (P<0.05) and within three of

Table 3 Bulk density of the O horizon and surface mineral soil

at five paired P. aquilinum and C. vulgaris stands in North
Wales. Mean and (in parentheses) one standard error of the
mean, and statistical test results for the difference between P.

aquilinum and C. vulgaris stands are shown, within each site for
the 12 individual plot values (9 for Penmaenmawr and Capelulo) per stand type, and across the sites for the 5 individual site
values per stand type; *, P<0.05; **, P<0.01

Site

Plant

Depth of O horizon
(cm)

Density O horizon
(gcm-3)

Density mineral soil

Abergwyn

C. vulgaris

4.33 (0.24)

0.16 (0.01)

1.22 (0.03)

Abergwyn

P. aquilinum

3.50 (0.31)

0.20 (0.01) **

1.36 (0.02) *

Moel y Ci

C. vulgaris

2.00 (0.00)

0.30 (0.02)

1.20 (0.02)

Moel y Ci

P. aquilinum

5.50 (0.50) **

0.18 (0.02) **

1.17 (0.03)

Holyhead

C. vulgaris

11.50 (0.87)

0.19 (0.02)

1.19 (0.04)

Holyhead

P. aquilinum

16.75 (2.14) **

0.16 (0.00) *

1.27 (0.03) **

Capelulo

C. vulgaris

4.89 (0.57)

0.20 (0.01)

1.32 (0.05)

Capelulo

P. aquilinum

8.67 (0.86) **

0.18 (0.00)

1.34 (0.01)

Penmaenmawr

C. vulgaris

5.22 (1.05)

0.22 (0.01)

1.33 (0.02)

Penmaenmawr

P. aquilinum

6.11 (0.59)

0.19 (0.01) *

1.33 (0.02)

Mean

C. vulgaris

5.41 (1.47)

0.21 (0.02)

1.25 (0.03)

Mean

P. aquilinum

7.87 (2.01) **

0.18 (0.01) *

1.29 (0.04)

Plant Soil (2013) 367:521534

527

the sites (although in a fourth site it was significantly

higher under P. aquilinum) (Table 3). Mineral soil pH
was slightly greater (by 0.09 units) under P. aquilinum
than C. vulgaris (P<0.05, Table 4). However, this
trend was also highly variable among sites; pH was
significantly higher under P. aquilinum at two sites
and significantly higher under C. vulgaris at two sites.

Nitrogen transformations in soils clearly differed by

vegetation type. We observed a consistent and impressive difference in net N mineralization and nitrification rates in surface mineral soils beneath neighboring
stands of P. aquilinum and C. vulgaris (Tables 4 and
5). Extractable (1 M KCl) inorganic N and amino acid
N concentrations were higher in soils under P. aquilinum than under C. vulgaris, with nearly five times
more NO3--N and twice as much NH4+-N and aminoN (all P<0.05 when averaged across sites). In all three
cases there were significant differences within at least

three of the sites all showing higher concentrations

under P. aquilinum than C. vulgaris (Table 4).
Interestingly, there was no consistent difference in potentially mineralizable N between the two vegetation
types (with just two sites showing (opposite) significant
differences) similar to the findings for total N.
Total NO3--N adsorbed to ionic resins over a 4month period was also found to be higher under P.
aquilinum than under C. vulgaris across all five sites
and within four of the sites (all P<0.01, Fig. 1a).
Average values were 475 g NO3--N capsule1 for P.
aquilinum compared with 17.6 g NO3--N capsule-1
for C. vulgaris. In contrast ammonification, as measured by NH4+ sorption to ionic resins, was not significantly different between P. aquilinum and C.
vulgaris when averaged across all five sites; however,
it was significantly higher under P. aquilinum than
under C. vulgaris at three of the sites (all P<0.01,
Fig. 1b). The magnitude of difference between the
two vegetation types averaged across all five sites
was more than two and a half times greater for NO3-

Table 4 Extractable (1 M KCl) NO3-, NH4+, amino acid-N,

potentially mineralizable N (PMN), and pH in surface soils (0
10 cm) collected under established stands of P. aquilinum and C.
vulgaris at five sites in north Wales. Mean and (in parentheses)
one standard error of the mean, and statistical test results for the

difference between P. aquilinum and C. vulgaris stands are

shown, within each site for the 12 (9 for Penmaenmawr and
Capelulo) individual plot values per stand type, and across the
sites for the 5 individual site values per stand type; *, P<0.05;
**, P<0.01

Nitrogen mineralization

NO3--N
Site/Plant

NH4+-N

Amino acid- N

PMN

pH

(mgkg1)

Abergwyngregyn
C. vulgaris

1.25 (0.41)

0.73 (1.38)

1.79 (0.27)

9.55 (1.62)

3.86 (0.08)

P. aquilinum

7.38** (3.27)

6.67** (2.44)

6.99** (1.39)

17.43**(3.95)

4.42** (0.03)

C. vulgaris

1.33 (0.36)

1.67 (0.95)

3.49 (0.57)

13.18 (0.78)

4.16 (0.02)

P. aquilinum

8.90** (1.94)

12.32**(3.35)

6.12** (0.96)

6.78** (3.09)

3.96* (0.02)

C. vulgaris

2.13 (1.38)

10.41 (9.52)

2.42 (1.13)

22.36 (6.43)

4.30 (0.02)

P. aquilinum

3.45 (1.79)

10.39 (5.91)

6.41* (2.31)

15.20 (3.90)

4.09* (0.04)

C. vulgaris

0.41 (0.60)

0.00 (0.00)

2.28 (1.17)

16.91 (16.27)

3.95 (0.08)

P. aquilinum

2.18 (4.17)

5.80** (4.29)

5.25* (1.11)

24.26 (28.56)

4.03 (0.03)

Moel-y-Ci

Holyhead

Capelulo

Penmaenmawr
C. vulgaris

0.65 (0.64)

18.43 (16.06)

7.67 (5.42)

22.62 (19.08)

3.88 (0.06)

P. aquilinum

4.64** (0.86)

20.15 (13.48)

4.38 (3.39)

43.33 (31.80)

4.20** (0.04)

Mean
C. vulgaris

1.16 (0.97)

6.25 (3.13)

3.53 (0.94)

16.92 (3.76)

4.03 (0.11)

P. aquilinum

5.31* (4.30)

11.07* (1.98)

5.83* (0.45)

21.40 (6.52)

4.14* (0.09)

528

Plant Soil (2013) 367:521534

Table 5 Analysis of variance F and P values for resin sorbed

inorganic N, net nitrification, and denitrification enzyme activity
(DEA) in mineral soils and for concentrations of total soluble
Variable

Resin NO3--N
F

phenols, total C, total N and C of senescent foliage associated

with P. aquilinum and C. vulgaris at five sites in North Wales

Resin NH4+-N
P

Net Nitrification
P

DEA
P

Plant

44.33

0.00

13.95

0.00

140.30

0.00

5.12

0.03

Site

2.80

0.03

28.94

0.00

23.78

0.00

6.86

0.00

SitePlant

3.77

0.01

6.96

0.00

24.00

0.00

8.19

0.00

Variable

Foliage phenols
F

Foliage C
P

Foliage N

Foliage C:N

Plant

15.24

0.00

85.27

0.00

3.31

0.08

2.38

0.13

Site

2.30

0.08

3.80

0.01

2.43

0.07

2.79

0.04

SitePlant

2.87

0.04

2.39

0.07

1.91

0.13

4.41

0.01

than NH4+ sorption to resins (320 versus 120 g capsule-1). Net nitrification as measured by 28-d aerobic
incubation was significantly higher under P. aquilinum
than C. vulgaris both across all five sites and within
each of the sites (all P<0.01, Fig. 2). Nitrifier activity
based on an aerated slurry method was low in all soils
and did not vary significantly between the two vegetation types across all five sites (0.0013, 0.0068
mean +/ 0.01, 0.007 SE for P. aquilinum and C. vulgaris respectively), and was only significantly (P<0.05)
different [P. aquilinum (0.12 mean +/ 0.009SE)>C.
vulgaris (0.0019 mean +/0.0022SE)] within one site,
Moel y Ci.
Surprisingly, DEA was significantly higher (P<
0.05, Table 5) under C. vulgaris (69 g N2O g-1 mean
+/ 47 SE) than under P. Aquilinum (26 g N2O g1
mean +/14 SE) despite the large differences in extractable soil NO 3- between the vegetation types
(Table 4).

In soil from under P. aquilinum or C. vulgaris arginine

mineralization followed a bi-phasic pattern of 14CO2
evolution (Fig. 4a) to which a double exponential
decay model conformed well (r2 00.9970.001 and
r2 00.9930.004 respectively). Although there was a
visible difference in cumulative arginine mineralization between the P. aquilinum and C. vulgaris soils,
the differences in their modeled parameter values
(Table 6), k1, k2, a1, a2 or CUE which described
14
CO2 evolution and microbial 14C partitioning were
not significant (P>0.05, n05) . Further, there was no
significant difference in net NH4+-N production during the 16-d incubation period; however, P. Aquilinum
soils exhibited rapid conversion of the mineralized
arginine to NO3-, while there was no measurable nitrification in the C. vulgaris soils leading to a highly
significant difference (P<0.01, Fig. 4b).

Foliage phenols and protein-polyphenol complexation

potential

Discussion

Senescent C. vulgaris foliage was found to have significantly higher total C concentration than P. aquilinum (52 and 48 % C respectively, P<0.001), but there
were no significant differences in foliage N concentration or C:N ratio (Table 5). P. aquilinum tissue
displayed significantly greater protein complexation
capacity than C. vulgaris across all five sites (P<
0.001, Fig. 3). Total soluble phenol concentration in
senescent leaf tissue was to be significantly greater (P
<0.05) in senescent P. aquilinum than C. vulgaris
foliage (Fig. 3 and Table 4).

Arginine mineralization

Effect of P. aquilinum on N cycling

Our findings indicate that P. aquilinum greatly alters
soil N cycling, creating an open N cycle wherein
soils exhibit high rates of net nitrification and the
accumulation of NO3- (Schimel and Bennett 2004).
Physical disturbance of soils as a result of tillage
(DeLuca and Keeney 1995; Voroney et al. 1981),
freeze-thaw events (DeLuca et al. 1992), or biopedoturbation (Baxter and Hole 1966; Schliemann and
Bockheim 2011) commonly exhibit similar conditions

Plant Soil (2013) 367:521534

Resin adsorbed NO3- (g NO3- -N capsule-1)

529

1000

P. aquilinum
C. vulgaris

800

600

400

**
**

200

**

**
**

er
Ab

Ho

ead
lyh

Ci
wr
ulo
ma
pal
el y
Ca
aen
Mo
nm
Pe

Me

an

Site

Resin sorbed NH4+ ( g NH4+ - N capsule-1)

1000
P. aquilinum
C. vulgaris

800

600

400

**
200

**
**

er
Ab

d
hea
oly

i
yC

wr
ulo
ma
pal
el
Ca
aen
Mo
m
n
Pe

an
Me

al. 2009) and exhibits extremely low rates of net

nitrification (Emmett et al. 2004).
The greater concentration of inorganic N and amino
acid N that we observed in soils under P. aquilinum
compared with C. vulgaris stands is potentially a result
of disturbance and aeration by rhizomes in P. aquilinum soils. These findings stand in contrast to previous
studies that demonstrated lower inorganic N concentrations under P. aquilinum compared with both
grasses and coniferous trees (Griffiths and Filan
2007; Smart et al. 2007). This contradiction in results
may partly be explained by confounding effects of
selective grazing in the Smart et al. (2007) study
(sheep avoiding P. aquilinum and spending most of
their time on grass plots). Furthermore, the extremely
wet conditions in their study would have resulted in net
NO3- leaching or denitrification of excess NO3-. Soil
moisture conditions are directly influenced by vegetation which in turn influences N turnover and the competition between species (Everard et al. 2010). The
moisture conditions associated with C. vulgaris were
generally similar or slightly drier than that under P.
aquilinum (data not shown); however, we did not monitor soil moisture potential differences between the different vegetation types. P. aquilinum has been found to
compete effectively for N in forest ecosystems and
exhibit high rates of N uptake compared to other species
with which it coexists when provided with an enriched,

Site

wherein net ammonification and nitrification occur.

This corresponds with the finding of Mitchell et al.
(1997) that concentrations of soil inorganic N increased with P. aquilinum succession. These results
also provide a mechanism to explain the results of
forest studies that demonstrate uniquely high concentrations of N in P. aquilinum tissue (Stams and
Schipholt 1990a) and high rates of NO3- and NH4+
in canopy throughfall associated with P. aquilinum
(Soulsby and Reynolds 1994). Our findings are in
keeping with earlier evidence that C. vulgaris retains
low concentrations of available N in its rooting zone
compared with grasses such as D. flexuosa (Nielsen et

60

50

Net nitrification (g g-1)

Fig. 1 Amounts of (a) soil NO3- and (b) NH4+ adsorbed to

ionic resin capsules buried beneath paired stands of P. aquilinum
and C. vulgaris for a 4 month period (June October) at five
locations in north Wales. Bars represent meansSEM (n09 for
individual sites and n05 for average across the five sites).
Significance indicated by **0P<0.01, *0P<0.05

P. aquilinum
C. vulgaris

40

30

20

**
**
**

10

**
**

**

0
er
Ab

y
el
Mo

Ci

r
d
aw
ea
lyh
nm
ae
Ho
m
n
Pe

lulo
pe
Ca

an
Me

Sites

Fig. 2 Net nitrification as measured using a 28-d aerobic incubation of soils collected under paired stands of P. aquilinum and
C. vulgaris at five locations in North Wales. Bars represent
meansSEM (n09 for individual sites and n05 for average
across the five sites). Significance indicated by **0P<0.01,
*0P<0.05

530

increase with increasing N availability (Devevre and

Horwath 2000). However, in this assay we found that
P. aquilinum soils rapidly converted NH4+ to NO3while there was no measurable nitrification in the C.
vulgaris soil despite it being greatly NH4+-enriched by
the arginine mineralization. This, along with our field
assay which also showed virtually no net nitrification
in soil under C. vulgaris stands, provide clear and
compelling evidence that soils under P. aquilinum
experience far more rapid nitrification of NH4+ than
soils under C. vulgaris.

3000

-1

P. aquilinum
C. vulgaris
2500

2000

**
**

**

**

1500

**
**

1000

500

Ab

er

el y
Mo

Ci
Ho

wr
ulo
ead
ma
pal
lyh
Ca
aen
m
n
Pe

Me

an

Impact of P. aquilinum on soil bulk density

Site

P. aquilinum
C. vulgaris

40

Soluble phenols (mg g-1)

P. aquilinum seems to possess a unique ability to

access N and therefore has not consistently responded
to N addition (Gordon et al. 1999b; Werkman and
Callaghan 2002). The great density of rhizomes of P.

50

30

**
**

20

er
Ab

el y
Mo

Ci

wr
ead
ma
lyh
aen
Ho
nm
e
P

lo
palu
Ca

an
Me

Sites

Fig. 3 Protein complexation potential (a) and total soluble

phenols (b) in senescent foliage of P. aquilinum and C. vulgaris
collected during early autumn 2011 at five sites in North Wales.
Bars represent meansSEM (n04 for individual sites and n05
for the average across the five sites). Significance indicated by
**0P<0.01, *0P<0.05

labile form of N (Stams and Schipholt 1990a) and

specifically increased NO3- concentration in P. aquilinum tissue, far in excess of that found in other plant
species within the same ecosystem .
In the arginine mineralization assay, we found no
significant difference in amino acid-C mineralization
(although there is a clear separation of rates) or microbial C partitioning between soils under P. aquilinum
and C. vulgaris providing no strong evidence of variation between the microbial communities in the soils
of the two vegetation types in terms of their efficiency
at utilizing this labile amino acid as a C resource. The
lack of difference in CUE is surprising given the large
differences in N availability and that CUE tends to

70
P. aquilinum
C. vulgaris

60

50

40

30

20

10

0
0

10

12

14

16

Time (days)

b
Concentration of NH4+-N or NO3- -N (g g-1)

14

14

10

CO2 evolution (% of total C-arginine added)

Protein complexation potential (g protein g )

Plant Soil (2013) 367:521534

140

120

P. aquilinum
C. vulgaris

100

80

60

40

20

**

0
Ammonium

Nitrate

Fig. 4 Nitrogen mineralization as indicated by (a) arginine

mineralization rate and (b) net ammonification and nitrification
during a 16-d period in soil collected from Moel-y-Ci in North
Wales under P. aquilinum and under C. vulgaris. Bars represent
meansSEM (n03). Significance indicated by **0P<0.01

Plant Soil (2013) 367:521534

531

Table 6 Model parameters describing the mineralization of

14
C-labelled arginine in soil collected from stands or either P.
aquilinum or C. vulgaris. Values represent meansSEM (n03).
P values describe the t-test for differences between P. aquilinum
Model parameter
a1 (% of total

14

53.7

14

47.1

C)

-1

k1 (d )
a2 (% of total

P. aquilinum

0.83
C)

and C. vulgaris. The model parameters are described in full in

the Materials and Methods section. r2 values describe the
goodness-of-fit of the model to the experimental data
C. vulgaris

2.8
0.09
2.4

42.6
0.79
57.2

P value
5.9

0.16

0.14

0.84

5.6

0.17

k2 (d-1)

0.013

0.001

0.011

0.001

0.26

CUE

0.47

0.03

0.57

0.06

0.16

r2

0.997

0.001

0.993

0.004

0.37

aquilinum (Marrs and Watt 2006) and their rapid development along the advancing front of P. aquilinum
encroachment (Whitehead and Digby 1997) may result in a virtual tilling of surface soils resulting in
increased N mineralization in soils occupied by P.
aquilinum. The depth of P. aquilinum rhizomes is
known to vary depending on type (long- versus
short-shoots, age of clonal fragment and disturbance),
but often results in rhizomatous soil disturbance to
depths of over a meter (Marrs and Watt 2006). In
contrast, heather species such as C. vulgaris produce
long-lasting suberized roots that turnover much more
slowly and are likely to have a lesser physical impact
on soil on an annual basis. Given the high total biomass associated with P. aquilinum stands in contrast to
heathland and that rhizomes have the capacity to
greatly increase N uptake in clonal species (Brooker
et al. 1999), the much higher concentrations of inorganic N found in P. aquilinum than C. vulgaris soils is
a striking result. It must be assumed that the potentially higher rate of N uptake by P. aquilinum is further
exceeded by its effect on soil N cycling, and in particular N mineralization rate, associated with soil
changes caused by the extensive horizontal and vertical growth of P. aquilinum rhizomes. It was also
notable that O horizon depth was significantly greater
and O horizon bulk density was significantly lower
under P. aquilinum compared to C. vulgaris stands
across the five sites. This is likely to be caused by a
combination of the physical impact of rhizome colonization of surface organic horizons, the rate of biomass turnover of P. aquilinum rhizomes and roots, the
quantity of frond litter produced annually by this deciduous fern, and the slow rate of decomposition of
this litter (Marrs and Watt 2006).

Foliage phenols and protein-polyphenol complexation

We hypothesized that the tannin-rich litter of C. vulgaris
(Jalala et al. 1982) could potentially result in N sequestration in complex organic forms (Httenschwiler and
Vitousek 2000) which might explain the low concentrations of inorganic N in surface soils under C. vulgaris
as observed here and in previous studies (Nielsen et al.
2009). Additionally, C. vulgaris exhibits the potential to
directly take up organic-N (Sokolovski et al. 2002),
perhaps preferentially taking up amino acids over inorganic N. Surprisingly, we observed much higher protein
complexation potential in senescent P. aquilinum foliage
than in that of C. vulgaris. To our knowledge there is no
evidence available to suggest that P. aquilinum takes up
significant quantities of N in an organic form. However,
the high inorganic N concentrations in the surface soil
under P. aquilinum (relative to C. vulgaris) could also
indicate a low rate of take-up of inorganic N by P.
aquilinum, but this notion is not supported by previous
findings that demonstrate high rates of N uptake in P.
aquilinum (Stams and Schipholt 1990a). The uptake of
NO3- by plants requires little energy, but the subsequent
reduction of NO3- to NH4+ is an energy intensive process which can be directly linked to photosystems in
plant leaves (Oaks 1994). Although there is this energetic cost associated with NO3- utilization, for plants
with a high photosynthetic capacity in particular, its use
(in contrast to uptake being restricted to other chemical
forms of N) can be explained by its potentially greater
availability at the root surface due to the rapid diffusion
rate of NO3- in soils compared with NH4+ or amino
acids (Jones et al. 2005).
It is known that, in contrast to neighboring plant
species, P. aquilinum concentrates NO3- in its fronds

532

(Fenn et al. 1996; Stams and Schipholt 1990b), and

this does provide evidence that P. aquilinum takes up
NO3- at a higher rate, which may reflect a high capacity for P. aquilinum to compete for this N resource in
the soil environment. Specifically, stimulation of nitrification by P. aquilinum that is invading an established
C. vulgaris heath, which had low concentrations of
available soil N combined with a likely preference for
organic-N and NH4+ uptake in C. vulgaris, would
ultimately benefit the invading P. aquilinum. The high
rate of NO3- uptake in P. aquilinum is partially due to its
rapid growth rate and high rate of chloroplast production, and perhaps due to its high rate of production of Nrich cyanogen glycosides, specifically prunasin an antiherbivory compound present in P. aquilinum fronds
(Vetter 2009). Increasing concentration of soil N has
been found to relate directly to increased prunasin production in a south Australian eucalypt, Eucalyptus cladocalyx (Simon et al. 2010). The concentration of NO3in P. aquilinum frond tissue may therefore be an important mechanism in its production of anti-herbivory compounds (Jones et al. 2011).
The total C concentration in senescent foliage was
slightly higher in C. vulgaris (520 g C kg-1 +/ 18) than
P. aquilinum (479 g C kg-1 +/ 14), but interestingly
there were not significant differences in total N concentration or C:N ratio. The high C content of the C. vulgaris
senescent foliage is likely to be related to its greater
lignin content than in P. aquilinum, which is likely to
results in rapid N immobilization upon decomposition in
the soil environment. However, as noted above, protein
complexation capacity was clearly greater for senescent
P. aquilinum foliage than for C. vulgaris foliage. This is
contrary to our hypothesis that C. vulgaris stands conserve N in an organic form to a greater extent than P.
aquilinum stands through polyphenol-protein complexation. The high C:N ratio in mineral soils associated with
C. vulgaris suggests that N immobilization is perhaps a
more likely mechanism limiting N availability under C.
vulgaris than polyphenolic N complexation.

Plant Soil (2013) 367:521534

nitrification assay were much greater under P. aquilinum than under C. vulgaris. Enrichment of extractable
amino N, and to a greater extent NH4+ and to yet a
greater extent NO3- under P. aquilinum reflect greater
rates of N accumulation at all stages of N mineralization but especially net nitrification (the percent greater
N pool enrichment under P. aquilinum compared to C.
vulgaris for PMN is 26 %, for amino N is 65 %, for
NH4+ is 77 % and for NO3- is 358 %).
These results underscore the role that competitive
or invasive species may play in perturbing ecosystem
N cycling (Rout and Callaway 2009). While this phenomenon has been well documented in a number of
cases of N-fixing invasive species (e.g. Myrica faya in
Hawaii (Vitousek and Walker 1989), U. europaeus in
New Zealand (Magesan et al. 2012), Sorghum halpense in North America (Rout and Chrzanowski
2009), the present study is one of the clearest example
that we are aware of for a non-N-fixing species.
Increased N availability in a P. aquilinum stand may
reduce the potential for C. vulgaris re-establishment in
the short term, but may also result in improved fertility
conditions for the establishment of desirable or domestic species. Further, acknowledgement of the influence of P. aquilinum on soil nutrient status may
influence the way we approach P. aquilinum management. Rather than attempting to eradicate P. aquilinum, achieving the level of control sufficient for
establishment of species planted or naturally regenerating in P. aquilinum stands may enable them to utilize the
improved fertility conditions in these sites. Provided that
these species can be successful in subsequent competition with P. aquilinum, e.g. if they are woody plants that
can shade the P. aquilinum to reduce its vigor (Marrs
and Watt 2006), this may provide a cost-effective solution for site management.
Acknowledgements The authors wish to thank Sanatan Das
Gupta, Kathi Ackermann, Rose Rusbridge, Emil DeLuca, Henry
DeLuca, Sarah Chesworth, Sophie Ackermann, and Prabi Basnet
for their assistance with field and laboratory work.

Conclusions
Nitrogen mineralization and nitrification are clearly
enhanced in soils associated with P. aquilinum compared with that under neighboring C. vulgaris. Levels
of net nitrification as measured by absorption to buried
ionic resins or by use of an aerobic 30-d net

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