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Oecologia
ISSN 0029-8549
Volume 167
Number 2
Oecologia (2011) 167:355-368
DOI 10.1007/s00442-011-1998-9
Effects of nutrient addition on
leaf chemistry, morphology, and
photosynthetic capacity of three bog
shrubs
Jill L. Bubier, Rose Smith, Sari Juutinen,
Tim R. Moore, Rakesh Minocha,
Stephanie Long & Subhash Minocha
1 23
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Oecologia (2011) 167:355–368
DOI 10.1007/s00442-011-1998-9
123
PHYSIOLOGICAL ECOLOGY - ORIGINAL PAPER
EVects of nutrient addition on leaf chemistry, morphology,
and photosynthetic capacity of three bog shrubs
Jill L. Bubier · Rose Smith · Sari Juutinen ·
Tim R. Moore · Rakesh Minocha · Stephanie Long ·
Subhash Minocha
Received: 7 June 2010 / Accepted: 5 April 2011 / Published online: 5 May 2011
© Springer-Verlag 2011
Abstract Plants in nutrient-poor environments typically
have low foliar nitrogen (N) concentrations, long-lived
tissues with leaf traits designed to use nutrients eYciently,
and low rates of photosynthesis. We postulated that
increasing N availability due to atmospheric deposition
would increase photosynthetic capacity, foliar N, and spe-
ciWc leaf area (SLA) of bog shrubs. We measured photo-
synthesis, foliar chemistry and leaf morphology in three
ericaceous shrubs (Vaccinium myrtilloides, Ledum groen-
landicum and Chamaedaphne calyculata) in a long-term
fertilization experiment at Mer Bleue bog, Ontario, Can-
ada, with a background deposition of 0.8 g N m¡2a¡1.
While biomass and chlorophyll concentrations increased
in the highest nutrient treatment for C. calyculata, we
found no change in the rates of light-saturated photosyn-
thesis (Amax), carboxylation (Vcmax), or SLA with nutrient
(N with and without PK) addition, with the exception of a
weak positive correlation between foliar N and Amax for
C. calyculata, and higher Vcmax in L. groenlandicum with
low nutrient addition. We found negative correlations
between photosynthetic N use eYciency (PNUE) and
foliar N, accompanied by a species-speciWc increase in
one or more amino acids, which may be a sign of excess N
availability and/or a mechanism to reduce ammonium
(NH4) toxicity. We also observed a decrease in foliar sol-
uble Ca and Mg concentrations, essential minerals for
plant growth, but no change in polyamines, indicators of
physiological stress under conditions of high N accumula-
tion. These results suggest that plants adapted to low-nutri-
ent environments do not shift their resource allocation to
photosynthetic processes, even after reaching N suY-
ciency, but instead store the excess N in organic com-
pounds for future use. In the long term, bog species may
not be able to take advantage of elevated nutrients, result-
ing in them being replaced by species that are better
adapted to a higher nutrient environment.
Keywords N deposition · Nutrient use eYciency ·
Amino acids · Ammonium toxicity · Peatland ·
Polyamines
Communicated by Robert Pearcy.
Electronic supplementary material The online version of this
article (doi:10.1007/s00442-011-1998-9) contains supplementary
material, which is available to authorized users.
J. L. Bubier (&) · R. Smith · S. Juutinen
Environmental Studies Program, Mount Holyoke College,
50 College Street, South Hadley, MA 01075, USA
e-mail: jbubier@mtholyoke.edu
T. R. Moore
Department of Geography, Global Environmental & Climate
Change Centre, McGill University,
805 Sherbrooke St. W, Montreal, QC H3A 2K6, Canada
R. Minocha · S. Long
US Department of Agriculture, Forest Service,
Northern Research Station, 271 Mast Road,
Durham, NH 03824, USA
S. Minocha
Department of Biological Sciences,
University of New Hampshire, Durham, NH 03824, USA
Present Address:
R. Smith
Ecosystems Center, 7 MBL St, Woods Hole, MA 02543, USA
Present Address:
S. Juutinen
Department of Forest Sciences, University of Helsinki,
P.O. Box 27, 00014 University of Helsinki, Finland
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356 Oecologia (2011) 167:355–368
123
Introduction
The anthropogenic release of nitrogen (N) into the atmo-
sphere has accelerated N cycling globally (Galloway et al.
2004). Atmospheric N deposition has the potential to
enhance plant productivity in N-limited ecosystems (Zaehle
et al. 2010 and references therein). However, the rate and
duration of N deposition impact cellular and soil N status,
and deposition rates can exceed the capacity for N uptake by
plants (Aber et al. 2003). The acidifying eVect of precipi-
tated NO3, and the consequent leaching of vital cations such
as calcium (Ca) and magnesium (Mg), can lead to nutrient
imbalances and ecosystem decline (Ågren and Bosatta 1988;
Aber et al. 1998, 2003; Fenn et al. 1998; Bauer et al. 2001).
The response to additional N supply depends largely on the
physiological adaptations of individual plant species. In peat-
lands, particularly nutrient-poor bogs, atmospheric N deposi-
tion changes plant community composition, enhancing
vascular plant growth to the detriment of the moss layer
(Bubier et al. 2007). However, mixed results have been
reported with respect to changes in whole ecosystem produc-
tivity, arising from diVerences in species composition, physi-
ology and resource utilization (Heijmans et al. 2001;
Tomassen et al. 2003; Bragazza et al. 2004; Limpens et al.
2008; Juutinen et al. 2010). Will atmospheric N deposition
alleviate the nutrient stress of bog plants and allow species to
become more productive, thus sequestering more carbon
(C)? Are these species able to shift their allocation strategies
and life history traits? Will these species be able to produce
more leaves or increase rates of photosynthesis and shift to
lower nutrient conservation?
Plants in nutrient-poor environments such as bogs and
arctic tundra are adapted in several ways to the slow turn-
over of N, phosphorus (P) and potassium (K). For example,
ericaceous evergreen shrubs make long-lived tissues,
including woody stems and roots, as well as leaves that live
for 1–4 years (Eckstein et al. 1999; Burns 2004; Wright
et al. 2004). Moreover, producing thick, waxy leaves is a
strategy to conserve nutrients, prevent frost damage, and
reduce water loss with high re-absorption from senescing
leaves (Aerts 1995; Burns 2004; Wright et al. 2004). How-
ever, nutrient use eYciency requires important trade-oVs
with productivity, resulting in slow growth rates and lower
foliar N concentrations, along with lower rates of maximum
photosynthesis (Berendse and Aerts 1987; Oberbauer and
Oechel 1989; Reich et al. 1998; Shipley et al. 2006). Theo-
retically, the light-saturated rate of photosynthesis (Amax)
increases with increasing foliar N allocation to proteins,
particularly to ribulose-1,5-bisphosphate carboxylase
(Rubisco), which determines the rate of carboxylation
(Vcmax) (Bowes 1991). Nutrient addition can also lead to
enhanced growth, producing more leaves, and competition
for light can result in increased leaf area per leaf mass (spe-
ciWc leaf area, SLA) (Shaver et al. 2000; Niinemets and
Kull 2003; Burns 2004).
However, there is evidence that plants can partition
excess N such that photosynthesis is downregulated rather
than enhanced after the overall nutrient balance reaches a
critical threshold (Bauer et al. 2004). For example, in peat-
lands, Bragazza et al. (2004) and Granath et al. (2009a)
found that 1–1.5 g N m¡2a¡1 was the optimal level for
Sphagnum moss photosynthesis, with lower growth rates at
higher N deposition levels. An overload of N, particularly
ammonium (NH4), can be toxic to plant cells, and the pres-
ence of amino acids and polyamines (PAs) in high concen-
trations could explain the strategy by which plants cope with
excess N if it is not invested into the primary processes of
CO2 assimilation. While several studies have examined the
role of these N-rich organic compounds in forest plant com-
munities, few have examined their role in peatland plants,
and most of those studies have focused on Sphagnum mosses
rather than vascular plants (e.g., Limpens and Berendse
2003; Tomassen et al. 2003; Wiedermann et al. 2009).
In the Mer Bleue bog fertilization study, we have found
increases in dwarf shrub growth and a loss of moss biomass,
but either no change or decreases in ecosystem photosynthesis
rates in response to 9 years of nutrient (N and NPK) addition
(Juutinen et al. 2010). The shift in community composition
along with changes in plant physiology could explain the eco-
system response. The goals of the current study were to exam-
ine the physiological responses of the dominant bog shrubs at
Mer Bleue to increased nutrient availability. Our research
questions were as follows. (1) How does nutrient addition
aVect the leaf chemistry and morphology, stress-related
metabolism, photosynthetic capacity, and photosynthetic N
use eYciency (PNUE) of the ericaceous shrubs Cham-
aedaphne calyculata Moench, Ledum groenlandicum Oeder,
and Vaccinium myrtilloides Michx? (2) How do leaf traits
relate to abundance of these species over the duration of the
experiment? We hypothesized that nutrient (N and NPK) addi-
tion would increase SLA, foliar N, chlorophyll, photosynthetic
capacity and PNUE. To address these issues, we studied shrub
abundance, leaf dimensions, foliar concentrations of nutrients,
soluble ions, free amino acids, free polyamines (PAs), soluble
proteins, chlorophyll, and photosynthetic parameters [light sat-
urated net photosynthesis (Amax), maximum carboxylation
capacity (Vcmax), electron transport (Jmax), and triose phosphate
utilization (TPU)], under diVerent nutrient treatments.
Materials and methods
Site description and experimental design
This study was conducted at Mer Bleue bog near Ottawa,
Ontario, Canada (45°40⬘N, 75°50⬘W), which has a cool
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Oecologia (2011) 167:355–368 357
123
continental climate and a mean annual temperature of
6.0°C (monthly range ¡11 to 21°C), as well as a mean
annual precipitation of 944 mm (Canadian Climate Nor-
mals 1971–2000). The experimental site is located in the
ombrotrophic part of the peatland, where vegetation is
dominated by dwarf ericaceous shrubs along with a ground
layer of the mosses Sphagnum magellanicum Brid., Sphag-
num capillifolium (Ehrl.) Hedw., and Polytricum strictum
Brid. Background inorganic wet N deposition in this region
is »0.8 g N m¡2a¡1 (Turunen et al. 2004). We chose N
amendments of 1.6 and 6.4 g m¡2a¡1 because they represent
the range of probable increases in N deposition in peatland
regions of North America and Europe in the twenty-Wrst
century (e.g., Reay et al. 2008). Thus, we experimentally
increased the ambient growing season wet N deposition by
factors of 5 and 20.
We began treatments (control, 5N, 5NPK and 20NPK) in
2000–2001; an additional treatment (20N) was initiated in
2005 (Table 1). Each treatment had three replicate plots
3£3 m in size. Fertilizer was given in soluble form, N as
NH4NO3 and PK as KH2PO4, dissolved in 18 L distilled
water (equivalent to the application of 2 mm of water), at
3 week intervals from early May to late August. Control
plots were treated with distilled water.
Aboveground growth and leaf morphology
The response of whole plant growth to fertilization was
examined by measuring the abundance of vascular plant
species in a 60 £60 cm quadrat in each treatment plot at
the beginning of the experiment in 2000 and again in 2008.
Stem number and stem height of each species were
recorded in 2000. The number of hits to a metal rod (radius
4 mm) in 61 grid points of a 60 £60 cm frame was
recorded in 2008. Owing to the diVerent methods of esti-
mating abundance, diVerences between control and nutrient
treatments were examined for each year separately.
In 2008, we measured the length, width, and thickness of
8–9 leaves per treatment for each of the three shrub species
from diVerent evenly spaced plants in each plot. These
leaves were Wrst measured for CO2 exchange. We only used
leaves from the top canopy, and performed the
measurements within 3 weeks during the growing season
from mid-July to mid-August. Leaves were frozen after
removal from the Weld, weighed, then oven-dried for 48 h at
50°C and re-weighed. We determined leaf moisture content
for each species and treatment and calculated speciWc leaf
area (SLA, cm2g¡1leaf). We compared these leaf measure-
ments with a larger set of randomly selected leaves and
found that treatment had a similar eVect on leaf area and
mass in both datasets.
CO2 exchange measurements and parameter estimation
We measured the CO2 exchange of intact leaves (same
leaves were measured for morphology) using a portable
photosynthesis system LI-6400 (Li-Cor, Lincoln, NE,
USA), including an infrared gas analyzer and a leaf cuvette
equipped with temperature, light, CO2 and humidity con-
trols. The response of net photosynthesis (A) to intercellular
CO2 concentration (Ci) was measured in 13 set points of
external CO2 concentration ranging from 50 to 2,100 ppm.
Chamber conditions other than CO2 were kept constant:
temperature, 25°C; Xow, 150 mol s¡1; humidity, »50%;
and photosynthetic photon Xux density (PPFD) 1,300 mol
photons m¡2s¡1. We found that CO2 uptake was light satu-
rated at 1,300 mol photons m¡2s¡1.
We used an application (Sharkey et al. 2007) based on
equations in Farquhar et al. (1980) to Wt A/Ci curves and
estimate parameters for maximum carboxylation capacity
(Vcmax), maximum RuBP regeneration (Jmax) and triose
phosphate utilization (TPU). We estimated parameters for
each leaf sample individually, and light saturated net photo-
synthesis (Amax) was measured at the ambient CO2 concen-
tration. Parameters are expressed mainly per unit leaf area
(mol CO2 m¡2s¡1), but were also calculated per unit leaf
mass (mol CO2 g¡1s¡1). We determined photosynthetic N
use eYciency (PNUE), expressing Amax per unit leaf N.
Leaf areas inside the cuvette, as well as whole leaf areas,
were determined from digital images of the leaves.
Biochemical analyses
Leaves measured for CO2 exchange were oven dried and
analyzed for total C and N concentrations using a Carlo
Erba (Milan, Italy) NC2500 elemental analyzer. A separate
set of leaves was collected for biochemical analyses con-
ducted at the US Forest Service, Durham, New Hampshire:
leaves from Wve evenly spaced plants from each of two rep-
licate plots (ten plants/treatment/species). The only excep-
tion to this was V. myrtilloides, as there were not enough
plants to sample equally in both plots: (1) a 20NPK treat-
ment, where eight plants were sampled from one plot and
two from a second plot; (2) a 20N treatment, where all ten
plants were sampled from one plot. Freshly excised leaves
Table 1 Experimental set-up with NPK fertilization levels equal to 5
and 20 times the ambient growing season wet N deposition
Treatment N (g m¡2 a¡1)P (gm
¡2 a¡1)K (gm
¡2 a¡1)
Control 0 0 0
5N 1.6 0 0
5NPK 1.6 6.3 5
20N 6.4 0 0
20NPK 6.4 6.3 5
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358 Oecologia (2011) 167:355–368
123
were cut into approximately 3 mm squares using sharp scis-
sors to create a pool of foliage sample for every plant. This
pool was divided into two subsamples: one (approximately
200 mg fresh weight) was placed in a preweighed micro-
fuge tube with 1 mL of 5% perchloric acid (HClO4); the
other was placed in a separate tube without anything. All
samples were transported to the laboratory on ice and
stored at ¡20°C until further analysis. The samples in
HClO4 were weighed, frozen and thawed three times, and
centrifuged at 13,000£gfor 10 min. The supernatants were
used for the analyses of HClO4-extractable free PAs, free
amino acids, and soluble inorganic ions (Minocha et al.
1994). The other set of subsamples was used for soluble
protein and total chlorophyll analyses. Leaf extracts from
each of ten plants/treatment/species were analyzed individ-
ually without pooling for all biochemical analyses.
Soluble inorganic ions were quantiWed using a simulta-
neous axial inductively coupled plasma emission spectro-
photometer (Vista CCD, Varian, Palo Alto, CA, USA) and
Vista Pro software (Version 4.0), following appropriate
dilutions with deionized water (Minocha et al. 1994). For
analysis of common amino acids and PAs (Putrescine,
Spermidine, Spermine), the supernatants from HClO4-
extracted samples were subjected to dansylation and quan-
tiWcation by HPLC according to Minocha and Long (2004).
The reaction was terminated by using 50 L of L-aspara-
gine (20mgmL
¡1 in water) instead of alanine. The proto-
col did not always separate glycine, arginine and threonine;
therefore, the peak areas for these three amino acids were
pooled for each standard to derive a combined calibration
curve for their quantiWcation.
For soluble protein analysis, 50 mg of leaf pieces were
placed in 0.5 mL of 100 mM Tris–HCl buVer (containing
20 mM MgCl2, 10 mM NaHCO3, 1 mM EDTA, and 10%
(v/v) glycerol; pH 8.0), frozen and thawed three times, and
the supernatant was used for protein analysis according to
Bradford (1976). For chlorophyll analysis, 10 mg of leaf
tissue was placed in 1.0 mL of 95% ethanol in the dark at
65°C for 16 h, and centrifuged (13,000£gfor 5 min). The
supernatant was scanned from 350 to 710 (U-2010, Hitachi
Ltd., Tokyo, Japan) and chlorophyll was quantiWed as per
Minocha et al. (2009). Results for leaf chemistry are
expressed per dry weight. We used the average percent
moisture for each species/treatment to calculate the dry
weight (DW) from fresh weight (FW).
Statistical analyses
We studied the eVect of treatments on the aboveground
abundance of C. calyculata and L. groenlandicum using
one-way ANOVA, analyzing years 2000 and 2008 sepa-
rately. DiVerences in V. myrtilloides abundance were not
analyzed, because it was not present in all survey plots, and
treatment 20N was excluded due to its diVerent experimen-
tal duration. Test variables were stem # £ height in 2000,
and number of point intercept hits in 2008; data were rank
transformed.
Leaf level variables were analyzed using multivariate
analysis of variance (MANOVA). The MANOVAs resulted
in highly signiWcant treatment, species, and species £ treat-
ment eVects (see Resource 1 of the Electronic supplemen-
tary material, ESM). We used one-way ANOVA to assess
the treatment eVects on variables with signiWcant between-
subject treatment or treatment £ species eVects. The set of
leaves measured for CO2 exchange, morphology and N
concentration was analyzed separately from the leaf sam-
ples used for biochemical analyses. All data were Wrst
tested for normality and equality of variances using
Levene’s test. Response variables with unequal variances
were rank transformed. Bonferroni adjustment was used to
evaluate statistical signiWcance (adjusted Pvalues were
0.003 for photosynthesis variables, and 0.001 for biochemi-
cal variables). We examined diVerences between treatments
and the control with Dunnett’s post-hoc test. Relationships
among photosynthetic parameters, leaf morphological and
biochemistry variables were quantiWed using Pearson’s cor-
relation and regression analyses. Statistical analyses were
performed using the SPSS statistical package 11.0 for MS
Windows (Lead Technologies, Inc. 2001).
Results
Aboveground growth and leaf morphology
Chamaedaphne calyculata was the most abundant vascu-
lar plant species in all plots (Fig. 1a, b). Analysis of vari-
ance did not show any diVerences between treatments and
control in the abundance of either C. calyculata or
L. groenlandicum in the Wrst year of the experiment
(2000). After 8–9 treatment years, we found no signiWcant
diVerences in the abundance of these species, but there
were trends for increased growth. For example, the abun-
dance of C. calyculata nearly doubled in the treatment
20NPK (Fig. 1).
We expected an increase in SLA with fertilizer treatment
for the plants to maximize light interception. We found no
changes, except for L. groenlandicum, which increased from
»60 cm2g¡1 SLA in the control plots to »100 cm2g¡1 with
5NPK treatment (Fig. 2a), perhaps due to a decrease in leaf
mass (Table 2), but these changes were not signiWcant.
Overall, the deciduous V. myrtilloides had a higher SLA
(»100–115 cm2g¡1) than the two evergreen species (»65–
100 cm2g¡1), and a higher moisture content (»50% DW)
than the evergreens (»10–30% DW) in control plots.
Compared with the control, percent moisture was lower in
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Oecologia (2011) 167:355–368 359
123
V. myrtilloides leaves in the 5N and 20N treatments (declin-
ing from »50–30%) and in L. groenlandicum leaves in the
20NPK treatment (declining from »30–15%). C. calyculata
had the lowest moisture content of all three species (»10–
15%), and did not change with treatment (Fig. 2b). A similar
trend was observed in soluble proteins (Fig. 3f).
Foliar chemistry
The total N concentration in the leaves of all three species
increased from »1% in the control to »1.5% in the highest
fertilizer treatments (Fig. 3a). Consequently, the C:N ratios
decreased from »52 in the control to »34 in 20NPK treat-
ments (Fig. 3b). Total chlorophyll ranged from approxi-
mately 0.9 to 1.8 mg g¡1 and followed a trend similar to
that of foliar N for C. calyculata, resulting in a twofold
increase in 20N compared to the control (Fig. 3c). Trends
for the other two species were not so clear. For example,
chlorophyll in L. groenlandicum leaves showed a small
(but insigniWcant) increase with 20N, but there was a sig-
niWcant decrease in the 20NPK treatment.
Species diVered in the partitioning of N into amino
acids, PAs, and soluble proteins. While none of the species
showed signiWcant diVerences between treatments and con-
trol in total levels of amino acids and levels of either total
or individual PAs, L. groenlandicum had higher total
amounts of amino acids and PAs than the other two species
in all treatments (Fig. 3d, e, Resources 2 and 3 of the ESM).
V. myrtilloides had the highest total amounts of soluble pro-
teins among the three species in the control plots (Fig. 3f),
but showed declines between the control and the two N-
only treatments (5N and 20N). Supplying P along with N
prevented this decline in soluble proteins. L. groenlandi-
cum had a small but insigniWcant decline in soluble proteins
between the control and 20NPK.
Individual amino acids showed species-speciWc patterns.
In the two evergreens, the combined concentrations of four
amino acids [GABA, alanine, glutamic acid, and arginine
(+ glycine and threonine)] constituted more than 50% of the
total amino acid pool under normal growth conditions, his-
tidine and tryptophan dominated the amino acid pool in the
deciduous V. myrtilloides. Since histidine and tryptophan
share a common pathway, an increase in histidine with 5N
signiWcantly reduced tryptophan (Resource 2 of the ESM).
More important than alterations in total amino acids were
the eVects of treatments on the relative ratios of diVerent
amino acids. Leaves of both evergreen species had higher
concentrations of glutamic acid, alanine, arginine (plus gly-
cine and threonine) and GABA in the 20N and/or 20NPK
treatments compared with the control, although some of
these increases were not signiWcant. V. myrtilloides had
higher concentrations of GABA accompanied by a decrease
Fig. 1 Abundance of Cham-
aedaphne calyculata and Ledum
groenlandicum in survey plots
(mean §SE, n=3) in a2000, at
the beginning of the experiment,
and b2008, after 9 years of fer-
tilization. ANOVA results for
diVerences among the treatments
are indicated in the panels. Test
variables were height £number
of stems in 2000 and number of
point intercept hits in 2008
Species
Cham caly Ledu groe
Abundance (height x #)
0
1000
2000
3000
Control
5N
5NPK
20NPK
Cham caly Ledu groe
Abundance (hits #)
0
50
100
150
200
250
300
F=1.802, P=0.225
F=1.802, P=0.225
F=3.551,
P=0.067
F=0.977, P=0.450
ab
Fig. 2 a SpeciWc leaf area (SLA) and bmoisture content in fresh
leaves (mean §SE, n=9)
Species
Cham caly Ledu groe Vacc myrt
Moisture content (%)
0
20
40
60
80
SLA (cm2 g-1)
0
40
80
120
Control
5N
5NPK
20N
20NPK
a
b
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360 Oecologia (2011) 167:355–368
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in tryptophan in 5NPK and 20NPK treatments compared
with the control (Fig. 4, Resource 2 of the ESM). These
amino acids are some of the most N-rich of the major
amino acids, in particular arginine and histidine with N:C
ratios of >0.5. All of these amino acids are derived from
glutamate. Consequently, there were signiWcant correla-
tions between foliar N and glutamic acid for L. groenlandi-
cum, alanine for C. calyculata, and GABA for
C. calyculata and V. myrtilloides (Fig. 4).
Fertilizer treatment had a signiWcant eVect on the accu-
mulation of several soluble ions in the foliage of all three
species (Fig. 5). Ca was lowered signiWcantly in all spe-
cies by almost all treatments; Mg concentration was low-
ered for C. calyculata and V. myrtilloides in the N-only
treatments. V. myrtilloides contained higher amounts of
Ca and Mg than the other two species in control plots
(Fig. 5a, b). Phosphorus (P) increased signiWcantly in
response to the NPK treatments, as expected, and the
increases were similar in the 5NPK and 20NPK treat-
ments, as these plots received the same amount of P. P
concentration was signiWcantly lower in N-only treated
plants as compared with controls in L. groenlandicum and
V. myrtilloides (Fig. 5c). Relative to P, changes in potas-
sium (K) concentration were smaller, but K increased in
C. calyculata in NPK treatments. V. myrtilloides leaves
had only about half the concentration of K in N-alone
treatments compared to the control (Fig. 5d). Manganese
(Mn) and aluminum (Al) concentrations in the foliage of
the evergreen species were generally smaller in treatment
plots than in the controls. Manganese declined from »6 to
»2mol g¡1 DW in C. calyculata and from »4 to
»0.5 mol g¡1 in L. groenlandicum (Fig. 5e, f). In con-
trast, V. myrtilloides had higher Mn in the 5NPK treat-
ment than in the control.
Photosynthetic parameters
Nutrient addition had few signiWcant eVects on photosyn-
thetic parameters. Rates of carboxylation (Vcmax per unit
mass) in L. groenlandicum in 5N and 5NPK treatments
were higher than in the control (Table 3). However, there
were no signiWcant diVerences between the treatments and
control for rates of light-saturated photosynthesis (Amax) or
for other photosynthetic parameters (Table 3). Amax ranged
from »8 to 13 mol CO2 m¡2s¡1 and Vcmax ranged from
»67 to 137 mol CO2m¡2s¡1 among the three species and
treatments, with C. calyculata having slightly higher rates
than the other species.
Table 2 SpeciWc leaf area SLA (cm2g¡1leaf), individual leaf area (cm2), leaf mass (g), and thickness (mm) (mean §SE) with test statistics from
one-way ANOVA
DiVerences are considered signiWcant at a Bonferroni adjusted Plevel of 0.003. Sample size was eight leaves per species per treatment. Either three
or two leaves were sampled from each plot
Species Treatment SLA Area Mass Thickness
C. calyculata Control 81.4 (6.1) 1.68 (0.16) 21.4 (2.5) 0.3 (0.03)
5N 86.8 (3.7) 2.00 (0.1) 22.5 (1.5) 0.36 (0.03)
5NPK 80.8 (3.5) 1.67 (0.12) 21.0 (1.7) 0.41 (0.04)
20N 78.7 (4.2) 2.13 (0.22) 27.3 (2.7) 0.39 (0.02)
20NPK 86.6 (4.3) 1.94 (0.14) 24.1 (2.2) 0.38 (0.02)
F0.45 1.57 1.67 1.15
P0.77 0.20 0.18 0.35
L. groenlandicum Control 65 (5.4) 1.78 (0.11) 28.3 (1.9) 0.76 (0.06)
5N 74.5 (4.1) 1.74 (0.22) 23.5 (2.7) 0.59 (0.03)
5NPK 103 (10.5) 1.64 (0.12) 16.8 (1.7) 0.63 (0.04)
20 N 72 (3.8) 1.78 (0.11) 27.6 (0.7) 0.66 (0.06)
20NPK 78.4 (6.2) 1.9 (0.11) 18.9 (2.4) 0.61 (0.06)
F5.23 0.73 6.36 1.82
P<0.001 0.58 1.67 0.15
V. myrtilloides Control 115.9 (7.0) 3.8 (0.51) 22.3 (4.9) 0.41 (0.04)
5N 120.9 (15.0) 3.1 (0.28) 27.1 (3.1) 0.36 (0.02)
5NPK 98.4 (8.3) 4.4 (0.49) 47.7 (7.5) 0.50 (0.04)
20N 110.8 (7.1) 3.8 (0.26) 35.1 (2.1) 0.48 (0.05)
20NPK 104.5 (10.8) 5.7 (0.58) 59.6 (8.9) 0.46 (0.03)
F0.96 4.59 6.36 1.69
P0.44 <0.001 1.67 0.18
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Oecologia (2011) 167:355–368 361
123
The highest rates of Amax or Vcmax seemed to co-occur
with intermediate leaf N concentrations (g m¡2 leaf)
(Fig. 6a, b, e, f, i, j). Only C. calyculata had a weakly posi-
tive linear relationship between foliar N and Amax. PNUE
had a signiWcantly negative relationship with foliar N for
V. myrtilloides and a weakly negative relationship
(although not signiWcant) with foliar N for the evergreens
(Fig. 6c, g, k). The Vcmax : foliar N ratio had a signiWcant
negative correlation with foliar N for all three species
(Fig. 6d, h, l). Foliar N tended to be higher in leaves with
low SLA for all three species combined, which likely is a
result of more N per unit leaf area in thicker leaves. Chloro-
phyll had a positive relationship with Amax (area) and a
negative relationship with Vcmax (mass), Jmax (area), and
TPU (area) (Table 4).
Discussion
N deposition at Mer Bleue was »0.2 g m¡2a¡1 in pre-
Industrial times, but has since increased to its current level
of »0.8 g m¡2a¡1. Contrary to our expectations, additions
of 1.6 and 6.4 g N m¡2a¡1 alone or in combination with P
resulted in few signiWcant responses in shrub biomass or
leaf biochemistry. C. calyculata was clearly the dominant
vascular species and had the largest growth increase after
Fig. 3 Concentrations (mean §SE) of foliar: anitrogen, cchlorophyll,
damino acids, epolyamines (Putrescine, Spermidine, Spermine),
fsoluble proteins, and bthe C:N ratios for Chamaedaphne calyculata,
Ledum groenlandicum and Vaccinium myrilloides. * and ** denote sig-
niWcant diVerences between the treatment and control conditions
(P< 0.05 and 0.01, respectively)
N concentration (%)
0
1
2
a
C:N (%)
0
20
40
60
80
b
Total Chlorophyll (mg g
-1
)
0
1
2
c
Total amino acids (µmol g
-1
)
0
1
2
3
4
5
d
Species
Polyamines (nmol g
-1
)
0
50
100
150
e
Cham caly Ledu groe Vacc myrt Cham caly Ledu groe Vacc myrt
Soluble proteins (mg g
-1
)
0
5
10
f
Control
5N
5NPK
20N
20NPK
*
**
*
**
**
*
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362 Oecologia (2011) 167:355–368
123
8 years of fertilization, but only in the 20NPK treatment
(Fig. 1). However, growth led to a larger investment into
stems and woody biomass than in leaves (Juutinen et al.
2010). Ledum groenlandicum was less abundant, but there
was a trend for increasing aboveground biomass of this spe-
cies with N treatment, particularly 5NPK. Nitrogen concen-
trations per unit leaf mass increased with nutrient addition,
and increases in chlorophyll were the largest in C. calycu-
lata leaves compared with other species (Fig. 3).
Relative to the control, we did not observe consistent
changes in chemistry, morphology and photosynthetic
capacity with fertilizer treatment. However, levels of Ca in
leaves were lowered signiWcantly in all three species, and a
declining trend was seen in Mg (Fig. 5). Loss of these cat-
ions from soil is a known consequence of excess N and
acidiWcation, and has been implicated as a primary cause of
forest decline in the US and in Europe (Schulze 1989;
Magill et al. 2004). Similar losses in Ca and Mg occurred
concomitant with a decline in Sphagnum productivity with
increased N deposition in European peatlands (Bragazza
et al. 2004). A decline in moss cover and biomass at Mer
Bleue bog upon high N additions has also been reported
earlier by Bubier et al. (2007) and Juutinen et al. (2010). In
the present study, we observed a species-speciWc increase
Fig. 4 Amino acid concentra-
tions (means §SE, n= 10) for
aglutamic acid, calanine,
earginine + glycin e + threonine,
and gGABA. SigniWcant
diVerences between fertilizer
and control treatments are
denoted by * (P< 0.05) or
** (P< 0.01). b, d, e, and
hshow plot means (n= 5) of
individual amino acid
concentrations versus plot
means of foliar nitrogen
concentration. Fitted regression
lines and r2 values denote linear
regressions with slopes that are
signiWcantly (P< 0.05) diVerent
from zero
Glutamic acid (nmol g
-1
)
0
100
200
300
400
500
Control
5N
5NPK
20N
20NPK
a
Alanine (nmol g
-1
)
0
200
400
600
c
Arginine + Glycine+
Threonine (nmol g
-1
)
0
400
800
1200
1600
2000
e
Species
Cham caly Ledu groe Vacc myrt
GABA (nmol g
-1
)
0
200
400
g
Nitrogen concentration (%)
1.0 1.5
b
d
f
h
*
**
*
Ledu groe, r2= 0.48
Cham caly
Vacc myrt
Ledu groe
Cham caly, r
2
=0.42
Vacc myrt
Vacc myrt
Vacc myrt, r
2
=0.54
Cham caly, r
2
=0.50
Ledu groe
Cham caly
Ledu groe
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Oecologia (2011) 167:355–368 363
123
in a few N-rich amino acids with treatments, which is
perhaps a strategy to avoid NH3 toxicity at the cellular
level. However, in the absence of signiWcant changes in
photosynthetic parameters, PAs and most other amino
acids, it is not evident at this time if the changes observed
in the few amino acids are large enough to indicate signiW-
cant physiological stress in these shrubs.
Photosynthetic capacity and foliar chemistry
In the present study, the lack of strong relationships
between foliar N and either Amax or Vcmax, coupled with
negative relationships between foliar N, SLA and PNUE
(Table 4; Fig. 6), are opposite to our hypotheses. The
results also disagree with earlier meta-analyses of natural
ecosystems predicting that increased foliar N should lead to
a corresponding increase in photosynthetic capacity (Reich
et al. 1998). We found no increase in photosynthetic param-
eters (Amax or Vcmax) to accompany the increases in foliar N,
with the exception of an increase in Vcmax in L. groenlandi-
cum at moderate N addition of 1.6 g N m¡2a¡1 (+ back-
ground deposition »0.8 g m¡2a¡1) (Table 3). We also
found a weakly positive correlation between Amax, foliar N,
and chlorophyll (Fig. 6; Table 4) and increased chlorophyll
in C. calyculata leaves (Fig. 3c), indicating that this species
is using some of the excess N to invest in light harvesting
and photosynthetic capacity. This was also the only species
to increase in biomass with fertilization, but only in the
Fig. 5 Foliar concentrations (mean §SE, n= 10) of soluble aCa, bMg, cP, dK, eMn, and fAl. * and ** denote signiWcant diVerences between
the treatment and control conditions (P< 0.05 and 0.01, respectively)
Ca (µmol g
-1
)
0
50
100
150
a
Mg (µmol g
-1
)
0
20
40
60
80
b
P (µmol g
-1
)
0
10
20
30
c
K (µmol g
-1
)
0
20
40
60
80
d
Species
Mn (µmol g
-1
)
0
2
4
6
8
e
Cham caly Ledu groe Vacc myrt Cham caly Ledu groe Vacc myrt
Al (µmol g
-1
)
0
1
2
f
Control
5N
5NPK
20N
20NPK
**
**
** **
*
** **
**
**
**
**
**
*
*
**
** **
**
**
**
**
**
*
**
**
**
***
*
**
*
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364 Oecologia (2011) 167:355–368
123
20NPK treatment (Fig. 1). In the other two species, we
observed either no change or a decline in chlorophyll con-
centrations, and no change in biomass. While it might be
expected that N addition would have an even stronger posi-
tive inXuence on photosynthetic capacity in nutrient-limited
ecosystems than in other ecosystems, results have been
mixed. For example, studies have found increases (St. Clair
et al. 2009), no change (Bowman et al. 1995; Starr et al.
2008), and decreases (Bigger and Oechel 1982; Bauer et al.
2004) in Amax in response to nutrient addition. The diVer-
ences in the species composition and their threshold for N
tolerance, initial N status of the site, and the duration of N
addition probably contribute to these varied results (Aber
1992).
Our results indicate that most of the additional N was
stored in simple organic compounds rather than invested in
photosynthetic machinery (i.e., Rubisco and chlorophyll).
Some of these metabolites, such as polyamines and amino
acids, N-rich compounds essential for growth and develop-
ment, in combination with chlorophyll and total soluble
proteins have been used as indicators of environmental
stress before the morphological symptoms of stress are vis-
ible (Näsholm et al. 1994; Bauer et al. 2004). PAs (speciW-
cally putrescine) and certain amino acids are indicative of
the physiological response of forest trees to an array of
environmental stress conditions, including a shortage of
soil-available Ca, excess Al, and chronic N accumulation
(Minocha et al. 1997, 2000; Wargo et al. 2002). It has been
suggested that, under conditions of stress, PAs impart stress
tolerance by lowering NH3 toxicity and scavenging free
radicals. Polyamines also act as signal molecules to regu-
late gene activity related to cellular N metabolism and
the metabolism of several amino acids: proline, argi-
nine, -aminobutyric acid (GABA) and glutamic acid, all
of which play important roles in plant responses to higher N
exposure (Näsholm et al. 1994; Bauer et al. 2004; Bouché
and Fromm 2004). Storing excess N in organic forms is
more favorable to plant health (reduces NH3 toxicity) and
growth, as the plant can access the stored N when supplies
diminish (Rabe 1990; Näsholm et al. 1994; Limpens and
Berendse 2003; Bauer et al. 2004).
We found increases in alanine and GABA with the highest
N treatment (Fig. 4), which, in boreal understory species, are
thought to indicate N saturation from the shoot to the root, and
therefore have the potential to inhibit further root uptake of
nutrients (Näsholm et al. 1994; Limpens and Berendse 2003;
Tomassen et al. 2003). SigniWcant changes in most amino
acids and all PAs were observed in a red pine stand at Harvard
Table 3 Vcmax and Amax per unit area (mol CO2 m¡2s¡1) and per unit mass (mol CO2 g¡1s¡1), Jmax, and TPU (mol CO2 m¡2s¡1) (mean §SE,
n= 7–8), along with test statistics of one-way ANOVA
DiVerences are considered signiWcant at a Bonferroni-adjusted Plevel of 0.003. SigniWcant (P<0.05) diVerences from the control conditions were
assessed with Dunnet’s test and are highlighted in boldface
Species Treat. Vcmax (area) Amax (area) Vcmax (mass) Amax (mass) Jmax (area) TPU (area)
Cham caly Control 132.1 (31.2) 10 (4.9) 1.1 (0.4) 0.08 (0.04) 93.4 (4.2) 7.2 (0.3)
5N 129.7 (30.6) 9.1 (4.3) 1.1 (0.4) 0.08 (0.03) 94.7 (7.7) 7.6 (0.6)
5NPK 122.9 (14.7) 11.8 (6.2) 1.0 (0.2) 0.09 (0.05) 122.7 (15.4) 9.1 (1.2)
20N 117.3 (23.1) 12.9 (3.2) 0.9 (0.1) 0.1 (0.03) 120.4 (9.7) 8.6 (0.7)
20NPK 129.7 (27.5) 8.6 (5.0) 1.1 (0.3) 0.09 (0.04) 117.2 (8.5) 8.7 (0.7)
F0.44 0.75 0.36 1.12 0.21 0.34
P0.78 0.57 0.84 0.37 0.93 0.85
Ledu groe Control 78.1 (13.4) 11.0 (2.6) 0.5 (0.2) 0.07 (0.02) 119.4 (12.1) 10.1 (1.0)
5N 137.4 (21.9) 9.6 (3.3) 1.0 (0.3) 0.07 (0.02) 177.8 (17.0) 14.4 (1.3)
5NPK 123.5 (49.1) 7.6 (3.0) 1.2 (0.4) 0.07 (0.02) 170.0 (18.5) 13.3 (1.3)
20N 118.4 (38.5) 9.5 (2.1) 0.8 (0.2) 0.07 (0.02) 141.5 (15.6) 11.5 (1.2)
20NPK 103.1 (34.2) 10.3 (4.7) 0.8 (0.4) 0.09 (0.03) 170.0 (16.6) 13.7 (1.3)
F3.57 2.06 6.80 0.91 2.04 1.76
P0.02 0.11 <0.001 0.47 0.11 0.16
Vacc myrt Control 84.6 (13.5) 9.8 (1.9) 0.8 (0.2) 0.11 (0.02) 164 (12.1) 13.4 (1.0)
5N 66.7 (14.3) 10.9 (4.3) 0.8 (0.2) 0.12 (0.04) 152.9 (6.3) 12.6 (0.5)
5NPK 96.6 (32.1) 10.8 (2.5) 1.0 (0.5) 0.11 (0.03) 159.2 (9.6) 12.7 (0.7)
20N 90.7 (25.6) 11.0 (4.3) 1.0 (0.4) 0.12 (0.06) 156.9 (11.8) 12.5 (1.0)
20NPK 92.4 (27.8) 10.1 (3.6) 1.0 (0.4) 0.1 (0.02) 152.1 (12.7) 12.0 (1.0)
F3.0 0.17 0.68 0.49 1.69 1.02
P0.03 0.95 0.61 0.74 0.17 0.41
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Oecologia (2011) 167:355–368 365
123
Forest, MA, when it was subjected to long-term chronic N
additions (Bauer et al. 2004). The metabolic costs of main-
taining high levels of free amino acids so as to avoid the toxic
eVects of free ammonia in our study would also considerably
increase maintenance respiration of the cells, thus removing
energy from growth processes (De Vries 1975).
Compared to the other species, total levels of PAs were
the highest in L. groenlandicum (Fig. 3d). However, we did
not Wnd signiWcant changes in the two major PAs (putres-
cine and spermidine) between the treatments and control in
this study (Resource 3 of the ESM). The data collected so
far indicate that these shrub species may be in the earlier
phases of N suYciency/saturation with 20N and/or 20NPK
additions. While all three species showed signiWcant
changes in some metabolites, we did not observe reductions
in photosynthetic capacity.
N use eYciency, leaf traits, and resource allocation
Higher SLA, as observed in L. groenlandicum in low-N
treatments, may indicate a change in nutrient allocation
with regard to leaf life span and morphological adjustments
(Shipley et al. 2005). SpeciWc leaf area is usually positively
correlated with light use eYciency; thinner leaves require
less photosynthetic machinery per unit area (Burns 2004),
while thicker or denser leaves have greater internal shading
and diVusion limitations, which may restrict the potential
for higher photosynthetic capacity because of the chloro-
plast stacking in thick leaves (Reich et al. 1998). In turn,
low SLA foliage tends to be longer lived but less produc-
tive than thinner leaves (Pornon and Lamaze 2007), indicat-
ing a trade-oV between photosynthetic capacity and leaf
persistence (Hikosaka 2004; Shipley et al. 2006).
Fig. 6 Amax (area), Vcmax (area), PNUE, and Vcmax : N ratio in relation
to leaf N content for Chamaedaphne calyculata (a–d), Ledum groen-
landicum (e–h), and Vaccinium myrtilloides (i–l). Fitted regressions
and coeYcients of determination are indicated only for signiWcant rela-
tionships (P< 0.05). Each point represents one leaf
Vcmax (µmol C m-2 s-1)
0
50
100
150
200
Amax (µmol CO2 m-2 s-1)
0
5
10
15
20
25
r2= 0.11
0.5 1.0 1.5 2.0 2.5
r2= 0.33
0.5 1.0 1.5 2.0 2.5
r2= 0.24
Leaf nitrogen content (g m
-2
)
0.5 1.0 1.5 2.0 2.5
Vcmax : N ratio
(µmol CO2 g N
-1
s
-1
)
0.00
0.05
0.10
0.15
0.20 r2 = 0.55
PNUE (µmol CO2 g N-1 s-1)
0.00
0.01
0.02 r2=0.21
a
d
c
b
e
h
g
f
i
k
j
l
Cham caly Ledu groe Vacc myrt
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366 Oecologia (2011) 167:355–368
123
Cost–beneWt models (Kikuzawa and Ackerley 1999;
Wright et al. 2004; Ellison 2006) suggest that better nutrient
availability and shorter leaf life spans allow the plant to rein-
vest nutrients in young, photosynthetically active tissues,
leading to higher PNUE. We found no correlation between
PNUE and SLA, and either no correlation or a negative cor-
relation between foliar N and SLA, and foliar N and PNUE
(Table 4; Fig. 6). These relationships are contrast with those
predicted by some cost–beneWt models (Hikosaka 2004;
Poorter and Evans 1998), but are analogous to those
observed by Ripullone et al. (2003) and Granath et al.
(2009b) under high N deposition. The latter study found a
unimodal relationship between foliar N and PNUE, with an
optimum N level of approximately 9 mg N g¡1 dry mass for
Sphagnum, although this level would also depend on N:P:K
ratios (Bragazza et al. 2004; Ellison 2006; Elser et al. 2007;
Hidaka and Kityama 2009). In our study, the weak negative
correlation (depending on the species) without an optimum
level between PNUE and foliar N suggests that any level of
N addition at Mer Bleue either has no eVect or decreases the
PNUE of these species (Fig. 6).
On the other hand, it can be argued that PNUE may not
be the most relevant measure of lifetime nutrient use
eYciency for bog species. The longer leaf life span and
mean residence time of leaf N may lead to longer lifetime
nutrient use eYciency in evergreens, particularly in nutri-
ent-limited environments (Small 1972; Berendse and Aerts
1987). These species can produce two- to threefold more
photosynthate using a given unit of N before it is returned
to the environment than do bog deciduous species, which
can produce about 60% more photosynthate per acquired
unit of N than do non-bog deciduous species (Small 1972).
Butler and Ellison (2007) observed that a predilection for
storing nutrients, rather than using them immediately, may
be one reason that photosynthetic rates of many wetland
plants are lower than expected given their foliar N concen-
trations.
The total mass of leaves per plant can be more important
than leaf photosynthetic rate in determining plant produc-
tivity, as observed in some arctic and peatland studies
(Chapin and Shaver 1996; Starr et al. 2008). Bartsch (1994)
found that biomass, Xower production, and shoot growth in
C. calyculata increased two to Wvefold with fertilizer treat-
ment in a Maine bog. Similarly, in our study, it appears that
nutrients have been invested mainly in woody biomass, and
less into new leaves; but total foliar N per unit area has
increased with N addition (Fig. 1; Juutinen et al. 2010). At
the leaf level, the current study shows that N has been allo-
cated to foliar storage compounds more than photosynthetic
processes (particularly in L. groenlandicum).
In addition to woody and foliar biomass, lifetime nutrient
use eYciency includes leaf lifespan. Shaver (1983) found
that Ledum palustre had decreased leaf longevity with nutri-
ent addition, possibly due to failure to survive the winter.
Leaves may turn over faster in order to eliminate potentially
toxic levels of ammonia. In fertilized and ambient environ-
ments, older leaves serve as storage organs, but have a lower
photosynthetic capacity than new leaves (Maier et al. 2008).
Our species at Mer Bleue may thus be shifting their alloca-
tion patterns to a shorter leaf lifespan in the highest nutrient
treatments as leaf litter accumulation has increased in these
plots (Juutinen, pers. comm.). Thus far, we have not
observed an invasion of deciduous and graminoid species,
which have been reported to have a competitive advantage
over evergreens under elevated atmospheric N and nutrient
addition in Europe and North America (Bowman et al. 1995;
Chapin and Shaver 1996; Van Wijk et al. 2003).
Finally, we observed lower foliar moisture content in
L. groenlandicum in the highest nutrient treatments, and in
the N-only treatments for V. myrtillloides (Fig. 2b). The
lower moisture content may be due to changes in nutrient
concentrations and consequently in root uptake mecha-
nisms, or due to drying of the surface soil, as observed
recently in the 20NPK plots with the loss of Sphagnum
(Humphreys, pers. comm.). Bowman et al. (1995) found
that photosynthetic rates in alpine tundra species were unre-
lated to variation in foliar N concentration, but instead corre-
lated with variations in stomatal conductance. Starr et al.
(2008) found lower stomatal conductance after drought peri-
ods, resulting in lower Amax values in arctic tundra. These
changes in soil and plant moisture will likely have stronger
eVects on the physiology of these plants in the future.
Conclusions
The varied responses of plant species to N deposition glob-
ally, and at Mer Bleue, are perhaps the result of physiological
Table 4 Pearson’s correlation coeYcients between leaf dimensions,
chlorophyll concentration and photosynthetic variables and N for all
species and treatments
Dry leaf mass (mg), speciWc leaf area (cm2g¡1), thickness (mm), area
(cm2), total chlorophyll (gg
¡1), leaf N (mg m¡2 leaf), Vcmax (area)
(mol CO2 m¡2s¡1), Vcmax (mass) (mol CO2g¡1s¡1), Amax (area)
(mol CO2 m¡2s¡1), Amax (mass) (mol CO2 g¡1s¡1). SigniWcant cor-
relations (P< 0.05) are highlighted in boldface
Mass Area SLA Thickness Chlorophyll
N0.134¡0.198 ¡0.669 0.251 0.340
Vcmax (area) ¡0.279 ¡0.380 ¡0.291 ¡0.004 ¡0.298
Vcmax (mass) ¡0.322 ¡0.128 0.354 ¡0.167 ¡0.590
Amax (area) 0.127 0.004 ¡0.300 ¡0.128 0.372
Amax (mass) 0.114 0.284 0.327 ¡0.307 0.220
Jmax (area) ¡0.370 ¡0.498 ¡0.355 0.095 ¡0.416
TPU (area) ¡0.425 ¡0.557 ¡0.376 0.103 ¡0.431
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Oecologia (2011) 167:355–368 367
123
diVerences and evolutionary adaptations to resource use and
allocation. Our biochemical data suggest that these bog shrub
species tolerate and store the additional N for future use rather
than invest it in increasing photosynthetic capacity. We do not
know how the decreased levels of essential plant nutrients
(e.g., Ca and Mg) will aVect these species in the future. More-
over, in the highest nutrient treatments, the whole-plant physi-
ology (e.g., the distributions of woody and foliar biomass and
nutrients, and shifts in life history traits such as leaf longevity)
and competition need to be studied to assess ecosystem
changes. While we have not yet seen a change in photosyn-
thetic capacity (either a reduction owing to nutrient stress or
an increase owing to a shift in N allocation to photosynthetic
processes), bog shrubs may not be able to adapt their evolu-
tionary strategies to take advantage of elevated nutrients in the
long term, resulting in replacement by species that are better
adapted to a higher nutrient environment.
Acknowledgments We appreciate the support from a National Sci-
ence Foundation award (DEB 0346625) to Jill Bubier, a Howard
Hughes Medical Institute research fellowship to Rose Smith, Natural
Sciences and Engineering Research Council discovery grants to Tim
Moore, and thank the National Capital Commission for access to Mer
Bleue Bog. This article was also supported by the New Hampshire
Agricultural Experiment Station and is scientiWc contribution no. 2426
from the NHAES. We thank Elyn Humphreys for sharing microclimate
data and providing laboratory facilities at Carleton University, and
Leszek Bledzki, Lisa Brunie, Mike Dalva, Meaghan Murphy, Nigel
Roulet, and Paliza Shrestha for assistance in the Weld and laboratory
work at Mount Holyoke College and McGill University. We thank
George Cobb, Martha Hoopes, Aaron Ellison and Kevin GriYn for
valuable discussions at various stages of this work.
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