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Nitrogen inputs and losses in response to chronic CO2 exposure in a sub-tropical oak woodland

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Nitrogen inputs and losses in response to chronic CO2 exposure in a sub-tropical oak woodland

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Rising atmospheric CO2 concentrations could alter the nitrogen (N) content of ecosystems by changing N inputs and N losses, but responses vary in field experiments, possibly because multiple mechanisms are at play. We measured N fixation and N losses in a subtropical oak woodland exposed to 11 yr of elevated atmospheric CO2 concentrations. We also explored the role of herbivory, carbon limitation, and competition for light and nutrients in shaping response of N fixation to elevated CO2. Elevated CO2 did not significantly alter gaseous N losses, but lower recovery and deeper distribution in the soil of a long-term 15N tracer indicated that elevated CO2 increased leaching losses. Elevated CO2 had no effect on asymbiotic N fixation, and had a transient effect on symbiotic N fixation by the dominant legume. Elevated CO2 tended to reduce soil and plant concentrations of iron, molybdenum, phosphorus, and vanadium, nutrients essential for N fixation. Competition for nutrients and herbivory likely contributed to the declining response N fixation to elevated CO2. These results indicate that positive responses of N fixation to elevated CO2 may be transient, and that chronic exposure to elevated CO2 can increase N leaching. Models that assume increased fixation or reduced N losses with elevated CO2 may overestimate future N accumulation in the biosphere.
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Biogeosciences, 11, 3323–3337, 2014
www.biogeosciences.net/11/3323/2014/
doi:10.5194/bg-11-3323-2014
© Author(s) 2014. CC Attribution 3.0 License.
Nitrogen inputs and losses in response to chronic CO2exposure in a
subtropical oak woodland
B. A. Hungate1, B. D. Duval1,5, P. Dijkstra1, D. W. Johnson2, M. E. Ketterer3, P. Stiling4, W. Cheng6, J. Millman7,
A. Hartley8, and D. B. Stover9
1Department of Biological Sciences and Center for Ecosystem Science Society, Northern Arizona University, Flagstaff,
AZ 86011, USA
2Department of Environmental and Resource Sciences, University of Nevada-Reno, Reno, NV 89557, USA
3Department of Chemistry and Biochemistry, Northern Arizona University, Flagstaff, AZ 86011, USA
4Department of Biology, University of South Florida, Tampa, FL, USA
5US Dairy Forage Research Center, USDA-ARS, Madison, WI 53706, USA
6Faculty of Agriculture, Yamagata University, 1–23, Wakaba-cho, Tsuruoka, Yamagata, 997-8555, Japan
7Horace Mann Bronx Campus, Bronx NY 10471, USA
8Department of Marine and Ecological Sciences, Florida Gulf Coast University, Fort Myers, FL 33965-6565, USA
9Department of Energy, Office of Biological and Environmental Research, US Department of Energy, Washington,
DC 20585, USA
Correspondence to: B. A. Hungate (bruce.hungate@nau.edu)
Received: 20 October 2013 – Published in Biogeosciences Discuss.: 2 January 2014
Revised: 29 April 2014 – Accepted: 8 May 2014 – Published: 23 June 2014
Abstract. Rising atmospheric CO2concentrations may alter
the nitrogen (N) content of ecosystems by changing N inputs
and N losses, but responses vary in field experiments, possi-
bly because multiple mechanisms are at play. We measured N
fixation and N losses in a subtropical oak woodland exposed
to 11 years of elevated atmospheric CO2concentrations. We
also explored the role of herbivory, carbon limitation, and
competition for light or nutrients in shaping the response of N
fixation to elevated CO2. Elevated CO2did not significantly
alter gaseous N losses, but lower recovery and deeper dis-
tribution in the soil of a long-term 15N tracer indicated that
elevated CO2increased leaching losses. Elevated CO2had no
effect on nonsymbiotic N fixation, and had a transient effect
on symbiotic N fixation by the dominant legume. Elevated
CO2tended to reduce soil and plant concentrations of iron,
molybdenum, phosphorus, and vanadium, nutrients essential
for N fixation. Competition for nutrients and herbivory likely
contributed to the declining response of N fixation to ele-
vated CO2. These results indicate that positive responses of
N fixation to elevated CO2may be transient and that chronic
exposure to elevated CO2can increase N leaching. Models
that assume increased fixation or reduced N losses with el-
evated CO2may overestimate future N accumulation in the
biosphere.
1 Introduction
Nitrogen (N) is the element most frequently limiting to plant
growth (LeBauer and Treseder, 2008). Nitrogen inputs and
losses from terrestrial ecosystems determine ecosystem N
pool size, and in turn influence the potential for carbon (C)
uptake when plant growth is N limited. Carbon uptake and
storage are thus sensitive to the balance of N inputs and
losses (Pepper et al., 2007; Gerber et al., 2010; Esser et al.,
2011). Here, we synthesize the effects of 11 years of chronic
exposure to increased CO2concentrations on N inputs and
losses from a subtropical oak woodland.
Nitrogen fixation is the major biological pathway through
which the biosphere accumulates N. Nitrogen fixation has a
high demand for reducing power to break the triple covalent
bond shared by the two atoms in the N2molecule (Benemann
and Valentine, 1972). Symbioses between bacteria capable
of N fixation and photosynthetic organisms have evolved
Published by Copernicus Publications on behalf of the European Geosciences Union.
3324 B. A. Hungate et al.: Nitrogen inputs and losses in response to chronic CO2exposure
multiple times, likely an adaptive pairing because of the high
energetic cost of N fixation and N being frequently limit-
ing to plant growth (Sprent, 1985). Increased photosynthe-
sis with rising concentrations of atmospheric CO2has been
postulated to shunt more labile C from the plant to bacte-
rial root symbionts, increasing rates of N fixation. In some
cases, elevated CO2has been found to disproportionately in-
crease the growth of N-fixing plants (Norby and Sigal, 1989;
Arnone and Gordon, 1990; Hartwig et al., 2000; Soussana
and Hartwig, 1996; Zanetti et al., 1996; Hebeisen et al., 1997;
Feng et al., 2004) and increase N fixation, though this poten-
tial is not always realized under field conditions (Schäppi and
Korner, 1997; Arnone, 1999; Hoosbeek et al., 2011).
Some symbiotic (i.e., free-living) heterotrophic bacteria
can also fix N. Elevated CO2can increase the amount of
C that plants produce belowground through root growth,
turnover, and exudation (Drake et al., 2011; Hagedorn et al.,
2013; Lagomarsino et al., 2013) and thereby alleviate C lim-
itation of nonsymbiotic N fixation. This may explain why
elevated atmospheric CO2has been found to stimulate non-
symbiotic N fixation by soil bacteria in a salt marsh (Dakora
and Drake, 2000) and a rice paddy soil (Hoque et al., 2001).
However, in a temperate pine forest and desert soil, elevated
CO2had no effect on nonsymbiotic N2fixation (Hofmockel
and Schlesinger, 2007; Billings et al., 2003). Thus, effects of
elevated CO2on nonsymbiotic N2fixation are equivocal.
N-fixing organisms require high concentrations of iron
(Fe), phosphate (P), and molybdenum (Mo), or in some in-
stances vanadium (V) (Williams, 2002). (Smith, 1992). Re-
sponses of N-fixation to elevated CO2can be limited by
availability of these nutrients (Niklaus et al., 1998;Jin et al.,
2012). The response of N fixation to elevated CO2across
multiple studies was only significant when non-N nutrients
were added as fertilizer; without nutrient amendments, the
effect of CO2on N fixation was negligible and not significant
(van Groenigen et al., 2006). Elevated CO2often increases
plant growth and element accumulation (Luo et al., 2006),
including in the subtropical woodland studied here (Duval et
al., 2013). Therefore, increased element uptake by non-fixing
plants could restrict nutrient availability for N-fixing organ-
isms, potentially limiting their response to elevated CO2.
Because of the high energy requirements of N fixation,
shading by the canopy can limit the growth of N-fixing plants
(Gutschick, 1987; Vitousek et al., 2002). Therefore, if el-
evated CO2promotes growth of the dominant species, en-
hancing its leaf area, the growth of N-fixing plants could be
suppressed. Herbivory could also influence the response of
N-fixing plants to elevated CO2. Herbivores often prefer the
tissue of N-fixing plants to that of other plants because N-
fixing plants have a higher protein content than non-fixing
plants (Ritchie and Tilman, 1995; Hulme, 1994, 1996). For
this reason, factors promoting the growth of N-fixing plants
could in turn stimulate selective herbivory.
With the exception of episodic losses during disturbance,
N losses from terrestrial ecosystems occur primarily as
gaseous products of nitrification and denitrification (NO,
N2O, and N2)and through leaching of NO
3and organic
N. Elevated CO2can alter N losses if input of labile C to
the rhizosphere enhances denitrification rates (Smart et al.,
1997; Robinson and Conroy, 1999; Baggs et al., 2003a, b),
or by altering soil water content because of reduced evap-
otranspiration (Hungate et al., 1997a; Arnone and Bohlen,
1998; Robinson and Conroy, 1999). Elevated CO2could also
reduce ammonium availability to nitrifiers, suppressing ni-
trification and N losses through gaseous fluxes (Hungate et
al., 1997b) and NO
3leaching (Torbert et al., 1998). Across
studies conducted to date, elevated CO2has been found to
increase N2O efflux from terrestrial ecosystems (van Groeni-
gen et al., 2011); effects on N leaching have not been synthe-
sized.
During the first 6years of the experimental treatment of
the subtropical woodland studied here, elevated CO2in-
creased N2fixation during the first year of treatment, but the
response subsequently disappeared (Hungate et al., 2004).
Here, we extend this record to include symbiotic N2fixa-
tion during the full 11years of the CO2experiment, and we
also assess responses of nonsymbiotic N2fixation to elevated
CO2. We also investigate possible mechanisms shaping the
responses to CO2, testing the hypotheses that selective her-
bivory, light competition, and changes in nutrient availability
modulate the response of N2fixation to elevated CO2. We
also report new data on rates of N gas losses and tracer 15N
recovery in deep soil to assess effects of CO2on N leaching.
2 Materials and methods
2.1 Site description
This work was conducted at the Smithsonian Environmen-
tal Research Center’s elevated CO2experiment at Kennedy
Space Center, Cape Canaveral, Florida, USA (28380N,
80420W). The experiment consisted of 16 open-top cham-
bers, each 2.5m high with an octagonal surface area of
9.42m2. Eight chambers were maintained at ambient atmo-
spheric CO2concentrations and eight chambers were main-
tained at approximately 350µL L1above ambient atmo-
spheric CO2concentration. The soils at the site were acidic
Spodosols (Arenic Haplohumods and Spodic Quartzipsam-
ments). The vegetation was Florida coastal scrub oak pal-
metto (Dijkstra et al., 2002; Johnson et al., 2003; Seiler et al.,
2009), dominated by three oaks (Quercus myrtifolia,Q. gem-
inata, and Q. chapmanii) and several less abundant species,
including saw palmetto (Seranoa repens), shiny blueberry
(Vaccinium myrsinites), rusty Lyonia (Lyonia ferruginea),
and tarflower (Befaria racemosa). A native vine, Elliott’s
milkpea (Galactia elliottii) constituted only 1% of above-
ground productivity (Hungate et al., 2004) but is important
for its ability to fix nitrogen.
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B. A. Hungate et al.: Nitrogen inputs and losses in response to chronic CO2exposure 3325
2.2 Symbiotic N2fixation by Galactia elliottii
We estimated G. elliottii annual aboveground production as
the annual flux of G. elliottii mass collected in litter traps
(Stiling et al., 1999, 2002, 2009). Litter of G. elliottii was
sorted and measured separately beginning in 1999; in 1997
and 1998, G. elliottii litter fall was measured together with
other “non-oak” species. As described previously (Hungate
et al., 2004), for 1997 and 1998, we estimated that G. elliot-
tii litter constituted 68% of the non-oak litter. This estimate
is the average of the proportion of G. elliottii litter mass in
in the total non-oak litter fraction as measured from 1999 to
2002 (63%) and the estimated proportion of G. elliottii litter
mass in 1997 and 1998 based on a mixing model using N
concentration in G. elliottii litter (high in nitrogen percent-
age) versus other species (lower in nitrogen percentage); this
mixing model yielded an estimate of 73%.
We measured N2fixation rates using isotope dilution. A
nitrogen-15 tracer was added on 19 June 1998 (0.18g Nm2
(NH4)2SO499.9atom% 15N). To the extent that G. elliot-
tii fixes N2via symbiotic bacterial fixation from the atmo-
sphere, G. elliottii leaves will be lower in δ15N than oak
leaves, whose N is derived from the soil, directly increased
by the δ15N value of the added tracer. The proportion of N in
G. elliottii that was derived from N2fixation (pf)was calcu-
lated using the standard model for 15N dilution (Shearer and
Kohl 1986), where the 15N signature of unlabeled G. elliottii
is the atmospheric end member:
pf=15NOδ15NG)/(δ15NOδ15NGo), (1)
where the subscript Orefers to the dominant oak (Q. myrtifo-
lia); Gto the N-fixer, G. elliottii, after adding the 15N tracer;
and Go to G. elliottii before adding the 15N tracer. This cal-
culation relaxes the common assumption that the δ15N value
of N obtained by fixation is equal to the δ15N of atmospheric
N2; it therefore accounts for biological fractionation during
N2fixation (Yoneyama et al., 1986) and provides a more
precise estimate of the end member of the mixing model.
The δ15N value of G. elliottii prior to label application was
2.2‰. A second assumption of this method is that the ref-
erence plant (Q. myrtifolia) obtains its N from the soil rather
than from N fixation. Departures from this assumption will
cause the mixing model to underestimate the proportion of N
derived from fixation, unlikely to be a serious error at our site
because the mixing model indicated that G. elliottii obtained
nearly all its N from fixation. The isotope dilution method
also requires that the δ15N tracer is sufficiently strong for the
δ15N value of the reference plant to remain distinct from the
atmospheric source. This was the case throughout the exper-
iment: the average δ15N in oak leaves was 131.0±5.3 ‰.
By the final harvest, this value declined to 84.3±4.2 ‰, still
clearly distinct from the N2-fixer value (2.2‰), providing
sufficient resolution in the mixing model such that the stan-
dard deviation for the proportion of N derived from fixation
(pf)was 0.012. The isotope dilution method also assumes
that G. elliottii, if it takes up N from the soil, accesses ap-
proximately the same soil N available to the reference plant.
Evidence suggests that this assumption is reasonable, as both
fine roots (67±13%) and G. elliottii nodules (74±9%)
were concentrated in the top 30cm of soil.
We measured N concentration and δ15N in G. elliottii fo-
liage and senesced litter on an elemental analyzer inline with
an isotope-ratio mass spectrometer. Total N2fixation was cal-
culated as the product of G. elliottii biomass production, N
concentration, and the proportion of N derived from fixa-
tion. To calculate pfusing Eq. 1, we used δ15N values from
senesced leaves because they were gathered in litter traps and
therefore captured an integrative sample of G. elliottii tissue
at the plot scale (Stiling et al., 2009). For N concentration, we
used the percentage of N in green leaves to avoid underesti-
mating N2fixation due to retranslocation during senescence.
We measured nodule biomass by handpicking soil from
cores taken in July 2007 (0–100cm) (Hungate et al., 2013b;
Day et al., 2013). Nodules were washed free of sand, oven
dried, and weighed. For comparison, we also report here data
obtained during the first year of the experiment (Hungate et
al., 1999), when we measured nodule mass and number in
buried columns of C-horizon sand placed in the top 15cm
of soil. The earlier assay using ingrowth cores captures new
nodule growth, whereas the cores at the final harvest mea-
sure the standing crop of nodules. Though not directly com-
parable, both assess responses of N2-fixing nodules to the
elevated-CO2treatment and thus are presented together here.
2.3 Herbivory
From 2000 to 2006, litter of the oaks and of G. elliottii was
scored for damage by insect herbivores and sorted, counted,
and weighed separately by species and insect damage cat-
egory as described previously (Stiling et al., 1999, 2003,
2009). The rate of litterfall of herbivore-damaged leaves, L
(gm2year1), was determined for each species as the to-
tal mass of damaged litter divided by the area of the lit-
ter traps in each chamber. Live green leaves of all species
were also sampled during the experiment in order to de-
termine the average mass of an undamaged green leaf for
each species. Seven plants were randomly chosen for each of
the three oak species in September 2004. Fifty leaves were
sampled haphazardly over the entire canopy of each plant.
For G. elliottii, total aboveground biomass was sampled in
1/6th of each chamber in September 1999, separated into
leaves and stems, and the leaves were counted. For all four
species, leaves were dried in a ventilated oven at 70C for
72h and weighed, and the average leaf mass was calculated.
We used these measurements to estimate the amount of tissue
consumed by insect herbivores. For each species, we multi-
plied the number of damaged leaves collected in the litter
traps (# leaves m2year1)by the average mass of a green
leaf of that species (g leaf1)to determine the production
of all the leaves of which insects consumed at least a part,
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3326 B. A. Hungate et al.: Nitrogen inputs and losses in response to chronic CO2exposure
P, (g m2year1). Note that this differs from total leaf pro-
duction in that P only includes leaves that were damaged
by insect herbivores. We then calculated herbivore consump-
tion (C) as the difference of PL. Our use of green leaf
mass in calculating Cis justified because herbivore dam-
age often causes early leaf abscission (Faeth et al., 1981;
Williams and Whitham, 1986; Stiling and Simberloff, 1989,
2002, 2003). Finally, we calculated total leaf production as
the total number of leaves in litterfall multiplied by aver-
age green mass and then calculated the proportion of total
leaf production that was consumed by herbivores, for each
species, as Cdivided by total leaf production.
2.4 Soil micronutrient analyses
In July 2005, October 2006, and July 2007, soil was collected
from the A (0–10cm) and E (10–30cm) horizons. Samples
were collected from five locations within each chamber. In
2007 we also analyzed samples from the E2 horizon (30–
60cm). The cores from each plot were composited, yielding
one A and E sample for each plot for 2005 and 2006, and one
A, E, and E2 sample in 2007. We sampled foliage of G. elli-
otii, collecting fully exposed and expanded leaves from five
plants per plot in May 2003 and in July 2007. Leaves were
oven dried at 60C. Total ecosystem element mass data for
2007 were reported previously (Duval et al., 2014); here, they
are shown as separate components (plant and soil), together
with data from 2003, 2005, and 2006.
Soils were air dried and passed through a 2 mm sieve. Soil-
available Mo, Fe, and V concentrations were determined af-
ter ammonium oxalate extraction (Liu et al., 1996), and soil-
available P was determined by extraction with NaOH (Carter,
1993). Extracts were filtered, diluted 10 times, suspended in
10mL of 0.32M trace metal grade HNO3, and analyzed by
Inductively coupled plasma mass spectrometry (ICP-MS) as
described below.
For 2007, plant samples were dried at 60 C and 750mg
was ashed at 600C. Five hundred milligrams of G. elliottii
leaves was run through a MARS microwave digester using
EPA protocol 3052x, consisting of a 30min cycle at 200C
with 9mL of HNO3, 3mL of HCl, and 2mL of HF. All
reagents were trace-metal-grade concentrated acids. The re-
sulting solutions were dried on a hot plate and resuspended
in 10mL of 0.32M trace-metal-grade HNO3prior to ICP-
MS analysis.
Element concentrations were determined on a Thermo X
Series quadrupole ICP-MS at the Keck Isotope Biogeochem-
istry Laboratory, Arizona State University, Tempe, Arizona,
and a Thermo X2 Series quadrupole ICP-MS at the Iso-
tope Geochemistry Laboratory, Northern Arizona University,
Flagstaff, Arizona. In these analyses, we used the standard
references cody shale (SCo-1) for soils and peach (NIST
1547) and apple leaves (NIST 1515) for plants. In the ICP-
MS analysis, we had >90% recovery of all elements mea-
sured.
2.5 Acetylene reduction for nonsymbiotic N2fixation
We measured acetylene reduction to ethylene in soil incuba-
tions to estimate N2fixation by free-living soil heterotrophs.
Soil samples were collected in January and March 2006. At
each date, five cores (4cm diameter x 15 cm deep) were col-
lected from each chamber, shipped overnight to Florida In-
ternational University (Miami, Florida), composited for each
plot, and sieved through a 2 mm sieve. Soils were composited
by treatment and 3 g subsamples were filled into 20mL glass
vials (n=4–5). In January 2006, the experimental design in-
cluded two levels of glucose addition (0 and 324µg C g1
soil) and two levels of soil moisture (3% and 10 % volumet-
ric). In March 2006, the experimental design included three
levels of glucose addition (0, 6.7, and 34.4µg C g1soil) and
two levels of phosphorus addition (0 and 6.5µg P g1soil).
For both experiments, treatments were crossed in a fully fac-
torial design with n=4 for January and n=5 for March.
Acetylene (1mL) was added to each vial, and the vials were
sealed and incubated for 6 h at room temperature. Headspace
samples were analyzed via gas chromatography for ethylene
production using an HP5890 gas chromatograph equipped
with a flame ionization detector.
2.6 Nitrous oxide and nitrogen oxide gas fluxes
We measured soil production of nitrous oxide (N2O) and ni-
tric oxide (NOx)using static chambers (Hutchinson and Liv-
ingston, 1993) during 2005–2007. For N2O, headspace sam-
ples were collected in syringes and analyzed by gas chro-
matography. For NOx, the static chamber was plumbed to
a chemiluminescent detector and the computer that logged
real-time [NOx]. Chambers (1.8L) were constructed from a
10.2 cm diameter PVC pipe closed with a PVC cap. The bot-
tom 3cm of each chamber was tapered to allow the cham-
ber to slide smoothly into PVC rings of similar diameter in-
stalled in each plot in 2004. Once the chamber was in place,
headspace air (15 mL) was sampled through a rubber septum
(fixed to the top of each chamber) using a 20mL nylon sy-
ringe equipped with a nylon stopcock and a 23-gauge needle.
Three subsequent headspace samples were taken at 15min
intervals. Syringes were maintained under pressure using a
rubber band until analysis, within 12h of sample collec-
tion. Samples were analyzed on a gas chromatograph system
(Shimadzu) with Haysep-Q60/80- and Porapack-Q60/80-
packed columns and equipped with an electron capture de-
vice to determine N2O concentrations. Field fluxes were cal-
culated using linear regression of concentrations over time.
The flux rates were expressed as µg N2O-Nm2d1.
2.7 Tracer 15N distribution and recovery in deep soils
During the final harvest of the experiment in 2007, each plot
was cored to the water table or to a depth of 3m (whichever
was deeper). Core depth averaged 260.6cm (SEM 13.5),
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B. A. Hungate et al.: Nitrogen inputs and losses in response to chronic CO2exposure 3327
not significantly different between the CO2treatments (P=
0.90; ttest). We modeled the depth distribution of tracer 15N
using the function
Y=1βd,
where dis depth, Yis the cumulative proportion of tracer 15N
recovered up to depth d, and βis a fitted parameter (Gale and
Grigal, 1987). We used Microsoft Excel’s Solver function to
find the values of β, minimizing the sum of squares of the
errors in Ybetween measured and modeled values for each
plot. We used a ttest to determine if elevated CO2altered
βand used nonlinear regression to explore relationships be-
tween the vertical distribution of recovered 15N and total 15N
recovery at the ecosystem scale (mg 15N m2).
2.8 Statistical analyses
We used analysis of variance (ANOVA) to test for effects of
elevated CO2, using repeated measures to test for effects of
time and interactions between CO2and time, and split-plot
ANOVA to test for differences among soil depths. When nec-
essary, data were log-transformed (N2fixation, soil element
concentrations) to meet assumptions of ANOVA. For nodule
biomass in 1996 and 1997, we used a Kruskal–Wallis test be-
cause data did not meet assumptions of ANOVA and values
of zero precluded log-transformation. We used an αthreshold
of 0.10. We omitted one data point from the analysis of foliar
Mo of G. elliottii in 2007 based on a Grubb’s outlier test;
omitting the data point did not influence the significance of
any statistical comparison. We used simple and multiple lin-
ear regressions to explore relationships between N2fixation
and possible drivers. We also used ANOVA for the acety-
lene reduction assay to test effects of CO2, glucose, and wa-
ter (in January 2006) or phosphorus (March 2006) on rates
of N2fixation by free-living soil microorganisms. We note
that compositing soils from plots by CO2treatment com-
promised the independence of replicates within CO2treat-
ments. The absence of any significant effects of CO2on non-
symbiotic N2fixation (see below) provides some protection
from the dangers of false inference caused by pseudorepli-
cation (Hurlbert, 1984). We assessed effects of N2fixation
as a function of time since disturbance by calculating time
since disturbance as the number of years that had elapsed be-
tween the date of the measurement and the most recent dis-
turbance, whether by fire at the beginning of the experiment
or by hurricane in September 2004 (Hungate et al., 2013a).
We expressed the effect of elevated CO2on N2fixation as the
absolute difference between elevated and ambient CO2plots.
Table 1. Proportion of N derived from fixation and N concentration
in Galactia elliottii during 11 years of exposure to elevated CO2
and results from repeated measures ANOVAs for effects of time,
elevated CO2, and their interaction.
Year pFixation %N
Ambient Elevated Ambient Elevated
1996 1.37±0.16 1.20 ±0.08
1997 2.20±0.16 2.33 ±0.16
1998 0.911±0.026 0.910 ±0.026 2.13 ±0.10 1.86 ±0.10
1999 0.939±0.008 0.918 ±0.005 1.86 ±0.12 1.48 ±0.05
2000 0.932±0.006 0.875 ±0.017 1.99 ±0.14 1.95 ±0.08
2001 0.953±0.008 0.924 ±0.025 2.13 ±0.10 1.83 ±0.07
2002 0.941±0.009 0.88 ±0.012 2.26 ±0.13 1.70 ±0.12
2003 0.965±0.006 0.958 ±0.009 1.98 ±0.05 1.75 ±0.07
2004 0.964±0.008 0.908 ±0.046 1.94 ±0.06 1.79 ±0.09
2005 0.992±0.004 0.99 ±0.008 1.92 ±0.04 1.75 ±0.08
2006 0.991±0.008 0.998 ±0.001 2.05 ±0.05 1.64 ±0.14
2007 0.994±0.002 0.987 ±0.004 1.76 ±0.09 2.12 ±0.11
CO20.005 0.006
Time 0.018 <0.001
CO2×Time 0.453 0.002
3 Results
3.1 N2fixation by G. elliottii
N2fixation by Galactia elliottii varied over time (P <
0.001), with high rates in 1998 and low rates in 2000 and
2005 (Fig. 1). Elevated CO2increased N2fixation by G. el-
liottii during the first six months of experimental treatment
(Fig. 1a and as reported in Hungate et al., 1999). However,
by the third year of exposure to elevated, CO2, N2fixation
was not different between the two treatments (CO2×time
interaction, P=0.070). Subsequently (years 5–7), elevated
CO2suppressed G. elliottii N2fixation. Rates equalized
again by year 9 (2005). Accumulated over the 11-year ex-
posure period, G. elliottii fixed 3.92±0.50 g N m2year1
in the ambient CO2treatment compared to 3.51±0.71 g
Nm2year1in the elevated-CO2treatment, a nonsignif-
icant difference (repeated measures ANOVA, P=0.376).
The effect of elevated CO2dissipated with time since dis-
turbance (Fig. 1b).
G. elliottii derived nearly all of its foliar N from fixation,
increasing from 92% in 1998 to 100% from 2005 to 2007
(Table 1). Given the small range of variation in reliance on at-
mospheric N2, temporal changes in N2fixation (Fig. 1) were
driven by effects of time and treatment on the productivity of
G. elliottii. Nevertheless, the proportion of N derived from
the atmosphere by G. elliottii was sensitive to temporal vari-
ation and to the CO2treatment. Across all years, elevated
CO2reduced the reliance of G. elliottii on N from the at-
mosphere, though the effect was small, from an average of
96.9% for the ambient CO2treatment to 94.7% for the ele-
vated treatment. Foliar nitrogen percentage of G. elliotti was
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3328 B. A. Hungate et al.: Nitrogen inputs and losses in response to chronic CO2exposure
Figure 1. Rate of N2fixation (a) and effect of elevated CO2on N2
fixation as a function of time since disturbance (b) for Galactia el-
liottii during 11 years of experimental exposure to increased carbon
dioxide concentrations. For panel (a) Values are means ±standard
error of the mean (n=8) for the ambient (open circles) and elevated
(filled circles) CO2treatments. For panel (b), values are differences
of means for each year of the experiment.
lower in the elevated-CO2treatment (Table 1), an effect that
varied over time. During the first year of the experiment,
elevated CO2stimulated nodule biomass (effect of CO2in
1996, P=0.062), but elevated CO2had no effect on nodule
mass at final harvest (Fig. 2, Table 2), a pattern similar to that
found for N2fixation.
N2fixation by G. elliottii (g Nm2year1)was positively
correlated with G. elliottii foliar N concentration, negatively
correlated with the total mass of the oak leaves, negatively
correlated with herbivory, and unrelated to leaf area index
(Table 3). Therefore, as the dominant plants grew larger, N2
fixation declined, suggesting competition. The absence of
any relationship with leaf area index argues against compe-
tition for light. N2fixation also declined as foliar nitrogen
percentage declined, which can indicate limitation by non-
N nutrients (Rogers et al., 2009). Finally, high proportional
consumption of G. elliottii tissue was associated with lower
rates of N2fixation, suggesting some control of N2fixation
by herbivory (Table 3).
Table 2. Nodule mass (gm2) recovered in soil cores at the final
harvest in 2007. P values from split-plot ANOVA are shown in the
last three rows.
Depth Ambient Elevated
0–10cm 108 ±56 66 ±29
10–30cm 70 ±62 71 ±60
30–60cm 122 ±98 35 ±35
60–100cm 32 ±13 10 ±5
0–100cm 332 ±143 182 ±108
CO20.416
Depth 0.498
CO2×Depth 0.812
Table 3. Multiple regression of rates of N2fixation
(gN m2year1) as a function of foliar N concentration in
G. elliottii (gg1×100 %), herbivory (% of leaf production con-
sumed), leaf area index (m2m2), and total oak biomass (kgm2).
The overall regression is significant (F4,103 =8.543; P < 0.001;
adjusted r2=0.22).
Effect Coefficient±SE P
Constant 0.0467±0.1682 0.782
N concentration 0.1594±0.0583 0.007
Ln(Herbivory) 0.0549±0.0147 <0.001
Leaf area index 0.0194±0.0474 0.683
Oak Biomass 0.0233±0.0104 0.027
3.2 Soil-extractable micronutrient concentrations
Concentrations of soil-extractable Fe, Mo, and V declined
from 2005 to 2007 (Fig. 3, Table 4). Elevated CO2signifi-
cantly reduced soil-extractable concentrations of Mo and V,
and tended to reduce Fe. Some assays of soil P availability
collected during the experiment indicated that elevated CO2
reduced soil P availability (ion exchangeable P in 1997, ex-
tractable P in 2001), though this effect was not apparent at
the final harvest (Fig. 4). Reductions in soil element avail-
ability under elevated CO2corresponded with reduced fo-
liar concentrations in G. elliottii: elevated CO2reduced fo-
liar concentrations of Fe in 2003 and 2007 (repeated mea-
sures ANOVA (RMA), P=0.013), and, for 2003, Mo (Hun-
gate et al., 2004), although foliar P was not affected in either
year (Fig. 5; RMA, P=0.886). However, the rate of N2fixa-
tion was not related to soil-extractable nutrient concentration
(data not shown, P > 0.10 for all regressions).
3.3 Herbivory
Herbivores in this subtropical woodland consumed a higher
percentage of G. elliottii leaf production (24.0 ±1.8%) than
of oak leaf production (14.1 ±0.6 %, P < 0.001, Fig. 6). The
percentage consumed of G. elliottii was not affected by the
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B. A. Hungate et al.: Nitrogen inputs and losses in response to chronic CO2exposure 3329
Table 4. Effect of elevated CO2, time, and soil depth on extractable nutrient concentrations for the last three years of the experiment (2005,
2006, and 2007). Values are P values from repeated measures, split-plot ANOVAs testing for the main effect of CO2treatment, repeated
measures effect of the year, and split-plot effect of the depth, as well as all interactions. All data were log-transformed before analysis.
Element CO2Year Depth CO2×CO2×Year×CO2×
year depth depth Y×D
Fe 0.223 <0.001 0.056 0.731 0.720 0.035 0.417
Mo 0.024 <0.001 0.012 0.150 0.674 0.088 0.796
V 0.010 <0.001 <0.001 0.376 0.209 0.036 0.844
Figure 2. Mass of G. elliottii nodules recovered in the top 10cm
of soil in 1996 (from ingrowth soil cores) and in 2007 (from intact
cores). Values are means ±standard errors.
elevated-CO2treatment (P=0.443), nor did it vary signif-
icantly from year to year (P=0.700; no interactions were
significant in the repeated measures ANOVA; P > 0.30 for
all).
3.4 Nonsymbiotic N2fixation
Elevated CO2had no effect on acetylene reduction in soil
laboratory incubations (Tables 5 and 6). Acetylene reduc-
tion was also unresponsive to phosphorus addition or soil
water content. Acetylene reduction increased with glucose
addition, consistent with carbon limitation of heterotrophic
nonsymbiotic N2fixation in these soils.
3.5 Nitrous oxide and nitric oxide fluxes
Across all measurements conducted during the CO2en-
richment experiment, N2O efflux averaged 0.346 mg N2O-
Nm2d1for the ambient CO2treatment and 0.377mg
N2O-Nm2d1for the elevated-CO2treatment, a nonsignif-
icant difference of 0.035mg N2O-N m2d1(5 and 9 %
confidence limits, 0.070 and 0.132). Scaled to the entire
11-year period and assuming that N2O constitutes 10% of
the total N losses in denitrification (with 90% as N2pro-
duction), our best estimate is that elevated CO2increased
Figure 3. Soil-extractable Fe, Mo, and V concentrations for 0–
10cm (left three panels) and 10–30 cm (right three panels) soil
depths over the last three years of the experiment. Values are
means±standard error of the mean. Units are µgg1soil (for Fe)
and ng g1soil (for Mo and V).
losses of N2O-N by 1.4g Nm2, though this difference (el-
evated – ambient) is not significant, with 5 and 95% confi-
dence limits that span zero (2.9 to 5.3). Across all mea-
surements, NOxlosses averaged 0.075mg N m2d1for
the ambient CO2treatment and were somewhat lower for
the elevated-CO2treatment at 0.027mg N m2d1, a differ-
ence between elevated and ambient treatments of 0.047mg
N m2d1(5 and 95% confidence limits, 0.116 to
0.002mg Nm2d1). Scaled to the entire 11-year period,
the elevated-CO2-treated plots lost 0.20g N m2less NOx
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3330 B. A. Hungate et al.: Nitrogen inputs and losses in response to chronic CO2exposure
Table 5. Rates of acetylene reduction (µmolC2H2g1h1) as a proxy for nonsymbiotic N2fixation, measured in soils from the ambient-
and elevated-CO2treatments from laboratory incubations with added glucose and either added water (January 2006) or added phosphorus
(March 2006). Values are means±standard errors of the mean (n=4, January 2006; n=5, March 2006).
Glucose Ambient CO2Elevated CO2Ambient CO2Elevated CO2
µgg1
Jan 2006 3 % H2O 10 % H2O
0 21.5±3.2 23.8±3.0 20.4±2.3 21.8±2.5
324 27.4±1.4 25.8±4.7 32.4±3.2 30.0±4.5
Mar 2006 0 P 6.5 P
0 36.0±1.7 37.7±4.1 37.2±4.2 37.5±1.6
6.7 42.0±3.8 42.7±4.5 34.6±1.2 36.0±2.7
34.4 42.4±3.1 44.9±3.1 43.6±3.5 41.0±2.8
Table 6. P values from ANOVAs testing the effects of chronic CO2
exposure and short-term additions of glucose, water, and phospho-
rus on acetylene reduction. Columns under each date show P val-
ues for main effects and interactions tested on that date, H2O for
January and phosphorus for March (note: the first P value in each
column is a main effect).
Jan 2006 Mar 2006
CO20.964 CO20.727
Glucose <0.001 Glucose 0.011
CO2×glucose 0.329 CO2×glucose 0.816
H2O 0.351 P 0.151
H2O×CO20.773 P×CO20.670
H2O×glucose 0.071 P ×glucose 0.604
H2O×CO2×glucose 1.000 P×CO2×glucose 0.673
compared to the controls (5% and 95% confidence limits,
0.01 to 0.47). If these rates are typical for N2O and NOx
losses over the experiment, the elevated-CO2-treated plots
lost 1.2g Nm2more N in gaseous fluxes compared to the
ambient CO2treatment, but this difference was not signifi-
cant (3.3 to 5.3; 5 and 95% confidence limits).
3.6 15 N tracer recovery and distribution as an indicator
of N leaching
Nitrogen movement from the O and A horizons into deeper
soil (>15cm) was measured directly during the first 3years
of the experiment using resin lysimeters (Johnson et al.,
2001), with a tendency for CO2to reduce vertical N move-
ment. Yet whole-system recovery and distribution of the 15N
tracer were consistent with increased 15N losses via leach-
ing in the elevated-CO2treatment. Elevated CO2reduced to-
tal 15N recovery (Hungate et al., 2013), and of 15N that re-
mained in mineral soil, more was found deeper in the pro-
file in the elevated-CO2treatment as indicated by a higher
βvalue (β=0.81±0.01 for ambient CO2and 0.89 ±0.03
for elevated CO2,P=0.03). These findings were associ-
ated such that reduced 15N recovery occurred in plots where
Figure 4. Effect of elevated-CO2concentration on available P in
soils over time using two methods, extractable soil P (open tri-
angles) and ion-exchange resins (filled circles). Values are the ef-
fect size of elevated CO2, expressed as a percentage: (E A)/A ×
100%. Bars denote 5 and 95 % confidence limits. (Data from 1997
to 2001 were calculated from raw data reported in Johnson et
al., 2001, 2003.).
the recovered 15N was distributed deeper in the soil profile
(Fig. 7). Thus, elevated CO2reduced 15N recovery and pro-
moted 15N movement down the soil profile, consistent with
increased N leaching losses.
4 Discussion
4.1 Effects of CO2on processes regulating ecosystem N
accumulation
Findings reported here explain the absence of any stimula-
tion of N accumulation in response to 11 years of exposure
of this subtropical oak woodland to elevated carbon diox-
ide concentration (Hungate et al., 2013b). Elevated atmo-
spheric CO2either elicited no response in processes favoring
N accumulation (nonsymbiotic N2fixation) or caused only
a transient and quantitatively negligible response (symbiotic
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B. A. Hungate et al.: Nitrogen inputs and losses in response to chronic CO2exposure 3331
Figure 5. Effects of elevated CO2concentration on Mo, Fe, and P
concentrations in leaves of G. elliottii sampled in 2003 and 2007
(values for Mo and Fe from 2003 are from Hungate et al., 2004).
N2fixation). Reduced gaseous N losses from the elevated-
CO2-treated plots were observed for NOx, but NOxwas a
minor component of the ecosystem N budget such that the
changes observed were insufficient to promote N accumu-
lation. Moreover, the reduced 15N recovery and pattern of
greater 15N recovery deep in the soil indicates that elevated
CO2enhanced leaching losses of N. Our results indicate that
processes promoting N loss were more responsive to elevated
CO2than were processes promoting N accumulation.
Figure 6. Percent of leaf productivity consumed by herbivores for
the N2-fixing vine, G. elliottii, and for the three co-dominant oak
species in the ambient- and elevated-CO2-treated plots.
Figure 7. Recovery of added tracer 15N as a function of its depth
distribution. Higher βvalues indicate relatively more 15N in deeper
soil layers, whereas low βvalues indicate concentration of 15N at
the soil surface.
4.2 Effects of CO2on N2fixation
N2fixation by the leguminous vine, G. elliottii, was only
temporarily responsive to the CO2treatment (Fig. 1). Growth
and N2fixation in N2-fixing plants can respond positively
to elevated CO2(Cernusak et al., 2011), but in many cases
these responses are absent, e.g., N-fixers in temperate grass-
lands (Garten et al., 2008; Zhang et al., 2011), Alnus (Tem-
perton et al., 2003; Millett et al., 2012), and ocean cyanobac-
teria (Czerny et al., 2009; Law et al., 2012). Chronic CO2
exposure was found to reduce cyanobacterial abundance in
desert crusts (Steven, 2012). Our finding that nonsymbiotic
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3332 B. A. Hungate et al.: Nitrogen inputs and losses in response to chronic CO2exposure
N2fixation in soil was also insensitive to the CO2treatment
is consistent with observations in other forest ecosystems
(Hofmockel and Schlesinger, 2007). In general, responses
appear to be more muted under field conditions in long-term
experiments than in short-term (van Groenigen et al., 2006;
Leuzinger et al., 2011), so our finding that elevated CO2did
not increase N2fixation is not unusual. Nitrogen availabil-
ity, light limitation, herbivory, and non-N nutrient limitation
are plausible explanations for the absence of a significant re-
sponse of N2fixation after the first year of the experiment.
4.3 Nitrogen availability
Increased N availability is well known to reduce N2fixation
(Streeter, 1988), as uptake of N from the soil is energeti-
cally favorable to fixation. In the scrub oak ecosystem stud-
ied here, elevated CO2increased plant N uptake from soil, in
part by enhanced turnover of soil organic matter (Carney et
al., 2007; Langley et al., 2009; Hungate et al., 2013b). The
slight reduction in the reliance of G. elliottii on atmospheric
N in response to elevated CO2(Fig. 1, Table 1) is consistent
with a CO2-stimulation of soil N availability. N2fixation can
decline with shading during canopy development, yet in this
system leaf area index was not a significant predictor of N2
fixation (Table 3), possibly because the N2-fixers are vines,
capable of climbing rapidly to the top of the short canopy to
alleviate light limitation (Kurina and Vitousek, 1999). On the
other hand, the negative relationship between N2fixation and
total oak biomass (Table 3) suggests competition for other
resources between the oaks and the N2-fixing vine, possibly
competition for nutrients.
4.4 Non-N nutrients
When supplied with sufficient non-N nutrients, N2-fixing
plants will increase nodule biomass and rates of N2fixation
(van Groenigen et al., 2006; Rogers et al., 2009), while at
the same time maintaining foliar N concentration in response
to elevated CO2. Under nutrient-limiting conditions, foliar N
declines, nodule growth diminishes, and rates of N2fixation
are depressed (Rogers et al., 2009), a pattern consistent with
the positive correlation between foliar N concentration and
N2fixation shown here (Table 3).
The responses of N2fixation and growth of N-fixing plants
to elevated CO2may depend on availability of non-N nutri-
ents. In our system, there was some evidence that the avail-
ability of soil P declined, particularly during the first 6years
of the experiment (Fig. 4, and see Johnson et al., 2001, 2003,
a response also observed in a rice–wheat rotation, see Ma et
al., 2007). However, reduced P availability is not a univer-
sal response to increased CO2concentration (Dijkstra et al.,
2012; Khan et al., 2008, 2010). Other elements critical for N2
fixation such as Mo, Fe, and V either declined significantly in
soil and in foliage or showed a tendency to decline (Figs. 3,
5), suggesting their role in modulating the response of N2
fixation to elevated CO2. Natali et al., 2009 found that total
soil metal content increased in the surface soils of a loblolly
pine and sweetgum plantations, though total metal concen-
tration may be a poor indicator of extractable metal concen-
tration and metal availability to plants. Foliar concentrations
of most metal elements were substantially lower at this scrub
oak site compared to the loblolly pine and sweetgum planta-
tions (Natali et al., 2009) (Duval et al., 2014). Sandy texture
and low pH are two soil properties typically associated with
low metal availability because sandy soils have low ion ex-
change capacity and because metal availability is very sensi-
tive to pH (Vlek and Lindsay, 1977; Sposito, 1984; Goldberg
et al., 1996; Kabata-Pendias, 2001). Thus, compared to other
ecosystems, this subtropical woodland may be more likely to
exhibit micronutrient limitation of ecosystem processes, such
as N2fixation.
Several experiments have demonstrated the importance of
P availability for the responses of N2fixation to elevated
CO2: the stimulation of growth and N2fixation by elevated
CO2was higher with P addition for clover (Edwards et al.,
2006), Azolla (Cheng et al., 2010), soybean (Lam et al.,
2012), chickpea, and field pea (Jin et al., 2012). The CO2-
stimulation of N-fixer growth and N2fixation in several
grasslands experiments was higher with P addition compared
to controls without supplemental P (Niklaus et al., 1998;
Grunzweig and Korner, 2003). In general, with supplemen-
tal nutrients added, elevated CO2often increases N2fixation
(Lee et al., 2003; Otera et al., 2011), but in experiments with-
out exogenous nutrient addition, N2fixation is generally not
responsive to elevated CO2(van Groenigen et al., 2006).
4.5 Herbivory
Herbivory has been postulated to reduce N2fixation in
ecosystems because of preferential feeding on the more nutri-
tious tissue of N2-fixers compared to other plants (Vitousek
and Howarth, 1991). This hypothesis is consistent with our
finding that herbivores in this subtropical woodland con-
sumed a larger proportion of G. elliottii leaf production than
leaf production of oaks (Fig. 6). Herbivory could also limit
the response of N2fixation to environmental change, if graz-
ing on the tissue of N2-fixers increases. Increasing herbivore
pressure on the dominant N2-fixing vine in our experiment
was associated with reduced rates of N2fixation (Table 3),
which supports this idea. Elevated CO2decreased the propor-
tion of leaves with herbivore damage in this scrub oak wood-
land (Stiling and Cornelissen, 2007), as has been found in
other systems (Lindroth, 2010; Robinson et al., 2012). Insect
herbivores often respond to reduced leaf nitrogen concentra-
tion by consuming more leaf tissue (Stiling et al., 2003). In
the scrub oak woodland studied here, this response resulted
in no effect of elevated CO2on the proportion of N-fixer leaf
production consumed by herbivory (Fig. 6).
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B. A. Hungate et al.: Nitrogen inputs and losses in response to chronic CO2exposure 3333
4.6 C limitation of nonsymbiotic N2fixation
Our finding that acetylene reduction increased with glucose
addition suggests C limitation of heterotrophic N2fixation in
soil (Tables 5 and 6), as has been found previously (Billings
et al., 2003). Neither P addition nor altered soil water content
influenced rates of acetylene reduction, indicating that these
factors were not limiting to soil N2fixation.
4.7 Temporal dynamics
Evidence for a single, dominating mechanism underlying the
response of N2fixation to elevated CO2was weak in our ex-
periment, possibly because the process is limited by multiple
factors whose influences shift over time. The experimental
site was struck by a hurricane in 2004 (Li et al., 2007a, b),
a disturbance that preceded the high soil nutrient concentra-
tions observed in 2005, which subsequently declined in 2006
and 2007 (Fig. 3). This disturbance also preceded a large pos-
itive response of oak production to elevated CO2found both
above- and belowground (Day et al., 2013; Hungate et al.,
2013a), as well as the disappearance of the CO2suppression
of G. elliottii growth and N2fixation (Fig. 1). Consistent
with this, the positive correlation between foliar Mo concen-
tration and N2fixation rates found in the two years before the
hurricane (Hungate et al., 2004) was no longer apparent by
the final harvest (regression between foliar [Mo] and N2fix-
ation for 2007, r2=0.138; P=0.638). Yet, rates of N2fix-
ation were low after the hurricane disturbance in both treat-
ments (Fig. 1), and there was no association between soil-
extractable micronutrient availability and rates of N2fixation
during the period 2005–2007; therefore, the availability of
micronutrients, if they played a role, were not the only factor
limiting N2fixation at this time.
The temporal variation in the response of N2fixation, in
particular the finding that initially strong positive responses
dissipate with time (Leuzinger et al., 2011), may be a gen-
eral feature of global change experiments. While elevated
CO2can stimulate N2fixation in some species and under
some conditions, responses in many field studies are far more
muted (van Groenigen et al., 2006) consistent with findings
reported here. Thus, increased N2fixation is not a certain
outcome of rising atmospheric CO2concentrations.
4.8 N Losses
N fixation is the major biological process mediating N inputs
to terrestrial ecosystems, but N losses through gaseous and
leaching pathways exert an equally important influence on
total N pool size. Our finding that elevated CO2had no sig-
nificant effect on N2O production in this subtropical wood-
land is consistent with several past studies finding no effect
of CO2on N2O production (Mosier et al., 2002;Phillips et
al., 2001), though increased N2O production has been de-
tected in others (Hagedorn et al., 2000; Ineson et al., 1998;
Kammann et al., 2008; Lam et al., 2010; Smith et al., 2010).
On average, elevated CO2tends to increase soil N2O emis-
sions by around 20% (van Groenigen et al., 2011), a larger
stimulation than the nonsignificant increase of 8% we ob-
served in this subtropical woodland. The trend for elevated
CO2to reduce NOxlosses from this subtropical woodland
indicates that NOxlosses were likely not the major path-
way of increased N loss from this system in response to el-
evated CO2. By contrast, the concurrence of reduced tracer
15N recovery (Hungate et al., 2013b) and deeper distribution
of tracer 15N throughout the soil profile (Fig. 7) supports the
notion that elevated CO2stimulated N leaching in this exper-
iment. Increased leaching with elevated CO2has been ob-
served (Korner and Arnone, 1992) and may be caused by a
combination of increased plant water-use efficiency result-
ing in greater downward water flux through the soil profile
(Jackson et al., 1998), along with increased turnover of soil
organic matter in response to rising CO2(Drake et al., 2011;
Hungate et al., 2013b). Some experiments have documented
reduced N leaching with elevated CO2(Johnson et al., 2004),
so our finding of increased leaching is likely not universal.
Furthermore, during the first three years of this experiment,
we found no effect of elevated CO2on vertical movement
of N from the surface to deeper soil layers (Johnson et al.,
2001). The use of the 15N tracer to integrate the cumula-
tive effects of elevated CO2on nitrogen losses in this exper-
iment may be both more temporally integrative and sensitive
to changes in the ecosystem-scale distribution and retention
of N in response to elevated CO2.
4.9 Summary
Eleven years of chronic exposure to increased CO2concen-
trations elicited disequilibrium in the N cycle, with increased
rates of internal N transformations, no change in N inputs,
and increased N losses. Elevated CO2accelerated the rate
of soil N mineralization (Langley et al., 2009; McKinley et
al., 2009), which likely contributed to increased N uptake by
plants (Hungate et al., 2013). Nitrogen losses also increased,
with increased turnover of N through plant tissues, as evi-
denced by the increased 15N dilution in plants (Hungate et
al., 2013) along with no change in net plant N capital (Hun-
gate et al., 2013). Elevated CO2also appeared to enhance N
losses at the scale of the soil profile: the pattern of lower 15N
recovery in plots exhibiting greater downward movement of
15N in the soil profile suggests increased leaching. Thus, pro-
cesses that make nutrients available to plants can also pro-
mote nutrient losses. Finally, we found no evidence that el-
evated CO2enhanced N inputs via N2fixation. Together,
these results describe an ecosystem in which more rapid cy-
cling of N with elevated CO2is unlikely to be sustained.
These empirical findings contrast with model projections in
which elevated CO2enhances N2fixation and reduces leach-
ing (Thornley and Cannell, 2000). Given the strong influence
of N cycling and N accumulation on the C cycle, the changes
www.biogeosciences.net/11/3323/2014/ Biogeosciences, 11, 3323–3337, 2014
3334 B. A. Hungate et al.: Nitrogen inputs and losses in response to chronic CO2exposure
in N cycling reported here, if general, would tend to dampen
the biosphere’s capacity to assimilate and store C in the face
of rising atmospheric CO2concentrations (Thornton et al.,
2007; Churkina et al., 2009; Arneth et al., 2010; Zaehle and
Dalmonech, 2011).
Acknowledgements. Funding for this long-term experiment was
provided by the US Department of Energy (DE-FG-02-95ER61993,
and DE-SC0008260) and the US National Science Foundation
(DEB-9873715, DEB-0092642, and DEB-0445324, and an NSF
IGERT fellowship to B. D. Duval), with technical and infrastructure
support from the National Aeronautics and Space Administra-
tion and the Kennedy Space Center. The Thermo X Series 2
quadrupole ICPMS at NAU was supported through funds from
the Arizona Technology Research and Innovation Fund (to M. E.
Ketterer). We thank Bert Drake for the vision to establish and over-
see the experiment, and David Johnson, Tom Powell, Troy Seiler
and Hans Anderson for their commitment to its upkeep and success.
Edited by: M. Williams
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... A full discussion of the limitations of the NA methodology including errors associated with the B value can be found in Unkovich et al. (2008). In a long-term experiment involving annual measurement for 10 years (1998 to 2007 inclusive) of P atm of a native leguminous vine, Elliott's milkpea, Hungate et al. (2014) used the δ 15 N of the legume (−2.2‰) measured at the start of the experiment as the B value. The experiment began by applying a single dose of ( 15 NH 4 ) 2 SO 4 (0.18 g N m −2 at 0.999 atom fraction 15 N; Hungate et al., 2004) but over the years the isotope became diluted so that at the last harvest the δ 15 N signatures in the leaves of the oak reference plants fell to 84.3 ± 4.2‰. ...
... On the other hand, one of the species (Amorpha) which showed a positive response at low N gave a negative response to e[CO 2 ] at high N (West et al., 2005). Although a native leguminous vine, Elliott's milkpea, showed year-to year variation in P atm , there was little effect of e[CO 2 ] on annual estimates of symbiotic dependence in a long term (10 y) experiment (Hungate et al., 2014). The forage legumes sub-clover (Lilley et al., 2001) and barrel medic (Guo et al., 2013; Lam et al., 2012a ) all showed marked positive responses to e[CO 2 ], while white clover had a marked negative response (Watanabe et al., 2013). ...
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Methodologies based on (15)N enrichment (E) and (15)N natural abundance (NA) have been used to obtain quantitative estimates of the response of biological N2 fixation (BNF) of legumes (woody, grain and forage) and actinorhizal plants grown in artificial media or in soil exposed to elevated atmospheric concentrations of carbon dioxide e[CO2] for extended periods of time, in growth rooms, greenhouses, open top chambers or free-air CO2 enrichment (FACE) facilities. (15)N2 has also been used to quantify the response of endophytic and free-living diazotrophs to e[CO2]. The primary criterion of response was the proportional dependence of the N2-fixing system on the atmosphere as a source of N. i.e. the symbiotic dependence (Patm). The unique feature of (15)N-based methods is their ability to provide time-integrated and yield-independent estimates of Patm. In studies conducted in artificial media or in soil using the E methodology there was either no response or a positive response of Patm to e[CO2]. The interpretation of results obtained in artificial media or with (15)N2 is straight forward, not being subject to the assumptions on which the E and NA soil-cultured methods are based. A variety of methods have been used to estimate isotopic fractionation attendant on the NA technique, the so-called 'B value', which attaches a degree of uncertainty to the results obtained. Using the NA technique, a suite of responses of Patm to e[CO2] has been published, from positive to neutral to sometimes negative effects. Several factors which interact with the response of N2-fixing species to e[CO2] were identified.
... Experimental field studies on BNF under eCO 2 are rare and inconclusive, presumably owing to the regulatory impacts of micronutrients and vegetation dynamics. Field experiments have found very large eCO 2 responses of BNF in fertilized grasslands but also moderate responses that declined and became negative over time in subtropical oak woodlands (Hungate et al., 2004(Hungate et al., , 2014. Heterotrophic fixation was shown not to be affected by eCO 2 at the Duke FACE experiment (Hofmockel and Schlesinger, 2007). ...
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Including a terrestrial nitrogen (N) cycle in Earth system models has led to substantial attenuation of predicted biosphere-climate feedbacks. However, the magnitude of this attenuation remains uncertain. A particularly important, but highly uncertain process is biological nitrogen fixation (BNF), which is the largest natural input of N to land ecosystems globally. In order to quantify this uncertainty, and estimate likely effects on terrestrial biosphere dynamics, we applied six alternative formulations of BNF spanning the range of process formulations in current state-of-the-art biosphere models within a common framework, the O-CN model: a global map of static BNF rates, two empirical relationships between BNF and other ecosystem variables (net primary productivity (NPP) and evapotranspiration), two process-oriented formulations based on plant N status, and an optimality-based approach. We examined the resulting differences in model predictions under ambient and elevated atmospheric [CO2] and found that the predicted global BNF rates and their spatial distribution for contemporary conditions were broadly comparable, ranging from 95 to 134 Tg N yr−1 (median 119 Tg N yr−1), despite distinct regional patterns associated with the assumptions of each approach. Notwithstanding, model responses in BNF rates to elevated levels of atmospheric [CO2] (+200 ppm) ranged between −4 Tg N yr−1 (−3 %) and 56 Tg N yr−1 (+42 %) (median 7 Tg N yr−1 (+8 %)). As a consequence, future projections of global ecosystem carbon storage (+281 to +353 Pg C, or +13 to +16 %), as well as N2O emission (−1.6 to +0.5 Tg N yr−1, or −19 to +7 %) differed significantly across the different model formulations. Our results emphasize the importance of better understanding the nature and magnitude of BNF responses to change-induced perturbations, particularly through new empirical perturbation experiments and improved model representation.
... We aimed to test the prediction that elevated CO 2 reduces N losses by measuring N accumulation (defined as N inputs—N losses) and retention (defined as the proportion remaining of a known amount of added 15 N) in a N-limited ecosystem with a relatively open N cycle, where external flux rates are large relative to internal flux rates and changes in N pools should be more readily detectable. Effects of elevated CO 2 on N pools are equivocal, with some empirical evidence supporting models that predict ecosystem N accumulation (Iversen et al., 2012) while other evidence indicates mixed (Reich & Hobbie, 2013) or even negative effects (Hungate et al., 2014). Many CO 2 enrichment studies focus on particular pools that may not necessarily capture the trajectory of total ecosystem N (e.g. ...
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Biogeochemical models that incorporate nitrogen (N) limitation indicate that N availability will control the magnitude of ecosystem carbon uptake in response to rising CO2 . Some models, however, suggest that elevated CO2 may promote ecosystem N accumulation, a feedback that in the long term could circumvent N limitation of the CO2 response while mitigating N pollution. We tested this prediction using a nine-year CO2 xN experiment in a tidal marsh. Although the effects of CO2 are similar between uplands and wetlands in many respects, this experiment offers a greater likelihood of detecting CO2 effects on N retention on a decadal timescale because tidal marshes have a relatively open N cycle and can accrue soil organic matter rapidly. To determine how elevated CO2 affects N dynamics, we assessed the three primary fates of N in a tidal marsh: (1) retention in plants and soil, (2) denitrification to the atmosphere, and (3) tidal export. We assessed changes in N pools and tracked the fate of a (15) N tracer added to each plot in 2006 to quantify the fraction of added N retained in vegetation and soil, and to estimate lateral N movement. Elevated CO2 alone did not increase plant N mass, soil N mass, or (15) N label retention. Unexpectedly, CO2 and N interacted such that the combined N+CO2 treatment increased ecosystem N accumulation despite the stimulation in N losses indicated by reduced (15) N label retention. These findings suggest that in N-limited ecosystems, elevated CO2 is unlikely to increase long-term N accumulation and circumvent progressive N limitation without additional N inputs, which may relieve plant-microbe competition and allow for increased plant N uptake.
... However, the human activities have led to a rapid increase in atmospheric CO 2 concentration currently reaching 400 ppm (Mauna Loa Observatory, 2014), with the possibility of disturbing the exudation rate of plants (Phillips et al., 2011). The mineralization of SOM in ecosystems exposed to elevated CO 2 is intensified (Langley et al., 2009;Phillips et al., 2012) leading, in several cases, to net decrease in SOM stock (Carney et al., 2007;Langley et al., 2009) and an increase in N leaching (Liu et al., 2008;Hungate et al., 2014). These findings Fig. 6. ...
... However, the human activities have led to a rapid increase in atmospheric CO 2 concentration currently reaching 400 ppm (Mauna Loa Observatory, 2014), with the possibility of disturbing the exudation rate of plants (Phillips et al., 2011). The mineralization of SOM in ecosystems exposed to elevated CO 2 is intensified (Langley et al., 2009;Phillips et al., 2012) leading, in several cases, to net decrease in SOM stock (Carney et al., 2007;Langley et al., 2009) and an increase in N leaching (Liu et al., 2008;Hungate et al., 2014). These findings Fig. 6. ...
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Molybdenum (Mo) is critical for the function of enzymes related to nitrogen cycling. Concentrations of Mo are very low in sandy, acidic soils, and biologically available Mo is only a small fraction of the total pool. While several methods have been proposed to measure plant-available Mo, there has not been a recent comprehensive analytical study that compares soil extraction methods as predictors of plant Mo uptake. A suite of five assays [total acid microwave digestion, ethylenediamenetetraaacetic acid (EDTA) extraction, Environmental Protection Agency (EPA) protocol 3050B, ammonium oxalate extraction, and pressurized hot water] was employed, followed by the determination of soil Mo concentrations via inductively coupled mass spectroscopy. The concentrations of soil Mo determined from these assays and their relationships as predictors of plant Mo concentration were compared. The assays yielded different concentrations of Mo: total digest > EPA > ammonium oxalate ≥ EDTA > pressurized hot water. Legume foliar Mo concentrations were most strongly correlated with ammonium oxalate–extractable Mo from soils, but an oak species showed no relationship with any soil Mo fraction and foliar Mo. Bulk fine roots in the 10- to 30-cm soil horizon were significantly correlated with the ammonium oxalate Mo fraction. There were significant correlations between ammonium oxalate Mo and the oxides of iron (Fe), manganese (Mn), and aluminum (Al). Results suggest that the ammonium oxalate extraction for soil Mo is the best predictor of plant-available Mo for species with high Mo requirements such as legumes and that plant-available Mo tracks strongly with other metal oxides in sandy, acidic soils.
Chapter
Trace gas exchange between soil-plant systems and the atmosphere is a complex phenomenon driven by a different set of physical, chemical, and biological processes for each chemical species and each environment. This chapter provides sufficient insight into the use of chamber systems to enable potential users to identify applications where the approach is appropriate. It examines the most consequential sources of error in chamber-based flux measurements, and suggests schemes for prioritizing and minimizing these errors. Both open- and closed-chamber systems have been criticized as susceptible to bias resulting from physical and biological disturbances associated with the measurement process. The exchange rate of a trace gas across the soil-atmosphere boundary is largely a function of its diffusion coefficient and concentration gradient between sites of production and the soil surface. Sources of bias associated with the analytical determination of trace gas concentration depend on both the properties of the gas and the technique chosen for its analysis. © 1993 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, 677 S. Segoe Rd., Madison, WI 53711, USA.
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Future high levels of atmospheric carbon dioxide will increase biomass production of terrestrial plants, however depletion of soil mineral nutrients may act as a negative feedback to increased growth. To test this, an ecosystem phosphorus budget was calculated in poplar grown under field conditions at ambient and elevated atmospheric CO2 for 5 years. The pools of total, plant available, weatherable and organic P were estimated, as well as the P storage in tree biomass components. While as a non-significant increase in amount of P taken up by the trees we observed, plant available P pools in the soil increased significantly. An increase in all soil P extractions was seen, with the greatest increase in an acid soluble P fraction which is considered to be the weatherable fraction. The formation of this P fraction may be biogenically driven and this additional P probably originates from weathering of occluded mineral pools.
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Sixteen legumes were grown in N-free media so that N was supplied entirely by symbiotic N2 fixation. The plant tissues were analyzed for natural ¹⁵N abundance (expressed as δ¹⁵N per mil relative to air N2) with a ratio mass spectrometer. The nodules of desmodium, centro, siratro, soybean and winged bean showed high enrichment in ¹⁵N (∼+9‰), while red clover showed slight enrichment (∼+2‰). The nodules of 9 other forage legumes (Townsville stylo, white clover, alsike clover, common vetch, Chinese milk vetch, senna, alfalfa, ladino clover, and hairy vetch) showed little enrichment in ¹⁵N. In all the legumes investigated, particularly in the ureide-transporting plants such as desmodium, centro, siratro, soybean, winged bean and field bean, the δ¹⁵N value of the shoots was negative (∼−3.2‰). The δ¹⁵N value of the shoots in winged bean and field bean varied by about 1‰ depending on the Rhizobium strains used. The isotopic mass balance of 13 legumes indicated that isotopic fractionation occurs during N2 fixation by the legume-rhizobia symbiosis with a preference for ¹⁴N over ¹⁵N, resulting in a δ¹⁵N value of −0.2 to −2‰ in the whole plant. The results indicate that ¹⁵N/¹⁴N isotopic discrimination with a preference for the lighter atom may occur in both N2 fixation and export of fixed N from nodules.