Environ. Sci. Technol. 2010, 44, 9265-9271 
Phosphorus Transformations during 
Decomposition of Wetland 
Macrophytes 
ALEXANDER   W.   CHEESMAN,*' + 
BENJAMIN   L.   TURNER,* 
PATRICK  W.   INGLETT,+  AND 
K.   RAMESH   REDDY + 
Wetland Biogeochemistry Laboratory, Soil and Water 
Science Department, University of Florida, Gainesville, 
Florida, United States, and Smithsonian Tropical Research 
Institute, Apartado 0843-03092, Balboa, Ancon, 
Republic of Panama 
Received  July   20,   2010.   Revised   manuscript   received 
October 12, 2010. Accepted November 4, 2010. 
The microbially mediated transformation of detrital P entering 
wetlands has important implications for the cycling and long- 
term sequestration of P in wetland soils. We investigated changes 
in P forms in sawgrass {Cladium jamaicense Crantz) and 
cattail {Typha domingensis Pers.) leaf litter during 15 months 
of decomposition at two sites of markedly different nutrient 
status within a hard-water subtropical wetland (Water 
Conservation Area 2A, Florida). Leaf litter decomposition at 
the nutrient enriched site resulted in net sequestration of P from 
the environment in forms characteristic of microbial cells 
(i.e., phosphodiesters and pyrophosphate). In contrast, low P 
concentrations at the unenriched site resulted in little or no net 
sequestration of P, with changes in P forms limited to the 
loss of compounds present in the initial leaf litter. We conclude 
that under nutrient-rich conditions, P sequestration occurs 
through the accumulation of microbially derived compounds 
and the presumed concentration of endogenous macrophyte P. 
Under nutrient-poor conditions, standing P pools within 
wetland soils appear to be independent of the heterotrophic 
decomposition of macrophyte leaf litter. These conclusions have 
important implications for our ability to predict the nature, 
stability, and rates of P sequestration in wetlands in response 
to changes in nutrient loading. 
Introduction 
Decomposition of macrophytes influences the biocycling, 
retention, and downstream release of nutrients in wetland 
systems. The often cited model of wetland macrophyte 
decomposition set out by Webster and Benfield (1) identifies 
three distinct yet overlapping phases of decomposition: (i) 
an initial rapid leaching of water-soluble components, (ii) 
microbial colonization and decomposition, and (ill) me- 
chanical and invertebrate mediated fragmentation of ma- 
terial. Of these, the second stage - microbial colonization 
and decomposition - involves the most dynamic alteration 
of nutrient forms. Whether microbes mineralize or sequester 
inorganic nutrients during decomposition of senesced bio- 
* Corresponding    author    phone:    +507    212-8236;    e-mail: 
CheesmanA@si.edu. 
+
 University of Florida. 
* Smithsonian Tropical Research Institute. 
10.1021/es102460h       ? 2010 American Chemical Society 
Published on Web 11/19/2010 
mass has implications for both the internal nutrient dynamics 
of wetlands (2) and overall nutrient sequestration, an 
important function of wetlands in the landscape (3). 
Phenological characteristics of the litter appear to influ- 
ence initial decomposition processes, after which decom- 
position rates are increasingly governed by gross nutrient 
ratios (4) and the nutrient status of the environment (5, 6). 
Anthropogenic perturbation of nutrient availability in aquatic 
systems can cause shifts in trophic status (7), changes in the 
composition of plant communities (8, 9), and alteration of 
microbial eco-physiological processes (e.g. rets 10 and 11). 
Observed alterations in catabolic processes appear to follow 
predicted changes in resource reallocation (12) with an 
increase in bioavailable P leading to a reduction in investment 
in P acquisition, e.g. a reduced release of extracellular 
phosphatase enzymes by microbes (13, 14). 
Although numerous studies have investigated factors that 
influence changes in tissue total P during wetland macrophyte 
decomposition (e.g. refs 15?17), transformations of the 
functional forms of P during decomposition are less well 
studied. Since the chemical nature of P forms impacts both 
abiotic stabilization in the environment (18) and biological 
turnover (19), it is vital to understand how environmental 
conditions impact the composition of P forms present during 
the decay continuum. For terrestrial systems, studies of 
temporal changes in P functional groups in decomposing 
plant tissue indicates both the accumulation of microbial 
phosphodiesters (20) and the synthesis of polyphosphates 
by fungi (21). Other studies have sought to partition biogenic 
P in soils into various microbial and plant sources (22), and 
transposed position within a soil profile for time, with the 
aim of tracking general transformations within the organic 
matter of forest soils (23). In wetlands, attempts have been 
made to characterize leachate from macrophyte leaves (24), 
but changes in P forms in the autochthonously derived 
organic matter of wetland systems remain poorly understood. 
The objective of this study was to use solution 31P nuclear 
magnetic resonance (NMR) spectroscopy to characterize the 
functional forms of P throughout a macrophyte decay 
continuum. In addition, we aimed to determine how litter 
quality and site characteristics regulate changes in both total 
P and its functional composition. It was hypothesized that 
in a P-enriched setting, the accumulation of microbial derived 
P and reduced catabolic breakdown of macrophyte P would 
result in net sequestration of certain P groups, whereas in 
an oligotrophic setting there would be close coupling of 
biogenic P production and its subsequent hydrolysis, limiting 
the accumulation of microbially derived P forms. 
Materials and Methods 
Site Description. Water Conservation Area 2A (WCA-2A) is 
a diked and hydraulically controlled 424 km2 portion of the 
northern Everglades, characterized as a freshwater peat 
system underlain by limestone bedrock. Historically, pro- 
ductivity in the northern Everglades has been limited by P 
availability, but anthropogenic loading from upstream 
agricultural practices has resulted in a distinct nutritional 
and concomitant vegetation gradient in WCA-2A (e.g. refs 25 
and 26). There is a distinct transition from native Everglades 
marsh dominated by sawgrass (Cladium jamaicenseCmntz), 
to dominance by cattail (Typha domingensis Pers.) in areas 
impacted by nutrient-rich inflow water (9). The nutrient- 
enriched areas have increased rates of heterotrophic de- 
composition (5,11) and a reduced extracellular phosphatase 
activity (10, 14). 
VOL. 44, NO. 24, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY ? 9265 
TABLE 1. Site Characteristics for Enriched and Unenriched 
Study Sites within WCA-2A" 
WCA-2A location 
enriched site      unenriched site 
latitude (N) 
longitude (W) 
distance from inflow 
structure (km) 
average water depth 
(cm; max, min)6 
overlying water 
26? 21.230' 
80? 20.967' 
1.93 
26? 16.382' 
80? 21.502' 
10.05 
12.8(11.9,14.0)   11.9(11.0,13.6) 
detritus 
soil (0- 1.5 cm) 
total P (fig P !_-') 
ortho P(jjg P L"1) 
total P (fig g-1) 
total C (mg g_1) 
total N (mg g~1) 
total P^gg- 
total C (mg g 
total N (mq q 
1) 
52.4 ? 6.7 
27.8 ? 4.4 
1334 ?904 
414 ?27 
26 ? 12 
1312 ?40 
421 ? 90 
26 ?3 
9.6 ?0.9 
1.7 ? 1.3 
206 ? 53 
387 ? 16 
14?3 
468 ? 40 
406 ? 21 
26 ?2 
a
 Detritus and soil samples average (n = 4) ? one 
standard deviation. Overlying water characteristics based 
upon published values. b Stage data from South Florida 
Water management (23/01/03 through 04/21/04). Sampling 
stations WCA2E1 and WCA2U1. "Site water characteristics. 
South Florida Water management (6/21/94 through 9/27/ 
94). Sampling staions WCA2E1 and WCA2E5. Available 
through DBHYDRO (http://my.sfwmd.gov/dbhydroplsql/). 
Two sites along a well documented P gradient were 
selected for this study: one enriched site 1.93 km from the 
northern inflow structure S10-C and a second within an area 
considered unenriched by P loading (Table 1). Site detritus 
and surface soil (core diameter 15 cm x 1.5 cm deep) were 
sampled {n = 4) from the study locations prior to experimental 
setup and analyzed for total C, N, P, and P composition by 
solution 31P NMR spectroscopy. 
Litterbag Study. Senescent leaf material of both Typha 
and Cladium were collected in bulk from locations proximate 
to both the enriched and unenriched sites. Material included 
only standing dead intact lamina of unknown age but 
presumed < 2 y. Bulk material was rinsed of adhering 
particulates, cut into 10 cm sections, and dried at 60 ?C to 
a constant weight. Litter was analyzed for total C, N, P, and 
P forms by solution 31P NMR spectroscopy. Litter quality was 
determined by a modified proximate forage analysis {27) 
utilizing the semiautomated Ankom A200 (Ankon Technol- 
ogy, Pairport, NY). 
Litterbags (15 cm x 15 cm) were constructed from gray 
polyethylene mesh (1 mm) allowing entry of micro fauna 
and local microbial assemblages. Approximately 15 g dry 
weight of litter was sealed into individual litter bags and 
placed (January 2003) on the surface of both the enriched 
and unenriched sites, secured in place with polyethylene 
stakes. Three replicate bags were recovered from each site 
after 16, 33, 75, 204, and 454 days. Litterbags were returned 
on ice to the laboratory, where they were gently washed with 
deionized water to remove surface debris, frozen, and 
lyophilized. Litter was then removed from the bags for mass 
loss determination and ground to pass a 2 mm mesh using 
a Wiley mini mill (Thomas Scientific, Swedesboro, NJ). 
Samples of coarsely ground material were then analyzed for 
total P and P composition and after further grinding, total 
C and N. All samples were stored in sealed containers in the 
dark at ambient lab conditions until analysis. 
Analysis of Biogeochemical Properties. Initial site de- 
tritus, surface soils, and recovered litter were analyzed for 
total N and C simultaneously using a Costech Model 4010 
Elemental Analyzer (Costech Analytical Industries, Inc., 
Valencia, CA). Loss on ignition (LOI) and total P were 
determined by a modified ashing method (28) using 550 ?C 
for 4 h, dissolution of the ash in 1 M H2S04, and subsequent 
P determination by discrete autoanalyzer (AQ2+, SEAL 
Analytical, Fareham, UK) and standard molybdate colorim- 
etry (USEPA, 1993). 
Phosphorus Composition. Initial site detritus, surface 
soil, and selected leaf litter samples recovered from the 
decomposition study were analyzed by solution 31P NMR 
spectroscopy. Given practical and financial constraints of 
analyzing multiple samples with 31P NMR spectroscopy, only 
Cladium and Typha sourced from the enriched site were 
analyzed, as these were expected to provide clear P signals 
during NMR spectroscopy. Samples were analyzed from time 
steps that were considered able to provide insight into 
mechanistic processes (i.e., initial and final material and after 
maximum total P leaching). Additional samples from the 
enriched site were analyzed to provide information on P 
accumulation rates. 
Phosphorus Extraction in NaOH-EDTA. A standard 
alkaline extraction [29] was applied to detritus, soil, and litter 
samples using combined field replicates and a 1:30 solid to 
solution (0.25 M NaOH plus 50 mM EDTA) ratio. Samples 
were shaken at ambient room temperature for 3 h and then 
centrifuged (Sorvall RC6 centrifuge, SLA 1500 Rotor; Thermo 
Fisher Scientific, Waltham, MA, USA) at 6500 rpm for 10 
min. After centrifugation a subsample (20 mL) of the 
supernatant was removed to a scintillation vial and combined 
with 1 mL of 50 mg P L ' methylenediphosphonic acid (MDP) 
as an internal standard. Mixed samples were immediately 
frozen (?80 ?C) and lyophilized prior to NMR spectroscopy. 
A second subsample was analyzed for total P (NaOHTP) by 
a modified double-acid digest using H2S04 and HN03 and 
a discrete molybdate colorimetric detection method. 
Residual P (total P - NaOHTP) is by definition unidentified, 
and its chemical stability is presumed to indicate its 
recalcitrance in the environment. For mineral soils it has 
been assigned as recalcitrant organic {29) and alkali-stable 
(acid-soluble) inorganic P (30). Although there is little 
information on its chemical nature in wetland soils, given 
relatively large concentrations of acid- extractable P recovered 
from detritus {31) and surface soils {32) in WCA-2A it seems 
likely that at least a proportion of the residual P was inorganic 
Ca-phosphates. 
Solution 31P Spectra Acquisition. Spectra were acquired 
using a Bruker Avance 500 Console with a Magnex 11.75 
T/54 mm magnet using a 10 mm BBO Probe. Lyophilized 
samples (~300 mg) were resuspended in 0.3 mL of D20 and 
2.7 mL of a solution containing 1 M NaOH and 0.1 M EDTA, 
vortexed, and then transferred to a 10 mm tube. Spectra 
acquisition was carried out at a stabilized 25 ?C with a 
calibrated (~30?) pulse length, a zgig pulse program, and a 
2 s Tl delay. Results presented here are of ~30,000 scans 
accumulated as three sequential experiments, with FIDs 
summed post acquisition by Bruker proprietary software. 
Spectra interpretation was conducted using NMR Utility 
Transform Software (Acorn NMR Inc., Livermore, CA). After 
applying 15 Hz line broadening, spectra were referenced and 
integrated against the internal standard, MDP, set as 17.46 
ppm (compared to externally held 85% phosphoric acid). 
Integration over set spectral windows were chosen to 
correspond with known P bonding environments {33). The 
region between 8 and 3 ppm was further elucidated on spectra 
processed using 3 Hz line broadening by a deconvolution 
subroutine applied to identify and quantify orthophosphate 
(6.21 ? 0.02 ppm) separately from phosphomonoesters. 
Data Analysis. All statistical tests were performed in SPSS 
for windows version 17.0.0 statistical software (SPSS Inc. 
2008). Mass remaining, P concentration, and mass of P were 
analyzed by a four-way univariate ANOVA using site of 
decomposition (site), species of litter (species), source of 
litter (source), and time as independent variables. Given the 
homogeneous nature of initial material,  time = 0 was 
9266 ? ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 24, 2010 
Enriched Site 
c 
1' 
co 
E 
m 
w 
w 
CD 
100- 
1 80- 
60- f * 
+ 1 
? 40- 
Unenriched Site 
oo- 
s 
80- ? E 
m 
60- ? a ? 
40- 
i 1 1                           ! 
? 
i             i 
? Enriched Cladium 
? Enriched Typha 
C Unenriched Cladium 
C Unenriched Typha 
400 CD 
0) 
200 CD E 
"co 
0 c 
O) 
-200 CL 
O) 
3. 
400 </) (/) 
CD 
E 
200 CL 
c 
0 CD 
c 
CD 
_c 
-200 O 
0   100  200  300  400  500  0  100  200  300  400  500 
Time (days) 
FIGURE 1. Time series of mass remaining (left panel) and change in total mass of P (right panel) in four litter types placed at 
distinct sites within WCA-2A. Symbols represent averages (i? = 3) with error bars showing one standard error. 
two 
excluded from the statistical analysis. Resulting model 
residuals were analyzed via P?P plot and visual inspection 
for normality. If required, original data were normalized using 
a natural log transformation. Significant results are reported 
alongside their calculated partial eta2 values. Given com- 
plications of interpreting multiple interaction factors, non- 
significant higher order terms are not presented here. Due 
to practical constraints, solution 31P NMR data were acquired 
for pooled samples and as such are without a measure of 
variance at individual time steps. All integrated results using 
MDP as an internal standard were within ?20% of determined 
NaOHTP concentrations. Calculated rates of accumulation 
of specific P forms are based upon simple linear regression 
after initial leaching and equilibration period. 
Results 
Initial Litter Material. Typha litter material from both the 
unenriched and enriched study sites contained greater 
concentrations of total N (4.5 and 5.4 mg g-1) and P (135 and 
261 ug g-1) and had a higher loss on ignition (indicating less 
biosilica) than Cladium (4.1 and 3.8 mg g-1 total N and 46 
and 171 fig g-1 total P in unenriched and enriched sites, 
respectively) (Table SI). Although Typha litter had a notice- 
ably larger proportion of neutral-detergent extractable C 
(Table SI), the proportion of lignin and cellulose was similar, 
with LCI (Lignin to Cellulose Index) between 0.170 and 0.197 
for all litters sampled. Within species, there was a distinct 
difference between material collected at the enriched and 
unenriched site, with Cladium showing an approximate 4-fold 
increase in total P and Typfta showing a 2-fold increase. Using 
both species, collected from enriched and unenriched settings 
allowed four litter types to be used within the subsequent 
decomposition study. 
Mass Loss. Between 36 and 70% of original mass of litter 
remained after 15 months in the field (Figure 1). Rates of 
decomposition varied significantly (ANOVA p < 0.001) 
between litter types, in terms of both macrophyte species 
and litter source, as well as between study sites (Table S2). 
In addition, decay rate constants based upon a simple 
exponential decay (Table S3) followed expected patterns, 
with increased rates of mass loss in response to increased 
litter quality and exogenous nutrients {16). However, de- 
composition rate constants were relatively constrained, 
varying from 0.00275 day ^ for enriched Typha material at 
the enriched site to 0.00093 day ^ for unenriched Cladium 
at the unenriched site. Significant interaction terms between 
time and both litter species and source demonstrated the 
continued and differential influence of initial material 
characteristics on the long-term stability of organic matter 
(Table S2). 
Phosphorus Content. Total P concentrations of decom- 
posing leaf litter (Figure SI) changed significantly with time. 
At the enriched site all litter types except unenriched Cladium 
showed an initial drop in P, as expected from a rapid leaching 
event, but then subsequently increased to between 5 and 10 
times the original P concentration. In contrast, at the 
unenriched site after an initial decrease in P, concentrations 
did rise but with the exception of unenriched Cladium never 
recovered to their initial levels (Figure SI). Univariate four- 
way ANOVA (Table S4) showed significant differences de- 
pendent not only upon the site of decomposition (p< 0.001) 
but also on the nature of the initial litter, as shown by highly 
significant effect of both litter species (p < 0.001) and source 
(p < 0.001). In addition, significant interaction terms (litter 
quality with time) suggest different responses in P concen- 
tration of litter types over time. 
Changes in P concentration during decomposition may 
result from two distinct processes: a change in litter mass 
that results in alteration of the endogenous P concentration 
or the loss or gain of P from the environment. This distinction 
was explored by plotting changes in the mass of P over time, 
standardized for the initial amount of litter used in each 
litter bag (Figure 1). A conservative breakpoint at 33 days 
(indicated by a dashed line in Figure 1) marked the switch 
from initial abiotic processes and site equilibration to long- 
term microbial action. From 33 days to the end of the study 
there was a distinction in changes in mass of P between 
sites. Linear regression demonstrated that the mass of P in 
all litter types at the enriched site showed a significant (p < 
0.001) increase of between 0.447 and 0.685 fig P g-1 initial 
material day-1 between day 33 and the end of the study (Table 
VOL. 44, NO. 24, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY ? 9267 
Initial Typha 
Chemical shift (ppm) Chemical shift (ppm) 
Enriched site Unenriched site 
FIGURE 2. Solution 31P NMR spectra of enriched material through the decay continuum at enriched and unenriched sites. From 
initial litter material, through to litter material after 445 days in the field, to site detritus and surface soils. Spectra plotted using 15 
Hz line broadening and scaled using height of internal standard MDP (i) = 17.46 ppm). Peaks were assigned to the following: A = 
orthophosphate, B = phosphomonoesters, C = phosphodiesters, D = DNA, E = pyrophosphate, F = midchain polyphosphate 
residues. 
TABLE 2. Phosphorus Forms As Determined by Solution 31P NMR Spectroscopy in Decomposing Leaf Litter, Site Detritus, and 
Surface Soils Sampled on the 10/20/03 at Both Enriched and Unenriched Sites within WCA-2A" 
% total P 
the field total P other 
site           sample (days) W P g ) phosphonate orthophosphate phosph omonoesters DNA phosphodiesters pyrophosphate polyphosphate residual 
enriched      Cladium 0 171 35 13 3 1 3 46 
16 104 27 26 2 2 1 42 
454 795 20 16 10 5 4 45 
Typha 0 261 39 23 5 4 6 25 
16 237 <0.5 18 33 2 12 1 2                 35 
454 1257 12 15 8 4 4 57 
detritus - 899 16 12 7 2 5 4                 58 
soil - 1311 18 10 5 1 1 64 
unenriched Cladium 0 171 35 13 3 1 3 46 
33 67 33 15 1 9 14 28 
454 85 29 29 7 5 nd 30 
Typha 0 261 39 23 5 4 6 25 
33 195 19 30 1 5 4 41 
454 239 16 18 4 1 2 59 
detritus - 221 27 9 13 3 5 56 
soil - 421 18 7 6 1 2 66 
a
 nd = not detected. 
S5). In contrast, at the unenriched site the mass of P in both 
enriched Cladium and unenriched Typha did not change 
significantly with time. The P content of unenriched Cladium 
increased slightly yet significantly (0.037 fig P g ^ initial 
material day-1; p = 0.001) during decomposition, whereas 
in enriched Typha there wasasignificant(p<0.001) decrease 
in the mass of P. 
Phosphorus Forms. Analysis of initial enriched leaf litter 
demonstrated a range of P forms present within both Cladium 
and Typha (Figure 2, Table 2). In both cases, NaOH-EDTA 
extractable P was dominated by orthophosphate (35 and 39% 
of P in Cladium and Typha, respectively). In addition, 
considerable amounts of phosphomonoesters (13 and 23% 
total litter P), phosphodiesters (4 and 9% total litter P), and 
inorganic pyrophosphate (2 and 6% total litter P) were 
identified. It should be noted that there was probably an 
inherent bias in the analysis, because some phosphodiesters 
(i.e., RNA and some phospholipids) decompose to phosph- 
9268 ? ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 24, 2010 
Cladium Typha 
0 
V) 
T3 
0 
LU 
0 
c/5 
0 
0 
c 
3 
o 5 & o o o 
co      ^r      LO 
Time (days) 
FIGURE 3. Changes in the proportions of major P pools identified by solution 31P NMR in enriched macrophyte leaf litter over the 
course of 454 days of decomposition in WCA-2A. 
omonoesters in alkaline solution during extraction and 
analysis (33). 
Comparison of initial P composition in the in situ detritus 
and soil at the two study sites (Figure 2, Table 2) demonstrated 
distinct differences in the forms and proportions of P 
identified. Standing detritus and surface soil from both sites 
contained orthophosphate, phosphomonoesters, phosphod- 
iesters (dominated by DNA), and pyrophosphate. However, 
only detritus from the enriched site contained long-chain 
inorganic polyphosphate (~3% of total P), indicated by mid- 
chain phosphate residues at approximately ?20 ppm (33). 
The proportion of total P identified by solution 31P NMR was 
higher in detritus than in soil at both sites. There was a distinct 
shift in the ratio of phosphomonoesters to phosphodiesters 
in material from the enriched and unenriched sites, from 
1.35 to 0.58 in detritus and 1.69 to 0.90 in surface soil, 
indicating a greater proportion of P identifiable as phos- 
phodiesters within detritus and at unenriched sites. Signals 
within the phosphomonoester regions indicative of the 
phosphomonoester myo-inositol hexakisphosphate were not 
detected in any sample. This is consistent with other studies 
of calcareous freshwater wetlands (34,35), although inositol 
phosphates could have been present at concentrations below 
the limit of detection by solution 31P NMR spectroscopy (36). 
Over the course of the decomposition study there was a 
convergence in the proportion of P forms present based upon 
the site of litter decomposition (Figures S2 and S3). Identifi- 
able P lost during equilibration (presumably by leaching) at 
both the enriched and unenriched site was dominated by 
orthophosphate, although in the enriched site this was offset 
by the accumulation of certain organic P forms. Since 
concentrations of phosphomonoesters increased or remained 
relatively stable in all litters between initial material and 
samples at days 16 and 33 (Figure 3), there was an increase 
in their proportional contribution to the total P of plant 
detritus (Table S6). 
Recovery of total litter P by NaOH-EDTA extraction 
averaged 56% across all samples. Residual P was a relatively 
stable proportion of the total P in Cladium litter and an 
TABLE 3 Coefficients ? One Standard Deviation of Linear 
Increases in Concentrations of Major P Forms Identified 
within Leaf Litter during Decomposition at the Enriched Study 
Site'' 
orthophosphate 
phosphomonoesters 
phosphodiesters 
pyrophosphate 
Cladium 
0.292?0.02c 
0.244?0.20fa 
0.265?0.39fa 
0.072?0.006fa 
Typha 
0.256?0.018h 
0.260?0.054fa 
0.285?0.057fa 
0.099?0.006h 
" Units fiQ P g~1 day"1. b p < 0.05. c p < 0.001. 
increasing proportion of P in Typha material at the enriched 
site, although in both cases the overall increase in total P 
resulted in an increase in the concentration of residual P. 
Trace concentrations of phosphonates and mid-chain poly- 
phosphate were detected in one sample (Typha from the 
enriched site after 16 d) but may have originated from 
adhering phytoplankton (37). After the initial equilibration, 
litter at the enriched site accumulated all forms of P identified 
by 31P NMR analysis, in direct contrast to the unenriched 
site, where there was little change in P composition after the 
initial leaching period (Figure 3). The increase in concentra- 
tion of P forms at the enriched site showed a significant 
linear response (Table 3), with most major forms showing 
a net change of between 26 and 107 mg P kg-1 y_1. Changes 
in pyrophosphate concentration appeared to be distinct from 
other major forms, showing a significant (p < 0.05) linear 
response at approximately a third of the rate of change for 
phosphomonoesters, phosphodiesters, or orthophosphate 
concentrations. 
Discussion 
Plant litter decomposition rates determined in this study 
over the course of 15 months are generally lower than values 
reported for other herbaceous litters (38) but correspond 
well with previous data from WCA-2A. For example, Debusk 
VOL. 44, NO. 24, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY ? 9269 
and Reddy (5) found simple decay rate constants of ap- 
proximately 0.003 d_1 for Typha decomposing under P-rich 
conditions. Rates of mass loss were similar to previously 
observed patterns, with higher litter quality (as determined 
by structural C and nutrient content) and higher environ- 
mental nutrient availability both resulting in an increased 
rate of mass loss. 
Over the course of the study, total P concentrations within 
litter at the two sites showed distinctly different trajectories. 
At the enriched site, total P concentration in all litters 
increased, resulting in an average molar C:P ratio of ~488 
after 15 months. In contrast, P concentrations at the 
unenriched site generally did not return to those of the initial 
material entering the system, with an average molar ratio of 
~4150 after 454 days. Although a conventional model of litter 
decomposition would assume a continued sequestration of 
P at both sites in response to C:P stoichiometry, analysis of 
changes in mass of P (Figure lb) suggests that while microbial 
activity at the enriched site resulted in the net sequestration 
of P from the endogenous environment, at the unenriched 
site there was little net gain. This interpretation assumes 
that mass loss due to fragmentation is minimal and may 
represent an underestimation of actual P accumulation in 
both sites. Yet previous studies have also suggested that 
oligotrophic systems such as the unenriched Everglades may 
present an environmental gradient in which P sequestration 
predicted from detritus C:P ratios is unlikely to occur. Indeed, 
Quails and Richardson (2000) (17) demonstrated that an 
environmental P concentration of 5 fig P L_1 in the water 
column resulted in the net loss of P to the environment from 
both Typha and Cladium leaf litter during decomposition. 
Initial changes in litter P composition as a result of labile 
P leaching corresponds to the identification of water extract- 
able forms by He et al. (2009) (39), who showed a predomi- 
nance of orthophosphate but additional leaching of organic 
P from plant material. The apparent stability of phosph- 
omonoesters during initial leaching may be due to the 
recalcitrant nature of phosphomonoesters remaining in plant 
material after nutrient resorption and senescence of leaves 
or due to the rapid synthesis of new phosphomonoesters in 
establishing microbial populations. In addition to leaching 
from the plant biomass, consideration should be given to 
the fact that some compounds (e.g., pyrophosphate) maybe 
lost from endophytic and saprotrophic fungi during initial 
preparation and site equilibration (40, 41). Indeed, the use 
of standing dead biomass of unknown age leaves us unable 
to determine alterations of P composition during these initial 
stages of the decay continuum (41). 
Initial abiotic leaching is often considered rapid (42), yet 
given an inability to interpolate data we drew a conservative 
breakpoint at 33 days, marking the switch from initial abiotic 
processes and site equilibration to long-term microbial 
processes. After 33 days this microbial activity at the enriched 
site resulted in the accumulation of all forms of P identified 
by solution 31P NMR spectroscopy. The estimated rates of 
change in P concentrations (Table 3) include contributions 
from both original and newly accumulated compounds. For 
example, the increase in recalcitrant P concentration (up to 
8-fold) is not accounted for by the passive accumulation of 
initial endogenous recalcitrant P and suggests a mechanism 
by which additional recalcitrant P is being accumulated. This 
may represent either recalcitrant biogenic P or alkaline stable 
(acid-soluble) inorganic P. Given the calcareous conditions 
of WCA-2A it seems likely that the later accumulated via the 
precipitation of Ca-phosphate (e.g., hydroxyapatite) (17). It 
is interesting to note that the proportion of total P determined 
as 'inorganic' in a previous study (31) of surface material in 
WCA-2A is quantitively similar (36-50%) to the residual 
fraction determined in this study. 
Differences in both C quality and total P of initial material 
appeared to influence the P composition of litter material 
collected at the enriched site, with Typha consisting of more 
labile forms. However, changes in P composition during 
decomposition appeared to be independent of initial ma- 
terial. Rather, changes were dependent upon site charac- 
teristics, with the most abundant P forms showing similar 
changes in concentration at the enriched site for both 
Cladium and Typha litter. Fungal biomass is a major 
contributor to heterotrophic decomposition in emergent 
herbaceous systems (41) and although litter quality can affect 
the composition of the microfungal community their gen- 
eralist nature may result in a similar response to the 
exogenous nutrient availability (43). 
Within the enriched study site, the P composition of 
decomposing litter appears to be on a trajectory toward that 
determined in standing detritus and surface soil. In contrast, 
litter material within the unenriched site after 454 days of 
decomposition did not appear to reflect standing detritus. 
This could reflect differences in the role of macrophyte litter 
across the northern Everglades. Within nutrient-impacted 
portions of WCA 2A, surface substrate originates from Typha 
detritus and a high standing population of heterotrophic 
microbes, while oligotrophic regions often include only 
minimal Cladium fragments but a significant contribution 
from the periphyton community (31). Further work would 
be needed to determine the role of these additional sources 
of biogenic P in determination of P forms seen within wetland 
substrates. 
Acknowledgments 
We thank Jim Rocca for NMR analytical support. The project 
was supported by a grant from the USDA-CREES National 
Research Initiative (No. 2004-35107-14918) and the External 
User Program of the National High Magnetic Field Laboratory 
administered through the Advanced Magnetic Resonance 
Imaging and Spectroscopy (AMRIS) facility of the McKnight 
Brain Institute University of Florida. 
Supporting Information Available 
Three figures and six tables, specifically, change in P 
concentrations, complete 31P NMR spectra, ANOVA tables, 
model coefficients, and additional leaf litter characterization. 
This material is available free of charge via the Internet at 
http://pubs.acs.org. 
Literature Cited 
(1) Webster, J. R.; Benfield, E. F. Vascular plant breakdown in fresh- 
water ecosystems. Annu. Rev. Ecol. Syst. 1986, 17, 567-594. 
(2) Reddy, K. R.; Wetzel, R. G.; Kadlec, R. H., Biogeochemistry of 
phosphorus in wetlands. In Phosphorus: agriculture and the 
environment, Sims, J. T., Sharpley, A. N., Eds.; American Society 
of Agronomy: Madison, USA, 2005; pp 263-316. 
(3) Alvarez, J. A.; Becares, E. Seasonal decomposition of Typha 
latifolia in a free-water surface constructed wetland. Ecol. Eng. 
2006, 28, 99-105. 
(4) Enriquez, S.; Duarte, C. M.; Sandjensen, K. Patterns in decom- 
position rates among photosynthetic organisms - the impor- 
tance of detritus C-N-P content. Oecologia 1993, 94, 457-471. 
(5) DeBusk, W. P.; Reddy, K. R. Litter decomposition and nutrient 
dynamics in a phosphorus enriched everglades marsh. Bio- 
geochemistty 2005, 75,217-240. 
(6) Rejmankova, E.; Sirova, D. Wetland macrophyte decomposition 
under different nutrient conditions: relationships between 
decomposition rate, enzyme activities and microbial biomass. 
Soil Biol. Biochem. 2007, 39, 526-538. 
(7) Khan, F. A.; Ansari, A. A. Eutrophication: An ecological vision. 
Bot. Rev. 2005, 71, 449-482. 
(8) Hagerthey, S. E.; Newman, S.; Rutchey, K; Smith, E. P.; Godin, 
J. Multiple regime shifts in a subtropical peatiand: community 
specific threshold to europhication. Ecol. Monogr. 2008, 78, 
547-565. 
9270 ? ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 24, 2010 
(9) Vaithiyanathan, P.; Richardson, C. J. Macrophyte species changes 
in the Everglades: examination along a eutrophication gradient. 
/. Environ. Qual. 1999, 28, 1347-1358. 
(10) Corstanje, R.; Reddy, K. R.; Prenger, J. P.; Newman, S.; Ogram, 
A. V. Soil microbial eco-physiological response to nutrient 
enrichment in a sub-tropical wetland. Ecol. Indie. 2007, 7,277- 
289. 
(11) Wright, A. L.; Reddy, K. R. Heterotrophic microbial activity in 
northern Everglades wetland soils. SoilSci. Soc. Am. J. 2001, 65, 
1856-1864. 
(12) Sinsabaugh, R. L.; Moorhead, D. L. Resource allocation to 
extracellular enzyme prodcution- A model for nitrogen and 
phosphorus control of litter decomposition. SoilBiol. Biochem. 
1994, 26, 1305-1311. 
(13) Newman, S.; McCormick, P. V.; Backus, J. G. Phosphatase 
activity as an early warning indicator of wetland eutrophi- 
cation: problems and prospects. /. Appl. Phycol. 2003, 15, 45- 
59. 
(14) Wright, A. L.; Reddy, K. R. Phosphorus loading effects on 
extracellular enzyme activity in Everglades wetland soils. Soil 
Sci. Soc. Am. J. 2001, 65, 588-595. 
(15) Brinson, M. M. Decomposition and nutrient exchange of litter 
in an alluvial swamp forest. Ecology 1977, 58, 601-609. 
(16) Corstanje, R.; Reddy, K. R.; Portier, K. M. Typha latifolia and 
Cladium jamaicense litter decay in response to exogenous 
nutrient enrichment. Aquat. Bot. 2006, 84, 70-78. 
(17) Quails, R. G.; Richardson, C. J. Phosphorus enrichment affects 
litter decomposition, immobilization, and soil microbial phos- 
phorus in wetland mesocosms. Soil Sci. Soc. Am. J. 2000, 64, 
799-808. 
(18) Cell, L.; Barberis, E., Abiotic stabilization of organic posphorus 
in the environment. In Organic phosphorus in the environment, 
Turner, B.L., Frossard, E., Baldwin, D.S., Eds.; CABI Publishing: 
Wallingford, UK, 2005; pp 113-132. 
(19) Quiquampoix, H.; Mousain, D., Enzymatic hydrolysis of organic 
phosphorus. In Organic phosphorus in the environment, Turner, 
B. L., Frossard, E., Baldwin, D. S., Eds.; CABI Publishing: 
Wallingford, UK, 2005; pp 89-112. 
(20) Miltner, A.; Haumaier, L.; Zech, W. Transformations of phos- 
phorus during incubation of beech leaf litter in the presence of 
oxides. Eur. J. Soil Sci. 1998, 49, 471-475. 
(21) Koukol, O.; Novak, P.; Hrabal, R.; Vosatka, M. Saprotrophic fungi 
transform organic phosphorus from spruce needle litter. Soil 
Biol. Biochem. 2006, 38, 3372-3379. 
(22) Makarov, M. I.; Haumaier, L.; Zech, W.; Marfenina, O. E.; 
Lysak, L. V. Can 31P NMR spectroscopy be used to indicate 
the origins of soil organic phosphates. SoilBiol. Biochem. 2005, 
37, 15-25. 
(23) Gressel, N.; McColl, ]. G; Preston, C. M.; Newman, R. H.; Powers, 
R. F. Linkages between phosphorus transformations and carbon 
decomposition in a forest soil. Biogeochemistry 1996, 33, 97- 
123. 
(24) Pant, H. K; Reddy, K. R. Hydrologic influence on stability of 
organic phosphorus in wetland detritus./. Environ. Qual. 2001, 
30, 668-674. 
(25) fensen, ]. R.; Rutchey, K; Koch, M. S.; Narumalani, S. Inland 
wetland change detection in the Everglades Water Conservation 
Area 2A using a time-series of normalized remotely-sensed data. 
Photogramm. Eng. Remote Sens. 1995, 61, 199-209. 
(26) Vaithiyanathan, P.; Richardson, C. I. Nutrient profiles in the 
Everglades: examination along the eutrophication gradient. Sci. 
Total Environ. 1997, 205, 81-95. 
(27) Goering, H. K; Soest, P. J. VAN Forage fiber analyses. (Apparatus, 
reagents, procedures, and some applications). In Agriculture 
Handbook, United States Department of Agriculture, U.S. 
Agricultural Research Service: WA, 1970; p 20. 
(28) Saunders, W. M. H.; Williams, E. G. Observations on the 
determination of total organic phosphorus in soils. /. Soil Sci. 
1955, 6, 254-267. 
(29) Cade-Menun, B. J., Using phosphorus 31 nuclear magnetic 
resonance spectroscopy to characterize organic phosphorus in 
environmental samples. In Organic phosphorus in the environ- 
ment, Turner, B. L., Frossard, E., Baldwin, D. S., Eds.; CABI 
Publishing: Wallingford, UK, 2005; pp 21-44. 
(30) Turner, B. L.; Condron, L. M.; Richardson, S. J.; Peltzer, D. A; 
Allison, V I. Soil organic phosphorus transformations during 
pedogenesis. Ecosystems 2007, 10, 1166-1181. 
(31) Wright, A. L.; Reddy, K. R. Catabolic diversity of periphyton and 
detritus microbial communities in a subtropical wetland. 
Biogeochemistry 2008, 89, 199-207. 
(32) Reddy, K. R.; Wang, Y.; Debusk, W. F.; Fisher, M. M.; Newman, 
S. Forms of soil phosphorus in selected hydrologic units of the 
Florida Everglades. Soil Sci. Soc. Am. J. 1998, 62, 1134-1147. 
(33) Turner, B. L.; Mahieu, N.; Condron, L. M. Phosphorus-31 nuclear 
magnetic resonance spectral assignments of phosphorus com- 
pounds in soil NaOH-EDTA extracts. Soil Sci. Soc. Am. J. 2003, 
67, 497-510. 
(34) Turner, B. L.; Newman, S. Phosphorus cycling in wetland soils: 
the importance of phosphate diesters. /. Environ. Qual. 2005, 
34, 1921-1929. 
(35) Turner, B. L.; Newman, S.; Newman, J. M. Organic phosphorus 
sequestration in subtropical treatment wetlands. Environ. Sci. 
Technol. 2006, 40, 727-733. 
(36) El-Rifai, H.; Heerboth, M.; Gedris, T. E.; Newman, S.; Orem, W.; 
Cooper, W. T. NMR and mass spectrometry of phosphorus in 
wetlands. Eur. J. Soil Sci. 2008, 59, 517-525. 
(37) Eixler, S.; Karsten, U; Selig, U. Phosphorus storage in Chlorella 
vulgaris (Trebouxiophyceae, Chlorophyta) cells and its depen- 
dence on phosphate supply. Phycologia 2006, 45, 53-60. 
(38) Fennessy, M. S.; Rokosch, A.; Mack, ]. ]. Patterns of plant 
decomposition and nutrient cycling in natural and created 
wetlands. Wetlands 2008, 28, 300-310. 
(39) He, Z. Q.; Mao, I.; Honeycutt, C. W.; Ohno, T.; Hunt, J. F.; Cade- 
Menun, BJ. Characterization of plant-derived water extractable 
organic matter by multiple spectroscopic techniques. Biol. Fertil. 
Soils 2009, 45, 609-616. 
(40) Kominkova, D.; Kuehn, K. A; Busing, N.; Steiner, D.; Gessner, 
M. O. Microbial biomass, growth, and respiration associated 
with submerged litter of Phragmites australis decomposing in 
a littoral reed stand of a large lake. Aquat. Microb. Ecol. 2000, 
22, 271-282. 
(41) Kuehn, K.A.; Lemke, M. L; Suberkropp, K; Wetzel, R. G. Microbial 
biomass and production associated with decaying leaf litter of 
the emergent macrophyte Juncus effusus. Limnol. Oceanogr. 
2000, 45, 862-870. 
(42) Davis, S. E.; Childers, D. L.; Noe, G. B. The contribution of 
leaching to the rapid release of nutrients and carbon in the 
early decay of wetland vegetation. Hydrobiologia 2006,569,87- 
97. 
(43) Thormann, M. N.; Currah, R. S.; Bayley, S. E. Succession of 
microfungal assemblages in decomposing peatland plants. Plant 
Soil 2003, 250, 323-333. 
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