Patterns and controls of anaerobic soil respiration and methanogenesis following extreme restoration of calcareous subtropical wetlands

Geoderma 245–246 (2015) 74–82

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Patterns and controls of anaerobic soil respiration and methanogenesis following extreme restoration of calcareous subtropical wetlands Cassandra A. Medvedeff ⁎, Kanika S. Inglett, Patrick W. Inglett 2181 McCarty Hall A, P.O. Box 110290, Department of Soil and Water Science, University of Florida, Gainesville, FL 32511, USA

a r t i c l e

i n f o

Article history: Received 18 July 2014 Received in revised form 23 January 2015 Accepted 27 January 2015 Available online 3 February 2015 Keywords: Carbon CO2 Everglades Marl CH4 Phosphorus

a b s t r a c t Wetland restoration is globally important to reestablish functions such as carbon (C) sequestration; however, restoration activities (e.g., land clearing) can affect anaerobic C cycling and greenhouse gas production. In this study, we compared enzyme activity (ß-glucosidase) and anaerobic production of carbon dioxide (CO2) and methane (CH4) in soils of reference wetlands and those restored (in 2000 and 2003) by complete soil removal (CSR) after farming in an area of Everglades National Park. We found elevated gaseous production and enzyme activity in restored relative to reference wetlands. Correlations with measured activities suggest soil phosphorus (P) and C content explain site differences in C processing. We tested the potential for P and labile C limitation through direct additions of P and glucose. Production of CO2 and CH4 in both systems was stimulated by glucose; however, P only stimulated CH4 production in reference wetlands. This suggests that decomposition is limited by labile C regardless of restoration, and that soil P concentrations potentially regulate CH4 production. Additional studies are necessary to establish CH4 production pathways and investigate the interaction between P and C availability. Following CSR, wetlands are hypothesized to shift from nitrogen (N) to P limitation; therefore, CH4 production may decrease with restoration age while approaching P limitation. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Wetlands have decreased by approximately 50% worldwide (Zedler and Kercher, 2005). Subsequently, restoration efforts have increased to reestablish essential wetland functions and achieve a “no net loss” standard. An assortment of restoration efforts have been tested and vary depending on site characteristics and restoration goals. These approaches range from less invasive techniques such as manipulating vegetation (Andersen et al., 2006; Smith et al., 2011), to more intensive techniques such as restoring hydrology (Acreman et al., 2007) or ecosystem maintenance through prescribed fire (Doren et al., 1991; Hamman et al., 2008). Wetlands which have accumulated excess nutrients, such as agricultural lands, are commonly targeted for mitigation (Aldous et al., 2007; Van Dijk et al., 2004). One of the restoration techniques to remove excess nutrients is topsoil removal (Klimkowska et al., 2007; Ross et al., 1982; Tallowin and Smith, 2001; Verhagen et al., 2001) which can include the extreme condition of complete soil removal (CSR) to bedrock (Dalrymple et al., 2003). Soil disturbance following this restoration technique can have drastic effects on ecosystem function. Changes in vegetation (composition and abundance) or organic matter (OM) structure during soil

⁎ Corresponding author at: Chapman University, Department of Earth and Environmental Sciences, 1 University Drive, Orange, CA 92866, USA. E-mail address: [email protected] (C.A. Medvedeff).

http://dx.doi.org/10.1016/j.geoderma.2015.01.018 0016-7061/© 2015 Elsevier B.V. All rights reserved.

disturbance may disrupt the ecosystem C balance (source/sink) (Höper et al., 2008; Kimmel and Mander, 2010) altering greenhouse gas (CO2 and CH4) emissions (Andersen et al., 2006; Bortoluzzi et al., 2006; Jauhiainen et al., 2008). With soil nutrient availability as a known control of CH4 production in some ecosystems (Smith et al., 2007; Wright and Reddy, 2001) changes in nutrient content following soil disturbance should be evaluated. Extensive research has been conducted and is ongoing in the greater Everglades (Florida, U.S.A.) to restore ecological function following alteration of hydrology and agricultural nutrient enrichment (Richardson, 2008). However, within Everglades National Park, the extreme restoration technique of complete soil removal is being tested in the Hole-inthe-Donut (HID) region as an attempt to remove and discourage regrowth of invasive vegetation (Dalrymple et al., 2003). The HID is a collection of calcareous marl prairie wetlands which were heavily farmed from the early 1900s through 1975 (Smith et al., 2011). Continuous fertilization and soil disturbance following the use of mechanical farming equipment altered the soil aeration status and structure while drastically increasing soil nutrient concentrations. Once farming ceased, these lands were invaded by Schinus terebinthifolius Raddi and vegetation typical of the oligotrophic Everglades such as Cladium ceased to exist (as described by Ewel et al., 1982). Sites were mechanically-cleared as an extreme restoration approach and were allowed to re-vegetate naturally and soils which were high in available P are now hypothesized to shift from nitrogen (N) to P limitation while approaching the native/reference status (Inglett et al., 2011; Liao et al., 2014).

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Previous work by Smith et al. (2007) investigated CH4 production following CSR in the HID, concluding that restored sites were elevated in CH4 production compared to those of undisturbed reference areas. Regulators of CH4 production including nutrient limitation warrants further investigation. In wetland soils, methanogens rely on simple substrates released from respiring or fermenting microbes (Conrad, 1999), a process which is often controlled by enzyme activity and the degradation of complex OM (Sinsabaugh et al., 1993). However, an additional control on microbial decomposition can be a nutrient limitation in some ecosystems (Hogg et al., 1994). Increasing P concentrations have been linked to elevated CH4 production (Pivničková et al., 2010) although this is not always the case (Bridgham and Richardson, 1992; Drake et al., 1996) suggesting the relationship between these parameters is complex and may depend on initial soil P concentrations and OM quality. The potential coupling of P biogeochemistry and C dynamics has important implications for wetland restoration and the subsequent ability for newly restored wetlands to function as a sink/source of C greenhouse gases. Wetlands are the largest natural source of CH4 (Lelieveld et al., 1998), with tropical and southern wetlands accounting for more than 70% of this CH4 source (Solomon et al., 2007). Therefore, understanding how restoration can affect the C balance and subsequent greenhouse gas release from these ecosystems is crucial. For this reason, we conducted the following study to investigate the patterns of anaerobic greenhouse gas production (CO2 and CH4) following the extreme restoration technique of complete soil removal. Following restoration, soils were enriched in P and organic carbon (OC) relative to the unamended reference soil. Thus, our objective was to investigate the relationship between soil nutrients (P and C) and anaerobic C gas production (CO2 and CH4). To determine if differences in microbial activity between restored and reference soils were related to anaerobic C gas production we investigated the importance of microbial biomass carbon (MBC) and β-glucosidase enzyme activity (as a source of available non-methanogenic substrates) as a predictor of site C gas production. In addition, we assessed the relationship between greenhouse gas production and nutrient limitation in both unamended soils and those incubated with C or P in two separate microcosm experiments. We hypothesized that CO2 and CH4 production would be elevated in restored relative to reference soils and elevated P (decreased microbial nutrient limitation) would correspond with heightened C processing.

2. Methods 2.1. Site description The Hole-in-the-Donut (HID) region of Everglades National Park (Miami Dade County, Florida, U.S.A.) consists of abandoned crop fields overlying a marl prairie wetland ecosystem (Fig. 1). These soils are Entisols which consist of Biscayne and Perrine marl with poor to poorly drained characteristics (USDA, 1996). Restoration of this area began in 1989 with mechanical removal of invasive vegetation followed by complete soil removal to bedrock (Dalrymple et al., 2003; Inglett and Inglett, 2013). In the current study, plots restored in years 2000 (Res00) and 2003 (Res03) were compared to a reference (never farmed) marl prairie wetland located within the HID vicinity. Based on restoration time and prior fertilization history each site varied in biogeochemical properties including soil nutrients, soil depth, and microbial parameters (Table 1). Vegetation in the reference site is typical of oligotrophic Everglades dominated by Cladium jamaicense Crantz (Loveless, 1959) with Muhlenbergia capillaris, Andropogon and Schoenus spp. In contrast, restored sites are dominated by a mixture of invasive and undesirable vegetation including Baccharis halimifolia and to a lesser extent Ludwigia spp., Salix sp. and Typha domingenesis.

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2.2. Soil sampling To encompass elevation variability, five approximately equidistant stations were selected along a 1.5 km transect within each of Res00, Res03, and reference wetlands (Fig. 1, Liao and Inglett, 2012). Soil depth at each site was measured with a soil probe (30 total readings per sampling location, Table 1). In October 2009 (wet season), three composite samples (5 grabs each) from 0 to 5 cm depth (or until bedrock) were collected using a spatula from each transect sampling location (15 composite samples per site). To estimate bulk density additional samples (n = 3) were taken using a sharpened steel tube. Soils were placed on ice and returned to the University of Florida for processing within two days of collection. Soil samples were sieved through a 2 mm mesh to remove rocks and roots. Representative aliquots of field-moist soil were removed from each sample and analyzed for multiple microbial properties including microbial biomass carbon (MBC), MBN and MBP, BGA, anaerobic respiration (CO2) and methanogenic potentials (CH4). Soil moisture content averaged 54 and 46% for restored and reference wetlands, respectively. Separate soil aliquots were dried at 105 °C for three days, hand ground with a mortar and pestle and used for determination of total carbon, nitrogen, and phosphorus (TC/TN/TP). 2.3. Soil nutrients and microbial biomass analyses Soil nutrient analyses were conducted by the Wetland Biogeochemistry Laboratory at the University of Florida (Gainesville, FL). Microbial biomass C/N/P was analyzed by extraction following chloroform fumigation (Vance et al., 1987). Briefly, soil samples were extracted with 0.5 M K2SO4 (MBC and N) or 0.5 M NaHCO3 (MBP) after being incubated with (fumigated) and without (control) chloroform. Filtered (0.2 μm) extracts were analyzed for total extractable organic carbon (TOC) using a 5050A TOC auto-analyzer (Shimadzu Corp., Columbia, MD; EPA method 415.1). The difference in extractable TOC between the fumigated and non-fumigated samples was considered MBC following correction with an extraction efficiency (KEF) of 0.37 (Sparling et al., 1990). For MBN determination, extracts were analyzed following Kjeldahl digestion on a Technicon™ auto-analyzer (EPA method 351.2). The difference between fumigated and non-fumigated TKN was considered MBN following correction with an extraction efficiency (KEF) of 0.54 (Brookes et al., 1985). Extracts were analyzed on a Technicon™ auto-analyzer (EPA method 365.1). Soil TN and TC were analyzed using a Thermo FlashEA 1112 series NC soil analyzer (Thermo Fisher Scientific, Inc., Waltham, MA). Loss on ignition (% LOI) of soil samples was determined after combustion at 550 °C for 3–4 h. Soil OC was estimated using LOI assuming 45% OC content factor derived from total OM (Wright et al., 2008). Soil TP was determined following sequential combustion at 550 °C for a 4 hour period and dissolution of remaining ash with 6 M HCl (Andersen, 1976). Extracts were analyzed colorimetrically for reactive P using a Technicon™ Autoanalyzer III (SEAL Analytical, Mequon, WI) (EPA method 365.1). Extractable inorganic P (Pi) was determined in soil samples using 0.5 M NaHCO3 according to method 365.1 (USEPA) (Kuo, 1996). 2.4. Anaerobic respiration and methanogenesis Anaerobic CO2 and CH4 potentials were quantified during laboratory incubations. Unfortunately, due to shallow soil depth in restored sites, field gas measurements (flux) were unfeasible. Approximately 2 g dry weight soil and 10 mL of nanopure water (treated with UV) were combined in 30 mL serum tubes (n = 15 per site), sealed with butyl rubber stoppers and aluminum crimp tops, flushed with N2 for 15 min to encourage anaerobic conditions and stored in the dark. The bottles remained sealed throughout the incubation and gas headspace (100 μL) was periodically measured (every 2 days prior to day 8 followed by weekly measurements) for CO2 and CH4 concentrations for 4 weeks.

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Miami Homestead

Hole-in-the-Donut

Florida, USA

Fig. 1. Hole-in-the-Donut sampling transect map for Res00, Res03 and reference sites (Liao and Inglett, 2012).

2.4.1. Experimental C and P addition Additional bulk soils were collected from one sampling station at each site during February 2010 (C addition) and 2011 (P addition) for independent experiments to determine the effect of C and P availability on CO 2 and CH4 production in soils of the three sites. These soils were processed as described previously and stored at 4 °C prior to initiating the experiment. To test the response of soils to the addition of an easily available C-source, soils were amended with 10 mL of a 3.3 mM glucose solution prior to anaerobic incubation in the dark at 25 °C. To test for P limitation, a 1 mM NaH2PO4 solution was added to reduce the soil C:P ratio similar to concentrations tested in peat Everglades soils (Amador and Jones, 1993). Throughout each experiment, anaerobic respiration and methanogenic potentials of amended soils were compared with those of control soils (with nanopure water) incubated concurrently. We acknowledge that gas production cannot be compared directly between treatments; however, the purpose of these individual additions was to determine how select nutrients (C and P independently) may affect CO2 and CH4 production.

2.4.2. Gas analysis Headspace CH4 from experimental tube incubations was detected using a Shimadzu gas chromatograph-8a fitted with a flame ionization detector (160 °C detector, 110 °C injection) using N2 as the carrier gas and a 1.6 m (45/60) Carboxyn 1000 column (Supelco Inc., Bellefonte, PA). Headspace CO2 was measured using a Shimadzu gas chromatograph-8a equipped with a thermal conductivity detector (120 °C injection, 40 °C detector), He as the carrier gas and a 1.9 m (80/100) Porapak-N column (Supelco Inc., Bellefonte, PA). Calibration curves were determined via standard gas mixtures (Scott Specialty Gases, Plumsteadville, PA) multiple times throughout each sampling event. Concentrations of CO2 and CH4 were determined based on calibration curves accounting for possible dissolved inorganic carbon fractions. Final values reported represent CO2 or CH4 accumulated over a four week period (mg C kg−1 soil). Methane production from surrounding marl Everglades soils was concentrated in the top 0–2 cm with negligible production deeper than 4 cm (Bachoon and Jones, 1992). For that reason reporting gas production per gram soil as opposed to reporting gas flux (areal basis) would be more representative given the deeper soil depths in reference wetlands.

C.A. Medvedeff et al. / Geoderma 245–246 (2015) 74–82 Table 1 Soil parameters from sites within the Hole-in-the-Donut collected October 2009. Parameter

Units

Lat./long. Soil depth⁎ Bulk density pH TN OC⁎⁎ TP Inorganic P Microbial biomass C Microbial biomass N Microbial biomass P Β-glucosidase

cm g cm−3 −1

g kg g kg−1 mg kg−1 mg kg−1 mg kg−1 mg kg−1 mg kg−1 nmol g−1 dw soil h−1

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represent the predictor with the second smallest residuals within groups and so forth (Quinn and Keough, 2002).

Study site Res00

Res03

Reference

N25.379° W80.675 2.2 (0.2)b 0.5 (0.0)a 7.6 (0.0)b 7.4 (0.5)a 80 (3.0)a 638 (45.0)b 8.6 (0.6)b 3388 (222.0)a 285 (22.0)a 22 (2.4)b 161 (15.0)b

N25.383° W80.700 1.8 (0.1)b 0.6 (0.1)a 7.6 (0.0)b 6.0 (1.4)b 80 (3.0)a 972 (83.0)a 14 (1.3)a 2850 (172.0)b 296 (16.0)a 30 (1.5)a 262 (28.0)a

N25.376° W80.672 12 (0.6)a 0.5 (0.0)a 8.0 (0.0)a 6.9 (0.3)ab 61 (3.0)b 143 (9.0)c 1.6 (0.1)c 2973 (121.0)ab 208 (15.0)b 17 (1.2)b 112 (10.0)b

Values are means ± standard deviation (n = 5; composite samples). Differences between means (α b 0.05) determined via Tukey–Kramer means comparison. ⁎ Soil depth range: Res00 (0–5 cm), Res03 (0–4.5 cm), and Reference (2–22.5 cm). ⁎⁎ OC calculated from LOI with a 45% OC from OM factor.

2.5. Enzyme activity Soil enzyme activity of β-glucosidase (4-methylumbelliferyl-betaD-glucoside) was measured fluorometrically with methylumbelliferone fluorophore on a Bio-tek® model FL600 plate reader (Biotek Instruments, Inc. Winooski, VT) following 100 fold soil dilution with nanopure water similar to Inglett et al. (2011); (Medvedeff et al., 2013). The end product from β-glucosidase activity is glucose. Thus, measurement of β-glucosidase enzyme activity is a proxy for C substrate release and the subsequent fueling of aerobic, anaerobic, and methanogenic processing. Briefly, a 500 μM substrate-fluorophore solution was incubated with soil for 2 h in the dark. After incubation, fluorescence was measured at an excitation of 350 nm and emission of 450 nm. To account for any soil quenching, quenching curves were prepared separately for each sample. Enzyme activity was determined as fluorescence based on methylumbelliferone standard curves. Results were reported as nmoles MUF g−1 dry weight soil h−1.

2.6. Data analysis All statistical analyses were performed in JMP vs. 8.0 (SAS institute, Cary NC). Site differences (microbial respiration and CH4 production, BGA, and soil and microbial parameters or concentrations) were determined via one way analysis of variance (ANOVA). Due to the high sample size (n = 45) analyses were run on untransformed data although normality was not achieved. The Levene's test was used to ensure homogeneity of variance was met for each parameter. The difference in gaseous production following amendment (C or P) was analyzed via one way ANOVA following log transformation to achieve normality. In addition, differences in parameters within sites at different elevations were compared via one way ANOVA. Differences between means (α b 0.05) were assessed via Tukey–Kramer means comparison. Regression analysis with Pearson product-moment correlation was used to evaluate relationships between anaerobic processing (CO2/CH4 production and BGA) and both soil P (total and extractable) and soil C (organic C and extractable C). Tree regression is a non-parametric exploratory data analysis technique which has been used to investigate the relationship between environmental factors and carbon gas production (Melling et al., 2005a, b). The first split within this predictive model represents the predictor which results in two groups with the smallest within-group sums of squares for the response (CH4 or CO2) variable. Subsequent splits

3. Results 3.1. Soil nutrients Restored and reference soils varied greatly in basic characteristics including soil depth, pH, OC, TP, and Pi (Table 1). As a result of CSR restored sites accumulated an average of 2 cm soil at the time of sampling in comparison to an average 11.5 cm in reference wetlands. However, no difference in bulk densities were observed between restored and reference sites. Reference soil pH averaged 8.0 in comparison to a more neutral (7.6) pH observed in restored wetlands. Soil OC in restored sites averaged 80 g kg−1 soil, 20 g kg−1 greater than OC in the reference soil. Soil TN was lowest in Res00 when compared to Res03 and reference soils on average although this was not significant. Differences in site TP resulted in a soil P gradient from Res03 (972 ± 83 mg kg− 1 soil) to Res00 (638 ± 45 mg kg−1 soil) and reference (143 ± 9 mg kg−1 soil) wetlands. Similar to soil TP, NaHCO3 extractable Pi was greatest in restored wetlands following the P concentration gradient Res03 (14.4 mg kg− 1 soil) N Res00 (8.6 mg kg− 1 soil) N reference (1.6 mg kg−1 soil) wetlands (Table 1). Ratios of OC:TP and OC:Pi were 3–5 times higher in reference when compared to restored soils. 3.2. Microbial parameters Microbial biomass C from the reference soil was similar to both restored sites, but when comparing restored sites, MBC in Res00 was greater than Res03 (Table 1). Microbial biomass N was elevated in restored wetlands compared to reference conditions (p b 0.01; Table 1). Microbial biomass P was greatest in Res03 compared to Res00 and reference soils (p b 0.05; Table 1). Similar to trends in MBP, BGA was greater from Res03 (262 nmol MUF g− 1 dw soil h− 1) when compared to Res00 (161 nmol MUF g−1 dw soil h− 1) and reference (112 nmol MUF g−1 dw soil h−1) soils. 3.3. Anaerobic C loss (CO2 and CH4) Both CO2 and CH4 production were greater from restored relative to the reference soil (p b 0.0001; Fig. 2). Production of CO2 averaged 2025 mg C kg−1 soil in restored sites in comparison to 430 mg C kg−1 soil from reference wetlands after 28 days (Fig. 2). Regardless of restoration age, CH4 production averaged 450 mg CH4–C kg−1 soil from restored wetlands in comparison to 90 mg CH4–C kg−1 soil from reference wetlands after 28 day incubation (Fig. 2). After 28 days the CO2–C:

Fig. 2. Accumulation of CO2, CH4, and CO2–C:CH4–C ratios (mean ± SE) from Res00, Res03, and reference wetlands within the HID region of Everglades National Park during the 2009 sampling season following 28 day anaerobic incubation.

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CH4–C ratio from restored soils (4.9) were higher than ratios from the reference wetland (3.8) although this was not statistically significant (Fig. 2). 3.4. Microbial and soil nutrient correlations Correlations were analyzed to determine the relationship of P to microbial C parameters. ß-glucosidase enzyme activity was positively correlated with both anaerobic CO2 and CH4 production (p b 0.001 and p b 0.0001, respectively). Strong positive correlations were observed between BGA and both TP and extractable inorganic P (Pi) (p b 0.0001; Fig. 3a, data not shown). Anaerobic CO2 and CH4 production were positively correlated with soil P (Fig. 3b, c); however, this relationship could not be explained by linear regression. Trends suggest a threshold between TP and both CO2 and CH4 production in which low gas production is observed at low soil P concentrations (~b333–450 mg kg−1; Fig. 3b, c). In contrast, at soil P concentrations above this threshold gas production is augmented. Similar to soil P trends, BGA was positively correlated with soil OC (p b 0.0001; Fig. 3d). Positive correlations were observed between anaerobic respiration potentials and soil C parameters (p b 0.01; Fig. 3e; R2 b 0.01). Strong correlations with soil OC (p b 0.0001, R2 = 0.41) suggest this parameter may be an important predictor of methanogenic trends (Fig. 3f). Correlations between P and both CO2 and CH4 did not conform to linear regression models; however, linear regression data suggests organic carbon to be a control on gas production. Poor regressions suggest a P

threshold exists in which both CO2 and CH4 production is augmented. Regression tree analysis determined P to be the most important variable in determining both CO2 and CH4 production (Fig. 4). However, the P threshold for elevated gas production increased from 381 (Fig. 4a) to 431 mg kg− 1 (Fig. 4b) with the higher P concentration at the first node increasing gas production by 4.7 fold on average regardless of gas end product. Surprisingly, OC was not an important factor in determining CO2 and CH4 production in reference soil; although, it was of importance in high P, restored soils. Elevated soil OC increased predictive CH4 production (Fig. 4a) in high P (N 381 mg kg−1) soils; however, in contrast, lower OC concentrations (b 71 g kg−1) coupled with high P (N431 mg kg−1) increased predictive CO2 production (Fig. 4b). 3.5. Effects of C and P amendments Glucose stimulated anaerobic respiration and methanogenic potentials from all sites (Fig. 5). Production of CO2 in glucose amended soils increased by 400–500 mg C kg−1 in restored soils and 800 mg C kg−1 in the reference soil after 12 day incubation (Fig. 5b). In contrast, a 10 and 3.5 fold increase in CH4 production was observed from restored (Res00 and Res03) and reference soils, respectively (Fig. 5a). As a result of C addition, the ratio of CO2–C:CH4–C decreased from 43 to 6 in Res00 and from 35 to 5 in Res03 (p b 0.0001) while increasing in the reference soil from 66 to 130 (Fig. 5c). When P was added to restored soils, there was no effect on anaerobic respiration or methanogenesis (Fig. 6). In contrast, both anaerobic respiration and methanogenic potentials were stimulated by P addition

Fig. 3. Relationships between ß-glucosidase enzyme activity (a.), anaerobic respiration (b.), methanogenesis (c.) and soil total P concentration (left side). Relationships between ß-glucosidase enzyme activity (d.), anaerobic respiration (e.), methanogenesis (f.) and soil organic carbon (right side) from Res00, Res03 and reference soils within the Hole-in-the-Donut region of Everglades National Park during the 2009 sampling season.

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Fig. 4. Regression tree for CH4 (a; R2 = 0.89) and CO2 (b; R2 = 0.70) production (mg C kg−1 soil). The CH4 production (a.) or CO2 production (b.) in the regression tree shown in each box (mean ± standard deviation). The number in each bracket represents the count of samples in the group.

in the reference soil, although the difference was only significant for methanogenesis (p b 0.05; Fig. 6b, a). Production of CH4 increased from 2 to 6 mg CH4–C kg−1 soil when amended with P in reference wetlands (p b 0.001; Fig. 6a). Ratios of CO2–C:CH4–C decreased from 222 to 116 in the reference soil amended with P although no significant difference in ratios were observed from restored sites (Fig. 6c). Ratios differed between restored sites (48 to 35 in Res00 and 8 to 5 in Res03); however, the reduction was uniform. 4. Discussion Among the key aspects of restoration is the initial loss of nutrients and OM contained in the soil (Badiou et al., 2011; Ross et al., 1982). In contrast, soils of the restored HID areas are rich in nutrients (residual un-removed P) with high productivity of pioneer species resulting in enhanced OM accumulation in the shallow young soils (Table 1) (Inglett et al., 2011; Inglett and Inglett, 2013; Smith et al., 2007). Site differences in OC and TP resulted in OC:TP ratios of 515 and 150 in the reference and restored soils, respectively. These values suggest that the reference site is P-limited while the restored sites are not (McGill and Cole, 1981). Additional support for nutrient limitation patterns includes the TN:TP ratio, which based on the global analysis by Cleveland and Liptzin (2007) suggest primarily N limitation in the Res03 site (N:P = 15), co-limitation of N and P in the Res00 site (N:P = 26), and strong limitation by P in the reference site (N:P = 108) (Inglett and Inglett, 2013; Liao and Inglett, 2012; Liao et al., 2014). Nutrient limitation can result in alteration of microbial activity such as enzyme expression, anaerobic CO2 production (Aerts and Toet, 1997), and methanogenic processing (Adhya et al., 1998; Aerts and Toet, 1997; Bridgham and Richardson, 1992). For example, soil P

concentrations have been shown to limit C mineralization in low-P peat (Amador and Jones, 1995; Wright and Reddy, 2001) and mineral (Medvedeff et al., 2014) soils. In the current study, BGA was significantly greater in restored (high-P) relative to reference (low-P) sites (p b 0.05; Table 1). This trend has been observed previously within the HID ecosystem (Inglett et al., 2011). Enzyme activities for all sites were similar to those reported by Inglett et al. (2011a), and were similar to enzyme activities from surrounding freshwater subtropical marshes (Corstanje et al., 2007). Regulation of decomposition by enzyme activity is common (Sinsabaugh et al., 1993). Elevated BGA in the restored soils suggest these sites may contain a greater labile C pool which is supported by a significant relationship between BGA and soil OC (Fig. 3d) (Sinsabaugh et al., 2008). However, OC content does not fully explain the difference in BGA between the soils of Res03 and Res00 where OC contents were similar (Table 1). One explanation for this could include differences in C quality (e.g., lignin and cellulose) (DeBusk and Reddy, 1998; Debusk and Reddy, 2005). In addition, high soil nutrient concentrations have been shown to correlate with BGA (Eivazi and Zakaria, 1993). For example, in subtropical wetlands Prenger and Reddy (2004) observed positive correlations between BGA and both soil extractable Pi and TP in a Florida freshwater marsh, as did Penton and Newman (2007) in other areas of the Everglades. Similarly, we observed a strong correlation between BGA and both soil TP and extractable Pi (p b 0.0001; Fig. 3a, data not shown) suggesting P availability may control BGA and subsequent decomposition rates. Increased C availability in response to elevated BGA should ultimately result in elevated CO2 and/or CH4 potentials (Freeman et al., 1997). In support of this hypothesis, we observed greater anaerobic CO2 and CH4 production in restored soils (p b 0.01; Fig. 2), as well as positive correlations between BGA and CO2/CH4 production (p b 0.0001, data not

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Fig. 5. Methanogenesis (a.), anaerobic respiration (b.), and CO2–C:CH4–C (c.) from Res00, Res03, and reference wetlands within the HID region of Everglades National Park during the dry season following glucose addition following 12 day anaerobic incubation (mean ± SE).

shown). The rates of methane production in the reference soil were greater than those reported for low-nutrient soils in the HID (Smith et al., 2007), low-nutrient tropical marshes (Pivničková et al., 2010), and other temperate wetlands soils (D'Angelo and Reddy, 1999), but were similar to the ranges reported for low-nutrient northern Everglades wetlands (Wright and Reddy, 2001) and Florida marshes dominated by Cladium (Inglett et al., 2011). In contrast, CH4 production in restored soils were greater than those of restored HID soils reported by Smith et al. (2007), and similar to the rates of the nutrient-impacted northern Everglades (Wright and Reddy, 2001). Based on positive correlations between microbial activities (BGA, CO2, and CH4) and soil TP (p b 0.0001; Fig. 3a, b, c) and Pi (p b 0.0001; data not shown), we conclude P availability is a major control on these processes. However, significant correlations between soil OC and BGA/CO2/CH4 (p b 0.01; Fig. 3d, e, f) suggest this parameter may also be an important control. Phosphorus has been shown to limit decomposition in bog ecosystems (Hogg et al., 1994), therefore we assessed limitation of anaerobic C processing by amending high- (restored sites) and low-P (reference site) soils with P. Stimulation of CO2 production occurred in the reference (40% increase), but not in the restored soils, while CH4 production increased in response to P addition in all soils, but was much higher in the reference soil (190% increase; p b 0.0001; Fig. 6). However, due to high variability, this increase in CO2 production following P addition was not significant. This suggests that anaerobic C processing is more

Fig. 6. Methanogenesis (a.), anaerobic respiration (b.), and CO2–C:CH4–C (c.) from Res00, Res03, and reference wetlands within the HID region of Everglades National Park during the dry season following phosphorus addition following 13 day anaerobic incubation (mean ± SE if visible).

strongly limited by P in the reference soil, but less so in the higher-P soils of the restored sites. A decreased CO 2 –C:CH4 –C ratio in Pamended reference soil (Fig. 6c) further suggest methanogens in these sites may be directly limited by P and an increase in hydrogenotrophy is possible (Smith et al., 2007). Future studies quantifying changes in carbon isotope dynamics would be necessary to determine CH4 production pathways. Previous studies conducted in both elevated- and low-P Everglades peat soils observed a negligible effect of P addition on CO2 (Amador and Jones, 1993) and CH4 production (Drake et al., 1996) indicating P was not limiting anaerobic respiration in the short-term. Similar results in CH4 production were observed by Bachoon and Jones (1992) in low-P marl soils, however in the marl soils of this study, we found P significantly enhanced CH4 production during short-term incubations (Fig. 6). In contrast, insignificant relationships between CH4 production and P addition in our high-P restored soils was consistent with shortterm results from impacted Everglades wetlands investigated by Drake et al. (1996) likely suggesting methanogenesis is not P-limited in high-P Everglades soils. This suggests another factor, such as C quality or phenolic compounds, may have been regulating methanogenesis in these sites. Phenolic compounds are more resistant to decomposition and contain anti-microbial properties (Swift et al., 1979). Richardson et al. (1999) reported phenolic concentrations of foliage to almost double across a soil P nutrient gradient, with the highest phenolics originating from oligotrophic peat Everglades wetlands suggesting incorporation of Cladium biomass into HID marl wetlands may be an additional control on microbial decomposition although this was not measured in the current study.

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While regression trees suggest soil P was driving site differences in anaerobic respiration and methanogenesis, soil C may also explain site differences to a lesser extent in soils of high P (i.e. restored sites). Similar to previous studies (Amador and Jones, 1995; Bachoon and Jones, 1992), both CO2 and CH4 production were stimulated by C addition (glucose) suggesting decomposition is regulated by C quality. Increased processing through primary fermentation pathways is possible evidenced by elevated CO2–C:CH4–C ratios in amended reference soil relative to controls. When glucose was amended to restored soils a stronger response was observed in CH4 production (7 and 14 times the control in the Res00 and Res03, respectively) (Fig. 5a) although stimulation of CO2 production was also significantly enhanced (2.5 times the control in both sites). The contrasting results in stimulation of CO2 versus CH4 production after addition of P and glucose indicates that P limitation dominates anaerobic C processing in the reference site, while C availability limits gaseous C production in restored soils. It has been hypothesized highP soils are limited to a greater extent by labile C substrates relative to low-P soils in Florida Everglades wetlands (Wright et al., 2009) suggesting anaerobic processing in restored (high-P) soils may be ultimately limited by C quality within the HID. A similar response of increased CO2 (not significant) and CH4 production following both C or P addition in the reference soil suggests that stimulation of primary fermentation as a control of substrate release for methanogenesis is limited by P. 5. Conclusions Nutrient availability is a key regulator of decomposition and thus a key determinant of C greenhouse gas production in wetlands. Potential methane flux from restored sites, calculated using bulk density and soil depth, averaged 116 (10) g CH4–C m−2 y−1 which is within the reported range of emissions from tropical wetlands (Mitsch et al., 2012). Based on regression tree analysis and independent nutrient addition studies, this study suggests P is a key regulator of CH4 production in this ecosystem. While available C is an additional control on anaerobic carbon cycling, the magnitude of the non-methanogenic relative to methanogenic response following addition of labile C to reference soil and lack of importance detected via regression tree analysis suggests this element to be a lessor control on methanogenic processing in this site. As a result of complete soil removal, wetlands are hypothesized to shift from soil N to P limitation with increased restoration time (Inglett and Inglett, 2013). Thus, if wetlands in this ecosystem follow this restoration trajectory, high CO2 and CH4 emissions from restored sites will likely decrease with time. Given that the Res00 site (the oldest restored site) was only 9 years post-restoration when sampled, caution must be used and extrapolation of this data to address questions regarding the long-term sustainability of restoring carbon dynamics within the Hole-in-the-Donut site following this extreme restoration approach may not be appropriate. For this reason, additional monitoring is necessary to increase the overall confidence in our findings over a longer restoration timescale. In order to better understand the hypothesized shift from C source to sink with age (Whiting and Chanton, 2001) additional studies in this ecosystem are necessary to target CH4 production pathways and C utilization to understand the interactions between soil P availability and C substrate acquisition. Based on extensive pathways which can control substrate availability to methanogenic microbes (Bridgham et al., 2013) understanding the relationship between these microbial communities and nutrient limitation/availability is crucial in predicting the effect of this restoration technique on CO2 and CH4 gas production. Acknowledgments We thank the following for their help in this work. Field support was provided by L. Serra, C. Fisher and A. S. McKinley (US National Park Service), and B. Hogue and X. Liao (University of Florida). Laboratory

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