Soil Phosphorus Forms Along a Strong Nutrient Gradient in a Tropical Ombrotrophic Wetland

Wetland Soils Soil Phosphorus Forms along a Strong Nutrient Gradient in a Tropical Ombrotrophic Wetland

Phosphorus cycling influences productivity and diversity in tropical wetlands, Alexander W. Cheesman* yet little is known about the forms of P found in the accreting organic matter Wetland Biogeochemistry Laboratory of these ecosystems. We used alkaline (NaOH–ethylenediamine tetraacetic Soil and Water Science Dep. acid [EDTA]) extraction and solution 31P nuclear magnetic resonance (NMR) Univ. of Florida 106 Newell Hall spectroscopy to characterize P in surface soils across a strong nutrient gradi- Gainesville, FL 32611 ent within a tropical ombrotrophic peat dome. From the interior bog plain to the marginal Raphia taedigera swamp, total soil P increased from 14.6 and to 70.9 g m−3 and resin-extractable P from 0.1 to 30 mg kg−1. Phosphatase Smithsonian Tropical Research Institute activity declined across the same transect (364–46 mmol methylumbellifer- Apartado 0843-03092 one kg−1 min−1), indicating an increase in P availability toward the periph- Balboa, Ancón, Republic of Panama ery of the wetland. Organic P identified by solution 31P NMR spectroscopy Benjamin L. Turner included phosphomonoesters (12–17%), phosphodiesters (10–14%), and Smithsonian Tropical Research Institute phosphonates (up to 3.3% of total P). Inositol phosphates were not detected Apartado 0843-03092 in these acidic peats. Inorganic P forms included orthophosphate (9–25% of Balboa, Ancón total P), pyrophosphate (up to 3%), and long-chain polyphosphates; the latter Republic of Panama occurred in concentrations (up to 24% of total soil P) considerably higher than previously found in wetland soils. The concentration of residual (unex- K. Ramesh Reddy tractable) P was similar among sites (mean 280 mg kg−1), resulting in an Wetland Biogeochemistry Lab. increase in its proportion of the total soil P from 29% at the P-rich margins to Soil and Water Science Dep. 55% at the P-poor interior. This is the first information on the P composition Univ. of Florida of tropical wetland soils and provides a basis for further study of the cycling 106 Newell Hall and contribution of P forms to the nutrition of plants and microorganisms. Gainesville, FL 32611 Abbreviations: AEM, anion exchange membrane; EDTA, ethylenediamine tetraacetic acid; NMR, nuclear magnetic resonance.

hosphorus is a key element limiting ecosystem processes in freshwater wetlands (Daniel et al., 1998; Rejmánková, 2001). Because much of the P in wetland soils occurs in organic forms (Davelaar, 1993; Newman and Robinson,P 1999; Reddy et al., 2005), P availability is dependent on the cycling of P from biological material. Biologically derived P in soils, however, represents a variety of compounds that differ markedly in their behavior and bioavailability (Celi and Barberis, 2005; Condron et al., 2005). The nature of these P forms is the result of the dynamic interplay among biological inputs (Makarov et al., 2005), abiotic stabilization (Celi and Barberis, 2005), and biological modification (Cheesman et al., 2010b), all of which are influenced by edaphic, environmental, and biological factors. Information on the role these factors have in determining P composition in wetland soils is therefore critical for understanding the cycling and productivity of wetland systems, as well as in explaining future responses to perturbations. Unlike quantitative extraction and determination of P pools based on chemical stability, techniques such as solution 31P NMR spectroscopy allow the assessment of actual P forms present in environmental samples (Cade-Menun, 2005; McKelvie, 2005; Turner et al., 2005). This has yielded valuable insight into the na-

Soil Sci. Soc. Am. J. doi:10.2136/sssaj2011.0365 Received 28 Oct. 2011. *Corresponding author ([email protected]). © Soil Science Society of America, 5585 Guilford Rd., Madison WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Soil Science Society of America Journal ture of P forms and their biogeochemical processing in wetlands. visible soil–vegetation catena, however, with current communi- For example, previous work has highlighted the diverse range of ties ranging from a monodominant Raphia taedigera Mart. palm biogenic P forms that can be present in wetland soils (Sundaresh- swamp at the periphery, through mixed and monodominant war et al., 2009), the importance of site conditions in determin- Campnosperma panamense Standl. swamp forest, to a central ing P composition (Cheesman et al., 2010b), and differences in “bog plain” community dominated by herbaceous species and P composition between wetland and terrestrial soils (Turner and stunted trees (Phillips et al., 1997; Sjögersten et al., 2011). Newman, 2005; Turner et al., 2006). Yet despite clear evidence that P availability influences both vegetation (Hagerthey et al., Sampling 2008; Sjögersten et al., 2011) and biogeochemical processes such We established nine sampling sites at 300-m intervals along as C fluxes (Wright et al., 2011) and N2 fixation (Šantrůčková a linear transect running perpendicularly from a bordering canal et al., 2010), little is known about P forms and their associated toward the geodesic center of the peat dome (Fig. 1; Table 1) (Co- cycling in tropical wetlands. hen et al., 1989; Sjögersten et al., 2011; Troxler, 2007). Sample Tropical peat domes are “self-emergent” organic wetlands Site 9 could not be located in the very center of the bog plain due within the humid tropics (Semeniuk and Semeniuk, 1997). to practical constraints but was considered to be just inside the Their upper surface shows a pronounced convex morphology central vegetation zone (Myrica–Cyrilla bog plain). In September leading to (or resulting from) their hydrologic isolation and an 2007, three replicate peat samples were collected from within 20 ombrotrophic hydrologic regime (Anderson, 1983; Andriesse, m of each site using a sharpened metal cutting head on a ridged 1988; Belyea and Baird, 2006). They are systems of environ- polycarbonate tube. Each replicate was an amalgamated sample mental, social, cultural, and economic importance, providing of three surface cores (7.5-cm diameter, 10-cm depth) collected numerous direct ecosystem services to local populations (Cen- from within 2 m of each other. Samples were immediately cooled tral American Commission for Environment and Development, (~10°C) and returned to the laboratory, where they were stored 2002; Ellison, 2004) as well as representing substantial and dy- at 4°C until processing within 72 h of collection. This processing namic pools in the global C cycle (Maltby and Immirzi, 1993; involved homogenization and the removal by hand of recogniz- Yu et al., 2010). Often associated with the swamps of maritime able roots (>1-mm diameter) and lignified structures (seeds, twigs, Southeast Asia (Anderson, 1983; Anderson and Muller, 1975), etc.). Due to the high concentration of fine roots from herbaceous significant peat deposits also occur throughout the Caribbean vegetation at Sites 8 and 9, however, a substantial amount of root coastal plain (Ellison, 2004; Phillips and Bustin, 1996). Al- biomass may have remained within these samples. The samples though these remain poorly studied in comparison with their were subsequently split, with half being air dried (~22°C for 10 Asian counterparts, there are strong similarities, including a vis- d) to constant weight and the remainder stored at 4°C in sealed ible soil–vegetation catena across the convex surface (Phillips et bags (subsequently referred to as fresh sample). Air-dried samples al., 1997) and distinct gradients in nutrient availability (Sjöger- were ground in a ball mill using tungsten carbide vessels and stored sten et al., 2011; Troxler, 2007). in airtight containers under ambient laboratory conditions until We used solution 31P NMR spectroscopy to assess the analysis. Additional soil was collected from Site 9 in November functional forms of P present within the surface soils of a tropi- 2007 for 31P NMR spectroscopy, for which the total P was not cal ombrotrophic wetland. By using a natural soil P gradient and significantly different from the September sample at this sitet ( - range of vegetation types while controlling for more general site test: P > 0.05). conditions, we aimed to derive novel information on the influence of nutrient status on the chemical nature of soil P found in Soil Properties tropical wetlands. Such data are critical to our understanding of Soil moisture was determined by gravimetric loss after dry- P cycling in wetlands, particularly given the extent to which an- ing at 105°C for 24 h and used to estimate the soil bulk density. thropogenic P enrichment affects such ecosystems (Cheesman Soil pH was determined on fresh soil using a 1:20 soil (oven-dry et al., 2010b; McDowell, 2009; Turner et al., 2006). weight)/water ratio and a glass electrode. Total elemental concentrations were determined on dried and ground samples. Total soil Methods C and N were determined by combustion and gas chromatogra- Study Site phy using a Flash EA1112 (Thermo Scientific), while total P was 2 The Changuinola peat deposit is an approximately 80-km determined by H2O2 + H2SO4 digestion (Parkinson and Allen, portion of the internationally recognized San San Pond Sak wet- 1975) and detection by inductively coupled plasma–optical emis- land in Bocas del Toro province, northwest Panama (Ramsar sion spectrometry (Optima 2100, PerkinElmer). Site 611, www.ramsar.org). Palynological evidence (Cohen et al., 1989; Phillips and Bustin, 1996; Phillips et al., 1997) sug- Phosphorus Characterization gests that Caribbean coastal systems such as the Changuinola Anion Exchange Membranes deposit developed by different mechanisms and contain distinct Readily extractable and microbial P were operationally deter- community types compared with the well-studied peat domes mined using anion exchange membranes (AEMs; BDH Prolabo of Southeast Asia (Anderson and Muller, 1975). They retain a 551642S, VWR International) using a method based on Kouno

www.soils.org/publications/sssaj 6 Fig. 1. Overview of study transect and sampling sites within the Changuinola peat deposit, San San Pond Sak, northwest Panama. Access route originates at a canal cut in 1908 and approximates previous leveling transects of the site (Cohen et al., 1989; Phillips and Bustin, 1996). Elevation increase from Site 1 to Site 9 is approximately 4 m. et al. (1995) and Myers et al. (2005), with modifications described some organic and condensed inorganic P forms might also be in- in Cheesman et al. (2010c). The difference in phosphate between cluded (Cheesman et al., 2010c). nonfumigated and hexanol-fumigated samples was attributed to microbial P, and we did not correct values for unrecovered biomass Solution Phosphorus-31 NMR Spectroscopy (Jenkinson et al., 2004). Extracted P in nonfumigated samples was Soil P composition was determined by standard alka- assumed to represent exchangeable inorganic phosphate, although line extraction and solution 31P NMR spectroscopy (Cade- Table 1. Soil characteristics from sampling stations across the Changuinola ombrotrophic peat dome.

Vegetation Total elements Site plot† pH Bulk density C N P Mg m−3 ————————— g k g −1 ————————— mg kg−1 1 1 3.6 ± 0.21‡ 0.069 ± 0.006 ab§ 498 ± 9.9 a 29 ± 0.5 a 1028 ± 43 a 2 3.8 ± 0.12 0.064 ± 0.009 ab 508 ± 7.3 ab 29 ± 1.0 a 1014 ± 51 a 3 3.9 ± 0.05 0.060 ± 0.008 abc 515 ± 6.7 ab 28 ± 0.7 ab 956 ± 69 a 4 2 3.7 ± 0.03 0.078 ± 0.012 a 535 ± 9.7 ab 26 ± 1.3 abc 655 ± 81 bc 5 3.9 ± 0.08 0.064 ± 0.007 ab 506 ± 6.2 ab 28 ± 0.2 ab 710 ± 21 b 6 3 3.6 ± 0.29 0.056 ± 0.005 bc 507 ± 4.2 ab 25 ± 0.5 bcd 659 ± 31 bc 7 4 3.7 ± 0.19 0.040 ± 0.005 cd 458 ± 22.7 c 23 ± 1.9 cd 672 ± 81 b 8 3.8 ± 0.34 0.050 ± 0.004 bcd 500 ± 3.1 a 19 ± 1.9 e 388 ± 23 d 9 5 3.6 ± 0.27 0.033 ± 0.005 d 417 ± 5.3 d 22 ± 1.7 de 442 ± 58 cd † Permanent vegetation sampling plot established by Sjögersten et al. (2011). ‡ Mean ± one standard deviation (n = 3). § Superscript letters denote homogenous subsets determined by post-hoc Tukey’s honestly significant difference test.

6 Soil Science Society of America Journal Menun and Preston, 1996; Turner et al., 2007). The extraction prepared in a 1:100 soil/water ratio (containing 1 mmol L−1 −1 −1 (0.25 mol L NaOH and 50 mmol L EDTA) was applied NaN3 to prevent microbial activity). The soil suspension (50 mL) to air-dried soils with shaking for 4 h, as well as fumigated and was then pipetted into wells on a micro-well plate (eight wells nonfumigated fresh soils after application of the AEM strips per substrate) containing 100 mL of 200 mmol L−1 substrate (Cheesman et al., 2010a) to assess the potential contribution of [4-methylumbelliferyl phosphate or bis(4-methylumbelliferyl) live microbial cells to the P composition of the air-dried soils. phosphate] and 50 mL of 200 mmol L−1 NaOAc–HOAc buffer All samples were extracted in a 1:30 soil (oven-dry weight)/solu- adjusted to pH 4.0 (the approximate mean pH of the peat). Plates tion ratio at ambient room temperature (~22°C) before being were incubated for 30 min at 26°C to approximate the daytime centrifuged at 7000 rpm (maximum relative centrifugal force soil temperature in the Changuinola peat deposit. The reaction ~7500 ´ g) (Sorvall RC6, SLA 1500 Rotor, Thermo Fisher was terminated by adding 50 mL of 0.5 mol L−1 NaOH (final Scientific) for 20 min and the supernatant decanted. Field rep- solution pH >11) and the plates were read immediately on a licates were combined on an equal-volume basis (15 mL), mixed FLUOstar Optima multidetection plate reader (BMG Labtech), with 1 mL of an internal standard (methylenediphosphonic acid with excitation at 360 nm and emission at 460 nm. Control −1 −1 [MDP], 50 mg P L ), frozen (−80°C), and lyophilized to await plates containing substrate, buffer, and 1 mmol L NaN3 (no resuspension and NMR spectroscopy. A second, independent soil suspension) were prepared and analyzed immediately before subsample was analyzed for total P by a double acid digest us- and after the analysis of the soil samples to account for initial ing concentrated H2SO4 and HNO3 (Rowland and Haygarth, fluorescence as well as pH-induced instability of the substrates. 1997), with P detection by automated molybdate colorimetry. Each soil had corresponding blanks, methylumbelliferone (MU) Spectra were acquired using a Bruker Avance 500 Console standards, and soil-specific quench standards. All enzyme activi- with a Magnex 11.75 T/51-mm magnet, using a 10-mm BBO ties are expressed as micromoles MU per kilogram of dry soil per probe. Lyophilized samples (~300 mg) were resuspended in 2.7 minute (mmol MU kg−1 min−1). mL of resuspension fluid (1 mol −1L NaOH and 0.1 mol L−1 Data Analysis EDTA) and 0.3 mL D2O in a 15-mL centrifuge tube. Samples were vortexed for 1 min, filtered through a prewashed 1-mm All statistical tests were performed in SPSS for Win- GF-B in-line syringe filter, and loaded into a 10-mm NMR tube dows version 17.0.0 statistical software (SPSS Inc.). Data were (1008-UP-7 Norell). Spectra were acquired at a stabilized 25°C checked for normality by application of a Shapiro–Wilk test with a 30° tip angle (calibrated on samples spiked with addition- and visual inspection. Natural logarithms were used for sta- al orthophosphate), a zgig pulse program (Berger and Siegmar, tistical analysis if the assumption of normality was improved. 2004), 0.4-s acquisition time (100-ppm spectral width), and a Differences between basic biogeochemical characteristics were 2-s pulse (d1) delay. Results presented here are of ~40,000 scans explored via a simple analysis of variance using a univariate gen- accumulated as four sequential experiments with free induction eral linear model (GLM), with site number as the fixed factor. decay signals summed post-acquisition by Bruker proprietary Post-hoc analysis (Tukey’s honestly significant difference) was software. Additional qualitative spectra from alkaline extrac- used to identify significant homogeneous subsets. The relation- tions after pre-extraction with AEM strips were acquired using a ship between hydrolytic enzyme activity and total soil P was ex- 5-mm NMR probe and a Bruker Avance 500-MHz spectrometer plored by use of the SPSS curve estimation of an inverse func- using a 45° pulse, a 1-s d1 delay, and a zgig pulse program. tion. Site biogeochemical characteristics were averaged among Spectra interpretation was performed using wxNUTS ver- samples (n = 3) and analyzed for correlation (Pearson’s r) with sion 1.0.1 for Microsoft Windows (Acorn NMR Inc.). Spectra P forms identified by31 P NMR. were referenced and integrated against the internal standard (MDP) set at d = 17.46 ppm after comparison to an externally Results held 85% H3PO4 set at d = 0 ppm. Spectra were integrated over Soil Biogeochemical Properties set spectral windows corresponding to known P bonding classes Surface peat from across the wetland transect was very (Turner et al., 2003b), with spectral deconvolution applied to acidic (pH 3.7 ± 0.4) and of low bulk density, ranging from the region between 8 and 3 ppm to separate orthophosphate 0.03 g cm−3 in the central bog plain to 0.08 g cm−3 in forested from phosphomonoesters and −3 to −5 ppm to separate pyro- portions of the transect (Table 1). Total C concentrations ranged phosphate and polyphosphate end residues. between 417 and 535 g kg−1, with significant differences among sites (P < 0.001) but with no clearly discernible pattern. Hydrolytic Enzyme Assays Total N and P concentrations declined (P < 0.001) from To assess biological investment in P acquisition from organ- the relatively nutrient-rich Site 1 (total N = 29 g kg−1, total P = ic compounds, the activities of two hydrolytic enzymes involved 1.0 g kg−1) to oligotrophic Site 9 (total N = 22 g kg−1, total P = in the P cycle (phosphomonoesterase and phosphodiesterase) 0.4 g kg−1), with total soil P showing the same gradient as that were determined using fluorogenic substrates based on a stan- observed by Sjögersten et al. (2011) (1.0–0.4 g kg−1). For both N dard microplate assay (Marx et al., 2001) as described in Turner and P, post-hoc analysis (Tukey’s honestly significant difference) and Romero (2010). For each sample, soil suspensions were showed a similar pattern of four homogeneous subsets of sites

www.soils.org/publications/sssaj 6 across the gradient, with proximate sites showing no significant differences with each other. When nutrient concentrations were expressed on a volumetric basis, the low bulk densities in the central region amplified the gradients in N and P across the transect (Fig. 2A and 2B), with a significant P( < 0.001) fivefold increase in total P (14.6 to 70.9 g m−3) and almost threefold increase in total N (0.73 to 2.01 kg m−3). Molar N/P ratios ranged from 62 at Site 1 to 111 at Site 9 (Fig. 2C), while the molar C/P ratio ranged from 1252 at Site 1 to 3335 at Site 8 (data not shown).

Phosphorus Biogeochemistry Concentrations of readily exchangeable P extracted by AEM strips were <3 mg kg−1 for Sites 5 to 9 but up to 30 mg kg−1 (2.8% of total P) at other sites (Table 2). Phosphorus released by hexanol fumigation differed significantlyP ( < 0.05) among sites, representing between 18 and 38% of soil total P, even without the use of a correction factor to account for unrecovered biomass (Bunemann et al., 2008; Jenkinson et al., 2004). There was a highly significant negative correlation (Pearson’s r = −0.55, P < 0.01) between total soil P and the proportion of total P released by fumigation. Phosphatase activity (both phosphomonoesterase and phosphodiesterase) increased from the nutrient-rich margin of the wetland to the oligotrophic interior, with central sites showing rates up to seven times that of the peripheral Site 1 (Fig. 3). Values differed significantly among sitesP ( < 0.001), with phosphatase activity showing a highly significant P( < 0.001) inverse relation- ship with total soil P (phosphomonoesterase, R2 = 0.55; phosphodiesterase, R2 = 0.74).

Phosphorus Recovery in Sodium Hydroxide– EDTA Extraction An average of 63% of total soil P was extracted in NaOH– EDTA from dried soils, with site averages ranging from 45 to 71% of total soil P. Extraction of P from dried soils was similar to that recovered from fresh soils by the combined use of AEM and NaOH–EDTA extraction after hexanol fumigation (Cheesman et al., 2010a). Although more P was recovered from dried soil (paired t-test, P < 0.001), the difference represented an average of only 0.3% of the total soil P, suggesting that alkaline extraction of air-dried soils recovers P in live microbial biomass (Turner et al., 2003a) and that unextractable residual P is a physically distinct pool. Extraction of air-dried soils for 16 h resulted in an average increase in recovery of 4.1% of total soil P across all samples (paired t-test, P < 0.001). Given the known hydrolysis of vari- ous phosphodiesters in alkaline solution (Turner et al., 2003b), however, we used a standard 4-h extraction for detailed 31P NMR Fig. 2. (A) Mass of total P, (B) mass of total N, and (C) N/P molar analysis. Replicate extractions using a 4-h extraction yielded a ratio from nine study sites (average ± SE, n = 3) across four different consistent recovery (±10% of total soil P) based on molybdate vegetation types found within the Changuinola peat dome. Samples colorimetry of digested alkaline solutions (data not shown). were taken along a transect between the periphery of the peat dome (Site 1) to near the geodesic center (Site 9), with general vegetation groupings based on “phasic communities” identified by Phillips et Solution Phosphorus-31 NMR Spectroscopy al. (1997). All attributes show significant overall differences among A diverse range of P forms was present in the soils, includ- sites (P < 0.001), with letters above bars indicating homogenous ing organic (phosphomonoesters, phosphodiesters, and phos- subsets as derived from Tukey’s honestly significant difference post- hoc analysis.

6 Soil Science Society of America Journal Table 2. Phosphorus identified by anion exchange membrane phonates) and inorganic (orthophosphate, pyrophosphate, and (AEM) extraction of fresh soils at sampling stations across the long-chain polyphosphate) forms (Fig. 4; Table 3). Changuinola ombrotrophic peat dome. Phosphomonoester concentrations ranged from 45 to Site AEM-extractable P Fumigation-released P 174 mg P kg−1 (11.5–17.1% of total soil P) (Fig. 4; Table 3) and ——————— m g k g −1 ——————— % total soil P did not include peaks characteristic of myo-, scyllo-, neo-, or D- 1 29.5 ± 9.6 a† 184 ± 40.5 abc 18 ± 4.6 a chiro-inositol hexakisphosphate (Fig. 5) (Turner and Richardson, 2 17.6 ± 4.2 ab 232 ± 20.1 cd 23 ± 0.4 ab 2004; Turner et al., 2003c; Turner et al., 2012). Concentrations a bcd ab 3 27.6 ± 7.9 193 ± 19.4 20 ± 2.1 of DNA ranged from 47 to 105 mg P kg−1, which represented 4 bc ab ab 5.4 ± 8.1 138 ± 52.5 21 ± 6.0 between 8.7 and 13.3% of the total soil P. Phosphodiesters other 5 0.1 ± 0.0 c 267 ± 24.2 d 38 ± 3.9 c than DNA were detected at low levels (0.6–3% of total soil P) but 6 0.9 ± 0.7 c 190 ± 25.3 bcd 29 ± 2.7 bc phosphodiesters such as phosphatidyl choline and RNA that are 7 2.9 ± 2.1 bc 148 ± 12.4 ab 22 ± 3.6 ab 8 0.2 ± 0.0 c 110 ± 8.5 a 28 ± 2.5 abc hydrolyzed in alkaline solution are likely to be underestimated 9 0.3 ± 0.2 c 151 ± 6.0 ab 35 ± 4.3 c (Makarov et al., 2002; McDowell and Stewart, 2005; Turner et al., −1 † Mean ± one standard deviation (n = 3); superscript letters denote 2003b). Phosphonates occurred at up to 31 mg P kg and ranged homogenous subsets determined by post-hoc Tukey’s honestly from 1.7 to 3.3% of total soil P. These compounds were not de- significant difference test. tected at the low-P Sites 8 and 9, although this may be due to a low signal/noise ratio and an inability to resolve their presence, as opposed to a true absence. Inorganic P identified by solution31 P NMR ranged between 100 and 400 mg P kg−1 (23–39% of total soil P) and included both orthophosphate and polyphosphates. Orthophos- phate concentrations ranged from 29 to 258 mg P kg−1 (7–25% of total soil P) and pyrophosphate from 2 to 32 mg P kg−1 (up to 3% of total soil P). Longer chain polyphosphates were found in surprisingly high concentrations from all study sites, ranging from 69 to 165 mg P kg−1 (10–24% of total soil P). Concen-

Fig. 3. Enzyme activity from nine study sites (average ± SE, n = 3) Fig. 4. Solution 31P NMR spectra showing a range of P forms across four different vegetation types found within the Changuinola present in surface soils from select sites across the study transect: peat dome. Samples were taken along a transect between the A = phosphonates, B = methylenediphosphonic acid (MDP, internal periphery of the peat dome (Site 1) to near the geodesic center (Site standard d = 17.46 ppm), C = orthophosphate, D = phosphomonoesters, 9), with general vegetation groupings based on “phasic communities” E = phosphodiesters, F = DNA, G = polyphosphate (end-chain residues), identified by Phillips et al. (1997). Both phosphomonoesterase and H = pyrophosphate, I = polyphosphate (mid-chain residues). Spectra phosphodiesterase activities show significant difference among sites plotted using 15-Hz line broadening referenced and scaled using (P < 0.001), with letters above bars indicating homogeneous subsets as internal standard MDP. ‡Due to a lack of sample material, spectra were derived from Tukey’s honestly significant difference post-hoc analysis. acquired on additional samples collected in November 2007. www.soils.org/publications/sssaj 6 Table 3. Phosphorus forms identified by solution31 P nuclear magnetic resonance (NMR) spectroscopy in soils from the Changuinola ombrotrophic peat dome. The concentration was determined by multiplying the proportion of spectral area by the total P determined by digest of alkaline extracts.

Total Other Polyphosphate‡ Residual Site P Phosphonate Orthophosphate Phosphomonoesters DNA Phosphodiesters† Pyrophosphate ER MR P ——————————————————————————————— m g P k g −1 s o i l ( % o f t o t a l P ) ——————————————————————————————— 1 1028 27 (3) 258 (25) 167 (16) 105 (10) 29 (3) 32 (3) 34 (3) 76 (7) 301 (29) 2 1014 31 (3) 233 (23) 174 (17) 93 (9) 30 (3) 20 (2) 14 (1) 107 (11) 310 (31) 3 956 18 (2) 170 (18) 143 (15) 91 (10) 26 (3) 11 (1) 25 (3) 138 (14) 333 (35) 4 655 15 (2) 129 (20) 79 (12) 57 (9) 10 (1) 26 (4) 19 (3) 61 (9) 260 (40) 5 710 16 (2) 82 (12) 86 (12) 72 (10) 14 (2) 4 (1) 18 (3) 147 (21) 271 (38) 6 659 16 (2) 83 (13) 75 (11) 61 (9) 9 (1) 3 (<0.5) 9 (1) 135 (20) 268 (41) 7 672 11 (2) 76 (11) 89 (13) 90 (13) 10 (1) 3 (<0.5) 9 (1) 124 (19) 260 (39) 8 388 ND§ 29 (7) 45 (11) 47 (12) 3 (1) 2 (1) 4 (1) 65 (17) 193 (50) 9¶ 578 ND 54 (9) 68 (12) 57 (10) 3 (1) 7 (1) ND 72 (12) 317 (55) † Includes RNA and phospholipids not hydrolyzed by alkaline extraction and solution 31P NMR spectroscopy. ‡ Polyphosphate ER = end-chain P residue; MR = mid-chain P residue. § ND, not detected or trace. ¶ Spectra acquired on samples collected in November 2007.

trations of polyphosphates determined in dried soils correlated monoesters, Pearson’s r = −0.80). In addition, there was a signifi- (Pearson’s r = 0.80, P < 0.05) with fumigation-released P in fresh cant positive correlation (Pearson’s r = 0.79, P < 0.05) between soils. Polyphosphate was also detected in alkaline extracts of fresh soil total P and the ratio of P identified as phosphomonoesters (undried) soil extracts (Fig. 6), indicating that their presence was and phosphodiesters, ranging from 1.40 at Site 2 to a low of 0.88 not an artifact of fungal growth during sample air drying (Kou- at Site 8. kol et al., 2008). When extracts of fresh soils were analyzed by solution 31P NMR after application of AEM strips but without Discussion the use of hexanol as a biocide, polyphosphates were detected. Surface peat at all sites was acidic and presumably derived When samples were pre-extracted with AEM strips and hexanol from the current standing vegetation. Given that mineral matter fumigation, however, polyphosphates were not detected or were deposited from external sources can be considered minimal in an found only in trace concentrations (Fig. 6). ombrotrophic system (Phillips et al., 1997), the significant dif- The concentration of residual P (i.e., P not extracted or iden- ferences in C concentrations may reflect differences in biosilica tified by NMR analysis) ranged from 193 to 333 mg P kg−1 and deposition from opal phytoliths and diatoms (Wüst et al., 2002) was correlated with total P (Pearson’s r = 0.75, P < 0.05). When or variance in C decomposition and relative peat accretion rates expressed as a proportion of the total P, residual P increased from across the transect (Craft and Richardson, 1993). 29% at the relatively enriched Site 1 to 55% at the oligotrophic Elemental analysis confirmed previous studies in identify- Site 9 (Table 3), with values correlated negatively with total P ing the presence of a strong nutrient gradient across the distinct (Pearson’s r = −0.87, P < 0.005). vegetation communities (Sjögersten et al., 2011; Troxler, 2007). The two forms of P with the largest absolute change in their Total P was at the high end of the range observed in other tropi- contribution to the total P both correlated negatively (P < 0.01) with residual P (orthophosphate, Pearson’s r = −0.83; phospho-

Fig. 6. Solution 31P NMR spectra of soils from Sites 1 and 9 after application of anion exchange membranes with (F) and without (NF) a Fig. 5. Detail of solution 31P NMR spectra from Site 7 soils. Spectra hexanol fumigation step. Spectra plotted using 15-Hz line broadening plotted using 2-Hz line broadening and referenced using internal referenced and scaled using internal standard methylenediphosphonic standard methylenediphosphonic acid (d = 17.46 ppm). acid (d = 17.46 ppm).

6 Soil Science Society of America Journal cal ombrotrophic systems, with sites in Kalimantan ranging from total organic P (Turner, 2007; Turner et al., 2012). This distinc- 272 to 373 mg kg−1 (Page et al., 1999) and the Peruvian low- tion has been attributed to differential stabilization in the or- land Amazonia ranging from 130 to 590 mg kg−1 (Lahteenoja ganic matter and redox conditions prevalent in wetlands (Turner et al., 2009). This may reflect the relatively young age and shal- and Newman, 2005; Turner et al., 2006), although the recent low depth of the study peatland (Phillips et al., 1997) or periodic detection of the phosphomonoester myo-inositol hexakisphos- deposition of P during its formation from either volcanic ash or phate within anaerobic sediments (McDowell, 2009; Turner and catastrophic flooding events (Cohen et al., 1989). Weckström, 2009) suggests a complex interplay between inputs Membrane-extractable and total P increased significantly and site-specific stabilization processes. The general distinction toward peripheral R. taedigera sites, yet the high total C/P molar between terrestrial soils and wetlands (Turner and Newman, ratios (range 1252–3335) and total N/P (62–111), in addition 2005) might also represent a difference in the proportion of to- to high rates of phosphatase enzyme activity at all sites, suggest tal soil P found within viable microbial biomass compared with the potential for P limitation of vegetation and microbes within the extracellular environment (Oberson and Joner, 2005), which the soils of all sample sites (Cleveland and Liptzin, 2007). Phos- seems likely here given the high proportion of total soil P found phomonoesterase activity at peripheral sites (Sites 1–3) were within the microbial biomass. similar to those reported in a Malaysian forested peat swamp Phosphonates, while not present in the highly studied cal- (Jackson et al., 2009), suggesting the potential for high organic P careous or soft-water peatlands of South Florida (Turner and turnover in tropical wetlands (Penton and Newman, 2008). The Newman, 2005; Turner et al., 2006), have been detected in acidic marked increase in phosphatase activity alongside a decreasing northern hemisphere blanket bogs and subarctic tundra (Bed- nutrient concentration toward the center of the dome indicates rock et al., 1994; Turner et al., 2004). It has been proposed that a clear increase in biological investment in the acquisition of P phosphonates are either more prevalent within the microbial from organic forms (Sinsabaugh and Moorhead, 1994; Wright biomass of these systems (Ternan et al., 1998) or that phospho- and Reddy, 2001). nates experience greater extracellular stability under cold, acidic, Across all sites, a substantial proportion of the total P was and saturated conditions (Condron et al., 2005). The presence of held in a pool released by fumigation. Although it is likely that phosphonates in the tropical wetland studied here demonstrates P from fine roots liberated by the action of hexanol contribut- that they may also be found in substantial quantities in acidic ed to this pool (Sparling et al., 1985), fumigation without the peats with a range of total P concentrations and a high soil tem- use of a correction factor underestimates the microbial biomass perature (26°C). (Brookes et al., 1982; Myers et al., 1999). It is therefore likely A large proportion of total P occurred as inorganic polyphos- that a substantial proportion of P identified in the NMR spectra phates from all sites, including those where P availability is expected represents live microbial biomass. A similar conclusion can be to be low and potentially limiting to both plant productivity and drawn when comparing the extraction efficiency of the coupled microbial activity. Polyphosphates play an integral role in archeal, AEM and alkaline extraction of fumigated fresh soil to that of prokaroyotic, and eukaryotic cells (Kornberg et al., 1999) and are air-dried soil. The large and significant increase in extraction ef- often associated with luxury microbial P uptake (i.e., high-P envi- ficiency between nonfumigated fresh and air-dried soil is con- ronments) (Hupfer et al., 2007; Khoshmanesh et al., 2002), includ- trary to reports of the influence of pretreatment on other peat- ing activated sludge processing (Reichert and Wehrli, 2007). Poly- based soils. For example, direct alkaline extraction of fresh and phosphates have also been detected in a range of natural wetland air-dried soils resulted in a similar recovery of total P for three and aquatic systems, however, including oligotrophic lake sediments sites in the Florida Everglades, with the notable exception of soft- (Ahlgren et al., 2006; Hupfer et al., 2004), Carolina bays (Sundar- water floc samples (pH 5.8) (Turner et al., 2007). We interpret eshwar et al., 2009), the humic-acid fraction of reseeded peatlands this as evidence of either a significant pH ´ pretreatment inter- (Bedrock et al., 1994), and subarctic tundra (Turner et al., 2004). action on the alkaline extraction efficiency or physical protection In this study, the large concentrations of inorganic polyphosphates of P pools and microbial biomass in the highly fibrous peats of determined by solution 31P NMR spectroscopy of dried soil extracts this study system. correlated strongly with fumigation-released (microbial) P in fresh The diversity of P forms identified by solution31 P NMR soils, as well as appearing to be released by hexanol fumigation of varied little among vegetation types or across the nutrient gradi- fresh soils (Fig. 6). Therefore, polyphosphate measured in this wet- ent, yet the relative proportions of different compounds showed land probably represent intracellular stores associated with P homeo- progressive alteration in relation to the basic biogeochemical stasis or microbe sporulation (Brown and Kornberg, 2004) under gradient and microbial P. This study showed that, similar to conditions of fluctuating redox (Davelaar, 1993), P storage associ- other organic wetlands, a significant proportion of organic P oc- ated with arbuscular or saprotrophic fungi (Koukol et al., 2008), or a curred as phosphodiesters or potential phosphodiester hydroly- generalized microbial metabolic response to environmental stress or sis products (Turner and Newman, 2005). This is in contrast to nutrient deficiency within an ombrotrophic environment (Seuffer- terrestrial soils, in which up to 90% of organic P occurs as phos- held et al., 2008). It should also be noted that polyphosphates, al- phomonoesters (Condron et al., 2005), with isomers of inositol though stable under the alkaline extraction conditions, are catalyti- hexakisphosphate often forming a substantial proportion of the cally degraded by the presence of divalent cations (Harold, 1966).

www.soils.org/publications/sssaj 6 The addition of EDTA improves the recovery of polyphosphates Ahlgren, J., K. Reitzel, R. Danielsson, A. Gogoll, and E. Rydin. 2006. Biogenic phosphorus in oligotrophic mountain lake sediments: Differences in (Cade-Menun and Preston, 1996) and reduces Fe-induced hydro- composition measured with NMR spectroscopy. Water Res. 40:3705–3712. lysis in alkaline extracts (Hupfer et al., 1995), yet some researchers doi:10.1016/j.watres.2006.09.006 interested in their role in lacustrine and marine sediments have ad- Anderson, J.A.R. 1983. The tropical peat swamps of western Malesia. In: A.J.P. Gore, editor, Mires: Swamp, bog, fen and moor. Ecosyst. of the World Vol. opted a pre-extraction with either buffered dithionite or EDTA to 4B. Regional studies. Elsevier Sci. Publ., Amsterdam. p. 181–199. further preserve polyphosphates (Ahlgren et al., 2007; Hupfer et al., Anderson, J.A.R., and J. Muller. 1975. Palynological study of a Holocene peat and 2004; Reitzel et al., 2007). Therefore, the presence of large quantities a Miocene coal deposit from NW Borneo. Rev. Palaeobot. Palynol. 19:291– of polyphosphates identified within this study when not using a pre- 351. doi:10.1016/0034-6667(75)90049-4 Andriesse, J.P. 1988. Nature and management of tropical peat soils. FAO, Rome. extraction step may be the result of intrinsically low divalent cation Bedrock, C.N., M.V. Cheshire, J.A. Chudek, B.A. Goodman, and C.A. Shand. concentrations and a reduced degradation rate compared with other 1994. Use of 31P-NMR to study the forms of phosphorus in peat soils. Sci. wetland extractions. Total Environ. 152:1–8. doi:10.1016/0048-9697(94)90545-2 Belyea, L.R., and A.J. Baird. 2006. Beyond “the limits to peat bog growth’’: In ombrotrophic wetlands, the vast majority of organic mat- Cross-scale feedback in peatland development. Ecol. Monogr. 76:299–322. ter in surface soils is autochthonously derived. Phosphorus inputs doi:10.1890/0012-9615(2006)076[0299:BTLTPB]2.0.CO;2 within this organic matter consist of a variety of forms dependent Berger, S., and S. Siegmar. 2004. 200 and more NMR experiments: A practical course. John Wiley & Sons, Hoboken, NJ. on the nature and structure of the biotic community (Harrison, Brookes, P.C., D.S. Powlson, and D.S. Jenkinson. 1982. Measurement of 1987; Makarov et al., 2005), yet microbial modification of P forms microbial biomass phosphorus in soil. Soil Biol. Biochem. 14:319–329. within the detrital material of organic wetland soils is strongly in- doi:10.1016/0038-0717(82)90001-3 fluenced by nutrient availability (Cheesman et al., 2010b). Given Brown, M.R.W., and A. Kornberg. 2004. Inorganic polyphosphate in the origin and survival of species. Proc. Natl. Acad. Sci. 101:16085–16087. doi:10.1073/ the rapid nature of microbially mediated processes (Oberson and pnas.0406909101 Joner, 2005), especially in a tropical setting, it is likely that soil P Bunemann, E.K., R.J. Smernik, P. Marschner, and A.M. McNeill. 2008. forms represent a balance among primary eukaryotic inputs mod- Microbial synthesis of organic and condensed forms of phosphorus in acid and calcareous soils. Soil Biol. Biochem. 40:932–946. doi:10.1016/j. erated by prokaryotic processing, as well as forms present within soilbio.2007.11.012 the live biomass (i.e., microbial cells, roots, and the microfauna). Cade-Menun, B.J. 2005. Using phosphorus-31 nuclear magnetic resonance The reduction in P availability toward the central portion of the spectroscopy to characterize organic phosphorus in environmental samples. In: B.L. Turner et al., editors, Organic phosphorus in the environment. wetland suggests an increase in the role of organic P cycling, which CABI Publ., Wallingford, UK. p. 21–44. corresponds with a pronounced decrease in the relative propor- Cade-Menun, B.J., and C.M. Preston. 1996. A comparison of soil extraction tions of total soil P identified as orthophosphate and phospho- procedures for 31P NMR spectroscopy. Soil Sci. 161:770–785. monoesters, as well as in the ratio of phosphomonoesters to phos- doi:10.1097/00010694-199611000-00006 Celi, L., and E. Barberis. 2005. Abiotic stabilization of organic phosphorus in phodiesters. The absolute concentration of residual P not identi- the environment. In: B.L. Turner et al., editors, Organic phosphorus in the fied by alkaline extraction and31 P NMR, however, was relatively environment. CABI Publ., Wallingford, UK. p. 113–132. consistent across the pronounced gradient in total P. This resulted Central American Commission for Environment and Development. 2002. Central American policy on the conservation and wise use of wetlands. CCAD, San in a gradient in the proportion of total P found as residual P, po- Jose, Costa Rica. tentially reflecting an inherently stable pool that comes to domi- Cheesman, A.W., E.J. Dunne, B.L. Turner, and K.R. Reddy. 2010a. Soil phosphorus nate soil P as turnover rates increase and other standing pools are forms in hydrologically isolated wetlands and surrounding pasture uplands. J. Environ. Qual. 39:1517–1525. doi:10.2134/jeq2009.0398 reduced. Further work is need to establish whether this pattern Cheesman, A.W., B.L. Turner, P.W. Inglett, and K.R. Reddy. 2010b. Phosphorus represents differences in extracellular stabilized P or changes in the transformations during decomposition of wetland macrophytes. Environ. constitutive components of the microbial biomass (Makarov et al., Sci. Technol. 44:9265–9271. doi:10.1021/es102460h 2005) that represent a major fraction of the soil P within this tropi- Cheesman, A.W., B.L. Turner, and K.R. Reddy. 2010c. Interaction of phosphorus compounds with anion-exchange membranes: Implications for soil analysis. cal ombrotrophic wetland. Soil Sci. Soc. Am. J. 74:1607–1612. doi:10.2136/sssaj2009.0295 Cleveland, C.C., and D. Liptzin. 2007. C:N:P stoichiometry in soil: Is there a Acknowledgments “Redfield ratio” for the microbial biomass? Biogeochemistry 85:235–252. We thank G. Jacome and P. Gondola of the Smithsonian Tropical doi:10.1007/s10533-007-9132-0 Cohen, A.D., R. Raymond, A. Ramirez, Z. Morales, and F. Ponce. 1989. The Research Institute, Bocas del Toro Research Station, for logistical Changuinola peat deposit of northwestern Panama: A tropical, back- support and Dr. Jim Rocca, Dr. Alex Blumenfeld, and Tania Romero barrier, peat (coal)-forming environment. Int. J. Coal Geol. 12:157–192. for analytical support. 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