DOI: 10.5061/dryad.cjsxksndg
title: Tree species controls over nitrogen and phosphorus cycling in a wet tropical forest

description: Wet tropical forests play an important role in the global carbon (C)
 cycle, but given current rates of land-use change, nitrogen (N) and
 phosphorus (P) limitation could reduce productivity in regenerating
 forests in this biome. Whereas the strong controls of climate and parent
 material over forest recovery are well known, the influence of vegetation
 can be difficult to determine. We addressed species-specific differences
 in plant traits and their relationships to ecosystem properties and
 processes, relevant to N and P supply to regenerating vegetation in
 experimental plantations in a single site in lowland wet forest in Costa
 Rica. Single tree species were planted in a randomized block design, such
 that climate, soil (Oxisol), and land-use history were similar for all
 species. In Years 15-25 of the experiment, we measured traits regarding N
 and P acquisition and use in four native, broad-leaved, evergreen tree
 species, including differential effects on soil pH, in conjunction with
 biomass and soil stocks and fluxes of N and P. Carbon biomass stocks
 increased significantly with increasing soil pH (P = 0.0184, previously
 reported) as did biomass P stocks (P = 0.0011). Despite large soil N
 pools, biomass P stocks were weakly dependent on traits associated with N
 acquisition and use (N2 fixation and leaf C:N, P &lt; 0.09).
 Mass-balance budgets indicated that soil organic matter (SOM) could supply
 the N and P accumulated in biomass via the process of SOM mineralization.
 Secondary soil P pools were weakly correlated with biomass C and P stocks
 (r = 0.47, P = 0.08), and were large enough to have supplied sufficient P
 in these rapidly growing plantations, suggesting that alteration of soil
 pH provided a mechanism for liberation of soil P occluded in
 organo-mineral soil complexes, and thus supply P for plant uptake. These
 results highlight the importance of considering species’ effect on soil pH
 for restoration projects in highly weathered soils. This study
 demonstrates mechanisms by which individual species can alter P
 availability, and thus productivity and C cycling in regenerating humid
 tropical forests, and the importance of including traits into global
 models of element cycling.  
description: <strong>How was the data set
 collected?</strong> We addressed the
 relationships between tree species and accrual of C, N, and P, using a
 long-term, replicated field experiment containing four native tree species
 grown in plantations under similar climate, parent material and land-use
 history in a tropical wet forest in Costa Rica. Our approach was to
 identify mechanisms that had two main criteria: (1) the potential to
 influence availability of N and P for plants, and thus productivity (as
 indicated by their biomass accumulation) and (2) a direct connection to
 traits that could differ among species in their attributes, and thus drive
 species-specific differences in ecosystem properties. We used various
 approaches to evaluate several mechanisms in relation to specific traits,
 while also considering that plant species differ in a plethora of traits
 that could interact to result in a non-significant correlation between a
 particular mechanism and trait. <strong>Field
 measurements </strong> The field experiment was
 established on a 12-ha site that had been mature wet tropical forest until
 deforestation in 1955, planted to pasture, and grazed until abandonment in
 1987. In 1988, a stratified randomized complete block experiment was
 established with individual species planted within each plot. The design
 contained twelve 0.25-ha (50 m × 50 m) plots in each of four blocks.  Each
 plot was divided into four quadrants to facilitate random sampling within
 plots. Eleven of the 12 plots within a block were planted with a single
 tree species, with a tree spacing of 3 × 3 m. The
 12<sup>th</sup> plot was left unplanted as a Control, with
 those plots receiving the same management, except that no trees were
 planted. We established a fifth block (150 x 200 m in size) in Mature
 Forest, &lt;150 m from the experimental site, such that soils,
 climate, and historical vegetation were similar to the adjacent
 experiment. By the time of this study, only four of the 11 tree species
 planted had survived with complete canopy coverage across at least 3 of
 the 4 blocks (four-letter codes in parentheses): <em>Hieronyma
 alchorneoides</em> Allemao (Hial); <em>Pentaclethra
 macroloba</em> (Willd.) Ktze, (Pema); <em>Virola
 koschnyi</em> Warb., (Viko); and <em>Vochysia
 guatemalensis</em> Donn. Sm. (Vogu). To evaluate
 differences among species related to N and P acquisition, use, and
 storage, we measured C, N, and P concentrations and stocks of these
 compartments: plant biomass (above- and belowground), surface litter, and
 mineral soil (five depth intervals, 0-100 cm). Components of the planted
 (overstory) biomass included: canopy (leaves, flowers and fruits); twigs
 (diameters &lt;1 cm); branches (diameters 1-10 cm); large branches and
 boles (diameters &gt; 10 cm); stumps; stump-roots; woody roots
 (&gt;2 mm diameter); and fine roots (≤ 2 mm diameter). Components for
 the regenerating (unplanted, understory) vegetation included the same
 components. See ‘Metadata’ sheet for full details. We
 measured above- and belowground fluxes of organic matter, C, N, and P of
 the planted species and the vegetation regenerating on its own, referred
 to as the ‘overstory’ and ‘understory’ respectively. Components measured
 included: fine litterfall; branchfall; aboveground biomass increments (Δ
 biomass) of the overstory and understory, coarse-root and stump root
 biomass increments and fine-root growth (overstory + understory). We
 assayed asymbiotic N<sub>2</sub> fixation in surface soil (0-5
 cm) and surface litter. Turnover time was calculated as the quotient of
 biomass and NPP within each component, expressed in years. Soil
 respiration was measured <em>in-situ</em> approximately
 monthly for two years (Nov 2011-Oct 2013) in 3-4 quadrats per plot.
 Sampling <strong>Laboratory
 methods</strong> Organic C and total N for both
 plants and soil were analyzed by dry combustion (DC) using a
 Thermo-Finnigan EA Flash (Series 1112, EA Elantech, Lakewood, NJ).
 Duplicates were run on all samples.<em> </em>Plant tissue P
 was measured by microwave-assisted acid digestion and analyzed using
 inductively coupled plasma optical emission spectroscopy (Kingston and
 Haswell 1997). In total, 2704 plant samples were analyzed for C, N, and P.
   We used the acetylene reduction assay to estimate
 nitrogenase activity and thus evaluate asymbiotic
 N<sub>2</sub> fixation. Gas analysis was conducted on a Tracor
 540 gas chromatograph (GC) fitted with a carbosphere column (Alltech,
 5' × 1/8" SS COL P/W ALUMINA F-1 60/80 1 EA, 5664PC). The oven
 and FID temperatures were 100 °C and 175 °C respectively. Regarding the
 incubation period, analysis of ethylene results gave no evidence of
 substrate depletion within 24 h, nor inhibition of activity over 7 d.
 Nitrogenase activity was low enough that a 7-d period was needed to detect
 activity; thus, data presented are for the 7-d incubation period. The
 C<sub>2</sub>H<sub>2</sub> reduced by the sample
 over the incubation period was calculated as:
 ΔC<sub>2</sub>H<sub>4</sub> (μmol
 C<sub>2</sub>H<sub>4</sub>
 L<sup>-1</sup> g<sup>-1</sup>
 hr<sup>-1</sup>) × headspace (ml/1000) / incubation period
 (hrs) / sample mass (g dry wt). Potential N<sub>2</sub>
 fixation was calculated using a molar ratio of 3:1 ethylene produced per N
 fixed. For soil, total P was extracted in
 HCl:HNO<sub>3</sub> (4:1 by volume) (Crosland et al. 1995) and
 P in the digests was measured using inductively coupled plasma atomic
 emission spectrometry (ICP-AES). For measurement of total organic P, we
 used the ignition method (Kuo 1996), in which organic P in the soil was
 converted to inorganic P by oxidation at 550° C. Phosphorus in ignited and
 unignited subsamples was then extracted with 0.5N
 H<sub>2</sub>SO<sub>4</sub> for 16 h. ICP-AES was
 used to measure P in the extracts. Organic P in the soil was calculated by
 subtracting P concentration in the unignited sample from P concentration
 in the ignited sample. Modified Olsen-extractable P was determined by
 extraction with NaHCO<sub>3</sub> and
 ethylenediaminetetraacetic acid (Kuo, 1996). Sequential P extractions were
 conducted to target secondary minerals following Chadwick et al. (1994) to
 quantify the P associated with these phases, and to determine whether they
 were altered by the experimental tree species. Following an initial
 extraction of labile nutrients with unbuffered 1 M ammonium acetate, we
 extracted soils with acid ammonium oxalate, which dissolves SRO Fe and Al
 phases, and then with citrate-dithionite, which dissolves most crystalline
 Fe oxyhydroxides (as well as substituted or co-precipitated Al). These
 extractions have long been used for assessing secondary mineralogy of
 highly weathered soils (Chadwick et al. 1994, Torn et al. 1997). The sum
 of P<sub>oxalate</sub> and P<sub>dithionite</sub>
 quantified in this procedure is hereafter referred to as ‘secondary soil
 P.’ It is important to note that the ‘organic’ and ‘secondary’ pools
 partially overlap because a portion of the organic P that is liberated by
 combustion will also likely be solubilized by the oxalate or dithionite
 extraction; that is, secondary mineral-bound P includes organic P that is
 bound with these   <strong>How
 has the dataset been processed?</strong> Field
 and laboratory data were either immediately entered into Excel
 spreadsheets from hand-written field data sheets, or from data-capturing
 apparati (e.g., LICOR 8100). Datasets were then formatted for analyses in
 SAS and R. All data sets were regularly backed up on storage
 devices. To confirm species variation in traits and
 better understand the relationships among traits, we summarized and
 visualized these multivariate traits with PCA. We used 40 traits for which
 we had data for every plot in the experiment. Data were centered and
 scaled data prior to principle components analysis. We also ran permanova
 as a mixed model with species, block, and their interaction using Type III
 sums of squares with residual randomization permutation
 procedures. For factorial analyses, the experimental
 unit was the plot. For variables with multiple measurements per plot, the
 mean was used in the analysis. The ‘treatment’ consisted of the vegetation
 type; for most variables, this consisted of the four planted tree species,
 but for some it included the Mature Forest and/or the Control. For
 variables in which measurements included only the four planted species,
 the design was a randomized complete block (n = 15, with 4 species × 4
 blocks, minus one <em>Vochysia</em> plot). For analyses
 including the Mature Forest, the design was an incomplete block design (n
 = 23 if it also included the Control). Blocks were considered a random
 effect. We tested for homogeneity of variances and normality of
 distributions. ANOVA was used to evaluate differences
 among vegetation types in element (C, N, and P) concentrations,
 stoichiometries, and stocks in vegetation (by component), surface litter
 (by component), soil (5 depth intervals, including a vegetation type ×
 depth interaction term), N<sub>2</sub> fixation (by substrate,
 including a vegetation × substrate interaction. We first conducted a
 MANOVA to determine whether all elements could be evaluated in one test
 for a given response variable. In tests for all variables, the
 interactions among elements were significant, thus individual ANOVAs were
 done for each element. To assess redistribution of N and P within the soil
 profile, we compared changes in N and P stocks within the rooting zone
 (0-30 cm) and below (30-100 cm) over the 8-y period of the project.
 Procedures were conducted in SAS (Littell et al. 1996).
 Multiple regression models were used to evaluate the relative
 effects of explanatory variables, i.e., species traits that could
 influence N and P acquisition and use, on the response variables of stocks
 of biomass C, N, and P.
description: # Tree species controls over nitrogen and phosphorus cycling in a wet
 tropical forest Give a
 brief summary of dataset contents, contextualized in experimental
 procedures and results. ## Description of the data and file structure This
 data set is organized as follows. The first sheet in this Excel file is
 labeled ‘Metadata’ and this sheet contains information about the data. The
 Metadata sheet is organized by these columns: Study name; General
 information; Components; Abbreviations; Description; Frequency/Dates of
 measurements; Sheet name within this file; and Notes. Each remaining sheet
 in this Excel file is labeled as per the Metadata column ‘Sheet name
 within this file’. The sheets include: * Biomass C,N,P conc * Biomass CN,
 CP, NP * C Budget * N Budget * P Budget * Fine-root biomass * Surface
 litter mass * Soil bulk density * Soil C * Soil N * Soil P * Litterfall OM
 * Litterfall C * Litterfall N * Litterfall P * Branchfall * Fine-root
 growth * Soil respiration * N fixation * Trait Ordination Data * Change in
 soil CNP  The ‘C budget’, ‘N budget’ and ‘P budget’ sheets incorporate
 many of the other data sheets. Abbreviations are defined in the ‘Metadata’
 sheet, but generally repeated within the other sheets. Across all sheets,
 missing data are denoted by a period (.). ## Sharing/Access information
 Data on soil pH from this study are available from the Dryad Digital
 Repository: The rDNA sequence
 file is available from the NCBI Sequence Read Archive (accession number
 SRX766152). ## Code/Software Data were centered and scaled data prior to
 principle components analysis (vegan::rda; v.2.6-4, Oksanen et al. 2022)
 in R (v.4.2.2; R Core Team 2022). SAS Programs used included: the MANOVA,
 MIXED, and REG procedures. For comparisons including the Mature Forest, we
 used the Satterthwaite adjustment for degrees of freedom. Pairwise
 comparisons for significant overall F-tests were conducted using
 *P*-values adjusted by Tukey’s Honestly Significant Difference method.