DOI: 10.5061/dryad.stqjq2cd0
title: Data from: Interspecific and intraspecific variations in root phosphatase activity among tropical tree species with different soil phosphorus associations

description: Variation in soil phosphorus (P) availability promotes niche
 differentiation among tropical tree species, yet the traits that underpin
 specialization on low-P and high-P soils (hereafter low-P and high-P
 species) remain poorly understood. Here, we examined interspecific and
 intraspecific variation in three types of root phosphatase enzymes and
 morphological traits among neotropical tree species in Panama with
 different habitat associations. We collected fine roots from 51
 individual trees of four congeneric pairs of low-P and high-P neotropical
 tree species in the genera Cordia, Hirtella, Inga, and Protium in
 forests on moderate to low-P soils. We determined root morphological
 traits (specific root length, diameter, and root tissue density), and the
 root-surface activities of phosphomonoesterase (PME), phosphodiesterase
 (PDE), and phytase (PHY) enzymes, which are synthesized to release
 inorganic orthophosphate from soil organic P. Soil P availability was
 determined by measuring resin-extractable P concentration for soils
 collected from the base of each tree. Low-P species allocated
 more resources to produce enzymes that decompose more complex forms of P,
 as indicated by greater PHY activity, and greater PHY:PME and PDE:PME
 ratios at a given soil P availability. A principal component analysis of
 fine-root traits showed a greater Euclidian distance among individuals of
 low-P species than among those of high-P species, supporting the
 hypothesis that fine-roots traits vary more among low-P species than among
 high-P species. Synthesis. These results suggest that the specialization
 of tropical tree species to low-P soil involves investment in the
 acquisition of complex soil organic phosphates such as phosphodiesters and
 phytic acid. This is possibly related to root trait divergence and
 indicates that variation in P acquisition strategies among tropical tree
 species could contribute to resource partitioning on low-P soils.
description: <em>Study site and species </em>
 We sampled leaves and fine roots from trees in three lowland
 forest census plots in the Panama Canal watershed (Pyke et al. 2001,
 Turner and Engelbrecht 2011, Condit et al. 2013). Vegetation is
 semi-deciduous, seasonally moist forest, with 2311-2848 mm of annual
 rainfall across the four plots and a mean annual temperature of 27°C. The
 four sites represent moderate (Barro Colorado Island) to low (Pipeline
 Road, Fort Sherman) soil P availability in this region (Table 1), based on
 a 2 mg P kg<sup>–1</sup> threshold at which there are marked
 differences in the response to P, including growth, distribution of low-P
 and high-P species, and soil phosphatase (Condit et al. 2013; Turner et
 al. 2018). Condit et al. (2013) showed that regional tree species
 distributions are structured by gradients of moisture and P. We selected
 moist forests to minimize effects of moisture availability on species
 traits. High-P and low-P species were determined by analyzing the
 responses of individual tree species to multiple environmental factors
 including soil P and moisture using the data of 72 sites and 550 species
 across Panama (Condit et al. 2013). This yielded  effect sizes for each
 species: species with P effect sizes &lt; 0 had a negative
 relationship between occurrence frequency and soil P availability (i.e.,
 low-P species) (Table 2). A pair of low-P and high-P species were selected
 for each of <em>Cordia</em> (<em>C. bicolor</em>
 and <em>C. alliodora</em>; Verbenaceae),
 <em>Hirtella</em> (<em>H. americana</em> and
 <em>H. triandra</em>; Chrysobalanaceae),
 <em>Inga</em> (<em>I. pezizifera</em> and
 <em>I. nobilis</em>; Fabaceae), and
 <em>Protium</em> (<em>P. panamense
 </em>and<em> </em><em>P.
 tenuifolium</em>;<em> </em>Burseraceae). The selected
 species are widespread subcanopy tree species in Panamanian neotropical
 forests. <em>Field sampling </em>
 We measured the activity of three phosphatases (PME, PDE; and
 PHY), mycorrhizal colonization, and root morphological traits. We included
 mycorrhizal colonization rate because it might affect plant P acquisition
 and phosphatase activities. At least six understory individuals were
 identified for each species, with diameter at breast height (DBH) between
 50 and 300 mm. Fine roots (i.e., roots &lt; 2-mm diameter) of each
 individual tree were sampled by tracing coarse roots from the trunk. Roots
 were immediately chilled in the field and transferred to the laboratory.
 We collected several root branches to determine physiological and
 morphological traits. Some individuals had few fine roots that could be
 tracked, so data for some parameters were incomplete, because we
 prioritized phosphatase activity data over mycorrhizal colonization data
 (e.g., for <em>Cordia bicolor</em>) and therefore omitted the
 data of mycorrhizal colonization from some analyses (see below). We
 collected bulk soil at 0–10 cm depth beneath the litter layer around each
 individual tree to determine local P availability. Finally, we collected
 shade leaves from the canopy of each tree to calculate leaf mass per area
 (g m<sup>–2</sup>) as an indicator of above-ground
 nutrient-use strategy. <em>Laboratory
 analyses</em> We measured root phosphatase
 activity (PME, PDE, and PHY), mycorrhizal colonization rate, and
 morphological traits (root diameter, specific root length, and root tissue
 density) for each fine-root sample and P availability for each soil
 sample. We determined fine-root PME activity using a modified method of
 Yokoyama et al. (2017). Each of two 10-mg subsamples of fine-root tissue
 was incubated with 2.25 ml of sodium acetate-acetic acid buffer adjusted
 to pH 5.0 and 0.25 ml of 25 mM <em>para</em>-nitrophenyl
 phosphate (<em>p</em>NPP) for 30 minutes in a water bath at 26
 °C (the mean soil temperature in lowland tropical forests of Panama). The
 buffer included 3 mM NaN<sub>3</sub> to inhibit microbial
 activity. We also prepared blanks that received buffer and substrate
 without fine roots. The enzymatic reaction was terminated by adding 0.5 mL
 of the assay solution to 4.5 mL of 0.11 M NaOH. The concentration of
 <em>para</em>-nitrophenol (<em>p</em>NP) was
 determined by measuring the absorbance at 405 nm on a spectrophotometer,
 after subtracting absorbance in blanks (roots but no substrate) and
 controls (substrate but no roots). Phosphodiesterase activity was
 determined by the same procedure, except using 25 mM
 bis-<em>para-</em>nitrophenyl phosphate
 (bis-<em>p</em>NPP) as substrate. The
 measurement of PHY activities was based on a modified method of Yokoyama
 et al. (2017). Five 10-mg subsamples of fine-root material were weighed
 into glass vials (indicated as S1, S2, C, P, N for each subsample). We
 also prepared glass vials without roots (indicated as B sample: see the
 later explanation for what these subsamples represent). Subsamples S1, S2,
 and B received 0.25 ml of 8 mM phytic acid (sodium salt) in acetate buffer
 (pH 5.0). Subsample C and N received 0.25 ml of acetate buffer. Subsample
 P received 0.25 ml of 2 mM
 KH<sub>2</sub>PO<sub>4</sub>. The subsamples were
 incubated for 14 h in a water bath at 26 °C and enzymatic reaction was
 terminated by adding 1 ml of 10% trichloroacetic acid (TCA). Subsequently,
 subsamples S1, S2, P, N received 1 ml of acetate buffer whereas subsample
 C received 0.25 ml of 2 mM phytic acid sodium salt in acetate buffer.
 Incubated subsamples were centrifuged, and orthophosphate was determined
 in the supernatants by molybdate colorimetry with the absorbance (712 nm)
 on a spectrophotometer using a 1-cm cell. PHY activity was calculated by
 the following equation: PHY activity =
 (S–C–B)/[T*(P–N)/P<sub>b</sub>] (µmol
 PO<sub>4</sub> g<sup>–1</sup>
 hr<sup>–1</sup>), (Equation 1) where S is
 the mean PO<sub>4</sub> of subsamples S1 and S2 after
 incubation per subsample root weight (μmol PO<sub>4</sub>
 g<sup>-1</sup>); C indicates PO<sub>4</sub> in
 subsample C after incubation per subsample root weight (μmol
 PO<sub>4</sub> g<sup>-1</sup>); P indicates
 PO<sub>4</sub> concentration in the subsample P after
 incubation (μg PO<sub>4</sub> mL<sup>-1</sup>); N
 indicates PO<sub>4</sub> concentration in the subsample N
 after incubation (μg PO<sub>4</sub>
 mL<sup>-1</sup>); T indicates Incubation time (hr); Pb
 indicates the added PO<sub>4</sub> concentration in the
 subsample P before incubation (μg PO<sub>4</sub>
 mL<sup>-1</sup>). Subsample C, B, and (P
 –N)/P<sub>b</sub> represent PO<sub>4</sub> release
 from fine roots during incubation, PO<sub>4 </sub>release from
 phytic acid sodium salt, and collection rate of
 PO<sub>4</sub>, respectively. The intensity
 of mycorrhizal colonization was determined by a modified method of
 Giovannetti and Mosse (1980). Root branches were cleaned with deionized
 water, soaked in 10% KOH in a 95°C water bath for 3 min, acidified with 2%
 HCl solution for 1 h, and stained with 0.02% Trypan blue in a 90°C water
 bath for 30 min, after which the stained root branches were placed in a
 solution of lactic acid, glycerol and water to remove excess stain
 (decolorization). If roots still had a dark color after boiling with KOH
 solution, they were soaked into 10%
 H<sub>2</sub>O<sub>2</sub> and 20%
 NH<sub>4</sub>OH for 10 min. The degree of root mycorrhizal
 colonization was quantified based on the method described by McGonigle et
 al. (1990). In brief, the cleaned and stained root
 branches were divided into fragments (ca. 1 cm) and mounted on slides.
 Root fragments were observed at 1-mm intervals at ×200 magnification using
 a microscope (100 to 200 points), and the proportion of root length
 containing fungal structure (arbuscules, vesicles and hyphae) was
 calculated. For morphological traits, roots were
 scanned at 300 dpi and average diameter and length determined using a
 semiautomated image-analysis software, Smart Root (Guillaume et al. 2011)
 based on Image J (Schneider et al. 2012). We manually tracked fine-roots
 used for PME analysis, calculating root length and average root diameter.
 All root branches were oven-dried at 65°C for two days and weighed.
 Specific root length and root tissue density were calculated as total
 length divided by dry mass, and dry mass divided by total volume,
 respectively. Leaves were scanned at 300 dpi and areas determined with
 Image J. Leaves were oven-dried at 65°C and weighed. Leaf mass per area
 was calculated as dry weight divided by leaf area.
 Readily available P was determined using anion-exchange membranes
 (hereafter “resin P”). Samples were analyzed immediately after soil
 collection, to eliminate artifacts caused by soil storage (Turner and
 Romero 2009). Briefly, 5-g soil was shaken for 24 h with 80 mL of
 deionized water and five anion-exchange membrane stripes (1 cm × 4 cm).
 The strips were rinsed in deionized water and then shaken for 1 h in 50 mL
 of 0.25 M H<sub>2</sub>SO<sub>4</sub>. Phosphate
 was determined in the extracts by automated molybdate colorimetry on a
 Lachat Quikchem 8500 (Hach Ltd, Loveland, CO).
description: # Data from: Interspecific and intraspecific variations in root
 phosphatase activity among tropical tree species with different soil
 phosphorus associations
 [https://doi.org/10.5061/dryad.stqjq2cd0](https://doi.org/10.5061/dryad.stqjq2cd0) ## Description of the data and file structure This is supplementary data used for analysis in the paper entitled “Interspecific and intraspecific variations in root phosphatase activity among tropical tree species with different soil phosphorus associations”. The csv files include the information on species characteristics (scientific name, habitat association) and values of root and leaf traits. There are some missing data because of limited sample availability, which are indicated as NA. ### Files and variables #### File: Data.csv ##### Variables * id: Identification number.  * site: Name of the site where root and leaf sampling was conducted. * genus: Genus name of the sampled individual. * species: Species name of the sampled individual. * phosphorus association: Habitat association of the species. Low-P and high-P indicate the species adapted to low-P and high-P environments, respectively. * resinP: Concentration of readily available phosphorus (P), determined with the resin extraction method (mg kg–1). * phytase: Fine-root phytase activity (µmol PO4* *g–1 hr-1) * phosphomonoesterase: Fine-root phosphomonoesterase activity (µmol *p*NP g–1 hr-1) * phosphodiesterase: Fine-root phosphodiesterase activity (µmol *p*NP g–1 hr-1) * lma: Leaf mass per area (g m–2) * srl: specific root length (cm mg–1) * Root diameter: fine-root diamter (mm) * RTD: root tissue density (mg cm–3) * am colonization rate: Arbuscular mycorrhizal fungi colonization rate on fine roots (decimal)