DOI: 10.5061/dryad.9kd51c5n0
title: Data for: Phosphorus limitation of early growth differs between nitrogen-fixing and non-fixing dry tropical forest tree species

description: Tropical forests are often characterized by low soil phosphorus (P)
 availability, suggesting that P limits plant performance. However, how
 seedlings from different functional types respond to soil P availability
 is poorly known but important for understanding and modeling forest
 dynamics under changing environmental conditions. We grew four nitrogen
 (N)-fixing Fabaceae and seven diverse non-N-fixing tropical dry forest
 tree species in a shade house under three P fertilization treatments, and
 evaluated carbon (C) allocation responses, P demand, P-use, investment in
 P acquisition traits, and correlations among P acquisition traits.
 N-fixers grew larger with increasing P addition in contrast to
 non-N-fixers, which showed fewer responses in C allocation and P-use.
 Foliar P increased with P addition for both functional types, while P
 acquisition strategies did not vary among treatments but differed between
 functional types, with N-fixers showing higher root phosphatase activity
 (RPA) than non-fixers. Growth responses suggest that N-fixers are limited
 by P, but non-fixers may be limited by other resources. However,
 regardless of limitation, P acquisition traits such as mycorrhizal
 colonization and RPA were non-plastic across a steep P gradient.
 Differential limitation among plant functional types has implications for
 forest succession and earth system models.
description: We initially selected seeds of 30 species that commonly occur
 across successional and soil fertility gradients (30 to 1272 total P mg
 kg<sup>-1</sup>) in ACG and Área de Conservación Arenal
 Tempisque (ACAT)-Parque Nacional Palo Verde (10.35 N, 85.35 W) (Powers
 & Tiffin, 2010; Werden <em>et al.</em>, 2018), and had
 seeds available at the time the experiment was established. Due to
 logistical constraints (i.e., low germination rates, pests, and high
 mortality), only 11 species of the original 30 were used in the
 experiment. We included four N-fixing species from the Papilionoideae
 subfamily, the most abundant Fabaceae subfamily in the area (Werden
 <em>et al.</em>, 2018) and the one with the highest species
 richness (Koenen <em>et al.</em>, 2021). Papilionoideae
 species primarily associate with arbuscular mycorrhizal fungi. We caution
 here that our results should not be extended beyond the Papilionoideae
 subfamily; however, the fixers and non-fixers used in this study do not
 differ significantly in distribution characteristics across successional
 or soil gradients compared to the regional species pool (Methods S1, Table
 S1, and Fig. S1). Non-fixers included one ectomycorrhizal and six
 arbuscular mycorrhizal species from seven different families, including
 one Fabaceae (Table S2 and Fig. S2). We collected most seeds in the ACG
 and ACAT and purchased seeds of <em>Tabebuia impegitinosa</em>
 (Mart. Ex DC.) Standl. from the Centro Agronómico Tropical de
 Investigación y Enseñanza (CATIE), Costa Rica. The
 experiment was conducted during the rainy season from May to October in a
 36 m<sup>2</sup> shade house that excluded approximately 55%
 of PAR (J. Powers, unpublished data). Prior to planting, all seeds were
 soaked in water overnight. <em>Hymenaea courbaril</em> L.
 seeds were scarified to accelerate germination (Smith-Martin <em>et
 al.</em>, 2017); seeds were placed in 100°C water for 30 s and then
 in ice water for one minute, this process was repeated three times. Then,
 between 2-15 seeds (depending on seed size and availability) of each
 species were planted per pot (11 species x 3 treatments x 7 replicates =
 231 pots). Pots were big enough to have a biomass volume ratio
 &lt;1gL<sup>-1</sup> (Poorter <em>et al.</em>,
 2012) (10 x 10 cm and 20 cm deep containing ~2 kg of soil). Pots were
 randomly arranged within plastic crates to avoid confounding positioning
 species and treatment effects (8 pots per crate). One week after
 germination, seedlings were thinned to one per pot. Because we obtained
 low germination rates for <em>Lonchocarpus rugosus</em> Benth.
 And <em>H. courbaril</em>, additional seeds were added. To
 minimize differences in soil microbial communities, all seedlings were
 inoculated with microbial inoculum from three different forest soils
 collected across the regional P gradient (Smith-Martin <em>et
 al.</em>, 2017; Waring <em>et al.</em>, 2019). We
 prepared the inoculum by combining equal weights of the three soils in 1 L
 water, shaking them, and adding 10 mL of this solution to each pot 3 weeks
 after sowing. This treatment was repeated the following week to ensure
 microbial colonization. When seedlings were ~1 month old they were
 fertilized every two weeks from July to October with 10 mL of either
 0.0016 g mL<sup>-1</sup> of phosphoric acid
 (H<sub>3</sub>PO<sub>4</sub>) (45 kg P
 ha<sup>-1 </sup>yr<sup>-1</sup>) (P++) (Waring
 <em>et al.</em>, 2019), 0.00016 g mL<sup>-1</sup>
 H<sub>3</sub>PO<sub>4</sub> (4.5 kg P
 ha<sup>-1</sup> yr<sup>-1</sup>) (P+), or tap
 water (P0) to emulate the regional soil P gradient and P application rates
 used in other fertilization experiments (Siqueira & Saggin-Júnior,
 2001; Alvarez-Clare <em>et al.</em>, 2013; Waring<em> et
 al.</em>, 2019). Soil moisture was measured in each pot to 5 cm
 depth three times over the course of the experiment with a SM150 Soil
 Moisture Sensor (Delta-T Devices, Cambridge, UK). When rainfall was
 scarce, i.e., lack of rain for &gt;24 h, seedlings were hand-watered
 every other day to prevent desiccation. At the conclusion of the
 experiment, we measured soil pH in distilled water on air-dried soils from
 all pots using a 1:2.5 soil to solution ratio. Finally, to quantify the
 distribution of soil P among different P pools, a modified Hedley
 fractionation was conducted on a subset of soils from the pots at the soil
 laboratory at CATIE. <em>Harvesting and
 biomass</em> We harvested all seedlings four
 months after germination over a 10-day period. None of the individuals
 showed evidence of being pot bound. At the time of harvest, each seedling
 was separated into root, stem, and leaf fractions. Roots were carefully
 removed, gently washed in water and weighed. In the case of nodulating
 species, nodules were removed from the roots (before weighing), counted,
 and weighed. Leaves were immediately scanned to calculate leaf area using
 ImageJ software (Schneider <em>et al.</em>, 2012). Leaves,
 stems, roots, and nodules of each seedling were dried at ~60°C for 5 days,
 and then weighed to calculate total dry biomass (TDB), leaf (LMF), stem
 (SMF), root (RMF) (Table S3), and nodule (NMF) mass fractions. Root
 biomass included the tap roots if they were present. Dry weights of leaves
 were used to calculate specific leaf area (SLA, cm<sup>2</sup>
 g<sup>-1</sup>). All statistical analyses were performed using
 dry weights. SLA did not vary between functional types or treatments (Fig.
 S3, SLA p&gt;0.05) and will not be further discussed.
 <em>Plant performance and nutrient
 limitation</em> We measured relative height
 growth rate (RGR), photosynthetic capacity, and water use efficiency as
 metrics of plant performance. Stem height was measured from the base of
 the stem to the apical meristem on each seedling every two weeks after the
 first true leaves emerged. We used the classic RGR
 (cmd<sup>-1</sup>) equation: (ln[final height]–ln[initial
 height])/(<em>final day</em>–<em>initial day</em>)
 (Hunt & Cornelissen, 1997; Rees <em>et al.</em>,
 2010). Photosynthetic capacity was quantified by measuring photosynthetic
 responses to carbon dioxide concentration (AC<sub>i</sub>
 curves). Gas exchange measurements were performed on one recently matured
 leaf of three seedlings per species per treatment in situ with a LI-6400XT
 portable photosynthesis system (LI-COR, Lincoln, NE, USA) at a constant
 temperature (30°C). Measurements were made at CO<sub>2</sub>
 concentrations ranging between 0 and 1200 ppm (9 points total), irradiance
 was 1000 μmol m<sup>-2</sup>s<sup>-1</sup>
 (unlikely to cause photoinhibition of photosynthesis when leaves are
 measured below or above optimum temperatures) and relative humidity ranged
 between 60-80%. Measurements were made between 07:00 and 14:00 h and the
 ambient air temperature generally increased from 25°C to 32°C. We then
 obtained maximum photosynthetic rates (A<sub>max</sub>) and
 rubisco maximum carboxylation rate (V<sub>cmax</sub>) using
 the ‘plantecophys’ R package (Duursma<em> et al.</em>,
 2015). Finally, we analyzed foliar nutrients, water use
 efficiency, and photosynthetic phosphorus-use efficiency (PPUE). We bulked
 all leaves per individual and transported them to the University of
 Minnesota (UMN) where they were ground and analyzed for total carbon (C),
 nitrogen (N) and C isotopic ratio (δ<sup>13</sup>C) on an
 Isoprime 100 isotope ratio mass spectrometer coupled with an Elementar
 Pyrocube combustion elemental analyzer (Elementar Americas Inc). Other
 foliar nutrients (aluminum, boron, calcium, chromium, copper, iron,
 phosphorus, potassium, magnesium, manganese, sodium, and nickel) were
 analyzed at the UMN Research Analytical Lab using standard methods in an
 iCAP™ 7600 ICP-OES (Inductively Coupled Argon Plasma Optical Emission
 Spectrometer) Analyzer (Thermo Fisher Scientific Inc. Waltham, MA, USA)
 following digestion in HNO<sub>3</sub>. We calculated PPUE
 from A<sub>max</sub> and foliar P concentrations (Veneklaas
 <em>et al.</em>, 2012).
 <em>Belowground traits</em>      
       We quantified plant investment in P acquisition using root mass
 fraction (RMF) and specific root length (SRL)—a morphological response,
 root phosphatase enzyme activity (RPA)—a physiological response, and %
 arbuscular mycorrhizal colonization (AMC)—a biotic response representing
 degree of collaboration with symbionts. SRL was calculated after scanning
 all roots with a transparency scanner (Epson Perfection V800, Suwa, Japan;
 1-4 images per individual). We placed the roots in a clear polycarbonate
 tray filled with water to ensure no root overlap and used the Rhizovision
 software (Seethepalli <em>et al.</em>, 2020) to calculate root
 length of fine root fractions (≤2 mm diameter), i.e., excluding any tap
 root. After scanning, root systems were separated into two subsamples: one
 sample for RPA, and the other for scoring AMC. RPA was determined using
 para-nitrophenyl phosphate (<em>p</em>NPP) as an analogue
 substrate for phosphomonoesterase using standard methods (Methods S2;
 Turner <em>et al.</em>, 2001). Finally, we scored arbuscular
 mycorrhizal colonization (AMC) by staining the roots with alanine blue
 (Koske & Gemma, 1989) and quantifying colonization microscopically
 using the magnified intersection method (McGonigle <em>et
 al.</em>, 1990) on ~100 intersections per root fragment. We
 calculated colonization as the percentage of root length colonized by AM
 fungi (Methods S3). Ectomycorrhizal colonization was measured using the
 gridline intersection method (Brundrett et al., 1996). This variable was
 not included in any of the analyses as only one species was
 ectomycorrhizal, and these data are not directly comparable to
 AMC. While quantifying N fixation activity was not a
 goal of this experiment, we assessed plant carbon construction investment
 in fixing structures by quantifying nodule mass and NMF (Methods S4, Table
 S4 and Fig S4).
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