Quantifying Nitrogen Uptake Preferences in Mature Tropical Plantation Trees with an In Situ Whole-Tree Paired ¹⁵N Labeling Method

Abstract

The nitrogen (N) uptake preferences of mature tropical trees remain poorly understood, largely because traditional hydroponic methods fail to adequately simulate field conditions. To address this, we applied an in situ whole-tree paired ¹⁵N labeling experiment to quantify N acquisition strategies in three common species of tropical plantations in southern China: Hevea brasiliensis, Pinus caribaea, and Acacia mangium. The in situ whole-tree paired ¹⁵N labeling experiment revealed distinct species-specific nitrogen uptake strategies: Hevea brasiliensis preferentially absorbed nitrate (contributing 76% to total N uptake), Pinus caribaea relied more heavily on ammonium (61%), while Acacia mangium exhibited no strong preference for either N form but demonstrated the highest N uptake rate. These findings indicate the significant role of mycorrhizal type and biological nitrogen fixation in shaping N uptake patterns. Importantly, even when accounting for the dilution by the soil nitrogen pool, nitrate still contributed 42–99% of the total nitrogen uptake across the three tree species. All three species showed a substantial capacity for nitrate assimilation, challenging the conventional view of ammonium dominance in tropical trees and providing a mechanistic basis for refining nitrogen management practices in plantation forestry.

1. Introduction

Planted forests serve as a significant component of China’s forest carbon sink. The expansion of planted forest areas is considered a major strategic initiative for achieving peak carbon and carbon neutrality [1]. Benefiting from favorable hydrothermal conditions and abundant solar radiation, southern China has developed into the primary plantation region in China, accounting for approximately 63% of the total plantation area nationwide [2]. However, the widespread practice of monoculture plantation management in this region has led to a decline in soil nitrogen (N) availability [3], resulting in a N supply deficit that struggles to meet the high demands of rapidly growing plantations. This N limitation restricts the sustained enhancement of productivity in plantation ecosystems. In this context, elucidating N acquisition strategies of common tree species in tropical plantations becomes a central scientific question for improving N use efficiency and maintaining long-term carbon sequestration capacity in plantations.

For tropical trees, the long-standing consensus holds a strong preference for ammonium (NH₄⁺) over nitrate (NO₃⁻) [4]. However, this view is not without uncertainties. Tree nitrogen uptake strategies are governed by a complex interplay of physiological trade-offs and biotic associations. Plants primarily take up ammonium (NH₄⁺) and nitrate (NO₃⁻) from the soil, while the physicochemical properties of inorganic N influence their assimilation: while ammonium assimilation is traditionally considered energetically less costly and faster [5,6,7], excessive NH₄⁺ absorption may induce phytotoxicity that partially offsets its energetic advantages [8]. In contrast, nitrate exhibits greater mobility in soil, thereby enhancing its accessibility to roots [9,10]. Additionally, mycorrhiza types significantly shape tree N uptake strategies. Arbuscular mycorrhizal (AM) tree species often exhibit a greater capacity for nitrate uptake [11,12,13], whereas trees associated with ectomycorrhizal fungi (ECM) tend to prefer ammonium [14,15,16]. Thus, the interactions among physiological trade-offs, such as assimilation cost versus toxicity, edaphic mobility, and mycorrhizal symbiosis, collectively determine N acquisition strategies, contributing to the uncertainty in predicting plant N uptake preferences.

The limitations of methodologies in previous studies have further exacerbated the current uncertainty regarding conclusions on tree nitrogen uptake preferences. Most prior research predominantly relied on hydroponic experiments, in which seedlings or excised roots from mature trees were exposed to ¹⁵N-labeled solutions to assess N uptake [17,18]. However, such hydroponic methodologies fail to account for soil microbial competition for ammonium and nitrogen immobilization mediated by organic matter [19,20]. In addition, this approach overlooks organ-specific variation in N translocation and assimilation within plants. For example, nitrate is often transported via the xylem to leaves for reduction [21], whereas ammonium is primarily assimilated in roots [22]. Consequently, hydroponic methods using excised roots tend to overestimate the NH₄⁺ uptake capacity of trees and inadequately reflect the N utilization strategies of mature trees under field conditions.

Recently developed in situ ¹⁵N paired-labeling techniques for mature trees provide a robust methodological framework for accurately determining tree N uptake preferences [23]. This approach integrates rhizospheric processes, nitrogen transformations, and whole-plant nitrogen allocation, having been successfully implemented in temperate conifers of northern China [23,24]. The results from these studies indicated that conifers assimilate 39%–90% of nitrogen as nitrate (NO₃⁻), contrasting sharply with the ammonium preference suggested by earlier hydroponic experiments. Nevertheless, this methodology has not yet been employed to investigate N uptake in mature trees within tropical plantations, significantly hindering the development of precision nitrogen management practices in this region. Importantly, the high temperature, high humidity, and intensified soil leaching dynamics characteristics of southern China may substantially alter soil nitrogen availability and cycling processes [25], potentially leading to fundamental divergences in tree nitrogen acquisition strategies compared with those in temperate forests [4]. Therefore, a systematic investigation of N acquisition strategies in southern plantation species is imperative for optimizing forest management and enhancing plantation productivity.

Integrating differences in mycorrhizal associations and physiological traits, three representative tree species from southern tropical plantations, Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. (H. brasiliensis, an AM-associated latex-producing economic species), Pinus caribaea Morelet (P. caribaea, an ECM-associated fast-growing timber species), and Acacia mangium Willd. (A. mangium, an ECM-associated N-fixing species), were selected to investigate interspecific variation in N acquisition strategies. We hypothesized that (1) nitrogen uptake preferences are influenced by tree species traits and mycorrhizal type, with ECM species (e.g., P. caribaea) preferentially acquiring NH₄⁺, AM species (e.g., H. brasiliensis) favoring NO₃⁻, and the nitrogen-fixing A. mangium exhibiting no strong preference due to access to multiple N sources; and (2) regional climate conditions further drive divergence in N acquisition, whereby the warm, humid environment and accelerated nitrogen cycling in southern plantations promote greater reliance on NH₄⁺ compared with northern temperate forests. To test these hypotheses, we employed an in situ whole-tree ¹⁵N labeling approach with the following specific aims: (1) to quantify and compare the N uptake strategies of these three species, and (2) to determine whether systematic differences in N acquisition correspond to mycorrhizal type and species traits.

2. Materials and Methods

2.1. Study Site

This study was conducted in the Jianfengling National Natural Reserve, on Hainan Island, southern China. The region is characterized by a tropical monsoon climate, with a wet season (from May to October) and a dry season (from November to April). Between 2009 and 2018, annual precipitation averaged 2414 mm (from 1637 mm to 3458 mm), and the mean annual temperature was 19.7 °C. The soil type is acidic lateritic. This experiment was performed in three plantations: H. brasiliensis, P. caribaea, and A. mangium. The plantation forests were all c. 30–40 yr. old.

2.2. Experimental Design

The labeling experiment was conducted in September 2024, during the late rainy season. This period corresponds to a key active growth phase for all species, yet with distinct phenological emphases: H. brasiliensis is in a phase of sustained vegetative growth, A. mangium is typically entering its flowering period, and P. caribaea is in a late growing and nutrient storage stage. Within each monoculture plantation, ten trees of similar diameter at breast height were selected and grouped into five pairs, with >10 m between any two pairs. Paired trees were spaced 5–10 m apart to minimize root system intermingling. For each pair, one tree received ¹⁵NH₄NO₃ (99.14 atom%, Shanghai Research Institute of Chemical Industry CO., LTD. Shanghai China) while the other received NH₄¹⁵NO₃ (99.21 atom%, Shanghai Research Institute of Chemical Industry CO., LTD. Shanghai China). The ¹⁵N labeling dose of 50 mg ¹⁵N m⁻² was added to a 1.5 m radius of each tree, thus each tree individual received 353.25 mg ¹⁵N (1.907 g N in total) dissolved in 7 L of tap water containing equimolar concentrations of ammonium nitrate, with either N form labeled (¹⁵NH₄NO₃ or NH₄¹⁵NO₃).

To reflect realistic N uptake by mature trees in situ, we tried to minimize disturbance by using a custom-made metal net of 1.5 m radius gridded at 10 cm intervals to locate positions for the injection of ¹⁵N tracer solutions. Prior to injection, understory shrubs and grasses were cleared within the labeling area to limit non-tree root uptake. The ¹⁵N tracer solutions were injected into the mineral soil at a depth of 10 cm using a 50 mL syringe equipped with a side-hole needle, with injections made at 5 cm intervals. After finishing tracer injections, another 7 L of tap water (pH = 7, 0.153 mg NO₃⁻ L⁻¹, 0.008 mg NH₄⁺ L⁻¹) was spread evenly over the labeling area (7.065 m²) to diffuse the ¹⁵N tracers. These tracer solutions and water additions were equivalent to 2 mm of precipitation.

Prior to ¹⁵N labeling, and 3 and 7 days after ¹⁵N tracer application, leaves, fine roots, and bulk soil within the ¹⁵N labeling area were sampled for analysis. For each sampling, at least one branch of the target trees was harvested from the middle crown; leaves and branches were separated. Mineral soil samples were extracted using an auger (5 cm in inner diameter) and divided into two layers (0–10 and 10–20 cm). Six soil cores were extracted randomly in each plot and mixed into one composite sample based on soil depth. Fine roots (0–20 cm) were hand-sorted from each composite soil sample and then cleaned with deionized water.

2.3. Chemical Analyses

All leaf and fine root samples were oven-dried at 65 °C to constant weight. Mineral soil from each plot was passed through a 2 mm mesh to remove fine roots and coarse fragments, and then the soil was air-dried at room temperature. After drying, the samples were ball-milled and analyzed for ¹⁵N natural abundance and total N and total carbon (C) concentrations using elemental analyzer–isotope ratio mass spectrometry (Elementar Analysen Systeme GmbH, Hanau, Germany; IsoPrime100, IsoPrime Ltd., Stockport, UK). Four reference compounds were calibrated: L-histidine, D-glutamic acid, glycine, and acetanilide. The analytical precision for both δ¹⁵N and δ¹³C was 0.2‰.

The soil samples were extracted with a 2 M KCl solution at a 1:4 soil-to-solution ratio. The KCl solution was pre-combusted at 450 °C for 48 h to eliminate background nitrate (NO₃^–) and ammonium (NH₄⁺) contamination. Extractions were performed within 12 h after soil sampling. The extracts were shaken for 1 h, filtered, and frozen at −36 °C until analysis. The ammonium-N concentrations were determined colorimetrically using the indophenol blue method, while nitrate-N concentrations were measured via hydrazine reduction spectrophotometry (SmartChem200, Rome, Italy).

Soil pH was determined by placing 10 g of air-dried soil into a 50 mL centrifuge tube. A soil suspension was prepared at a soil-to-water ratio of 1:2.5 (dry weight basis) using CO₂-free deionized water. The mixture was shaken on an orbital shaker for 10 min and then allowed to settle for 30 min. Subsequently, the pH of the supernatant was measured with a PHS-3E pH meter (INESA (Group) Co., Ltd. Shanghai China).

Soil bulk density was determined by collecting soil samples using a 100 cm³ soil core sampler (ring knife), followed by oven-drying and calculation based on dry soil mass and core volume.

2.4. Calculation of ¹⁵N Uptake and Biomass Metrics

The ¹⁵N atom% excess (APE) was calculated as the difference in atom% ¹⁵N between ¹⁵N-labeled plant organs and prior to ¹⁵N labeling samples (control). The ¹⁵N enrichment value was calculated using the following formula: [(sample abundance (δ¹⁵N_sample) − natural abundance of the control (δ¹⁵N_ref))/(control abundance (δ¹⁵N_ref) − 1000)] × 1000 [26].

The ¹⁵N uptake and assimilation rates (µg ¹⁵N g⁻¹ dry mass d⁻¹) were calculated by multiplying fine root and leaf N content (µg g⁻¹ dry mass) by the corresponding (APE/100), and then dividing by the labeling time (in days) and the atom% ¹⁵N/100 of the applied ¹⁵N-labeled N form (99.14 atom% for ¹⁵NH₄NO₃ and 99.21 atom% for NH₄¹⁵NO₃) [18]. The contribution percentage of each inorganic nitrogen source was calculated as follows: absorption rate of ammonium nitrogen or nitrate nitrogen/sum of absorption rates of two nitrogen sources × 100%.

Leaf biomass was estimated using species-specific allometric equations for H. brasiliensis, A. mangium, and P. caribaea [27,28,29], as shown below, where W represents the leaf biomass and D represents the diameter at breast height:

W_{A. mangium} = 0.0228 × D^1.7535

(1)

W_{H. brasiliensis} = 0.007 × D^2.215

(2)

W_{P. caribaea} = 0.332 × D^0.855

(3)

while fine root biomass within labeled areas was extrapolated from collected root samples.

2.5. Data Compilation

We identified 12 articles from the Web of Science reporting on the N uptake strategies of tropical tree species. The selected studies were categorized based on the key methodological consideration of whether they accounted for soil microbial competition, resulting in two main groups (including considering microbial competition result and no microbial competition results), each encompassing two prevalent experimental subtypes (including seedling nitrogen-15 (¹⁵N) labeling experiment; natural ¹⁵N abundance methods; seedling root ¹⁵N labeling and mature tree root ¹⁵N labeling). Studies were selected based on three criteria: (1) the experiment directly assessed plant N uptake (excluding long-term atmospheric deposition fate studies); (2) it was conducted in a (sub)tropical region; (3) the contribution of NO₃⁻ to total N uptake was reported or calculable. The NO₃⁻ contribution was standardized as: NO₃⁻ uptake/(NO₃⁻ uptake + NH₄⁺ uptake). Details are in Supplementary Table S1.

2.6. Statistical Analysis

To test the hypothesis that nitrogen uptake strategies differ among species, one-way ANOVA followed by LSD post hoc tests and independent t-test were used to compare the three tree species across key response variables: soil physicochemical properties, plant traits, ¹⁵N abundance, and uptake/assimilation rates of ¹⁵NH₄⁺ and ¹⁵NO₃⁻. Effect sizes (Eta-squared, η², for ANOVA; Cohen’s d for t-tests) were calculated to quantify the magnitude of observed differences beyond mere statistical significance. All analyses were performed at a significance level of p < 0.05 using SPSS 19.0 (version 19.0; SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Soil and Stand Properties

Soil ammonium and nitrate concentrations differed significantly among the three plantations. Ammonium is the dominant N form in H. brasiliensis and P. caribaea plantations, with concentrations ranging from 1.0 to 8.4 mg N kg⁻¹ (Figure 1, η² = 0.566, η² = 0.247). By contrast, A. mangium plantations were nitrate-dominated, showing nitrate-N concentrations of 2.5–10.8 mg N kg⁻¹ (Figure 1, η² = 0.386).

Soil C:N ratios were significantly higher in P. caribaea plantations than in the other two forest types (

Table 1. Soil physicochemical properties in the 0–20 cm mineral soil for the Hevea brasiliensis, Acacia mangium, and Pinus caribaea forest plantations.

	C (%)	N (%)	C/N	pH	Bulk Density (g/cm3)	NH4+/NO3−
Hevea brasiliensis	0.89 ± 0.15 b	0.08 ± 0.01 b	11.13 ± 0.82 b	4.80 ± 0.92 a	2.40 ± 0.10 b	24.90 ± 5.71 a
Acacia mangium	1.52 ± 0.19 a	0.12 ± 0.01 a	12.67 ± 0.84 b	4.50 ± 0.63 b	2.60 ± 0.14 a	0.50 ± 0.06 c
Pinus caribaea	1.85 ± 0.16 a	0.11 ± 0.02 a	16.82 ± 1.91 a	4.50 ± 0.74 b	2.60 ± 0.12 a	1.83 ± 0.31 b

Values are presented as mean ± SE. Different superscript letters indicate significant differences in soil properties across the Hevea brasiliensis, Acacia mangium, and Pinus caribaea forest plantations (one-way ANOVA, p < 0.05).

). All soils were consistently acidic, with pH values ranging from 4.5 to 4.8 and exhibiting significant differences among the plantations (Table 1). Leaf biomass of sampled trees ranged from 6.3 to 10.9 Mg ha⁻¹, while fine root biomass per tree in the labeled area varied between 0.55 and 1.64 kg (

Table 2. Plant traits in the Hevea brasiliensis, Acacia mangium, and Pinus caribaea forest plantations.

	Age	DBH (cm)	Leaf			Fine Root
			C(%)	N(%)	Biomass (Mg/ha)	C (%)	N (%)	Biomass (kg)
Hevea brasiliensis	40	24.60 ± 0.22 c	49.49 ± 0.14 a	2.27 ± 0.07 a	8.44 ± 0.17 b	43.96 ± 0.37 a	1.39 ± 0.05 c	1.28 ± 0.06 a
Acacia mangium	30	33.70 ± 0.79 a	45.99 ± 4.25 a	2.80 ± 0.27 a	10.91 ± 0.45 b	44.59 ± 0.57 a	2.06 ± 0.06 b	0.40 ± 0.04 b
Pinus caribaea	30	31.42 ± 0.41 b	49.34 ± 0.49 a	1.04 ± 0.05 b	6.33 ± 0.70 a	41.28 ± 0.80 b	1.28 ± 0.10 a	0.42 ± 0.06 b

Values are presented as mean ± SE. Different superscript letters indicate significant differences in plant traits across the Hevea brasiliensis, Acacia mangium, and Pinus caribae forest plantations (one-way ANOVA, p < 0.05). Diameter at breast height, DBH.

). Significant differences in leaf N concentration and fine root C/N content were found among the three plantations (Table 2).

3.2. The ¹⁵N Enrichment and ¹⁵N Uptake Rates

Before ¹⁵N labeling, fine root and leaf exhibited δ¹⁵N values of −0.4‰ to 9.1‰ and −2.4‰ to 3.7‰, respectively (Figure S3). After ¹⁵N labeling, a marked contrast in fine root ¹⁵N enrichment dynamics was observed: whereas H. brasiliensis and P. caribaea showed progressive enrichment peaking at day 7, A. mangium reached its maximum by day 3 and then declined slightly (Figure 2). In contrast, leaf δ¹⁵N continued to increase over the entire measurement period.

The three plantation species exhibited distinct responses to ¹⁵NO₃⁻ and ¹⁵NH₄⁺ labeling in terms of ¹⁵N enrichment. In fine roots, H. brasiliensis showed significantly higher ¹⁵N enrichment under ¹⁵NO₃⁻ labeling than under ¹⁵NH₄⁺ labeling on day 7 (Figure 2, p < 0.01, Cohen’s d = 2.57), whereas P. caribaea displayed the opposite pattern, with higher enrichment following ¹⁵NH₄⁺ labeling on day 7. In leaves, the enrichment of ¹⁵NO₃⁻ was significantly higher than that of ¹⁵NH₄⁺ in A. mangium on day 7 (p < 0.05, Cohen’s d = 2.03), and a consistent nitrogen preference was observed in the other two species, mirroring the patterns observed in their fine roots.

After ¹⁵N labeling, the three studied species exhibited clear organ-specific and species-specific patterns in nitrogen uptake dynamics. Fine root uptake peaked uniformly on day 3 before declining across all species (Figure 3), with rates ranging from 0.50 to 4.92 μg ¹⁵N g⁻¹ d⁻¹. Fine roots of H. brasiliensis showed a pronounced preference for ¹⁵NO₃⁻ (0.91–5.3 μg ¹⁵N g⁻¹ d⁻¹), significantly exceeding ¹⁵NH₄⁺ uptake (Figure 3, p < 0.01, Cohen’s d = 2.24). A. mangium exhibited no statistically significant difference between nitrogen forms, though ¹⁵NO₃⁻ uptake (0.61–4.55 μg ¹⁵N g⁻¹ d⁻¹) was numerically higher than ¹⁵NH₄⁺ uptake (0.53–3.03 μg ¹⁵N g⁻¹ d⁻¹). In contrast, P. caribaea fine roots demonstrated a marked dependence on ¹⁵NH₄⁺ (0.76–3.56 μg ¹⁵N g⁻¹ d⁻¹), which became significantly greater than ¹⁵NO₃⁻ uptake by day 7 (p < 0.05, Cohen’s d = 2.1).

Leaf ¹⁵N uptake rates remained consistently lower than those in fine roots, ranging from −0.03 to 0.65 μg ¹⁵N g⁻¹ d⁻¹, and displayed a gradual increase over time. A. mangium exhibited the highest foliar uptake capacity, followed by H. brasiliensis, while P. caribaea showed the lowest rates (Figure 3). By day 7, clear species-specific nitrogen form preferences emerged: A. mangium leaves absorbed ¹⁵NO₃⁻ (0.08–0.45 μg ¹⁵N g⁻¹ d⁻¹) at rates higher than ¹⁵NH₄⁺ (Figure 3); H. brasiliensis displayed similar uptake for both forms with marginally elevated ¹⁵NO₃⁻ assimilation; and P. caribaea demonstrated significantly greater ¹⁵NH₄⁺ uptake (0.0003–0.018 μg ¹⁵N g⁻¹ d⁻¹) compared with ¹⁵NO₃ (0.001–0.007 μg ¹⁵N g⁻¹ d⁻¹) (p < 0.05, Cohen’s d = 1.81).

3.3. Contribution of Nitrogen Uptake

Since preliminary results showed that obvious ¹⁵N enrichment in leaves could not be detected on day 3, nitrogen contribution percentages were derived from ¹⁵N uptake rates measured at day 7. At the whole-plant level, H. brasiliensis exhibited a clear preference for nitrate, with ¹⁵NO₃⁻ accounting for 76% of total nitrogen uptake. In contrast, A. mangium showed comparable assimilation of both nitrogen forms, though with a slightly higher contribution from ¹⁵NO₃⁻ (53%), while P. caribaea demonstrated a marked dependence on ammonium, with ¹⁵NH₄⁺ contributing 61% to total uptake (Figure 4). Additionally, even after accounting for the dilution effect of the soil inorganic N pool on the ¹⁵N-labeled sources, the proportional nitrate uptake by the three tree species remained between 42% and 99% (Figure 5).

A similar nitrogen contribution pattern of nitrogen contribution at the whole-plant level was observed at the plant organ level. For fine roots, ¹⁵NO₃⁻ uptake substantially exceeded that of ¹⁵NH₄⁺ in H. brasiliensis and A. mangium, contributing 68% and 47% of total root uptake, respectively. This contrasted sharply with P. caribaea, which showed a 59% contribution from ¹⁵NH₄⁺ (Figure 4). For leaves, the nitrogen uptake mirrored the strategies observed in fine roots. Both H. brasiliensis and A. mangium maintained higher ¹⁵NO₃⁻ contributions (83% and 61%, respectively), whereas P. caribaea continued to rely predominantly on ¹⁵NH₄⁺ (62% contribution) (Figure 4).

4. Discussion

4.1. Efficient Nitrate Uptake by Tropical Tree Species

Our in situ whole-tree paired ¹⁵N labeling experiment reveals clear interspecific divergence in N acquisition strategies among three species in tropical plantations. Contrary to the prevailing view that tropical tree species exhibit a predominant preference for ammonium [4], our results indicated that tropical tree species assimilated nitrate efficiently, with ¹⁵NO₃⁻ contributing 39% to 76% of total ¹⁵N tracer uptake (Figure 4). Even considering the dilution effect, all three tree species still demonstrated a capacity for nitrate absorption (Figure 5).

Specifically, H. brasiliensis and A. mangium exhibited a clear tendency toward nitrate uptake, accounting for 76% and 53% of their respective tracer uptake. This finding challenges conclusions derived primarily from hydroponic studies (Figure 6), which may overlook critical field complexities such as microbial competition for ammonium and soil N immobilization [19,20]. The observed nitrate preference may represent an ecological strategy to circumvent these competitions. Nitrate’s higher mobility enhances its diffusion to root surfaces in soil [30], thereby enhancing its plant availability [10,31]. Moreover, NO₃⁻ can be assimilated in both roots and leaves, offering metabolic flexibility. In contrast, NH₄⁺ is prone to adsorption by soil organic matter [20] and must be assimilated rapidly in roots to avoid toxicity [32]. It is also preferentially consumed by soil microorganisms [20,33,34]. Thus, although nitrate assimilation is energetically more costly [35,36], it may represent a strategy to circumvent microbial competition [37].

Notably, P. caribaea derived 61% of the total N uptake from ammonium, indicating a distinct NH₄⁺ preference. This contrasts with findings from in situ ¹⁵N labeling studies in temperate coniferous forests of northeastern China, where NO₃⁻ contributed 39%–90% to N acquisition in conifers [49]. Climatic influences may explain this discrepancy [4]. Compared with temperate regions, the tropical monsoon climate at our site, characterized by high temperature and humidity, likely accelerates N cycling and alters N bioavailability. Elevated temperatures can enhance the diffusion rate of NH₄⁺ in soil [6], increasing its mobility and thus improving root accessibility. Concurrently, evidence suggests that shifts in precipitation regimes also modulate plant nitrogen uptake preference [18,43]. Under combined thermal and hydrological drivers, NH₄⁺ in tropical forest soils may reach root surfaces more rapidly than in temperate systems, thereby facilitating plant acquisition. This mechanism offers a plausible explanation for the high ¹⁵NH₄⁺-N uptake rates observed in the P. caribaea, underscoring the role of regional climate in shaping nitrogen uptake strategies.

4.2. Nitrogen Uptake Regulated by Mycorrhizal Types and Nitrogen-Fixing Capacity

Our results indicated significant interspecific differences in inorganic nitrogen acquisition strategies among the three tropical tree species. H. brasiliensis, commonly associated with AM fungi, exhibited a strong preference for NO₃⁻, while A. mangium (an ECM-associated nitrogen-fixing species) also showed a tendency toward NO₃⁻ uptake, albeit to a lesser extent. In contrast, P. caribaea (commonly associated with ECM fungi) demonstrated a clear preference for NH₄⁺. Although the present study did not experimentally quantify mycorrhizal colonization in the study trees, the observed nitrogen acquisition preferences align closely with the known mycorrhizal associations of these species in natural and cultivated settings, supporting the well-established framework of mycorrhizal-mediated niche differentiation in N nutrition [45].

Under field conditions, AM tree species often exhibit a preference for NO₃⁻ [11,12,13,47], which may be attributed to the enhanced nitrate uptake capacity mediated by AM fungal symbiosis. Genomic analyses indicate that AM fungi rarely assimilate organic nitrogen but primarily acquire inorganic nitrogen from soil [50,51]. Furthermore, their high nitrate reductase activity enables efficient assimilation of environmental nitrate [52], which may explain the generally high nitrate uptake observed in AM-associated trees. In contrast, ECM fungi can secrete extracellular hydrolytic enzymes [52], facilitating access to both organic and inorganic nitrogen sources. However, the expression of genes involved in nitrate assimilation is often suppressed in the presence of ammonium [53]. Cultivation experiments have confirmed that most ECM fungi grow more efficiently and exhibit higher nitrogen uptake rates with ammonium than with nitrate [54,55], suggesting a physiological bias toward ammonium utilization in ECM systems.

Consistent with our hypothesis, A. mangium, an ECM species with nitrogen-fixing capacity, displayed no strong preference for either nitrogen form. This flexibility likely stems from its reduced dependence on soil inorganic N due to symbiotic biological nitrogen fixation. This finding is consistent with a previous pot experiment showing that nitrogen-fixing tree species exhibit no specific preference for inorganic nitrogen forms [56]. The slight tendency toward NO₃⁻-N observed in A. mangium may be attributed to the higher mobility of nitrate in soil [31]. Furthermore, A. mangium exhibited higher foliar nitrogen uptake rates than the other two species (Figure 3), which is consistent with the observation that nitrogen-fixing plants often exhibit elevated rates of inorganic nitrogen uptake [57,58]. This may be linked to the upregulation of nitrogen assimilation pathways by symbiotic rhizobia, which enhance the expression of key genes involved in nitrogen uptake and metabolism, such as ammonium transporters (AMTs), nitrate transporters (NRTs), nitrate reductase (NR), and glutamine synthetase (GS) [59,60]. In plant transport systems, AMTs and NRTs play central roles in regulating ammonium and nitrate uptake, respectively [61], while NR and GS are key enzymes in nitrate and ammonium assimilation [62,63].

5. Conclusions

This study employed an in situ ¹⁵N labeling technique to quantify NH₄⁺-N and NO₃⁻-N uptake preferences in three common tropical plantation species in southern China: H. brasiliensis (AM-associated), P. caribaea (ECM-associated), and A. mangium (ECM-associated and N-fixing). Our results demonstrate that the AM-associated species H. brasiliensis showed a tendency toward NO₃⁻-N assimilation, while the ECM-associated species P. caribaea depended more heavily on NH₄⁺-N. In contrast, A. mangium did not exhibit a strong preference for either nitrogen form but instead showed higher nitrogen uptake and greater metabolic flexibility. This supports our first hypothesis that both mycorrhizal type and nitrogen fixation capacity are important factors influencing plant nitrogen acquisition preferences. Notably, our results also challenge the conventional view that tropical trees predominantly favor ammonium. Contrary to our second hypothesis, all three species exhibited measurable NO₃⁻-N uptake, with H. brasiliensis and A. mangium assimilating nitrate as a major nitrogen source. This suggests that mature tropical trees retain physiological adaptations for nitrate utilization, likely reflecting the rapid nitrogen cycling characteristic of warm, humid ecosystems. These mechanistic insights provide a scientific basis for refining nitrogen management in plantation forestry, including the strategic selection of tree species and mycorrhizal types to optimize nitrogen use efficiency and long-term productivity.