Increasing phosphorus limitation with tree age in tropical forests

Increasing phosphorus limitation with tree age in tropical forests | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Increasing phosphorus limitation with tree age in tropical forests Nan Hu, Qinggong Mao, Liang Zheng, Xibin Sun, Yixue Hong, Yi Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5904638/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 May, 2025 Read the published version in Plant and Soil → Version 1 posted 6 You are reading this latest preprint version Abstract Aims Phosphorus (P) availability commonly limits the growth of tropical plants, yet how this limitation changes with tree age remains uncertain. Methods Here we investigated the effect of tree age on P limitation in a tropical forest by examining three functional plant groups: fast-growing, slow-growing, and nitrogen (N)-fixing tree species. We measured leaf N and P resorption efficiency (NRE and PRE), and used the ratio of PRE to NRE (PRE:NRE) as an indicator of plant P limitation. Results Our results revealed a significant increase in both PRE and PRE:NRE with tree age across all functional plant groups, indicating a widespread intensification of P limitation as plants mature. Furthermore, such increase in P limitation was more pronounced in slow-growing and N-fixing species compared to fast-growing species. Conclusions These findings underscore the crucial role of tree age in influencing P limitation in tropical forests, a factor that should be incorporated into terrestrial biogeochemical models, which have traditionally overlooked this effect. nutrient limitation nitrogen fixation growth type stand age plant strategy model development Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Phosphorus (P) is a critical nutrient essential for plant growth, function, and reproduction. However, in most soils, P is not readily available to plants (Holford 1997 ; Vitousek et al. 2010 ), making it a key limiting factor for plant productivity in terrestrial ecosystems (Hou et al. 2020 ). Tropical forests, in particular, are characterized as P-limited ecosystems, largely due to the prevalence of older, weathered soils (Walker and Syers 1976 ; Wardle, Walker and Bardgett 2004 ), higher plant nitrogen-to-phosphorus (N:P) ratios (Townsend et al. 2007 ), greater plant P-use efficiencies (Paoli, Curran and Zak 2005 ), and lower P concentrations in both plants and soil (Hidaka and Kitayama 2011 ). Factors that aggravate (Richardson et al. 2004 ) or alleviate (Tipping et al. 2014 ) this P limitation can substantially affect nutrient cycling and ecosystem functioning in tropical forests. Given that P limitation is integrated into ecological models to predict plant production and carbon storage in terrestrial ecosystems (Wieder et al. 2015 ; Sun et al. 2017 ; Du et al. 2021 ), understanding the factors regulating P limitation is crucial for improving our knowledge of P cycling and enhancing the predictive accuracy of ecological models. Tree age is an important yet often overlooked factor influencing various physiological processes in plants. Despite its potential significance, our understanding of how P limitation varies with tree age, particularly in tropical forests, remains limited. Theoretically, as plant biomass increases with age (O'Brien et al. 1995), P demand is expected to rise, potentially aggravating P limitation in tropical forests (Perakis and Pett-Ridge 2019 ; Wu et al. 2020 ; Zhao et al. 2023 ; Lai et al. 2024 ). However, some studies suggest that older plants may develop adaptive strategies to enhance P acquisition, such as increasing root phosphatase activity (Brooks et al. 2013 ; Huang et al. 2013 ), producing root secretions (Hinsinger 2001 ), and fostering associated fungal biomass (Liu et al. 2021 ), which could alleviate P limitation. Additionally, field experiments have reported increased growth rates in smaller trees but no significant effect in intermediate or larger trees under P fertilization, implying a decrease in P limitation with increasing tree age (Alvarez-Clare, Mack and Brooks 2013 ; Li et al. 2018 ; Aoyagi et al. 2024 ). However, these studies often compare different growth stages across different species or forests, complicating direct interpretation. These theoretical ambiguity and experimental uncertainty underscore the need for further investigation into how P limitation shifts with tree age in tropical forests. The variation in P limitation with tree age may also differ among functional plant types, such as fast-growing, slow-growing, and nitrogen (N)-fixing species. Fast-growing species generally have higher nutrient demands (Arias et al. 2011 ), and these demands may increase with growth. If soil P supply is insufficient to meet the needs of these species, P limitation may aggravate over time. In contrast, slow-growing species maintain relatively lower nutrient requirements throughout their lifespan (Sayer and Banin 2016 ), potentially resulting in less pronounced P limitation compared to fast-growing species. For N-fixing trees, their enhanced N-fixation capacity as they mature can create a substantial imbalance between N and P (Toro et al. 2023 ; Zhang et al. 2023 ), further exacerbating P limitation. Despite these observations, previous studies have not systematically compared how plant P limitation changes with age across various functional plant groups, thereby hindering our understanding of P limitation dynamics at the community level in tropical forests. The most direct method to assess plant P limitation is P fertilization, where relative plant growth rates before and after fertilization are compared. A significant increase in growth following fertilization indicates P limitation (Alvarez-Clare, Mack and Brooks 2013 ; Li et al. 2018 ). However, this method is not well-suited for examining the temporal progression of P limitation, as the limited area of fertilization plots and the slow growth rates of trees makes it challenging to track P limitation within the same species over time. Alternative methods, such as the leaf N:P ratio or root phosphatase activities, have been commonly used in previous studies (Koerselman and Meuleman 1996 ; Gusewell 2004; Vitousek et al. 2010 ; Brooks et al. 2013 ; Janes-Bassett et al. 2022 ). Recently, a global study on plant nutrient limitation introduced the ratio of leaf P and N resorption efficiencies (PRE:NRE) as a novel indicator for assessing P limitation (Du et al. 2020 ). This metric effectively captures plant responses to P availability and correlates well with results from field fertilization experiments (Han et al. 2013 ; Yan et al. 2018 ; Tong et al. 2022 ), making it a widely accepted indicator in the literature. In this study, we investigated the effect of tree age on plant P limitation in a tropical forest, using leaf PRE:NRE as an indicator of P limitation. To examine the potential influence of functional plant types, we selected three groups, i.e. fast-growing, slow-growing, and N-fixing plants, from 12 different species. We hypothesized that (1) plant P limitation may increase with tree age across all functional plant groups, and (2) this increase would be more pronounced in fast-growing and N-fixing species compared to slow-growing species. Materials and Methods Study site This study was conducted in Dinghushan Biosphere Reserve (DHSBR) (23°10′12″ N, 112°32′42″ E), the first nature reserve established in China. The area is characterized by a subtropical monsoon climate (Mao et al. 2021a ). The mean annual temperature is 21℃ with the coldest month in January (12.6°C) and the warmest month in July (28.0°C). The mean annual precipitation is 1927 mm, 75% of which occurs between March and August. The area primarily consists of subtropical evergreen broad-leaved forests (~ 80%, old-growth), well-protected secondary forests established since the 1960s (~ 15%), and plantations (~ 5%). The soil type throughout the sampling area is lateritic red earth (Oxisol, according to the U.S. soil taxonomy system) (Mao et al. 2021b ). Experimental design This experiment employs a two-factor design, with tree age and plant type as the main factors. We selected twelve tree species, categorized into three groups: four typically fast-growing species, four slow-growing species, and four N-fixing species. Fast-growing species include Triadica cochinchinensis (Euphorbiaceae), Mallotus paniculatus (Euphorbiaceae), Pinus massoniana (Pinaceae), and Eucalyptus urophylla (Myrtaceae). Slow-growing species include Psydrax dicocca (Rubiaceae), Nageia nagi (Podocarpaceae), Aidia canthioides (Rubiaceae), and Syzygium rehderianum (Myrtaceae). N-fixing species include Cycas revoluta (Cycadaceae), Archidendron lucidum (Fabaceae), Acacia mangium (Fabaceae), and Ormosia glaberrima (Fabaceae). To ensure similar environmental conditions for each species across different age stages, all selected plants were situated on sun-exposed slopes within a narrow elevation range (440–500m). Due to the variations in growth rates among species, each species was classified into five age groups by using diameter at breast height (DBH) as a proxy for tree age (Table 1 ): young, middle, near-mature, mature, and over-mature trees (Jiang et al. 2021 ). In addition to DBH, different age groups were characterized in the field by a simple way (Ling et al. 2015 ): 1) young trees: distinguished by recent germination and the emergence of leaves; 2) middle trees: characterized by broad leaves in the upper canopy and narrow leaves in the lower canopy, without blossoms or fruit setting; 3) near-mature trees: characterized by the growth of broad leaves throughout the canopy, with some new branches emerging from the trunk. These individuals begin to bloom and bear fruit; 4) mature trees: characterized by the growth of broad leaves throughout the canopy, with minimal or no withering branches. These trees are at their peak period of blossom and fruit production; 5) over-mature trees: the largest individuals characterized by broad leaves in the canopy, accompanied by a significant number of withered branches. Given that the photosynthetic tissue of Pinus massoniana consists of needles rather than broad leaves, its age classification follows the methodology established by Yang et al. ( 2010 ). Additionally, Cycas revoluta exhibits distinct morphological characteristics compared to typical tropical evergreen trees. Since standardized age group classifications for Cycas revoluta have not yet been established, we consulted local botanical experts to guide the age categorization in the field based on breast diameter. Table 1 Traits of twelve studied species in this study. Functional type Species Angiosperms or gymnosperms Growth type DBH growth rate (cm/yr) Maturity age (yr) DBH (cm) Young Middle Near-mature Mature Over-mature Fast-growing Eucalyptus urophylla Angiosperms Broadleaf > 0.5 16 40 Triadica cochinchinensis Angiosperms Broadleaf > 0.5 30 35 Mallotus paniculatus Angiosperms Broadleaf > 0.5 30 35 Pinus massoniana Gymnosperms Coniferous > 0.5 36 35 Slow-growing Syzygium rehderianum Angiosperms Broadleaf < 0.2 80 20 Psydrax dicocca Angiosperms Broadleaf < 0.2 80 20 Aidia canthioides Angiosperms Broadleaf < 0.2 80 20 Nageia nagi Gymnosperms Broadleaf < 0.2 60 22 Nitrogen-fixing Ormosia glaberrima Angiosperms Broadleaf > 0.5 40 25 Archidendron lucidum Angiosperms Broadleaf 0.2–0.5 40 25 Acacia mangium Angiosperms Broadleaf > 0.5 30 35 Cycas revoluta Gymnosperms Coniferous < 0.2 100 20 Collection of leaf and soil samples Fresh and senescent leaves were collected during the peak litterfall period in November 2021. Three replicate trees were sampled for each age stage of every species, with selected trees spaced at least 20 m apart to prevent interactions among individuals of the same age group. Fresh leaves were obtained from the upper canopy using high pruning shears. To collect senescent leaves, long sticks were used to shake the trunk or branches in the upper canopy. For young trees with fewer senescent leaves, mesh bags were placed over the branches to capture recently fallen leaves. Following collection, both fresh and senescent leaves were placed in envelopes and transported to the laboratory for pretreatment on the same day. The leaf samples in the envelopes were then dried in an oven at 65°C for 72 hours. Soil sampling was conducted concurrently with leaf collection. Soil was collected from the root zone of the trees using a soil auger to a depth of 0–10 cm. Three 5-cm diameter soil cores were taken from each tree and combined into a composite sample. The soil samples were air-dried in a shaded and well-ventilated environment for approximately one month until a constant weight was achieved. During soil sieving, living fine roots were hand-sorted. Both plant and soil samples were then ground in a ball mill, passed through a 100-mesh sieve, and stored in a cool, dry location for subsequent chemical analysis. Determination of N and P concentrations of soils and leaves Total N concentrations in both fresh and senescent leaves and soils were determined using the Kjeldahl acid-digestion method (Jackson, Peltzer and Wardle 2013 ). Total P concentrations in leaves and soils were determined using the molybdenum blue colorimetric method following digestion in a H 2 SO 4 + HClO 4 solution (Bouma and Dowling 1982 ). Determination of N fixation rates of N-fixing species Nitrogen fixation rates of N-fixing species were estimated using the 15 N natural abundance (δ 15 N) methods (Bedard-Haughn, Van Groenigen and Van Kessel 2003 ). Eucalyptus urophylla , a non-N-fixing species growing adjacent to the four N-fixing species studied, served as a reference plant to calculate the δ 15 N value of soil-derived N. Fresh leaves of the four N-fixing species and Eucalyptus urophylla were collected for δ 15 N determination. The ratio of 15 N to 14 N (δ 15 N, ‰) in leaves was measured using isotope ratio mass spectrometry (IRMS; Thermo Fisher Scientific, USA). The N fixation rate was characterized by the proportion of plant N derived from the atmosphere (%Ndfa), calculated using the following formula (Bedard-Haughn, Van Groenigen and Van Kessel 2003 ): where δ 15 N reference plant and δ 15 N legume refer to the 15 N natural abundance in Eucalyptus urophylla and four N-fixing species. The B value is − 1.4‰ in the current study, obtained from the average of several previous studies (Jacot et al. 2000 ; Png et al. 2017 ). This value represents the δ 15 N when atmospheric N is the sole N source for the N-fixing plants, as determined by planting the N-fixing seedlings in an N-free medium for three months (Andrews et al. 2011 ). Calculation of leaf N and P resorption efficiency Leaf N and P resorption efficiencies (NRE and PRE) were calculated as follows (Vergutz et al. 2012 ). $$\:\text{NRE}\left(\text{%}\right)\text{=[}\left({\text{N}}_{\text{g}}\text{-}{\text{N}}_{\text{s}}\text{×MLCF}\right)\text{/}{\text{N}}_{\text{g}}\text{]×100}$$ 2 $$\:\text{PRE}\left(\text{%}\right)\text{=[(}{\text{P}}_{\text{g}}\text{-}{\text{P}}_{\text{s}}\text{×MLCF)/}{\text{P}}_{\text{g}}\text{]×100}$$ 3 where N g and N s represent N concentrations in green and senescent leaves, respectively, and P g and P s denote P concentrations in green and senescent leaves, respectively. MLCF is a mass loss correction factor used to compensate for the loss of leaf mass during the senescence period (Van Heerwaarden, Toet and Aerts 2003 ). NRE and PRE were corrected using different MLCF values according to our previous study (Chen et al. 2021 ). We used leaf PRE:NRE as an indicator of plant P limitation. A PRE:NRE greater than 1 indicates P limitation, with higher ratios reflecting increasing severity of the limitation (Du et al. 2020 ). Statistical analysis Prior to statistical analyses, the normality of residuals was assessed using the Kolmogorov-Smirnov test, and homogeneity of variance was evaluated using Levene’s test. To compare differences in the studied variables, including NRE, PRE, PRE:NRE, soil total P, and N fixation rate across plant types and age groups, we conducted a one-way analysis of variance (ANOVA) followed by the least significant difference test (LSD). To compare the variation in P limitation with age between different plant types, we performed a linear regression analysis using DBH as the proxy for tree age. We calculated the regression slopes and determined that slopes are significantly different if their 95% confidence intervals do not overlap. A steeper slope indicates a more rapid intensification of plant P limitation with age, while a shallower slope suggests the opposite (Deng et al. 2019 ). All analyses were conducted using SPSS 19.0 (SPSS Inc.). Results Nutrient limitation status of the studied species Our results showed that all studied species were P-limited, as indicated by the mean PRE:NRE consistently exceeding 1 across all functional plant types (Fig. 1 a). Effects of tree age on plant P limitation Plant P limitation increased with tree age, as evidenced by the consistent increase in both leaf PRE:NRE and PRE. Specifically, NRE showed no significant change with tree age in most species, except for a significant decrease observed in Cycas revoluta (Fig. S3, Fig. 2 b-d). In contrast, PRE increased significantly with tree age for all individual species (Fig. S2) and across functional plant types (Fig. 2 f-h). Consequently, the leaf PRE:NRE also exhibited a significant increase with tree age in all individual species (Fig. S1 ) and across functional plant types (Fig. 1 b-d). Factors controlling the age effect on plant P limitation The magnitude of increasing P limitation varied among fast-growing, slowing growing, and N-fixing trees, as evidenced by their varying regression slopes (Fig. 3 ). The most pronounced increase in P limitation was observed in slow-growing trees (Slope S = 0.05), followed by N-fixing trees (Slope N = 0.03) and fast-growing trees (Slope F = 0.01). Significant differences were noted between slow-growing and fast-growing trees, as well as between N-fixing and fast-growing trees, but not between slow-growing and N-fixing trees. Soil P content and N fixation rate were important factors explaining the changes in plant P limitation with tree age (Fig. 4 ). A significant decrease in soil total P was observed with increasing age across all species (Fig. 4 a). Additionally, the N fixation rate of N-fixing trees significantly increased with age (Fig. 4 b). Discussion In this study, we provide the first evidence of a universal increase in P limitation with tree age in tropical forests, based on field observations of three functional plant groups comprising 12 tree species. (Fig. 1 a). Our investigation further revealed that slow-growing and N-fixing trees exhibit a greater increase in P limitation with age compared to fast-growing trees (Fig. 3 ). These empirical results substantially enhance our understanding of ecosystem P cycling. Given that terrestrial biogeochemical models have yet to incorporate the influence of tree age, our study highlights the importance of considering age-related effects on plant nutrient cycling to improve the accuracy of global nutrient cycling projections within such models. Increasing P limitation with tree age We observed a consistent increase in PRE:NRE across fast-growing, slow-growing, and N-fixing trees, indicating a widespread trend of aggravating P limitation with tree age in tropical forests. This finding supports our initial hypothesis. However, it contrasts with several field studies (Alvarez-Clare, Mack and Brooks 2013 ; Li et al. 2018 ; Aoyagi et al. 2024 ), which suggested that young trees experience greater P limitation than mature trees based on relative growth rates following P addition. These studies compared young and mature trees but focused on different species within broader functional groups, rather than examining the same tree species across their entire lifespan. In contrast, our research investigated the same tree species throughout their lifespans, providing more direct evidence of the age-related dynamics of P limitation. Moreover, previous studies may have been influenced by the use of relative growth rates post-fertilization as the primary metric for assessing P limitation. While young trees typically exhibit faster growth, older trees naturally slow down, meaning that the enhanced growth of young trees following P addition may not necessarily reflect greater P-limited, but rather their intrinsic growth characteristics. In this context, the leaf P and N resorption ratio (PRE:NRE) more accurately captures the demand for P, making it a more reliable indicator of P limitation. Overall, our findings emphasize the need to reconsider how P limitation varies across different growth stages in tropical forest ecosystems. Two mechanisms may underlie the observed increase in P limitation with tree age. The first mechanism is related to the growing demand for P as trees mature. As trees age, their photosynthetic capacity improves due to enhanced leaf exposure to optimal light conditions (Bond 2000 ; Forrester et al. 2017 ). This increase in photosynthetic activity drives a higher demand for P, which is a critical component in the light reaction of photosynthesis, essential for the synthesis of adenosine triphosphate (ATP) (Li et al. 2016 ). Additionally, as trees continue to grow, they require additional P for the development of structural components, such as wood, and reproductive structures, including flowers and seeds (Kerkhoff et al. 2006 ; Sardans and Penuelas 2015 ; Fortier and Wright 2021 ). Indeed, research has observed increases in the wood P:N ratio with tree growth, suggesting that storing P in wood is a strategy that tropical plants employ to adapt to low-P environments (Elser et al. 2010 ; Sardans and Penuelas 2015 ). Furthermore, substantial amounts of P are necessary for the development of reproductive structures, particularly for synthesizing phospholipid and nucleotide that are critical for seed formation (Kerkhoff et al. 2006 ; Fortier and Wright 2021 ). During the early growth stages, trees prioritize efficient nutrient use and vertical growth to maximize sunlight exposure (Martinez-Vilalta, Vanderklein and Mencuccini 2007 ). However, as trees mature, growth priorities shift toward reproduction, characterized by vigorous flowering and fruiting. This transition in growth priorities further exacerbates P limitation with increasing tree age. The second mechanism contributing to increased P limitation over time is the gradual depletion of soil P. Soil P primarily originates from the weathering of parent materials, a process that occurs at a slow rate (Holford 1997 ; Shen et al. 2011 ). As soil develops, P levels typically decrease due to processes such as leaching and plant uptake (Djodjic, Börling and Bergström 2004 ). In this study, the sampled trees ranged from 1 to 60 years of age, a period during which total soil P levels may decrease, leading to a reduced P supply for plant growth. This mechanism is supported by our observation of a decrease in soil total P with increasing tree age (Fig. 4 a). Plant type mediates the age effect on P limitation We found that slow-growing trees are more limited by P than fast-growing trees with increasing age, which contradicts our second hypothesis. This unexpected result may be attributed to differences in growth strategies and physiological traits between these two groups. Fast-growing trees, which typically have shorter lifespans (Black, Colbert and Pederson 2008 ), are likely to develop more efficient P acquisition strategies throughout their lives (Comas, Bouma and Eissenstat 2002 ). These strategies include increasing root surface area (Comas and Eissenstat 2004 ), secreting phosphatases (Yaffar et al. 2021 ), and enhancing P resorption from senescent leaves (Xu et al. 2021 ). In contrast, slow-growing trees may have less efficient P acquisition mechanisms due to slower root growth and a reduced ability to uptake P from the soil (Comas, Bouma and Eissenstat 2002 ; Comas and Eissenstat 2004 ). Additionally, slow-growing trees often face greater competition from fast-growing trees within the same ecosystem, which may exacerbate their P limitation over time. Furthermore, we observed that N-fixing trees are more limited by P than fast-growing trees with increasing age, partially supporting our hypothesis. Although many N-fixing trees are also fast-growing, they have a greater P demand due to increasing N-fixing capacity with age (see Fig. 4 b). Indeed, biological N fixation relies on P to produce nitrogenase, the enzyme responsible for catalyzing the conversion of atmospheric nitrogen (N 2 ) into ammonia (NH 3 ) (Reed, Cleveland and Townsend 2011 ). Additionally, as the amount of fixed N increases, N-fixing trees require additional P to maintain the stoichiometric balance between N and P (Pearson and Vitousek 2001 ). This increased P demand highlights the unique nutrient dynamics of N-fixing trees in tropical forests. Implications for model development The impact of tree age has long been neglected in previous ecological models, potentially leading to biased assessments of phosphorus (P) limitation's effects on ecosystem functioning. For instance, some models predict that future P limitation on plant growth could turn ecosystems into net CO 2 sources by the end of this century (Wieder et al. 2015 ; Sun et al. 2017 ; Luo et al. 2022 ). However, these models do not consider tree age, introducing significant uncertainties into their projections (Reed, Yang and Thornton 2015 ; Jiang et al. 2019 ). When tree age is considered, it becomes clear that young and old plants may play distinct roles in future carbon sequestration due to their different levels of P limitation. Our results suggest that neglecting the impact of tree age could lead to either an overestimation or underestimation of the potential for vegetation to mitigate climate change. Thus, incorporating tree age into ecological models is crucial for improving the accuracy of predictions regarding ecosystem responses to P limitation. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This study is supported by Shenzhen Science and Technology Program (Grant No. JCYJ20220530150015035) and Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, Chinese Academy of Sciences (VRMDE2301). Liang Zheng was supported by National Natural Science Foundation for Regional Innovation and Development (U21A20189) and Hunan provincial Natural Science Foundation of China (2022JJ50032). Author contributions HC, NH, and QM designed the research. NH, and QM collected data, and conducted fieldwork. NH performed lab analysis. NH conducted all data analyses and drafted the initial manuscript with significant contribution from HC and QM. All authors contributed to manuscript revisions. 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For Ecol Manag 460:117896. http://doi.org/10.1016/j.foreco.2020.117896 Xu M, Zhu Y, Zhang S, Feng Y, Zhang W, Han X (2021) Global scaling the leaf nitrogen and phosphorus resorption of woody species: Revisiting some commonly held views. Sci Total Environ 788:147807. http://doi.org/10.1016/j.scitotenv.2021.147807 Yaffar D, Defrenne CE, Cabugao KG, Kivlin SN, Childs J, Carvajal N, Norby RJ (2021) Trade-offs in phosphorus acquisition strategies of five common tree species in a tropical forest of Puerto Rico. Front Global Change 4:698191. http://doi.org/10.3389/ffgc.2021.698191 Yan T, Lü XT, Zhu JJ, Yang K, Yu LZ, Gao T (2018) Changes in nitrogen and phosphorus cycling suggest a transition to phosphorus limitation with the stand development of larch plantations. Plant Soil 422:385–396. https://doi.org/10.1007/s11104-017-3473-9 Yan Z, Tian D, Han W, Tang Z, Fang J (2017) An assessment on the uncertainty of the nitrogen to phosphorus ratio as a threshold for nutrient limitation in plants. 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Sci Total Environ 889:164047. https://doi.org/10.1016/j.scitotenv.2023.164047 Supplementary Files SupportinginformationPS.docx Cite Share Download PDF Status: Published Journal Publication published 07 May, 2025 Read the published version in Plant and Soil → Version 1 posted Editorial decision: Major revisions 17 Mar, 2025 Reviewers agreed at journal 10 Feb, 2025 Reviewers invited by journal 06 Feb, 2025 Editor invited by journal 30 Jan, 2025 Editor assigned by journal 30 Jan, 2025 First submitted to journal 29 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5904638","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":411988095,"identity":"e9538326-b87e-401c-908b-479c74ac34dd","order_by":0,"name":"Nan Hu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Hu","suffix":""},{"id":411988096,"identity":"08a0cb25-1d33-4945-a797-8ee435550cee","order_by":1,"name":"Qinggong Mao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qinggong","middleName":"","lastName":"Mao","suffix":""},{"id":411988097,"identity":"4811709e-16e9-4fac-a548-88093551bb07","order_by":2,"name":"Liang Zheng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Zheng","suffix":""},{"id":411988098,"identity":"5cd469da-04a0-4629-844e-81dbd782547b","order_by":3,"name":"Xibin Sun","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xibin","middleName":"","lastName":"Sun","suffix":""},{"id":411988099,"identity":"c955133d-867a-42e5-8aba-01f5aeade592","order_by":4,"name":"Yixue Hong","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yixue","middleName":"","lastName":"Hong","suffix":""},{"id":411988100,"identity":"d2ab0026-d180-46d4-b056-df5a62b5cbd6","order_by":5,"name":"Yi Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Yang","suffix":""},{"id":411988101,"identity":"26fa80c0-a5ba-4c71-b6f0-37aa1d01f7db","order_by":6,"name":"Jiarong Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jiarong","middleName":"","lastName":"Chen","suffix":""},{"id":411988102,"identity":"754f9fa8-dbce-4a03-ade4-3412742cb03f","order_by":7,"name":"Hao Chen","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-3577-2345","institution":"Sun Yat-Sen University","correspondingAuthor":true,"prefix":"","firstName":"Hao","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-01-26 05:37:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5904638/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5904638/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11104-025-07521-4","type":"published","date":"2025-05-07T15:57:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75887940,"identity":"9f3b38fa-3fe3-4061-a036-85944f387cee","added_by":"auto","created_at":"2025-02-10 09:25:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":610819,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVariations in PRE:NRE across different plant types and age stages\u003c/strong\u003e. (a) mean PRE:NRE across different plant types; (b, c, d) mean PRE:NRE across age stages. “*” denotes significant difference compared to young stage. The dashed horizontal lines represent the P limitation threshold (PRE:NRE=1), with mean PRE:NRE values above this line indicating P limitation.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5904638/v1/521425d593b570f4f4f2829e.png"},{"id":75886588,"identity":"5b761741-f21d-49b3-bd84-f9e3449f03b2","added_by":"auto","created_at":"2025-02-10 09:17:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1084539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVariations in NRE and PRE across different plant types and age stages\u003c/strong\u003e. (a) mean NRE across different plant types; (b, c, d) mean NRE across different age stages; (e) mean PRE across different plant types; (f, g, h) mean PRE across different age stages. “*” denotes significant difference compared to young stage.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5904638/v1/c71fdc2eec4c44663b805f95.png"},{"id":75886585,"identity":"222404b9-35f6-4db4-a95c-7554bbdb4094","added_by":"auto","created_at":"2025-02-10 09:17:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1262177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationships between diameter at breast height (DBH) and leaf PRE:NRE across three studied plant types.\u003c/strong\u003e Slope\u003csub\u003eF\u003c/sub\u003e, Slope\u003csub\u003eS\u003c/sub\u003e, and Slope\u003csub\u003eN\u003c/sub\u003e represent the regression slopes for fast-growing, slow-growing, and N-fixing trees, respectively. Different letters represent significant differences between slopes for different plant types.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5904638/v1/b88f80b0b75884af22e7a172.png"},{"id":75886583,"identity":"c164cec5-7556-4046-b455-8b305167a750","added_by":"auto","created_at":"2025-02-10 09:17:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1851795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVariations in soil available P (a) and N fixation rate (b) across different age stages. \u003c/strong\u003eDifferent letters represent significant differences among age stages. Boxes represent the 25\u003csup\u003eth\u003c/sup\u003e–75\u003csup\u003eth\u003c/sup\u003e percentiles (median shown as the solid lines), with the 5\u003csup\u003eth\u003c/sup\u003e and 95\u003csup\u003eth\u003c/sup\u003e percentiles represented by the whiskers.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5904638/v1/5ca43c3b938c46c7682e5716.png"},{"id":82537502,"identity":"3a68a149-f63d-47c6-a312-180f8794d2ca","added_by":"auto","created_at":"2025-05-12 16:07:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5244031,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5904638/v1/53f5ed79-cff9-4186-81e5-85dc04ca2cf2.pdf"},{"id":75886599,"identity":"85fa3e37-3901-4317-a326-3455d3ecfa86","added_by":"auto","created_at":"2025-02-10 09:17:28","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":781484,"visible":true,"origin":"","legend":"","description":"","filename":"SupportinginformationPS.docx","url":"https://assets-eu.researchsquare.com/files/rs-5904638/v1/d2a97ea04c4edc51efcfadfb.docx"}],"financialInterests":"","formattedTitle":"Increasing phosphorus limitation with tree age in tropical forests","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePhosphorus (P) is a critical nutrient essential for plant growth, function, and reproduction. However, in most soils, P is not readily available to plants (Holford \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Vitousek et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), making it a key limiting factor for plant productivity in terrestrial ecosystems (Hou et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Tropical forests, in particular, are characterized as P-limited ecosystems, largely due to the prevalence of older, weathered soils (Walker and Syers \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Wardle, Walker and Bardgett \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), higher plant nitrogen-to-phosphorus (N:P) ratios (Townsend et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), greater plant P-use efficiencies (Paoli, Curran and Zak \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and lower P concentrations in both plants and soil (Hidaka and Kitayama \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Factors that aggravate (Richardson et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) or alleviate (Tipping et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) this P limitation can substantially affect nutrient cycling and ecosystem functioning in tropical forests. Given that P limitation is integrated into ecological models to predict plant production and carbon storage in terrestrial ecosystems (Wieder et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Du et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), understanding the factors regulating P limitation is crucial for improving our knowledge of P cycling and enhancing the predictive accuracy of ecological models.\u003c/p\u003e \u003cp\u003eTree age is an important yet often overlooked factor influencing various physiological processes in plants. Despite its potential significance, our understanding of how P limitation varies with tree age, particularly in tropical forests, remains limited. Theoretically, as plant biomass increases with age (O'Brien et al. 1995), P demand is expected to rise, potentially aggravating P limitation in tropical forests (Perakis and Pett-Ridge \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lai et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, some studies suggest that older plants may develop adaptive strategies to enhance P acquisition, such as increasing root phosphatase activity (Brooks et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), producing root secretions (Hinsinger \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and fostering associated fungal biomass (Liu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which could alleviate P limitation. Additionally, field experiments have reported increased growth rates in smaller trees but no significant effect in intermediate or larger trees under P fertilization, implying a decrease in P limitation with increasing tree age (Alvarez-Clare, Mack and Brooks \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Aoyagi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, these studies often compare different growth stages across different species or forests, complicating direct interpretation. These theoretical ambiguity and experimental uncertainty underscore the need for further investigation into how P limitation shifts with tree age in tropical forests.\u003c/p\u003e \u003cp\u003eThe variation in P limitation with tree age may also differ among functional plant types, such as fast-growing, slow-growing, and nitrogen (N)-fixing species. Fast-growing species generally have higher nutrient demands (Arias et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and these demands may increase with growth. If soil P supply is insufficient to meet the needs of these species, P limitation may aggravate over time. In contrast, slow-growing species maintain relatively lower nutrient requirements throughout their lifespan (Sayer and Banin \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), potentially resulting in less pronounced P limitation compared to fast-growing species. For N-fixing trees, their enhanced N-fixation capacity as they mature can create a substantial imbalance between N and P (Toro et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), further exacerbating P limitation. Despite these observations, previous studies have not systematically compared how plant P limitation changes with age across various functional plant groups, thereby hindering our understanding of P limitation dynamics at the community level in tropical forests.\u003c/p\u003e \u003cp\u003eThe most direct method to assess plant P limitation is P fertilization, where relative plant growth rates before and after fertilization are compared. A significant increase in growth following fertilization indicates P limitation (Alvarez-Clare, Mack and Brooks \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, this method is not well-suited for examining the temporal progression of P limitation, as the limited area of fertilization plots and the slow growth rates of trees makes it challenging to track P limitation within the same species over time. Alternative methods, such as the leaf N:P ratio or root phosphatase activities, have been commonly used in previous studies (Koerselman and Meuleman \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Gusewell 2004; Vitousek et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Brooks et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Janes-Bassett et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recently, a global study on plant nutrient limitation introduced the ratio of leaf P and N resorption efficiencies (PRE:NRE) as a novel indicator for assessing P limitation (Du et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This metric effectively captures plant responses to P availability and correlates well with results from field fertilization experiments (Han et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tong et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), making it a widely accepted indicator in the literature.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the effect of tree age on plant P limitation in a tropical forest, using leaf PRE:NRE as an indicator of P limitation. To examine the potential influence of functional plant types, we selected three groups, i.e. fast-growing, slow-growing, and N-fixing plants, from 12 different species. We hypothesized that (1) plant P limitation may increase with tree age across all functional plant groups, and (2) this increase would be more pronounced in fast-growing and N-fixing species compared to slow-growing species.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eStudy site\u003c/h2\u003e\n \u003cp\u003eThis study was conducted in Dinghushan Biosphere Reserve (DHSBR) (23\u0026deg;10\u0026prime;12\u0026Prime; N, 112\u0026deg;32\u0026prime;42\u0026Prime; E), the first nature reserve established in China. The area is characterized by a subtropical monsoon climate (Mao et al. \u003cspan class=\"CitationRef\"\u003e2021a\u003c/span\u003e). The mean annual temperature is 21℃ with the coldest month in January (12.6\u0026deg;C) and the warmest month in July (28.0\u0026deg;C). The mean annual precipitation is 1927 mm, 75% of which occurs between March and August. The area primarily consists of subtropical evergreen broad-leaved forests (~\u0026thinsp;80%, old-growth), well-protected secondary forests established since the 1960s (~\u0026thinsp;15%), and plantations (~\u0026thinsp;5%). The soil type throughout the sampling area is lateritic red earth (Oxisol, according to the U.S. soil taxonomy system) (Mao et al. \u003cspan class=\"CitationRef\"\u003e2021b\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cp\u003eThis experiment employs a two-factor design, with tree age and plant type as the main factors. We selected twelve tree species, categorized into three groups: four typically fast-growing species, four slow-growing species, and four N-fixing species. Fast-growing species include \u003cem\u003eTriadica cochinchinensis\u003c/em\u003e (Euphorbiaceae), \u003cem\u003eMallotus paniculatus\u003c/em\u003e (Euphorbiaceae), \u003cem\u003ePinus massoniana\u003c/em\u003e (Pinaceae), and \u003cem\u003eEucalyptus urophylla\u003c/em\u003e (Myrtaceae). Slow-growing species include \u003cem\u003ePsydrax dicocca\u003c/em\u003e (Rubiaceae), Nageia nagi (Podocarpaceae), \u003cem\u003eAidia canthioides\u003c/em\u003e (Rubiaceae), and Syzygium rehderianum (Myrtaceae). N-fixing species include \u003cem\u003eCycas revoluta\u003c/em\u003e (Cycadaceae), \u003cem\u003eArchidendron lucidum\u003c/em\u003e (Fabaceae), \u003cem\u003eAcacia mangium\u003c/em\u003e (Fabaceae), and \u003cem\u003eOrmosia glaberrima\u003c/em\u003e (Fabaceae). To ensure similar environmental conditions for each species across different age stages, all selected plants were situated on sun-exposed slopes within a narrow elevation range (440\u0026ndash;500m).\u003c/p\u003e\n\u003cp\u003eDue to the variations in growth rates among species, each species was classified into five age groups by using diameter at breast height (DBH) as a proxy for tree age (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e): young, middle, near-mature, mature, and over-mature trees (Jiang et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition to DBH, different age groups were characterized in the field by a simple way (Ling et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e): 1) young trees: distinguished by recent germination and the emergence of leaves; 2) middle trees: characterized by broad leaves in the upper canopy and narrow leaves in the lower canopy, without blossoms or fruit setting; 3) near-mature trees: characterized by the growth of broad leaves throughout the canopy, with some new branches emerging from the trunk. These individuals begin to bloom and bear fruit; 4) mature trees: characterized by the growth of broad leaves throughout the canopy, with minimal or no withering branches. These trees are at their peak period of blossom and fruit production; 5) over-mature trees: the largest individuals characterized by broad leaves in the canopy, accompanied by a significant number of withered branches. Given that the photosynthetic tissue of \u003cem\u003ePinus massoniana\u003c/em\u003e consists of needles rather than broad leaves, its age classification follows the methodology established by Yang et al. (\u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). Additionally, \u003cem\u003eCycas revoluta\u003c/em\u003e exhibits distinct morphological characteristics compared to typical tropical evergreen trees. Since standardized age group classifications for \u003cem\u003eCycas revoluta\u003c/em\u003e have not yet been established, we consulted local botanical experts to guide the age categorization in the field based on breast diameter.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTraits of twelve studied species in this study.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eFunctional type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eAngiosperms or gymnosperms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eGrowth type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eDBH growth rate\u003c/p\u003e\n \u003cp\u003e(cm/yr)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eMaturity age (yr)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" align=\"left\"\u003e\n \u003cp\u003eDBH (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYoung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMiddle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNear-mature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOver-mature\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" align=\"left\"\u003e\n \u003cp\u003eFast-growing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eEucalyptus urophylla\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAngiosperms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBroadleaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u0026ndash;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u0026ndash;20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u0026ndash;40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eTriadica cochinchinensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAngiosperms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBroadleaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026ndash;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u0026ndash;18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u0026ndash;35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMallotus paniculatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAngiosperms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBroadleaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026ndash;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u0026ndash;18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u0026ndash;35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePinus massoniana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGymnosperms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConiferous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026ndash;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u0026ndash;18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u0026ndash;35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" align=\"left\"\u003e\n \u003cp\u003eSlow-growing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eSyzygium rehderianum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAngiosperms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBroadleaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026ndash;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026ndash;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u0026ndash;20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePsydrax dicocca\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAngiosperms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBroadleaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026ndash;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026ndash;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u0026ndash;20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAidia canthioides\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAngiosperms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBroadleaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026ndash;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026ndash;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u0026ndash;20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNageia nagi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGymnosperms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBroadleaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u0026ndash;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u0026ndash;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u0026ndash;22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" align=\"left\"\u003e\n \u003cp\u003eNitrogen-fixing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eOrmosia glaberrima\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAngiosperms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBroadleaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u0026ndash;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u0026ndash;16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u0026ndash;25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eArchidendron lucidum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAngiosperms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBroadleaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2\u0026ndash;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u0026ndash;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u0026ndash;16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u0026ndash;25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAcacia mangium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAngiosperms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBroadleaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026ndash;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u0026ndash;18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u0026ndash;35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCycas revoluta\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGymnosperms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConiferous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026ndash;5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026ndash;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u0026ndash;20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003eCollection of leaf and soil samples\u003c/h3\u003e\n\u003cp\u003eFresh and senescent leaves were collected during the peak litterfall period in November 2021. Three replicate trees were sampled for each age stage of every species, with selected trees spaced at least 20 m apart to prevent interactions among individuals of the same age group. Fresh leaves were obtained from the upper canopy using high pruning shears. To collect senescent leaves, long sticks were used to shake the trunk or branches in the upper canopy. For young trees with fewer senescent leaves, mesh bags were placed over the branches to capture recently fallen leaves. Following collection, both fresh and senescent leaves were placed in envelopes and transported to the laboratory for pretreatment on the same day. The leaf samples in the envelopes were then dried in an oven at 65\u0026deg;C for 72 hours.\u003c/p\u003e\n\u003cp\u003eSoil sampling was conducted concurrently with leaf collection. Soil was collected from the root zone of the trees using a soil auger to a depth of 0\u0026ndash;10 cm. Three 5-cm diameter soil cores were taken from each tree and combined into a composite sample. The soil samples were air-dried in a shaded and well-ventilated environment for approximately one month until a constant weight was achieved. During soil sieving, living fine roots were hand-sorted. Both plant and soil samples were then ground in a ball mill, passed through a 100-mesh sieve, and stored in a cool, dry location for subsequent chemical analysis.\u003c/p\u003e\n\u003ch3\u003eDetermination of N and P concentrations of soils and leaves\u003c/h3\u003e\n\u003cp\u003eTotal N concentrations in both fresh and senescent leaves and soils were determined using the Kjeldahl acid-digestion method (Jackson, Peltzer and Wardle \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). Total P concentrations in leaves and soils were determined using the molybdenum blue colorimetric method following digestion in a H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;HClO\u003csub\u003e4\u003c/sub\u003e solution (Bouma and Dowling \u003cspan class=\"CitationRef\"\u003e1982\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eDetermination of N fixation rates of N-fixing species\u003c/h3\u003e\n\u003cp\u003eNitrogen fixation rates of N-fixing species were estimated using the \u003csup\u003e15\u003c/sup\u003eN natural abundance (\u0026delta;\u003csup\u003e15\u003c/sup\u003eN) methods (Bedard-Haughn, Van Groenigen and Van Kessel \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e). \u003cem\u003eEucalyptus urophylla\u003c/em\u003e, a non-N-fixing species growing adjacent to the four N-fixing species studied, served as a reference plant to calculate the \u0026delta;\u003csup\u003e15\u003c/sup\u003eN value of soil-derived N. Fresh leaves of the four N-fixing species and \u003cem\u003eEucalyptus urophylla\u003c/em\u003e were collected for \u0026delta;\u003csup\u003e15\u003c/sup\u003eN determination.\u003c/p\u003e\n\u003cp\u003eThe ratio of \u003csup\u003e15\u003c/sup\u003eN to \u003csup\u003e14\u003c/sup\u003eN (\u0026delta;\u003csup\u003e15\u003c/sup\u003eN, \u0026permil;) in leaves was measured using isotope ratio mass spectrometry (IRMS; Thermo Fisher Scientific, USA). The N fixation rate was characterized by the proportion of plant N derived from the atmosphere (%Ndfa), calculated using the following formula (Bedard-Haughn, Van Groenigen and Van Kessel \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e):\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" style=\"width: 738px;\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\u003cp\u003ewhere \u0026delta;\u003csup\u003e15\u003c/sup\u003eN reference plant and \u0026delta;\u003csup\u003e15\u003c/sup\u003eN legume refer to the \u003csup\u003e15\u003c/sup\u003eN natural abundance in \u003cem\u003eEucalyptus urophylla\u003c/em\u003e and four N-fixing species. The B value is \u0026minus;\u0026thinsp;1.4\u0026permil; in the current study, obtained from the average of several previous studies (Jacot et al. \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e; Png et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). This value represents the \u0026delta;\u003csup\u003e15\u003c/sup\u003eN when atmospheric N is the sole N source for the N-fixing plants, as determined by planting the N-fixing seedlings in an N-free medium for three months (Andrews et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCalculation of leaf N and P resorption efficiency\u003c/h2\u003e\u003cp\u003eLeaf N and P resorption efficiencies (NRE and PRE) were calculated as follows (Vergutz et al. \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv id=\"FileID_Equ1\" class=\"mathdisplay\"\u003e$$\\:\\text{NRE}\\left(\\text{%}\\right)\\text{=[}\\left({\\text{N}}_{\\text{g}}\\text{-}{\\text{N}}_{\\text{s}}\\text{\u0026times;MLCF}\\right)\\text{/}{\\text{N}}_{\\text{g}}\\text{]\u0026times;100}$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\n \u003cdiv id=\"FileID_Equ2\" class=\"mathdisplay\"\u003e$$\\:\\text{PRE}\\left(\\text{%}\\right)\\text{=[(}{\\text{P}}_{\\text{g}}\\text{-}{\\text{P}}_{\\text{s}}\\text{\u0026times;MLCF)/}{\\text{P}}_{\\text{g}}\\text{]\u0026times;100}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003cp\u003ewhere N\u003csub\u003eg\u003c/sub\u003e and N\u003csub\u003es\u003c/sub\u003e represent N concentrations in green and senescent leaves, respectively, and P\u003csub\u003eg\u003c/sub\u003e and P\u003csub\u003es\u003c/sub\u003e denote P concentrations in green and senescent leaves, respectively. MLCF is a mass loss correction factor used to compensate for the loss of leaf mass during the senescence period (Van Heerwaarden, Toet and Aerts \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e). NRE and PRE were corrected using different MLCF values according to our previous study (Chen et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe used leaf PRE:NRE as an indicator of plant P limitation. A PRE:NRE greater than 1 indicates P limitation, with higher ratios reflecting increasing severity of the limitation (Du et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003ePrior to statistical analyses, the normality of residuals was assessed using the Kolmogorov-Smirnov test, and homogeneity of variance was evaluated using Levene\u0026rsquo;s test. To compare differences in the studied variables, including NRE, PRE, PRE:NRE, soil total P, and N fixation rate across plant types and age groups, we conducted a one-way analysis of variance (ANOVA) followed by the least significant difference test (LSD). To compare the variation in P limitation with age between different plant types, we performed a linear regression analysis using DBH as the proxy for tree age. We calculated the regression slopes and determined that slopes are significantly different if their 95% confidence intervals do not overlap. A steeper slope indicates a more rapid intensification of plant P limitation with age, while a shallower slope suggests the opposite (Deng et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). All analyses were conducted using SPSS 19.0 (SPSS Inc.).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eNutrient limitation status of the studied species\u003c/h2\u003e \u003cp\u003eOur results showed that all studied species were P-limited, as indicated by the mean PRE:NRE consistently exceeding 1 across all functional plant types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffects of tree age on plant P limitation\u003c/h2\u003e \u003cp\u003ePlant P limitation increased with tree age, as evidenced by the consistent increase in both leaf PRE:NRE and PRE. Specifically, NRE showed no significant change with tree age in most species, except for a significant decrease observed in \u003cem\u003eCycas revoluta\u003c/em\u003e (Fig. S3, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d). In contrast, PRE increased significantly with tree age for all individual species (Fig. S2) and across functional plant types (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-h). Consequently, the leaf PRE:NRE also exhibited a significant increase with tree age in all individual species (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and across functional plant types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFactors controlling the age effect on plant P limitation\u003c/h2\u003e \u003cp\u003eThe magnitude of increasing P limitation varied among fast-growing, slowing growing, and N-fixing trees, as evidenced by their varying regression slopes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The most pronounced increase in P limitation was observed in slow-growing trees (Slope\u003csub\u003eS\u003c/sub\u003e= 0.05), followed by N-fixing trees (Slope\u003csub\u003eN\u003c/sub\u003e= 0.03) and fast-growing trees (Slope\u003csub\u003eF\u003c/sub\u003e= 0.01). Significant differences were noted between slow-growing and fast-growing trees, as well as between N-fixing and fast-growing trees, but not between slow-growing and N-fixing trees.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSoil P content and N fixation rate were important factors explaining the changes in plant P limitation with tree age (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A significant decrease in soil total P was observed with increasing age across all species (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Additionally, the N fixation rate of N-fixing trees significantly increased with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we provide the first evidence of a universal increase in P limitation with tree age in tropical forests, based on field observations of three functional plant groups comprising 12 tree species. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Our investigation further revealed that slow-growing and N-fixing trees exhibit a greater increase in P limitation with age compared to fast-growing trees (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These empirical results substantially enhance our understanding of ecosystem P cycling. Given that terrestrial biogeochemical models have yet to incorporate the influence of tree age, our study highlights the importance of considering age-related effects on plant nutrient cycling to improve the accuracy of global nutrient cycling projections within such models.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIncreasing P limitation with tree age\u003c/h2\u003e \u003cp\u003eWe observed a consistent increase in PRE:NRE across fast-growing, slow-growing, and N-fixing trees, indicating a widespread trend of aggravating P limitation with tree age in tropical forests. This finding supports our initial hypothesis. However, it contrasts with several field studies (Alvarez-Clare, Mack and Brooks \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Aoyagi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which suggested that young trees experience greater P limitation than mature trees based on relative growth rates following P addition. These studies compared young and mature trees but focused on different species within broader functional groups, rather than examining the same tree species across their entire lifespan. In contrast, our research investigated the same tree species throughout their lifespans, providing more direct evidence of the age-related dynamics of P limitation. Moreover, previous studies may have been influenced by the use of relative growth rates post-fertilization as the primary metric for assessing P limitation. While young trees typically exhibit faster growth, older trees naturally slow down, meaning that the enhanced growth of young trees following P addition may not necessarily reflect greater P-limited, but rather their intrinsic growth characteristics. In this context, the leaf P and N resorption ratio (PRE:NRE) more accurately captures the demand for P, making it a more reliable indicator of P limitation. Overall, our findings emphasize the need to reconsider how P limitation varies across different growth stages in tropical forest ecosystems.\u003c/p\u003e \u003cp\u003eTwo mechanisms may underlie the observed increase in P limitation with tree age. The first mechanism is related to the growing demand for P as trees mature. As trees age, their photosynthetic capacity improves due to enhanced leaf exposure to optimal light conditions (Bond \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Forrester et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This increase in photosynthetic activity drives a higher demand for P, which is a critical component in the light reaction of photosynthesis, essential for the synthesis of adenosine triphosphate (ATP) (Li et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, as trees continue to grow, they require additional P for the development of structural components, such as wood, and reproductive structures, including flowers and seeds (Kerkhoff et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sardans and Penuelas \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Fortier and Wright \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Indeed, research has observed increases in the wood P:N ratio with tree growth, suggesting that storing P in wood is a strategy that tropical plants employ to adapt to low-P environments (Elser et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Sardans and Penuelas \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, substantial amounts of P are necessary for the development of reproductive structures, particularly for synthesizing phospholipid and nucleotide that are critical for seed formation (Kerkhoff et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Fortier and Wright \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). During the early growth stages, trees prioritize efficient nutrient use and vertical growth to maximize sunlight exposure (Martinez-Vilalta, Vanderklein and Mencuccini \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, as trees mature, growth priorities shift toward reproduction, characterized by vigorous flowering and fruiting. This transition in growth priorities further exacerbates P limitation with increasing tree age.\u003c/p\u003e \u003cp\u003eThe second mechanism contributing to increased P limitation over time is the gradual depletion of soil P. Soil P primarily originates from the weathering of parent materials, a process that occurs at a slow rate (Holford \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). As soil develops, P levels typically decrease due to processes such as leaching and plant uptake (Djodjic, B\u0026ouml;rling and Bergstr\u0026ouml;m \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In this study, the sampled trees ranged from 1 to 60 years of age, a period during which total soil P levels may decrease, leading to a reduced P supply for plant growth. This mechanism is supported by our observation of a decrease in soil total P with increasing tree age (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePlant type mediates the age effect on P limitation\u003c/h2\u003e \u003cp\u003eWe found that slow-growing trees are more limited by P than fast-growing trees with increasing age, which contradicts our second hypothesis. This unexpected result may be attributed to differences in growth strategies and physiological traits between these two groups. Fast-growing trees, which typically have shorter lifespans (Black, Colbert and Pederson \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), are likely to develop more efficient P acquisition strategies throughout their lives (Comas, Bouma and Eissenstat \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). These strategies include increasing root surface area (Comas and Eissenstat \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), secreting phosphatases (Yaffar et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and enhancing P resorption from senescent leaves (Xu et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In contrast, slow-growing trees may have less efficient P acquisition mechanisms due to slower root growth and a reduced ability to uptake P from the soil (Comas, Bouma and Eissenstat \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Comas and Eissenstat \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Additionally, slow-growing trees often face greater competition from fast-growing trees within the same ecosystem, which may exacerbate their P limitation over time.\u003c/p\u003e \u003cp\u003eFurthermore, we observed that N-fixing trees are more limited by P than fast-growing trees with increasing age, partially supporting our hypothesis. Although many N-fixing trees are also fast-growing, they have a greater P demand due to increasing N-fixing capacity with age (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Indeed, biological N fixation relies on P to produce nitrogenase, the enzyme responsible for catalyzing the conversion of atmospheric nitrogen (N\u003csub\u003e2\u003c/sub\u003e) into ammonia (NH\u003csub\u003e3\u003c/sub\u003e) (Reed, Cleveland and Townsend \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Additionally, as the amount of fixed N increases, N-fixing trees require additional P to maintain the stoichiometric balance between N and P (Pearson and Vitousek \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). This increased P demand highlights the unique nutrient dynamics of N-fixing trees in tropical forests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImplications for model development\u003c/h2\u003e \u003cp\u003eThe impact of tree age has long been neglected in previous ecological models, potentially leading to biased assessments of phosphorus (P) limitation's effects on ecosystem functioning. For instance, some models predict that future P limitation on plant growth could turn ecosystems into net CO\u003csub\u003e2\u003c/sub\u003e sources by the end of this century (Wieder et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Luo et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, these models do not consider tree age, introducing significant uncertainties into their projections (Reed, Yang and Thornton \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Jiang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). When tree age is considered, it becomes clear that young and old plants may play distinct roles in future carbon sequestration due to their different levels of P limitation. Our results suggest that neglecting the impact of tree age could lead to either an overestimation or underestimation of the potential for vegetation to mitigate climate change. Thus, incorporating tree age into ecological models is crucial for improving the accuracy of predictions regarding ecosystem responses to P limitation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study is supported by Shenzhen Science and Technology Program (Grant No. JCYJ20220530150015035) and Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, Chinese Academy of Sciences (VRMDE2301). Liang Zheng was supported by National Natural Science Foundation for Regional Innovation and Development (U21A20189) and Hunan provincial Natural Science Foundation of China (2022JJ50032).\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eHC, NH, and QM designed the research. NH, and QM collected data, and conducted fieldwork. NH performed lab analysis. NH conducted all data analyses and drafted the initial manuscript with significant contribution from HC and QM. All authors contributed to manuscript revisions.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe sincerely thank Dehong Cheng, Yu Zou, Xiujin Cai, and Xin Xie for the assistance in fieldwork.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eData available from the ScienceDB at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.scidb.cn/en/s/bIBrem\u003c/span\u003e\u003cspan address=\"https://www.scidb.cn/en/s/bIBrem\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlvarez-Clare S, Mack M, Brooks M (2013) A direct test of nitrogen and phosphorus limitation to net primary productivity in a lowland tropical wet forest. 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Sci Total Environ 889:164047. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2023.164047\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2023.164047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"nutrient limitation, nitrogen fixation, growth type, stand age, plant strategy, model development","lastPublishedDoi":"10.21203/rs.3.rs-5904638/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5904638/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eAims\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePhosphorus (P) availability commonly limits the growth of tropical plants, yet how this limitation changes with tree age remains uncertain.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHere we investigated the effect of tree age on P limitation in a tropical forest by examining three functional plant groups: fast-growing, slow-growing, and nitrogen (N)-fixing tree species. We measured leaf N and P resorption efficiency (NRE and PRE), and used the ratio of PRE to NRE (PRE:NRE) as an indicator of plant P limitation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur results revealed a significant increase in both PRE and PRE:NRE with tree age across all functional plant groups, indicating a widespread intensification of P limitation as plants mature. Furthermore, such increase in P limitation was more pronounced in slow-growing and N-fixing species compared to fast-growing species.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThese findings underscore the crucial role of tree age in influencing P limitation in tropical forests, a factor that should be incorporated into terrestrial biogeochemical models, which have traditionally overlooked this effect.\u003c/p\u003e","manuscriptTitle":"Increasing phosphorus limitation with tree age in tropical forests","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-10 09:17:23","doi":"10.21203/rs.3.rs-5904638/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-03-17T09:43:07+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-02-10T07:01:35+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-06T09:31:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2025-01-30T07:06:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-30T07:04:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2025-01-29T21:22:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"962d54a1-54ef-461d-ba81-d542d7705bab","owner":[],"postedDate":"February 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-12T16:01:26+00:00","versionOfRecord":{"articleIdentity":"rs-5904638","link":"https://doi.org/10.1007/s11104-025-07521-4","journal":{"identity":"plant-and-soil","isVorOnly":false,"title":"Plant and Soil"},"publishedOn":"2025-05-07 15:57:26","publishedOnDateReadable":"May 7th, 2025"},"versionCreatedAt":"2025-02-10 09:17:23","video":"","vorDoi":"10.1007/s11104-025-07521-4","vorDoiUrl":"https://doi.org/10.1007/s11104-025-07521-4","workflowStages":[]},"version":"v1","identity":"rs-5904638","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5904638","identity":"rs-5904638","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}