Tropical forest carbon sequestration accelerated by nitrogen

Domain: earth_science
Published: 2026-01-13
Editorial outcome: major_revision

Abstract
Understanding forest carbon sequestration is crucial for predicting and managing the carbon cycle, yet we lack evidence for whether, when and how the carbon sink in tropical forests recovering from land use change is nutrient limited. Here we show how the tropical forest recovery rate responds to experimental nutrient manipulation over a secondary succession gradient in a naturally recovering Central American landscape. Nutrient limitation of aboveground biomass accumulation shifts from strong nitrogen limitation in young forests to no evidence of nitrogen or phosphorus limitation in older secondary or mature forests. Nitrogen addition increases aboveground biomass accumulation by 95% in recently abandoned pasture and 48% in 10-year-old forests. Conversely, we observe no influence of nitrogen on older forests and no evidence of phosphorus limitation at any stage. If our findings of nitrogen limitation extend to young tropical forests globally, nitrogen could prevent the sequestration of 0.69 (0.47-0.84) GtCO2 each year.

Introduction
Tropical forests play a key role in the terrestrial carbon sink by sequestering atmospheric carbon dioxide (CO2) and thus slowing the rate of global climate change1–3. Mature tropical forests harbor substantial quantities of biomass carbon, and young secondary tropical forests (i.e., those naturally regenerating from disturbance) offer a large potential carbon sink as a natural climate solution1,4, owing to their increasing importance in modern tropical landscapes and their potential for high rates of carbon accumulation relative to mature forests1,5–7.

While phosphorus is often thought to limit carbon sequestration by tropical forests on highly weathered lowland soils8,9 (but see refs. 10–13), we know little about how nutrients and carbon interact throughout the course of tropical forest recovery from disturbance. One possibility is that nutrient limitation of tree growth is independent of forest successional age. Alternatively, nutrient limitation may depend on forest age, with a shift from nitrogen to phosphorus limitation due to differences in nutrient losses, sources and accumulation rates for each nutrient following disturbance. Disturbances like blowdowns, logging, fire and agriculture cause losses of soil nitrogen through leaching and gaseous emissions, exacerbating nitrogen limitation14–17. As secondary succession proceeds, symbiotic nitrogen fixation can build up ecosystem nitrogen over time18,19, which, in turn, can alleviate nitrogen limitation and shift the forest to limitation by another resource, usually assumed to be phosphorus. Soil phosphorus may or may not decrease following disturbance – and can even increase after forest cutting and burning20,21 – and is unlikely to increase with secondary succession since there are few inputs of new phosphorus to tropical forests over such short timescales15,22. Thus, tropical forests may – in theory – shift from nitrogen to phosphorus limitation over secondary succession.

Evidence that soil nutrients are likely to limit the tropical forest carbon sink and that there could be shifts over succession derives from multiple sources. Nutrient addition experiments find that nutrients can limit tropical tree growth23–28, with nutrient limitation stronger in young forests24. Several experiments suggest nitrogen limitation of young forests (refs. including25,29) and that nitrogen or phosphorus limitation can occur in mature forests (refs. including25,26,28,30–32). A meta-analysis across 44 experiments found that both nitrogen and phosphorus limit young but not mature tropical forests24. None of these experiments included both young and mature forests in the same area, limiting inference about shifts in limitation. Direct observations of tropical forest nitrogen cycles generally show decreasing symbiotic nitrogen fixation and increasing nitrogen losses to the atmosphere and groundwater as forest mature, suggesting nitrogen limitation in young forests that declines with forest age15,18,19,33 (but see refs. 21,34–36). Biogeochemical model predictions provide indirect evidence for a similar nitrogen limitation decline and shift to phosphorus limitation14,37, and dynamic global vegetation models indicate the future land carbon sink will be constrained by nitrogen and/or phosphorus38–41. Thus, although some evidence suggests tropical forest nutrient limitation shifts from nitrogen to phosphorus over secondary succession, experimental evidence shows the potential for both types of limitation, and evaluating nutrient limitations across disparate experimental sites could be confounded by other variables. It therefore remains unclear whether and how nutrient limitation evolves over secondary succession.

To address this knowledge gap, we established a landscape-scale experiment in lowland moist tropical forests of Panama. The experiment was designed to directly test whether nitrogen, phosphorus or the two nutrients combined affect stand-scale aboveground biomass and rates of biomass net change, gain and loss, and whether the effects change over forest successional age. Because our 76 (0.1–0.16 ha) plots were located across a ~16 km2 landscape, our experiment was able to control for metacommunity species pool, land use history, soils and climate while avoiding pseudo-replication. We distributed our plots across a secondary succession gradient that included newly regenerating forests (“0-year-old forests”), two middle stages (“10-” and “30-year-old forests”) and a mature forest occurring on land that has had limited human disturbance (“600-year-old forests”; Supplementary Fig. 1, “Methods”). In our sites, soil total nitrogen ranges between 0.28–0.35%, and soil total phosphorus ranges between 246 to 286 mg P kg−1 in the top 30 cm and does not change over forest succession (Supplementary Tables 1–3). The early successional forest sites were previously clear-cut for cattle pasture, with no burning since the initial deforestation 30–40 years ago. We monitored forest changes over four censuses (four years in secondary forests and 21 years in the mature forest). In total, we monitored the responses of 88,843 trees to nutrient manipulation. A resolution to tropical nutrient limitation, even at one site, would aid the implementation and management of tropical reforestation to increase carbon sequestration as a natural climate solution. Our experiment also offers an opportunity to validate dynamic global vegetation models (DGVMs) and improve predictions of the future of tropical forests.

Results
Stand-scale aboveground biomass (determined by individual biomass and stem density) recovered rapidly following the abandonment of cattle pasture, independent of the addition of nutrients (black line in Fig. 1). By 14 years, our forests contained 25% of the aboveground biomass of mature forests, and by 30 years, they had accumulated half the aboveground biomass of mature forests. These biomass recovery rates are similar to mean rates for secondary forests across the American tropics5. With added nutrients, however, the aboveground biomass in our forests recovered even more rapidly. Across treatments, nitrogen had the largest and only statistically significant effect on aboveground biomass, with four years of nitrogen addition increasing biomass by 95% and 33% in the 0- and 10-year-old forests, respectively (green and blue lines versus black and red lines in Fig. 1).

Nutrient limitation of net aboveground biomass accumulation (measured as the change in plot biomass from one census to the next) shifted over secondary succession. The 0- and 10-year-old tropical forests were strongly limited by nitrogen, while the 30- and 600-year-old forests were not (Table 1, Fig. 2). The interaction between nitrogen addition and forest age was statistically significant for net aboveground biomass change (Table 1). Nitrogen addition caused 95% and 48% increases in net aboveground biomass accumulation rates for the 0- and 10-year-old forests, respectively (Fig. 2, Supplementary Table 4 and Supplementary Figs. 2, 3). Nitrogen limitation in the control plots, therefore, prevented the sequestration of ~4.1 tons CO2 ha−1 year−1 over the first decade of tropical forest recovery (“Methods”). In contrast, there was no nitrogen effect on net aboveground biomass change in the 30-year-old secondary forest or after twenty years of sustained nutrient addition in the mature forest (Fig. 2, Supplementary Table 4 and Supplementary Figs. 4, 5). Thus, we conclude that nitrogen limits the net accumulation of aboveground biomass in our young successional forests but not in forests 30 years and older.

Next, we sought to understand the mechanism by which nutrient addition influences the dynamical balance between the growth and mortality of aboveground biomass across our forest successional gradient. To do this, we decomposed net aboveground biomass change into two demographic components: First, the gain in aboveground biomass caused by the growth of trees that survived between census intervals, plus any trees that recruited into the plots between censuses. And second, the loss of aboveground biomass caused by the mortality of trees between censuses.

We found that, similar to net aboveground biomass change, the addition of nitrogen interacted with forest age (Fig. 3a, Table 1), triggering a 99% increase in aboveground biomass gain in 0-year-old forests (Fig. 3a, Supplementary Table 4) and a 23% gain in 10-year-old forests. Also similar to our results for net aboveground biomass change, we observed no statistically significant nitrogen effect on aboveground biomass gain in forests 30 years and older (Fig. 3a, Supplementary Table 4 and Supplementary Figs. 4, 5). Nitrogen addition also influenced aboveground biomass loss, with substantial biomass loss due to mortality in the 0-year-old forests (Table 1, Supplementary Table 4), likely caused by self-thinning, resulting from increased competition between the rapidly growing plants42. We found no statistically significant effect, however, of nitrogen on aboveground biomass loss in forests 10 years and older (Fig. 3b, Supplementary Table 4 and Supplementary Figs. 3–5).

We found little evidence of phosphorus limitation on stand-scale aboveground biomass dynamics in our tropical moist forests, either across forest ages or in interaction with forest age (Table 1). Furthermore, there was no significant effect of phosphorus on aboveground biomass net change, gain or loss, with the exception of aboveground biomass loss in the 0-year-old forests (Fig. 3, Table 1 and Supplementary Table 4). Note these findings are counter to expectations from biogeochemical theory8,9 (but see ref. 37) and consistent with analyses of individual tree growth in our mature forest11.

Taken together, our findings provide direct support for the hypothesis that nutrient limitation on aboveground biomass shifts over secondary succession in tropical forests, from nitrogen limitation early in succession to limitation by another resource later in succession. Our findings emerge from a landscape-scale experiment that spans a secondary succession gradient from abandoned pasture to mature forests and captures the effect of nutrients on stand-scale net aboveground biomass accumulation. Although they derive from one site, these findings advance our understanding of the role that nitrogen and phosphorus can have in tropical forests.

Methods
Research sites
A factorial nitrogen and phosphorus fertilization experiment was established at Agua Salud (9o13’N, 79o47’W, 330 meters above sea level) and Gigante (9o06’31”N, 79o50’37”W, 60 meters above sea level) in the Republic of Panama (Supplementary Fig. 1). These two sites lie within the Panama Canal Watershed and include forests at different stages of succession (from young secondary to mature forest). Both forest sites are classified as lowland tropical moist forests, receiving similar annual precipitation (~2700 mm) with a dry season (contributing ~10% of total rainfall) from December to April, having a similar mean annual temperature (26°C), and sharing a similar diverse metacommunity of tree species18,23,72,73. Soils across all forests are highly weathered with generally low plant-available soil nutrient concentrations18,74. Gigante contains deep soils on relatively flatter rain and has been classified as Oxisols (Typic Kandiudox), and Agua Salud soils are classified as Inceptisols (Typic Dystrudept; five of six soil pits) and Oxisols (Typic Haplodux; one of six pits). The soil physical and chemical properties of Agua Salud and Gigante forests can be found in the Supplementary Table 1. The soil classification reports can be found in ref. 75.

In Agua Salud, the landscape consists of cattle pastures and cultivated fields, fallows, plantations and different age secondary forests which recovered naturally following cattle ranching and small-scale clear-cutting18,73. Topography in this area varies, consisting of narrow streams and steep but short slopes73. In Gigante, which is a part of the Barro Colorado Nature Monument, the land is covered by a protected mature forest on relatively flatter rain76.

Experimental design
The Gigante fertilization experiment (named ‘600-year-old forest’) was established in a mature forest of at least 600 years post-human disturbance, as determined with charcoal dating in 199777. It consists of four nutrient addition treatments (control, nitrogen, phosphorus and nitrogen plus phosphorus) with each replicated four times (1 forest age × 4 treatments × 4 replicates)23. The area of each plot is 0.16 ha (40 × 40 m). The Agua Salud Project fertilization experiment consists of experimental plots at three different successional stages: very young secondary forests established immediately after the abandonment of cattle pastures (named “0-year-old forests”) and two middle aged secondary forests (named “10-year-old forests” and “30-year-old forests”). The experiment began in 2015 with plot establishment of four replicates per treatment for the 10- and 30-year-old forests and the establishment of four replicate former pasture plots that were clear of trees as 0-year-old forests. In 2016, we established a fifth replicate set of plots for all forest ages and began fertilization treatments with the same nutrient addition treatments as the mature forests. In both sites, within each replicate, we blocked the control, nitrogen, phosphorus and nitrogen plus phosphorus plots within sites on the landscape to minimize the effects of small-scale variations in climate, soils and surrounding forest tree diversity and seed source (Supplementary Fig. 1 in Supplementary Information 1). Plots are located on the landscape to eliminate the possibility of the flow of nutrients from one plot to another based on slope, and the minimum distance between plots is 40 m. The area of fertilized plots is 0.16 ha (40 × 40 m), and the control plots are 0.1 ha (20 × 50 m) since they do not need a fertilization buffer. In every Agua Salud fertilization plot, trees are monitored only within the inner 0.1 ha, leaving a buffer zone on four sides that was fertilized.

Fertilizer was added as coated urea ((NH2)2CO) and triple superphosphate (Ca(H2PO4)2·H2O) in nitrogen and phosphorus-treated plots, respectively. Annual doses were 125 kg N ha−1 yr−1 and 50 kg P ha−1 yr−1, and fertilizers were added by hand in four equal doses at regular intervals beginning approximately two weeks after the beginning of regular, heavy rains (approximately 15–30 May, 1–15 July, 1–15 September and 15–30 October)11.

We also measured the annual rainfall when the Agua Salud fertilization experiment was established (see the annual rainfall variation in the Supplementary Fig. 6). We found that changes in rainfall do not affect biomass accumulation and its dynamics.

Forest inventory
We monitored all 76 plots since the start of the nutrient fertilization (i.e., 2015 in Agua Salud forests and 1997 in Gigante forest). All free-standing woody plants (trees, palms and lianas – hereon referred to as trees since 75% of plants were trees) within the plots were identified, but the monitoring protocols differed slightly between the two sites. In Agua Salud, in the center 0.1 ha of plots, all stems of trees and palms with diameters ≥ 5 cm and all lianas with diameter ≥ 1 cm were measured, as well as 50% of all tree and palm stems with diameters between 1 and 5 cm. All trees of the same size cutoffs were measured in the control plots. In Gigante, trees with a diameter ≥ 10 cm were measured in the whole plot, and trees with a diameter between 1 and 10 cm were measured in the central 20 × 30 m subplot. We use the nested design to capture the dynamics of small trees. For the large trees, diameters were measured above any buttresses or other deformities of the lower trunk23,73. All diameters were measured at 1.3 meters height.

In Agua Salud, plots were censused prior to fertilization in 2015, with the exception of the plots established in 2016. Plots were then censused yearly after fertilization from 2016 to 2019. In Gigante, plots were censused every five years from 1997 to 2018; however, we focused on the census prior to fertilization in 1997 and the censuses after fertilization from 2003 to 2018. We divided our analysis into two census intervals to capture (1) the most recent forest dynamics that would be comparable to the climatic conditions at the Agua Salud experiment (2013–2018) and (2) the forest characteristics prior to fertilization and the subsequent post-fertilization dynamics (the 1997 to 2013 censuses).

Biomass at the plot scale
We first estimated the aboveground biomass (kg stem−1) of all recorded stems in Agua Salud and Gigante plots. We applied different allometric functions to estimate the aboveground biomass of each stem of each tree, liana and palm. For trees, we estimated the aboveground biomass of each stem using the following allometric function78:

AGB=exp[−1.803−0.976E+0.976ln(WD)+2.673ln(DBH)−0.0299[ln(DBH)]2]:

where AGB represents aboveground biomass (kg stem−1), E is the local climatic index79, and WD is wood density (g cm−3). DBH represents the diameter at 1.3 meter (cm). The climate index, E=0.05645985, near our study site, represent the effect of the environment on tree height allometry79. Species-specific wood density (in g cm−3) was estimated from the most common species in Agua Salud and Gigante (S. Joseph Wright unpublished data and ref. 79).

For lianas, the aboveground biomass of each stem was calculated using a liana-specific allometric equation80,81:

AGB=exp[−0.999+2.682*ln(DBH)]:

For palms, we calculated the aboveground biomass using a palm-specific allometric equation79,82:

AGB=0.0417565*(DBH)2.7483:

We summed the aboveground biomass of all stems one centimeter and above in each plot and scaled to one hectare to get the plot scale per hectare biomass (Mg ha−1, Fig. 1). To account for differences in the methods of inventorying all stems five centimeters and above and stems one to five centimeters in half of the plot at Agua Salud, we cloned the aboveground biomass of stems one to five centimeters to double the aboveground biomass of stems in that size class. At Gigante, where we inventoried all stems greater than ten centimeters and stems one to ten centimeters in 37.5% of the plot area, we summed the aboveground biomass of stems one to ten centimeters and multiplied by 2.667.

Forest dynamics at the plot scale
We calculated the mean annual net change of aboveground biomass for each plot and census interval (between 2015 and 2016, 2016 and 2017, 2017 and 2018, and 2018 and 2019 for Agua Salud plots, and between 1997 and 2013, and 2013 and 2018 for Gigante plots; Mg ha−1 year−1; Fig. 2, Supplementary Figs. 2–5).

We then calculated plot scale aboveground biomass gains and losses for each census interval in Agua Salud plots (the same intervals as described above), and for longer intervals in the Gigante plots (between 1997 and 2003, between 2003 and 2008, between 2008 and 2013, and between 2013 and 2018; Mg ha−1 year−1; Fig. 3, Supplementary Figs. 2–5). For Gigante, we then took the mean of the dynamics between 1997 and 2003, between 2003 and 2008, and between 2008 and 2013 to get the dynamics for the interval 1997 to 2013. We then took the mean dynamics of these census intervals to represent the full fertilization period. Growth was calculated as the gains of the trees recorded in the first census year that survived until the next census, divided by the time between the two censuses. Recruitment was calculated as the total aboveground biomass gain of trees that were recorded in the later census but not in the previous, divided by the time between the two censuses. Mortality was calculated as the aboveground biomass loss because of the loss of trees recorded in the initial census by the second census, divided by the period between the two censuses. Due to potential systematic error of overestimating growth and underestimating recruitment because of the cutoff of minimum tree size, we combined growth and recruitment into one measure of biomass gain and considered mortality as biomass loss.

Finally, for forest ages where we found significant effects of nutrients, we converted the net aboveground biomass change due to nutrients into CO2 equivalent by multiplying by 0.47 (the carbon:biomass ratio83) and 3.66 (the number of CO2 molecules per carbon). We then took the mean of the values at 0 and 10 years to calculate the mean CO2 accumulation due to nutrient addition over the first ten years of forest recovery (tons CO2 ha−1 year−1).

Statistical analysis
All analyses were performed in R (4.0.2)84. We used linear mixed-effects models (‘lmer’ function in “lme4” package) to test for the effects of nutrient addition and forest age on aboveground biomass net change, aboveground biomass gain and aboveground biomass loss at the plot scale as a mean across censuses. The mixed-effects models included nitrogen, phosphorus, forest age and their interactions, with block – the grouping of treatments across the landscape – as a random effect. In each model, all fixed effects were treated as factors. We log-transformed aboveground biomass loss to meet the model assumptions. For all models, we used the “anova” function for results and checked the residual and Q-Q plots to evaluate model assumptions. We evaluated whether annual rainfall influenced forest dynamics at Agua Salud; however, the effects were minimal (Supplementary Fig. 6).

We use the ‘Holm’ adjustment to avoid the problem of multiple comparisons85. When we found a significant interaction of a nutrient and age, we used contrasts (the ‘emmeans’ function from the ‘emmeans’ package) with the ‘Holm’ adjustment to determine the specific age at which the nutrient was limiting. We considered an effect to be significant if p<0.05. The marginal and conditional R2 were obtained for the models with the ‘r2’ function from the ‘r2mlm’ package.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "I reviewed an earlier version of this manuscript submitted to Nature. Compared to the earlier version there are several things that have improved substantially. Several earlier, somewhat exaggerated claims have disappeared and language usage is now much more concise compared to the earlier version. The authors also better acknowledge and discuss earlier work on N limitation in tropical forests which also improves the clarity on what is new and noteworthy in the current study. When considering the critical question how well the soils in Panama represent soils under tropical forests, it was interesting to learn that the authors now acknowledge that both sites (Gigante and Agua Salud) also have less weathered soils than Oxisols. The authors however argue that the total P values are more important than the classification and I agree with that. In their answer to my earlier questions regarding the total P values of 600 mg/kg the authors explain that the high number came from the paper by (Mirabello et al., 2013) which apparently overestimated the P in the ‘hot’ fractions. I looked up this paper and indeed they suggest that silica interference in phosphate detection by molybdate colorimetry may have led to an overestimation. Fortunately, total P was also analyzed in the (Koehler et al., 2009)) paper, which was mentioned by the authors in their rebuttal. Koehler et al. (2009) was conducted in the Gigante Experiment and they used pressure digestion in concentrated HNO3 followed by analysis of the digests using inductively coupled plasma-atomic emission spectrometer and report them in Table 1 of their paper. This total P method does not use colorimetry for analyses and thus does not suffer from potential silicon interference. The numbers reported by Koehler et al, 2009 for the control plots of the Gigante experiment are 550 mg kg-1 in the top 0.05m of mineral soil and still as much as 400 mg kg-1 in the 0.05 – 0.5m depth interval. If we take the lower value of 400 mg kg-1, this would still put the Gigante site in the top 10% values of total P in the Quesada et al., 2010 paper. So even when I accept that the 600ppm reported by Mirabello et al., 2013 were overestimated because of methodological problems, the total P values of Gigante are more comparable to the top 10% of Amazonian forests. And as I pointed out in my review of the earlier version, this was exactly what I would expect of soils developed from basaltic parent materiel in tectonically active regions such as Panama. However, the real question for this manuscript is whether the total P levels are so important to extrapolate the result to other parts of the tropics and below I will argue, why I think that is not the case. In their rebuttal the authors also write that it may be possible that the younger secondary forests could have a stronger nutrient (P) limitation compared to mature forests due to the rapid relative growth rates of young trees. In the manuscript, theoretical biogeochemical models are also mentioned as the basis for this expectation (l. 44 – l.49). Furthermore, they write counter to expectations from biogeochemical theory, they found little evidence of P limitation in the experiment (L. 122). Although this may be true, I think that the authors miss to appreciate the potential importance of land use history in this case (and in general with secondary forests). A strong P limitation in young secondary forest is unlikely because the secondary forest grows on areas that were previously cleared which probably involved slash-and-burn procedures and potentially agricultural practices. The results of slash and burn is that an important part of the rock derived-nutrients, including P, end up in the soil and the soil pH increases, effects that can last for decades. A higher pH leads to higher P availability and a higher base saturation which means that it is quite unlikely that rock-derived nutrients are limiting in the earlier successional stages. Something that was more or less shown for P by (Wong et al., 2024). To my knowledge, the Walker and Syers model considers natural systems without considering previous land use. However, taking the previous land use into account will go a long way to explain at least some of the findings in your study and I think that at the moment this is not sufficiently done. You discuss the possibility that P limitation is not observed in wood productivity (l 177 and further), you discuss that it is possibly less common than previously thought (L 189 and further), but you never discuss that forest clearing and burning leads to substantial increases in rock-derived nutrients and their availability, which I think is currently missing. You should also not forget that the data from the (Quesada et al., 2010) paper were collected in old-growth forest, where presumable no slash and burn has been taken place in the last centuries. This is presently ignored in the manuscript when you compare your soils with soil of the Amazon Basin, and I really think it is misleading and causes a lot of confusion (at least it took me two reviews to realize what was wrong). I expect that young secondary forests and abandoned agricultural areas will have higher values in total and available nutrients because of former slash-and-burn activities, and that should be highlighted in your discussion. It should not be too difficult to back this up with some studies. BTW, thank you for providing the full soil analysis for the Agua Salud project. Your soil chemical analyses show pH-water values around 5 which is a pH value when soil P is relatively well available. Also, base saturation is regularly above 50% in the top soil which may be the result of ashes from earlier forest clearing and also indicates that these are not very nutrient poor soils. I am still not convinced about your implications, in your rebuttal you write that you especially focus on tree planting when suggesting to increase the abundance of N fixing species. I have no doubt that there are some examples of secondary forests with little N fixing trees. However, the (Gei et al., 2018) paper, which was mentioned in the rebuttal, also shows that the majority of secondary forests have a relative basal area of 10% or (much) more. I am not sure whether adding more N-fixing legumes would make a big difference and as far as I know the vast majority of secondary forests is the result of spontaneous regeneration and succession and not from planting. In summary, I think the study presented here definitely gives new, noteworthy and exciting insights in to shifts in nitrogen and phosphorus limitations over a secondary succession gradient in soils that are at the higher end of total phosphorus content of soils under (secondary) forests, and as such is worth publishing. I think your claim that your soils are close to the median value for tropical forests soils globally is simply not true and frankly, I don’t think it is important for your manuscript. What is important for your message is that in secondary forests, N limitation is decreasing over time, and in my interpretation, it is unlikely to be replaced in the first decades of a secondary forest by P limitation (not even on heavily weathered Oxisols in the Amazon) because of the higher and more available rock-derived nutrient stocks, including soil P, which is a remnant of former slash and burn activities. References Gei, M., et al., 2018. Legume abundance along successional and rainfall gradients in Neotropical forests. Nat. Ecol. Evol. 2, 1104–1111. https://doi.org/10.1038/s41559-018-0559-6 Koehler, B., et al. 2009. Immediate and long-term nitrogen oxide emissions from tropical forest soils exposed to elevated nitrogen input. Glob. Change Biol. 15, 2049–2066. https://doi.org/10.1111/j.1365-2486.2008.01826.x Mirabello, M.J., et al. 2013. Soil phosphorus responses to chronic nutrient fertilisation and seasonal drought in a humid lowland forest, Panama. Soil Res. 51, 215. https://doi.org/10.1071/sr12188 Quesada, C.A., et al., 2010. Variations in chemical and physical properties of Amazon forest soils in relation to their genesis. Biogeosciences 7, 1515–1541. https://doi.org/10.5194/bg-7-1515-2010 Wong, M.Y., et al. 2024. Trees adjust nutrient acquisition strategies across tropical forest secondary succession. New Phytol. 243, 132–144. https://doi.org/10.1111/nph.19812", "reviewer_2": "This is the second time I have reviewed this paper (albeit for a different journal). So I am copying out my previous review below since it addresses many of the stock questions asked by NCOMMS. The authors now present the carbon numbers with appropriate nuance. They have also directly addressed my concern about nitrogen being confounded with age at the beginning. And the methods are now clearly documented, and are sound. I have no additional critiques beyond the two minor grammatical catches below. Line 39: “increasing predominance in modern tropical landscapes” would be better than “increasing importance” since the latter is normative Line 194: in LATIN America? Hawaii is also USA. ------------------- The paper by Tang et al examines impacts of fertilization on a network of tropical forest plots within Panama. They provide supplemental nitrogen and phosphorus to forests that vary in age (~0, 10, 30, and 600 years old) and find that the nitrogen addition greatly augments growth in the young forests only and that – despite standard biogeochemical theory – woody biomass accumulation does not appear to be limited by phosphorus. The paper is very well written. I have no editorial critiques for clarity, and this never happens when I review a manuscript. As I was reading, multiple questions arose in my mind but they addressed these later in the manuscript. For example, they find that phosphorus is not limiting which is a surprising result, but then provide a very nice discussion to explore potential mechanisms underlying their results. I don’t think the paper’s findings are enough to upend theory, but it certainly suggests that more work is needed to determine the universality of phosphorus limitation in the tropics. I was concerned that the interaction between nitrogen and forest age was confounded by nitrogen conditions in the soil (i.e., more nitrogen intact forest soils), but they provide baseline soil fertility conditions in a supplemental information (Extended Data 2) that shows this is not the case. I suggest making that point explicitly within the main manuscript. I was also concerned that the applied take home from the paper would be that people should apply fertilizer to young regrowing forests to augment growth and carbon removal. But the paper very clearly states that this is not recommended (I agree!) and provide alternative options suggest as prioritizing regrowth in places with N deposition. However, the largest issue is reproducibility. The methods are non-existent. The paragraph located lines 68-78 seems written to make the experiment sound larger than it actually is. They say there are 76 plots, but I think this includes both the treatment and the control so actually the number of experimental units is smaller. Sample size per treatment appears to be restricted to the figure legends. They say that these cover 88,843 trees, but those are not independent replicates. They report that they monitored these plots for up to 21 years and then mention in the parentheses that secondary forests have been monitored for four, but in fact the main results of the paper come from those secondary forests. How large are the plots? They say they cover 16 km2, but what is the range of sizes for the individual plots? Where are they located (i.e., can the geolocations be made available)? How much fertilizer did they add? What dosage, what frequency? How did they quantify biomass (i.e., which allometric equations were used)? This is all easily remedied by including a clear methods section – in the supplement if no space is available in the main text. My only other critique is that their carbon numbers require some nuance. I appreciate that they estimated mitigation potential at a cost-constrained rather than unconstrained level. However, their number remains quite theoretical. It is highly unlikely/impossible to achieve 1.1 GtCO2/yr because there are only so many locations with N deposition or higher fertility soils and only so much N that can be provided by nitrogen fixing species. Adding fertilizer to relieve nutrient limitations would produce upstream and in-field emissions that would offset the climate benefit of enhanced forest growth. I suggest the co-authors nuance that result more appropriately. For example, in the abstract instead of “could sequester up to an additional 1.1 GtCO2/yr” it might say “N limitations prevent up to 1.1 GtCO2/yr”. In other words, that 1.1 GtCO2/yr shouldn’t be presented as an opportunity. Optional suggestions: (1) Table 1 could be supplemental (2) Figure 1 should be checked for color blind friendliness. And would a log-scale for the y-axis better illustrate the differences among treatments (as an alternative to the inset approach).", "reviewer_3": "This study evaluates the effects of fertilization on aboveground biomass accumulation in early, mid and late successional forests in Panama. The work itself is incredibly ambitious, and the results are very robust and of interest. This study was a major undertaking. The manuscript is also beautifully written, and the authors certainly present some intriguing ideas. I read an earlier draft of this manuscript, and there are substantial improvements. I appreciate the broader acknowledgement prior work, and the more developed discussion of alternate explanations for the lack of response to N and P fertilization in mature forests. That said, many of my prior reservations hold. 1. Abstract: I appreciate that in the discussion, the authors compare this work to forests in the Amazon, but I am still extremely hesitant about generalizing findings from a single forest site in Panama to the tropics as a whole, especially given the wide range of tropical forest types (i.e., seasonal, evergreen, flooded, etc) and that the neotropics may not reflect what we would see in Asian forests or other less studied systems. 2. Regarding the following statement – “To date, we are aware of no direct ecosystem experiment that investigates how nutrient limitation on stand-scale aboveground carbon dynamics evolves over the course of a complete tropical forest secondary succession gradient, from recently abandoned pastures to mature forests.” And this matters why? Being the first, or only, in and of itself isn’t that interesting, but something that helps fill a specific knowledge gap, is. Please elaborate on why we as a scientific community should care about having this additional detail, and if the experiment just supports what is already generally accepted amongst tropical forest ecologists (i.e., early successional forests are N limited), is this really transforming our understanding? Definitely this is a really well-designed study, and the findings are very robust, but it just supports what others have shown - although maybe not as well. The authors suggest that some element other than P is what is really limiting mature tropical forests, but what do they think it is?. Also -- why would we expect mature trees to respond in the same way as fast growing early successional trees that are working to access the forest canopy? 3. The following statement “These findings advance our understanding of the potential roles of both nitrogen and phosphorus in tropical forests.” Once again, this study is specific to one study in Panama. There is a limit to how much these results can be generalized beyond this site, although I do recognize that the authors do a much better job of putting these findings in the broader context when referring to prior work. This was not a cross-site comparison across a range of tropical forest and thus has limited ability to generalize beyond other very similar forest types. 4. I don’t find the results all that surprising. We know that within 15 years of forest clearing that LAI cannot be distinguished between secondary and old growth forests. We expect N to be limiting in fast growing early successional trees in the tropics and adding N just gets the trees to the “saturation” point a few years faster. To say this is the full successional gradient, is not quite correct since it is still just early, mid and late succession. I fully agree that this work is of high value and whole heartedly commend the authors for undertaking such a substantial effort to evaluate effects of fertilization. It just really seems to support what other work has already shown – they just haven’t shown it in high-profile journals. 5. Line 145 – Phosphorus may not have generated a response in the mature forests of THIS system, but it is also important to recognize that the form, timing, and amount of nutrients added is important, as is the forest site. There is evidence in Costa Rica that P is important. For example, a litter manipulation experiment in Costa Rica found a positive effect of litter addition on forest productivity that was positively correlated with the total P in the added litter (Work by Dr. Wood). This held for some forest sites included, but not all (i.e., low P, secondary versus mature). In contrast, the long-term litter addition experiment in Panama (work by Dr. Sayer) found no effect of litter addition and P didn’t seem to be important. I am not sure of the location of this site in Panama versus the litter manipulation experiment of Dr. Sayer, but it seems that these are important considerations that the forest in Panama may not represent other tropical forest site that have more rainfall or other considerations. Further, evaluation of nutrient resorption across a P gradient in Costa Rica found that resorption of P was greater during periods of greater reproductive effort (work by Dr. Tully), and in that same forest, there was a mid-day draw down of available soil P that was linked with stem flow. Perhaps that was passive uptake, but it suggests that at least for other forest that P may be important in mature forests, and that as the authors suggest the effect of P may manifest in components other than woody growth. Honestly – it would help to know what the typical rate of woody growth is for these forests and whether they have the capacity to grow any faster than they already are. I appreciate that the authors touch on these alternate explanations, as well as the discussion of the community composition. My sentiment holds that if an organism is adapted to low P environment, then its competitive advantage is being able to acquire these components under limiting conditions. If we add tons of fertilizer to plant that is adapted to low nutrients and the plant doesn't grow faster or growth declines, does that mean that it doesn’t exist in low nutrient environment and that the system is not limited? or does it really mean the plant itself has limited capacity to respond to more? Mature tropical trees tend to be much slower growing. And there are adaptive strategies for getting rock derived P that may not provide advantageous when tons of it are dumped as fertilizer on the soil. We know from agricultural work and mass production of plants that different species have different nutrient, soil and water requirements. So really, what do we mean when we discuss limitation? Is it really that the organism grows faster when more is added? Or is it that they are adapted to limiting conditions and there are strategies that allow that organism to out compete others under specific limiting conditions that also prevents them from being able to respond to excess. Examples of efficient cycling could be efficient resorption of that element (i.e., Dr. Vitousek, Dr. Killingbeck, among others), changes in allocation belowground, changes in leaf lifespan, changes in phenology or reproductive efforts. While woody growth and aboveground biomass are definitely important for carbon storage, and if that's all we care about, then knowing that mature trees in Panama don't grow faster with N and P is of value. But if the goal is to fully evaluate nutrient limitation, there are other ways that plants can respond and this is just one of them. 6. Fundamentally, when substituting space for time with manipulation experiments, the capacity for adaptation, evolution, etc. is missed, especially when considering the long lifespan of mature tropical trees (as the authors rightly note). How plastic is the ability of these trees to respond to nutrients? Definitely in young, fast-growing trees it makes sense that they would respond, but already mature trees? I don’t doubt the results, but I am not convinced by the interpretation, as much as I do appreciate that further discussion is given to these ideas. 7. Regarding the suggestions for managing tropical forest reforestation. Basically, what the authors are saying is that forests reach “saturated” above ground biomass faster when fertilized, which means this effect is a transient boost that goes away in maybe 5 years given that at 15-20 years tropical forests are pretty well developed (i.e., Dr. Chazdon’s work). The authors go from 10 years to 30 years, so we don’t really know, but I feel like the benefits might be overstated. It isn’t that these forests add an additional Megaton of C carbon forever. 8. The entire discussion of forest management doesn’t feel appropriate for this paper given that this is not a reforestation study. I think the suggestion that this forest might be limited by some other nutrient – with no example or suggested alternate listed – is indeed interesting, but it doesn't provide us with a way forward for solving this major question in tropical research. And given that focus, I don't think this discussion of management is appropriate in this manuscript. I think it should be a separate commentary or included in a study that is actually studying reforestation and carbon sequestration. Overall, I think this work is of high value scientifically. However, it failed to provide me with enough convincing evidence for me to say it's time to put the nail in the coffin for P limitation in mature tropical forests, especially when there isn’t any evidence of what the “other” limiting factor or element might be and the limited response variables considered. Perhaps this forest is growing as fast as it possibly can given the physiological constraints of mature tropical hardwoods, or potentially the timescale of research isn't long enough to capture increased growth given the slow rate of diameter growth. Further, once a forest is mature and reaches the forest canopy, maybe it is more beneficial to invest excess nutrients elsewhere, for example - turnover of leaves, or in reproduction. Younger leaves tend to have more nutrients and thus higher rates of photosynthesis and with added nutrients the response might instead be reducing leaf lifespan and increasing turnover, which may not be captured in traditional measures of diameter growth, or that less energy is put into resorption and more into other components. The authors touch on these subjects, but I think other lines of evidence are needed for the scientific community to fully rule out the importance of P in favor of searching for an elusive \"other\" limiting element. The mystery of what drives mature tropical forest productivity remains, and ultimately, this work didn't move us closer to solving it. Further, while the experimental design is very robust for this site in Panama and certainly a major undertaking, I tend to be very cautious when it comes to generalizing single tropical forest sites too broadly. Finally, as I mentioned above, I don’t think the management discussion works in this paper. The paper is focusing on evaluating nutrient limitation during natural succession, and to then translate this to how we grow trees and where in the tropics is well beyond the scope, especially given the number of people that are actively working on reforestation projects."}, "author_response": "RESPONSE TO REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nI reviewed an earlier version of this manuscript submitted to Nature. Compared to the\nearlier version there are several things that have improved substantially. Several earlier,\nsomewhat exaggerated claims have disappeared and language usage is now much\nmore concise compared to the earlier version. The authors also better acknowledge and\ndiscuss earlier work on N limitation in tropical forests which also improves the clarity on\nwhat is new and noteworthy in the current study.\nThanks for reading our draft again and for providing insightful comments to help improve\nour paper. We appreciate that you recognize that we have improved our manuscript –\nyour feedback has been very valuable.\nWhen considering the critical question how well the soils in Panama represent soils\nunder tropical forests, it was interesting to learn that the authors now acknowledge that\nboth sites (Gigante and Agua Salud) also have less weathered soils than Oxisols. The\nauthors however argue that the total P values are more important than the classification\nand I agree with that.\nWe appreciate that the reviewer agrees that total P values are more important than the\nsoil classification, as total P values may vary a lot within one soil order. Interestingly, the\ntotal P values in our young Agua Salud soils are lower than the older Gigante soils. For\nclarification, while the Agua Salud soils are now correctly reported as less weathered\nthan Oxisols, the soils at Gigante are classified as Oxisols – the data can be found here\n(https://figshare.com/s/1d048546bfa4efcc9cb6).\nIn their answer to my earlier questions regarding the total P values of 600 mg/kg the\nauthors explain that the high number came from the paper by (Mirabello et al., 2013)\nwhich apparently overestimated the P in the ‘hot’ fractions. I looked up this paper and\nindeed they suggest that silica interference in phosphate detection by molybdate\ncolorimetry may have led to an overestimation. Fortunately, total P was also analyzed in\nthe (Koehler et al., 2009)) paper, which was mentioned by the authors in their rebuttal.\nKoehler et al. (2009) was conducted in the Gigante Experiment and they used pressure\ndigestion in concentrated HNO3 followed by analysis of the digests using inductively\ncoupled plasma-atomic emission spectrometer and report them in Table 1 of their paper.\nThis total P method does not use colorimetry for analyses and thus does not suffer from\npotential silicon interference. The numbers reported by Koehler et al, 2009 for the\ncontrol plots of the Gigante experiment are 550 mg kg-1 in the top 0.05m of mineral soil\nand still as much as 400 mg kg-1 in the 0.05 – 0.5m depth interval. If we take the lower\nvalue of 400 mg kg-1, this would still put the Gigante site in the top 10% values of total P\nin the Quesada et al., 2010 paper. So even when I accept that the 600ppm reported by\nMirabello et al., 2013 were overestimated because of methodological problems, the total\nP values of Gigante are more comparable to the top 10% of Amazonian forests. And as I\npointed out in my review of the earlier version, this was exactly what I would expect of\nsoils developed from basaltic parent materiel in tectonically active regions such as\nPanama. However, the real question for this manuscript is whether the total P levels are\nso important to extrapolate the result to other parts of the tropics and below I will argue,\nwhy I think that is not the case.\nThe reviewer raises further questions about the different values of total phosphorus\nreported for our Gigante soils which has prompted us to take a full inventory and critical\nanalysis of differences in the values of soil phosphorus measured at Gigante over the\ncourse of the experiment. We have now compiled all total phosphorus data that has\nbeen collected for soils at Gigante, which is reported in Extended Data Table 2.\nSoils in the top 10-15 cm of control plots and nearby soil pits at Gigante ranged from 303\nto 680 mg P kg-1. There could be two sources of inconsistency. First, soils can be highly\nvariable in total phosphorus in central Panama. Second, the four different methods used\nto measure total phosphorus can result in different values. We previously discounted the\nvalue of 680 mg kg-1 reported in Mirabello et al. 2013 because of the issue of the Hedley\nfractionation method overestimating total phosphorus content when summing all\nfractions as well as the interference issues discussed above. The reviewer raises the\nvalues of 400 to 550 mg kg-1 reported in Koehler et al. 2006. As we write in the Extended\nData Table 2, “this soil was measured using pressure digestion with nitric acid followed\nby analysis with inductively coupled plasma-atomic emission spectrometry89 (henceforth\npressure digestion). Pressure digestion has an array of operational concerns and safety\nlimitations that can cause errors90,91.” In addition, this point is much higher than the other\nvalues, and thus we also discount it. Soils were measured with ignition (550C) and\nextraction on four occasions; however, as we write in Extended Data Table 2, ignition\nand extraction is known to underestimate total phosphorus. Finally, on two occasions we\nmeasured soils with nitric acid extraction. This is the gold standard estimate of total\nphosphorus, and we believe provides the best estimate of total phosphorus at Gigante.\nThus, we use these values to compare to Agua Salud and the Amazon. Extended Data\nTable 2 summarizes all of the methods and data.\nNow that we have evaluated the data sources and determined the total phosphorus\nlevels at Gigante, we re-evaluate the fractional land area of the Amazon with total\nphosphorus higher than our sites. We realize that we did not explain how we estimated\nthis in our previous response, and why our estimate (25%+) differs from that of the\nreviewer (10%). We believe that the latter method considers the fraction of sites,\nwhereas we used the fraction of forested land area across the Amazon with different soil\ntypes. To do this, we combined the mean total phosphorus for each soil type for forests\nacross the Amazon Basin (Quesada et al 2010) and the fraction of the Amazon Basin\nforests that has each soil type (Quesada et al 2011). We find that ~25% of the\nAmazonian forests have more mean phosphorus than our forests. We conclude that our\nsites are relatively high in total phosphorus compared to some Amazonian forests, but\nthere is substantial land area in the Amazon with even more total phosphorus than our\nsites.\nWe have updated the Extended Data Table 3.\nWe have updated the soils data for our experimental sites in Extended Data Table 1,\nupdated the text in the manuscript in lines 89 and 195-200, included the data for total\nphosphorus in Extended data table 2, made open a table with calculations to scale total\nphosphorus in surface to deep soil layers, and provided the comparison of Amazon soils\nto ours in Extended Data Table 3.\nReferences\nC.A. Quesada, J. Lloyd, M. Schwarz, Patiño, S., T.R. Baker, C. Czimczik, N.M. Fyllas, L. Martinelli,\nG.B. Nardoto, J. Schmerler, A.J.B. Santos, M.G. Hodnett, R. Herrera, F.J. Luizão, A. Arneth, G. Lloyd,\nN. Dezzeo, I. Hilke, I. Kuhlmann, M. Raessler, W.A. Brand, H. Geilmann, J.O. Moraes Filho, F.P.\nCarvalho, R.N. Araujo Filho, J.E. Chaves, O.F. Cruz Junior, T.P. Pimentel, R. Paiva, Variations in\nchemical and physical properties of Amazon forest soils in relation to their genesis. Biogeosciences 7,\n1515–1541 (2010). https://doi.org/10.5194/bg-7-1515-2010\nQuesada, C. et al. Soils of Amazonia with particular reference to the RAINFOR sites. Biogeosciences\n8, 1415–1440 (2011). https://doi.org/10.5194/bgd-6-3851-2009\nIn their rebuttal the authors also write that it may be possible that the younger secondary\nforests could have a stronger nutrient (P) limitation compared to mature forests due to\nthe rapid relative growth rates of young trees. In the manuscript, theoretical\nbiogeochemical models are also mentioned as the basis for this expectation (l. 44 –\nl.49). Furthermore, they write counter to expectations from biogeochemical theory, they\nfound little evidence of P limitation in the experiment (L. 122). Although this may be true,\nI think that the authors miss to appreciate the potential importance of land use history in\nthis case (and in general with secondary forests). A strong P limitation in young\nsecondary forest is unlikely because the secondary forest grows on areas that were\npreviously cleared which probably involved slash-and-burn procedures and potentially\nagricultural practices. The results of slash and burn is that an important part of the rock\nderived-nutrients, including P, end up in the soil and the soil pH increases, effects that\ncan last for decades. A higher pH leads to higher P availability and a higher base\nsaturation which means that it is quite unlikely that rock-derived nutrients are limiting in\nthe earlier successional stages. Something that was more or less shown for P by (Wong\net al., 2024). To my knowledge, the Walker and Syers model considers natural systems\nwithout considering previous land use. However, taking the previous land use into\naccount will go a long way to explain at least some of the findings in your study and I\nthink that at the moment this is not sufficiently done. You discuss the possibility that P\nlimitation is not observed in wood productivity (l 177 and further), you discuss that it is\npossibly less common than previously thought (L 189 and further), but you never discuss\nthat forest clearing and burning leads to substantial increases in rock-derived nutrients\nand their availability, which I think is currently missing. You should also not forget that\nthe data from the (Quesada et al., 2010) paper were collected in old-growth forest,\nwhere presumable no slash and burn has been taken place in the last centuries. This is\npresently ignored in the manuscript when you compare your soils with soil of the\nAmazon Basin, and I really think it is misleading and causes a lot of confusion (at least it\ntook me two reviews to realize what was wrong). I expect that young secondary forests\nand abandoned agricultural areas will have higher values in total and available nutrients\nbecause of former slash-and-burn activities, and that should be highlighted in your\ndiscussion. It should not be too difficult to back this up with some studies. BTW, thank\nyou for providing the full soil analysis for the Agua Salud project. Your soil chemical\nanalyses show pH-water values around 5 which is a pH value when soil P is relatively\nwell available. Also, base saturation is regularly above 50% in the top soil which may be\nthe result of ashes from earlier forest clearing and also indicates that these are not very\nnutrient poor soils.\nYour point that total phosphorus can increase following slash and burning of forests is\nvery important. We have added this to the manuscript in lines 50-58 and 204-213\nincluding references to the recent literature. However, our young forests were pastures\nfor the past 30 or 40 years prior to transitioning to forests and did not experience slash\nand burn during that time since initial deforestation. It is possible that total and available\nrock derived nutrients may be higher in individual Agua Salud plots than they otherwise\nwould have been in the absence of slash and burn agriculture. Unfortunately, we cannot\ntest this because we do not have pre-deforestation phosphorus or pH values to compare\nwith our recent measurements in the same plots. We now recognize this in the text.\nHowever, we can compare our young forests to our old forests with the expectation that\nphosphorus and pH would be higher in the young forests. The data for total and\navailable phosphorus (Extended data table 1) do not show clear patterns of elevated\nphosphorus in the young forests relative to our mature forests as might be expected if\nthey were impacted by biomass burning. There was no significant difference in pH or\ntotal and resin available phosphorus in the young forests compared to older forests\n(Extended data Table 1). We also compared the resin available phosphorus in our\nyoung plots to the wide range of available phosphorus across dozens of sites in Panama\n(samples for both young and old forests were analysed with the same methods in the\nsame lab; Condit et al 2013). We found that the available phosphorus levels are\nrelatively low compared to the mature forests (Extended Data Table 1). This adds further\nevidence that our sites are not particularly phosphorus rich.\nAs we argue above, regardless of whether or not our Agua Salud forests were impacted\nby slash and burn agriculture in decades past, we still find it useful to compare these\nnumbers to the Amazon and globally to give context for where our study sits in terms of\nphosphorus.\nWe have added an evaluation of the potential for slash and burn agriculture to affect\nphosphorus in our forests in lines 204-213.\nReference\nR. Condit, B.M.J. Engelbrecht, D. Pino, R. Pérez, B.L. Turner, Species distributions in response to\nindividual soil nutrients and seasonal drought across a community of tropical trees. Proceedings of\nthe National Academy of Sciences 110, 5064–5068 (2013). https://doi.org/10.1073/pnas.1218042110\nI am still not convinced about your implications, in your rebuttal you write that you\nespecially focus on tree planting when suggesting to increase the abundance of N fixing\nspecies. I have no doubt that there are some examples of secondary forests with little N\nfixing trees. However, the (Gei et al., 2018) paper, which was mentioned in the rebuttal,\nalso shows that the majority of secondary forests have a relative basal area of 10% or\n(much) more. I am not sure whether adding more N-fixing legumes would make a big\ndifference and as far as I know the vast majority of secondary forests is the result of\nspontaneous regeneration and succession and not from planting.\nThanks for your comments. We recognize the large variation in relative abundance of\nnitrogen-fixing trees over succession in tropical wet forests; however, note that there are\nmany sites with less than 10% nitrogen-fixers and mean basal area in the Gei et al study\nis 8.5% at 1 year and 10% at 10 years (from Fig 1c). This suggests 1) nitrogen fixers\nincrease in abundance in the first few years of forest recovery, potentially because of\nnutrient limitation, competition or dispersal limitation; and 2) forests potentially could\nsupport more nitrogen fixers than 10% abundance and/or fix more nitrogen. Thus,\nincreasing fixer abundances either in active reforestation efforts or supplemental\nplanting in managed natural regeneration could make a difference, even in sites with\nonly 10% fixers. We agree that increased nitrogen fixer abundance may not enough to\nrelieve all nitrogen limitation and discuss this point in our draft (line 307).\nReference\nM. Gei, D.M.A. Rozendaal, L. Poorter, F. Bongers, J. I. Sprent, M.D. Garner, T. Mitchell\nAide et al. \"Legume abundance along successional and rainfall gradients in Neotropical\nforests.\" Nature ecology & evolution 2, no. 7 (2018): 1104-1111.\nhttps://doi.org/10.1038/s41559-018-0559-6\nIn summary, I think the study presented here definitely gives new, noteworthy and\nexciting insights in to shifts in nitrogen and phosphorus limitations over a secondary\nsuccession gradient in soils that are at the higher end of total phosphorus content of\nsoils under (secondary) forests, and as such is worth publishing.\nWe appreciate your enthusiasm and thank you again for your insightful comments which\nwe believe have substantially improved our manuscript.\nI think your claim that your soils are close to the median value for tropical forests soils\nglobally is simply not true and frankly, I don’t think it is important for your manuscript.\nWhat is important for your message is that in secondary forests, N limitation is\ndecreasing over time, and in my interpretation, it is unlikely to be replaced in the first\ndecades of a secondary forest by P limitation (not even on heavily weathered Oxisols in\nthe Amazon) because of the higher and more available rock-derived nutrient stocks,\nincluding soil P, which is a remnant of former slash and burn activities.\nWe agree with the reviewer’s conclusion that our soils are relatively high in phosphorus\ncompared to tropical forests. We revised the sentence, please see lines 193-200. We\nalso appreciate the reviewer’s summary of what is the important finding from our study –\nthat nitrogen limitation decreases over time and is unlikely to be replaced by phosphorus\nlimitation, at least in our study area. However, we respectfully disagree that the reason\nfor our lack of phosphorus limitation is because of phosphorus inputs from slash and\nburn practices in secondary forests. We recognise that this is a possibility; however, our\nforests have had limited slash and burn treatment decades ago and soil indicators\nsuggest that there was not a substantial influx of phosphorus. Nonetheless, we now\nacknowledge this possibility in the discussion (lines 204-213).\nReferences\nGei, M., et al., 2018. Legume abundance along successional and rainfall gradients in\nNeotropical forests. Nat. Ecol. Evol. 2, 1104–1111. https://doi.org/10.1038/s41559-018-\n0559-6\nKoehler, B., et al. 2009. Immediate and long-term nitrogen oxide emissions from tropical\nforest soils exposed to elevated nitrogen input. Glob. Change Biol. 15, 2049–\n2066. https://doi.org/10.1111/j.1365-2486.2008.01826.x\nMirabello, M.J., et al. 2013. Soil phosphorus responses to chronic nutrient fertilisation\nand seasonal drought in a humid lowland forest, Panama. Soil Res. 51,\n215. https://doi.org/10.1071/sr12188\nQuesada, C.A., et al., 2010. Variations in chemical and physical properties of Amazon\nforest soils in relation to their genesis. Biogeosciences 7, 1515–\n1541. https://doi.org/10.5194/bg-7-1515-2010\nWong, M.Y., et al. 2024. Trees adjust nutrient acquisition strategies across tropical forest\nsecondary succession. New Phytol. 243, 132–144. https://doi.org/10.1111/nph.19812\nReviewer #2 (Remarks to the Author):\nThis is the second time I have reviewed this paper (albeit for a different journal). So I am\ncopying out my previous review below since it addresses many of the stock questions\nasked by NCOMMS.\nThe authors now present the carbon numbers with appropriate nuance. They have also\ndirectly addressed my concern about nitrogen being confounded with age at the\nbeginning. And the methods are now clearly documented, and are sound. I have no\nadditional critiques beyond the two minor grammatical catches below.\nThank you very much for reading our paper and provide constructive comments. We\naddress your new comments below. We addressed all of the comments from the\nprevious review in our last revision, and appreciate that the reviewer recognizes this.\nLine 39: “increasing predominance in modern tropical landscapes” would be better than\n“increasing importance” since the latter is normative\nDone\nLine 194: in LATIN America? Hawaii is also USA.\nThank you for catching this. We have changed it to “Central and South America” since\nwe prefer to avoid the use of the term “Latin” American due to it’s Eurocentric and\ncolonial origins. Please see the line 241.\n-------------------\nWe do not respond to the comments from Reviewer 2 below because we addressed\nthem in full in the last revision.\nThe paper by Tang et al examines impacts of fertilization on a network of tropical forest\nplots within Panama. They provide supplemental nitrogen and phosphorus to forests that\nvary in age (~0, 10, 30, and 600 years old) and find that the nitrogen addition greatly\naugments growth in the young forests only and that – despite standard biogeochemical\ntheory – woody biomass accumulation does not appear to be limited by phosphorus.\nThe paper is very well written. I have no editorial critiques for clarity, and this never\nhappens when I review a manuscript. As I was reading, multiple questions arose in my\nmind but they addressed these later in the manuscript. For example, they find that\nphosphorus is not limiting which is a surprising result, but then provide a very nice\ndiscussion to explore potential mechanisms underlying their results. I don’t think the\npaper’s findings are enough to upend theory, but it certainly suggests that more work is\nneeded to determine the universality of phosphorus limitation in the tropics.\nI was concerned that the interaction between nitrogen and forest age was confounded\nby nitrogen conditions in the soil (i.e., more nitrogen intact forest soils), but they provide\nbaseline soil fertility conditions in a supplemental information (Extended Data 2) that\nshows this is not the case. I suggest making that point explicitly within the main\nmanuscript.\nI was also concerned that the applied take home from the paper would be that people\nshould apply fertilizer to young regrowing forests to augment growth and carbon\nremoval. But the paper very clearly states that this is not recommended (I agree!) and\nprovide alternative options suggest as prioritizing regrowth in places with N deposition.\nHowever, the largest issue is reproducibility. The methods are non-existent. The\nparagraph located lines 68-78 seems written to make the experiment sound larger than\nit actually is. They say there are 76 plots, but I think this includes both the treatment and\nthe control so actually the number of experimental units is smaller. Sample size per\ntreatment appears to be restricted to the figure legends. They say that these cover\n88,843 trees, but those are not independent replicates. They report that they monitored\nthese plots for up to 21 years and then mention in the parentheses that secondary\nforests have been monitored for four, but in fact the main results of the paper come from\nthose secondary forests. How large are the plots? They say they cover 16 km2, but what\nis the range of sizes for the individual plots? Where are they located (i.e., can the\ngeolocations be made available)? How much fertilizer did they add? What dosage, what\nfrequency? How did they quantify biomass (i.e., which allometric equations were used)?\nThis is all easily remedied by including a clear methods section – in the supplement if no\nspace is available in the main text.\nMy only other critique is that their carbon numbers require some nuance. I appreciate\nthat they estimated mitigation potential at a cost-constrained rather than unconstrained\nlevel. However, their number remains quite theoretical. It is highly unlikely/impossible to\nachieve 1.1 GtCO2/yr because there are only so many locations with N deposition or\nhigher fertility soils and only so much N that can be provided by nitrogen fixing species.\nAdding fertilizer to relieve nutrient limitations would produce upstream and in-field\nemissions that would offset the climate benefit of enhanced forest growth. I suggest the\nco-authors nuance that result more appropriately. For example, in the abstract instead of\n“could sequester up to an additional 1.1 GtCO2/yr” it might say “N limitations prevent up\nto 1.1 GtCO2/yr”. In other words, that 1.1 GtCO2/yr shouldn’t be presented as an\nopportunity.\nOptional suggestions:\n(1) Table 1 could be supplemental\n(2) Figure 1 should be checked for color blind friendliness. And would a log-scale for the\ny-axis better illustrate the differences among treatments (as an alternative to the inset\napproach).\nReviewer #2 (Remarks on code availability):\nCode and metadata are appropriate.\nReviewer #3 (Remarks to the Author):\nThis study evaluates the effects of fertilization on aboveground biomass accumulation in\nearly, mid and late successional forests in Panama. The work itself is incredibly\nambitious, and the results are very robust and of interest. This study was a major\nundertaking. The manuscript is also beautifully written, and the authors certainly present\nsome intriguing ideas. I read an earlier draft of this manuscript, and there are substantial\nimprovements. I appreciate the broader acknowledgement prior work, and the more\ndeveloped discussion of alternate explanations for the lack of response to N and P\nfertilization in mature forests. That said, many of my prior reservations hold.\nThank you very much for reading our draft and providing so many thoughtful comments.\nWe appreciate that you recognize the ambitiousness and robustness of our study. We\nalso value that you see our improvements following your helpful comments from a\nprevious review. We address your reservations below.\n1. Abstract: I appreciate that in the discussion, the authors compare this work to forests\nin the Amazon, but I am still extremely hesitant about generalizing findings from a single\nforest site in Panama to the tropics as a whole, especially given the wide range of\ntropical forest types (i.e., seasonal, evergreen, flooded, etc) and that the neotropics may\nnot reflect what we would see in Asian forests or other less studied systems.\nWe take your point and have now specified that we are discussing results from our\nforests in the abstract and add a caveat to the line about scaling up. We write (new text\nin bold): “Central American landscape” and “Nutrient limitation of aboveground\nbiomass accumulation shifts over succession in our forests” in the abstract (lines 27-\n29). Finally, we modified the last sentence of the abstract to read, “Our findings suggest\nthat, if nitrogen limitation is widespread in young recovering tropical forests,\nnitrogen limitation could prevent up to 0.84 Gt CO uptake by recovering tropical forests\n2\neach year” (lines 33-35). We further reduce generalization in other parts of the\nmanuscript besides the abstract, including lines 198-200, and discuss the possibility of\nheterogeneity in nutrient limitation in young forests (164-167).\n2. Regarding the following statement –\n“To date, we are aware of no direct ecosystem experiment that investigates how nutrient\nlimitation on stand-scale aboveground carbon dynamics evolves over the course of a\ncomplete tropical forest secondary succession gradient, from recently abandoned\npastures to mature forests.”\nAnd this matters why? Being the first, or only, in and of itself isn’t that interesting, but\nsomething that helps fill a specific knowledge gap, is. Please elaborate on why we as a\nscientific community should care about having this additional detail, and if the\nexperiment just supports what is already generally accepted amongst tropical forest\necologists (i.e., early successional forests are N limited), is this really transforming our\nunderstanding? Definitely this is a really well-designed study, and the findings are very\nrobust, but it just supports what others have shown - although maybe not as well.\nWe now understand your point, and have deleted this sentence and rearranged the\nintroduction to make a clearer argument identifying the knowledge gap and how we\naddress it in lines 53-82.\nThe authors suggest that some element other than P is what is really limiting mature\ntropical forests, but what do they think it is?. Also -- why would we expect mature trees\nto respond in the same way as fast growing early successional trees that are working to\naccess the forest canopy?\nThanks for your suggestion. We have added a sentence that mature forest carbon sink\ncould be limited by other resources like potassium (Wright et al., 2011; Manu et al.\n2022), calcium (Bauters et al., 2022) or water (Tao et al., 2022) (lines 256-257).\nYou have a good point about the possibility that the mature forest trees may not be able\nto respond to nutrients in the same way that young forest trees can. For example, the\nmature forest trees may be adapted to lower phosphorus conditions and may not be\nable to respond to added phosphorus. We have added some sentences about this in\nlines 224-238, 240-249.\nReferences\nS.J. Wright, J.B. Yavitt, N. Wurzburger, B.L. Turner, E.V.J. Tanner, E.J. Sayer, L.S. Santiago, M.\nKaspari, L.O. Hedin, K.E. Harms, M.N. Garcia, M.D. Corre, Potassium, phosphorus, or nitrogen limit\nroot allocation, tree growth, or litter production in a lowland tropical forest. Ecology 92, 1616–1625\n(2011). https://doi.org/10.1890/10-1558.1\nR. Manu, M.D. Corre, A. Aleeje, M.J.G. Mwanjalolo, F. Babweteera, E. Veldkamp, O. van Straaten,\nResponses of tree growth and biomass production to nutrient addition in a semi-deciduous tropical\nforest in Africa. Ecology 103, e3659 (2022). https://doi.org/10.1002/ecy.3659\nM. Bauters, I.A. Janssens, D. Wasner, S. Doetterl, P. Vermeir, M. Griepentrog, T.W. Drake,\nJ. Six, M. Barthel, S. Baumgartner, K. Van Oost, I.A. Makelele, C. Ewango, K. Verheyen, P.\nBoeckx. Increasing calcium scarcity along Afrotropical forest succession. Nature Ecology &\nEvolution 6, 1122–1131 (2022).\nS. Tao, J. Chave, P.L. Frison, T.L. Toan, P. Ciais, J. Fang, J.P. Wigneron, M. Santoro, H.\nYang, X. Liu, N. Labriere, Increasing and widespread vulnerability of intact tropical\nrainforests to repeated droughts. Proceedings of the National Academy of Sciences 119,\ne2116626119 (2022). https://doi.org/10.1073/pnas.2116626119\n3. The following statement “These findings advance our understanding of the potential\nroles of both nitrogen and phosphorus in tropical forests.” Once again, this study is\nspecific to one study in Panama. There is a limit to how much these results can be\ngeneralized beyond this site, although I do recognize that the authors do a much better\njob of putting these findings in the broader context when referring to prior work. This was\nnot a cross-site comparison across a range of tropical forest and thus has limited ability\nto generalize beyond other very similar forest types.\nWe agree our study is specific to one area and therefore may not reflect limitation in\nother tropical forests. Nonetheless, we still believe that the findings may have relevance\nto other tropical forests, especially because the results follow what has previously been\nfound in several studies (as you mention above). We have deleted this sentence and\nreplaced it with, “Although they derive from one site, these findings advance our\nunderstanding of the role that nitrogen and phosphorus can have in tropical forests”\n(lines 153-154). We also added a sentence in lines 202-203, “Future work should\nexamine the degree to which these patterns are consistent in other tropical forests,\nincluding African and Asian tropical forests.”\n4. I don’t find the results all that surprising. We know that within 15 years of forest\nclearing that LAI cannot be distinguished between secondary and old growth forests. We\nexpect N to be limiting in fast growing early successional trees in the tropics and adding\nN just gets the trees to the “saturation” point a few years faster.\nAlthough there is some experimental and observational evidence that suggests nitrogen\nlimits young forests, a metanalysis with all field-based fertilization experiments found that\neither nitrogen or phosphorus can be limiting young tropical forests (Wright, 2019).\nFurthermore, comparison of results from different experiments in different sites may\nsuffer from confounding factors that hinder inference. Thus, nitrogen limitation in our\nyoung forests was not a forgone conclusion, and the shift in nutrient limitation within one\nforest area is novel. We have substantially reorganized our introduction to more clearly\nhighlight the novelty of our experiment. Please see lines 60-77.\nReference\nS.J. Wright, Plant responses to nutrient addition experiments conducted in tropical\nforests. Ecological Monographs 89, e01382 (2019). https://doi.org/10.1002/ecm.1382\nTo say this is the full successional gradient, is not quite correct since it is still just early,\nmid and late succession. I fully agree that this work is of high value and whole heartedly\ncommend the authors for undertaking such a substantial effort to evaluate effects of\nfertilization. It just really seems to support what other work has already shown – they just\nhaven’t shown it in high-profile journals.\nWe have removed the word “complete” in lines 27, 85, 189.\n5. Line 145 – Phosphorus may not have generated a response in the mature forests of\nTHIS system, but it is also important to recognize that the form, timing, and amount of\nnutrients added is important, as is the forest site. There is evidence in Costa Rica that P\nis important. For example, a litter manipulation experiment in Costa Rica found a positive\neffect of litter addition on forest productivity that was positively correlated with the total P\nin the added litter (Work by Dr. Wood). This held for some forest sites included, but not\nall (i.e., low P, secondary versus mature). In contrast, the long-term litter addition\nexperiment in Panama (work by Dr. Sayer) found no effect of litter addition and P didn’t\nseem to be important. I am not sure of the location of this site in Panama versus the litter\nmanipulation experiment of Dr. Sayer, but it seems that these are important\nconsiderations that the forest in Panama may not represent other tropical forest site that\nhave more rainfall or other considerations. Further, evaluation of nutrient resorption\nacross a P gradient in Costa Rica found that resorption of P was greater during periods\nof greater reproductive effort (work by Dr. Tully), and in that same forest, there was a\nmid-day draw down of available soil P that was linked with stem flow. Perhaps that was\npassive uptake, but it suggests that at least for other forest that P may be important in\nmature forests, and that as the authors suggest the effect of P may manifest in\ncomponents other than woody growth.\nWe do not intend to argue that phosphorus is not important in other tropical forests or\neven in our sites (see, for example, Wong et al 2024 showing that phosphorus\ninfluences some nutrient acquisition strategies in our experiment). In fact, we see our\nstudy as contributing to the increasing evidence that nutrient limitation in tropical forests\nis heterogeneous and more complex than just influencing aboveground woody growth\n(see lines159-170 and 231-238). We agree with the reviewer that there are many factors\nthat could determine whether a response to phosphorus is generated including site, and\nthat other components of trees may be affected by phosphorus while woody growth is\nnot (lines 215-217). For reference, the litter manipulation experiment that Dr. Sayer\nmaintains is adjacent to our mature forest fertilization experiment on the same Gigante\npeninsula. At that experiment, the current hypothesis is that litter removal induced\nnitrogen rather than phosphorus limitation because litter removal carried away 25 times\nmore nitrogen than phosphorus (Tanner et al 2024, The Gigante Litter Manipulation\nExperiment, from The First 100 Years of Research on Barro Colorado: Plant and\nEcosystem Science, vol. 2). We have expanded our discussion of potential\nheterogeneity in nutrient limitation in tropical forests (lines 231-238) and have explicitly\nacknowledged that some tropical forests may be phosphorus limited by adding the\nsentence that “Nonetheless, evidence suggests that phosphorus may limit some tropical\nforests” (lines 255-260).\nReference\nC_Muller-Landau, Helene, and S. Joseph Wright. \"The First 100 Years of Research on Barro\nColorado: Plant and Ecosystem Science (Volumes 1 and 2).\" (2024).\nHonestly – it would help to know what the typical rate of woody growth is for these\nforests and whether they have the capacity to grow any faster than they already are. I\nappreciate that the authors touch on these alternate explanations, as well as the\ndiscussion of the community composition. My sentiment holds that if an organism is\nadapted to low P environment, then its competitive advantage is being able to acquire\nthese components under limiting conditions. If we add tons of fertilizer to plant that is\nadapted to low nutrients and the plant doesn't grow faster or growth declines, does that\nmean that it doesn’t exist in low nutrient environment and that the system is not limited?\nor does it really mean the plant itself has limited capacity to respond to more? Mature\ntropical trees tend to be much slower growing. And there are adaptive strategies for\ngetting rock derived P that may not provide advantageous when tons of it are dumped\nas fertilizer on the soil. We know from agricultural work and mass production of plants\nthat different species have different nutrient, soil and water requirements. So really, what\ndo we mean when we discuss limitation? Is it really that the organism grows faster when\nmore is added? Or is it that they are adapted to limiting conditions and there are\nstrategies that allow that organism to out compete others under specific limiting\nconditions that also prevents them from being able to respond to excess. Examples of\nefficient cycling could be efficient resorption of that element (i.e., Dr. Vitousek, Dr.\nKillingbeck, among others), changes in allocation belowground, changes in leaf lifespan,\nchanges in phenology or reproductive efforts. While woody growth and aboveground\nbiomass are definitely important for carbon storage, and if that's all we care about, then\nknowing that mature trees in Panama don't grow faster with N and P is of value. But if\nthe goal is to fully evaluate nutrient limitation, there are other ways that plants can\nrespond and this is just one of them.\nYou raise a good question about whether these forests have the capacity to grow more\nthan they currently do, and thus if we expect them to be able to respond to resource\nadditions. We believe that they do have this capacity, although we agree with the\nreviewer that we may not be able to observe a response to phosphorus in woody growth\nbecause of mismatches in the timescale of our experiment and species turnover.\nAboveground woody productivity (AWP) in our mature forest plots (5.3 Mg ha-1 year-1) is\nat the lower end for forests in Panama. There is high small-scale variability in AWP at\nthe nearby 50 ha plot at BCI ranges, where AWP ranges from from 6.4 to 9.2 Mg ha-1\nyear-1 across a small-scale topographic gradient within the 50 ha (Piponiot et al 2024)50.\nFurthermore, there is a two-fold range across 39 mostly 1 ha plots located across the\nIsthmus of Panama, ranging from 3 to 7 Mg ha-1 yr-1 (Muller-Landau et al. 2024) 49. Thus,\nnearby forests can grow up to twice as fast as our forests. We now discuss this on lines\n228-231.\nWe agree with you that the timescales at which a response might occur could exceed\nthat of our experiment because the current trees may be adapted to low nutrient\nconditions and therefore do not have capacity to respond to phosphorus addition. Over\nlonger timescales, response to phosphorus may occur through changes in species\ncomposition from low to high phosphorus adapted species. We have added a paragraph\ndiscussing this on lines 224-235. We also agree that trees may adjust nutrient\nacquisition and use strategies which could make them more efficient in limiting\nconditions. In fact, we have found that root phosphatase activity increases in the 30-\nyear-old and mature forests and is suppressed by phosphorus in our experiment (Wong\net al 2024)34 (lines 244-246). We mention responses of other tissues that have been\nobserved in our mature forests, like increased reproduction (Fortier & Wright, 2021)48, in\nlines 216.\nReferences\nC. Piponiot, R. Condit, S.P. Hubbell, R. Pérez, S. Lao, S. Aguilar, H.C. Muller-Landau,\nWoody Biomass Stocks and Fluxes in the Barro Colorado Island 50-ha Plot. Smithsonian\nInstitution Scholarly Press. Chapter (2024). https://doi.org/10.5479/si.26882404\nH. Muller-Landau, J. Wright, Looking Forward to the Next 100 Years of Plant and Ecosystem\nScience at Barro Colorado. Smithsonian Institution Scholarly Press. Chapter (2024).\nhttps://doi.org/10.5479/si.26882656\nM.Y. Wong, N. Wurzburger, J.S. Hall, S.J. Wright, W. Tang, L.O. Hedin, K. Saltonstall, M. van\nBreugel, S.A. Batterman, Trees adjust nutrient acquisition strategies across tropical forest secondary\nsuccession. New Phytologist (2024). https://doi.org/10.1111/nph.19812\nR. Fortier, S.J. Wright, Nutrient limitation of plant reproduction in a tropical moist forest. Ecology 102,\ne03469 (2021). https://doi.org/10.1002/ecy.3469\n6. Fundamentally, when substituting space for time with manipulation experiments, the\ncapacity for adaptation, evolution, etc. is missed, especially when considering the long\nlifespan of mature tropical trees (as the authors rightly note). How plastic is the ability of\nthese trees to respond to nutrients? Definitely in young, fast-growing trees it makes\nsense that they would respond, but already mature trees? I don’t doubt the results, but I\nam not convinced by the interpretation, as much as I do appreciate that further\ndiscussion is given to these ideas.\nThe plasticity question is very important and we appreciate the opportunity to add\ndiscussion of flexibility in lines 224-235.\nWe do have some evidence that the nutrient strategies in our experimental forests are\nplastic, including root phosphatase activity and mycorrhizal colonization (Wong et al\n2024), and this plasticity occurs in all forest ages. However, it is a good question\nwhether this flexibility in nutrient strategies translates to an ability to be plastic in growth\nrates over short time periods.\nReference:\nM.Y. Wong, N. Wurzburger, J.S. Hall, S.J. Wright, W. Tang, L.O. Hedin, K. Saltonstall, M. van\nBreugel, S.A. Batterman, Trees adjust nutrient acquisition strategies across tropical forest secondary\nsuccession. New Phytologist (2024). https://doi.org/10.1111/nph.19812\n7. Regarding the suggestions for managing tropical forest reforestation. Basically, what\nthe authors are saying is that forests reach “saturated” above ground biomass faster\nwhen fertilized, which means this effect is a transient boost that goes away in maybe 5\nyears given that at 15-20 years tropical forests are pretty well developed (i.e., Dr.\nChazdon’s work). The authors go from 10 years to 30 years, so we don’t really know, but\nI feel like the benefits might be overstated. It isn’t that these forests add an additional\nMegaton of C carbon forever.\nYes, you are correct that we are finding that nutrients boost forest recovery rates and\nthat the carbon sequestration boost is temporary. We recognize that the rate of forest\ncarbon sequestration due to alleviation of nutrient limitation for aging forests declines\nover time and that the land area available for reforestation theoretically would also\neventually decline. However, this initial boost is important and valuable because natural\nclimate solutions that allow us to sequester more carbon quickly albeit temporarily will\nbuy us time to switch to slower-to-scale solutions that sequester carbon for the long-\nterm. We focus on the next 30 years because this time period is critical for our carbon\ntrajectories and amount of warming.\nWe have now added nuance to our estimate of how the maximum potential carbon\nsequestration could prevented by nitrogen limitation in the future when reforestation is\nvalued as a natural climate solution (e.g., $100 tCO -1). Note that our estimate is an\n2\nupper bound as noted by the word maximum, since our scaling assumes that all newly\nregenerating tropical forests have the same type and strength of nutrient limitation as\nour site, despite the evidence that there could be heterogeneity in the strength and type\nof limitation in young forests.\nWe also add nuance to our estimate by considering the aging forest and declining\ncarbon sequestration rate with age. We fine-tune our carbon multiplier by using our data\nat 4, 14 and 30 years to estimate the carbon multiplier at 10, 20 and 30 years. The 10-\nyear increments align with the forest ages used in model projections from Busch et al\n2019 and therefore allows for more precise scaling. We calculate the additional carbon\nsequestered at 10, 20 and 30 years, assuming a constant rate from 0-4 years and linear\ndeclines between 4 and 14 years and 14 and 30 years. We then track the proportion of\nland area in each decade of forests that are 0-10, 10-20 and 20-30 years old by\nconsidering the land area reforested in each decade. The addition of this nuanced\nmethod reduces the projection of carbon sequestration prevented by nitrogen limitation,\nas the reviewer implies, from a maximum of 1.1 Gt CO year-1 to a maximum of 0.84 Gt\n2 2\nDespite this reduction, the amount of reforestation prevented by nutrient limitation is still\nsubstantial because the majority of land newly reforested in each time period is in forests\nthat are less than 10 years old, when the carbon multiplier is highest. From 2020-2030,\n100% of projected reforested areas would have forests that are 0-10 years old. From\n2030-2040, 53% of projected reforested areas would have forests that are 0-10 years\nold and 47% would have forests that are 10-20 years old. From 2040-2050, 38% of\nprojected reforested areas would have forests that are 0-10 years old, 33% would have\nforests that are 10-20 years old and 29% would have forests that are 20-30 years old.\nWe have expanded our discussion of methods and caveats for this projection in the\nSupplemental Information 2 and in lines 295-302, which we hope adds clarity.\nReference\nJ. Busch, J. Engelmann, S.C. Cook-Patton, B.W. Griscom, T. Kroeger, H. Possingham, P.\nShyamsundar, Potential for low-cost carbon dioxide removal through tropical reforestation.\nNature Climate Change 9, 463–466 (2019). https://doi.org/10.1038/s41558-019-0485-x\n8. The entire discussion of forest management doesn’t feel appropriate for this paper\ngiven that this is not a reforestation study. I think the suggestion that this forest might be\nlimited by some other nutrient – with no example or suggested alternate listed – is\nindeed interesting, but it doesn't provide us with a way forward for solving this major\nquestion in tropical research. And given that focus, I don't think this discussion of\nmanagement is appropriate in this manuscript. I think it should be a separate\ncommentary or included in a study that is actually studying reforestation and carbon\nsequestration.\nWe appreciate the reviewer’s perspective but respectfully disagree that we should cut\nour management recommendations from the manuscript. Our projection of how much\nadditional carbon could be sequestered because of management practices that alleviate\nnutrient limitation hinge on all regrowing tropical forests being limited by nitrogen – a\nsubstantial uncertainty that we now acknowledge in the main text. Nonetheless, we think\nit important to provide a maximum bound for how much nitrogen limitation on carbon\nsequestration there may be – and strategies for alleviating that limitation – in recovering\nforests reforested as natural climate solutions.\nFurthermore, we disagree with the reviewer’s argument that we should not include\nmanagement recommendations because we do not resolve what limits older tropical\nforests. The type of nutrient limitation in older forests does not influence our carbon\nsequestration estimates because, as the reviewer acknowledges, several other lines of\nevidence along with our study provide substantial evidence that nitrogen limits young\nforests, and we remain nutrient limitation type-neutral in our projections for older forests,\nassuming no nutrient limitation as forests age (as per our experimental results), which is\nconservative. We recognize that our experimental results reported here were not able to\ndetermine whether a different nutrient besides nitrogen or phosphorus limited older\nforests, but any limitation by a different nutrient would provide an opportunity for the\nsequestration of even more carbon if forests were managed to alleviate that nutrient\nlimitation. Thus, we believe that our carbon projections and management\nrecommendations are valuable, despite the fact that we did not resolve what limits\nmature tropical forests.\nWe also believe that our recommendations for management are valuable even though\n“this is not a reforestation study” because we indeed designed it as a reforestation study\n– one of natural regeneration. Our main goals were to understand how nutrient limitation\npatterns shift along forest succession and provide recommendations for policymakers to\nincrease carbon storage potential of reforestation projects as a natural climate solution.\nWe have now updated our introduction to specifically mention tropical reforestation as a\nnatural climate solution and how our study could benefit the implementation and\nmanagement of tropical reforestation (lines 40-44, 94-98). Indeed, understanding\ncontrols of natural regeneration rates is critical for reforestation practices because it is\nthe main method of reforestation used in the tropics today (Gao et al 2025), is pledged\nto be used in over a third of total area to be reforested under the Bonn Challenge (Lewis\net al 2019), and could be used in much more land area in the future, including in many of\nthe areas pledged for reforestation with plantations under the Bonn Challenge (Chazdon\nand Guariguata 2016, Crouzeilles et al 2017, Lewis et al 2019, Williams et al 2024). We\nare conservative in our carbon projections because we only consider additional land\nreforested when using reforestation as a natural climate solution (that resulting from a\ncarbon price), but reforestation that occurs in the future under business-as-usual\nscenarios could also sequester more carbon, which would also be additional, if it is done\nintentionally with our management recommendations.\nResolving the role of nutrients in limiting natural forest regeneration is an important\nknowledge gap. Previous studies of controls of carbon sequestration rates over\nsecondary succession in American tropical forests have failed to identify nutrients as a\nkey factor (e.g., Poorter et al 2016). Our findings about nitrogen’s effect can inform\nreforestation efforts and estimates of the carbon sequestration potential of tropical\nreforestation. Thus, we find it relevant and important to raise the broader implications in\nour manuscript. Nature Communications appeals to a broad and diverse audience, and\nthus our study – if published there – has the potential to reach, amongst others, the\nrestoration and natural climate solutions communities, including those actively managing\nrestoration and those making projections of future carbon sequestration potential.\nReference:\nX. Gao, P.B. Reich, J.R. Vincent, M.E. Fagan, R.L. Chazdon, S. Fritz, D. Schepaschenko, M.D. Potts,\nM.C. Hansen, M. Jung, P.H.S. Brancalion, M. Uriarte, T.F. Keenan, T.W. Crowther, R.O. Dubayah, M.\nLesiv, S. Liang, D. Wang. The importance of distinguishing between natural and managed tree cover\ngains in the moist tropics. Nature Communications 16, 6092 (2025). https://doi.org/10.1038/s41467-\n025-59196-1\nS. L. Lewis, C. E. Wheeler, E. T. Mitchard, A. Koch. Regenerate natural forests to store carbon.\nNature 568, 25–28 (2019). https://doi.org/10.1038/d41586-019-01026-8\nR. L. Chazdon, & M. R. Guariguata, Natural regeneration as a tool for large‐ scale forest restoration in\nthe tropics: prospects and challenges. Biotropica 48, 716-730\n(2016). https://doi.org/10.1111/btp.12381\nR. Crouzeilles, M.S. Ferreira, R.L. Chazdon, D.B. Lindenmayer, J.B.B. Sansevero, L. Monteiro, A.\nIribarrem, A.E. Latawiec, B.B.N. Stassburg. Ecological restoration success is higher for natural\nregeneration than for active restoration in tropical forests. Science advances 3, e1701345 (2017).\nhttps://DOI: 10.1126/sciadv.1701345\nB. A. Williams, H.L. Beyer, M.E. Fagan, R.L. Chazdon, M. Schmoeller, S. Sprenkle-Hyppolite, B.W.\nGriscom, J.E.M. Watson, A.M. Tedesco, M. Gonzalez-Roglich, G.A. Daldegan, B. Bodin, D.\nCelentano, S.J. Wilson, J.R. Rhodes, N.S. Alexandre, D.H. Kim, D. Bastos, R. Crouzeilles, Global\npotential for natural regeneration in deforested tropical regions. Nature 636, 131–137 (2024).\nhttps://doi.org/10.1038/s41586-024-08106-4\nL. Poorter, F. Bongers, T.M. Aide, A.M. Almeyda Zambrano, P. Balvanera, J.M. Becknell, V. Boukili,\nP.H.S. Brancalion, E.N. Broadbent, R.L. Chazdon, D. Craven, J.S. de Almeida-Cortez, G.A.L. Cabral,\nB.H.J. de Jong, J.S. Denslow, D.H. Dent, S.J. DeWalt, J.M. Dupuy, S.M. Durán, M.M. Espírito-Santo,\nM.C. Fandino, R.G. César, J.S. Hall, J.L. Hernandez-Stefanoni, C.C. Jakovac, A.B. Junqueira, D.\nKennard, S.G. Letcher, J.-C. Licona, M. Lohbeck, E. Marín-Spiotta, M. Martínez-Ramos, P. Massoca,\nJ.A. Meave, R. Mesquita, F. Mora, R. Muñoz, R. Muscarella, Y.R.F. Nunes, S. Ochoa-Gaona, A.A. de\nOliveira, E. Orihuela-Belmonte, M. Peña-Claros, E.A. Pérez-García, D. Piotto, J.S. Powers, J.\nRodríguez-Velázquez, I.E. Romero-Pérez, J. Ruíz, J.G. Saldarriaga, A. Sanchez-Azofeifa, N.B.\nSchwartz, M.K. Steininger, N.G. Swenson, M. Toledo, M. Uriarte, M. van Breugel, H. van der Wal,\nM.D.M. Veloso, H.F.M. Vester, A. Vicentini, I.C.G. Vieira, T.V. Bentos, G.B. Williamson, D.M.A.\nRozendaal, Biomass resilience of Neotropical secondary forests. Nature 530, 211–214 (2016).\nhttps://doi.org/10.1038/nature16512\nOverall, I think this work is of high value scientifically. However, it failed to provide me\nwith enough convincing evidence for me to say it's time to put the nail in the coffin for P\nlimitation in mature tropical forests, especially when there isn’t any evidence of what the\n“other” limiting factor or element might be and the limited response variables considered.\nPerhaps this forest is growing as fast as it possibly can given the physiological\nconstraints of mature tropical hardwoods, or potentially the timescale of research isn't\nlong enough to capture increased growth given the slow rate of diameter growth.\nFurther, once a forest is mature and reaches the forest canopy, maybe it is more\nbeneficial to invest excess nutrients elsewhere, for example - turnover of leaves, or in\nreproduction. Younger leaves tend to have more nutrients and thus higher rates of\nphotosynthesis and with added nutrients the response might instead be reducing leaf\nlifespan and increasing turnover, which may not be captured in traditional measures of\ndiameter growth, or that less energy is put into resorption and more into other\ncomponents. The authors touch on these subjects, but I think other lines of evidence are\nneeded for the scientific community to fully rule out the importance of P in favor of\nsearching for an elusive \"other\" limiting element. The mystery of what drives mature\ntropical forest productivity remains, and ultimately, this work didn't move us closer to\nsolving it. Further, while the experimental design is very robust for this site in Panama\nand certainly a major undertaking, I tend to be very cautious when it comes to\ngeneralizing single tropical forest sites too broadly.\nFinally, as I mentioned above, I don’t think the management discussion works in this\npaper. The paper is focusing on evaluating nutrient limitation during natural succession,\nand to then translate this to how we grow trees and where in the tropics is well beyond\nthe scope, especially given the number of people that are actively working on\nreforestation projects.\nThank you again for reading our draft and provide so many constructive comments. We\nvery much appreciate that you recognize that our study has high scientific value. We\nbelieve we have vastly improved our manuscript in response to your feedback. We\nsummarize our responses to your conclusions below.\nOur goal was not to “put the nail in the coffin for P limitation.” It was to examine limitation\nat our site over a short time scale, recognizing many caveats that may prevent us from\ndetermining whether there is ultimate nutrient limitation on our site (discussed across\nlines 204-261). In fact, we believe that the possibility of lack of phosphorus limitation at\nour site adds to the growing body of evidence that nutrient limitation may be much more\nheterogeneous in the tropics than previously assumed, including that some sites are\nlimited by phosphorus, as some studies show. We discuss this in the paragraph of lines\n204-261, with new text on lines 204-213, 224-235, 245-260. We have added discussion\nthat higher growth of our forests seems biophysically possible since nearby forests have\nproductivity up to twice that of our forests (lines 228-233), although we agree with the\nreviewer that the timescale of our experiments may not be able to capture turnover in\ntree community, limiting our ability to completely out-rule limitation, especially in the\nolder forests (more completely discussed in lines 233-235). We agree with the reviewer\nthat it may be beneficial for trees in older forests to allocate carbon to other tissues\nrather than wood, and we discuss this in lines 216-217. We have taken the reviewer’s\nadvice and have provided even more caution in generalizing our study to other tropical\nsites broadly, including on lines 27-28, 34-36, 94-96, 200-204, 274-276. Finally, we\nappreciate the reviewer’s perspective, but respectfully disagree and believe we should\ninclude the management implications for multiple reasons outlined in our response to\ncomment #8."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This is the third round of reviews that I have conducted on this manuscript, and I believe it is now ready for publication. Compared to the previous revision, I believe the manuscript has improved further. The authors are more concise and specific, and they have toned down the generalisation of their results even further. They put a lot of work into analysing the available soil analytical data, especially the total phosphorus analyses, and I can accept the choices they made, even if I would have made different ones. The authors now correctly claim that Agua Salud soil are less weathered than Oxisols, but insist that the soils at Gigante are Oxisols and refer in their rebuttal to the classification made by Ben Turner in 2009. To be clear: I have no doubt that you can find Oxisols in Gigante, but I maintain that a substantial part is also less weathered as was shown in the Koehler et al (2009) paper. Actually, Ben Turner’s sampling in 2009 was a direct reaction to the Koehler et al. (2009) paper to prove that he was correct that the soils were Oxisols. And frankly, any soil scientist will know where to sample in the Gigante landscape if you want to find Oxisols. So, in the end the authors decide to make a different choice as I would have made. That does not make them more correct but I can accept this. Another problem the authors encountered is that, in general, phosphorus analytical data, including total phosphorus, are difficult to compare due to the variety of existing methods. In their response, the authors refer to digestion with nitric acid as the 'gold standard'. Perhaps that is true where they live, but it is not true where I am from. In my understanding, a method that measures 'total phosphorus' tries to measure as much phosphorus as possible in a sample, so if conducted correctly, a method that measures more is probably better for 'total' phosphorus than a method that fails to measure some of the phosphorus present. Nevertheless, I think we can now all agree that the Panama sites are not representative of the part of the Amazon basin with the most weathered soils. The fact that 25% of Amazonian forests have more total phosphorus than the Panama sites is not actually surprising, since a substantial proportion of the region is covered by soils that are less weathered than Oxisols (see Quesada et al., 2009). I was glad to see that the authors had now given serious thought to the effects of deforestation and slash-and-burn management on nutrient availability. Initially, I was surprised that they did not observe an effect of soil pH, base saturation or total phosphorus (P), but examining the soil chemical data from Gigante (from Ben Turner's sampling mentioned earlier) reveals that this is probably not so surprising, given that the soils in Gigante, even in areas classified as Oxisols, have a relatively high pH (above 5) and high base saturation (above 50% in the top 50 cm and above 80% in the topsoil). Given these initial conditions, it is not surprising that the impact of ash input from a few decades earlier was not measurable against these background values. I think these effects will be much clearer and long-lasting in sites where the original forest has much lower pH, base saturation and total P values. Nevertheless, I still expect that, under such conditions, the nutrient input from ashes following slash-and-burn will have a significant impact on the regrowth of secondary tropical forests and potential nutrient limitations. In summary, as I mentioned in my earlier review, the study presents new, noteworthy and exciting insights in shifts in nutrient limitation across a secondary succession gradient on soils at the high end of total phosphorus content. I recommend to publish is, as it will make an important contribution to the discussion on nutrient limitation of secondary tropical forests."}, "author_response": "Response:\nThanks again for sending our draft back to the same reviewers, and we appreciate\nthe reviewer’s feedback in vastly improving our manuscript in previous revisions."}]

Decision letter