The impact of insect herbivory on biogeochemical cycling in broadleaved forests varies with temperature

Domain: earth_science
Published: 2024-07-17
Editorial outcome: minor_revision

Abstract
Herbivorous insects alter biogeochemical cycling within forests, but the magnitude of these impacts, their global variation, and drivers of this variation remain poorly understood. To address this knowledge gap and help improve biogeochemical models, we established a global network of 74 plots within 40 mature, undisturbed broadleaved forests. We analyzed freshly senesced and green leaves for carbon, nitrogen, phosphorus and silica concentrations, foliar production and herbivory, and stand-level nutrient fluxes. We show more nutrient release by insect herbivores at non-outbreak levels in tropical forests than temperate and boreal forests, that these fluxes increase strongly with mean annual temperature, and that they exceed atmospheric deposition inputs in some localities. Thus, background levels of insect herbivory are sufficiently large to both alter ecosystem element cycling and influence terrestrial carbon cycling. Further, climate can affect interactions between natural populations of plants and herbivores with important consequences for global biogeochemical cycles across broadleaved forests.

Introduction
Herbivory is an important mediator of ecosystem nutrient cycling and occurs via transfer of labile nutrients from green leaves to the soil in primary production across biome types1,2. A wide diversity of herbivores shape the form, function, and biochemistry of plants, exhibiting deep and taxonomically diverse co-evolutionary linkages to plants3. The impacts of mammalian herbivores and those of a small group of insects that cause extensive but rare mass defoliation events have received significant attention4,5. However, a more cryptic, diverse, and extensive community of insects is responsible for near-continuous and ubiquitous background levels of herbivory. The seemingly minor contributions of background insect herbivory to ecosystem processes under non-outbreak conditions may be substantial over the long term and over large spatial scales, with ecosystem consequences that likely differ from the more charismatic yet sporadic outbreak events6. The magnitude of these impacts, the variation of these impacts across the world’s forests, and importantly for terrestrial ecosystem modeling, the drivers of this variation all remain poorly quantified6,7.

An expanded focus on insect herbivory is also warranted because they create important feedbacks between plants and soils mediated by a wide variety of mechanisms8. One key direct, immediate feedback occurs via transfer of labile nutrients from green leaves to the soil in the form of excreta, cadavers, leachate, unconsumed leaf fragments, and prematurely abscised leaves (Fig. 1)8,9. Relative to leaf litter, herbivory-related insect deposits are typically enriched with labile forms of nutrients, and in many forests, insect-mediated nutrient fluxes are comparable to or even exceed fluxes from other inputs of relatively labile, mineral forms of nutrients9,10. In contrast, most of the nutrients in leaf litter are resorbed and retained within plant biomass, or they are released in relatively recalcitrant forms11. When folivores alter the fluxes of limiting nutrients such as nitrogen (N) and phosphorus (P), they also have the potential to influence plant growth and ecosystem carbon (C) cycling11,12. Silicon (Si) is increasingly investigated in plant science research because silica enhances plant structural integrity, reduces the impact of stressors such as herbivory and drought, and correlates with C sequestration13,14. However, the biogeochemical dimensions of herbivory impacts on Si remain understudied15. Though global analyses of herbivory exist e.g.16, the flux of nutrients associated with insect herbivory and insect deposits remains poorly understood8, as is the potential impact of climate on these fluxes.

H1—The flux of N and P from plant to soil mediated by insect herbivores would meet or exceed other major labile fluxes of these elements, such as atmospheric deposition (for N and P) and bedrock weathering (for P).

H2—Insect-mediated element fluxes would increase with increasing mean annual temperature, decrease with increasing dryness (potential evapotranspiration/mean annual precipitation), and increase with increasing soil nutrient concentration.

H3—Observed responses of insect mediated element fluxes to MAT, dryness and soil nutrient concentrations would be driven in equal measure by similar responses from foliar herbivory, biomass production and foliar element concentrations.

Here, we show that background levels of insect herbivory can have profound impacts on biogeochemical cycling in broadleaved forests. For some localities, these fluxes exceed atmospheric deposition inputs, showing that background levels of insect herbivory are large enough to both alter ecosystem element cycling and, because primary productivity in most forests is nutrient-limited, influence terrestrial C cycling. Insect-mediated fluxes of N and P are especially high in tropical forests compared to temperate and boreal forests. Further, we show that all insect-mediated element fluxes increase strongly with MAT. These results reveal how climate can affect interactions between natural populations of plants and herbivores with important consequences for C and nutrient cycling across global broadleaved forest biomes.

Results
Forest characteristics across our global network of research plots
MAT across our global network of plots ranged from −1.4–26.9°C (Supplementary Table 2), which represents nearly the full range of mean annual temperature (MAT) for broadleaved trees on Earth. Similarly, dryness, represented by potential evapotranspiration/mean annual precipitation, ranged from 0.21–1.30, capturing a very broad range of moisture conditions for broadleaved forests (Supplementary Table 2). Mean soil element concentrations were 0.8–27.2% for C, 0.04–1.81% for N, 0.0004–0.2785% for P, and 0.1–24.7% for Si (Supplementary Table 2). Capturing most of the global range for foliar biomass production in broadleaved forests (Table 1, Supplementary Table 3, and Supplementary Fig. 2), the mean for tropical forests (539.3±42.6 gm−2 y−1) was 66% larger than temperate foliar biomass production (323.4±28.6 gm−2 y−1) and 240% greater that boreal forest foliar biomass production (158.8±47.6 gm−2 y−1). We found that foliar herbivory (henceforth herbivory) was widespread, with an average of 48.8±2.4% of all leaves globally showing some level of damage from folivores. Mean herbivory rate per leaf was 4.1±0.1% globally, with herbivory rate across plots ranging from 0.6±0.1% – 13.6±0.8% (Supplementary Table 2). Herbivory showed a strong gradient across latitude zones, ranging from a high of 5.4±0.2% in tropical forests, to 3.3±0.1% in temperate forests, to the low of 2.5±0.2% in boreal forests (Table 1, Supplementary Table 3 and Supplementary Fig. 2). Resorption efficiency for N was significantly greater in boreal (61.8±3.3%) than in tropical (46.5±2.7%) forests while resorption efficiency for P was significantly greater in tropical (66.5±2.1%) than in temperate (57.9±2.1%) or boreal (55.2±4.5%) forests (Supplementary Table 3 and Supplementary Fig. 2). Resorption efficiency for C globally was 19.7±0.4% (Supplementary Table 2).

Insect-mediated fluxes compared to other major sources of labile nutrients
Net insect-mediated element fluxes (Hi) exceeded that of some other labile element sources in some locations. The global mean for HiC was 1.7±0.1 gm−2 y−1, 0.17±0.02 gm−2 y−1 for HiN, and 0.014±0.002 gm−2 y−1 for HiP (Supplementary Table 2). HiN and HiP were greatest at tropical sites, averaging 68% of N deposited atmospherically, 440% of P deposited atmospherically, and 41% of estimated mean bedrock weathered P for the region (Table 2). In contrast, HiN and HiP were lowest at boreal sites, averaging 10% of the N deposited atmospherically, matching mean atmospheric P deposition rates, and 4% of mean bedrock weathered P for the region (Table 2). HiN was greater than the regional mean for atmospheric N deposition at 29% of the tropical plots but none of the temperate and boreal plots. HiP exceeded the regional mean of atmospheric P deposition at 73% of the tropical plots, 69% of the temperate plots, and 33% of the boreal plots.

Background levels of insect herbivory represented a small but persistent pathway for the return of foliar organic material and elements from broadleaved forest canopies to the soil (Table 2, Supplementary Table 2). In partial agreement with Hypothesis 1, net insect-mediated nutrient fluxes from this pathway exceeded that of atmospheric nutrient deposition in some localities (Table 1). For some sites, HiN and HiP exceeded that of atmospheric N (21% of locations) and atmospheric P (72% of locations). Insect-mediated N and P fluxes could have particular importance in systems that are limited by the availability of these nutrients. For example, in the tropics where soil P availability can be very low21,22, the relative importance of insect-mediated P could have greater implications for P cycling compared to other regions. That we found HiP and soil C:P to be greatest in tropical forests (Table 2, Supplementary Table 2) underscores the potentially important role of insect-mediated P fluxes in this zone. Further, HcP:Litter P was significantly higher in tropical forests than in either temperate or boreal forests. Resorption is a mechanism for addressing high soil P fixation, especially in acidic and heavily weathered soils22. Therefore, HiP might be more easily fixed by soil (into non-available inorganic forms), which over time would drive down P availability. Meanwhile, productivity especially in cooler regions is generally projected to increase as are forest disturbances (e.g., drought, fire) because of climate change23,24. Therefore, greater foliar biomass production and herbivory due to temperature increases could amplify the importance of some insect-mediated element fluxes in cooler forests (Supplementary Table 6).

Abiotic influences on insect-mediated element fluxes
When evaluating the effects of abiotic variables on insect-mediated fluxes, MAT consistently contributed significantly and positively to Hc and Hi models (Figs. 2 and 3, Supplementary Table 4). In addition, soil C:N (CI = −0.35, −0.04) and soil C:P (CI = −0.47, −0.11) negatively correlated with HcN and HcP, respectively, while soil Si concentration (CI = 0.10, 0.48) correlated positively with HcSi (Figs. 2 and 3). PET/MAP was not a significant contributor to variation in insect-mediated element fluxes at the global level (Fig. 2, Supplementary Table 4), but significantly explained variance of HcN and HcP between tropical forests and HcN between temperate forests (Supplementary Table 5). Although annual contributions of elements (gm−2 y−1) derived from litter exceeded that of Hc overall and especially in boreal forests, mean HcP:Litter P in tropical forests was 1.8 times greater than that of temperate forests and 3.2 times greater than that of boreal forests (Supplementary Table 3).

Relative to other abiotic factors and in line with Hypothesis 2, MAT exerted the strongest positive influence on insect-mediated element fluxes (Fig. 2). Therefore, plant-insect herbivore interactions tend to interrupt efficient plant recycling of elements to a greater degree in warmer locations. We expect that insect-driven increases in the release of labile nutrients (insect frass, cadavers, greenfall, leachate, early abscised leaves) to the soil would exert strong effects on soil fertility and biogeochemical cycling, with the extent of these effects dependent on site-specific characteristics8,9. We might also expect greater insect-mediated element fluxes to result from climate change-driven increases in insect outbreaks33, which would amplify trends in insect-mediated fluxes from background herbivory. Soil C:N, soil C:P, and soil Si concentrations played additional roles for Hc, in support of Hypothesis 2, and also indicates that insect folivores move more nutrients in locations with nutrient-rich soils (Fig. 2, Supplementary Table 5). In partial support of Hypothesis 2, PET/MAP and soil nutrient concentrations played significant roles for some insect-mediated nutrient fluxes within some latitude zones (Supplementary Table 5). Contrary to Hypothesis 2, however, PET/MAP did not have a significant effect on insect-mediated nutrient fluxes under non-outbreak conditions at the global level (Fig. 2). That the effects of PET/MAP and soil nutrient concentrations were inconsistent between scales and between latitude zones suggests that local to regional conditions can have an outsized influence over insect-mediated element fluxes (Fig. 2, Supplementary Table 5). For example, PET/MAP positively correlated with HcN and HcP in forests within the tropical zone (Supplementary Table 5), suggesting that overly wet conditions can reduce herbivory34 and thus insect-mediated nutrient fluxes. Solar radiation and water availability can also be important drivers of insect activity during outbreaks, with droughts, which are typically local in scale, weakening tree defenses35,36. Finer scale measurements of temporal and spatial patterns such as seasonal and interannual precipitation or aspect may uncover differences in insect-mediated element fluxes generated by microclimatic variation37,38. High-resolution, locally sourced climate data might also better reveal potential roles of seasonal or more localized interannual water stress on insect-mediated element fluxes.

Biotic influences on insect-mediated element fluxes (Hc and Hi)
In estimating relative contributions of biotic variables to insect-mediated element fluxes, we found that foliar biomass production and herbivory explained more variation in Hc and Hi than did foliar element concentrations for C, N and P (Fig. 2, Supplementary Table 6). In contrast, foliar Si concentrations explained more variation in HcSi than foliar biomass production or herbivory. Resorption efficiency played a smaller role than foliar biomass production or herbivory in explaining variation in Hi.

Because leaves in this study were passively collected in litter traps of a known area at the end of the leaf’s lifespan, we were able to avoid some biases in estimating herbivory related to timing (e.g., snapshot versus repeated), scale (e.g., single versus multiple species), and selection (e.g., systematic versus haphazard) described in prior syntheses6. Our 4.12% global mean for foliar herbivory (Supplementary Table 2) is consistent with the 4.30% global estimate for herbivory of woody plant leaves in an extensive meta-analysis16 when herbivory data were collected in a “blinded way” where leaf collectors were unaware of the hypothesis being tested. However, the variation of herbivory across our plots was substantial, with leaf area loss ranging from 0.6±0.1% to 13.6±0.8% (Supplementary Table 3).

Links between abiotic variables and biotic precursors to fluxes
Soil C:N (CI = −0.48, −0.18) was negatively correlated with foliar biomass production, whereas MAT (CI = 0.47, 0.83) was positively correlated with foliar biomass production (Figs. 2 and 3). Tropical forests (Table 1) produced more foliar biomass than temperate forests, which produced more foliar biomass than boreal forests (F(2, 71) = 17.71, P<0.001). Only MAT (CI = 0.20, 0.67) positively correlated with herbivory globally (Figs. 2 and 3). Tropical forests (Table 1) exhibited greater herbivory than temperate or boreal forests (F(2, 71) = 5.22, P=0.008). While soil C:N (CI = −0.44, −0.00) and soil Si concentration (CI = 0.09, 0.62) were correlated with their respective foliar nutrient concentrations (Fig. 2), foliar P concentration positively correlated with MAT (CI = 0.10, 0.66; Fig. 3). Foliar N concentrations in tropical forests (Supplementary Table 3) were lower than those in temperate and boreal forests (F(2, 71) = 11.73, P=0.003). Foliar P concentrations (Supplementary Table 3) were also significantly lower in tropical than in temperate or boreal forests (F(2, 71) = 26.80, P<0.001). MAT (Figs. 2 and 3) positively correlated with N resorption efficiency (CI = 0.14, 0.62) while PET/MAP was negatively correlated with N resorption efficiency (CI = −0.48, −0.03). N resorption efficiency (Supplementary Table 3) was significantly greater in boreal forests than in tropical or temperate forests (F(2, 70) = 12.07, P=0.002) whereas P resorption was significantly greater in tropical forests than in temperate or boreal forests (F(2, 70) = 14.05, P<0.001). Soil C:P (Fig. 2) was positively correlated with P resorption efficiency (CI = 0.08, 0.44), which was significantly greater in tropical than in temperate or boreal forests (F(2, 70) = 6.79, P=0.002; Supplementary Table 3). Foliar Si concentration was positively and weakly correlated with foliar biomass production (F(1, 67) = 4.68, R2 = 0.05, P=0.034) but was not significantly correlated to herbivory.

MAT and soil C:N explained 49% of the variability in foliar biomass production, where higher MAT and lower soil C:N were associated with higher foliar biomass production (Fig. 2). Additionally, only MAT helped to explain herbivory among variables we studied (Fig. 2). We might expect then that Hc and Hi would be greatest in the tropics, where foliar biomass production, herbivory and MAT are the greatest and soil C:N is the lowest (Table 1, Supplementary Fig. 2). The effects of abiotic factors on foliar element concentrations were mixed, where soil element concentration played a significant role in explaining variability in foliar element concentrations for all elements except C, and MAT positively correlated with foliar P concentration (Figs. 2 and 3). The results of this investigation are in line with a study on Quercus garryana which determined that climate and leaf traits explained variation in plant-herbivore interactions37. Ultimately, different plant life forms and species are likely to exhibit different strategies to cope with herbivory based on local conditions37,38.

Interestingly, foliar nutrient content did not significantly correlate with herbivory in this study, indicating that nutrients measured in plants may not correspond to what is available to insect herbivores. For example, leaf age can affect leaf toughness and thus nutrient extraction efficiency41. Further, improving plant quality can benefit early stages of insect development but may not improve insect survivorship or escape from natural enemies41. Alternatively, other variables such as MAT (Figs. 2 and 3) or defense strategies not explored in this study (e.g., volatiles, leaf toughness) may have played greater roles than foliar nutrient concentrations in explaining variability in herbivory39. Interactions between nutrients and other variables could also explain the negative relationship between foliar nutrient concentrations and herbivory. For example, because P availability is generally lower in tropical biomes where soils are more weathered21, low foliar P concentrations could simply be incidental to local nutrient availability in warmer climates where herbivory is greatest. Our study suggests that both broad-scale variables and local-scale conditions, as well as plant traits may all simultaneously shape plant-herbivore interactions. Future work would be needed to disentangle these effects.

Resorption efficiency was highest in tropical broadleaved forests while N resorption efficiency was highest in boreal forests, supporting the broadly appreciated and supported perspective that P limits tree growth in the tropics while N limits growth in higher latitudes (Supplementary Table 3)22,42. Furthermore, though soil C:N did not differ appreciably across biomes, soil C:P in tropical forests was significantly higher than in temperate and boreal forests, highlighting again the potential importance of labile compounds derived from insect herbivory in nutrient-limited systems (Supplementary Table 3). Our results are consistent with a recent meta-analysis on global-scale patterns of nutrient resorption of woody plants in which N resorption efficiency significantly decreased with MAT whereas P resorption efficiency marginally increased with MAT (Figs. 2 and 3)43. As with insect-mediated element fluxes, this suggests that although the extent of nutrient resorption efficiency might be regulated by local factors such as substrate43, large-scale variables such as MAT can drive broad spatial patterns of ecosystem processes.

We investigated the effects of entire naturally occurring insect assemblages in mature, undisturbed broadleaved tree communities to develop an integrated description of insect herbivore-mediated element fluxes in tropical, temperate and boreal forests. We found compelling evidence that MAT plays a strong global role in insect-mediated element fluxes. The complexity embodied in this study, which includes an extraordinary diversity of plants representing a tremendous range of defense strategies interacting with diverse insect communities across sites with highly variable resource conditions, makes the generalization more remarkable. These results provide contemporary baseline data to better inform Earth system modeling on the myriad interactions among herbivores, plants, and soil. Next generation models could integrate these different processes and predict the overall effects of herbivores on ecosystem structure and function over longer time scales44,45.

Methods
Study sites
We established a network of 74 measurement plots in mature, undisturbed forests at 40 sites across 6 continents (Supplementary Data 1). In the global network (Supplementary Fig. 1), 16 of the sites (33 plots) are tropical, 18 sites (32 plots) are temperate, and 6 sites (9 plots) are boreal46, representing 12 of the 14 major broadleaved forest types17.

The method used to assess stand-level herbivory and element flux relies on the visual estimation of leaf area removal from passively collected leaves10. Visual quantification of incremental removal from needle shaped leaves was not possible, so we focused our measurements on sites dominated by broadleaved tree species. That we examined only broadleaved-dominated forests also provided some level of constraint on the global study. To minimize the confounding influence of disturbance history, we set up plots in mature forest stands where there was no documented evidence or visible indications of recent human activity. All of the forests in this study could be classified as primary or old-growth forests47. Of the 74 forest plots, 68 plots spanned an area of 1 ha each, and 64 plots were situated at least 450 m from major anthropogenic disturbances such as major roads or settlements (Supplementary Data 1). During site selection, we also consulted local experts and literature for information on current and past natural (e.g., insect outbreaks) and anthropogenic (e.g., logging) histories (Supplementary Data 1). Plots ultimately included in the analysis did not show evidence of recent insect outbreak activity. Plots were not randomized, and some plots were in site clusters along elevation or precipitation gradients (Supplementary Data 1).

Green leaf, leaf litter and soil collections
To characterize green leaf nutrients, we sampled and pooled 150 or more (to obtain at least 25 g dry weight) green, healthy (not to minimal signs of damage or discoloration) leaves at multiple heights in the canopy from at least ten randomly selected trees during the growing season for deciduous forest plots, and every dry and wet season for evergreen forest plots. We dried all green leaves at 70°C until constant mass and finely ground them for chemical analysis.

We installed 9–25 litter traps (0.1–0.5 m2 in area) in each of the 74 plots such that leaf litter traps were 0.3–1.0 m above the surface of the ground and spaced at ~20-m intervals (one site exception to interval distance is described in Supplementary Data 1). We collected litter for 1 or 2 years (between 2018 and 2021, see two site exceptions to year of collection described in Supplementary Data 1); for each plot, litter was collected every 14–31 days. In cases where 2 years of data were collected, we calculated an annual mean for regression analyses. We dried all leaves at 70°C until constant mass and weighed them to estimate total leaf litter fall as gm−2 y−1. We then subsampled leaves from each litter trap and pooled ~160 leaves or more by plot (to obtain at least 25 g dry weight), which we then finely ground for chemical analysis.

To characterize soil, we homogenized ten cores (0–15 cm depth) once during the growing season at each plot. After sieving through 2 mm mesh, we dried soils at 65–70°C until constant mass and ground them for chemical analysis. We report the soil nutrient concentrations for C and Si, and the soil stoichiometric ratios C:N and C:P derived from soil nutrient concentrations.

Chemical analyses
For total C and N analyses, we combusted finely ground and homogenized green leaf, litter, and soil samples in an elemental analyzer at Copenhagen University, Denmark (Flash 2000, Thermo Scientific, Bremen, Germany). To estimate total P, we calculated the difference between the amounts of inorganic P extracted by sulfuric acid for ignited and unignited samples using an autoanalyzer (Seal AA500 Continuous Flow Analyzer; Seal Analytical GmbH, Norderstedt, DE) at Copenhagen University, Denmark48. To obtain Si concentrations, we digested samples in a sodium carbonate solution for 3 h (leaves) and 5 h (soils) before analyzing them with a SmartChem© 200 Discrete Analyzer (AMS Alliance, KPM Analytics, Westborough, MA, USA) at Lund University, Sweden49. In the case of the Malaysian and Brazilian sites, soil and foliar C, N, and P concentrations in this study were derived from previously published estimates (Supplementary Data 1).

Estimates of foliar herbivory
We scanned and visually assessed insect damage to half of all collected broadleaved litter before processing for chemical analysis using a classification system described by Alliende50. A single observer scored insect damage according to six levels of leaf area removal by herbivores (0-1%, 1–5%, 5–25%, 25–50%, 50–75%, and >75%) from quarterly scans of leaf litter. This method for visually estimating damage and assessing leaf area consumption is efficient and accurate6,51, with resulting estimates of herbivory from abscised leaves providing a metric of leaf-level insect defoliation accumulated over the entire lifespan of leaves. The approach provides an unbiased, community-level estimate of background level foliar herbivory.

Calculations of foliar biomass production and insect herbivore-related element fluxes
To estimate gross and net insect-mediated release of elements from the canopy via foliar consumption, we used the approach described in Metcalfe et al.10. Specifically, we calculated this release as the product of: (i) foliar biomass production (FP), (ii) green leaf element concentration (FE), (iii) foliar herbivory rate (H), and (iv) element resorption (RE, for net release estimates of C, N and P), where subscript E could be the elements C, N, P, or Si (Fig. 1).

We estimated annual leaf production at each plot using 1 or 2 years of litterfall collections and calculated nutrient fluxes due to insect defoliation from these estimates. We converted the total dry mass of leaf litter accumulated over the year divided by known trap area (LH, gm−2 y−1) to annual foliar biomass production (FP) calculated as LH/(1-H), where H is the proportion of leaf litter removed by insect folivores. We converted FP to element production with foliar element concentration (FE) data. FE was then multiplied by H, yielding total leaf elements consumed by insect herbivores (Hc), or the gross insect-mediated element flux (see Supplementary Table 1 for equations, based on methods described in Metcalfe et al.10). We assume Hc is approximately equal to the quantities of the same elements released by herbivores to the ground via excreta, bodies, moults, and unconsumed leaf fragments under steady-state assumptions, which should be reasonable for these relatively intact mature forests where herbivore populations, and the balance between ingoing and outgoing migratory herbivores, should be relatively stable over time.

To further understand the potential importance of the herbivory-mediated fluxes resulting from insects intercepting green leaves before resorption (Fig. 1), we also estimated resorption efficiency (% withdrawal of an element during senescence) of C, N and P following Vergutz et al.52. That is, we utilized mass loss correction factors 0.78 and 0.784 for predominantly evergreen angiosperm forests and predominantly deciduous angiosperm forests, respectively52. Using this approach, we assumed nominal nutrient leaching from litterfall traps between collections and that herbivores exclusively target foliage before initiation of resorption. By combining Hc with plot-level resorption estimates for C, N and P, we estimated this net insect-mediated element flux (Hi)—defined here as the additional C, N or P released via green leaf herbivory from plants to soil prior to senescence-related resorption (Fig. 1; Supplementary Table 1). That is, Hi is the difference in total (litter + herbivore-mediated) nutrient inputs between a scenario with herbivores and a scenario without herbivores (Fig. 1). Hc is the total amount of a foliar element consumed and ultimately released to the ecosystem by herbivores. If we assume that leaves abscise at the end of their lifetime after resorption has occurred, and that the vast majority of herbivory occurs before this resorption26, then Hi is designed to calculate the foliar elements released by herbivores that would not otherwise have been released via litterfall. For this reason, Hi is more closely comparable to external inputs of relatively labile, inorganic nutrients derived from atmospheric deposition, biological fixation or bedrock weathering. In this analysis, we do not distinguish between C allocated to biomass versus respiration by herbivores because this allocation is poorly constrained for most geographies, biome types, and herbivore groups10,53. We report only gross (Hc) and net (Hi) C removed from the foliage by herbivory.

Estimating other major sources of labile nutrients
To compare the amounts of elements passing through insect folivores with other major sources of labile nutrients, we derived atmospheric N and P-values from models developed by Brahney et al.54 and we estimated mineral-weathered P-values from the model described by Hartmann et al.55.

Climate variables
To obtain air temperature estimates, we installed TMS-4 data loggers (Tomst S.R.O, Czech Republic) at the plots during the collection period. In case of gaps in temperature data, we supplemented our dataset with other local measurements (e.g. on-site instruments from other researchers, local weather station data), scientific literature, and consulted with local experts to finalize MAT estimates (Supplementary Data 1). Dryness or climate ratios56,57 depict the ratio of potential evapotranspiration (PET) to mean annual precipitation (MAP). We derived mean annual PET at each plot after summing the monthly means (Penman-Monteith method) for each year during the 2018–2021 collection period using the dataset from Singer et al.57. In the few cases where collections were made during different times (2017 for the Malaysian site, 2010 for the Brazilian site), we obtained PET for their associated years57. We consulted local weather station data, scientific literature, and local experts to estimate MAP for all plots (Supplementary Data 1).

Statistics and reproducibility
We applied linear mixed models to assess global patterns in insect herbivore-mediated fluxes across latitude using R programming language v. 4.2.1 computer58. To rule out severe multicollinearity, we ensured that variance inflation factors (vif function in the car v. 3.1–1 package) for all predictors fell below 2 before proceeding with each model59. We fitted the linear mixed models in the lme4 v. 1.1–030 package60, where site or site:plot was treated as a random factor for all models to account for unique geographical and floristic characteristics. Because we did not detect a significant difference in herbivory across latitude between northern and southern hemispheres in this dataset, we converted latitude to absolute values. We first determined the effect of abiotic variables on biotic variables with simple models (single fixed variable + site as random factor). The main variables used to construct full models (all fixed variables + site as random factor) are summarized in Table 3. Before fitting the full models with correlated variables from simple regressions, we produced standardized regression coefficient values for the global analysis by converting all variables to z scores across each dataset after log- or logit-transforming variables as necessary. However, we could not fully resolve moderate unequal variances in the full model for foliar biomass production61. We constrained 95% confidence intervals (CI) of the effect sizes with 1000 parametric bootstrap simulations to obtain a conservative estimate of significance62 and considered effects significant when CI did not cross zero. We report marginal and conditional R2GLMM values as goodness-of-fit statistics using the MuMIn v. 1.47.1 package63. We constructed pathway diagrams between abiotic, biotic precursor, and insect-mediated element fluxes to summarize results of the linear mixed models and depict potential causal relationships.

To test for differences in variable means between latitude zones, we performed two-way Kruskal-Wallis non-parametric tests (kruskal.test function in stats v. 4.2.1 package) followed by Dunn pairwise comparisons with Bonferroni-Holm adjustments (dunnTest function in FSA v. 0.9.5 package).

Though observations in this study were likely dependent on spatial and temporal context and were thus difficult to reproduce, computational reproducibility can be achieved by using the same datasets, codes and software as this study. As a result of shared workflow, transparency allows for other researchers to build directly on this primary work.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study addressed the variations of nutrient fluxes associated with background-level\ninsect herbivory in global broadleaved forest ecosystems with standardized methods.\nAuthors reported that mean annual temperature is an important abiotic factor in driving the\ninsect herbivory mediated nutrient fluxes across tropical, temperate, and boreal forests.\nWhile I found the results are meaningful, I have several concerns on this work.\nThe first hypothesis (H1) is not appropriate. There are many nutrient fluxes in forest\necosystems. While insect-mediated nutrient flux is important, it cannot exceed all other\nmajor fluxes. For example, the nutrient absorption of plants from soil will be much higher\nthan any other fluxes. At least, it is not clear for the statement ‘other major fluxes’ in H1.\nFor the second hypothesis (H2), authors did not provide enough background information.\nFor example, the core words ‘temperature’, ‘dryness’, and ‘soil nutrient concentration’\nactually did not present in the earlier paragraphs of the Introduction section.\nFor the third hypothesis (H3), it is clearly that this is a hypothesis based on the results.\nLine 1 As this study collected samples from broadleaved forests, it is important to show the\ninformation of ‘broadleaved’ in the title.\nLine 96 Many ecosystem processes would be altered by herbivorous. It would be better to\nclearly state which process this study will focus on.\nLine 104 It is not clear for the meaning of ‘Insect-mediated nitrogen fluxes’\nLine109-111 The findings from large spatial scale, such as this study, would have limited\nimplications for the temporal changes of temperature at local scale. The same process (as\ninsect herbivory in this study) would be controlled by quite different ecological factors at\nglobal and local scales, and between temporal and spatial scales.\nLine 143 What do you mean by ‘deposition’?\nLine 228 Table 2. Please check the value and the unit of mineral weathered P. Are more than\n50 g P m-2 being mineralized annually for all the forests averaged globally?\nFigure 2. P values for the regressions should be shown in the figure.\nLine 289-290 and Figure 2d, If foliar N was higher in tropical forests than other two forest\ntypes (Line 289-290), why Fn was negatively correlated with MAT as shown in Figure 2d?\nThe similar problem with Fp (figure 2d and Lines 290-291).\nLine 306-307 The role of insect-mediated fluxes is overstated, as authors only compared\nthat with one flux (atmospheric nutrient deposition) in Table 1. Actually, only in 20%\nlocation the insect-mediated N fluxes was higher than atmospheric N deposition, that\nmeans insect mediated N flux in most sites was much lower than N deposition.\nLine 315-316 Reference is needed for this statement.\nLine 352-353 the impacts of in situ warming would be hardly inferred from space-time\nsubstitution.\nLine 480 broadleaved forests", "reviewer_2": "This is a very interesting study where leaf samples from forests all over the world were\ncollected and analyzed for chemical composition and herbivory. Soil chemical analysis was\nalso performed and the impact of \"background herbivory\" on element cycling was\ncalculated. The amount of data is impressive and I consider that the message that\n\"background herbivory\" has a significant impact on element cycling is important and\ninteresting for a broad audience.\nI am not an expert on insect herbivory effects on element cycling in forests but i consider\nthis work original. Overall the work supports the conclusions but I have a few points that can\nbe addressed in a possible revision.\nFirst, the confidence intervals for figure 3, (named figure 3 erroneously) are extremely small.\nThis seems wrong as they are supposed to be 95% C.I. according to the legend. also, the R2\nis very low in a number of graphs and i wonder whether these relationships are significant. it\nis not presented.\nSecond, I have a question about the calculations. both the Hc and Hi are based on\ncalculations, and not measured in real life. What conclusions can be drawn from the Hi\nvalues, if these are calculations, and do you also have information from some sites based on\nactual nutrient cycling data? Herbivory now in the SEM leads to Hc and Hi, but because this\nis based on calculations, with the same data, in particular the Hi link is questionable I think.\nThird, leaf N is measured after drying at 70%, but it is well described that the N\nconcentration is reduced when drying at such high temperature, and that N is usuallyt\nmeasured after drying at lower temperatures, max 60 C i believe.\nLine 185 says: we anticipate that soil nutrient concentration (SE) remains unchanged with H,\neven as element cycling increases. I wonder if this is true. If i understand it correctly,\nnutrient cycling is thought to be affected but it does not change the actual concentration in\nthe soil, ie all the nutrients are taken up? Why is this true and why is this important. Please\nexplain.\nMinor comment: values in Appendix D are not presented in percentages (e.g. for leaf N)\neven though it says so in the table"}, "author_response": "Reviewer #1 (Remarks to the Author):\n(Comment) This study addressed the variations of nutrient fluxes associated with background-\nlevel insect herbivory in global broadleaved forest ecosystems with standardized methods. Authors\nreported that mean annual temperature is an important abiotic factor in driving the insect herbivory\nmediated nutrient fluxes across tropical, temperate, and boreal forests. While I found the results\nare meaningful, I have several concerns on this work.\n(Response) We thank the editor and reviewers for their generally positive appraisal of the\nimportance and broad interest of the manuscript topic, and their comments on the text. We have\nnow revised the manuscript in response to these comments, which has further improved the\nmanuscript. We believe that the resulting manuscript provides important insights on herbivores\nthat will guide further research and help refine biogeochemical modelling efforts.\n(Comment) The first hypothesis (H1) is not appropriate. There are many nutrient fluxes in forest\necosystems. While insect-mediated nutrient flux is important, it cannot exceed all other major\nfluxes. For example, the nutrient absorption of plants from soil will be much higher than any other\nfluxes. At least, it is not clear for the statement ‘other major fluxes’ in H1.\n(Response) Indeed, there are numerous internal ecosystem fluxes of nutrients. In contrast to\nlitterfall, many other fluxes are derived from relatively recalcitrant organic forms (e.g., soil organic\nmatter) so flux rates from these pools are low. As a result, they have less potential to affect\nplant/ecosystem processes, relative to insect derived inputs, which like the typically larger litterfal\nflux, tend to be in more labile forms (Lovett et al., 2002). To clarify, we have revised H1 to specify\nthe fluxes to which we compare studied insect-mediated fluxes: “H1 – The flux of N and P from\nplant to soil mediated by insect herbivores would meet or exceed other major labile fluxes of these\nelements, such as atmospheric deposition (for N and P) and bedrock weathering (for P). (Lines\n162-164) We have also mentioned these specific fluxes earlier in the introduction: “Furthermore,\nrelative to leaf litter, herbivory-related insect deposits are typically enriched with labile forms of\nnutrients, and in many forests, insect-mediated nutrient fluxes are comparable to or even exceed\n1\nfluxes from other inputs of relatively labile, mineral forms of nutrients such as atmospheric\ndeposition or bedrock weathering9,10.” (Lines 134-137)\nLovett, G.M., Christenson, L.M., Groffman, P.M., Jones, C.G., Hart, J.E. and Mitchell, M.J., 2002.\nInsect defoliation and nitrogen cycling in forests: laboratory, plot, and watershed studies indicate that\nmost of the nitrogen released from forest foliage as a result of defoliation by insects is redistributed\nwithin the ecosystem, whereas only a small fraction of nitrogen is lost by leaching. BioScience, 52(4),\npp.335-341.\n(Comment) For the second hypothesis (H2), authors did not provide enough background\ninformation. For example, the core words ‘temperature’, ‘dryness’, and ‘soil nutrient\nconcentration’ actually did not present in the earlier paragraphs of the Introduction section.\n(Response) We have now introduced the terms “mean annual temperature”, “dryness”, and “soil\nnutrient concentration” to the existing background information earlier in the introduction. The\nsection now reads: “High nutrient availability, warm temperatures, and low water stress can\npositively influence foliar nutrient concentrations, foliar biomass production, and insect abundance\nwith positive synergistic effects on overall insect-mediated element fluxes 17,18,19. Consequently,\nwarm mean annual temperatures and high soil nutrient concentrations could increase insect\nherbivory and thus insect-mediated element fluxes in nutrient-limited but not water-limited (i.e.,\nlow dryness) systems.” (Lines 155-160)\n(Comment) For the third hypothesis (H3), it is clearly that this is a hypothesis based on the results.\n(Response) We have now revised this hypothesis to clarify our reasoning, and how it is\nindependent of the findings in H1 and H2. New text in the introduction describes how the\nprecursors to H /H tend to respond in the same direction to warming/moisture/fertility (Lines 155-\nc i\n160). H3 aims to assess how each of the precursors to H/H respond to these factors, which help\ni c\nto explain the broader results of H2. The new text outlining H3 is as follows: “Observed responses\nof insect mediated element fluxes to MAT, dryness and soil nutrient concentrations would be\ndriven in equal measure by similar responses from foliar herbivory, biomass production and foliar\nelement concentrations.” (Lines 168-170)\n(Comment) Line 1 As this study collected samples from broadleaved forests, it is important to\nshow the information of ‘broadleaved’ in the title.\n2\n(Response) We thank the reviewer for the title suggestion. We have altered the title to read:\n“Temperature mediates insect herbivore impact on biogeochemical cycling in broadleaved\nforests”. (Lines 1-2)\n(Comment) Line 96 Many ecosystem processes would be altered by herbivorous. It would be\nbetter to clearly state which process this study will focus on.\n(Response) We have replaced “ecosystem processes” with “biogeochemical cycling”. (Line 97)\n(Comment) Line 104 It is not clear for the meaning of ‘Insect-mediated nitrogen fluxes’\n(Response) We have replaced “Insect-mediated nitrogen fluxes” with “Nitrogen that was released\nfrom plants by insect herbivores”. (Lines 104-105)\n(Comment) Line109-111 The findings from large spatial scale, such as this study, would have\nlimited implications for the temporal changes of temperature at local scale. The same process (as\ninsect herbivory in this study) would be controlled by quite different ecological factors at\nglobal and local scales, and between temporal and spatial scales.\n(Response) There is a long and well-established history of using latitudinal variation to make\ninferences about ecosystem responses to temperature (e.g., De Frenne et al., 2013). However, we\nacknowledge that the primary strength and focus of our data lies in documenting and explaining\ncontrols over global biogeographical patterns in ecosystem processes, rather than inferring\ntemporal change. We have rephrased the abstract and other relevant sections of the text to make\nthis clearer (Lines 325-327, 354-355). The relevant section in the abstract has been revised to the\nfollowing: “Further, these results show how climate affects interactions between natural\npopulations with important consequences for biogeochemical cycles.” (Lines 110-112)\nDe Frenne P, Graae BJ, Rodríguez‐Sánchez F, Kolb A, Chabrerie O, Decocq G, De Kort H, De\nSchrijver A, Diekmann M, Eriksson O, Gruwez R. Latitudinal gradients as natural laboratories to infer\nspecies' responses to temperature. Journal of Ecology. 2013 May; 101(3):784-95.\n(Comment) Line 143 What do you mean by ‘deposition’?\n(Response) To avoid confusion, we have chosen to replace “deposition” with “insect deposits”\nfrom the sentence which now reads: “Though global analyses on herbivory existe.g.,16, the flux of\n3\nnutrients associated with insect herbivory and insect deposits is poorly understood8, as is the\npotential impact of climate on these fluxes.” (Lines 143-145)\n(Comment) Line 228 Table 2. Please check the value and the unit of mineral weathered P. Are more than\n50 g P m-2 being mineralized annually for all the forests averaged globally?\nFigure 2. [sic] P values for the regressions should be shown in the figure.\n(Response) We thank the reviewer for pointing out the error; we have made the units correction\nin Table 2. (Line 237) We have also added p-values and other regression statistics to Fig. 3. (Line\n314)\n(Comment) Line 289-290 and Figure 2d, If foliar N was higher in tropical forests than other two\nforest types (Line 289-290), why Fn was negatively correlated with MAT as shown in Figure 2d?\nThe similar problem with Fp (figure 2d and Lines 290-291).\n(Response) We thank the reviewer for pointing out these discrepancies. We have corrected the\ntext to read: “Foliar N concentrations in tropical forests (Appendix D) were lower than those in\ntemperate and boreal forests (F = 11.73, P = 0.003). Foliar P concentrations (Appendix D)\n(2, 71)\nwere also significantly lower in tropical than in temperate or boreal forests (F = 26.80, P <\n(2, 71)\n0.001).” (Lines 401-404)\n(Comment) Line 306-307 The role of insect-mediated fluxes is overstated, as authors only\ncompared that with one flux (atmospheric nutrient deposition) in Table 1. Actually, only in 20%\nlocation the insect-mediated N fluxes was higher than atmospheric N deposition, that means insect\nmediated N flux in most sites was much lower than N deposition.\n(Response) Indeed, we have claimed only partial agreement with our hypothesis. However, we\nhave rephrased the sentence to further avoid overstating our results: “In partial agreement with\nHypothesis 1, net insect-mediated nutrient fluxes from this pathway exceeded that of atmospheric\nnutrient deposition in only some localities (Table 1).” (Lines 241-243)\n(Comment) Line 315-316 Reference is needed for this statement.\n(Response) We have added reference 22 to the statement. (Line 252)\n4\n(Comment) Line 352-353 the impacts of in situ warming would be hardly inferred from space-\ntime substitution.\n(Response) This section has been revised in response to your earlier, related comment. While there\nis a long and well-established history of using latitudinal variation to make inferences about\necosystem responses to temperature (De Frenne et al., 2013), we acknowledge that the primary\nstrength and focus of our data lies in documenting and explaining controls over global\nbiogeographical patterns in ecosystem processes, rather than inferring temporal change. We have\nnow revised relevant text in this section to make this clearer, as follows: “Therefore, plant-insect\nherbivore interactions tend to interrupt, otherwise highly efficient, plant recycling of elements to\na greater degree in warmer locations.” (Lines 325-327)\nDe Frenne P, Graae BJ, Rodríguez‐Sánchez F, Kolb A, Chabrerie O, Decocq G, De Kort H, De\nSchrijver A, Diekmann M, Eriksson O, Gruwez R. Latitudinal gradients as natural laboratories to infer\nspecies' responses to temperature. Journal of Ecology. 2013 May; 101(3):784-95.\n(Comment) Line 480 broadleaved forests\n(Response) We thank the reviewer for pointing out this typo. The sentence now reads: “Visual\nquantification of incremental removal from needle shaped leaves was not possible, so we focused\nour measurements on sites dominated by broadleaved tree species.” (Lines 474-476)\nReviewer #2 (Remarks to the Author):\n(Comment) This is a very interesting study where leaf samples from forests all over the world\nwere collected and analyzed for chemical composition and herbivory. Soil chemical analysis was\nalso performed and the impact of \"background herbivory\" on element cycling was calculated. The\namount of data is impressive and I consider that the message that \"background herbivory\" has a\nsignificant impact on element cycling is important and interesting for a broad audience.\nI am not an expert on insect herbivory effects on element cycling in forests but i consider this work\noriginal. Overall the work supports the conclusions but I have a few points that can be addressed\nin a possible revision.\n5\n(Response) We thank the reviewer for a generally positive appraisal of the breadth of work and\ninterest as well as the importance of the manuscript topic. Our responses to specific comments\nfollow.\n(Comment) First, the confidence intervals for figure 3, (named figure 3 erroneously) are extremely\nsmall. This seems wrong as they are supposed to be 95% C.I. according to the legend. also, the R2\nis very low in a number of graphs and i wonder whether these relationships are significant. it is not\npresented.\n(Response) We thank the reviewer for pointing out the discrepancy. We have changed the figure\nname from Fig. 2 to Fig. 3 and presented all regression statistics on individual plots to clarify\ninputs and significant differences. To prevent confusion, we removed the standard error bars from\nthe plot means and depict 95% confidence intervals around the regressions (74 plots). We also\nindicate in the caption that both the means and the confidence intervals are at the 74-plot level.\n(Lines 321-323)\n(Comment) Second, I have a question about the calculations. both the Hc and Hi are based on\ncalculations, and not measured in real life. What conclusions can be drawn from the Hi values, if\nthese are calculations, and do you also have information from some sites based on actual nutrient\ncycling data? Herbivory now in the SEM leads to Hc and Hi, but because this is based on\ncalculations, with the same data, in particular the Hi link is questionable I think.\n(Response) We did not directly measure H and Hi in this study. Instead, H (gross insect-mediated\nc c\nelement flux) was calculated by direct measurements of foliar nutrient concentration, herbivory,\nand litterfall (to estimate foliar production) from all 74 plots in this study. H represents the\ni\nadditional input of elements to the soil released by insects consuming green leaves prior to\nresorption and was calculated by estimating the flux of elements with and without herbivory.\nResorption efficiency and litterfall were also directly measured from every plot to derive estimates\nof H. It is true that the biotic precursors are not fundamentally independent of Hi and H since\ni c\nthey are included in the calculations used to derive H and H. The purpose of including them in\nc i\nthe SEM is to test H3 – the relative amount and direction of effects that each biotic precursor exerts\non the estimates of H and H. We now clarify this in the text and captions (Lines 168-170, 187-\nc i\n188).\nc i\nthe methods to motivate the importance of this distinction (Lines 188-189, 561-569). “H\nc\nquantifies the total amount of foliar elements consumed and ultimately released to the ecosystems\nby herbivores. However, to some extent these elements would have been released anyway to the\necosystem when the leaves abscise. If we assume that leaves abscise at the end of their lifetime\nafter resorption has occurred, and that the vast majority of herbivory occurs before this\nresorption26, then H is designed to calculate the foliar elements released by herbivores that would\ni\nnot otherwise have been released via litterfall. For this reason, H is more closely comparable to\ni\ntruly external inputs of relatively labile, mineral nutrients from atmospheric deposition, biological\nfixation and bedrock weathering.” (Lines 557-565)\n(Comment) Third, leaf N is measured after drying at 70%, but it is well described that the N\nconcentration is reduced when drying at such high temperature, and that N is usuallyt measured\nafter drying at lower temperatures, max 60 C i believe.\n(Response) Although drying at 60 degrees C is also a reasonable option, drying plant material for\nnutrient analysis at 70 degrees C is a well-cited and standard method in plant nutrient studies:\nWolf B. A comprehensive system of leaf analyses and its use for diagnosing crop nutrient status.\nCommunications in Soil Science and Plant Analysis. 1982 Jan 1; 13(12):1035-59.\nLitton CM, Giardina CP, Selmants PC, Sparks JP. Impact of mean annual temperature on nutrient\navailability in a tropical montane wet forest. Frontiers in Plant Science. 2020 Jun 12; 11:500586.\n(Comment) Line 185 says: we anticipate that soil nutrient concentration (SE) remains unchanged\nwith H, even as element cycling increases. I wonder if this is true. If i understand it correctly,\nnutrient cycling is thought to be affected but it does not change the actual concentration in the soil,\nie all the nutrients are taken up? Why is this true and why is this important. Please explain.\n(Response) Given that this is not tested in our study and represents a small part of the figure, we\nhave chosen to remove the sentence, “While not tested here, we anticipate that soil nutrient\nconcentration (SE) remains unchanged with H, even as element cycling increases.” (Lines 189-\n190)\n7\n(Comment) Minor comment: values in Appendix D are not presented in percentages (e.g. for leaf\nN) even though it says so in the table\n(Response) Thanks to the reviewer for pointing out this error. We have converted proportions to\npercentages in Appendix D. (Line 870)\n8"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Lines 147-152 The information for the time and frequency of measurement should be given.\nDid you measure one time in a particular year in each plot? Or, did you measure the\nvariables several times in a whole year, and then quantify the annual fluxes?\nLine 241-247 I challenge the assertion here. While the insect mediated nutrient flux is\nquantitatively larger than nutrient deposition in some areas, that does not mean the insect-\nmediated nutrient would be important for the nutrient balance or soil availability in those\nareas. For any ecosystem, nutrient deposition represents the external nutrient sources. In\ncontrast, insect-mediated nutrient is the internal nutrient, without any external inputs for\nthe ecosystem. All (or most of) the nutrients mediated by insects exist in the ecosystem,\neven if there is no insect in the ecosystem. Insects do not provide any new nutrient for the\necosystem. If there is no insect, such amount of nutrients would be present in other parts of\nthe ecosystem, such as the litters.\nLines 261-263 This case study from European Russia was based on boreal forest, which is\nquite different from broadleaved forest. Thus, the lower annual loss rate led by insect\noutbreak in boreal forest does not provide strong evidence for the importance of\nbackground levels of insect herbivory in broadleaved forests.\nLine 292 the comma should be moved after the ()\nLine 315 Using ‘P’ instead of ‘PValue’ in all the figures\nLine 488 ‘leaf litter’ instead of ‘litter leaf’\nLine 548 Authors have reported the data of C resorption. The method used to calculate\ncarbon resorption should be added.", "reviewer_2": "I have read the revision and the questions i had have all been answered. I have no further\nquestions. As stated before, i find this an interesting and important study and recommend\npublication in nature communications. The topic addressed and the insights merit\npublication in this top journal in my opinion."}, "author_response": "We thank the reviewers for continued support of our manuscript and additional comments. We have\nresponded to new comments from the reviewers below.\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\n(Comment) Lines 147-152 The information for the time and frequency of measurement should be given.\nDid you measure one time in a particular year in each plot? Or, did you measure the variables several\ntimes in a whole year, and then quantify the annual fluxes?\n(Response) Thank you for pointing out this need for clarification in the introduction. We have tried to\nclarify our methods without being verbose in the following way: “We used standardized methods to\nregularly collect and analyze green and freshly senesced leaves throughout the growing season for one\nor two years at each plot for: (i) foliar biomass production, (ii) foliar herbivory, and (iii) foliar C, N, P and\nSi concentrations. We then used these measures to calculate annual stand-level element fluxes\ngenerated during leaf consumption by entire, natural communities of insect herbivores under non-\noutbreak conditions10.” (Lines 150-155)\n(Comment) Line 241-247 I challenge the assertion here. While the insect mediated nutrient flux is\nquantitatively larger than nutrient deposition in some areas, that does not mean the insect-mediated\nnutrient would be important for the nutrient balance or soil availability in those areas. For any\necosystem, nutrient deposition represents the external nutrient sources. In contrast, insect-mediated\nnutrient is the internal nutrient, without any external inputs for the ecosystem. All (or most of) the\nnutrients mediated by insects exist in the ecosystem, even if there is no insect in the ecosystem. Insects\ndo not provide any new nutrient for the ecosystem. If there is no insect, such amount of nutrients would\nbe present in other parts of the ecosystem, such as the litters.\n(Response) The statement here refers to H1 which makes the distinction not between internal or\nexternal fluxes, but between labile and recalcitrant fluxes (Lines 163-165). We have now carefully\nscreened the text throughout to ensure that this comparison is clear (Lines 133-138, 155-156, 234-236,\n283-285, 520-522, 572-577). We believe this is a more ecologically relevant comparison because there is\nample ecological evidence that most ecosystem nutrients occur in relatively inaccessible organic forms,\nand that ecosystems respond not to overall nutrient abundance, but to the abundance of labile,\nplant/microbe accessible nutrients (Walker & Syers, 1976; Vitousek & Reiners, 1975; Reiners et al., 1981;\nMenge et al., 2012). So, while it is true that the presence of insects will not alter the total quantity of\nnutrients in a system (soil and plants), this metric is poorly related to ecosystem processes. Instead, we\nargue (Lines 130-140, 178-193) that the important effects of herbivorous insects derive from altering\npathways of nutrients within the system and the relative proportion of labile versus recalcitrant\nnutrients. There is ample evidence that herbivores produce more labile material than the leaves they\nconsume (Bardgett & Wardle, 2010; Hartley & Jones, 2008; Frost & Hunter, 2004; Kaukonen et al.,\n2013). We thus believe that the more appropriate fluxes to compare herbivore mediated fluxes with are\nother labile fluxes such as deposition and weathering.\nFurthermore, insect herbivory can impact host-specific nutrient conservation by transferring foliar\nnutrients to soil before translocation mechanisms can conserve nutrients for the host species. This\nshortcutting hurts host species while making a large pool of nutrients available to other species,\nespecially beneficial to species not impacted by herbivory (Bardgett et al., 1999). This redistribution of\nnutrients might either increase diversity if host species are dominant (i.e., herbivory enhances nutrient\nstatus of less common species) or reduce diversity if impacted species are rare. Insect herbivores thus\nhave the capacity to influence plant community composition, which in turn affects decomposer activity\nand consequently the supply of nutrients to plants from the soil (Hartley & Jones, 2008; Bardgett &\nWardle, 2010).\nBardgett, R. D., Cook, R., Yeates G. W., Denton, C. S. The influence of nematodes on below-ground processes in\ngrassland ecosystems. Plant Soil 212, 23-33 (1999).\nBardgett, R. D. & Wardle, D. A. Aboveground–belowground Linkages: Biotic Interactions, Ecosystem Processes, and\nGlobal Change (Oxford University Press, Oxford, 2010).\nFrost, C. J. & Hunter, M. D., Insect canopy herbivory and frass deposition affect soil nutrient dynamics and export\nin oak mesocosms. Ecology, 85, 3335-3347 (2004).\nHartley, S. E. & Jones, T. H. Insect Herbivores, Nutrient Cycling and Plant Productivity. In Insects and Ecosystem\nFunction Ch. 2 (Springer–Verlag, Heidelberg, 2008).\nKaukonen, M., Ruotsalainen, A. L., Wäli, P. R., Männistö, M. K., Setälä, H., Saravesi, K., Huusko, K. & Markkola, A.\nMoth herbivory enhances resource turnover in subarctic mountain birch forests? Ecology, 94, 267-272 (2013).\nMenge, D\nMenge, D. N., Hedin L. O., Pacala, S. W. Nitrogen and phosphorus limitation over long-term ecosystem\ndevelopment in terrestrial ecosystems. PLoS One 7, e42045 (2012).\nReiners, W. A. Nitrogen cycling in relation to ecosystem succession. Ecol. Bull., 33, 507-528 (1981).\nVitousek, P. M. & Reiners, W. A. Ecosystem succession and nutrient retention: a hypothesis. BioSci, 25, 376-56\n(1975).\nWalker, T. W., & Syers, J. K. The fate of phosphorus during pedogenesis. Geoderma, 15, 1-19 (1976).\n(Comment) Lines 261-263 This case study from European Russia was based on boreal forest, which is\nquite different from broadleaved forest. Thus, the lower annual loss rate led by insect outbreak in boreal\nforest does not provide strong evidence for the importance of background levels of insect herbivory in\nbroadleaved forests.\n(Response) We thank the reviewer’s evaluation of this statement. We have decided to remove these\nlines from the manuscript because the preceding text and references are sufficient to make the point.\n(Comment) Line 292 the comma should be moved after the ()\n(Response) Thank you for pointing out this typo. We have removed the comma after the parentheses.\n(Line 304)\n(Comment) Line 315 Using ‘P’ instead of ‘PValue’ in all the figures\n(Response) We have replaced “PValue” with “P” in the micrographs. (Line 326)\n(Comment) Line 488 ‘leaf litter’ instead of ‘litter leaf’\n(Response) We have replaced “litter leaf” with “leaf litter” in the subheading. (Line 497)\n(Comment) Line 548 Authors have reported the data of C resorption. The method used to calculate\ncarbon resorption should be added.\n(Response) Thank you for noticing this oversight. We have added methods of calculating C resorption in\nthe text alongside existing methods of calculating N and P resorption. (Lines 559-569)\nReviewer #2 (Remarks to the Author):\n(Comment) I have read the revision and the questions i had have all been answered. I have no further\nquestions. As stated before, i find this an interesting and important study and recommend publication in\nnature communications. The topic addressed and the insights merit publication in this top journal in my\nopinion.\n(Response) We thank the reviewer for continued support of the study."}]

Decision letter