Climate warming and elevated CO_2 alter peatland soil carbon sources and stability

Domain: earth_science
Published: 2023-11-20
Editorial outcome: accept

Abstract
Peatlands are an important carbon (C) reservoir storing one-third of global soil organic carbon (SOC), but little is known about the fate of these C stocks under climate change. Here, we examine the impact of warming and elevated atmospheric CO2 concentration (eCO2) on the molecular composition of SOC to infer SOC sources (microbe-, plant- and fire-derived) and stability in a boreal peatland. We show that while warming alone decreased plant- and microbe-derived SOC due to enhanced decomposition, warming combined with eCO2 increased plant-derived SOC compounds. We further observed increasing root-derived inputs (suberin) and declining leaf/needle-derived inputs (cutin) into SOC under warming and eCO2. The decline in SOC compounds with warming and gains from new root-derived C under eCO2, suggest that warming and eCO2 may shift peatland C budget towards pools with faster turnover. Together, our results indicate that climate change may increase inputs and enhance decomposition of SOC potentially destabilising C storage in peatlands.

Introduction
The capacity of peatlands to store more than one-third of the global SOC pool under waterlogged conditions1,2 has been touted as a powerful natural form of C sequestration3. However, there is concern that rising global temperatures and atmospheric CO2 concentrations, along with corresponding changes in hydrology and biology, have the potential to destabilize SOC stocks and increase the flux of CO2 and CH4 from peat soils to the atmosphere2,4, amplifying the drivers of climate change. Yet processes regulating the formation and stability of peat SOC, and their responses to interactive climate change factors remain unclear5,6, making it difficult to forecast changes in C dynamics in peatland ecosystems7. In peatlands, SOC stability is controlled by a suite of environmental conditions, including temperature, hydrology (water saturation), and pH8,9, and biotic factors such as plant and microbial community composition and functioning10,11 as well as the chemical characteristics of the peat itself12,13. These biotic and abiotic factors regulate the current rates and pathways of enzymatically catalysed SOC decomposition8,10, and they are expected to determine the response of SOC decomposition under future environmental change5,7.

A key question for future peatland C balance is whether a portion of SOC that is currently stable may become more accessible to microbial decomposition2,4,7, via direct effects of rising temperature and CO2 concentration, indirect effects of altered water table9,14,15, or due to changes in plant production and thus litter inputs to SOC11,16,17. Soil warming, occurring more rapidly at high compared to low latitudes18,19, can lead to lower water-tables and increases in peat aeration9,15. Changes in moisture availability combined with the fertilization effects of elevated CO2 can alter plant biomass production and the quality of litter inputs10,16,20. Such a change in peat aeration and plant litter (including root exudates) inputs can impact the microbially-mediated SOC turnover because soil water content and litter chemistry regulate microbial community composition, function, and enzymatic activity8–11. Differences in litter chemistry can also affect the sensitivity of organic matter decomposition to soil temperature, with further effects on C cycling10,21. Consequently, if altered organic matter sources persist over extended periods, they are likely to stimulate the mineralisation of currently stable C22. In short, climate-driven changes in water-table and plant litter inputs are likely to modify C storage5,6, but the underlying mechanisms and their interactions, particularly those resulting in shifts in organic matter inputs and composition are still unknown in peatlands7. The role of such plant-soil interactions in regulating future SOC storage merits attention because a much larger proportion of SOC may be susceptible to climate-mediated losses than previously assumed7,19.

Here, we investigate the effects of warming and elevated atmospheric CO2 concentration (eCO2) on peatland SOC molecular composition to infer SOC sources, stability, and implications for storage. To describe SOC dynamics, we partitioned bulk soil C into classes of molecular compounds comprising SOC defined operationally by organic matter origins (plant-, microorganism- and fire-derived) and potential decomposition rates23–25. Specifically, we targeted SOC compounds with distinct turnover times. Firstly, we targeted solvent-extractable compounds (alkanoic acids, alkanols, alkanes, steroids, and terpenoids, derived from plant- and microbial-material26) that are thought to turn over predominantly on a timescale of bulk SOC or faster24,27. Secondly, we targeted SOC components from different plant origins, hydrolysable biopolymers distinct to either leaf/needle (cutin) or root/bark (suberin) compartments (and presumably slowly cycling)26–29. Lastly, we targeted lignin and pyrogenic carbon (PyC) representing the most slowly cycling C23,30–32. We assessed the stability of these SOC compound classes following 4 years of warming and 2 years of eCO2 at an in-situ climate manipulation experiment (SPRUCE: Spruce and Peatland Responses Under Changing Environments). The SPRUCE experiment provides a powerful climate change gradient whereby above- and below-ground warming (whole-ecosystem warming; +0, +2.25, +4.5, +6.75, +9 °C above ambient) are crossed with ambient and elevated CO2 concentrations (+500 ppm above ambient)33. Given that high-latitude soils are projected to experience temperature increases of up to 8.3 ± 1.9 °C by 2100 under a high CO2 emissions scenario (RCP8.5)19, this unique experimental setup allowed us to explore the fate of SOC across mild to extreme future warming scenarios. From the perspective of long-term peatland C sequestration, we are particularly interested in the response of polymeric SOC components because kinetic theory predicts that decomposition and turnover of complex SOC molecules (greater chemical stability) should be more sensitive to temperature changes than simple molecules34. In peatlands, warming is typically accompanied by a lower water-table coupled with peat aeration, which can further impact the apparent temperature sensitivity of organic matter decomposition8,9,14,15. Consequently, if temperature and oxygenation effects predominate over other biotic and abiotic factors that influence decomposition dynamics9,25,34, we hypothesize that complex SOC molecules such as lignin, and PyC that have been proposed to turn over much slower than bulk C in soils due to their polyaromatic chemical structure23,30,35, would degrade faster. Overall, our study rejects this hypothesis that complex SOC molecules are more responsive to temperature changes. Instead, we found that all molecular compounds comprising SOC, regardless of source and complexity, were vulnerable to shifts in climate drivers, demonstrating the high sensitivity of peatlands to climate change.

Results
Warming and eCO2 have divergent effects on plant- and microorganism-derived SOC
First, we assessed how whole-ecosystem warming under ambient CO2 modified SOC molecular composition. Four years of experimental warming decreased SOC molecules content in the surface peat (0–30 cm; Fig. 1; Fig. S2). Solvent-extractable compounds of plant and microbial origins26,27,29 declined by 30% between the 0 and 9 °C treatments which corresponds to a slope of -0.79 ± 0.2 mg g-1 per °C increase in temperature (r2 = 0.60, p = 0.0004; Fig. 1a, Fig. S2a). Similarly, plant-derived hydrolysable biopolymers that presumably cycle more slowly26,28,36, decreased by 14% between the 0 and 9 °C treatments, corresponding to -0.65 ± 0.2 mg g-1 per °C increase in temperature (r2 = 0.52, p < 0.0001; Fig. 1b, Fig. S2b), likely due to accelerated microbial decomposition of soil C4,37. Surprisingly, the concentration of lignin phenols, which are made up of phenolic units that are expected to decompose faster under aerobic conditions8,9,15, instead increased significantly in the surface peat by 12% between the 0 and 9 °C treatments (0.51 ± 0.2 mg g-1 per °C warming; r2 = 0.18, p = 0.01; Fig. 1d, Fig. S2d), indicating that warming might have induced lignin stabilization. Indeed, it is plausible that more lignin phenols entered soils via increased root litter production or that a shift in plant species composition toward shrubs richer in these compounds16,17 may explain this increase. It is also possible that this apparent increase in lignin phenols may be due to accelerated decomposition of more rapidly cycled labile C given that warming has increased the availability of labile sugars (and protein) in this experiment38. In contrast, ratios of commonly used lignin oxidation (degradation) indices; acid-to-aldehyde ratios of vanillyl and syringyl phenols, which typically serve as proxies for oxidation32, increased linearly with increasing temperatures (Fig. 2a-c), suggesting that warming, as expected, induced lignin degradation. The observed relative increase in lignin phenols (Fig. 1d; Fig. S1) thus reflects a combination of increased organic matter inputs via plant litter and root exudates, enhanced degradation of more labile SOC molecules induced by higher temperatures16,38 and a better resilience of these molecules to warming as compared to the more labile ones.

Overall, the above findings indicate that warming stimulated a loss of SOC molecules, irrespective of their origins and potential decomposition rates – although lignin phenols showed a lower response than expected. These transformations likely reflect extensive decomposition driven by low water tables during summer dry periods17, compounded by warm temperatures and increased above- and below-ground plant productivity16,17,20, and labile C inputs to SOC38,39. Warming is typically accompanied by a lower water-table and enhanced peat aeration, which can affect the temperature sensitivity of organic matter decomposition8,9,14,15. Here, warming resulted in a substantial water level drawdown during summer dry periods (30 cm below hollow surface in warmer plots)17. Since soil microbial activity is intrinsically temperature sensitive5,40, the lowered water table, together with additional plant litter and labile C inputs10,16,17,38 potentially stimulated microbial SOC transformation (priming), leading to measurable loss in SOC components.

We then assessed how warming combined with an elevated atmospheric CO2 concentration (eCO2) modified key SOC molecules. We tested the hypothesis that eCO2 would result in increased plant litter production and C transfer from plant litter into SOC, thereby buffering warming-induced SOC losses as temperatures rise17,41,42. Our results support this hypothesis and provide evidence that the interaction of warmer temperatures and eCO2 led to increased plant C inputs to SOC in the surface peat, potentially offsetting the warming-induced C losses (Fig. 1; Fig. S2b). We observed that concentrations of hydrolysable biopolymers under eCO2 increased by 18% between the 0 and 9 °C treatments (0.86 ± 0.3 mg g-1 per °C warming; r2 = 0.42, p < 0.0001; Fig. 1b; Fig. S2b) (indicating plant contribution), despite a non-significant increase in the concentration of solvent-extractable compounds (from 19.8 ± 0.9 mg g-1 in the +0 °C plot to 23.3 ± 1.1 mg g-1 in the +9 °C plot; p > 0.05; Fig. 1a). The source of hydrolysable biopolymers could be roots, as evidenced by elevated concentrations of root-specific suberin monomers (Fig. 3b) and root litter production16. Surprisingly, the concentration of lignin phenols decreased linearly with increasing temperatures (-0.38 ± 0.2 mg g-1 per °C warming; r2 = 0.36, p = 0.004; Fig. 1d), raising possibilities of enhanced incorporation of other SOC compounds (i.e., hydrolysable biopolymers) into soil, leading to the decrease (dilution) of lignin phenols. Previous work at the study site demonstrated increased above- and belowground vascular plant biomass in response to warming and eCO2 treatments16,17,20. As the plant litter input increases, so do the concentrations of SOC components in our results (Fig. 1; Fig. S2b), which suggest that warming and eCO2 could stimulate SOC incorporation via increased plant litter inputs17,42, whereas general impacts on C storage remain unclear. However, it is plausible that our observed increase in SOC components under eCO2 reflect a transient adjustment period to the 2 years of eCO2 treatment, whereby plant and microbial growth and traits, and decomposition rates have not yet reached a new equilibrium43. Alternatively, more rapid C cycling likely occurred within extant SOC components41, although an increase in SOC turnover appear to have been offset by additional plant productivity16,17. Therefore, we posit that SOC will be partially preserved under warmer temperatures and greater CO2 concentration in the short-term, though this may change in the future.

The above responses in SOC components to warming (only) and warming plus eCO2 were much greater in the surface peat (0–30 cm) than at deeper depths (>30 cm; deep peat) (Figs. 1, 3; Fig. S5-6). We observed a rapid turnover of SOC molecules in the surface peat (Figs. 1–3), a high degree of transformation that likely reflects a combination of altered plant C inputs and decomposition driven by low water tables during summer dry periods, compounded by warmer temperatures (Fig. 4). Warming was accompanied by a substantial water level drawdown during summer dry periods (30 cm below hollow surface in warmer plots)17 in our study, and water table depth was correlated with SOC molecules (Fig. 4), suggesting that the warming and eCO2 effect on SOC components was influenced by water level and downstream effects of peat oxygenation such as increased nutrient availability44. However, it is worth noting that previous studies have not yet observed a direct effect of warming on soil moisture in this experiment17. Four years into the treatment, deep anaerobic peat appears to be stable (Fig. S5-6) likely due to the slower decomposition that occurs under anoxic conditions2,9 and low-quality C substrates in these layers13,35. It is also plausible that the deep anaerobic peat underwent extensive decomposition when it was part of the surface peat (1580-9200 BP)45, creating peat that is less susceptible to the changes that we observed in the surface layers. Together, our results suggest that warming and eCO2 induce depth-dependent SOC responses, which should be carefully assessed to improve our mechanistic understanding and predictions of SOC dynamics under changing climate.

Warming and eCO2 enhanced the contribution of root-derived C inputs into SOC
To further delineate the contribution of plant litter inputs into SOC, we assessed changes in the incorporation and degradation of biopolymers distinct to either leaf/needle (cutin) or root/bark (suberin) compartments26–29 in the surface peat. The concentration of cutin monomers decreased linearly with increasing temperatures under both ambient CO2 and eCO2 (by ~20% between the 0 and 9 °C treatments, corresponding to -0.03 mg g-1 per °C warming) (r2 = 0.23, p = 0.004 and r2 = 0.18, p = 0.01 respectively; Fig. 3a; Fig. S3a). By contrast, the concentration of suberin monomers increased linearly with increasing temperatures in both ambient CO2 and eCO2 plots (by ~30% between the 0 and 9 °C treatments, corresponding to 0.19 mg g-1 per °C warming) (r2 = 0.36, p = 0.0003 and r2 = 0.46, p < 0.0001 respectively; Fig. 3b; Fig. S3b). Thus, warming and eCO2 led to the loss of needle/leaf-derived C and gain of root-derived C in these soils (Fig. 3; Fig. S3). Importantly, root-derived C (inferred from suberin monomers) was positively correlated with SOC, fine root biomass and water table depth (Fig. 4) and fine root biomass significantly increased under warming and eCO2 (Table S1)16, implying increased root-derived C inputs within the surface peat. In corroboration of our results, litter manipulation experiments showed root-derived organic matter to be a source of SOC with greater relative stability and longer turn-over times28, whereas leaf-derived C was found to be turned over more rapidly in (mineral) soils27,36,46. Our study provides evidence that future climate change may lead to increased retention of root-derived inputs into SOC and a decline in leaf-derived C inputs in ecosystems such as boreal forested peatlands. As warming is likely to be accompanied by the decimation of peat-forming mosses and increases in fine-root biomass in peatland ecosystems16,47, it is likely that root-driven accrual of SOC will represent an important mechanism for a continued C sequestration in these ecosystems in the short-term. However, the extent to which root-derived C can be incorporated and stabilized into deeper peat layers will depend on the depth at which root inputs increase. Thus, while we show that warming and eCO2, in the short term, enhanced incorporation of root-derived C into peat soils; it remains to be seen what the longer-term fate of this root-derived C will be.

There is no inherently stable peat SOC
We observed a rapid turnover of complex SOC molecules (such as lignin, cutin and suberin) and plant- and microbe-derived SOC components under warming-only and warming plus eCO2 (Figs. 1, 3; Fig. S2), supporting the paradigm already observed in mineral soils that there is no inherently stable SOC24,25. Complex SOC constituents such as lignin have traditionally been considered to cycle more slowly (on a multi-decadal time scale) relative to bulk SOC23,32. However, the expectation that the enzymatic breakdown of complex SOC molecules is more sensitive to temperature in comparison to simple molecules34, based in part on the kinetic theory, is not consistent with our results. We show rapid turnover of SOC molecules irrespective of their origins and potential decomposition rates (Fig. 1; Fig. S2). This high degree of transformation likely reflects extensive decomposition driven by low water tables17 and was compounded by warm temperatures along with increased labile organic matter38 and plant inputs (Fig. 4). Our results are consistent with previous studies reporting a decline in aromatic compounds and soluble aromatics (indicative of lignin and pyrogenic C) in the water-extractable organic matter with warming and eCO2 38,39. These results combined, show that microbial decomposition dynamics in this peatland are controlled by a range of biotic and abiotic ecosystem properties and not only by the kinetics of individual enzymatically catalysed reactions24,25.

Unsurprisingly, molecules derived from historical pyrolysis (fire-derived carbon or pyrogenic C; PyC) were unaffected by warming or eCO2 (Fig. 1c; Fig. S2c). We did not observe significant changes in PyC concentrations under ambient CO2 or eCO2 (p > 0.05; Fig. 1c; Fig. S4a), despite the peat soils containing PyC above global averages (~1.4% of the SOC; Fig. S1)48. PyC did show an increasing trend with warming under eCO2 (Fig. 1c; p > 0.05) probably due to previous biomass burning and incorporation of residues before the SPRUCE experiment49. While these results are expected given that PyC is viewed as ‘highly resistant organic matter’23,30,32, recent evidence suggests that the increased stability of these SOC components does not guarantee long-term persistence23. To the best of our knowledge, no previous in-situ peatland manipulation studies have reported on the fate of PyC under changing climate drivers.

Collectively, our study provides evidence that rising temperatures and atmospheric CO2 levels will have cascading effects on the turnover rates and controls of SOC compounds that dictate peatland C storage. Specifically, climate-induced warming and associated water table drawdown may increase oxygen availability and accelerate microbial decomposition of SOC. We also highlight a rapid turnover of complex SOC molecules in response to warming and eCO2, supporting the paradigm that SOC stability depends on ecosystem properties24,25. Thus, SOC decomposition and turnover are predominantly determined by environmental and belowground plant-input constraints rather than temperature sensitivity of individual molecules25. While our results indicate that ecosystem responses at SPRUCE are largely driven by surface peat, it remains to be seen whether a system shift characterized by more rapid C cycling to deeper depths in the peat column will be created by extended water-table drawdown and associated changes in plant and microbial community composition4,16,17,38 with further warming. Furthermore, we do not know whether the long-term SOC balance will be affected by plant–soil interactions or changes in soil microbial communities as they adapt to warmer temperatures5. Therefore, it is important to complement our results with long-term and time-resolved analyses. We also note that the results described here do not necessarily reflect the expected or observed responses for other peatland habitats. Nonetheless, the environmental gradients of SPRUCE provide us with model functions and parameters that can be applied to other similar peatland systems.

Methods
Site description
The Spruce and Peatland Response Under Changing Environments (SPRUCE) experiment is located at the southern boundary of the boreal region on the Marcell Experimental Forest in northern Minnesota, USA (47°30'20.5“N, 93°27'12.6“W; http://mnspruce.ornl.gov/). Mean annual temperature and precipitation is 3.4°C and 780 mm respectively49. The bog is ombrotrophic with peat depths of ~3 m (which accumulated over the last 11,000 years), with a pH ranging from 4.1 at the surface to 5.1 at 2 m depth45,49. The bog is dominated by overstory trees, Picea mariana (black spruce) and Larix laricina (larch), and ericaceous shrubs, Rhododendron groenlandicum and Chamaedaphne calyculata, and bryophyte layer, primarily Sphagnum mosses. This ombrotrophic bog has a perched water table which fluctuates about 10–20 cm above the hollows after snowmelt, receding deeper later in the growing season17,45.

Experimental design and sample collection
The SPRUCE experimental design is described in detail by Hanson, et al.33. Briefly, the experiment consists of ten octagonal transparent open‐top enclosures of 12 m diameter and 7 m height. Whole-ecosystem warming is maintained at a series of increasing temperatures (regression design) to five levels (+0, +2.25, +4.5, +6.75 and +9 °C) down to a depth of ~3 m using concentric arrays of electrical resistance heaters installed in peat and forced air warming. Peat warming was initiated between June and July 2014, following 3 months of a gradual treatment equilibration. Air warming was established in August 2015, thereby achieving whole ecosystem warming. In June 2016, eCO2 treatment was introduced to the duplicate warming enclosures completing the planned experimental setup in this project. The eCO2 treatment consists of elevating the local ambient atmospheric CO2 concentration by +500 ppm (~900 ppm, with δ13C–CO2 isotope value of ~54‰).

In August 2018, we randomly collected two soil cores from each of the plots in hollow microtopography where the surface of the hollow was defined as 0 cm. Surface samples (0–30 cm) were cut using a stainless-steel knife and extracted by hand, while a Russian peat corer was used to sample deeper peat (30–200 cm). Once collected, duplicate cores from the same plot were sectioned by depth (into 10 cm increments over 0 to 50 cm depth and 25 cm intervals from 50 to 200 cm), homogenized and combined to form a mixed sample. Dominant vascular plants and Sphagnum mosses were also randomly collected from each plot. Peat and plant samples were stored frozen at -20°C immediately after sampling until further analyses. To avoid disturbance within the treatment plots, roots were collected from the dominant plants outside the enclosures and additionally picked from the soil manually. Peat sections and plant materials were later freeze-dried to constant weight. We sieved dried peat samples through a 5 mm mesh to remove larger litter fragments (2 mm sieve would have caused a loss of peat moss biasing the results). A sub-sample of the sieved peat and plant material was ground using a ball mill (MM400, Retsch, Haan, Germany) and then analysed for C concentration by an elemental analyser-isotope ratio mass spectrometer (EA-IRMS, Thermo Fisher Scientific, Bremen, Germany). Overall, SOC concentration increased with increasing depth from 44.0% at the surface to 52.5% at 2 m depth, but there was no effect of temperature or eCO2 on the SOC concentration43. Additionally, investigations of SOC stock at the site so far have shown that the mean effect of warming on soil C stocks was indistinguishable from zero17. Our observations are not contradictory with this results since the specific compounds we followed represent 20% of the total SOC (see Fig. S1) and should be seen as tracers of SOC. Bulk density measurements were taken from soil cores taken from each of the experimental plots in 2013 and 2020. Soil cores 5.2 cm in diameter were carefully excavated using a Russian corer and soil bulk density was calculated using the freeze-dried weights of the volumetric slices. Bulk density was used to estimate the mass of SOC molecular components (stocks) in the top 30 cm (g m-2). We calculated the mass of SOC molecular components by multiplying bulk density value (g soil cm-3) by concentrations of individual SOC compounds (solvent-extractable compounds, hydrolysable biopolymers, lignin phenols and pyrogenic C) (g g peat-1) from each peat depth and summarized the values for the mentioned depth intervals.

Solvent-extractable compounds
Solvent-extractable lipids (alkanes, and alkanoic acids) in these soils have been previously reported43. Here, we extend our previous study by including alkanols, steroids and terpenoids in our analysis in order to assess the stability of total solvent-extractable lipids (herein, solvent-extractable compounds) with varied degradability. These soils were analysed using the protocol reported in Wiesenberg and Gocke50. Briefly, ~1 g of plant materials and ~2 g of milled peat was extracted using Soxhlet extraction with dichloromethane (DCM):methanol (MeOH) (93:7; v/v). The extracts were sequentially separated into low-polarity and acid (alkanoic acid) fractions using a KOH-coated (5%) silica gel column by eluting with DCM, and DCM:formic acid (99:1; v/v), consecutively. The low-polar fractions were further separated into aliphatic hydrocarbons (including alkanes, steranes and hopanes), polycyclic aromatic compounds, and hetero-compounds (including alcohols, sterols and steranes) using an activated silica gel column by eluting with hexane, hexane:DCM (1:1; v/v) and DCM:MeOH (93:7; v/v), consecutively. An aliquot of the alkanoic acid fraction was spiked with an internal standard (eicosanoic acid; D39C20) and derivatized to fatty acid methyl esters (FAMEs) prior to analysis using boron trifluoride:MeOH solution (BF3:MeOH). Aliphatic hydrocarbons, polycyclic aromatic compounds, and hetero-compounds were spiked with tetracosane (D50C24), D10-phenanthrene and octadecanol (D37C18), respectively, as an internal standard prior to analysis. Overall, analytical errors were typically <10% based on replicate analysis (n = 6). Given that alkanes, alkanoic acids, alkanols, steroids and terpenoids showed similar responses to both warming and eCO2, we summed them to represent solvent-extractable compounds.

Hydrolysable biopolymers
After pre-extraction of the solvent-extractable compounds, soil/plant residues were subjected to alkaline hydrolysis to extract ester-bound hydrolysable lipids (including cutin and suberin markers) as described by Mendez-Millan et al.51. Solvent‐extracted residues (~0.5 g of plant and ~1 g of peat) were refluxed at 85°C for 18 h with methanolic potassium hydroxide (KOH) (MeOH:water; 9:1, v/v; with 6% KOH). The extracts were filtered and acidified to pH 2.0 with 6 N hydrochloric acid (HCl). Compounds were recovered by liquid‐liquid phase separation with DCM. The extracts were passed over sodium sulphate and dried under nitrogen gas. An aliquot of the extracts was spiked with an internal standard (eicosanoic acid; C20) and derivatized with N,O-Bis(trimethylsilyl)-acetamide (BSA; Merck, Germany) for 1 h at 80°C. Samples were analysed in duplicates and analytical errors were typically <10% (we report mean values n = 2). Biopolymers distinct to either leaf/needle (cutin) or root/bark (suberin) were selected according to their occurrence in the analysed leaves, stems, and roots of the dominant plant species at SPRUCE; Picea mariana, Larix laricina, Rhododendron groenlandicum, Chamaedaphne calyculata, and Sphagnum mosses (Table S2). Leaves, stems, and roots were characterised by different abundances and chain lengths of n-alcohols, n-carboxylic acid, α-hydroxyalkanoic acid, ω-hydroxy alkanoic acid, α,ω-alkanedioic acid and mid-chain-substituted hydroxy and epoxy alkanoic acids. The hexacosanoic acid (n-C26:0), octacosanoic acid (n-C28:0), dihydroxyoctadecanoic acid (9,10-diOH C18:0) and dihydroxyoctadecenoic acid (x,18-diOH C18:1) were used as markers for leaf/needle-derived C as they were either not detected or occurred in trace abundance in plant roots (Table S2). The ω-hydroxyalkanoic acids with a chain length of C20, C24 and C26 and α,ω-alkanedioic acid with a chain length C20, C22 and C24 (also α,ω‐octadecenoic acids, C18:1 diacid) were used as markers for root/bark-derived C as they were either not detected or occurred in trace abundance in leaf/needle (Table S2). These compounds correspond to previously suggested cutin and/or suberin monomers27,51. The unspecific monomers were treated as compounds derived from cutin or suberin, and they were summed to represent hydrolysable biopolymers (herein, hydrolysable compounds)27,29.

Lignin phenols
Bulk peat samples (milled) were subjected to copper (II) oxide (CuO) oxidation to extract lignin phenols52,53. Briefly, peat material equivalent to ~2 mg of organic carbon was mixed with 500 mg CuO, 50 mg of ammonium iron (II) sulphate hexahydrate and 20 mL of 2 M NaOH solution in Teflon-lined bombs. The bombs were then flushed with nitrogen for 10 min and heated (oxidized) in the microwave for 90 min at 150°C. Once cooled, the oxidation products were spiked with ethylvanillin (3-ethoxy-4-hydroxy-benzaldehyde) and cinnamic acid (3-phenyl-2-propenoic acid) as the recovery and internal standards, respectively, and subsequently acidified to pH 2.0 using 6 M HCl. The oxidation products were then extracted from the aqueous phase with ethyl acetate, dried under nitrogen and redissolved in p-Anisic acid. An aliquot of the oxidation products was converted to trimethylsilyl derivatives with N,O-bis (trimethylsilyl) trifluoroacetamide/ tetramethylchlorosilane (BSTFA+TCMS; 99:1; Sigma-Aldrich, USA) by heating for 20 min at 60°C. Two analytical replicates were measured for all samples (analytical errors <20%; we report mean values n = 2). Eight characteristic oxidation products were summarized to represent the concentration of lignin phenols, including vanillyl (vanillin, acetovanillone, vanillic acid), syringyl (syringaldehyde, acetosyringone, syringic acid) and cinnamyl (p-coumaric acid, ferulic acid) phenols (Fig. S4b). In addition, CuO oxidation released salicylic acid, salicylaldehyde, and piceol phenols (see Fig. S4b for individual fractional abundances). Lignin-specific oxidation products were used to calculate lignin oxidation (degradation) indices; acid-to-aldehyde ratios of vanillyl and syringyl phenols32. We note that while the CuO method is not interfered by any other organic component in peat soils, the method may significantly reduce the yield of phenolic units upon CuO oxidation54,55.

The above SOC molecular compounds were quantified using internal standards on gas chromatography (GC) equipped with a multi-mode injector and a flame ionization detector (GC-FID, Agilent 7890B). Compound identification was performed on an Agilent 6890N GC equipped with split/splitless injector coupled to an Agilent 5973 mass selective detector (MS) using external standard mixtures and by interpretation of the mass spectra. On the GC-FID/MS, the samples (1 μl) were injected in splitless mode. Both GC instruments were equipped with DB-5MS column (50 m x 0.2 mm × 0.33 μm), with helium as the carrier gas (1 ml min-1). For solvent extractable compounds, details of the GC operating conditions are described elsewhere50. For hydrolysable biopolymers, the GC oven temperature was held at 50°C for 4 min, increased to 150°C at a rate of 4°C min-1, then increased to 320°C at a rate of 3°C min-1, with final isothermal hold at 320°C for 40 min. Quantification of the biopolymers was achieved by comparison with an external calibration with 16-hydroxyhexadecanoic acid. For lignin phenols, the temperature was held at 80°C for 5 min, increased from 80°C to 110°C at a rate of 2°C min-1, then increased to 170°C at a rate of 0.5°C min-1, and at a rate of 15°C min-1 to 320°C, with final isothermal hold at 320°C for 10 min.

Pyrogenic carbon
Benzene polycarboxylic acids (BPCAs) were used as an approximation of pyrogenic carbon (PyC) and they were analysed following the protocol by Wiedemeier et al.56. Briefly, milled peat samples containing ~10 mg of organic carbon were digested with 65% nitric acid in the digestion tubes at 170°C for 8 h. The extracts were filtered and passed over a cation exchange resin for further cleaning and subsequently freeze-dried. The dried extracts were redissolved in MeOH:water (1:1; v/v) and passed over a solid phase extraction cartridge (C18 SPE tube, Supelco, U.S.A), then dried under nitrogen and redissolved in deionized water. Compounds were quantified on a high-performance liquid chromatograph (HPLC) (Agilent 1290 Infinity HPLC system, Santa Clara, U.S.A.), equipped with an Agilent InfinityLab Poroshell 120 SB-C18 column (100 mm × 4.6 mm × 2.7 mm), and measured using a photodiode array detector (DAD). Ortho-Phosphoric acid (Honeywell, U.S.A.) dissolved in water (pH 1.2) was used as mobile phase A and pure acetonitrile (Scharlau, Spain) as mobile phase B. Samples were analysed in duplicates and analytical errors were typically <10% (we report mean values n = 2). All carboxyl functional groups (B3CA, B4CA, B5CA and B6CA) were summed to represent total concentration of PyC (see Fig. S4a for individual fractional abundances).

Data analyses
General linear mixed-effect models were used to determine the effects of temperature, elevated CO2 concentrations and peat depth on individual SOC compounds (solvent-extractable compounds, hydrolysable biopolymers, lignin phenols and pyrogenic C). We checked for bivariate relationships between all variables to ensure that a linear model was appropriate. Normality and homoscedasticity in the models were checked using residuals and Q-Q plots (none of the dataset was adjusted to fit parametric assumptions). In all cases, linear regression models included plot as a random effect, and all other predictor variables (temperature, elevated CO2 concentrations and peat depth) as fixed effects. For instances in which both temperature and CO2 effects were significant, we evaluated separate regressions against temperature for ambient and elevated CO2 treatments (Table S3). When significant differences among depths were detected, we conducted pairwise comparisons using Tukey’s honest significant difference test. In the models, we used the actual temperature measured at -0.3 m below the hollows averaged over the period 2016 to 2018 (Table S1). We chose 0.3 m as a representative depth because the water table has fluctuated about 30 cm relative to hollow during the growing seasons17.

Correlation heatmap (Pearson correlation) was calculated between individual SOC compounds (solvent-extractable compounds, hydrolysable biopolymers, lignin phenols and pyrogenic C) and possible predictors (SOC concentration, nitrogen concentration, carbon:nitrogen ratio, net primary productivity, aboveground net primary productivity, fine-root biomass, and maximum distance to the water table). Stepwise multiple linear regression with Akaike Information Criterion (AIC) as the model selection condition was used to investigate relationships between individual SOC compounds and the most important predictor variable(s). Only variables showing significant correlations with the investigated parameter (p < 0.1) were included as independent variables in the model. After the establishment of regressions, candidate models were carefully investigated for collinearity among the selected predictors and assumptions of normality by calculating variance inflation factors (a cut-off value of 1 when collinearity was considered not to exist) and graphically examining plots of residuals (log-transformation did not improve the overall distribution). In the multiple linear regression model, potential explanatory variables included: SOC concentration, nitrogen concentration, net primary productivity, fine-root biomass, and maximum distance to the water table (Fig. 4; Table S4).

Overall, the chosen level of significance was 5% (p < 0.05) in all statistical tests (unless stated otherwise). All data analyses were performed using the R v. 4.2.0 (R Core Team, Vienna, Austria) using the RStudio interface v. 1.2.5033 (RStudio Team, PBC, Boston, MA).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study used a warming x eCO2 manipulafion to measure changes in soil organic mafter molecular composifion in peat soils. They found that surface peat soil responded fairly quickly, with different compounds increasing or decreasing depending on if solely temperature changed or temperature combined with CO2. They found roots contributed more to SOC, and leaves contribute less, with warming regardless of CO2 level, and that deeper peat did not show changes in SOC composifions over the four-year period examined.\nOverall I found this an interesfing study that elucidated how the molecular composifion of peat may shift under global change. We sfill lack general conceptual models describing soil organic mafter molecular composifion, and carefully collected datasets like this one spanning climate manipulafions help reveal the drivers and constraints on SOC composifion. I appreciate that the authors highlight that this was a short-term response – it is interesfing how fast the peat responded but does not indicate the long-term dynamics (as they acknowledge). The finding relates to root vs shoot inputs was especially interesfing.\nI think this manuscript could be improved by constraining the focus to these interesfing and novel findings and not making too many extrapolafions to SOC stocks overall, which were not the focus of the manuscript. Throughout the paper and especially the last Discussion paragraph contained a good deal of text related to overall SOC storage, for which no data are provided in this paper. I feel the manuscript would be stronger if the authors sfick more closely to what their data show. Changes in molecular composifion can have implicafions for stocks and that is worth discussing, but themselves are interesfing for the insights they provide into plant and microbial dynamics.\nAnother general comment is that at many points it was unclear when the authors were drawing conclusions from the data in this study vs results of others. It would be befter if this was clearer throughout. I also found many points where the language was not as precise as I wanted it to be, making it hard to follow the logic (specific lines below). One last general comment is that I found the second paragraph of Introducfion a bit muddled. It seems this is the main jusfificafion for the study but as wriffen is too vague and doesn’t clearly lay out what we know and remaining key unknowns.\nBelow I provide specific suggesfions for areas to strengthen the text.\nLine 34 – ‘sequestrafion’ implies acfion, uptake into a pool leading to long-term storage. I think you simply mean that peatland C is a large (and important) stock. I suggest you remove this word/rephrase this sentence.\nLine 53 – ‘by unknown mechanisms’ is quite vague. What exactly do you mean here, that we know how the components of the system will respond individually but it’s difficult to predict the net effect? Or something else? Please clarify.\nLines 63-67 – sentence is too long and rambling, it is difficult to understand what was done. This should be improved. Also, which of these components are assumed to be microbial-derived?\nLines 78-83 – Not arguing with the Arrhenius framework overall, but specifically for lignin, degradafion proceeds primarily by the acfion of oxidafive enzymes. I would think that oxygen availability is therefore the main boftleneck, as in reference 8. In wetlands/peatlands, might apparent temperature sensifivity with warming in fact be due to increased oxygenafion under hofter, drier condifions? How will you be able to see this ‘simple kinefic effect’?\nLine 91 – again, which are the microbial-derived compounds, what underpins that assumpfion?\nLines 99-104 – you seem to be posing several explanafions for the increase in lignin – increased stabilizafion, more inputs, and ‘faster cycling labile C’. Please clarify the rafionale for this last one (I do not understand), and if possible state whether you think there is more evidence for one or the other of these (or some combinafion) before concluding the lignin discussion.\nLine 108 – ‘an overall loss of SOC molecules’ – given the previous discussion about lignin this does not seem accurate. Lignin decomp rates might be increasing (I’m convinced by Figure 2), but overall/on balance you are gaining lignin.\nLine 126 – ‘enhanced incorporafion of plant C inputs to SOC’ I’m confused here, lignin also comes from plants, do you mean plant C inputs other than lignin?\nLines 128-129 – enhanced C storage? But this manuscript addresses molecular composifion, I don’t see any evidence for increased C stocks overall. Need to either aftribute that finding to other papers or make a clearer link.\nLines 132-135 – You lost me with this long sentence, I can’t follow the logic.\nLine 163 – I think by ‘vegetafion composifion’ you mean the molecular composifion of vegetafion? Or do you actually mean abundance and cover of specific species? Please clarify.\nLines 169-170 – your (great!) study is one site, one biome. I advise caufion in over-extrapolafing, perhaps say ‘in peatlands’ or ‘in boreal forest’\nFigure 4 – Not sure I got much from this heat map. Did you consider instead providing a table describing best-fit models for each of the SOC components?\nLine 185 – what do you mean by disproporfionately lost? Lignin concentrafion increased with warming. I am also sfill left to wonder, which (non-measured) compounds increased with warming, if SOC overall did not change as stated in the Methods?\nLine 210 – to show that they are occurring at the same rate, wouldn’t you need to discuss the slopes? This might be something helpful to add to the figures that are cited.\nLine 272 – there was no effect of the treatments on SOC concentrafion? This is surprising given the statement in lines 85-86 that all compounds are vulnerable to loss under warming. If all compounds decline, how is total SOC unchanged?\nLine 318 – there are some known limitafions of the CuO method for measuring lignin in soil (for instance, the two references below). Since in this study, you are assessing treatment effects on otherwise idenfical soils, I don’t see a major issue as relafive differences should be robust, but do think you should acknowledge that the method often underesfimates lignin overall.\nHernes, P. J., Kaiser, K., Dyda, R. Y. & Cerli, C. Molecular trickery in soil organic mafter: hidden lignin. Environ. Sci. Technol. 47, 9077–9085 (2013).\nKlotzbücher, T., Kalbitz, K., Cerli, C., Hernes, P. J. & Kaiser, K. Gone or just out of sight? The apparent disappearance of aromafic lifter components in soils. SOIL 2, 325–335 (2016).\nTable S1 – why do the Elevated CO2 plots tend to have higher soil temperatures within the same nominal warming class?\nFigure S7 – it’s nice to see the soil bulk density data since compound abundances are presented as stocks. Please briefly address how and when bulk density was measured in the Methods\nData access – when submifting a revised version, will you have submifted datasets and thus can provide a DOI? This would be ideal. If not, can you describe how interested parfies can find your datasets within the named repositories?", "reviewer_2": "This manuscript presents results on the effect of warming and elevated atmospheric CO concentrafions in experimental enclosures in a peatland in Minnesota, over a four-year period. The study provides strong evidence of changes in the chemical composifion of the upper layers of the peat associated with both warming and elevated CO2; the former leads to decreases in selected organic components, associated with enhanced decomposifion, while the doubling of CO2 concentrafion increased components associated with plant growth, such as roots and leaves/needles. The manuscript is generally well wriften and structured and presents evidence of possible changes in organic soil components resulfing from ‘climate change’. The SPRUCE study was designed to determine the effects of warming and elevated CO2 concentrafion, with a clear recognifion that the experimental manipulafions (warming up to 9oC and CO2 concentrafion increased by about 300 ppm) do not represent ‘real’ climate change but give some indicafion of the expected changes in direcfion, rather than in magnitude. SPRUCE has been very successful in its operafion and the wide range of experfises that have been assembled at the site.\nHowever, I do think that the results, although impressive, need to be presented with some caufion: the environmental changes occurred more or less overnight, the range of change is beyond what might be expected to occur (though 2023 may seem to be a portent of rapid changes…), the fall in water table may be moderated by lateral flow in open systems and bogs have many adapfive mechanisms which has allowed them to survive changes in the past. Thus, I suggest the authors tone down some of the statements made, less dramafic and more caufionary in whether the changes shown at SPRUCE will occur in the vast area of northern bogs. The caufionary tone appears in lines 216-219 and it might be applied in the text. Parficularly important is the recognifion that increases in some SOC components (suberin) are linked to below-ground plant acfivifies (roots), which are expected to have a slower turnover fime, with vegetafion responding posifively to a warmer and drier peat soil.\nI provide some specific comments, by line #:\n1 ‘rapidly’ seems a bit strong given the heavy jolt the system was exposed to and the short durafion of the study (4 years temperature change, 2 years CO2 change).\nI am not sure whether the above is correct (happy to be corrected), but I wonder how the figure of 7.6 mg/g decrease in solvent-extractable compounds per oC increase in temperature was derived. If the temperature rose by 7oC, then all the original component would be gone. Please correct me if I have this wrong.\n112. It seems that the lowering of the water table could be a significant response to the warming and elevated CO2, in terms of the increase in the aerobic zone, possible increase in available nutrients through accelerated decomposifion and potenfial increase in roofing zone. The only metric cited appears to be the maximum depth of the water table over three years, which occurs in August-September. The maximum depth increases from 17 cm to 44 cm (Table S1), a lowering of 27 cm. Is there a more useful metric to indicate the drying of the peat, from the available data? There are few warming experiments of the scale of SPRUCE, but quite a few studies have examined the effect of drainage on peat chemistry and vegetafion pafterns.\n160. Reference might be made to Table S1 in which fine root biomass is presented and shows a six-fold increase from the ‘control’ to the +9 plots (55 and 327 g/m2, respecfively) and there seems to be a general increase among the plots.\nThe data are presented primarily as concentrafions per gram of peat, though in Fig. S2 an esfimate is made of the mass of components in the top 30 cm. Is there any evidence of the loss of SOC (e.g. g/m2) from the four years of treatments or over a longer period (e.g. the 2012 and 2020 bulk density data in the Supplementary Informafion)?\nThe components measured (Fig. 1&3) contribute a total of about 95 mg per gram of peat in the ambient plots. How might the remaining 90% of the peat react to the changes?\nFig. 1. I assume the regression is driven through the data for all three depths, and there is liftle differenfiafion among the 0-10 cm, 10-20 cm and 20-30 cm depths.\nTim Moore"}, "author_response": "The present manuscript was previously submitted to Nature Communications (NCOMMS-23-30015-T) and was assessed by two reviewers. We appreciate the extensive comments provided by the reviewers. We have substantially revised the manuscript based on the feedback we received; major changes are as follows:\n1) The reviewers suggested constraining the focus of the manuscript on changes in SOC molecular composition and reducing broad extrapolations to SOC stocks. Furthermore, they suggested introducing caveats around some statements and discuss uncertainties and limitations. We have removed the more speculative aspects of our interpretation and have ensured that our work cannot be misconstrued as a study of soil C stocks. We have also reduced focus on broad extrapolations and toned down the strength of language throughout.\n2) We have also clarified that the aims of the study were to investigate whether warming and elevated atmospheric CO concentration (eCO ) affects SOC stability via changes in molecular compounds comprising SOC and to identify which SOC molecules are susceptible to climate driven changes. We have clarified that we are talking about SOC molecular components that are best suited to infer mechanisms and not SOC stocks. These corrections have not altered our conclusions but have improved the coherence of the results.\nWe are confident that the revisions have addressed all of the reviewers’ concerns and we give our detailed responses to each point below, along with references to the revised manuscript text when applicable.\nReviewers' comments:\nReviewer #1 (Remarks to the Author):\nThis study used a warming x eCO manipulation to measure changes in soil organic matter molecular 2 composition in peat soils. They found that surface peat soil responded fairly quickly, with different compounds increasing or decreasing depending on if solely temperature changed or temperature combined with CO . They found roots contributed more to SOC, and leaves contribute less, with 2 warming regardless of CO level, and that deeper peat did not show changes in SOC compositions over 2 the four-year period examined.\nOverall, I found this an interesting study that elucidated how the molecular composition of peat may shift under global change. We still lack general conceptual models describing soil organic matter molecular composition, and carefully collected datasets like this one spanning climate manipulations help reveal the drivers and constraints on SOC composition. I appreciate that the authors highlight that this was a short-term response – it is interesting how fast the peat responded but does not indicate the long-term dynamics (as they acknowledge). The finding relates to root vs shoot inputs was especially interesting.\nI think this manuscript could be improved by constraining the focus to these interesting and novel findings and not making too many extrapolations to SOC stocks overall, which were not the focus of the manuscript. Throughout the paper and especially the last Discussion paragraph contained a good deal of text related to overall SOC storage, for which no data are provided in this paper. I feel the manuscript would be stronger if the authors stick more closely to what their data show. Changes in 1 molecular composition can have implications for stocks and that is worth discussing, but themselves are interesting for the insights they provide into plant and microbial dynamics.\nResponse:We thank the reviewer for the constructive and insightful comments.\nThere is compelling evidence from observational studies across climate gradients, and field warming experiments, that more rapid decomposition is not synonymous with reductions in total soil C stocks, at least in the short-term (<1to ~10years) (Lu et al., 2013; Bradford et al., 2016; Melillo et al., 2017; Song et al., 2019). The lack of direct evidence for reductions in soil C stocks, which is the case in our experiment (Hanson et al., 2020; Ofiti et al., 2022) makes it difficult to quantify the effects of warming on total soil C. Thus, this encouraged the use of molecular compounds comprising SOC (indirect measurements), to understand warming effects, and implications for overall SOC storage. As pointed out by the reviewer, “changes in molecular composition can have implications for stocks and that is worth discussing”, we therefore aimed to investigate the effects of warming and elevated atmospheric CO concentration on peatland SOC molecular composition and use the observed changes in molecular composition to infer SOC sources (insights on plant and microbial dynamics), stability, and implications for storage. Given that several peatland climate change experiments exist (e.g., Bragazza et al., 2013; Walker et al., 2016; Liu et al., 2022), and biomarkers are an established tool to understand ecosystem processes (Feng et al., 2008; Pisani et al., 2015; Pold et al., 2016; Cheng et al., 2017; Bailey et al., 2018; Jia et al., 2019; Zosso et al., 2023), investigations on peat SOC molecular composition responses to changing environment are scarce. This makes our study unique as our biomarker technique is applied in SPRUCE’s whole-ecosystem warming setting. We are also unaware of any large-scale studies that has measured the changes we demonstrate in our study.\nWe have revised the manuscript to focus solely on SOC molecular components that are best suited to infer mechanisms and not SOC stocks and we acknowledge discussing the implications these changes have on C storage.\nAnother general comment is that at many points it was unclear when the authors were drawing conclusions from the data in this study vs results of others. It would be better if this was clearer throughout. I also found many points where the language was not as precise as I wanted it to be, making it hard to follow the logic (specific lines below). One last general comment is that I found the second paragraph of Introduction a bit muddled. It seems this is the main justification for the study but as written is too vague and doesn’t clearly lay out what we know and remaining key unknowns.\nResponse:We have removed speculative discussions and streamlined the results and discussion sections of the manuscript, by focusing on discussing the meanings indicated by our measurements, for example in line 108-112, 115-120, 142-148, 160-165. We now believe that the novelties of our study are strengthened in the revised manuscript. We have also clarified wording where it was unclear what was our study vs previous work.\nWe have revised the second paragraph of the Introduction to focus solely on what we know and remaining key unknowns in the stability of peat SOC, and their responses to climate change factors. It now reads:\n“For projections of peatland C balance under future climate scenarios, a key question is whether a portion of SOC that is currently stable may become more accessible to microbial decomposition2,4,7, via direct effects of rising temperature, indirect effects of altered water table9,14,15, or due to changes in plant production and thus litter inputs to SOC11,16,17. Soil warming is occurring more rapidly at high compared 2 to low latitudes18,19, which can lead to lower water-tables and increases in peat aeration9,15, with alterations in plant biomass production and in the quality of litter inputs10,16,20. Such a change in peat aeration and plant litter (and root exudate) inputs can impact the microbially-mediated turnover of belowground soil carbon because soil water content and litter chemistry regulate the composition and activity of the microbial community in peat soils8–11. Differences in litter chemistry can also affect the sensitivity of organic matter decomposition to soil temperature, with further effects on C cycling10,21. Consequently, if these alterations in organic matter source persist over extended periods, they are likely to stimulate the mineralisation of currently stable C22. In short, climate-driven changes in water-table and plant litter inputs are likely to modify C storage5,6, but the underlying mechanisms and their interactions, particularly those resulting in shifts in organic matter inputs and composition are still unknown in peatlands7. The role of such plant-soil interactions in regulating future SOC storage merits attention because a much larger proportion of SOC may be susceptible to climate-mediated losses than previously assumed7,19.” (Line 46-62).\nBelow I provide specific suggestions for areas to strengthen the text.\nComment 1: Line 34 – ‘sequestration’ implies action, uptake into a pool leading to long-term storage.\nI think you simply mean that peatland C is a large (and important) stock. I suggest you remove this word/rephrase this sentence.\nResponse: We disagree with the reviewer. Radiocarbon data in particular show that peatlands than nearly any other ecosystems have been active in sequestering carbon for over 10,000 years (Hobbie et al., 2016; Griffiths et al., 2017; Wilson et al., 2021a). We have now added the statement that “The capacity of peatlands to store more than one-third of the global SOC pool under waterlogged conditions1,2 has been touted as a powerful natural form of C sequestration3.”(Line 33-34).\nComment 2: Line 53 – ‘by unknown mechanisms’ is quite vague. What exactly do you mean here, that we know how the components of the system will respond individually but it’s difficult to predict the net effect? Or something else? Please clarify.\nResponse: We agree and have removed this statement. We have added a paragraph to explain that climate-driven changes in water-table and plant litter inputs have the potential to modify C storage, but the underlying mechanisms are still unknown in peatlands, particularly around the sources and stability of organic matter (Chaudhary et al., 2020; Wilson et al., 2022). We have revised this sentence it now reads “In short, climate-driven changes in water-table and plant litter inputs are likely to modify C storage5,6, but the underlying mechanisms and their interactions, particularly those resulting in shifts in organic matter inputs and composition are still unknown in peatlands7.” (Line 57-60).\nComment 3: Lines 63-67 – sentence is too long and rambling, it is difficult to understand what was done. This should be improved. Also, which of these components are assumed to be microbial-derived?\nResponse: We have now changed the sentence completely, breaking it into three parts and specifying which compounds are plant- and microorganism-derived. This sentence it now reads:\n“Specifically, we targeted SOC compounds with distinct turnover times. Firstly, we targeted solvent- extractable compounds (alkanoic acids, alkanols, alkanes, steroids, and terpenoids, derived from plant- and microbial-material26) that are thought to turn over predominantly on a timescale of bulk SOC or faster24,27. Secondly, we targeted SOC components from different plant origins, hydrolysable biopolymers distinct to either leaf/needle (cutin) or root/bark (suberin) compartments (and presumably 3 slowly cycling)26–29. Lastly, we targeted lignin and pyrogenic carbon (PyC) representing the more slowly cycling C23,30–32.” (Line 67-73).\nWe do have components that are only microbial derived. Among the myriad of SOC constituents, solvent-extractable compounds, including alkanoic acids, alkanols, alkanes, steroids, and terpenoids, may yield information on the relative contribution of plants and microbes to SOC as they are derived from plant tissues and microorganisms (Kögel-Knabner, 2002; Otto et al., 2005; Angst et al., 2016). Plant-derived organic matter is characterized by long-chain alkanoic acids, alkanols, alkanes, diacids, and terpenoids whereas compounds such as iso- and anteiso-branched alkanoic acids are only derived from microorganisms (Harwood and Russell, 1984; Kramer and Gleixner, 2006; Wiesenberg et al., 2008). Additionally, with the decomposition of plant materials, soil microorganisms convert available carbon (including plant-derived organic matter) into microbial necromass or microbial processed compounds along with their own biomass (Miltner et al., 2012; Cotrufo et al., 2015; Kallenbach et al., 2016). We thus refer to solvent-extractable compounds as plant- and microorganism-derived as compounds such as iso- and anteiso-branched alkanoic acids and the microbial processed compounds contributed to the solvent-extractable portion of SOC.\nAt best, we can get at plant-only through the other category of hydrolysable polymers where we target biopolymers distinct to either leaf/needle (cutin) or root/bark (suberin) compartments. As some of the specific biomarker terms may not be familiar to the broad readership of Nature Communications, we have tried to keep the text accessible.\nComment 4: Lines 78-83 – Not arguing with the Arrhenius framework overall, but specifically for lignin, degradation proceeds primarily by the action of oxidative enzymes. I would think that oxygen availability is therefore the main bottleneck, as in reference 8. In wetlands/peatlands, might apparent temperature sensitivity with warming in fact be due to increased oxygenation under hotter, drier conditions? How will you be able to see this ‘simple kinetic effect’?\nResponse: Good point! Sphagnum peatlands have been suggested to possess a particularly effective mechanism for restricting enzymic decomposition under the “enzyme latch” hypothesis, whereby oxygen constraints associated with waterlogging suppress the activity of phenol oxidase enzymes (Freeman et al., 2001; Ise et al., 2008; Fenner and Freeman, 2011). This, in turn, allows an accumulation of enzyme-inhibiting phenolic compounds that impede nutrient cycling and decomposition. However, rather than just waterlogging, multiple constraints including temperature, pH, and biotic factors such as plant and microbial community composition and functioning as well as the chemical characteristics of the peat itself control SOC stability (Wang et al., 2015). As warming is typically accompanied by lower water-table and increased oxygenation, we agree that lower water-table and increased oxygenation have an increasing influence on the apparent temperature sensitivity of organic matter decomposition. We have modified this sentence it now reads:\n“From the perspective of long-term peatland C sequestration, we are particularly interested in the response of polymeric SOC components because kinetic theory predicts that decomposition and turnover of complex SOC molecules (greater chemical stability) should be more sensitive to temperature changes than simple molecules34. In peatlands, warming is typically accompanied by a lower water-table coupled with peat aeration, which can further impact the apparent temperature sensitivity of organic matter decomposition8,9,14,15. Consequently, if temperature and oxygenation effects predominate over other biotic and abiotic factors that influence decomposition dynamics9,25,34, we hypothesize that complex SOC molecules such as lignin, and PyC that have been proposed to turn over 4 much slower than bulk C in soils due to their polyaromatic chemical structure23,30,35, would degrade faster.” (Line 81-90).\nComment 5: Line 91 – again, which are the microbial-derived compounds, what underpins that assumption?\nResponse:See previous comment 3 (Line 67-73).\nComment 6: Lines 99-104 – you seem to be posing several explanations for the increase in lignin – increased stabilization, more inputs, and ‘faster cycling labile C’. Please clarify the rationale for this last one (I do not understand), and if possible state whether you think there is more evidence for one or the other of these (or some combination) before concluding the lignin discussion.\nResponse: The slowly cycling compounds (e.g., lignin) can increase in relative abundance, if more labile compounds decrease in concentration, “dilution effect”.At SPRUCE, warming has increased the availability of labile sugars (and protein) (Wilson et al., 2021b). We thus posit that the relative increase in lignin phenols may be due to preferential decomposition of labile sugars and dilution of slowly cycling lignin phenols by an enhanced plant input. We have added a paragraph explaining why the increase in lignin implies increased stabilization, more inputs, and faster cycling labile C. This sentence now reads:\n“Indeed, it is plausible that more lignin phenols entered soils via increased root litter production or that a shift in plant species composition toward shrubs richer in these compounds16,17 may explain this increase. It is also possible that this apparent increase in lignin phenols may be due to accelerated decomposition of more rapidly cycled labile C and dilution of slowly cycling lignin phenols, given that warming has increased the availability of labile sugars (and protein) in this experiment38.” (Line 107- 112).\nWe have also concluded on what we believe is the reason for increase in lignin as per the reviewer’s suggestion. We have added the following statement “The observed relative increase in lignin phenols (Fig.1d; Fig. S1) thus reflects a combination of increased organic matter inputs via plant litter and root exudates, enhanced degradation of more labile SOC molecules induced by higher temperatures16,38 and a better resilience of these molecules to warming as compared to the more labile ones.” (Line 115-118).\nComment 7: Line 108 – ‘an overall loss of SOC molecules’ – given the previous discussion about lignin this does not seem accurate. Lignin decomp rates might be increasing (I’m convinced by Figure 2), but overall/on balance you are gaining lignin.\nResponse:We changed the formulation and hope we can now avoid any confusion. “Overall, the above findings indicate that warming stimulated a loss of SOC molecules, irrespective of their origins and potential decomposition rates.” (Line 118-120).\nComment 8: Line 126 – ‘enhanced incorporation of plant C inputs to SOC’ I’m confused here, lignin also comes from plants, do you mean plant C inputs other than lignin?\nResponse:Apologies for this confusion. We have modified this sentence it now reads “Surprisingly, the concentration of lignin phenols decreased linearly with increasing temperatures (-0.38 ± 0.2 mg g-1 per °Cwarming; r2 = 0.36, p = 0.004; Fig. 1d), raising possibilities of enhanced incorporation of other SOC compounds (i.e., hydrolysable biopolymers) into soil, leading to the decrease (dilution) of lignin phenols.” (Line 140-143).\nComment 9: Lines 128-129 – enhanced C storage? But this manuscript addresses molecular composition, I don’t see any evidence for increased C stocks overall. Need to either attribute that finding to other papers or make a clearer link.\nResponse: Our study investigated the consequences of warming and elevated atmospheric CO 2 concentration on peatland SOC components and our results provide key evidence to suggest that C losses in soil are increasing via enhanced degradation, and C inputs are increasing because of increase of plant biomass, but the net change in SOC is not clear. We asked which molecular compounds comprising SOC were vulnerable to shifts in climate drivers and whether changes in decomposition rates and plant-derived inputs would alter the overall stability of SOC. As mentioned previously in our response to the reviewer’s general comment, due to the lack of direct evidence for reductions in soil C stocks, which is the case in our experiment (Hanson et al., 2020; Ofiti et al., 2022) and a synthesis of field data (Lu et al., 2013; Song et al., 2019), we opted for a different approach. We partitioned bulk soil C into classes of molecular compounds as they can provide unparalleled insight into SOC pools that are differently vulnerable to changing climate.\nBased on reviewer comments, we have revised the manuscript and made it clearer that we are talking about SOC molecular components that are best suited to infer mechanisms and not SOC stocks. This sentence now reads:\n“Previous work at the study site demonstrated increased above- and belowground vascular plant biomass in response to warming treatments16,17,20. As the plant litter input increases, so do the concentrations of SOC components in our results (Fig. 1; Fig. S2b), which suggest that warming and eCO could stimulate SOC incorporation via increased plant litter inputs17,42, whereas general impacts 2 on C storage remain unclear.” (Line 143-148).\nComment 10: Lines 132-135 – You lost me with this long sentence, I can’t follow the logic.\nResponse: We have modified this sentence it now reads “Alternatively, more rapid C cycling likely occurred within extant SOC components41, although an increase in SOC turnover appears to have been offset by additional plant productivity16,17.” (Line 150-152).\nComment 11: Line 163 – I think by ‘vegetation composition’ you mean the molecular composition of vegetation? Or do you actually mean abundance and cover of specific species? Please clarify.\nResponse: Thank you for pointing this out. We have added the following statement to acknowledge the fact that neither warming nor CO led to changes in molecular composition of the overlying plant 2 biomass. “Thus, warming and eCO led to the loss of needle/leaf-derived C and gain of root-derived C 2 in these soils (Fig. 3; Fig. S3). These losses are not related to changes in the chemical quality of the vegetation since neither warming nor eCO led to changes of the overlying plant quality16,43.” (Line 183- 2 186).\nComment 12: Lines 169-170 – your (great!) study is one site, one biome. I advise caution in over-extrapolating, perhaps say ‘in peatlands’ or ‘in boreal forest’\nComment 13: Figure 4 – Not sure I got much from this heat map. Did you consider instead providing a table describing best-fit models for each of the SOC components?\nResponse:We used stepwise multiple linear regression to investigate relationships between individual SOC compounds and the most important predictor variable(s). The most important predictor variables were those that showed significant correlations with the investigated SOC components; this is why we decided to report correlations and not best-fit models. We would prefer to hold on to the presentation of heat map (Figure 4), as we think this helps to focus on the biogeochemical drivers of SOC molecules. However, we do agree that the best-fit models should be shown in the manuscript. In the revised manuscript we provide a table describing best-fit models for each of the SOC components (Supplementary Table 4).\nElevated 18.21 0.32 0.29 0.28\nHydrolysable compounds Ambient 36.07 -0.65 0.17 0.002\nElevated 22.41 0.86 0.26 0.006\nCutin monomers Ambient 1.10 -0.03 0.01 0.046\nElevated 1.08 -0.02 0.01 0.07\nSuberin monomers Ambient 1.86 0.18 0.06 0.01\nElevated 1.78 0.20 0.05 0.002\nPyrogenic carbon Ambient 6.52 0.03 0.28 0.922\nElevated 4.47 0.20 0.18 0.304\nLignin phenols Ambient 25.51 0.51 0.22 0.04\nElevated 31.83 -0. 38 0.15 0.026\nResponse variable CO2 Significance Predictor Coefficient Predictor Coefficient Predictor Coefficient Predictor Coefficient Adjusted\ntreatment criterion R2\nSolvent- Ambient p < 0.01 Intercept 16.72*** ANPP 0.02* (0.01) Fine-root -0.01 -- -- 0.67\nextractable (2.64) biomass (0.005)\ncompounds Elevated p < 0.01 Intercept -23.31 SOC 0.10* (0.03) Water table 0.19* (0.08) Soil N -0.55 (0.37) 0.52\n(15.80) depth\nAmbient p < 0.05 Intercept 37.90*** Fine-root -0.02** Soil N -0.32 (0.24) -- -- 0.51\nHydrolysable (3.26) biomass (0.004)\ncompounds Elevated p < 0.05 Intercept -12.42 Fine-root 0.01* SOC 0.07 (0.04) Soil N 0.06 (0.35) 0.59\n(16.89) biomass (0.005)\nAmbient p < 0.05 Intercept 1.01*** Fine-root -0.001* -- -- -- -- 0.25\nCutin monomers (0.06) biomass (0.000)\nElevated p > 0.05 Intercept -5.27* Water table -0.30* Fine-root 0.21* (0.07) ANPP 0.71* (0.24) 0.44\n(1.84) depth (0.10) biomass\nAmbient p < 0.05 Intercept 1.81** Water table 0.06* (0.02) Fine-root -0.002 -- -- 0.45\nSuberin (0.42) depth biomass (0.002)\nmonomers Elevated p < 0.01 Intercept -8.63** Soil N 0.15* (0.06) SOC 0.02** Fine-root 0.002** 0.72\n(2.92) (0.01) biomass (0.001)\nAmbient p > 0.05 Intercept -0.77 (3.91) Soil N 0.64 (0.33) 0.16\nPyrogenic\nElevated p < 0.05 Intercept -0.96 (2.07) Soil N 0.70** -- -- -- -- 0.45\ncarbon\n(0.20)\nAmbient p < 0.01 Intercept 71.11** SOC -0.14** NPP 0.05** Water table 0.38*** 0.80\nLignin phenols (16.77) (0.04) (0.01) depth (0.06)\nElevated p < 0.01 Intercept 56.87**** SOC -0.06** Water table -0.12** ANPP 0.01 (0.01) 0.72\n(7.42) (0.02) depth (0.03)\nResponse: As previously mentioned in Comment 6 (Lines 99-104), slowly cycling compounds (e.g., lignin) can increase in relative abundance if more labile compounds decrease in concentration (dilution effect). Since lignin decreased with increasing SOC (Figure 4), we posit that the apparent increase in lignin phenols may be due to dilution by other SOC components such as microbial-processed degradation products. We have modified this sentence it now reads “However, the expectation that the enzymatic breakdown of complex SOC molecules is more sensitive to temperature in comparison to simple molecules34, based in part on the kinetic theory, is not consistent with our results. We show rapid turnover of SOC molecules irrespective of their origins and potential decomposition rates (Fig. 1; Fig. S2).” (Line 206-209).\nComment 15: Line 210 – to show that they are occurring at the same rate, wouldn’t you need to discuss the slopes? This might be something helpful to add to the figures that are cited.\nResponse: For this and for reviewer 2 comment on rates of change, we have added a Supplementary Table 3 with slope estimate for each of the SOC components.\nComment 16: Line 272 – there was no effect of the treatments on SOC concentration? This is surprising given the statement in lines 85-86 that all compounds are vulnerable to loss under warming. If all compounds decline, how is total SOC unchanged?\nResponse: The difficulties involved in detecting changes in the total size of soil C stocks (Lu et al., 2013; Bradford et al., 2016; Melillo et al., 2017; Song et al., 2019) encouraged the use of molecular compounds comprising SOC, to understand warming effects. However, the issues with using such techniques to infer change in stock sizes echoes those for decomposition; environmental change can alter the sizes of individual C pools or fluxes without altering the total stock (Lu et al., 2013; Bradford et al., 2016; Melillo et al., 2017). Throughout the manuscript now, based on reviewer comments, we have made it clearer where we are referring to SOC molecular components (that are best suited to infer mechanisms and not SOC stocks) vs where we are referring to SOC concentration data derived from solvent-extractable compounds, hydrolysable biopolymers, lignin phenols, and pyrogenic carbon (see methods section) vs where we are referring to other studies like Hanson et al. (2020) that have done an in depth (and independent to our study) analysis of SOC stocks at the same study site.\nThis sentence now reads “Overall, SOC concentration increased with increasing depth from 44.0% at the surface to 52.5% at 2 m depth, but there was no effect of temperature or eCO on the SOC 2 concentration43. However, investigations of SOC stock at the site so far have shown that the mean effect of warming on soil C stocks was indistinguishable from zero17. Our observations are not contradictory with this result since the specific compounds we followed represent 20% of the total SOC (see Fig. S1) and should be seen as tracers of SOC.” (Line 296-301).\nWe have also modified our statement in lines 85-86 that all compounds are vulnerable to loss under warming. This sentence now reads “Overall, our study found support for this hypothesis wherein all molecular compounds comprising SOC, regardless of source and complexity, were vulnerable to shifts in climate drivers, demonstrating the high sensitivity of peatlands to climate change.” (Line 90-92).\nHernes, P. J., Kaiser, K., Dyda, R. Y. & Cerli, C. Molecular trickery in soil organic matter: hidden lignin. Environ. Sci. Technol. 47, 9077–9085 (2013).\nKlotzbücher, T., Kalbitz, K., Cerli, C., Hernes, P. J. & Kaiser, K. Gone or just out of sight? The apparent disappearance of aromatic litter components in soils. SOIL 2, 325–335 (2016).\nResponse: We agree that an impediment to lignin phenol analysis is that copper (II) oxide (CuO) oxidation method may ‘hide’ lignin and significantly reduce the yield of lignin phenols upon CuO oxidation (Hernes et al., 2013; Klotzbücher et al., 2016). As demonstrated by Hernes et al. (2013), sorption to iron oxides and minerals may significantly reduce the yield of lignin phenols upon CuO oxidation. We add the following text “We note that while the CuO method is not interfered by any other organic component in peat soils, the method may significantly reduce the yield of phenolic units upon CuO oxidation 54,55.” (Line 370-372).\nComment 18: Table S1 – why do the Elevated CO plots tend to have higher soil temperatures within 2 the same nominal warming class?\nResponse: A key reason for plot-to-plot variation in achieved temperatures and CO concentrations is 2 variable cover by the existing or treatment modified (in some cases) shrub and tree canopies. This variability leads to different energy balance conditions within each enclosure and impacts how diurnal and season temperatures are experienced by the vegetation and microbial communities at depths (Hanson et al., 2017, 2020).\nComment 19: Figure S7 – it’s nice to see the soil bulk density data since compound abundances are presented as stocks. Please briefly address how and when bulk density was measured in the Methods.\nResponse: Thank you for pointing this out. We have added the following statement to address how and when bulk density was measured. “Bulk density measurements were taken from soil cores taken from each of the experimental plots in 2013 and 2020. Soil cores 5.2 cm in diameter were carefully excavated using a Russian corer and soil bulk density was calculated using the freeze-dried weights of the volumetric slices. Bulk density was used to estimate the mass of SOC molecular components (stocks) in the top 30 cm (g/m2). We calculated the mass of SOC molecular components by multiplying bulk density value (g soil cm-3) by concentrations of individual SOC compounds (solvent-extractable compounds, hydrolysable biopolymers, lignin phenols and pyrogenic C) (mg g peat-1) from each peat depth.” (Line 301-307).\nComment 20: Data access – when submitting a revised version, will you have submitted datasets and thus can provide a DOI? This would be ideal. If not, can you describe how interested parties can find your datasets within the named repositories?\nResponse: Yes. Data will be openly available through the SPRUCE repository. We will provide the DOI as soon as the paper is accepted.\nThis manuscript presents results on the effect of warming and elevated atmospheric CO concentrations 2 in experimental enclosures in a peatland in Minnesota, over a four-year period. The study provides strong evidence of changes in the chemical composition of the upper layers of the peat associated with both warming and elevated CO ; the former leads to decreases in selected organic components, 2 associated with enhanced decomposition, while the doubling of CO concentration increased 2 components associated with plant growth, such as roots and leaves/needles. The manuscript is generally well written and structured and presents evidence of possible changes in organic soil components resulting from ‘climate change’. The SPRUCE study was designed to determine the effects of warming and elevated CO concentration, with a clear recognition that the experimental manipulations (warming 2 up to 9oC and CO concentration increased by about 300 ppm) do not represent ‘real’ climate change 2 but give some indication of the expected changes in direction, rather than in magnitude. SPRUCE has been very successful in its operation and the wide range of expertise that have been assembled at the site.\nHowever, I do think that the results, although impressive, need to be presented with some caution: the environmental changes occurred more or less overnight, the range of change is beyond what might be expected to occur (though 2023 may seem to be a portent of rapid changes…), the fall in water table may be moderated by lateral flow in open systems and bogs have many adaptive mechanisms which has allowed them to survive changes in the past. Thus, I suggest the authors tone down some of the statements made, less dramatic and more cautionary in whether the changes shown at SPRUCE will occur in the vast area of northern bogs. The cautionary tone appears in lines 216-219 and it might be applied in the text. Particularly important is the recognition that increases in some SOC components (suberin) are linked to below-ground plant activities (roots), which are expected to have a slower turnover time, with vegetation responding positively to a warmer and drier peat soil.\nResponse: We thank Prof. Tim Moore for his constructive review as well as for his positive inputs on how to additionally improve our manuscript and we believe these revisions have strongly improved the coherence of the results. We have streamlined the results and discussion sections of the manuscript, by focusing more on the strengths of these responses and discussing the meanings indicated by our measurements. We have also reduced focus on broad extrapolations and toned down the strength of language throughout.\nIn the future, we hope to complement our results with long-term, time-resolved analysis to see further changes that will have occurred and whether the current findings are generalizable.\nI provide some specific comments, by line #:\nComment 1: Line 1 – ‘rapidly’ seems a bit strong given the heavy jolt the system was exposed to and the short duration of the study (4 years temperature change, 2 years CO change).\nComment 2: Line 26 – I prefer ‘may’ rather than ‘will’, and it seems that the lowering of the water table may be a significant contributor to the observe changes.\nComment 3: Line 65 – What would be the timescale of turnover of these SOC components in northern peat soils: the components are placed in three cycling groupings.\nResponse: Turnover of organic matter in peat sequences has been rarely described. It is rather assumed that a majority of the newly formed organic matter in peat is preserved through “humification” processes (Tfaily et al., 2014; Zaccone et al., 2018). Some studies refer to the selective preservation of biomacromolecules (González et al., 2003). Due to the anoxic conditions and the low temperature in the northern peat soil, turnover of these SOC components can be assumed to be much slower than that of well aerated soils that is mostly studied (Schmidt et al., 2011), probably on the centennial timescale in the acrotelm (Hobbie et al., 2016).\nComment 4: Line 72 – The planned increase of 500 ppm CO seems not to have been achieved, from 2 Table S1, more like 300 pp increase.\nResponse: The experiment only seeks to achieve +500 ppm CO during active seasons and daytime 2 periods (Hanson et al., 2017). In Table S1 the calculated mean CO levels are low because they were 2 averaged from day- and night-time CO data. Also, a key reason for plot-to-plot variation in achieved 2 temperatures and CO concentrations is variable cover by the existing or treatment modified (in some 2 cases) shrub and tree canopies. This variability leads to different energy balance conditions within each enclosure and impacts how diurnal and season temperatures are experienced by the vegetation and microbial communities at depths (Hanson et al., 2017, 2020).\nComment 5: Line 93 – The title implies speed of change and one way to indicate that is to express the rate of change in the SOC components not just as a function of the change in temperature but over the four years of warming. Fig. 1a shows a regression in which there appears to be a decline in ‘solvent-extractable compounds’ from about 23 mg/g at ambient temperature (4oC), to about 16 mg/g at 11.4oC (9oC warming treatment): a decline of about 7 mg/g or about 30% of original content over 4 years. Thus, at the warmest treatment, that would amount to an average loss of about 8% per year (over the four years) and at a more ‘realistic’ warming treatment of 2.25oC (resulting in a soil temperature of about 5.5oC by eyeballing Fig. 1a) of about 1.5 mg/g or 6% of the original over four years, or 1.5% per year. Of course, the changes may be fast at the outset and slow down.\nI am not sure whether the above is correct (happy to be corrected), but I wonder how the figure of 7.6 mg/g decrease in solvent-extractable compounds per oC increase in temperature was derived. If the temperature rose by 7oC, then all the original components would be gone. Please correct me if I have this wrong.\nResponse: Corrected- thank you for pointing out this mistake! We have corrected the change in ‘solvent- extractable compounds’ to -0.79 ± 0.2 mg g-1 per degree Celsius increase in temperature over four years. In the regression we compare the temperature responses (rate of change) between +0, +2.25, +4.5, +6.75, and +9 °C plots assuming the plots were at equilibrium at the beginning of the experiment (Tfaily et al., 2014). However, this is a dynamic system in that our observed decrease in SOC components may reflect a transient adjustment period to the applications of the experimental treatments, where our system has not yet reached a new equilibrium.\nComment 6: Line 112 – It seems that the lowering of the water table could be a significant response to the warming and elevated CO , in terms of the increase in the aerobic zone, possible increase in 2 available nutrients through accelerated decomposition and potential increase in rooting zone. The only metric cited appears to be the maximum depth of the water table over three years, which occurs in August-September. The maximum depth increases from 17 cm to 44 cm (Table S1), a lowering of 27 cm. Is there a more useful metric to indicate the drying of the peat, from the available data? There are few warming experiments of the scale of SPRUCE, but quite a few studies have examined the effect of drainage on peat chemistry and vegetation patterns.\nResponse: We agree that lowering of the water table does change the subsurface environment from anaerobic to aerobic (oxygenated) at the water table boundary. Such changes will alter the activity of the microbial community and change the mesotelm zone into aerobic functions. The sharp boundary for the water table was documented early in the project and was found to be a clear boundary between aerobic and anaerobic conditions (Tfaily et al., 2014, 2018). As such, we did not invest in depth specific oxygen measurements. Surface moisture measurements are collected at hollow bottoms and for the 0- 20 cm peat depth but are quantitatively problematic due to the strong influence of peat bulk density variation near the surface. Nevertheless, they verify unsaturated peat conditions above the water table with strong precipitation events representing the only transient perturbation to this pattern.\nComment 7: Line 160 – Reference might be made to Table S1 in which fine root biomass is presented and shows a six-fold increase from the ‘control’ to the +9 plots (55 and 327 g/m2, respectively) and there seems to be a general increase among the plots.\nResponse: Thank you for pointing this out. To date, findings at SPRUCE experiment suggest that warming and associated peat drying have increased above-ground growth in shrubs and fine-root growth (McPartland et al., 2019; Hanson et al., 2020; Malhotra et al., 2020). We have added the following statement “Importantly, root-derived C (inferred from suberin monomers) was positively correlated with SOC, fine root biomass and water table depth (Fig. 4) and fine root biomass significantly increased under warming and eCO (Table S1)16, implying increased root-derived C inputs within the 2 surface peat. In corroboration of our results, litter manipulation experiments showed root-derived organic matter to be a source of SOC with greater relative stability and longer turn-over times28, whereas leaf-derived C was found to be turned over more rapidly in (mineral) soils27,36,46.” (Line 186-191).\nComment 8: The data are presented primarily as concentrations per gram of peat, though in Fig. S2 an estimate is made of the mass of components in the top 30 cm. Is there any evidence of the loss of SOC (e.g. g/m2) from the four years of treatments or over a longer period (e.g. the 2012 and 2020 bulk density data in the Supplementary Information)?\nResponse: Previous studies at the site report no evidence for reductions in soil C stocks based on Hanson et al. (2020). Also see responses above to reviewer 1 general comment and comment 9 and 16 on SOC stocks. We have also added new methods section to address how and when bulk density was measured and estimate of the mass of components in the top 30 cm. “Bulk density measurements were taken from 14 soil cores taken from each of the experimental plots in 2013 and 2020. Soil cores 5.2 cm in diameter were carefully excavated using a Russian corer and soil bulk density was calculated using the freeze- dried weights of the volumetric slices. Bulk density was used to estimate the mass of SOC molecular components (stocks) in the top 30 cm (g/m2). We calculated the mass of SOC molecular components by multiplying bulk density value (g soil cm-3) by concentrations of individual SOC compounds (solvent-extractable compounds, hydrolysable biopolymers, lignin phenols and pyrogenic C) (mg g peat-1) from each peat depth.” (Line 301-307).\nComment 9: The components measured (Fig. 1&3) contribute a total of about 95 mg per gram of peat in the ambient plots. How might the remaining 90% of the peat react to the changes?\nResponse: The main source of SOC is plant- and microbe-derived organic matter (Miltner et al., 2012; Cotrufo et al., 2015; Kallenbach et al., 2016). Here, we investigate SOC molecules defined by organic matter origins (plant-, microorganism- and fire-derived). These molecules carry information on sources and transformation process and are typically regarded to be representative of SOC components of similar origins (Kögel-Knabner, 2002; Otto et al., 2005; Angst et al., 2016; Jansen and Wiesenberg, 2017). 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Rapid loss of complex polymers and pyrogenic carbon in subsoils under whole-soil warming. Nature Geoscience 2023 1–5. doi:10.1038/s41561-023-01142-1\nReviewer #1 (Remarks to the Author):\nI reviewed a previous version of this manuscript. I appreciate the author’s work to revise the text and find it much improved. However, I think there remain areas where the language can be clarified to improve precision of their arguments and ensure readers can follow the methods, key findings, and proposed rafionale.\nLines 27-29 – I wonder if the second to last sentence of the abstract can be re-formulated. ‘The observed decline in SOC’ sounds like stocks, perhaps change to ‘SOC molecules’? And the reference to ‘younger’ makes me expect radiocarbon, is this word necessary?\nLines 53-54 – ‘composifion and acfivity of the microbial community’ could you menfion enzymes since the oxidafive enzymes are likely a key factor? Perhaps you could add ‘and their enzymes’ to encompass this?\nLine 81 – in the Response to Reviewers doc, you menfion this would be a new paragraph but this isn’t how it appears in the submission. I think this would befter, consider updafing.\nLine 90 – But the stated hypothesis is that more chemically complex compounds would decompose faster, how did you ‘find support’ for this if all the compounds decreased? Suggest you acknowledge this nuance in the text.\nLines 118-120 – but lignin was lost less so compared to other types of compounds, which is a bit surprising no? I think it’s worth re-iterafing this interesfing point by amending this sentence to something like, ‘Overall, the above findings indicate that warming sfimulated a loss of SOC molecules, irrespecfive of their origins and potenfial decomposifion rates – although lignin declined less relafive to other compound classes.’ Also, I would consider starfing a new paragraph with this sentence, just my opinion but I find shorter paragraphs easier to read and follow.\nLine 145 – warming treatments? I’m surprised, do you mean eCO2? Since this paragraph is about how eCO2 plus warming responses are different from warming alone.\nLine 165 – should ‘Nevertheless’ be revised to ‘Likely because the deeper soils remained waterlogged’ or similar? I like the new text about water table effects, but since the drop was about 30 cm (if I’m understanding correctly), it’s not surprising that the deep peat was unaffected, since it was not oxygenated like the surface peat, correct?\nLine 186 – by ‘plant quality’ do you mean plant fissue chemistry? Might say this, or at least give a very brief definifion of what quality means in this context\nLine 220 – Given Figure S1, it seems ‘13% of the SOC’ is incorrect. Maybe 13% of the molecules examined in this study, but from looking at the Y axis of Figure S1 it looks like only 1-2% of total SOC. Please revise.\nLine 298 – it seems like ‘However’ should be ‘Addifionally’ since this sentence agrees with the one before, there was no change with treatment.\nLines 305-306 and Figures S2 and S3 – I want to make sure of the units here. To get to g/m2 from the starfing units indicated (g/cm3, mg/g, cm), I believe you need to mulfiply by 10. Is that what you did? If so, please menfion it or include an equafion.\nTable S3 – ‘from a single bivariate linear regression’ – with what variables, each compound regressed against temperature? Please clarify in the table capfion. It might also be helpful to reference this table in the methods secfion, perhaps Line 410?\nLine 426 – what is the ‘global model’? Not seeing that in Table S4. Also, there are more variables listed in the Predictor column of that table compared to what is listed in the text.\nReviewer #2 (Remarks to the Author):\nI have reviewed the response of the authors to my comments (though the format of the text is difficult to decipher in parts). I found the responses are adequate and the manuscript is more 'reasonable' and clearer in parts. I did not evaluate the responses to Reviewer 1.\nReviewers' comments:\nReviewer #1 (Remarks to the Author):\nI reviewed a previous version of this manuscript. I appreciate the author’s work to revise the text and find it\nmuch improved. However, I think there remain areas where the language can be clarified to improve\nprecision of their arguments and ensure readers can follow the methods, key findings, and proposed rationale.\nResponse: We thank the reviewer for the constructive and insightful review of the earlier version as\nwell as for the positive inputs on how to additionally improve our manuscript. We have now clarified\nwording where it was unclear as per reviewer suggestions. We now believe that the novelties of our\nstudy are strengthened in the revised manuscript.\nComment 1: Lines 27-29 – I wonder if the second to last sentence of the abstract can be re-formulated.\n‘The observed decline in SOC’ sounds like stocks, perhaps change to ‘SOC molecules’? And the\nreference to ‘younger’ makes me expect radiocarbon, is this word necessary?\nResponse:We agree and have removed this statement. Based on reviewer suggestions, we have revised\nthis sentence it now reads “The decline in SOC compounds with warming and gains from new root-\nderived C under eCO , suggest that warming and eCO may shift peatland C budget towards pools with\n2 2\nfaster turnover.” (Line 26-28).\nComment 2: Lines 53-54 – ‘composition and activity of the microbial community’ could you mention\nenzymes since the oxidative enzymes are likely a key factor? Perhaps you could add ‘and their enzymes’\nto encompass this?\nResponse:Good point! We have added the following statement to acknowledge the fact that oxidative\nenzymes are likely a key factor in the microbially-mediated turnover of belowground soil carbon under\nlower water-tables and increased peat aeration. This sentence now reads “Such a change in peat aeration\nand plant litter (including root exudates) inputs can impact the microbially-mediated SOC turnover\nbecause soil water content and litter chemistry regulate microbial community composition, function,\nand enzymatic activity8–11.” (Line 51-53).\nComment 3: Line 81 – in the Response to Reviewers doc, you mention this would be a new paragraph,\nbut this isn’t how it appears in the submission. I think this would better, consider updating.\nResponse:Thank you for pointing this out. This section is now a new paragraph (Line 81).\nComment 4: Line 90 – But the stated hypothesis is that more chemically complex compounds would\ndecompose faster, how did you ‘find support’ for this if all the compounds decreased? Suggest you\nacknowledge this nuance in the text.\nResponse: We initially stated this ‘support’ with the perspective that even the complex compounds\nresponded. However, as per your comment, it indeed makes more sense to state this as a rejected\nhypothesis. We have now changed the sentence, specifying that our results reject the hypothesis that\ncomplex SOC molecules are more responsive to temperature changes as all molecular compounds\n1\ncomprising SOC, regardless of source and complexity, were vulnerable to shifts in climate drivers. We\nhave revised this sentence it now reads “Overall, our study rejects this hypothesis that complex SOC\nmolecules are more responsive to temperature changes. Instead, we found that all molecular compounds\ncomprising SOC, regardless of source and complexity, were vulnerable to shifts in climate drivers,\ndemonstrating the high sensitivity of peatlands to climate change” (Line 90).\nComment 5: Lines 118-120 – but lignin was lost less so compared to other types of compounds, which\nis a bit surprising no? I think it’s worth re-iterating this interesting point by amending this sentence to\nsomething like, ‘Overall, the above findings indicate that warming stimulated a loss of SOC molecules,\nirrespective of their origins and potential decomposition rates – although lignin declined less relative to\nother compound classes.’ Also, I would consider starting a new paragraph with this sentence, just my\nopinion but I find shorter paragraphs easier to read and follow.\nResponse: Good point! This sentence was revised as suggested and now reads “Overall, the above\nfindings indicate that warming stimulated a loss of SOC molecules, irrespective of their origins and\npotential decomposition rates – although lignin phenols showed a lower response than expected.” (Line\n120-122). This section is also a new paragraph.\nComment 6: Line 145 – warming treatments? I’m surprised, do you mean eCO2? Since this paragraph\nis about how eCO plus warming responses are different from warming alone.\nelevated atmospheric CO concentration (eCO ) treatment. This sentence now reads: “Previous work at\n2 2\nthe study site demonstrated increased above- and belowground vascular plant biomass in response to\nwarming and eCO treatments16,17,20.” (Line 147).\nwaterlogged’ or similar? I like the new text about water table effects, but since the drop was about 30\ncm (if I’m understanding correctly), it’s not surprising that the deep peat was unaffected, since it was\nnot oxygenated like the surface peat, correct?\nResponse: We have removed this statement and changed the formulation to show that surface peat had\nstronger responses than deeper peat and that this is likely due to the water table. This sentence now\nreads “Four years into the treatment, deep anaerobic peat appears to be stable (Fig. S5-6) likely due to\nthe slower decomposition that occurs under anoxic conditions2,9 and low-quality C substrates in these\nlayers13,35.” (Line 167-169).\nComment 8: Line 186 – by ‘plant quality’ do you mean plant tissue chemistry? Might say this, or at\nleast give a very brief definition of what quality means in this context.\nResponse: Here, we were referring to specific vegetation chemistry of overstory trees, ericaceous\nshrubs, and bryophyte layer. Based on reviewer comment, we have removed this statement as we\nrealized that it was tangential to increased Suberin in SOC and corresponding increases in root inputs\n(Line 184).\nComment 9: Line 220 – Given Figure S1, it seems ‘13% of the SOC’ is incorrect. Maybe 13% of the\nmolecules examined in this study, but from looking at the Y axis of Figure S1 it looks like only 1-2%\nof total SOC. Please revise.\nResponse: Corrected- thank you for pointing out this mistake! We have corrected proportion of total\npyrogenic carbon normalized to organic carbon concentration in the surface peat to 1.4%. This sentence\nnow reads “We did not observe significant changes in PyC concentrations under ambient CO or eCO\n2 2\n2\n(p > 0.05; Fig. 1c; Fig. S4a), despite these peat soils containing PyC above global averages (~1.4% of\nthe SOC; Fig. S1)48.” (Line 218).\nComment 10: Line 298 – it seems like ‘However’ should be ‘Additionally’ since this sentence agrees\nwith the one before, there was no change with treatment.\nResponse: Revised as suggested. This sentence now reads “Additionally, investigations of SOC stock\nat the site so far have shown that the mean effect of warming on soil C stocks was indistinguishable\nfrom zero17.” (Line 296).\nComment 11: Lines 305-306 – and Figures S2 and S3 – I want to make sure of the units here. To get\nto g/m2 from the starting units indicated (g/cm3, mg/g, cm), I believe you need to multiply by 10. Is\nthat what you did? If so, please mention it or include an equation.\nResponse: Good point! We calculated the mass of SOC molecular components by multiplying bulk\ndensity value (g soil cm-3) by concentrations of individual SOC compounds (g g peat-1) from each peat\ndepth. We have modified this sentence it now reads: “We calculated the mass of SOC molecular\ncomponents by multiplying bulk density value (g soil cm-3) by concentrations of individual SOC\ncompounds (solvent-extractable compounds, hydrolysable biopolymers, lignin phenols and pyrogenic\nC) (g g peat-1) from each peat depth and summarized the values for the mentioned depth intervals.”\n(Line 303-306).\nComment 13: Table S3 – ‘from a single bivariate linear regression’ – with what variables, each\ncompound regressed against temperature? Please clarify in the table caption. It might also be helpful to\nreference this table in the methods section, perhaps Line 410?\nResponse:For each SOC compound, we evaluated separate regressions against temperature for ambient\nand elevated CO treatments. We have modified this sentence it now reads: “Each reported slope is from\n2\na single bivariate linear regression of each compound regressed against temperature under ambient or\nelevated CO treatment in the surface peat (0-30 cm depth) following 4 years of warming and 2 years\n2\nof elevated atmospheric CO concentrations” (see Table S3).\nComment 14: Line 426 – what is the ‘global model’? Not seeing that in Table S4. Also, there are more\nvariables listed in the Predictor column of that table compared to what is listed in the text.\nResponse: We changed the formulation and hope we can now avoid any confusion. “In the multiple\nlinear regression model, potential explanatory variables included: SOC concentration, nitrogen\nconcentration, net primary productivity, fine-root biomass, and maximum distance to the water table\n(Fig. 4; Table S4).” (Line 425-427).\nI have reviewed the response of the authors to my comments (though the format of the text is difficult to\ndecipher in parts). I found the responses are adequate and the manuscript is more 'reasonable' and clearer in\nparts. I did not evaluate the responses to Reviewer 1.\nResponse: We thank the reviewer for the constructive review of the earlier version.\n4"}]

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