Sphagnum increases soil’s sequestration capacity of mineral-associated organic carbon via activating metal oxides

Domain: earth_science
Published: 2023-08-19
Editorial outcome: accept

Abstract
Sphagnum wetlands are global hotspots for carbon storage, conventionally attributed to the accumulation of decay-resistant litter. However, the buildup of mineral-associated organic carbon (MAOC) with relatively slow turnover has rarely been examined therein. Here, employing both large-scale comparisons across major terrestrial ecosystems and soil survey along Sphagnum gradients in distinct wetlands, we show that Sphagnum fosters a notable accumulation of metal-bound organic carbon (OC) via activating iron and aluminum (hydr)oxides in the soil. The unique phenolic and acidic metabolites of Sphagnum further strengthen metal-organic associations, leading to the dominance of metal-bound OC in soil MAOC. Importantly, in contrast with limited MAOC sequestration potentials elsewhere, MAOC increases linearly with soil OC accrual without signs of saturation in Sphagnum wetlands. These findings collectively demonstrate that Sphagnum acts as an efficient ‘rust engineer’ that largely boosts the rusty carbon sink in wetlands, potentially increasing long-term soil carbon sequestration.

Introduction
Sphagnum is a well-known ‘peat builder’1–4. More than half of northern wetlands (including peatlands) develop from Sphagnum-dominated landscapes, contributing to approximately 15% of soil carbon pool globally5–7. Cultivation and restoration of Sphagnum are advocated in many countries to recover ecosystem functions including carbon sequestration8. The standing paradigm considers that the tremendous carbon storage in Sphagnum-dominated wetlands (abbreviated as ‘Sphagnum wetlands’ hereafter) is mainly attributed to the inhibited microbial decomposition as well as low decomposability of the recalcitrant Sphagnum litter9–11, leading to soil organic carbon (SOC) accumulation mainly as poorly degraded plant detritus or particulate organic carbon (POC)12. By comparison, organic carbon (OC) associated with minerals (i.e., silt/clay-sized aluminosilicates and metal oxides) is a more persistent SOC pool with longer turnover times than POC13,14, since the accessibility of mineral-associated organic carbon (MAOC) to decomposers is limited15. However, the buildup of MAOC has rarely been investigated in Sphagnum wetlands, which may underpin long-term SOC accrual and dynamics under global changes.

Emergent evidence suggests that MAOC constitutes a significant fraction of SOC in wetlands16,17. In particular, phenolic metabolites of Sphagnum (such as sphagnum acid) may induce the transformation of pedogenic iron (Fe) (hydr)oxides to reactive (i.e., poorly crystalline or short-range-ordered, SRO) species and promote strong mineral protection of SOC18. Sphagnum-induced acidic and water-saturated environments9 may also facilitate mineral weathering and enhance the production of SRO Fe and aluminum (Al) (hydr)oxides19,20, which can effectively bind OC21–23 and potentially contribute to MAOC accrual. More importantly, Sphagnum-induced transformation and production of SRO Fe and Al (hydr)oxides may increase the sequestration capacity of MAOC, which is shown to saturate in upland (e.g., forest and grassland) soils due to limited availability of reactive minerals (including metal oxides)24,25. However, metal-bound OC (abbreviated as “bound OC” hereafter) is rarely examined in Sphagnum wetlands, and its contribution to SOC or MAOC has not been assessed in comparison to other terrestrial ecosystems26. Hence, it is essential to clarify whether Sphagnum has a prevalent enhancement impact on metal-organic associations at the landscape scale and whether Sphagnum may elevate MAOC sequestration potentials via activating metal oxides. Answering these questions may not only reveal an underappreciated mechanism for SOC sequestration in Sphagnum wetlands, but also aid in the effort to increase the saturation level of MAOC to stabilize soil carbon in terrestrial ecosystems24.

Reactive Fe and Al (hydr)oxides in soils are ultimately derived from Fe- or Al-bearing minerals in parent materials13,20, which show contrasting distributions in different geological settings, e.g., igneous vs. sedimentary rocks. Vast areas of wetlands worldwide are located in volcanic and tephra-receiving areas27, which facilitate Sphagnum establishment28,29. Soils in such areas (i.e., Andosols) are replenished with Fe and Al minerals30,31, resulting from parent material weathering and eolian deposition of volcanic glass32. Alternatively, Sphagnum-dominated wetlands are also commonly found in sedimentary landscapes such as karst regions that are relatively deprived of SRO Fe and Al (hydr)oxides but rich in less weatherable phyllosilicates33. There is little information, however, on how Sphagnum’s enhancement of metal-organic associations (if present) varies across these different geological settings.

Here, we employed two complementary approaches to prove Sphagnum’s control on mineral protection of SOC by metal oxides. We first compared the contents of bound OC and reactive metals in the surface soils (0–25 cm) of Sphagnum wetlands with non-Sphagnum wetlands and other major terrestrial ecosystems by compiling published data (Fig. 1a, b; Supplementary Data 1). Considering the paucity of related investigations in wetlands, especially Sphagnum wetlands, we supplemented the database by surveying 20 Sphagnum wetlands and 29 non-Sphagnum wetlands with diverse climatic, geological and soil characteristics across China (Fig. 1c), with 12 pairs located in the same area with similar geological and climatic conditions. Second, we selected three spatial gradients (coverage from 0 to 100%) and one temporal gradient (cultivation time from 0 to 20 years) of Sphagnum in four distinct wetlands to track dynamic changes of metal-organic associations under Sphagnum expansion (Fig. 1d). In all of our surveyed wetlands, we examined environmental variables that potentially regulated bound OC content, and assessed the influence of bound OC on the accumulation of SOC and its slow-cycling fraction (MAOC). With these approaches, we aim to elucidate Sphagnum’s effects on metal-organic associations and the sequestration capacity of MAOC among terrestrial ecosystems.

Results
Large-scale comparison of metal-organic associations among terrestrial ecosystems
Using compiled published data and a national survey of wetlands in China, we generated a dataset of 512 measurements of bound OC in the surface soils (0–25 cm) of diverse terrestrial ecosystems globally (Fig. 1a, b; Supplementary Data 1). The examined sites spanned climatic zones from arctic tundra to the tropics, and included wetlands, forests and grasslands mainly distributed in Northern America, East Asia and Europe. Given a large number of data from permafrost-dominated tundra and boreal regions with unique hydrogeographic settings (frozen soils with a high moisture content), we also categorized permafrost as a special ecosystem (excluding Sphagnum-dominated landscapes). Wetlands were further divided into Sphagnum and non-Sphagnum wetlands.

Among these major types of terrestrial ecosystems, Sphagnum wetlands had the highest SOC content (287±5 mg g–1, mean±SE, n=118), followed by non-Sphagnum wetlands (125±7 mg g–1; n=144) and permafrost soils (114±5 mg g–1; n=129) based on one-way ANOVA (p<0.05; Fig. 2a). Bound OC had the highest proportion in SOC in Sphagnum wetlands (22.5±0.6%; n=118), in comparison with either non-Sphagnum wetlands (13.8±0.4%; n=144) or the average value of all terrestrial ecosystems estimated by ref. 26 which barely included Sphagnum-influenced landscapes (14.8±1.0%; n=191; p<0.05; Fig. 2b). This result indicated a notable reservoir of bound OC in Sphagnum wetlands. In addition, Sphagnum wetlands had the lowest soil pH, the highest content of oxalate-extractable Al and Fe (Alo and Feo, i.e., SRO; representing the poorly crystalline or amorphous phases with a strong association with OC)34, the highest reactivity of Fe (hydr)oxides indicated by the ratio of Feo to dithionite-extractable Fe35 (Fed, representing both SRO and crystalline phases)34, and the lowest clay content among all terrestrial ecosystems (p<0.05; Fig. 2c–f and Supplementary Fig. 1a). By contrast, Sphagnum wetlands had similar geographical distributions in latitude, net primary productivity (NPP) and mean annual temperature (MAT) as non-Sphagnum wetlands, which were comparable to some other examined ecosystems (Supplementary Fig. 1b–d). Sphagnum wetlands also had similar mean annual precipitation (MAP) as forests in our dataset (Supplementary Fig. 1e). Given the strong influence of SRO Fe and Al (hydr)oxides on SOC preservation36 especially at low pH22, the above results suggested that the high abundance of reactive metals in the acidic Sphagnum wetlands may have led to a remarkable accumulation of bound OC therein.

To ascertain the main drivers leading to the storage of bound OC in Sphagnum vs. other wetlands, we measured a comprehensive list of soil properties (Supplementary Data 2) that potentially affected bound OC content in our surveyed 49 wetlands, including soil moisture content, water-soluble phenols (compounds exerting a high affinity to SRO Fe and Al)22, the reactive fraction of total Fe and Al (i.e., Feo:Fetotal and Alo:Altotal), molar ratio of bound OC to weight-normalized contents of Fe and Al (i.e., 0.5Fed+Ald) as a proxy for metal-organic association strength37, and sulfate-extractable Ca (Cas; representing organic-associated Ca)38. Compared with non-Sphagnum wetlands, Sphagnum wetlands had higher molar ratio of bound OC:(0.5Fed+Ald), Feo:Fetotal and Alo:Altotal, suggesting a stronger metal-organic association and a higher proportion of reactive species in total Fe and Al (p<0.05; Fig. 3a–c). Sphagnum wetlands also had higher soil moisture and water-soluble phenol contents owing to the incredible water-holding capacity9 and high exudation of phenolic metabolites11 of Sphagnum (p<0.05; Fig. 3d, e), while soil Cas did not differ between the two types of wetlands (Fig. 3f). A principal component analysis separated Sphagnum and non-Sphagnum wetlands (Fig. 3g) and, corroborated by correlation analysis (Supplementary Fig. 2), revealed that bound OC most strongly aligned with weight-normalized contents of SRO Fe and Al (i.e., 0.5Feo+Alo)37 in our surveyed wetlands (r=0.81; p<0.05), followed by water-soluble phenols (r=0.73; p<0.05) and soil moisture content (r=0.63; p<0.05). Soil pH negatively affected bound OC (r=–0.64; p<0.05), while other soil properties (such as clay+silt and Ca) had no influence. These results suggested that Sphagnum induced strong metal-organic associations primarily by activating Fe and Al (hydr)oxides in the soil, which was likely further strengthened by increasing phenolic metabolites, acidity and moisture18. These variables were, however, correlated in the field, and their separate influences on metal-organic associations need to be confirmed with future experimental approaches.

Dynamic increases of metal-organic associations under Sphagnum expansion
To complement the large-scale comparison across terrestrial ecosystems, Sphagnum’s effect on metal-organic associations was further investigated with both spatial and temporal gradients of Sphagnum in four distinct wetlands. The spatial gradient included natural vegetation successions in wetlands with varied coverage of Sphagnum from 0 to 100%, representing a gradual shift from Carex to Sphagnum as the dominant species (in Jinchuan, Hani, and Dajiuhu). The temporal gradient was based on a cultivation project in Dushan, involving varying cultivation time of Sphagnum (0–20 years) in rice paddy soils. Sphagnum expansion (reflected in an increasing coverage or cultivation time) significantly increased SOC contents (by 11–20%), the proportion of bound OC in SOC (by 5–16%) as well as SRO Fe and Al (i.e., 0.5Feo+Alo; by 20–60%) at two surface depths (0–10 and 10–20 cm) of all sites (p<0.05; Fig. 4a–c). The molar ratio of bound OC:(0.5Fed+Ald) also increased at a similar magnitude among sites (from ~5 to ~12; p<0.05; one-way ANOVA; Fig. 4d), suggesting an elevated metal-OC association strength under Sphagnum expansion39, likely owing to decreasing soil pH and increasing phenolic compounds that can effectively ‘glue’ SRO Fe and Al (hydr)oxides and organic matter22,37 (p<0.05; Fig. 4e, f). Notably, Sphagnum-induced increase of Feo:Fetotal (15–34%) outpaced that of Alo:Altotal (3–6%; Supplementary Fig. 3a, b), likely because Sphagnum metabolites (e.g., sphagnum acid) may reductively dissolve ferric Fe to ferrous Fe (Supplementary Fig. 3c) and further induce the transformation of crystalline Fe to SRO counterparts18. Nonetheless, the transformation of Fe and Al (hydr)oxides was accompanied (and likely facilitated) by Sphagnum-induced acidity and water saturation (p<0.05; one-way ANOVA; Fig. 4e and Supplementary Fig. 3d). Hence, the Sphagnum expansion gradients reinforced our findings from the large-scale comparison in that Sphagnum increased metal-organic associations by gradually activating soil Fe and (to a lesser extent) Al (hydr)oxides.

Geological influence on Sphagnum’s enhancement of metal-organic associations
As Sphagnum primarily enhanced metal-organic associations by activating soil Fe and Al (hydr)oxides, we further tested whether Sphagnum’s effect varied in geological landscapes with different contents of transformable Fe and Al minerals. We selected a subset of 12 Sphagnum wetlands among our surveyed sites (6 each in igneous and sedimentary rock-based regions, respectively), and paired them with the non-Sphagnum wetlands in the same area. A response ratio (RR) of bound OC and related variables was calculated, i.e., the natural logarithm-transformed ratio of a specific variable in the Sphagnum wetland relative to that in the paired non-Sphagnum wetland, to reflect Sphagnum’s influence under similar geological and climatic settings. A positive value of RR indicated an enhancement of the examined variable in the Sphagnum than non-Sphagnum wetlands, and vice versa. Given different variances of the calculated RR for different sites, the weight of each site was estimated based on the reciprocal of the variance for individual RRs. We further evaluated site-weighted response (RR++) by weighting the RR of six individual sites with the inverse variance (see details in Methods) for the igneous and sedimentary rock-based wetlands, respectively.

Despite similar magnitudes of change (i.e., RR++) in soil pH and Al reactivity in response to Sphagnum dominance in the igneous and sedimentary rock-based wetlands, the increase of bound OC was larger in the igneous than sedimentary rock-based wetlands (Fig. 5a). The latter result was closely related to the stronger response of Fe reactivity to Sphagnum dominance in the igneous rock-based wetlands (Fig. 5a), where Fe-bearing minerals such as olivine and pyroxene containing more transformable Fe (indicated by Fed; p<0.05; Supplementary Fig. 4) were more prone to transformation compared to phyllosilicates in sedimentary rocks33. These results were consistent with observations that very high levels of bound OC were found in the sediments of volcanic and tephra-rich locations26,40, possibly due to a high availability of reactive Fe (and Al) species therein. Additionally, bound OC was more responsive to Feo change in the igneous rock-based wetlands (reflected in the steeper slope of correlation; Fig. 5b), presumably due to larger increases of water-soluble phenols therein (Fig. 5a) as complexing reagents between organics and SRO Fe (hydr)oxides22. Hence, igneous rock-based wetlands replenished in transformable Fe (hydr)oxides may be a hotspot for Sphagnum’s enhancement on metal-organic associations.

Sequestration capacity of MAOC under the influence of Sphagnum in wetlands.
To probe whether Sphagnum-enhanced metal-organic associations may affect the accumulation of soil functional pools and elevate MAOC sequestration capacity, soils from the spatial and temporal gradients of Sphagnum expansion were physically separated into light fraction, POC and MAOC using the standard protocol41 (Supplementary Fig. 5). As expected, POC contributed the largest proportion (54±11%) of surface SOC stocks (0–20 cm) in these wetlands. However, MAOC increased more (by an average of 188%) than POC (by ~89%) from 0 to 100% coverage of Sphagnum (p<0.05; Fig. 6a), and contributed to 18–33% of surface SOC stocks in the Sphagnum wetlands (100% coverage; Supplementary Fig. 5). In the Sphagnum cultivation project in Dushan, MAOC also showed a marked increase (by ~166%; Supplementary Fig. 6a), despite loss of fine-sized particles (Supplementary Fig. 6b) due to irrigation-related soil erosion at the site. These results suggested that MAOC played an increasingly important role in surface SOC stocks in wetlands under Sphagnum expansion. Interestingly, MAOC and bound OC stocks showed similar increasing trends under Sphagnum expansion (Supplementary Fig. 5), suggesting a dominance of bound OC in MAOC in these wetlands. Indeed, bound OC and MAOC contents were closely related (r=0.93; p<0.05; n=144) and evenly distributed along the 1:1 line in all our surveyed Sphagnum-influenced wetlands, while bound OC had overall much lower contents than MAOC in non-Sphagnum wetlands (Fig. 6b). These results demonstrated that bound OC dominated MAOC in Sphagnum wetland soils due to strong mineral protection of SOC therein.

Given the paucity of published MAOC data in wetlands, we further examined the MAOC–SOC relationship in our studied wetlands in comparison with published results from uplands including forests and grasslands25 to better understand the limits of MAOC sequestration in different ecosystems. Our studied wetlands (especially Sphagnum wetlands) encompassed a much wider range and higher values of both SOC and MAOC contents (per gram of soil) than uplands (Fig. 6c). Similar to upland soils, contents of MAOC plateaued with SOC increases in non-Sphagnum wetlands (Fig. 6c), suggesting that MAOC saturated and ceased to increase when SOC reached ~150 mg SOC g–1 soil, which was higher than in upland soils (~50 mg SOC g–1 soil25). However, MAOC increased linearly with SOC in the Sphagnum-influenced wetlands within our examined range (up to 400 mg SOC g–1 soil), so that saturation of MAOC was not observed. This result suggested that Sphagnum largely increased the sequestration capacity of MAOC via promoting the formation of reactive Fe and Al (hydr)oxides in wetlands.

Methods
Synthesis of the literature data
To compare contents of bound OC and reactive Fe and Al (hydr)oxides in the surface soils (0–25 cm) of Sphagnum wetlands with other major terrestrial ecosystems, we compiled related data from the National Ecological Observatory Network (NEON) database58. We supplemented the database by searching on the Web of Science and Google Scholar with the following keywords: “wetland” OR “peatland” OR “bog” OR “fen” OR “marsh” OR “swamp” OR “permafrost” OR “forest” OR “grassland” AND “metal-organic association” OR “bound OC” OR “reactive metal” OR “oxalate-extractable Fe and Al” OR “dithionite-extractable Fe and Al”. Only references using the same extraction protocols for bound OC, i.e., by the citrate-bicarbonate-dithionite (CBD) method21, and reactive Fe and Al species (by oxalate and dithionite extractions) were included. We compiled a final dataset containing 272, 650, 520 and 714 measurements of bound OC, Feo, Alo, and Fed, respectively (Supplementary Data 1). Ancillary data including soil pH, SOC, location (longitude and latitude), clay, NPP and climate (MAT and MAP) were also collected from the literature. When data for these variables were unavailable from the original literature, the global climate database (Worldclim, Version 2.0) and high-resolution (250-m) gridded soil property database (http://data.isric.org) were used to obtain related information.

Survey of wetlands in China
Considering the paucity of investigations on bound OC in wetlands, especially Sphagnum wetlands, we supplemented the global dataset by surveying 20 Sphagnum wetlands and 29 non-Sphagnum wetlands with diverse climatic, geological and soil characteristics across China between 2019 and 2022 (Fig. 1c). The surveyed sites represent typical wetlands in China developed on the Great and Small Khingan Mountains, Sanjiang Plain, Changbai Mountain, Subalpine Basin in Central China, Qinghai-Tibet Plateau, Yunnan-Guizhou Plateau, Yangtze River and Yellow River Basins, respectively. The sites span diverse climatic zones and landscapes. The MAT ranges from –5.8 to 18.9 °C, and MAP ranges from 150 to 1635 mm. Twelve Sphagnum wetlands were paired with a non-Sphagnum wetland in the same area (six each in igneous rock- and sedimentary rock-based regions, respectively). The distance between the Sphagnum and paired non-Sphagnum wetlands varied between 500 m and 2 km. It should also be mentioned that the majority of our surveyed Sphagnum wetlands (14 out of 20) were minerotrophic, while the rest were ombrotrophic. Most of our surveyed Sphagnum wetlands also had a thin layer of peat (Supplementary Fig. 7), which differs from some Sphagnum-dominated ombrotrophic bogs characterized by thick peat layers made up of plant residuals with low mineral contents, e.g., at higher northern latitudes in North America and Europe52.

To further track the dynamic change of metal-organic associations with Sphagnum expansion, we selected three spatial gradients and one temporal gradient of Sphagnum (Fig. 1d). The spatial gradients represented natural successions of Sphagnum (coverage of 0–100%) in Jinchuan, Hani and Dajiuhu wetlands. Both Jinchuan (42°20′N, 126°21′E) and Hani wetlands (42°13′N, 126°30′E) are located on the flank of Changbai Mountains, Northeast China, developed from basalt platforms along the Longgang Volcanic field59 with the upland soils classified as Histic Andosols (WRB, 2015). MAT is 3.3 and 4 °C, and MAP is 1054 and 750 mm for Jinchuan and Hani, respectively. The dominant plant species are Sphagnum palustre, Carex schmidtii, Carex lasiocarpa and Polytrichum strictum, etc. in Jinchuan, and Sphagnum palustre, Sphagnum magellanicum and Carex lasiocarpa in Hani. Dajiuhu wetland (31°28′N, 110°00′E) is located in a subalpine basin (1700 m a.s.l.) of Mt. Shen Nong Jia, Hubei Province, surrounded by steep mountains developed from igneous and carbonate rocks60. It was formed by the combined effects of glaciers and karstification in the Late Pleistocene. The region has an MAT of 7.2 °C and MAP of 1560 mm, with upland soils classified as Histosols (WRB, 2015). The dominant plant species are Sphagnum palustre, Carex argyi, Juncus effuses and Sanguisorba officinalis. All three wetlands were sampled in July 2020 or 2021. Four Sphagnum coverage gradients were selected at each location (i.e., 0%, 20–30%, 70–80% and 100%, n=3), representing a gradual shift from Carex to Sphagnum as the dominant species.

A temporal gradient of Sphagnum cultivation was selected in Dushan (25°54′N, 107°39′E), Guizhou Province, one of the largest Sphagnum cultivation bases in Southwest China61. The region has an MAT of 15 °C, MAP of 1430 mm, and widespread Sphagnum wetlands developed on karst landforms with soils classified as Histosols (WRB, 2015). Historically, pristine Sphagnum wetlands were drained for agriculture (mainly rice paddies). In the past 20–30 years, Sphagnum has been cultivated for restoration and business purposes, producing temporal gradients from 0 to 20 years of Sphagnum cultivation (Fig. 1d). The Sphagnum cultivation fields as well as rice paddies were periodically irrigated with stream water from nearby, and the above-ground biomass was harvested every two years in autumn. In April 2021, we selected a time sequence of Sphagnum cultivation (2, 10 and 20 years; n=3) for soil sampling. For comparison, we also collected soils from pristine Sphagnum wetlands and rice paddies managed for >60 years nearby (i.e., Sphagnum cultivation time of 0 years; n=3).

At each sampling site, litter or peat layer was collected in a quadrat (20 cm × 20 cm) with the layer depth measured. The underneath mineral soil was sampled down to 20 cm with PVC pipes (diameter of 7 or 10 cm, depth of 25 cm) and transported (cooled by ice bags) to the laboratory immediately (Supplementary Fig. 7). For Sphagnum expansion gradients in Jinchuan, Hani, Dajiuhu and Dushan, mineral soils were sampling using PVC pipes (diameter of 10 cm, depth of 25 cm) with both ends sealed by plastic film. The collected soil columns were further divided into two subsamples (0–10 and 10–20 cm). For all samples, a small aliquot of the soil was immediately mixed in 0.5-M hydrochloric acid (HCl) to extract ferrous iron [Fe(II)] (described below) and saved for moisture determination, while the rest was freeze-dried and sieved (<1 mm) with roots removed by hand before further analysis.

Bulk soil properties
The field moisture content and bulk density of soil were determined by thermogravimetric desiccation at 105 °C for 24 h. Soil pH was measured at a soil:water ratio of 1:5 (w:v). The OC contents of bulk soils and litter were analyzed by an elemental analyzer (Vario EL III, Elementar, Hanau, Germany) after fumigation by concentrated HCl for 96 h to remove carbonates (no inorganic carbon was detected in fumigated soils)62. Quality control on the OC analysis was performed via repeated measurements of certified soil reference materials from the National Research Centre of China (GBW07448) and acetanilide. The accuracy (percent difference relative to the certified values; RPD%) and precision (the relative standard deviation of the sample mean value; RSD%) for the OC analysis were ±0.21% and 0.28% determined by the soil standard, respectively (n=6), and were ±0.14% and 0.17% determined by acetanilide, respectively (n=6). Organic carbon stocks (g cm–2) in the litter layer or surface soils (0–20 cm) were calculated as:

OCstock = OC × BD × D / 1000 (1)

where OC, BD and D represent the OC contents (mg g–1), bulk density (g cm–3) and depth (cm) of litter and surface mineral soils, respectively.

Water-soluble phenols were extracted by mixing 20 mL of Milli-Q water with 1 g of freeze-dried soil for 2 h, followed by centrifugation and filtration (0.45 μm), and determined using the Folin-Ciocaltu method63 at 750 nm by a Multi-Mode Microplate Reader (synergy Mx, BioTek Instruments Inc., USA). Total contents of major metals in bulk soils, including Fe, Al and manganese (Mn) were analyzed using an X-ray fluorescence analyzer (XRF; Panalytical AXIOS MAX) and denoted with a subscript ‘total’. Soil samples were prepared as pressed pellets (40-mm diameter) with a semi-automatic pressor (PrepP-01, Ruishenbao, China). Contents of the examined metals were calibrated against calibration models created with 19 certified reference soil materials (GSS1-GSS19) from the National Research Centre of China. The accuracy and precision of the applied method were checked by measurements of certified reference GSS2, GSS5 and GSS7. The accuracy (RPD%) for total Fe, Al and Mn analysis was ±1.5%, ±1.2% and ±2.2%, respectively (n=9). The averaged precision (RSD%) for total Fe, Al and Mn analysis was 1.8%, 1.4% and 2.9%, respectively, based on three certified materials.

For soil texture (clay and silt) measurement64, dried soils were treated repeatedly with hydrogen peroxide solution (10%) to remove organic matter until no bubbles were produced, and then boiled with HCl (10%) to remove lime. The residues were repeatedly rinsed with deionized water and then dispersed in sodium hexametaphosphate by sonication. Soil texture was measured using a laser diffraction using a Malvern Mastersizer 2000 particle analyzer (Malvern Instruments Ltd., UK) with particles <2 μm and of 2–50 μm defined as clay and silt, respectively.

Reactive metal species and bound OC
To probe Fe transformation and speciation in wetlands, acid-extractable ferrous iron [Fe(II)HCl] was isolated from 1 g of fresh soil using 5 mL of 0.5-M HCl immediately upon sampling. After centrifugation, an aliquot of the supernatant reacted with 5-mM ferrozine solution, and Fe(II) was measured using the ferrozine-ultraviolet absorbance method65 at 562 nm on a UV-Vis spectrometer (Shimadzu UV-2550; Japan). A standardized calibration curve of ferrous ammonium sulfate (0–50 mg L–1) was made using the same procedure described above.

Reactive pedogenic Fe and Al (hydr)oxides, which are recognized important players in SOC stabilization66,67, were examined using a selective dissolution procedure involving extraction by ammonium oxalate-oxalic acid34 and the CBD method21, respectively. Feo and Alo represent poorly crystalline or SRO Fe and Al (hydr)oxides, while Fed and Ald represent SRO and crystalline Fe and Al not bound in silicates34. The suspensions were filtered through 0.45-μm filters, and dissolved metals were quantified on an inductively coupled plasma-optical emission spectrometer (ICP-OES; ICAP 6300, Thermo Scientific, USA). Calibration solutions were prepared by dilution of certified standard solutions of Fe and Al (1000 μg mL–1). The blank control was assessed by deionized water. The determinations were performed in triplicate to guarantee quality control procedures. The accuracy (RPD%) for Fe and Al were ±1.3% and ±1.1%, respectively. The precision (RSD%) for Fe and Al were 1.5% and 1.2%, respectively (n=9). When assessing the role of reactive Fe and Al as a whole, we summarized the weight-normalized contents of Fed and Ald as “0.5Fed+Ald” to normalize the atomic mass difference between Fe and Al for graphing and statistical purposes37. To further explore the potential effect of Ca bridging on bound OC, we analyzed extractable Ca with sodium sulfate (Cas)38. Mn is also an important redox-active metal in soils. However, both total Mn and reactive (oxalate- and dithionite-extractable) Mn were two orders of magnitude lower in the soil compared with Fe and Al in Sphagnum wetlands (Supplementary Fig. 8). Mn oxides are hence not considered in this study.

A modified CBD method16,21 was used to extract Fed and Ald from soils and to release OC bound to reactive metals. Dry soils (0.25 g) were reacted with 15 mL of CBD buffer solution (containing 0.27 M trisodium citrate, 0.11 M bicarbonate and 0.25 g of dithionite) at 80 °C in a water bath for 15 min twice. After cooling, the supernatant was separated after centrifugation at 2100 × g for 10 min, and the CBD-treated soil residues were rinsed with sodium chloride (NaCl; 1 M) solution four times. An aliquot of dry soil (0.25 g) was also extracted with NaCl (0.25 M) in tandem as a control with another buffer solution (containing 1.6 M NaCl and 0.11 M sodium bicarbonate). The OC contents of soil residues after extraction by NaCl (OCNaCl) and CBD (OCCBD) were determined after freeze-drying. Bound OC (in percentage) was determined as:

Bound OC = (OCNaCl - OCCBD) / SOC × 100% (2)

The molar ratio of bound OC to metal oxides can indicate the association strength between metal oxides and organic matter, with lower values (<1) indicating sorptive interactions and high values (>1) indicating complexation or co-precipitation68. Therefore, we calculated the molar ratio as:

Bound OC:(0.5Fed+Ald) molar ratio = (bound OC / Mc) / [(0.5Fed + Ald) / MAl] (3)

where Mc and MAl represent the molar mass of carbon and Al, respectively.

Soil fractionation
To differentiate carbon accumulation in various pools of SOC under Sphagnum expansion, soils in Jinchuan, Hani, Dajiuhu and Dushan were physically separated into three fractions41. Briefly, dried and sieved soils were shaken in sodium polytungstate (SPT) solutions (1.6 g cm–3) at a soil:solution ratio of 1:10 (w:v) for 30 min at 180 rpm to gently disperse aggregates, and subsequently centrifuged at 2800 × g for 15 min to isolate light fraction. The light fraction and high-density soil residues were washed with Milli-Q water until the solution conductivity was <50 μS cm–3. The high-density residues were then wet sieved to obtain particulate organic matter (POM; >53 μm) and mineral-associated organic matter (MAOM; <53 μm). All three fractions were freeze-dried, weighed and ground to <0.25 mm in an agate mortar. The OC content of each fraction was analyzed as described previously.

To compare bound OC and MAOC contents, we further separated MAOM from the remaining soils of our surveyed 49 wetlands according to ref. 69. Briefly, dried and sieved soils were wet sieved through 53 μm. Size fractions of <53 μm were further separated by density using SPT solutions (1.6 g cm–3), removing light fraction and POM from MAOM. The resulting fraction (MAOM<53μm) was repeatedly rinsed with Milli-Q water until the electric conductivity was <50 μS cm–3. Samples were subsequently collected by centrifugation, freeze-dried, weighed and ground to <0.25 mm in an agate mortar. The OC content of MAOM was analyzed as described previously.

Statistical analysis
Data analysis was performed using the R software (version 4.1.3; R Core Team, 2022). Difference between groups was analyzed by one-way ANOVA. Two-way ANOVA was used to examine the effects of Sphagnum coverage and sites on the investigated parameters along the Sphagnum expansion gradient, followed by one-way ANOVA to test the effect of Sphagnum coverage or cultivation time for a specific site. Relationships between the tested variables were explored using Spearman correlation. PCA was conducted to examine the variance of bound OC with its potential influencing factors across different wetlands. Differences and correlations were considered to be significant at a level of p<0.05.

To compare soil’s responses to Sphagnum’s influence, i.e., in Sphagnum relative to the paired non-Sphagnum wetland, RR was calculated for individual site70:

RR = ln(Xt) - ln(Xc) = ln(Xt / Xc) (4)

where Xt and Xc are the reported means for each soil variable in the Sphagnum and paired non-Sphagnum wetlands for each site, respectively. The variance (v) of the logarithmic effect size was calculated as follows:

v = S2c / (Nc X2c) + S2t / (Nt X2t) (5)

where Sc and St are the standard deviations of Xc and Xt, and Nc and Nt are the sample sizes of data. The weighting function was calculated based on the reciprocal of the variance in individual RRs:

w = 1 / v (6)

We further consolidated the RR of individual site to evaluate site-weighted response (RR++) for the igneous- and sedimentary rock-based wetlands, respectively. The weighted RR (RR++) was calculated from the individual RRij (i=1, 2…, m; j=1, 2…, k) by pairwise comparison between the Sphagnum and paired non-Sphagnum wetlands, where m is the number of groups and k is the number of comparisons in the ith group:

RR++ = (Σmi=1 Σki j=1 wij RRij) / (Σmi=1 Σki j=1 wij) (7)

The standard error (S) of RR++ was estimated as follows:

S(RR++) = 1 / sqrt(Σmi=1 Σki j=1 wij) (8)

The 95% confidence interval (95% CI) was RR++ ± 1.96 S(RR++). The difference was considered to be significant, if the 95% confidence interval did not overlap between the igneous and sedimentary rock-based wetlands.

Data availability
All data (including compiled data) supporting the findings are available online in the Supplementary Data or the Figshare data repository (https://doi.org/10.6084/m9.figshare.23545557)71. Additionally, the raw data of National Ecological Observatory Network (NEON) database is available at https://portal.edirepository.org/nis/mapbrowse?packageid=edi.999.158.

Code availability
Data analysis was carried out using R v. 4.1.3. which is publicly available at https://www.r-project.org. The supporting code is provided at Figshare (https://doi.org/10.6084/m9.figshare.23545707)72.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Dear Editor,\nThank you for the opportunity to contribute and comment on the manuscript NCOMMS-23-15886-T\nentitled \"Sphagnum lifts soil's sequestration capacity of mineral-associated organic carbon via\nactivating metal oxides,\" written by Dr. Feng and colleagues.\nIn this study, the researchers conducted a field and literature survey of terrestrial ecosystems to\nevaluate the contribution of mineral-associated organic carbon (MAOC) to the ecosystem's SOC\npool. MAOC refers to organic carbon physically bound or associated with mineral particles in the\nsoil. It is more stable and resistant to decomposition than particulate organic carbon (POC), with a\nlifetime of approximately 50 years. MAOC can persist for hundreds of years, playing a crucial role\nin carbon sequestration and nutrient cycling. However, the formation of metal-bound organic\ncarbon in Sphagnum-dominated ecosystems is seldom investigated.\nThe authors emphasize the significance of understanding how Sphagnum enhances MAOC\nsequestration by activating metal oxides. This knowledge would unveil a previously overlooked\nmechanism for SOC sequestration in Sphagnum-dominated wetlands. To support these claims, in\nthis well design study, the authors collected surface soil from 49 wetlands sites across China, forty\npercent of them dominated by Sphagnum peat moss. The sampled sites represent a wide range of\nclimatic zones with annual average temperatures ranging from -5 to 19 C and precipitation\nregimes from 150 to 1635 mm. Additionally, the authors collected metadata from previously\npublished observations across the globe. A combined dataset included 1000 observations.\nThe authors concluded that Sphagnum-dominated ecosystems exhibit notably higher levels of\nSOC, including MAOC, and phenolic compounds, than other ecosystems. Additionally, the authors\nobserved a significant correlation between Sphagnum surface coverage, phenolic compounds, and\nMAOC.\nOverall, current studies not only support the ecological role of Sphagnum as an ecosystem\nengineer but also propose a new role as a \"rust engineer\" for peat moss, influencing the carbon\npool in Sphagnum-dominated wetlands. Nevertheless, the author might be willing to correct/clarify\na few points below.\nLine 27: \"significant implications for long-term soil carbon sequestration.\"\nPlease clarify which implications do you refer.\nLine 73: Please add \"s\" wetland Â wetlands\nLine 119-121: \"By contrast, Sphagnum-dominated wetlands did not differ from other examined\necosystems\nin geographical distributions, soil clay content, net primary productivity (NPP), mean annual\ntemperature (MAT), or precipitation (MAP; Supplementary Fig. 1).\"\nThe authors indicate no significant difference from other examined ecosystems; however, based\non Figure S1, this is not always the case. Therefore the authors consider revising the text to\nprovide a more accurate description of the observations.\nLine 251: Please remove the word \"both\"\nLine 326: Please split it into two sentences.\n…National Ecological Observatory Network (NEON) database. We supplemented the database…\nLine 350: please remove \"Among them\"\nLine 383: please remove the word \"from\"", "reviewer_2": "Summary. Sphagnum is known to play a critical role in soil carbon storage on a global scale, with\nsoils in Sphagnum-dominated wetland currently storing massive amounts of carbon. It is well\nknown that much of this carbon storage is regulated by slow decomposition of Sphagnum under\nanaerobic conditions in wetlands, but the current project demonstrates that Sphagnum can also\nincrease mineral-associated organic carbon storage by activating metal oxides (via phenolic and\nacid metabolites). The work uses published data as well as comparison of multiple spatial\ngradients (transition from Carex to Sphagnum coverage) and a temporal gradient (time since\nSphagnum cultivation) to arrive at this finding. Overall, this is a well-written manuscript, and there\nis strong, central story implicating Sphagnum as a key species in increasing MAOC in wetlands. I\ndo, however, have a number of comments that the authors might consider.\nGeneral Comments.\nMy largest concern with the current paper reflects my own experiences working a Sphagnum-\ndominated wetlands. In particular, much of my own research occurs in ombrotrophic bogs in the\nboreal zone of North America. I had a hard time conceptualizing how the current work would apply\nto these systems. These wetlands typically have meters of peat above the basal mineral layer and\nthat amount of minerals in these precipitation-fed systems is by definition very small. Maybe I’m\nshowing my ignorance with this comment, but I have a really hard time believing that up to 33%\nof SOC could be in MAOC in these systems – I just don’t see how the mineral content could be this\nhigh. Similarly, these bogs almost certainly have far lower clay content than the Sphagnum-\ndominated systems in the current study (Supp Fig 1).\nI do realize that many of the wetlands explored in the current manuscript are very different with\nsignificantly higher mineral content than the systems that I work in. Perhaps my concern is just a\nsystem-familiarity issue (or a systems bias), but I think it needs to be addressed. The reality is\nthat true ombrotrophic bogs in the boreal zone are almost exclusively “Sphagnum-dominated” and\nthat these wetlands are responsible for a massive chunk of the global soil organic carbon stored in\nwetlands (this larger carbon stock is appropriately used to frame and justify the current work). The\n“conventional wisdom” that the authors describe in L35-39 seems to come close to the definition of\npeat that makes up the soils in these histosols.\nAt a minimum, I would encourage the authors to clarify if these types of wetlands were covered in\ntheir survey and what this means for the interpretation of their results. The fact that pH of\nSphagnum-dominated wetlands was between 4-5 makes me think that true bogs were likely not\nthe majority of systems studied, and their spatial gradient include Carex-dominated systems which\nare almost certainly more minerotrophic. I don’t think that this would invalidate the results of the\ncurrent work in any way, but it would provide important context.\nI am not sure that “lifts” is the right word for the title (although I confess that this may be a\nmatter of personal preference in writing). I would if “increases” would be more effective. The\nauthors are consistent with using “lift” throughout the manuscript. If the title is changed, it should\nbe changed throughout (e.g., L51, L60, L92, L234, L282).\nI wonder if there would be value in exploring next steps for this work. The authors ascribe the\nincreased MAOC associated with Sphagnum coverage to acidity, phenolics and increased moisture\ncontent. These seems like reasonable hypotheses based on the correlative approach used in the\ncurrent work; but, these hypothesis can (and should) be tested with future experimental\napproaches. These factors are all co-correlated and there could be additional co-correlates as well.\nSpecific Comments.\nL20. Consider removing “outstanding”.\nL34. I think that saying that our understanding of why carbon is accumulated in Sphagnum-\ninfluenced wetlands in “rudimentary” is an overstatement. While there are still knowledge gaps,\nwe do know quite a bit about how these systems work, right?\nL47. I believe that environment should be plural, i.e., “environments”\nL60. I believe that wetland should be plural, i.e., “wetlands”\nL75. Consider removing “To bridge the above knowledge gaps, here”. I think you can be more\nconcise and simply say “Here, we employed two complementary…”\nL76. Consider replacing “Firstly” with “First”, i.e., “We FIRST compared…”\nL84. Consider replacing “Secondly” with “Second”, i.e., “SECOND, we selected…”\nL410. Should you spell out “CBD” here? I believe this is the first time you use the acronym.\nL688. In the legend of Fig 3, you use “(e)” instead of “(g)” to describe the final panel. Simple typo\nto be correct.", "reviewer_3": "Review of Zhao et al. 2023, Nature Comms\nI read this manuscript with interest, and overall, I think it’s a great piece of work, and deserves\nto be published in Nature Communications. The results are extremely interesting, and the\ndemonstration of mineral associated organic carbon variability in wetlands is very useful. Of\nparticular interest to me are the conclusions relating to Fe oxide supply from volcanic\nenvironments, but then I’m a little biased as that’s my specific area of interest! The data as\npresented support the conclusions, although I think a bit more detail on statistical methods is\nnecessary. The methodology appears sound, but the description of the approaches is lacking much\nof the information needed to be certain of this. Below I have a number of comments and\nsuggestions, but nothing which would preclude publication once the changes have been made.\nGeneral comments\nLanguage in the abstract is a little exaggerated – I would tone it down. E.g. using terms such as\n‘outstanding’, ‘unique’, ‘incredible’ makes it sound like the authors need to oversell the story.\nThe data will speak for themselves, without the need to exaggerate anything.\nIntroduction has too many hypotheses, and gives away the findings of the main discussion prior to\neven explaining how the work will be done. I explain in more detail below, but I would save the\ndiscussion of MAOC hotspots until after the data which shows peatland MAOC levels has actually\nbeen presented.\nI think the results are presented in a manner which is a little too certain. The conclusions from the\ndata appear reasonable, but are based on correlations and PCA alone- I would maybe just tone\ndown the certainty of the language. I include a couple of specific points in my comments below.\nI think I understand what ‘response ratio’ means, but it was not clear. As this is a primary result\nof your work, I would include simple steps to explain exactly how you got the numbers in the text,\nand not just in the methods. Similarly, how is the site-weighting done?\nUse of stats throughout is a little unclear as to what is being presented. Often a p-value is shown\nwith no n, and no indicator of which test/measure it relates to.\nFormat is a little strange. The results end and are followed by what looks like a series of\ndiscussion paragraphs, but then a final discussion is added after this. I would adjust it so that all the\nparagraphs of discussion are after the heading discussion, or change what is currently headed\n‘discussion’ to ‘implications’ as that’s what it is basically.\nThe methodology is light on detail for many of the techniques used here. Further information on a\nnumber of the approaches (especially XRF and ICP-OES) is necessary. Without this I cannot be\ncertain the results are reliable.\nSpecific comments\nI think the term ‘lift’ in the title is misused. I would prefer ‘raises’ or ‘increases’\nL29: Use of ‘remarkable’ is an example of what I mention above. No need to oversell the work,\nespecially in the introduction.\nL29: I think a sentence setting the scene for the readers would be good here. How important is\nSOC for global carbon cycling? How about peatlands? Comparisons to other carbon sinks would be\nnice. As I say, just a sentence, but something big-picture.\nL39-42: It would be nice to have a but more info on what MAOC exactly is. Which minerals? What\nare the primary processes? I know you explain a little more in the following paragraph, but in this\none it would be good to have a basic explainer which means I can then move into the detail in the\nfollowing paragraph with a clear idea of what the subject of this paper is.\nL45: Where do the oxides come from? Are they lithogenic?\nL46: ‘metallic’ or ‘mineral’ protection? You use the term mineral-associated OC, surely Fe\noxides are the mineral you’re talking about, so I would say mineral.\nLine 56-57: Re-order the sentence – ‘Hence, it is essential…’\nL62-74: This is all speculative and I would rewrite it without the first and last sentences. The\ndiscussion of where Fe and Al may come from is important to include, but not a hypothesis that\nvolcanic regions are best suited to MAOC enrichment – this is your hypothesis and should not be in\nthe introduction. It very much sounds like the results are presented before the analyses have even\nbegun… We would say this is the cart leading the horse – wrong order!\nL89-92: I don’t like the presentation of a conclusion at the end of the introduction. I would rather\nyou make a general comment on how your approach is intending to study the topic, not that it is\ncertain your results will prove the point you want to make.\nL107: I would use the abbreviation ‘SE’ not s.e.m. as this confuses it with the technique\nL113-114: Again, I wouldn’t say ‘extraordinary’. Also, the tense seems to have changed – keep in\npresent tense or change everything to past!\nL115: Need to explain SRO, and briefly re-iterate what it means when OC is oxalate-extractable.\nWhat fraction is this?\nL118: What are the p-values relating to? I assume from the figure it’s ANOVA, but in the text this\nis not clear. As you use them quite a bit in this section, just clarify somewhere exactly what they\nrefer to and what they are showing.\nL125: Again, I would not use ‘extraordinary’.\nL141-144: Can you show these data and associated scatter plots somewhere?\nL145-147: really nice results, on first glance, but this is just correlation and PCA-based. I think\ntherefore this line is a little too ‘certain’. I would like you to at least consider the possibility here\nthat some of the correlations are not entirely causation driven. Don’t get me wrong- I think the\nresults are solid, just that a little more caution in the language would be nice .\nL165: do you mean ‘between’ the percentages rather than ‘by’?\nL170: Again, what’s the p-value referring to? Make it clearer for the reader…\nL170-174: I don’t really like this summation sentence – the use of ‘unique’ is a little excessive,\nand I feel you’re just repeating what you say above.\nL177: I think the word order/choice is wrong here, I would say ‘As Sphagnum primarily enhanced\nmetal-organic associations by…’\nL178: I am not a fan of asking hypotheses in the text. Rephrase this so it’s just making a point\nabout an aspect which needs to be tested.\nL180: Should be ‘igneous’ not ‘volcano’\nL183-185: The idea of a response ratio needs explaining further. How exactly did you calculate\nit? Further, how is the weighting done for the site-weighted response. This is all a little ‘black box’ as\nit is.\nL191-193: Very interesting finding. We see something similar in marine sediment (e.g.\nhttps://doi.org/10.1029/2021GB007140) – very high levels of OC bound to FeR in and around\ntephra layers. I assume reactivity, and potentially speciation of Fe explain this. Or is it just\nbecause more FeR is available generally? Maybe add a line or two to discuss this point.\nL234: What does ‘activating’ mean in this context?\nL237: I think ‘implications’ is a better title for this section. The previous 3 paragraphs were\ndiscussion already.\nL240: again, I wouldn’t use ‘outstanding’ here, or ‘extraordinary’ in L241. The data are great- they\ndon’t need to be over-sold!\nL263: Maybe again use ‘mineral’ rather than ‘metallic’\nL280-283: repeating the same words here – always an ‘incredible rust engineer’. I would vary the\nlanguage a bit more.\nL284: Could you speculate on what would happen if a volcanic ash input event (not a long-term\nprocess, but a similar end result) were to happen? This would supply highly reactive volcanic\nmaterial – maybe worth looking at for future study.\nL336: how did you find locations which had a Sphagnum wetland right next to a non-sphagnum\none?\nL372: How was the soil sampled? Did you take precautions not to contaminate with (potentially\nrusty) metal tools?\nL385: how was accuracy and precision estimated for the EA analyses? Reference materials?\nFurther info needed here.\nL387; More detail on the fumigation needed. How did you test for carbonate removal?\nL395-398: A lot more detail on the XRF analyses needed here. How were samples prepared\n(pressed pellets/glass beads)? How were accuracy & precision measured and quantified?\nWhich reference materials/standards were used?\nL398: Do you mean grain size? Not ‘texture’? If so, where were these data presented? How did\nyou interpret them? Which aspects of the output were used?\nL407: Can you provide more info on the UV analysis method? How was calibration done? Any\nexternal reference materials used to check for accuracy?\nL410: Define CBD\nL412-415: Again, a lot more detail for the ICP-OES analysis is needed. How was calibration done?\nHow were blank levels monitored? Accuracy and precision? Any estimate of extraction efficiency?\nL423: By which method exactly? Do you present the control experiment data anywhere?", "reviewer_author_response": "Response to Reviewers of NCOMMS-23-15886-T\nReport from the Editor\nDear Dr Feng,\nThank you again for submitting your manuscript \"Sphagnum lifts soil’s sequestration capacity\nof mineral-associated organic carbon via activating metal oxides\" to Nature Communications.\nWe have now received reports from 3 reviewers and, on the basis of their comments, we have\ndecided to invite a revision of your work for further consideration in our journal.\nAll reviewers make a number of constructive comments to help you increase the impact of\nyour study. In particular you will need to provide more detail on methods (particularly\nstatistical techniques), add discussion on how your findings may (or may not) apply to areas\nwith thick peat layers, and tone down your language where appropriate adding discussion on\nuncertainty/alternative possibilities.\n[…]", "reviewer_including": "n(1) Provide more details on the methods (in response to Reviewers #3);\n(2) Add discussion on the implications/applicability of our findings to Sphagnum-dominated\nombrotrophic bogs with thick peat layers and low mineral contents (in response to\nReviewer #2);\n(3) Adjust and tone down our language (in response to Reviewer #1, #2 and #3).\nAs detailed below, we provide response to each of the reviewer’s comments/concerns on\na point-by-point basis. The original comments are listed below in black and our replies follow\nin blue font. Line numbers listed below corresponds to the revised version with changes\nhighlighted in red in the main text. We sincerely hope that the revised version addresses all\nissues raised by three reviewers. Thank you again for your consideration.\nReviewer #1 (Remarks to the Author):\nDear Editor,\nThank you for the opportunity to contribute and comment on the manuscript NCOMMS-23-\n15886-T entitled \"Sphagnum lifts soil's sequestration capacity of mineral-associated organic\ncarbon via activating metal oxides,\" written by Dr. Feng and colleagues.\nIn this study, the researchers conducted a field and literature survey of terrestrial ecosystems\n1 / 19\nto evaluate the contribution of mineral-associated organic carbon (MAOC) to the ecosystem's\nSOC pool. MAOC refers to organic carbon physically bound or associated with mineral\nparticles in the soil. It is more stable and resistant to decomposition than particulate organic\ncarbon (POC), with a lifetime of approximately 50 years. MAOC can persist for hundreds of\nyears, playing a crucial role in carbon sequestration and nutrient cycling. However, the\nformation of metal-bound organic carbon in Sphagnum-dominated ecosystems is seldom\ninvestigated.\nThe authors emphasize the significance of understanding how Sphagnum enhances MAOC\nsequestration by activating metal oxides. This knowledge would unveil a previously\noverlooked mechanism for SOC sequestration in Sphagnum-dominated wetlands. To support\nthese claims, in this well design study, the authors collected surface soil from 49 wetlands sites\nacross China, forty percent of them dominated by Sphagnum peat moss. The sampled sites\nrepresent a wide range of climatic zones with annual average temperatures ranging from -5 to\nmetadata from previously published observations across the globe. A combined dataset\nincluded 1000 observations.\nThe authors concluded that Sphagnum-dominated ecosystems exhibit notably higher levels of\nSOC, including MAOC, and phenolic compounds, than other ecosystems. Additionally, the\nauthors observed a significant correlation between Sphagnum surface coverage, phenolic\ncompounds, and MAOC.\nOverall, current studies not only support the ecological role of Sphagnum as an ecosystem\nengineer but also propose a new role as a \"rust engineer\" for peat moss, influencing the carbon\npool in Sphagnum-dominated wetlands. Nevertheless, the author might be willing to\ncorrect/clarify a few points below.\nReviewer #2 (Remarks to the Author):\nSummary. Sphagnum is known to play a critical role in soil carbon storage on a global scale,\nwith soils in Sphagnum-dominated wetland currently storing massive amounts of carbon. It is\nwell known that much of this carbon storage is regulated by slow decomposition of Sphagnum\nunder anaerobic conditions in wetlands, but the current project demonstrates that Sphagnum\ncan also increase mineral-associated organic carbon storage by activating metal oxides (via\nphenolic and acid metabolites). The work uses published data as well as comparison of\nmultiple spatial gradients (transition from Carex to Sphagnum coverage) and a temporal\ngradient (time since Sphagnum cultivation) to arrive at this finding. Overall, this is a well-\nwritten manuscript, and there is strong, central story implicating Sphagnum as a key species\nin increasing MAOC in wetlands. I do, however, have a number of comments that the authors\nmight consider.\nReviewer #3 (Remarks to the Author):\nReview of Zhao et al. 2023, Nature Comms\nI read this manuscript with interest, and overall, I think it’s a great piece of work, and deserves\nto be published in Nature Communications. The results are extremely interesting, and the\ndemonstration of mineral associated organic carbon variability in wetlands is very useful. Of\nparticular interest to me are the conclusions relating to Fe oxide supply from volcanic\nenvironments, but then I’m a little biased as that’s my specific area of interest! The data as\npresented support the conclusions, although I think a bit more detail on statistical methods is\nnecessary. The methodology appears sound, but the description of the approaches is lacking\nmuch of the information needed to be certain of this. Below I have a number of comments and\nsuggestions, but nothing which would preclude publication once the changes have been made.\n9 / 19", "reviewer_respectively\nreference": "nSchwertmann, U. & Cornell, R. M. Iron oxides in the laboratory: preparation and characterization. John\nWiley & Sons (2008).\nL423: By which method exactly? Do you present the control experiment data anywhere?\nReviewer #2 (Remarks to the Author):\nThe authors have done a fine job of addressing my comments and concerns from the first\nsubmission and those of the other 2 Reviewers. This effort has resulted in a stronger manuscript\nand I only have minor editorial comments on the revised manuscript.\nSpecific Comments:\nL29. Peatland should be plural, add an ‘s’.\nL72-74. I found the added sentence beginning “It will be interesting” to be awkward as written and\nwould suggest a rewrite. Perhaps something like “There is little information, however, on how\nSphagnum’s enhancement of metal-organic associations (if present) vary across these different\ngeological settings.”\nL113-114. Consider moving Al and Fe before the lengthy parenthetical in this sentence. i.e.,\n…content of oxalate-extractable Al and Fe (Alo and Feo, i.e., SRO; representing…. As written the\nminerals that were extracted in oxalate get lost.\nL126. Consider removing “notable”.\nL154. Consider replacing “proven by” with “investigated with”. You investigated this effect, you\ndidn’t prove it.\nL181. Consider rewriting “we wanted to further text” with “we further tested”.\nL227-232. This section is awkward. I wonder if you could tighten this up a bit by removing the\nsentence beginning “To confirm this…”. Consider something like, “Indeed, bound OC and MAOC\ncontents were closely related….”\nL233. Should “metallic protection” be “mineral protection” for consistency here?\nL236. Consider replacing “to glean on the limit” with something like “to better understand the\nlimits of”.\nL311. Replace “may differ” with “differs”. There is no uncertainty here – boreal bogs by\ndefinition have a thick layer of peat (for example, in the US histosols have a minimum of 40 cm of\norganic matter).\nL360. Replace “rests” with the singular “rest”.\nL363. Consider removing the parentheses and saying “at higher northern latitudes in North\nAmerica and Europe”.\nReviewer #3 (Remarks to the Author):\nI have read through the resubmission of Zhao et al., and am happy to see they have implemented\nall suggested changes.\nI am happy to recommend acceptance as is, but would suggest a final read through by the authors\nto double check grammar.\nRESPONSETOREVIEWERS' COMMENTS\nReviewer#2(RemarkstotheAuthor):\nThe authors have done a fine job of addressing my comments and concerns from the first\nsubmission and those of the other 2 Reviewers. This effort has resulted in a stronger\nmanuscript and Ionlyhaveminoreditorialcommentsontherevisedmanuscript."}, "author_response": ""}]

Decision letter