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Variable names, units and timestampsStreamflow is runoff routed along a drainage network, in m3/s, also known as discharge, which is the variable name used in the files. Water temperature is given in units of Kelvin. Filenames include the variable name, GCM, scenario (hist for historical, or one of the RCPs) and the time period (years). The timestamps in the files reflect the last date of the period over which the output was averaged, so the first timestamp of the weekly averages is January 7th 1976.Ecologically-relevant variablesThe ecologically-relevant streamflow and water temperature variables derived from the weekly values are established based on a combination of classification frameworks, i.e., indicators of hydrologic alteration19, terrestrial bioclimatic variables in the worldclim dataset20 as well as the CMCC-BioClimInd dataset21, aggregated accordingly: 1976–2005 (1979–2005 for E2O); 2021–2040; 2041–2060; 2061–2080; 2081–2099. The scripts used to compute these derived variables can be found under Code Availability.For files containing information on timing (see Tables 2–3), note that the counting is 0-indexed. So week numbers run from 0 through 51, months from 0 to 11. For timing of quarters, 0 is DJF, 1 is MAM, 2 is JJA, 3 is SON. The week number (for WT-wmin, WT-wmax, Q-wmin, Q-wmax) is determined as the mode, i.e. the most frequent week number within a period. For each period (20, 25 or 30 years) we looked for the week number in which the minimum or maximum water temperature or discharge occurs. If that happens most often in week X, that week number is stored. It can however occur that a certain minimum/maximum temperature or discharge occurs equally often in multiple weeks – then we assign a missing value.The variables Q-bfi and Q-vi are calculated according to Pastor et al.30. The baseflow index is an indicator of the importance of stored sources; a high index indicates that flow is mostly sustained by stored sources such as groundwater.Scripts used to create the derived variables are available through the FutureStreams GitHub repository (see Code Availability below).Multi-model set-upWe provide future scenarios for four RCPs (representative concentration pathways; 2.6, 4.5, 6.0 and 8.5 W/m2 in 2100) for the five ISI-MIP GCMs. Projections differ across RCPs due to differences in greenhouse gas forcing, and across GCMs due to differences in e.g model parameterization and resolution. Generally the spread across GCMs is larger than that across RCPs7,31. When interested in the general effect of climate change, users are advised to use the mean or median across the GCMs, rather than selecting a specific GCM. When interested in the spread across GCMs, users can explore or represent that in various ways, such as color intensity indicating agreement amongst models5, bar or violin plots7 etc.Warming levelsTo facilitate assessments and comparisons of streamflow and water temperature at a certain air temperature rise rather than specific years5,7, we provide a table with the years in which each GCM/RCP reaches the global mean temperature rises 1.5°, 2.0°, 3.2°, 4.5° compared to pre-industrial temperatures (as used by Barbarossa et al.7) with our scripts (see Code Availability). These years represent the central value of a 30-year running mean, so users should evaluate the 30-year mean (or other statistic) of discharge or water temperature centered around the year that a certain warming level is reached, which is specific to each RCP and GCM combination. For instance, if 1.5° warming is reached in 2040, the 30-year period 2025–2054 should be considered.GCMs, bias-correction and reanalysis dataThe majority of our simulations are forced with meteorological time series from GCMs. Those are bias-corrected27 before being applied to impact models such as PCR-GLOBWB, which corrects for systematic deviations of the simulated historical data from observations. For instance, for temperature the offset in average temperature in the historical GCM simulation with respect to observations is subtracted from temperatures in all scenarios of that GCM. The bias-corrected GCM forcing should thus well represent climatology, but not necessarily timing of actual events such as floods and droughts. Reanalysis data is created by assimilating observations into weather models, to obtain consistent and globally complete time series. The output of the simulation forced with meteorological time series from the (E2O) reanalysis data should therefore reflect not only the average streamflow and water temperatures, but also timing of actual events such as droughts.If users want to check for themselves how the GCM-forced historical simulations discussed here deviate from reanalysis-forced simulations, they can use the output from the E2O-forced simulation provided here, the monthly output linked to Wanders et al.13 (see also Code Availability) or the daily output of those simulations which are available from Niko Wanders upon request. The latter are forced with ERA-40/ERA-Interim reanalysis data.Notes of cautionBeware of temperature in grid cells where streamflow is low, which can cause temperatures to become unrealistically high due to strong fluctuations in the water level. The computational timesteps currently implemented in DynWat are not sufficiently small to provide stable solutions for these conditions. For some lakes and reservoirs we observe a similar problem when lakes expand or shrink as a result of water levels changes. These locations can be masked and we can assume that water temperature follows the air temperature for these very shallow water layers. A file with locations of lakes and reservoirs is provided in the data repository (under indicators/mask) so users can mask these if desired.Furthermore, we provide masks for each GCM-RCP-period which users can apply to the derived variables if desired. These masks are based on Q-mean and WT-mean and thresholds of 10 m3/s and 350 K, respectively. They can be found in the data repository (i.e. indicators/waterTemperature/WT-mask). The scripts used to create these masks are provided through the FutureStreams GitHub repository (see Code Availability below), which can be used to create masks with different thresholds. These scripts are called mask_unrealistic_values.py and maskFunctions.py.We also provide scripts to mask out unrealistic values directly in the weekly Q and WT files, these scripts are mask_unrealistic_values_weekly.py and maskFunctions_weekly.py. In all these scripts the threshold for discharge is set to 10 m3/s and for water temperature to 350 K, but users can change those to their preferred values. The threshold value will be included in the resulting output file name.Furthermore, we encountered spin-up issues in some pixels for the future RCP simulations. Instead of following the temperatures from the end of the historical simulation, temperatures drop at the beginning of the future simulation, so the first few weeks of 2006 temperatures can be unrealistically low. In Fig. 2, output of the year 2007 is used for the year 2006 .Fig. 2Water temperature [°C] anomaly. The maps show the difference between the mean water temperature over the period 2070–2099 (RCP8p5) and the historical period 1975–2005. The map shows values only for rivers with streamflow greater than 50 m3/s and the width of the rivers is scaled based on the streamflow values for clarity of representation. Insets below the map show the original gridded resolution at 5 arcminute for cells with streamflow values greater than 10 m3/s. The bottom insets show water temperature time series sampled at specific grid-cell locations (white crosses in the insets) for the Amazon (−57.2083° longitude, −2.625° latitude), Danube (20.125° lon, 45.2083° lat) and Ganges (88.375° lon, 24.375° lat). Time series are represented for each GCM and RCP available within FutureStreams; thin lines represent weekly values, thick lines represent 10 year rolling means.Full size imageFig. 3Streamflow [m3/s] anomaly. The maps show the difference between the log10 transformed mean streamflow over the period 2070–2099 (RCP8p5) and the log10 transformed mean streamflow over historical period 1975–2005. The map shows values only for rivers with streamflow values greater than 50 m3/s and the width of the rivers is scaled based on the streamflow values for clarity of representation. Insets below the map show the original gridded resolution at 5 arcminute for cells with streamflow values greater than 10 m3/s. The bottom insets show water temperature time series sampled at specific grid-cell locations (white crosses in the insets) for the Amazon (−57.2083° longitude, −2.625° latitude), Danube (20.125° lon, 45.2083° lat) and Ganges (88.375° lon, 24.375° lat). Time series are represented for each GCM and RCP available within FutureStreams; thin lines represent weekly values and thick lines represent 10 year rolling means.Full size imageFig. 4Anomalies for selected ecologically relevant derived variables (bioclimatic indicators) for the same areas in the Amazone (left), Danube (middle) and Ganges (right) basins as used in Figs. 2 and 3. Differences are shown between RCP8.5 2080–2099 and 1976–2005. WT-cq is the water temperature of the coldest quarter, WT-range is temperature range, Q-max is maximum streamflow, Q-dm is streamflow of the driest month (see also Tables 2 and 3 below). For streamflow we show the difference between log10-transformed flow.Full size image More

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The water table depth, surface water body area, and surface ecological processes have all changed significantly during the 20 years the ecological water conveyance projects have been underway in the lower reaches of the Tarim River. Specifically, there has been a notable increase in the water table, surface water body area, vegetation density and coverage, the vegetation index (NDVI), Net Primary Production (NPP) of natural vegetation, and ecosystem function and health. The following sections provide details on these changes.Changes in groundwater table depthGroundwater (soil water) is the most important water source for maintaining natural vegetation in the lower reaches of the Tarim River, as the climate is extremely arid and atmospheric precipitation has little ecological significance. The changes in water table depth are directly related to the composition, distribution, and growth of the natural vegetation of the desert riparian forest, which in this case is mainly P. euphratica5. During the past 20 years, the ecological water conveyance in the lower reaches of the Tarim has been intermittent, and the groundwater table elevation has been closely related to the water conveyance. From the analysis of the groundwater table’s rise in the upper, middle, and lower reaches of the Tarim River (Fig. 1), the magnitude of the uplift is clearly related to four crucial factors: the groundwater table depth prior to the water conveyance, the volume of water discharge, the duration of the transfer, and the water head location.Figure 1Changes in groundwater depth of typical monitoring cross-sections pre- and post-conveyance of water in the lower reaches of Tarim River from 2000 to 2020. Yengsu, Karday, Argan and Yikanbujima are four monitoring sections in the lower reaches of Tarim River. “#1”is the No. 1 groundwater level monitoring well on each monitoring section, which is located 50 m away from the river.Full size imageIn the early stages of the water conveyance projects (2000–2010), the groundwater table in the upper and middle segments of the lower reaches of the Tarim River rose to a relatively large extent, while the groundwater table in the lower segment of the river only showed an increasing rising trend after 2011. The monitoring results reveal that after nearly 20 years of ecological water conveyance, the groundwater table in three sections of the lower reaches of the Tarim has been affected at a range of more than 1000 m. The three sections are the Yengsu section in the upper segment, the Karday section in the middle segment, and the Yiganbujima section in the lower segment. Furthermore, the groundwater table has risen by 2.69, 1.38 and 1.59 m, respectively, in these three sections22. Within 100 m from the river, the water table depth rose from 7.76, 9.31 and 7.82 m prior to ecological water conveyance to 3.70, 4.48, and 2.69 m, and 4.06, 4.83, and 5.13 m, respectively, after it. Within 500 m from the river, the water table rose by 1.6, 3.99, and 5.26 m, respectively. The shallow groundwater in the lower reaches of the Tarim River has also been recharged to a certain extent, and the lateral influence range is still gradually expanding.Changes in water body areaThe changes in water body area in the lower reaches of the Tarim River are closely related to the amount of water delivered via conveyance. During the past 20 years, the surface water body area, seasonal water body area, and permanent water body area all decreased to the lowest point in 2009, with the river water failing to reach Taitema Lake, the river’s terminal, in 2006, 2007, and 200923. The surface water body area, seasonal water body area and permanent water body area in the river’s lower reaches fluctuated and increased during the ecological water conveyance process. In particular, the seasonal water body area in the upstream section showed a significant expansion. The area increase rate of surface water, seasonal water, and permanent water in the middle section from Yengsu to Argan is 1.75 km2 a−1, 1.58 km2 a−1, and 0.16 km2 a−1, respectively. Similarly, the area of surface water bodies, seasonal water bodies, and permanent water bodies in the lower section (below Argan) increased at the rate of 13.48 km2 a−1, 8.24 km2 a−1, and 5.23 km2 a−1, respectively. It is worth mentioning that the area of surface permanent water body and seasonal water body in Taitema Lake significantly increased, with the area of the lake waters expanding 417.08 km2, from 38.19 km2 in 2000 to 455.27 km2 in 2019. This represents a nearly 12-fold increase (Fig. 2).Figure 2Spatial distribution of water surface area in lower reaches of Tarim River in 2000 and 2019. The subfigures were generated in R 4.0.2 (https://cran.r-project.org/bin/windows/), and then merged in Microsoft PowerPoint 2013 (https://www.microsoft.com/).Full size imageVegetation sample site monitoring analysisThe vegetation species in the lower reaches of the Tarim River were sparsely distributed, with P. euphratica and Tamarix sp. as the main established species. In the longitudinal direction, surface vegetation coverage and species number decreased as the water table depth increased from the upper and middle segments to the lower segment. In the lateral direction, surface vegetation shows the same trend, with groundwater table depth increasing the greater the distance from the river13.The surface ecological processes in the lower reaches of the Tarim River have responded positively to the water conveyance project, with density, coverage and the number and diversity of species significantly increasing. However, the response of surface ecological processes to the changes in groundwater table uplift has varied from section to section. In the lateral direction, the groundwater table in areas nearer to the river had a more prominent rise and the response of surface vegetation was stronger, whereas the groundwater table rise in areas farther from the river was smaller and so the response of surface vegetation was weaker. In the longitudinal direction, the same trend was observed from the upper to the lower segments in response to changes in the groundwater table. In this paper, we analyze the changes in detail by taking a closer look at the Yengsu section, which is located at the beginning of the middle section of the lower reaches of the Tarim River. In so doing, we apply sample site investigation and dynamic monitoring of the groundwater table to the study area.Changes in vegetation density and coverageThe results of our sample site monitoring show notable positive changes in groundwater depth between 2000 and 2021 as a direct result of the ecological water conveyance initiative. At 150 m from the river, the groundwater table depth rose from 8.47 m to 4.34 m, respectively, representing an uplift of 4.13 m (Fig. 3c). Moreover, the vegetation coverage and density increased from 18.77% and 0.016 plants/m2 to 46.51% and 0.049 plants/m2, and the number of species doubled from three to six.Figure 3Changes in vegetation coverage, density and number of species (a), species diversity indices (b), and groundwater depth (c) for each site at Yengsu section in the lower reaches of Tarim River.Full size imageAt 250 m from the river, the groundwater table depth rose from 8.07 m in 2000 to 4.85 m in 2021, representing an uplift of 3.22 m. The vegetation coverage and density increased from 10.89% and 0.020 plants/m2 to 31.24% and 0.160 plants/m2, respectively, and the number of species jumped from five to seven.At 350 m from the river, the water table rose 2.48 m between 2000 and 2021. The vegetation coverage and density increased from 3.69% and 0.010 plants/m2 to 22.27% and 0.022 plants/m2, respectively, and the number of species increased from two to three. It is worth noting that the expansion in vegetation cover in the first three sample sites was mainly due to the increase in the number and canopy width of herbs and shrubs that occurred as a direct result of the ecological water conveyance process.At 750 m from the river, the groundwater table depth rose from 5.96 m to 4.98 m between 2005 and 2021, respectively, representing an uplift of 0.64 m, while the vegetation coverage and density increased from 20.07% and 0.011 plants/m2 to 26.43% and 0.019 plants/m2, respectively.At 1050 m from the river, the sample site had an elevated water table of 1.22 m. The vegetation coverage and density increased from 2.41% and 0.004 plants/m2 in 2005 to 5.89% and 0.0148 plants/m2 in 2021, respectively (Fig. 3a). Among them, the increase in canopy area of Tamarix sp. and P. euphratica in the sample site was the main reason for the expansion in coverage.Changes in species diversity indicesPlant richness and evenness in the lower reaches of the Tarim River were low, with species diversity indices showing significant changes in response to the ecological water conveyance and the rise in the groundwater table (Fig. 3b). For example, at the Yengsu section, the Simpson dominance index, McIntosh evenness index and Margalef richness index, which reflect changes in species diversity, decreased from 0.58, 0.45 and 0.74 in 2005 to 0.46, 0.03 and 0.03, respectively. These changes occurred in response to the increase in groundwater depth from the first sample site at 150 m to the sixth sample site at 1050 m from the river channel. After 20 years of ecological water conveyance, the Simpson dominance index, McIntosh evenness index and Margalef richness index had increased on average by 0.33, 0.35 and 0.49, respectively, in the first three sample sites (Fig. 3b).Vegetation index (NDVI) changesThe Normalized Difference Vegetation Index (NDVI) is an important indicator of vegetation growth24. The study results reveal that the NDVI of the lower reaches of the Tarim River increased from 0.14 in 2000 to 0.21 in 2020, representing a rise of about 33.3%. The ecological water conveyance expanded the river region’s natural vegetation 188%, from 492 km2 in 2000 to 1423 km2 in 2020. Specifically, the area of low, medium, and high vegetation cover expanded by 277 km2, 537 km2 and 132 km2, representing increases of 20.8%, 448% and 190%, respectively. Further analysis of changes in vegetation coverage at different river sections indicate that the area of low vegetation coverage in the upper and middle segments showed a decreasing trend, whereas the area of medium and high vegetation coverage in the upper and middle segments showed an increasing trend. This latter trend was especially prominent in the middle segment, where the increase in the area covered by medium and high vegetation was relatively large.In the downstream segment, the area covered by all types of vegetation showed an upward trend, with the area covered by low vegetation expanding significantly (Fig. 4). In the lateral direction, the NDVI within 2 km of the water conveyance channel showed a more obvious response with greater increases, while NDVI beyond 2 km from the channel revealed smaller increases25. These differences reflect the influence range of the ecological water conveyance.Figure 4Variation of vegetation cover in the lower reaches of Tarim River. Spatial distribution of fraction of vegetation cover in (a) 2000, (b) 2010 and (c) 2020. Trends of (d) high fraction of vegetation cover, (e) middle fraction of vegetation cover and (f) low fraction of vegetation cover in different river sections. (g) Vegetation area and (h) change trend at different distances from the river.Full size imageChanges in net primary production (NPP) of natural vegetationNet primary production (NPP) is a key parameter of carbon cycling and energy flow in terrestrial ecosystems. NPP not only reflects terrestrial ecosystem productivity, but also characterizes the quality of terrestrial ecosystems and plays an important role in global change and carbon balance26,27. The results of our study show that the area of natural vegetation in the lower reaches of the Tarim River with highly significant and significant increases in NPP during the study period accounted for 31.93% (P  herbaceous community. The largest increase in NPP was observed in the Tamarix spp. community, rising 350.20% from 2001 to 201928.Area changes in vegetation carbon sink areaThe ecological water conveyance project in the lower reaches of the Tarim expanded the vegetation coverage and enhanced the carbon sequestration capacity of the region through photosynthesis. The lower reaches of the river are dominated by desert and sparse vegetation, and the ecosystem carbon sinks are mainly low carbon sinks. The monitoring results of the study show that the vegetation carbon sink area in the river’s lower reaches indicate a gradual expansion under the influence of the ecological water conveyance29, increasing from 1.54% of the study area in 2001 to 7.8% in 2020. As well, the Net Ecosystem Productivity (NEP) of the area’s vegetation showed an increasing trend at a rate of 0.541 g C·m−2·a−1, with the largest increase – 0.406 g C·m−2·a−1 – occurring in summer29and no significant carbon sink area in winter.Furthermore, in order to quantitatively investigate the degree of influence of ecological water conveyance on the carbon sink area in the lower reaches of the Tarim, a linear fit of cumulative water conveyance and carbon sink area was performed (Fig. 5). Based on the results, a strong linear correlation was found between cumulative water conveyance and carbon sink area (R2 = 0.958, p  More

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Experimental siteAll procedures for the experiment involving animals were carried out in accordance with relevant guidelines and regulations and they were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Florida (protocol #201509019). The experiment was conducted at the University of Florida North Florida Research and Education Center (NFREC) located in Marianna, FL (30° 52ʹ N, 85° 11ʹ W, 35 m asl) during 2018 and 2019.The study site was an existing mixed RP-bahiagrass grazing study where ‘Ecoturf’ RP was strip-planted into ‘Argentine’ bahiagrass on 12 June 2014. Rhizoma peanut strips were approximately 2-m wide, making it possible to harvest RP forage, roots, and rhizomes free of bahiagrass contamination3,4. The RP was collected from a nursery at the University of Florida—NFREC, whereas the bahiagrass seeds were bought from a seed company. All plants were collected, purchased, managed, and the research was conducted in compliance with relevant institutional, the corresponding national, and international guidelines and legislation.Soils at the experimental site were classified as Orangeburg loamy sand (fine-loamy, kaolinitic, thermic Typic Kandiudults24. At the beginning of the study, soil pH was 5.7 and soil OM was 15.4 g kg−1. Additionally, Mehlich-I extractable soil P, K, Mg, and Ca concentrations at the beginning of the experiment were 26, 99, 43, and 224 mg kg−1, respectively. Total annual rainfall and average annual temperature at the experimental site were 1889 and 602 mm, and 19 and 21 °C, for 2018 and 2019, respectively, and their monthly averages are shown in Fig. 5.Figure 5Monthly weather conditions at North Florida Research and Education Center (NFREC) Marianna, FL, during the experimental years.Full size imageTreatments and experimental designTreatments were two defoliation regimes applied to RP, continuously stocking and 56-days interval between clipping harvests. At the continuous stocking, stocking rates were variable to maintain similar herbage allowance among pastures, which was assessed every 14 days as described by Sollenberger et al.25. Two tester Angus crossbred steers (Bos spp.) remained on each pasture throughout the experimental period. Put-and-take cattle were allocated as needed to maintain a target herbage allowance of 1.5 kg DM kg−1 bodyweight3. Treatments were situated adjacent to each other (i.e., paired sites) in monoculture strips of RP within each of three 0.85-ha pastures. Each pasture was considered a block, thus the experiment consisted of three replicates of each treatment in a randomized complete block design. Within each replicate, treatments had three repetitions (pseudo replicates). To prohibit animal access to the non-grazed treatment, three 2 × 2-m exclusion cages were placed on RP strips in each pasture. Rhizoma peanut herbage mass was determined at both the grazed and non-grazed sites three times each year, at days 56, 112, and 168 of the experimental period by using a 0.25-m−2 quadrat. Two quadrats were collected in each repetition by clipping all the biomass within each quadrat at 2-cm stubble height. After each herbage mass sampling, the non-grazed residual dry matter inside the cages was clipped to a 2-cm stubble height using a weed eater and the herbage removed by raking. On average, across sampling dates and years, herbage mass at the grazed and non-grazed sites was 1050 and 1810 kg of organic matter (OM) ha−1, respectively.Long-term and short-term decomposition studiesThere were two types of root-rhizome decomposition trials. The first is referred to as the long-term decomposition study, and the second is the short-term decomposition study. The long-term study had an incubation period of 168 days, with a single in-situ incubation per year starting in May. The short-term study had in-situ incubation periods of 56 days and there were three incubations per year, occurring in May, June, and August. In all cases, only roots and rhizomes attached to the plant were used in both trials.Long-term studyOn 26 Apr. 2018 and on 23 Apr. 2019, right after RP emergence after breaking dormancy, RP roots and rhizomes were collected from an existing mixed RP-bahiagrass grazing study where RP had been planted in strips into bahiagrass (Paspalum notatum Flüggé) in 2014. Rhizoma peanut strips were approximately 2.75-m wide, alternating with similar wide bahiagrass strips. A pure stand of RP had been maintained in the strips during previous years using herbicides3, making it possible to harvest RP roots and rhizomes free of bahiagrass contamination. Roots and rhizomes were collected at 24 different points in each of three blocks of the original experiment. Roots and rhizomes were collected at 20-cm depth using shovels. As defoliation treatments had not being applied at this time of the year, the same material was used to perform the incubation inside and outside the exclusion cages. After harvesting, excess soil was removed by shaking from the root-rhizome mat using a 1.4-cm diameter sieve. Thereafter, the existing aboveground material was clipped, and the roots and rhizomes were then washed over the same sieve to remove the remaining soil. After washing, roots and rhizomes were dried to constant weight in a forced-air drying oven at 55 °C.To perform the decomposition study, approximately 12 g of dry roots and rhizomes were placed in Ankom bags (10 by 20 cm, 50 µm porosity; ANKOM Technology) and sealed17. Roots and rhizomes were aimed to be placed intact into Ankom bags, nonetheless, when they could not fit inside the bags, they were cut in the middle before being placed. On 2 May 2018 and 1 May 2019, the incubation period began. For each treatment, bags were incubated in situ in the field at 10-cm depth in the same blocks from which they were collected. Bags were removed from the field after 0, 3, 7, 14, 28, 56, 112, and 168 days. For each treatment within each block, three bags were incubated for each incubation time. Additionally, empty bags (one bag per treatment per time per block) were placed in the field. After removal of the in-situ bags from the field, samples and empty bags were dried at 55 °C for 72 h, cleaned with a brush, and weighed. Thereafter, samples were ground to pass a 2-mm screen using a Wiley Mill (Model 4, Thomas-Wiley Laboratory Mill, Thomas Scientific) and analyzed for DM and OM. Subsamples of the 2-mm ground samples were ball milled in a Mixer Mill (MM 400, Retsch) at 25 Hz for 9 min. Ball-milled samples were analyzed for C and N by dry combustion using an elemental analyzer (Vario Micro cube, Elementar). Additionally, samples ground at 2-mm were used to determine ADF in aboveground samples26. The N concentration in the ADF was determined using the above protocol to obtain the ADIN.Short-term studiesThe short-term studies were performed following the same procedures as the long-term study, except that the incubation period was only 56 days, and these studies were repeated three times each year. Roots and rhizomes were incubated in situ on 2 May, 27 June, and 23 Aug. 2018 and on 1 May, 26 June, and 21 Aug. 2019, following the same protocol as described above, except that bags were removed from the field after 0, 3, 7, 14, 28, and 56 days of incubation. The incubations occurring in May, June, and August will be referred as early, middle, and late season, respectively.The early-season incubation period uses the data from the first 56 days of the long-term study described above. For the middle- and late-season incubations each year, roots and rhizomes were harvested approximately 7 days days prior to incubation. Approximately six points in each repetition were collected at 20-cm depth using shovels. For the grazed treatment, roots and rhizomes were collected in the grazed area nearby the exclusion cages, whereas for the non-grazed treatment, the material was collected inside the exclusion cages. After removal of the bags from the field, they were processed and analyzed for DM, OM, C, and N following the protocol described above.Statistical analysesLong-term studyRemaining biomass, remaining N, C:N ratio, ADF, and ADIN were analyzed using the PROC GLIMMIX from SAS27, with treatment and days of incubation as fixed effects, and years and blocks as random effects. Days of incubation were considered repeated measures. Means were compared using the PDIFF procedure at the 5% significance level. When treatment or the interaction of treatment × day of incubation were statistically significant in the ANOVA, nonlinear models were tested to fit the data for each variable and treatment. Nonlinear models were selected for a given response based on data distribution and type of response. If only days of incubation was significant, the same model was applied for all treatments.Remaining biomass (OM basis), remaining N, and C:N ratio were explained by the single exponential decay model14,17,28. The equation describing this process is:$$X=B0, {exp}^{-kt},$$
(1)
where X is the remaining biomass, remaining N, or C:N ratio at day t, B0 is the disappearance coefficient, and k is the relative decay rate (g g−1 day−1). The model used to describe ADF and ADIN was the two-stage model “linear plateau”15,29. The equation describing this process is:$$begin{gathered} Xt = A + b1 times t, {text{if t }} le {text{ T}}, hfill \ {text{and}},{ } Xt = A + b1 times T, {text{if t }} > {text{ T}}, hfill \ end{gathered}$$
(2)
where X is the concentration of ADIN, t is the day of incubation, A is the initial concentration, b1 is the rate of increase in concentration from the beginning of incubation until plateau is reached; and T is the day in which concentration reaches the plateau.Short-term studiesThe single exponential model was applied in the remaining OM and remaining N, for each experimental unit, to obtain individual values for B0 and k. The data for initial N concentration, initial C:N ratio, and B0 and k for remaining OM and remaining N were analyzed using the PROC GLIMMIX from SAS27, with treatment and period as fixed effects, and years and blocks as random effects. Means were compared using the PDIFF procedure at the 5% significance level.Arrive guidelinesThis is study is reported in accordance to ARRIVE guidelines. More

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