No long-term effect of land-use activities on soil carbon dynamics in tropical montane 3 grasslands 4 5

1. Title page 1 2 No long-term effect of land-use activities on soil carbon dynamics in tropical montane 3 grasslands 4 5 Viktoria Oliver1,2*, Imma Oliveras3, Jose Kala4, Rebecca Lever5,2, Yit Arn Teh 1,2 6 7 1 Institute of Biological and Environmental Sciences, University of Aberdeen, Cruickshank 8 Building, St. Machar Drive, AB24 3UU Aberdeen, UK. 9 2 Formerly at the School of Geography and Geosciences, University of St Andrews, UK 10 3 Environmental Change Institute, School of Geography and the Environment, University of 11 Oxford. South Parks Road, OX13QY Oxford, UK. 12 4 Universidad de Santo Antonio Abad del Cusco, Cusco, Peru. 13 5 Department of Life & Environmental Sciences, University of California, Merced 5200 North 14 Lake Rd. Merced, CA 95343, United States. 15 16 * Corresponding author: v.oliver@abdn.ac.uk 17 18 Running title: Tropical montane grassland soil carbon dynamics 19


Abstract 22
Montane tropical soils are a large carbon (C) reservoir, acting as both a source and a sink of 23 CO2. Enhanced CO2 emissions originate, in large part, from the decomposition and losses of 24 soil organic matter (SOM) following anthropogenic disturbances. Therefore, quantitative 25 knowledge of the stabilization and decomposition of SOM is necessary in order to understand, 26 assess and predict the impact of land management in the tropics. In particular, labile SOM is 27 an early and sensitive indicator of how SOM responds to changes in land use and 28 management practices, which could have major implications for long term carbon storage 29 and rising atmospheric CO2 concentrations. The aim of this study was to investigate the 30 impacts of grazing and fire history on soil C dynamics in the Peruvian montane grasslands; an 31 understudied ecosystem, which covers approximately a quarter of the land area in Peru. A 32 density fractionation method was used to quantify the labile and stable organic matter pools, 33 along with soil CO2 flux and decomposition measurements. Grazing and burning together 34 significantly increased soil CO2 fluxes and decomposition rates and reduced temperature as a 35 driver. Although there was no significant effect of land use on total soil C stocks, the 36 combination of burning and grazing decreased the proportion of C in the free LF, especially at 37 the lower depths (10-20 and 20-30 cm). In the control soils, 20 % of the material recovered 38 was in the free LF, which contained 30 % of the soil C content. In comparison, the burnt-39 grazed soil, had the smallest recovery of the free LF (10 %) and a significantly lower C content 40 (14 %). The burnt soils had a much higher proportion of C in the occluded LF (12%) compared 41 to the not-burnt soils (7%) and there was no significant difference among the treatments in 42 the heavy F (~ 70%). The synergistic effect of burning and grazing caused changes to the soil 43 C dynamics. CO2 fluxes were increased and the dominant temperature driver was obscured 44 by some other process, such as changes in plant C and N allocation. In addition, the free LF 45 was negatively affected when these two anthropogenic activities took place on the same site. 46 Most likely a result of reduced detritus being incorporated into the soil. A positive finding 47

Introduction 54
High altitudinal montane grasslands (3200 -4500 m a.s.l) account for a major proportion of 55 land cover in the Andes, particularly in Peru, where they make-up approximately 25 % of land 56 cover (Feeley and Silman 2010). Every year, especially in the dry season, large areas of these 57 grasslands are burned to support traditional cattle grazing, which has been apparent since 58 the early 1500s (Luteyn 1992). Fires for agricultural clearing and maintenance of these highly 59 productive forage grasses is of considerable importance in these ecosystems and for the 60 livelihood of the local people (Sarmiento and Frolich 2002). To some extent, this natural 61 system is tolerant of these management practices (Ramsay 1992 Oliveras et al. 2014b). However, despite the concern on the effects of 70 land management practices, there are very few studies on soil C dynamics in this tropical 71 region of the Peruvian Andes. It is particularly unclear how land management affects the soil 72 C dynamics and sequestration potential under the influence of grazing and burning. For 73 example, (Oliveras et al. 2014b) , found that grazing and fire in montane grasslands resulted 74 in decreased net primary productivity, but there were no differences between these two 75 disturbances. Studies in other montane grasslands have found that an increase in the 76 frequency of fire events can reduce the amount of soil organic matter (SOM) in the top soil 77 (Knicker 2007), or it may increase the biomass growth period afterwards, causing more 78 detritus to accumulate in the upper soil layers (Ojima et al. 1994). 79 80 SOM influences many soil functions and occupies a key position in the global C cycle (Lal 81 2004). It is a highly heterogeneous and dynamic composite of organic molecules (such as: 82 polysaccharides, lignin, aliphatic biopolymers, tannins, lipids, proteins and aminosugars) 83 derived from progressively decomposed plant, animal and microbial material (Zimmermann 84 et al. 2007a;Totsche et al. 2010). 85 The inert pool (passive pool) contains highly carbonized organic material resistant to 117 microbial mineralisation and has a turnover time of decades to thousands of years. Charcoal 118 or black C tends to reside in this pool and is considered to have a recalcitrant structure due 119 to its high degree of aromaticity, which causes it to have an estimated residence time of 5000 120 to 10 000 years (Derenne and Largeau 2001). Although this pool has a low C concentration, it 121 is the largest and conceptually unaffected by land-management or climate, making it the most 122 stable and relevant for long-term C storage (Falloon and Smith 2000). It is also central to the 123 stabilization of humus and soil aggregation (Brodowski et al. 2006). 124 125 Land-use change and land management studies have found that even when the bulk soil C 126 does not appear to be affected, the distribution of SOM pools may change due to their 127 differing sensitivities to environmental forcing or external perturbation (Zimmermann et al. In addition, soil temperature (at 5 cm and 10 cm depth) and soil moisture (at 10 cm depth) 219 were simultaneously measured in three locations adjacent to the collars using a ML2x 220 ThetaProbe equipped with 12 cm rods (Delta-T Ltd., UK) and type K thermocouples (Omega 221 Engineering Ltd., Manchester, UK). 222 223

Soil sampling and analysis 224
Soil sampling: 50 g soil samples were taken in July 2012 with six replicates at 0-5, 5-10, 10-20 225 and 20-30 cm depths on each site. In many instances, the soil depths were shallow before 226 reaching the bedrock, so samples were only taken at 20-30 cm where possible. Soil samples 227 were air-dried and sieved with a 2 mm mesh sieve before being shipped to the University of 228 St Andrews for all further analysis (Brown and Lugo 1982). 229 230 Bulk density: soil bulk density was determined by the soil core method (Klute 1986). 231 Undisturbed soil cores (30 cm 3 ) were taken from three soil pits at 0-10, 10-20 and 20-30 cm. 232 The samples were dried at 105 °C for 48 hours and bulk density was estimated as the mass of 233 oven-dry soil divided by the core volume. The air-dried soil material (15 g) was sieved in a 2mm mesh sieve to remove any living roots 240 and larger organic material and was then saturated with 60 mL sodium polytungstate solution 241 (NaPT, Na6 [H2W12O40], Sometu-Germany) at a density of 1.85g/mL and centrifuged for 45 242 minutes at 3600 rpm and allowed to settle overnight. The floating free light fraction (free LF) 243 was aspirated via a pump and rinsed with 500 mL of deionised water through a 0.4 µm 244 polycarbonate filter (Whatman Nuclepore Track Etch Membrane) to remove residual NaPT. 245 The remaining slurry was further saturated with 60 mL sodium polytungstate solution (1.4 g 246 cm -3 ), mixed using a benchtop mixer (Mixer/Vortexer -BM1000) for 1 minute at 3200 rpm 247 and dispersed ultrasonically (N10318 Sonix VCX500 sonicator Vibra-cell ultrasonic processor) 248 for 3 min at 70 % pulse for a total input of 200 J/mL. Centrifugation (45 minutes at 3600 rpm) 249 was used to separate the occluded light fraction (occluded LF) from the mineral residue and 250 allowed to sit overnight to achieve further separation by flotation of organic debris and 251 settling of clay particles in solution. The occluded LF was then aspirated via a pump and rinsed. 252 In order to remove the NaPT from the heavy fraction (heavy F), deionised water was mixed 253 with the material and centrifuged for 15 minutes at 4000 rpm 5 times. All fractions were oven 254 dried at 100 °C overnight, weighed and physically ground to a fine powder before C analysis 255 and isotope analysis. The recovery of the soil C density fractions was 96 %. Differences in soil C between the areas were analaysed using a one-way ANOVA and 287 TukeyHSD post-hoc test, after testing for normality and homogeneity of variances. 288 289 290

Soil respiration and environmental drivers 292
The overall annual CO2 mean for the pooled data set, including all types of land management, 293 was 1.39 ± 0.05 µmol m -2 s -1 . The combination of grazing and burning significantly increased 294 soil CO2 fluxes at Wayqecha (2003) but not at Acjanaco (Fig 2). Regardless of land use, the 295 plots at Wayqecha (2003) had greater variability and overall higher mean annual soil 296 temperature (15 °C) and CO2 flux (1.34 ± 0.09 µmol m -2 s -1 ) compared to the sites in Acjanaco 297 (2005) (12 °C and 0.79 ± 0.03 µmol m -2 s -1 ) ( Table 2). The highest measured temperatures and 298 CO2 fluxes at Wayqecha were synchronously recorded during July-11, November-12 and 299 March-12, whereas at Acjanaco the changes in CO 2 flux with season and temperature were 300 less pronounced. 301 302 Season (which run from October to March), soil and air temperature were the main drivers 303 of soil respiration (p-values = 0.031, 9.3 x 10 -7 and 0.0001, respectively), with higher 304 temperatures having a positive effect on soil CO2 fluxes. However, when analyzing the grazed-305 burnt plots at both Wayqecha and Acjanaco, there was no relationship between CO2 fluxes 306 and temperature or any of the other environmental variables measured. 307 308

Decomposition rates 309
The decomposition of the birch wood sticks was slow, with an overall average weight loss of 310 ~ 20 % in one year. Grazing alone appeared to slightly increase the rate of decomposition 311 when all the data were pooled together (grazed: y = 104.53 + -4.23x, R 2 = 0.98, not grazed: y 312 = 103.63 + -3.11, R 2 0.94), but burning alone did not affect decomposition rate (burnt: y = 313 103.34 + -3.57, R 2 = 0.96, not burnt: y = 104.82 + -3.76x, R 2 = 0.97) (Fig 3). Site-specific 314 differences were observed for decomposition rates; for example, decomposition was 315 generally faster at Wayqecha compared to Acjanaco. In particular, the grazed -not burnt plot 316 at Wayqecha showed the fastest overall rate of decomposition (y = 101.98 + -0.19x, R 2 = 0.77) 317 and the not grazed -not burnt plots (controls) had the slowest decomposition rates (Fig 3) on 318 both sites. 319 320 Decomposition was not a strong overall predictor for CO2 fluxes for the pooled dataset, 321 although there were some strong correlations between these two variables at specific study 322 sites. For example, there was a strong relationship between decomposition and soil CO 2 fluxes 323 at Acjanaco (y = 0.38 + -0.18x, R² = 0.99) (i.e. faster mass loss = higher soil respiration), 324 whereas at Wayqecha, this relationship was weak (y = 1.56 + 0.06x, R² = 0.07). Land-use did 325 not appear to influence the decomposition rate-soil CO2 flux relationship. 326 327

Soil C stocks 328
Grazing, burning and the combination of burning and grazing did not significantly alter total 329 soil C at any depth down to 30 cm on either of the sites (Table 3). The overall sum of all the 330 measured depths showed signs of a decrease in C stocks on the grazed soils, from 189 ± 32 331 Mg C ha -1 on the undisturbed sites to 130 ± 20 Mg C ha -1 on the grazed-burnt sites, but this 332 was not statistically significant at the P < 0.05 level. On average, Acjanaco (2003)  The pooled dataset demonstrated that these soils have a notably large free LF (~20 %). When 336 looking at the different treatments and averaging the data across the soil profile (0-30 cm), 337 burning and grazing had a significant negative effect on the proportion of C in the free LF 338 (Table 4). The free LF in the control soils made 20 % of the bulk soil mass and 30 % of the soil 339 C content compared to the burnt-grazed soils, which had the smallest recovery of free LF (10 340 %) and had significantly lower C content (14 %). However, when analysing the depths 341 individually, there was only a significant loss of C in the free LF at 10-20 and 20-30 cm depth, 342 with a reduction of ~ 16 % (Fig 4). When analysing the two sites separately, the burnt-grazed 343 soils at Wayqecha had a significantly smaller proportion of C in the free LF at 0-5 cm (p-value 344 = 0.002), whereas at Acjanaco there were no significant differences among the land uses. rather Acjanaco being at a slightly higher elevation and on average 4 °C cooler. Despite the 367 variance in mean annual temperature, the two sites both showed a positive correlation 368 between temperature and soil respiration. Interestingly though, the decomposition rates at 369 Acjanaco correlated with the CO2 fluxes, suggesting that decay was a good predictor of CO2 370 flux. This was in contrast to the lower elevation site in Wayqecha, where CO2 fluxes did not 371 correlate with decomposition rates, implying that autotrophic respiration or other 372 environmental factors may have had a stronger influence on soil respiration. 373 374 Burning alone or grazing alone enhanced soil respiration and decomposition rates when these 375 land management practices were considered separately, with soil temperature identified as 376 the main environmental driver in each of these treatment types. However, when plots had 377 been exposed to both burning and grazing together, soil temperature no longer correlated 378 well with soil respiration. The combination of burning and grazing also produced higher soil 379 respiration rates than the two treatments independently. While this pattern has been 380 identified before in other studies (Ward et al. 2007 175 Mg C ha -1 reported here), perhaps reflecting within site spatial heterogeneity. There was 415 no significant effect of either burning or grazing but grazing had a more negative effect that 416 burning on the total soil C stocks. This negligible effect of burning may be a consequence of 417 low intensity fires, fire-resilient grasses, and potentially low fuel loads at the time of burning 418 (Knicker 2007). Grassland fires on slopes can move very quickly, so even when intense, the 419 transfer of heat to the soil is less damaging due to low residence times (Rollins, Cohen and 420 Durig 1993). As a result, surface temperatures do not typically exceed 100 °C or 50 °C at 5 cm 421 depth (Campbell et al. 1995), and organic matter can only be fully volatilized between 200 422 and 315 °C (Knicker 2007). Even if the soils were dry at the time of burning which is possible 423 during the dry season, then belowground temperatures would rise very slowly because of the 424 insulating properties of air-filled pores, which curtail heat transfer belowground (Neary et al. 425 1999). 426 427 Grazing on the other hand, had a more negative impact on total SOC content than burning 428 but there was not a significant loss of total soil C. One explanation is that the grazing pressure 429 in these sites may have been below the threshold required to cause severe degradation, 430 supporting previous studies in the Peruvian Andes, where they also found no significant effect 431 of grazing or burning on total SOC stocks (Gibbon et al. 2010;Oliveras et al. 2014b). 432 433 Overall, the free LF was larger than in other tropical systems (30 % of total soil C). By 434 comparison, studies in Puerto Rico found the free LF was only 10 % of total soil C content 435 (Marin-Spiotta et al. 2009). As a consequence, loss of the free LF due to disturbance may have 436 a greater proportional impact on net ecosystem C loss in these systems. In addition, the larger 437 free LF suggests that the decomposition of labile material may be slower in these montane 438 grasslands than in other tropical environments. Grazing had a negative impact on the free LF. recalcitrance. Because the fires took place almost ten years ago, the charcoal may no longer 452 be resident the free LF but may have become occluded into soil micro-aggregates due to its 453 high sorptive capacity (Qayyum et al. 2014 Table 2 Annual and seasonal mean soil temperature, VWC and CO2 flux for Wayqecha and Table 3. Mean soil C content (Mg C ha -1 ) for each depth and total C stocks (0-30 and 0-20 cm) on all the land  Table 4 Mean mass recovery of density fractions and proportion of total C residing in the three density fractions (%) from the total soil profile (0-30 cm). Different letters down the columns represent significant differences.