This is the accepted version of the article: Chen, Xiaoxuan; Wiesmeier, Martin; Sardans i Galobart, Jordi; [et al.]. «Effects of crabs on greenhouse gas emissions, soil nutrients, and stoichiometry in a sub- tropical estuarine wetland 260 2 020ˇ.BiologyandF ertilityof Soils,(September2020).DOI10.1007/s00374−020−01512−6 This version is available at https://ddd.uab.cat/record/232757 under the terms of the license 1 Effects of crabs on greenhouse gas emissions, soil nutrients and 2 stoichiometry in a subtropical estuarine wetland 3 4 Xiaoxuan Chen1,2 · Martin Wiesmeier3 · Jordi Sardans4,5* · Lukas Van Zwieten6 · 5 Yunying Fang7 · Albert Gargallo-Garriga4,5 · Youyang Chen1,2 · Shuyun 6 Chen1,2 · Congsheng Zeng1,2 · Josep Peñuelas4,5 · Weiqi Wang1,2* 7 1 8 Key Laboratory of Humid Subtropical Eco-geographical Process, Ministry of Education, Fujian 9 Normal University, Fuzhou 350007, China 2 10 Institute of Geography, Fujian Normal University, Fuzhou 350007, China 3 11 TUM School of Life Sciences Weihenstephan, Technical University of Munich, Freising, 12 Germany 4 13 CSIC, Global Ecology CREAF-CSIC-UAB, Bellaterra, 08193 Barcelona, Catalonia, Spain 5 14 CREAF, Cerdanyola del Valles, 08193 Barcelona, Catalonia, Spain 6 15 NSW Department of Primary Industries, Wollongbar Primary Industries Institute, Wollongbar 16 NSW 2477, Australia 7 17 NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, 18 NSW 2568, Australia 19 20 21 * Corresponding authors 22 W. Wang: Key Laboratory of Humid Subtropical Eco-geographical Process, Ministry of Education, 23 Fujian Normal University, Fuzhou 350007, China, Tel: +86 591-83465214, E-mail: 24 wangweiqi15@163.com. 25 J. Sardans: CSIC, Global Ecology CREAF-CSIC-UAB, Bellaterra, 08193 Barcelona, Catalonia, 26 Spain. Tel: +34 93 581 46 73, E-mail: j.sardans@creaf.uab.cat. 27 28 1 29 Abstract 30 Crabs may elicit effects on wetland carbon (C), nitrogen (N) and phosphorus (P) 31 concentrations and associated ecological stoichiometry. In this study, we assessed effects 32 of crabs on carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) emissions, 33 soil C, N, and P concentrations, and stoichiometry in upper and mid tidal flats of an 34 estuarine wetland in China. The results showed that averaged CO2, CH4, and N2O fluxes 35 were greater in the upper and mid-tidal flats in the presence of crabs, being 46.4, 66.7, 36 and 69.7% and 53.6, 143, and 73.1% greater than control, respectively. Mixed model 37 analyses showed overall positive relationships between wetland soil CO2 CH4 and N2O 38 emissions (F=4.65, P=0.033; F =42.42, P=0.042 and F=10.2, P=0.0018, respectively) in 39 the presence of crabs, taking into account season, flooding intensity, and plot effects. This 40 may be related to the direct effects of respiration and the indirect effects of feeding, 41 excretion, and disturbance of soil on microorganisms and/or plant roots. There were no 42 effects of crabs on total C or N concentrations, whereas decreased soil total P 43 concentrations, especially in the upper tidal flats (P=0.04). Crab presence was positively 44 associated with soil C:P and N:P ratios (P<0.0001 and P<0.0001, respectively), taking 45 into account season, flooding intensity, and plot effects. In the upper and mid-tidal flats, 46 soil CO2 emissions were negatively correlated with total soil C; CH4 emissions were 47 positively correlated with ratios of C:N and C:P; and N2O emissions were positively 48 correlated with N content. In general, global warming potential (GWP) of the upper tidal 49 flats in the presence of crabs increased by 138% compared with the absence of crabs, and 50 GWP of the mid-tidal flats in the presence of crabs increased by 99.3% compared to the 51 absence of crabs. Global warming and associated flooding rise in several coastal wetland 52 areas is favoring benthic fauna number enhancement and this in turn increase GWP of 53 overall gas emissions further contributing to future warming rise. 2 54 Keywords Greenhouse gas emission · Carbon · Nitrogen · Phosphorus · Elemental 55 stoichiometry · Wetlands 56 57 Introduction 58 The global average temperature increased by 0.58 °C during the period between 1880 and 59 2012 (IPCC 2014), and although many factors contributed to this rise in temperature, 60 increasing concentrations of the key greenhouse gases (carbon dioxide: CO2; methane: 61 CH4; and, nitrous oxide: N2O) in the atmosphere contributed about 80% to global 62 warming (IPCC 2014). Globally, wetlands account for 5–8% of the land surface area 63 (RoyChowdhury et al. 2018), yet despite this relatively small area, they represent 64 important sources and sinks of greenhouse gases (Altor and Mitsch 2008; Chen et al. 2010; 65 Bridgham et al. 2013), due to high levels of primary productivity and soil carbon (C) and 66 nutrient storage (Mander and Shirmohammadi 2008). Wetlands are increasingly 67 important in the retention and filtration of excess reactive niN from surrounding soils, 68 and may produce N2O by nitrification and denitrification processes (Burgin and Groffman 69 2012; Cui et al. 2013). Moreover, CH4 emissions are non-linearly increased with 70 temperature (Peltola et al. 2015) that may elicit a positive feedback on climate change in 71 wetland. 72 Recent studies of soil C, N, and P have explored the relative balance in 73 concentrations of different elements and turnover processes (Elser et al. 2000) as part of 74 the concept of ecological stoichiometry, which is based on the relationships between 75 ecosystem structure and function, stoichiometry and use of C:N:P in different ecosystem 76 compartments (Elser et al. 2000; Sterner and Elser 2002; Sardans et al. 2012). The 77 relationship between ecosystem energy and multi-elemental balance described using 78 ratios (Elser et al. 2000) has become a focus on the study of elemental ecological 3 79 stoichiometry (Elser et al. 2000; Tessier and Raynal 2003; Allen and Gillooly 2009). 80 While spatial variability in ecological stoichiometry and implications for plant growth 81 and ecosystem function have been studied (Han et al. 2005), the effects of benthic 82 organisms on soil nutrient stoichiometry and greenhouse gas emissions are unknown, 83 particularly in estuarine wetlands. The influence of factors at the land-sea interface, 84 periodic tidal inundation, and the effects of benthic fauna on soil and plants are known to 85 affect soil C, N, and P cycling in estuarine wetlands (Liu et al. 2014). Therefore, 86 quantification of nutrient ecological stoichiometry in these ecosystems may increase 87 understanding of drivers and mechanisms of their ecological function and service 88 provision. 89 As secondary producers in estuarine wetlands, benthic fauna (Stahl et al. 2014) is 90 essential for a healthy ecosystem (Vermeiren and Sheaves 2015), where their bioturbation 91 (Suren et al. 2011), excretion and other physiological activities, and biological irrigation 92 effects impact biogeochemical cycling processes in wetlands (Hu et al. 2016), especially 93 for soil C and N (Liu et al. 2005). Studies have shown that benthic species diversity is 94 greater in the intertidal zone than in the upper mid-tidal zone (Wang et al. 2011; Liu et al. 95 2015), mainly due to flooding frequency and associated changes in salinity. For example, 96 flooding of the mid-tidal zone brings rapid influxes of nutrients beneficial to benthic 97 organisms and higher levels of salinity that support the growth of algae and other 98 organisms, leading to increased abundance of crabs (Chen et al. 2008). 99 While the effects of benthic fauna on C and N cycling in wetlands has been studied 100 (Mereta et al. 2013), effects on C, N, and P stoichiometry remain unclear. Crabs 101 (Arthropoda: Brachyura) are commonly occurring zoobenthos in wetlands and they may 102 directly affect soil properties, such as acidity, aeration, and texture, through burrowing 103 activity that subsequently affects C, N, and P cycling (Simioni et al. 2014). For example, 4 104 CO2 emissions from burrows in which the crab Upogebia yokoyai was present were 105 greater than in the absence of the crab (Sasaki et al. 2014). CO2 fluxes at the soil- 106 atmosphere interface were greater in burrows containing crabs (Kristensen et al. 2008), 107 and crab activity in upper tidal flats may promote the production and release of N2O as a 108 result of digging behavior, excretion, and sediment resuspension (Liu et al. 2008). Other 109 types of benthos have been shown to elicit CH4 by nutrient-mediated changes to the 110 microbial cycle and coupling between the benthic upper food chain and this can lead to 111 increased CH4 emissions (Figueiredobarros et al. 2009). Predicted rises in sea levels due 112 to greenhouse gas-meditated global warming are the intensity predicted to lead to 113 increases in the intensity and frequency of flooding that may result in greater crab activity 114 in wetlands, along with associated changes in soil properties, and production and 115 emissions of greenhouse gases (Chen et al. 2008; Wang et al. 2011; Liu et al. 2015). 116 Therefore, it is important to increase the understanding of the role of crabs in the emission 117 of greenhouse gases so as to pormote mitigation. The impacts of crab activities on soil 118 stoichiometry and C, N and P concentrations and further on greenhouse gas emissions in 119 tidal marshes remain, however, to be studied. 120 We hypothesized: 1) crabs presence increases organic matter and aeration in soil, 121 with associated increases in CO2 and N2O emissions and decreases in CH4 emissions, 2) 122 crab presence changes physicochemical properties with effects on CO2 ,CH4 and N2O 123 emissions; and, 3) crab presence increases soil C and nutrient concentrations and leads to 124 lower soil C:N and C:P ratios and stimulates emissions of greenhouse gases. To test these 125 hypotheses and to solve these gaps: 1) we determine the effects of the presence of crabs 126 on soil CO2, CH4, and N2O emissions in the upper and middle tidal flats, 2) we measured 127 the impact of crab presence on soil nutrient levels and their stoichiometry, and 3) we 128 evaluated the relationship between greenhouse gas emissions and soil nutrient 5 129 concentrations and associated stoichiometry in the presence of crabs. 130 131 Materials and methods 132 Study sites 133 The study sites were located in a tidal Spartina alterniflora wetland in the Minjiang River 134 estuary, China (Fig. 1), where there is a subtropical monsoon climate, with a mean annual 135 temperature of 19.9 °C and a mean annual precipitation of 1380 mm. Precipitation is 136 concentrated between March and September, with bimodal peaks in June in the rainy 137 season and August in the typhoon period (Liu et al. 2006). The dominant plant species in 138 the study area included Kandelia candel, Casuarina equisetifolia, Phragmites australis, 139 Cyperus malaccensis, Scirpus striqueter, and Spartina alterniflora. 140 Three replicates of 1 × 1 m sampling plots were established in four experimental 141 treatments that comprised combinations of presence (P) and absence of crabs (D, control) 142 at upper (G) and mid-tidal (Z, control) flats (GP, GD, ZP, and ZD). Mid-tidal flat is the 143 high-flooding habitat, which is flooded by intermediate tides ca. 240 d y-1 and is 144 submerged beneath 10-120 cm of water for 0.5-4 h during each tidal inundation (average 145 of 540 h y-1 of inundation). Upper-tidal flat is the intermediate-flooding habitat nearer to 146 coastal line, which is flooded by intermediate tides ca. 220 d y-1 and is submerged beneath 147 10-100 cm of water for 0.5-3 h during each tidal inundation (average of 385 h y-1 of 148 inundation). All measurements have been performed +/- 1h from low tide. Presence of 149 crabs at the sampling plots was confirmed by the presence of flat holes and chimney 150 burrows (>4 cm, and our in situ investigation showing that these holes were crab holes): 151 in the upper tidal flats, crab burrows were larger in diameter, but fewer in number than in 152 the mid-tidal flats (Table S1). To avoid the effects of isolated crabs, control plots (D) 153 were covered with rust-proof barbed wire, polypropylene random (PPR) tubes, and 60- 6 154 mesh nylon mesh that was installed 70 cm above ground level and 30 cm below ground 155 level. 156 157 Greenhouse gas sampling 158 Greenhouse gas emissions from the plots were collected using static chambers that 159 consisted of an upper chamber and lower base made of opaque PVC (polyvinyl chloride), 160 with diameter and heights of 0.2 × 0.4 m, and 0.2 × 0.1 m, respectively. There was a 161 extraction gas and temperature measurement hole in the top of the upper chamber (Fig. 162 S1). The base of the static chamber used to collect gas samples in the crab-free plots, 163 whereas there was a ≥4 cm- hole in the center of the base of the static chamber used in 164 the crab plots to facilitate normal crab activity. 165 We collected gas samples at low tide in 2015 on January 10 (winter), April 18 166 (spring), June 27 (summer), and September 13 (autumn) at 09:00, 12:00, and 15:00 hrs. 167 At each time step, three samples were collected consecutively for 15 min; 40 ml of gas 168 were sampled from the upper chamber and injected into a 50mL aluminum foil gas sample 169 bag. The temperature in the chamber was recorded during gas collection by the 170 thermometer. 171 We used gas chromatography to determine CO2 and CH4 (Shimadzu GC-2010, 172 Kyoto, Japan) and N2O (Shimadzu GC-2014, Kyoto, Japan) concentrations in the gas 173 samples using a Porapak Q stainless steel column (2 m in length, 4 mm OD, 80/100 mesh). 174 A methane conversion furnace, flame ionization detector (FID) and electron capture 175 detector (ECD) were used for the determination of the CO2, CH4 and N2O concentrations, 176 respectively. The operating temperatures of the column, injector and detector for the 177 determination of CO2, CH4 and N2O concentrations were adjusted to 45, 100 and 280 °C; 178 to 70, 200 and 200 °C and to 70, 200 and 320 °C, respectively. These temperatures were 179 the optimum values for the different parts of the instrument. The gas chromatograph was 7 180 calibrated before and after each set of measurements using 503, 1030, and 2980 μL of 181 CO2 L-1 in He; 1.01, 7.99, and 50.5 μL of CH4 L-1 in He, and 0.2, 0.6, and 1.0 μL of N2O 182 L-1 in He (CRM/RM information center of China, Beijing) as primary standards (Wang 183 et al. 2015a, b). 184 CO2 CH4 and N2O fluxes and the global warming potential (GWP) estimation 185 The CO2 CH4 and N2O fluxes were estimated by the following equation: M dc 273 186 F=   H ( ) V dt 273 + T 187 where F is the CO2, CH4 or N2O flux (mg/μg CO2/CH4/N2O m−2 h−1), M is the molecular 188 weight of the gas (44, 16 and 44 g mol-1 for CO2, CH4 and N2O, respectively), V is the 189 molar volume of gas in a standard state (22.4 mol-1), dc/dt is the variation ratio of CO2, 190 CH4 and N2O concentrations (μmol mol h-1), H is the height of the chamber above the 191 water surface (m), and T is the air temperature inside the chamber (°C). 192 To estimate GWP, CO2 is typically taken as the reference gas, and a change in the 193 emission of CH4 or N2O is converted into “CO2-equivalents” (Hou et al. 2012): 194 f = F ·t 195 Where f (kg/hm2) represents the amount of different greenhouse gas emissions during the 196 sampling period. t represents the time of the period. According to Ahmad et al (2009) the 197 equation used to calculate GWP is: 198 𝐺𝑊𝑃 = 𝑓𝐶𝑂2 + 34𝑓𝐶𝐻4 + 298𝑓𝑁2 𝑂 199 The GWP for CH4 is 34 (based on a 100-year time horizon and a GWP for CO2 of 1), and 200 the GWP for N2O is 298 (Myhre et al. 2013). 201 Soil sampling 202 More than 95% of large benthic fauna is distributed in the upper 10 cm of soil. The 203 diameter of a crab hole is greater at the soil surface and extend to around 10 cm. 8 204 Therefore, we collected five soil samples from the upper 10 cm of soil of each plot; these 205 samples were mixed to reduce heterogeneity and interference from crab holes (Wang et 206 al. 2008), placed in closure pockets and transported to the laboratory. Impurities, such as 207 stones, wood, and plastic, were removed from the soil, and after air-drying, the samples 208 were passed through a 2-mm sieve prior to measurement of physicochemical soil 209 properties (total C, N and P concentrations, dissolved organic C, soil exchangeable NH4+– 210 N, NO3-–N concentrations, soil bulk density, water content, pH, salinity, and temperature). 211 Half of the samples were subsequently passed through a 0.149-mm sieve prior to 212 measurement of soil nutrient. 213 Total C and N (TC and TN, respectively) concentrations were measured using a 214 Vario MAX CN Elemental Analyzer (Elementar Scientific Instruments, Hanau, Germany), 215 and total P (TP) concentration was determined using Mo-Sb colorimetry (Lu 1999; Ruban 216 et al. 1999). Soil DOC was determined by extracting the soils with deionized water (1:5 217 ratio) and measuring the total C concentration using a TOC-V CPH total C analyzer 218 (Shimadzu Scientific Instruments, Kyoto, Japan). Soil exchangeable NH4+–N, NO3-–N 219 concentrations were measured using a continuous flow injection analyzer (Skalar 220 Analytical SAN ++ Instruments, Breda, Netherlands). 221 Bulk density was measured from three 5 × 3-cm cores, while soil water content and 222 soil pH were determined using the drying method and a portable IQ 150 pH meter in a 223 1:5 ratio of soil:water (IQ Scientific Instruments, Carlsbad, USA), respectively. Salinity 224 and soil temperature in the field were measured using a 2265FS salinity/temperature 225 meter (Spectrum Technologies Inc., Paxinos, USA). 226 227 Statistical analyses 228 We used general mixed models (GLM) in the “nlme” (Pinheiro et al. 2016) R package 9 229 with the “lme” function, with crab presence, tidal flat (upper or mid to represent flooding 230 intensity) and season as independent fixed categorical variables, plot as independent 231 random factor and, CO2, CH4, and N2O soil gas emissions and soil physicochemical 232 properties as continuous dependent variables. When a variable did not follow normal 233 distribution, it was log transformed to reach normal distribution before statistical analyses. 234 We chose the best model for each dependent variable based on the Akaike information 235 criterion, and we used the MuMIn package (Barton 2012) in R to estimate variance 236 explained by the mixed models. Tukey’s post hoc tests in the “multcomp” (Hothorn et al. 237 2013) R package, with the “glht” function, were used to test for differences between the 238 eight combinations of crab presence × season. 239 Associations between environmental factors and CO2, CH4, and N2O emissions, and 240 between C, N, and P concentrations were tested using Pearson correlation analysis in 241 SPSS 20.0 (StatSoft, Inc. Tulsa, USA). 242 Effects of crab presence and flooding intensity on soil greenhouse gas emissions and 243 soil physicochemical properties were tested using general discriminant analysis (GDA) 244 in Statistica 8.0 (StatSoft, Inc. Tulsa, USA), with a supervised statistical algorithm that 245 derived an optimal separation between groups, which was established a priori by 246 maximizing between-group variance while minimizing within-group variance, to control 247 for effects of season (Raamsdonk et al. 2001). 248 We used major axis (MA) and standardized major axis (SMA) (SMATR package; 249 http://www.bio.mq.edu.au/ ecology/SMATR) regression to compare the slopes of the 250 regressions of the relationships among the C, N and P with and without crab. 251 252 Results 253 General effects of crab presence on greenhouse gas emissions 10 254 CO2 emissions were globally higher with than without crabs (P<0.05, Table S2). Average 255 CO2 emissions from soils in the presence and absence of crabs in the upper tidal flats 256 were 125 ± 14.8 and 67.1 ± 7.67 mg m-2·h-1, respectively (CVs: 23.7 and 22.9%, 257 respectively), and in the mid-tidal flat, average emissions were 128 ± 62.4 and 58.8 ± 258 20.2 mg m-2·h-1, respectively (CVs: 97.2 and 68.7%, respectively) (Fig. 2). There was no 259 effect of crabs on CO2 emissions, regardless of degree of tidal inundation (P>0.05). CO2 260 emissions were higher in winter and summer than in spring and autumn (Table S2, Fig.4) 261 Cumulative CH4 emissions were significantly higher with than without crabs (Table 262 S2). Mean CH4 emissions in the upper tidal flats with than without crabs were 0.35 ± 0.22 263 and 0.10 ± 0.05 mg m-2·h-1, respectively (CVs: 128 and 98%, respectively), and in the 264 mid-tidal flats, mean CH4 emissions were 0.29 ±0.26 and -0.11 ±0.18 mg m-2·h-1, 265 respectively (CVs: 180 and 320%, respectively) (Fig. 3). There was no effect of crabs on 266 soil CH4 emissions, regardless of degree of tidal inundation. 267 N2O emissions were globally higher with than without crabs (P<0.05, Table S2). 268 N2O emissions in the upper tidal flats were greater in the presence of crabs, especially 269 during the autumn (Fig. 4), and overall, average N2O emissions in the tidal flats were 270 significantly greater in the presence of crabs (Table S2, P<0.05). N2O emissions were 271 higher in autumn than in the other three seasons and were higher under high tidal intensity 272 (Table S2, Fig.4) 273 Under the action of crabs, the CO2 and N2O global warming potential (GWP) of the 274 upper tidal and mid-tidal flats were significantly higher than those without crabs (P<0.05). 275 GWP of the upper tidal flats in the presence of crabs increased by 138% compared with 276 the absence of crabs, and GWP of the mid-tidal flats in the presence of crabs increased by 277 99.3% compared to the absence of crabs (Tables S2 and S3). 278 11 279 Crab presence and soil properties 280 In general, crab presence was associated with greater levels of soil water content, pH. 281 Maximum and minimum soil temperatures were recorded in June and January, 282 respectively, maximum and minimum soil salinities were recorded in January and 283 September, respectively, and maximum and minimum soil pH values were recorded in 284 September and June, respectively. Soil bulk density was greater in the upper tidal flat than 285 middle tidal flat. There was no effect of crab presence on seasonal variations in soil 286 temperature, salinity, pH, water content, or salinity, but soil water content was greater 287 with than without crabs (Fig. S2). 288 There was an overall effect of crab presence on soil TP, but not on soil TC or TN 289 through the year (Table S2, S4). Concentrations of soil TC and TN were greater in the 290 upper tidal flats in the winter and spring than in the upper tidal flats (P<0.05). Crab 291 presence was associated with greater levels of soil C:P and N:P ratios (Table S2). 292 Season had a significant influence on the total soil nutrients and their stoichiometric 293 ratios. TN varied greatly for different tidal flats and was related to the frequency of soil 294 flooding (P<0.05). TC and C:N were significantly influenced by different seasons, 295 different tidal flats and presence or absence of crabs. The change of C: P was significant 296 under the combined effect of seasons and crabs and TP in different seasons (P <0.001). 297 The ratio of soil C:N was stable among the seasons in the upper tidal flats in the presence 298 of crabs, while that of N:P varied; ratios of soil C:N and N:P in the upper tidal flat in the 299 presence of crabs was stable (Fig. 5). Nutrient ratios were greater with than without crabs, 300 and the ratio of soil C:N ratio varied with season (P<0.01, Table S2). 301 The y1 and y2 functions represent the linear fitting relationship of total nutrients with 302 and without crab activity, respectively. The R2 value in the y1 function was significantly 303 higher than the y2 function, indicating that the crab activity helps to improve the 304 correlation between the total soil nutrients (Fig. 6). We observed that none of the slopes 12 305 of the regression lines of the TN-TC, TP-TC and TP-TN differed significantly between 306 with and without crabs (P>0.05, SMA test of common slopes). Concentrations of soil TC, 307 TN, and TP were related (Fig. 6, P<0.01), but the relationship between TC and TP 308 concentrations was weaker than between TC and TN concentrations. Overall, differences 309 in soil concentrations of TC and TN were more distinct than differences in TP during the 310 year. 311 312 Relationship between greenhouse gas emissions and soil properties 313 Overall, CO2 emissions were positively correlated with soil temperature (R=0.35, P<0.05) 314 and negatively associated with soil bulk density (R=-0.36, P<0.05), CH4 and N2O 315 emissions were positively correlated with soil temperature (R=0.60, R=0.57, P<0.01, 316 respectively), and N2O emissions were positively correlated with soil pH (R=0.44, P<0.01) 317 and water content (R=0.39, P<0.05). However, the relationships changed in function of 318 the presence of crabs depending on the level of tidal intensity. N2O emissions in the upper 319 tidal flat in the presence of crabs was positively correlated with pH (R=0.82, P<0.01) and 320 water content (R=0.59, P<0.05), negatively correlated with salinity (R=-0.59, P<0.01) 321 (Table 1). In the upper tidal flat without crabs, there was a negative correlation between 322 CH4 emissions and salinity (R=-0.86, P<0.01), and N2O emissions were positively 323 correlated with soil temperature (R=0.86, P<0.01) and water content (R=0.70, P<0.05). 324 Fluxes of CO2, CH4, and N2O were positively correlated with soil temperature (R=0.84, 325 R=0.89, R=0.88, P<0.01, respectively) and in the presence of crabs (P<0.005), while N2O 326 emission flux was negatively correlated with soil bulk density (R=-0.88, P<0.01). 327 We found that N2O fluxes in the upper tidal flats were positively correlated with 328 soil concentration of TN, TP (R=0.75, R=0.79, P<0.01, respectively), and TC (R=0.63, 329 P<0.05) in the presence of crabs and with soil concentration of TC, TN, and TP (R=0.77, 13 330 R=0.83, R=0.78, P<0.01, respectively) in the absence of crabs (Table 2). CO2 emissions 331 in the mid-tidal flats were negatively correlated with soil TC (R= -0.58, P<0.05) in the 332 presence of crabs and with soil TC concentration and ratio of N:P (R= -0.62, R= -0.68, 333 P<0.05, respectively) in the absence of crabs. CH4 emissions were positively correlated 334 with C:N (R=0.71, P<0.01) and C:P ratios (R=0.60, P<0.05), and N2O flux was positively 335 correlated with ratio of C:N (R=0.77, P<0.01). Overall CO2 emissions were negatively 336 correlated with soil concentration of TC (R= -0.37, P<0.05), while CH4 flux was 337 positively correlated with C:N and C:P ratios (R=0.36, R=0.29, P<0.05, respectively) and 338 N2O flux was positively correlated with soil TN concentration (R=0.35, P<0.05). The 339 relationships between soil physicochemical factors on soil TC, TN and TP and soil 340 stoichiometry are provided in supplementary material (Table S4). 341 342 Overall effects of crabs and flooding intensity on greenhouse gas emissions and soil 343 physicochemical properties 344 The GDA showed that N2O emissions, soil TC, C:N ratio, water content, bulk density and 345 temperature clustered in the four combinations of crab presence/absence and flooding 346 intensity (Tables S5, S6). This GDA detected that the presence of crabs in upper tidal had 347 higher overall effects in the studied variables than in the middle tidal intensity (Figure 348 S4). Plots in the upper-tidal flats were associated with N2O emissions, bulk density and 349 soil C:P and N:P ratios more with than without crab presence. These great differences due 350 to crab presence were instead not observed in the middle tidal intensity plots (Figure S4). 351 Plots in the middle tidal flats were though correlated with higher soil water content and 352 TP more with than without crab presence (Fig.S4). 353 354 Discussion 14 355 Effects of crab presence on greenhouse gas emissions 356 CO2 emissions in wetland soils largely derives from respiration of soil animals and plant 357 roots, and the decomposition of organic C by soil microorganisms (Blagodatsky and 358 Smith 2012). This study showed that CO2 emissions were greater with than without crabs, 359 possibly due to the direct effects of respiration and the indirect effects feeding, excretion, 360 and disturbance of soil on soil microorganisms and/or plant roots. The burrowing activity 361 of crabs may increase the diffusion of gas and increase soil concentration of O2 and this 362 increases soil oxidation capacity. Similar increases in organic C mineralization due to 363 high litter decomposition rates in crab burrows observed in other wetlands (Daleo and 364 Iribarne 2009; Weissberger et al. 2009) depended on the stimulation aerobic bacteria 365 respiration (Liang et al. 2015). 366 Estuarine wetlands are an important source of CH4 (Du et al. 2016) and emissions 367 of CH4 mainly depend on the relative balance its production and oxidation processes. In 368 this study, greater CH4 emissions were correlated with presence of crabs throughout the 369 year. Consumption of plant and animal litter by crabs is converted to detritus and 370 promotes the accumulation of organic C (Moseman-Valtierra et al. 2011) and this 371 stimulates the growth and reproduction of methanogens (Kammann et al. 2009) leading 372 to increased CH4 production and emission. Burrowing activities of crabs increase the 373 sediment-water-gas contact interface, and the diffusion of inorganic N in sediments from 374 the overlying water bodies and accelerates the rate of ammonia formation (Mereta et al. 375 2013) and its accumulation in soil. Studies have found that an increase in NH4+ inhibits 376 methanotrophic activity and this promotes CH4 emissions (Hu et al. 2015). 377 Our results showed that N2O fluxes were greater in the presence of crabs. Production 378 of N2O was the result of a combination of soil nitrification and denitrification (Harley et 379 al. 2015), which are microbial processes, so factors that affect soil microbial activity also 15 380 impact soil nitrification and denitrification (Angar et al. 2016). Crab burrowing activity 381 results in the heterogenous creation of anaerobic and aerobic micro-sites in soils that are 382 used by a diverse range of microbes; soils surrounding burrows were compacted and 383 anaerobic, while burrows facilitate advection and diffusion of O2. Microbial activity in 384 these contrasting soil environments generates N2O through nitrification and 385 denitrification (Liu et al. 2008). Crabs may affect microbial diversity through feeding on 386 bacteria and fungi, mechanical action on organic matter, propagation of microbial 387 propagules, and changes in nutrient availability that leads to increases in soil N2O 388 emissions (Cragg and Bardgett 2001). The O2 exchange interface between soil and water 389 or the atmosphere is increased in crab burrows. This may lead to increased soil 390 nitrification at the burrow wall, while soil behind this wall may remain anaerobic 391 (Kristensen et al. 2008) and support denitrification leading to greater production and 392 release of N2O. Studies have shown that crabs in upper tidal flats may elicit environmental 393 effects. For example, high-densities of crabs at small spatial scales accelerate N2O release 394 in flooded tidal flats (Liu et al. 2008), and crabs fragmentation of surface accelerates litter 395 decomposition with the release of N to the soil environment (Chen et al. 2010). In addition, 396 direct input of excreta by crabs increases soil N concentration (Van et al. 2015) that 397 triggers N2O emissions due to the increased abundance and activity of nitrifying and 398 denitrifying soil bacteria (Welsh et al. 2015). 399 Global warming potential (GWP) of the upper tidal flats increased by 138% with than 400 without crabs, and GWP of the mid-tidal flats increased by 99.3% % with than without 401 crabs (Tables S2, S3). This a very noticeable result because highlight the importance that 402 the direct impacts of global warming on ecosystem processes can in turn exert a powerful 403 feed-back effect on future climate change. In this case a clear positive feed-back has been 404 detected. Rises in sea levels due to global warming are predicted and under this scenario 16 405 further increases in the intensity and frequency of flooding are expected that may result 406 in greater crab activity in wetlands, along with associated changes in soil properties (Chen 407 et al. 2008; Wang et al. 2011; Liu et al. 2015). We have demonstrated that higher crab 408 presence favors the production and emissions of greenhouse gases closing a positive feed- 409 back effect. 410 411 Effects of crab presence on soil C, N, and P concentrations 412 Soil C, N, and P in wetlands may be affected by multiple factors, such as microbial activity, 413 animal disturbance, decomposition of animals and plants, tidal movement, and human 414 activities (Liu et al. 2014). In this study, the disturbance by crabs was correlated with 415 changes in the concentrations of soil C, N, and P that varied with season. The greatest 416 concentrations of soil C, N, and P were recorded in autumn, correlated with plant 417 senescence and higher inputs of litter that results in larger inputs of food to be consumed 418 and decomposed by the crabs. Overall soil TP concentrations were significantly lower in 419 the presence of crabs than in the control. These results were not consistent with the 420 findings of Mortimer et al. (1999) who did not observe changes in soil TP in the presence 421 of crabs. Animal excreta (feces + urine) drive soil N:P ratios (Sitters et al. 2017). 422 Nevertheless, the greater range in soil TC and TN than TP concentration values 423 throughout the year is consistent with lower mobility and transformation rates of soil P 424 than C and N. 425 426 Ecological stoichiometry of soil C, N, and P under the presence of crabs 427 Concentrations of soil C, N, and P vary spatio-temporally with soil factors, such as animal 428 activity, vegetation, climate, and human interference (Cleveland and Liptzin 2007; Liu et 429 al. 2014). We found that C:N, C:P, and N:P ratios were lowest in summer and these lower 17 430 ratios were correlated with higher temperatures in summer when microbial activity is also 431 higher, favoring nutrient use and organic mineralization. The greater release of soil C 432 during the summer reduces its soil concentration and thus favors the drop of its ratios with 433 nutrients, indicating that soil C:nutrient ratios are also controlled by soil C retention and 434 input (Xiao et al. 2014). The decrease in the N:P ratio during the summer is likely due to 435 the slower mineralization rates of P than N, while N responded more rapidly to 436 environmental changes than P (Cleveland and Liptzin 2007). These results confirm the 437 observed nutrient limitation that occurs during S. alterniflora growth in the estuarine 438 wetlands of the Minjiang River (Wang et al. 2015c; Mactavish and Cohen 2017) that lead 439 to greater plant N and P-uptake, especially during summer. 440 The ratios of C:N, and N:P were greater with than without crabs, and these ratios 441 were greater in the crab-free upper tidal flats than in the crab-free mid-tidal flats. Thus, 442 the presence of crabs was correlated with greater N:P ratios and thus specially with the 443 lower TP concentration. 444 Nutrient ratios and C concentration have important implications for C storage. In 445 this study, we found the ecological stoichiometric ratios (C:N, C:P, and N:P) were 446 positively correlated with C stocks, supporting findings of Wang et al. (2016). The N:P 447 ratio in soils of our study without and with crabs (2.03 and 2.14) were lower than the 448 national wetland N:P ratio (13.6) (Zhang et al. 2016); in general, a lower N:P ratio 449 indicates greater primary productivity capacity, but N-limitation may become more 450 critical for plant growth in this wetland area under increasing temperatures due to climate 451 change (Wang et al. 2015c). 452 453 Effect of presence of crabs on relationships between soil greenhouse gas emissions 454 and soil nutrient and ecological stoichiometry 18 455 The availability of soil organic C is an important factor for microbial-mediated 456 decomposition processes and CO2 flux (Vieux et al. 2013). Crabs convert litter into debris 457 through feeding and digestion and this debris is subsequently incorporated into the soil in 458 the form of excrement; this process increases input to the soil of organic substances and 459 abundance and activity of soil microbes that altogether lead to accelerated C-cycling 460 (Žifčáková et al. 2016), accumulation of organic C in the soil, and increased CO2 461 production and emissions (Mehnaz et al. 2018). When oxidation of organic matter is 462 incomplete, as expected under flooding conditions, the production of a series of pyrolytic 463 compounds increases and this increase the resistance to chemical and biological 464 degradation (Shahbaz et al. 2017). However, crab activity increases aeration and destroys 465 large aggregates, allowing greater decomposition of soil organic matter until complete 466 oxidation occurs, resulting in greater CO2 formation (Lehmann and Kleber 2015; Murphy 467 et al. 2017). 468 Consistently, in mid flooding plots without crabs we found a positive correlation 469 between CH4 emission flux and ratios of C:N and C:P , indicating that increasing amounts 470 of organic C substrates with lower nutritional quality favored growth and development of 471 soil methanogens, and an increase in CH4 production and emissions in absence of crabs. 472 This was not observed in the presence of crabs suggesting the role of crabs in increasing 473 soil aeration and destroying large aggregates. 474 475 Other drivers of greenhouse gas emissions from wetland soils 476 In this study, the CO2 flux of soil was positively correlated with soil temperature, likely 477 as a result of associated increases in soil microbial abundance and activity (Andrews et 478 al. 2000). Increases in soil temperature to 35 °C drive increases in the abundance and 479 activity of soil microorganisms and CO2 emission flux (Pugh et al. 2018). Soils contain 19 480 autotrophic and heterotrophic respiring microorganisms (Murdiyarso et al. 2010), and 481 temperatures of c. 15–31 °C enhance wetland soil microbial activity and accelerate the 482 rate of degradation of soil organic C, leading to greater releases of CO2 (Yang et al. 2017). 483 We found that emissions of CH4 from the soil were positively correlated with soil 484 temperature, supporting a previous study in a wetland (Yang et al. 2017). The associated 485 increase in soil microbial activity with temperature increases soil O2 consumption and 486 this stimulates the growth of methanogens (Zheng et al. 2018), thus observing a positive 487 correlation between soil N2O flux and soil temperature consistent with Tian et al (2015). 488 As soil temperatures rise, soil microbial nitrification and denitrification and N2O 489 emission rate similarly increase (Castaldi 2000). The optimum soil temperature range for 490 nitrification is c. 15–35 °C, while that for denitrification is c. 5–75 °C (Sun et al. 2010), 491 so it is likely that these processes were active in this study, with greater levels of microbial 492 activity and associated N2O emissions from soil. The soil water content, which was 44.2– 493 59.1%, was positively correlated with N2O flux, probably because of nitrification 494 processes are favored in this range of soil water content (Bramley and White 1989; 495 Szukics et al. 2010). These results support a previous study that showed N2O production 496 and emissions increased with increasing soil water content (Di et al. 2014). N2O flux was 497 positively correlated with soil pH, consistent with what observed in previous reports 498 (Nkongolo et al. 2008; Duan et al. 2018), associated with the microbial growth 499 (Castellano-Hinojosa et al. 2018). Moreover, the wetland anaerobic environment was rich 500 in ammonium substrate, and then N2O flux here is mainly produced by nitrification, and 501 its emissions increase with the increase of soil pH. 502 503 Conclusion 504 This study, at the best of our knowledge is the first that have studied the shift of gas 20 505 emissions and nutrient stoichiometry due to the presence of crabs in a subtropical 506 estuarine wetland ecosystem. The presence of crabs mainly accelerates the C and N 507 cycling probably due to increasing aeration, allowing greater decomposition of soil 508 organic matter with greater CO2 formation. However, crab presence, specially in up-tidal 509 sites, decreased soil P concentration, thus rising soil C:P and N:P ratio. 510 Crab activity (feeding and digestion) also favored the incorporation of litter into soil 511 in the form of excrement; this process increased the input of organic substances to soil 512 and the abundance and activity of soil microbes, both leading to accelerated C-cycling 513 and increased CO2 production and emissions. Moreover, crab burrowing activity resulted 514 in the heterogeneous creation of anaerobic and aerobic micro-sites, where soils 515 surrounding burrows were compacted and anaerobic, while burrows facilitate advection 516 and diffusion of O2. This soil environment generates the conditions favorable for CH4 517 production and for N2O emission. Cumulative CH4 emissions were significantly higher 518 with than without crabs under both tidal intensities, whereas cumulative CO2 and N2O 519 emissions were also higher with than without crabs but the impact was significantly 520 higher under up- than under middle-tidal intensity. 521 The rise of sea level due to global warming may increase the intensity and 522 frequency of flooding, thus promoting the activity of crabs in wetlands, along with 523 associated changes in soil properties, thus inducing a positive feed-back effect on global 524 warming. 525 526 Acknowledgements 527 The authors would like to thank Dengzhou Gao, Xu Song, and Kang Xu for their 528 assistance with field sampling. Funding was provided by the National Science Foundation 529 of China (41571287), the Outstanding Youth Scientific Research Talents Cultivation Plan 21 530 in Colleges and Universities of Fujian Province 2017, Spanish Government grant 531 CGL2016-79835, Catalan Government grant SGR 2017-1005, and European Research 532 Council Synergy grant ERC-SyG-2013-610028, IMBALANCE-P. 533 534 Conflicts of Interest 535 The authors declare no conflicts of interest. 536 537 References 538 Ahmad S, Li C, Dai G, Zhan M, Wang J, Pan S, Cao C (2009) Greenhouse gas emission 539 from direct seeding paddy field under different rice tillage systems in central China. 540 Soil Till Res 106: 54-61 541 Allen AP, Gillooly JF (2009) Towards an integration of ecological stoichiometry and the 542 metabolic theory ecology to better understand nutrient cycling. 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Environ 780 Microbiol 18: 288-301 31 Tables Table 1 Correlation between greenhouse gas emission fluxes and environmental factors (Pearson correlation coefficient) Treatment Index pH Salinity Bulk density Temperature Water content CO2 -0.445 0.14 -0.347 -0.274 0.117 GP CH4 -0.258 0.407 0.315 -0.234 -0.082 N2O 0.816** -0.742** -0.162 0.02 0.591* CO2 -0.107 0.007 -0.331 0.224 -0.228 GD CH4 0.459 -0.857** -0.169 0.51 0.178 N2O -0.454 -0.114 -0.308 0.856** 0.701* CO2 -0.452 -0.16 -0.430 0.654* -0.308 ZP CH4 0.235 -0.254 -0.624* 0.636* 0.267 N2O -0.482 -0.052 -0.246 0.616* -0.238 CO2 -0.254 -0.224 -0.532 0.839** 0.012 ZD CH4 0.037 -0.21 -0.451 0.887** 0.534 N2O -0.247 -0.138 -0.875** 0.883** 0.374 CO2 -0.257 -0.091 -0.358* 0.352* -0.113 Total CH4 -0.039 -0.113 -0.186 0.598** 0.134 N2O 0.439** -0.143 0.005 0.568** 0.386* GP: upper tidal flat in presence of crabs; GD: upper tidal flats in absence of crabs; ZP: mid-tidal flats in presence of crabs; and, ZD: mid-tidal flats in absence of crabs. *P<0.05, **P<0.01. 32 Table 2 Correlation between emissions of greenhouse gases and soil content of nutrients in the presence and absence of crabs (Pearson correlation coefficient) Treatment Index TC TN TP C:N C:P N:P CO2 -0.282 -0.172 -0.106 -0.154 0.170 0.344 GP CH4 0.055 -0.064 -0.086 0.352 0.301 0.139 N 2O 0.633* 0.746** 0.787** -0.093 -0.097 -0.103 CO2 -0.300 -0.151 -0.060 0.039 -0.116 -0.206 GD CH4 -0.283 0.025 0.102 -0.440 -0.448 -0.315 N 2O 0.773** 0.833** 0.778** 0.150 -0.227 -0.429 CO2 -0.576* -0.115 0.301 -0.175 0.095 0.232 ZP CH4 0.473 0.476 0.488 0.055 0.111 0.114 N 2O -0.468 -0.071 0.340 -0.034 0.173 0.244 CO2 -0.616* -0.441 0.338 0.468 0.066 -0.675* ZD CH4 -0.273 -0.180 0.068 0.710** 0.601* -0.118 N 2O -0.568 -0.066 0.267 0.769** 0.482 -0.187 CO2 -0.374** -0.153 0.099 0.059 0.093 -0.022 Total CH4 0.026 0.058 0.157 0.355* 0.289* -0.009 N 2O 0.260 0.351* 0.283 0.082 0.097 0.042 TC: soil total C; TN: soil total N; soil TP: total P; C:N: soil C:N ratio; C:P: soil C:P ratio; N:P: soil N:P ratio; GP: upper tidal flat in presence of crabs; GD: upper tidal flats in absence of crabs; ZP: mid-tidal flats in presence of crabs; and, ZP: mid-tidal flats in absence of crabs. *P<0.05, **P<0.01. 33 Figure Captions Fig. 1 Location of the study area and sampling sites in southeastern China Fig. 2 Effect of presence of crabs on CO2 emission fluxes from soils of a Spartina alterniflora wetland. Different uppercase letters indicate within season treatment differences and different lowercase letters indicate between season treatment differences (P<0.05) Fig. 3 Effect of presence of crabs on CH4 emission fluxes from soils of a Spartina alterniflora wetland. Different uppercase letters indicate within season treatment differences and different lowercase letters indicate between season treatment differences (P<0.05) Fig. 4 Effect of crabs on N2O emission fluxes from soils of a Spartina alterniflora wetland. Different uppercase letters indicate within season treatment differences and different lowercase letters indicate between season treatment differences (P<0.05) Fig. 5 Effect of crabs on soil TC, TN, and TP concentrations and their stoichiometric ratios. Different uppercase letters indicate within season treatment differences and different lowercase letters indicate between season treatment differences (P<0.05) Fig. 6 Linear regression of the effect of crabs on relationships between soil C, N, and P. The y1 and y2 functions represent the linear fitting relationship of total nutrients with and without crab activity, respectively. 34 1 2 3 4 5 6 7 8 9 10 11 12 Fig. 1 13 14 15 16 17 18 19 35 20 21 Upper tidal flat with crabs Upper tidal flat without crabs Mid-tidal flat with crabs Mid-tidal flat without crabs 500 250 Winter Spring 400 200 CO2 flux（mg m h ） CO2 flux（mg m h ） Aa ABab -1 -1 300 150 Ba Bab -2 -2 Bb Bab Bab 200 100 Bb 100 50 0 0 -100 -50 GP GD ZP ZD GP GD ZP ZD 600 350 Aa Summer Autumn Ab 280 CO2 flux（mg m h ） CO2 flux（mg m h ） 450 Ab -1 -1 210 -2 -2 300 ABb ABa 140 Bb 150 Bb Bb 70 0 0 GP GD ZP ZD GP GD ZP ZD 22 23 Fig. 2 24 25 26 36 Upper tidal flat with crabs Upper tidal flat without crabs Mid-tidal flat with crabs Mid-tidal flat without crabs 2.0 0.15 Winter Spring 1.5 0.10 CH4 flux（mg m h ） CH4 flux（mg m h ） Ba -1 -1 1.0 Aa Aa -2 -2 0.05 Aa Aa 0.5 Aa Ba Aa 0.00 0.0 -0.5 -0.05 -1.0 -0.10 GP GD ZP ZD GP GD ZP ZD 250 16 Summer Autumn 200 12 CH4 flux（mg m h ） CH4 flux（mg m h ） -1 -1 150 8 -2 -2 Aa 100 Aa 4 Aa 50 Ba Aa Ab Ab Ab 0 0 -4 -50 GP GD ZP ZD GP GD ZP ZD 27 28 29 Fig. 3 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 37 63 64 65 Upper tidal flat with crabs Upper tidal flat without crabs Mid-tidal flat with crabs Mid-tidal flat without crabs 90 120 Winter Spring Ba 60 80 N2O flux（μg m h ） N2O flux（μg m h ） Bab Ba Ba -1 -1 Ba Ba 40 Ba -2 -2 30 Bb 0 0 -40 -30 -80 GP GD ZP ZD GP GD ZP ZD 250 1000 Aa Aa Summer Autumn 200 800 N2O flux（μg m h ） Aa N2O flux（μg m h ） -1 -1 150 600 -2 -2 Aab 100 Bb 400 50 Bb 200 Bb Bb 0 0 -50 -200 GP GD ZP ZD GP GD ZP ZD 66 67 Fig. 4 68 38 69 Upper tidal flat with crabs Upper tidal flat without crabs Mid-tidal flat with crabs Mid-tidal flat without crabs 16 10.5 A A a AB a A A a a 10.0 A AB a 15 concentration (g kg-1) ab a A AB AB a a 9.5 A B A ab 14 bc A a ab A AB B b b A B ab AB b b A AA ab 9.0 b C N AB bc bc c A b A b 13 BC b c C 8.5 C A B b c b c A c 12 8.0 TC 7.5 11 Winter Spring Summer Autumn Winter Spring Summer Autumn A 2.0 24 A a AB A AB a a a AB AB concentration (g kg-1) 1.8 A ab A a a B 22 A a a A AB AB a ab b b AB AB A b B 1.6 B 20 a A b AB b B b A A c a a b AB ab C P B c c A A AB 1.4 18 a ab b A A a A ab b B 1.2 16 b TN 1.0 14 Winter Spring Summer Autumn Winter Spring Summer Autumn 1.0 2.7 A A A AA a a A ab A A a a a a a A concentration (g kg-1) a A A A A A 2.4 A a a A a A ab A a a a A 0.8 ab A A a a A A b A AB b A A b A b a a A a A b B b a 2.1 N P b b A a 0.6 1.8 TP 0.4 1.5 Winter Spring Summer Autumn Winter Spring Summer Autumn 70 71 Fig. 5 39 High tidal flat with crabs Middle tidal flat with crabs Linear regression with crabs High tidal flat without crabs Middle tidal flat without crabs Linear regression without crabs 2.0 1.0 1.0 y2=0.02827x2+0.3942 y1=0.1184x1-0.0482 2 2 R2=0.1388, P2<0.05 y2=0.37853x2+0.18372 TN concentration (g kg ) TP concentration (g kg ) TP concentration (g kg ) R1=0.6821, P1<0.01 -1 -1 -1 0.9 0.9 2 R2=4096, P2<0.01 1.8 0.8 0.8 1.6 0.7 0.7 1.4 y2=0.09699x2+0.25892 y1=0.0504x1-0.0553 y1=0.539x1-0.106 2 0.6 0.6 2 R2=0.6685, P2<0.01 2 R1=0.2008, P1<0.01 R1=0.5198, P1<0.01 1.2 12 14 16 18 12 14 16 18 1.2 1.4 1.6 1.8 2.0 -1 -1 -1 TC concentration (g kg ) TC concentration (g kg ) TN concentration (g kg ) Fig. 6 40