Scott Johnson
email: scott.johnson@scri.ac.uk

Adam T Barton

Katherine E Clark

Peter J Gregory

Lindsay S Mcmenemy

Rob D Hancock

Elevated atmospheric CO2 impairs the

Keywords: black vine weevil, carbon dioxide, induced responses, phenolics, roots, soils, secondary metabolites black vine weevil, carbon dioxide, induced responses, phenolics, roots, soils, 34 secondary metabolites

concentration following herbivory. There was an interactive effect of CO2 and root herbivory on root phenolic concentrations, whereby weevils induced an increase at ambient CO2, suggestive of defensive response, but caused a decrease under eCO2. Contrary to predictions, there was a positive relationship between root phenolics and weevil performance. We conclude that impaired root growth underpinned the negative effects of eCO2 on vine weevils and speculate that the plants failure to mount a defensive response at eCO2 may have intensified these negative effects. Increasing crop production to achieve food security in the face of global climate change has become a 65 priority as the world's population continues to grow by 1.2% each year (The Royal Society, 2009).

66

Predicting how crop pests will be affected by global climate change is vital for realising such 67 production goals, and will increasingly rely on empirically based research [START_REF] Gregory | Integrating pests and pathogens into 393 the climate change/food security debate[END_REF]. In 68 particular, there is a considerable amount of research describing how insect herbivores may respond to 69 elevated carbon dioxide (eCO 2 ) concentrations (reviewed by Bezemer & Jones, 1998), yet this 70 information is almost exclusively concerned with shoot-feeding insects. In a recent review, Staley and 71 Johnson (2008) were only able to identify two studies that investigated the effects of eCO 2 on root-72 feeding insects [START_REF] Salt | Interspecific herbivore interactions in a high CO 2 462 environment: root and shoot aphids feeding on Cardamine[END_REF][START_REF] Johnson | Elevated CO 2 and aboveground-belowground herbivory by 419 the clover root weevil[END_REF], despite the considerable pest status of 73 root-feeding insects in many agro-ecosystems [START_REF] Blackshaw | Root herbivory in agricultural ecosystems[END_REF].

75

The effects of eCO 2 on root herbivores are unlikely to simply mirror those effects seen for shoot 76 herbivores due to the contrasting habitats of the two [START_REF] Staley | Climate change impacts on root herbivores[END_REF]. In particular, the 77 effects of eCO 2 are likely to be largely plant-mediated as CO 2 concentrations in the soil are already 78 considerably higher than atmospheric concentrations, making soil invertebrates pre-adapted to eCO 2 79 conditions [START_REF] Haimi | 396 Impacts of elevated CO 2 and temperature on the soil fauna of boreal forests[END_REF]. Moreover, soil-dwelling herbivores are likely to be more buffered 80 from the direct effects of temperature increases [START_REF] Bale | Herbivory in global climate change 364 research: direct effects of rising temperature on insect herbivores[END_REF]. Changes in patterns of root growth, 81 nutritional status and secondary metabolism are therefore more likely to drive root-herbivore 82 interactions [START_REF] Staley | Climate change impacts on root herbivores[END_REF]. The effect of eCO 2 on root traits vary depending on plant taxa, 83 but there is a general trend for roots to increase in mass relative to shoots [START_REF] Rogers | Plant responses to atmospheric CO 2 enrichment 458 with emphasis on roots and the rhizosphere[END_REF]Rogers 84 et al., 1996) and to have decreased nitrogen concentrations with a corresponding increase in C:N ratios 85 [START_REF] Luo | Predicting responses of photosynthesis and root 441 fraction to elevated CO 2 : interactions among carbon, nitrogen and growth[END_REF]Bezemer & Jones, 1998;[START_REF] Cotrufo | Elevated CO 2 reduces the nitrogen concentration of 385 plant tissues[END_REF]. Responses of secondary metabolites 86 to eCO 2 are more variable [START_REF] Hartley | Biosynthesis of plant phenolic 407 compounds in elevated atmospheric CO 2[END_REF]. For carbon-based secondary metabolites, such as 87 phenolics, studies have shown both increases (e.g. [START_REF] Lindroth | Responses of deciduous trees to elevated 438 atmospheric CO 2 -productivity, phytochemistry, and insect performance[END_REF][START_REF] Roth | Effects of CO 2 mediated changes in paper birch and white pine 460 chemistry on gypsy moth performance[END_REF] and88 decreases (e.g. [START_REF] Hartley | Biosynthesis of plant phenolic 407 compounds in elevated atmospheric CO 2[END_REF][START_REF] Veteli | Effects of 485 elevated CO 2 and temperature on plant growth and herbivore defensive chemistry[END_REF] [START_REF] Zvereva | Consequences of simultaneous elevation of carbon dioxide 490 and temperature for plant-herbivore interactions: a metaanalysis[END_REF]. Instead, this study suggested that elevated temperatures 92 caused decreases in phenolic concentrations, irrespective of CO 2 conditions. However, the majority of 93 the research effort has focussed on secondary metabolites in shoots and has not, to our knowledge, 94 addressed how eCO 2 affects induction of secondary compounds by root herbivores. Given recent 95 evidence that roots deploy an array of defences against root herbivores [START_REF] Rasmann | In defense of roots: a research agenda for studying plant 454 resistance to belowground herbivory[END_REF], 96 this constitutes an important, but overlooked, aspect of how global climate change will affect plant-97 herbivore interactions [START_REF] Van Noordwijk | 483 Global change and root function[END_REF].

99

The objective of this study was to determine how eCO 2 affected a root feeding herbivore via 100 changes to plant growth and concentrations of carbon, nitrogen and phenolics. We focussed 101 on phenolics as they are ubiquitous in terrestrial plants, they have a well characterised role in 102 plant defence, and their concentrations are known to respond to many environmental factors 103 [START_REF] Harborne | Phenolics. In: Natural Products. Their chemistry and biological 402 significance[END_REF][START_REF] Hartley | Plant chemistry and herbivory, or why the world is green[END_REF]. 104 105

The study system used for this research was a perennial crop, blackcurrant (Ribes nigrum L. 106 Saxifragales: Grossulariaceae) and the vine weevil (Otiorhynchus sulcatus L. Coleoptera: 107 Curculionidae). The vine weevil is a polyphagous parthenogenetic herbivore and has a 108 lifecycle that takes place above-and belowground. The adult lives aboveground and lays eggs 109 that fall into the soil. Eggs hatch and give rise to root-feeding larvae, which can cause 110 substantial economic damage to horticultural and nursery crops [START_REF] Moorhouse | A review of the biology and control of the 446 vine weevil, Otiorhynchus sulcatus (Coleoptera, Curculionidae)[END_REF]. 111

This study specifically compared the effects of eCO 2 (700 µmol mol -1 ) and ambient carbon 112 dioxide (aCO 2 ) concentrations (375 µmol mol -1 ) on (i) vine weevil larvae performance, (ii) 113 shoot and root biomass, (iii) leaf and root water concentration, (iv) leaf and root C and N 114 concentrations and (v) root phenolic concentrations. The eCO 2 concentration of 700 µmol 115 mol -1 represents current predictions for the late twenty-first century (IPCC, 2007). We 116 hypothesised that eCO 2 would cause vine weevil performance (abundance and mass) to 117

Materials and Methods 120

Plant growth chambers 121

In this study, replicating soil temperature gradients seen in the field was of particular 122 importance to accurately determine how roots and herbivores would respond to eCO 2 123 [START_REF] Johnson | Elevated CO 2 and aboveground-belowground herbivory by 419 the clover root weevil[END_REF]. To this end, this study used specially constructed chambers that 124 allowed soil and air temperature patterns to be regulated in a spatially and temporally realistic 125 manner (see Gordon et al., 1995 for full details). Experiments were conducted in two plant 126 growth chambers in which CO 2 was maintained at either aCO 2 (375 µmol mol -1 ) or eCO 2 (700 127 µmol mol -1 ). The experiment was therefore repeated three times to overcome the issue of 128 pseudo-replication and chamber effects (discussed by Bezemer et al., 1998). Air temperature 129 followed a daily sine function with a midday peak of 20°C (± 0.5°C) and nocturnal low of 130 10°C (± 0.5°C). Soil temperature at depths of 0.55m and 1.0m remained constant at 131 approximately 12°C whereas soil temperature at 0.1m followed a damped lag function of air 132 temperature. Plants were grown in long vertical rhizotubes (120 × 2.5 × 5cm) which were 133 surrounded by cooling coils and thermal insulation. [CO 2 ] and temperature measurements 134 were monitored with thermocouples (placed in the soil) and an infra red gas analyser (IRGA 135 225, ADC Ltd, UK), respectively, and relayed to a data logger (CR21X, Campbell Scientific 136 Ltd) at 2 min intervals, which then directed injections of CO 2 and the coolant as appropriate 137 (for full details, see [START_REF] Gordon | Plant growth chambers for the 390 simultaneous control of soil and air temperatures, and of atmospheric carbon dioxide 391 concentration[END_REF]. Each rhizotube was installed with an irrigation tube 138 that delivered 28ml of water every 24hr. The chambers were located in a glasshouse and 139 received supplemental lighting (16:8 light: dark) from overhead lamps (Philips 400 W SON-T 140 AGRO). 141 142

Experiments 143 F o r R e v i e w O n l y
For each run of the experiment, forty similar sized blackcurrant plants (cv. Ben Connan) were 144 established from stem cuttings in sand (Silver sand, J. Arthur Bowers, Lincoln, UK) and 145 acclimatised to the appropriate CO 2 treatment in the chambers after being transplanted to 146 compost (peat-sand-perlite mix containing 17N:10P:15K; William Sinclair Horticulture Ltd, 147 Lincoln, UK). When plants were a further 6 weeks old (6-8 cm in height) they were 148 transferred to the rhizotubes filled with 1.35kg of an air dried cambisol that had been sieved 149 to <2mm and then watered to 20% gravimetric water content. Vine weevil eggs were 150 harvested from an established culture at SCRI that originated from adult vine weevils 151 collected from a field site (56°447'N, 3°012'W) comprising multiple cultivars of raspberry, 152 strawberry and blackcurrant (see Johnson et al., 2009 for full details). One week after 153 introducing plants to tubes, 35 vine weevil eggs were applied to 10 randomly assigned plants 154 in each chamber. Eggs were ca. 5 days old and had melanised (i.e. were viable) by the time 155 tubes were inoculated. Each experiment ran for a further five weeks before all plants were 156 harvested. This represents the substantive period that vine weevils are root feeding (i.e. in 157 larval stages), with most generally going on to adulthood after this period of larval 158 development. Larvae were carefully separated from the root system, counted and weighed on 159 a microbalance. In order to attribute individuals to larval instar, two larvae were selected at 160 random from each tube and examined under a microscope at ×40 magnification to ascertain 161 the size of the head capsule using a micrometer (see La [START_REF] Lone | Larval development of Otiorhynchus sulcatus (Coleoptera, 432 Curculionidae) and effects of larval density on larval mortality and injury to 433 Rhododendron[END_REF]. Fresh plant 162 material was separated and weighed, snap frozen in liquid nitrogen and then stored at -18°C. 163

Material was then freeze-dried and weighed to determine dry mass and water content prior to 164 chemical analysis. The experiment was repeated three times, so that each of the factorial 165 treatments (aCO 2 and eCO 2 , with and without weevils) was repeated 30 times over the three 166 Freeze-dried leaf material was ball-milled to a fine powder for all extractions and chemical 170 analyses. The C and N concentration of leaves was determined using an Exeter Analytical 171 CE440 Elemental Analyzer (EAI, Coventry, UK). Combustion of the weighed sample (ca. 3 172 mg) occurred in pure oxygen using helium to carry the combustion products through the 173 analytical system. The C and N concentrations of samples were calculated using standards 174 (Acetanilide) with known C and N concentrations. Benzoic acid was also used as a standard 175 and to check the nitrogen blanks (see [START_REF] Anon | Technical Note 232 -CE440 Elemental Analyzer -Theory of operation[END_REF] for full details). 176 177

Phenolic analysis 178

Analysis was carried out using the enzymatic method described by [START_REF] Stevanato | Enzymatic method for the determination of total 478 phenolic content in tea and wine[END_REF], 179 which is more specific than the commonly used Folin-Ciocalteu procedure (Waterman & 180 Mole, 1994) having the advantage that it is not affected by interfering substances such as 181 ascorbate, citrate and sulphite [START_REF] Stevanato | Enzymatic method for the determination of total 478 phenolic content in tea and wine[END_REF]. In summary, phenolics were extracted 182 in a 10:1 ratio from 50 mg freeze dried root material by incubating in 0.5 ml 50% methanol at 183 80°C for 2.5 hr. The aqueous phase was removed and cleared by centrifugation. A 1 ml 184 enzymatic reaction was set up using 50 µl of the resultant supernatant mixed with 740 µl 100 185 mM potassium phosphate buffer (pH 8.0), 100 µl 30 mM 4-aminophenazone, 100 µl 20mM 186 hydrogen peroxide and 1U horse radish peroxidise dissolved in 10 µl potassium phosphate 187 buffer. The reaction was incubated at room temperature for 15 min and absorbance read at a 188 wavelength of 500 nm. Absorbance data were converted to catechin equivalents using a 189 standard curve produced by serial dilution (0 -0.10 mg ml -1 catechin). Vine weevil larvae were significantly less abundant (F 1,2 = 21.46, P = 0.044) on the roots of 207 plants grown in eCO 2 compared with those grown in aCO 2 (Fig. 1a). In terms of the original 208 inoculation with eggs, this represents survival rates of 24% at aCO 2 and 16% at eCO 2 . Larvae 209 were attributed to fourth-fifth instar by assessment of head capsule size (La Lone & Clarke, 210 1981) in both CO 2 environments. However, the average mass of larvae was lower (F 1,2 = 211 17.12, P = 0.054, which approached statistical significance at the 95% confidence interval) 212 when they had fed on eCO 2 plants compared to aCO 2 plants (Fig. 1b). Larvae were typically 213 recovered from the top 10cm of the soil profile and there were no apparent differences in 214 vertical distribution of larvae between the two CO 2 treatments. 215 216 Elevated CO 2 concentrations did not significantly affect shoot mass in blackcurrant (Table 1) 217 but significantly impaired root growth (Fig. 2a). Maximum rooting depth was similar between 218 the treatments (data not shown). Shoot-to-root ratios and leaf water concentration were also 219 unaffected by either eCO 2 or weevils (Table 1), but water concentrations in roots were 220 reduced by root herbivory and to a lesser extent by eCO 2 (Fig. 2b; Table 1). Concentrations of 221 C and N were not significantly affected by CO 2 in blackcurrant foliage or roots (Table 1). 222 There were, however, significant changes in plant minerals following root herbivory by vine 223 weevils under both CO 2 scenarios (Table 1). Weevils significantly reduced leaf C 224 concentrations (Fig. 2c) and reduced the C:N ratio in roots of blackcurrant (Fig. 2d). This 225 increase in root C:N ratio arose through a decrease in root C and increase in root N, although 226 these differences were not statistically significant when considered individually (Table 1). 227

There was a significant interactive effect of CO 2 and weevil herbivory in terms of root 228 phenolic concentrations, with concentrations rising following root herbivory under aCO 2 but 229 decreasing with herbivory under eCO 2 (Fig. 2e, Table 1). Apart from root phenolics, there 230 were no significant interactive effects between CO 2 and weevil herbivory (Table 1). 231 232Relationships between all plant traits listed in Table 1 andweevil performance were 233 examined. There was a strong positive correlation between root mass and vine weevil larval 234 mass (Fig. 3; F 1,52 = 14.75, P < 0.001) under both CO 2 conditions, which did not differ in 235 terms of statistical significance (F 1,52 = 1.57, P = 0.215). Unexpectedly, phenolic 236 concentration in roots was also positively correlated with larval mass (Fig. 4a; F 1,52 = 14.73, P 237 < 0.001) at both CO 2 concentrations (F 1,52 = 0.14, P = 0.710). A similar positive association 238 was seen between root phenolics and weevil population size (Fig. 4b; F 1,53 = 6.09, P = 0.017) 239 at aCO 2 , but CO 2 significantly affected this relationship (F 1,53 = 4.28, P = 0.043) and the 240 correlation was less apparent at eCO 2 (Fig. 4b). 241 242 Overall, there were no consistent statistically significant relationships between plant traits and 243 plant chemistry, although there was a very weak correlation between root dry mass and root 244 phenolics on control plants (r s = 0.222, P = 0.088), which was stronger at aCO 2 (r s = 0.459, P 245 = 0.006) than eCO 2 (r s = 0.119, P = 0.073). 246 This study set out to investigate how eCO 2 affected a root herbivore via changes to root 249 growth and chemistry and found that vine weevil performance was impaired under eCO 2 , both 250 in terms of population size and body mass (being 33% and 20% lower, respectively). In R. 251 nigrum, eCO 2 impaired root growth by 16%, but had no significant effect on C and N 252 concentrations. Reductions in root growth were strongly associated with lower body mass in 253 weevils, which probably underpinned the indirect effects of eCO 2 on weevil performance. 254 Phenolic concentrations increased in R. nigum roots challenged by root herbivores under 255 aCO 2 , consistent with an induced defensive response, but phenolic concentrations did not 256 increase following herbivory under eCO 2 which suggested that plants could not mount this 257 response at eCO 2 . 258 259

The effects of eCO 2 on root growth have been studied for over 150 plant species (Rogers et 260 al., 1994), with most responding to eCO 2 by increasing dry root mass [START_REF] Rogers | Root to shoot ratio of crops as 456 influenced by CO 2[END_REF]. 261

Our study suggests that this is unlikely to happen in R. nigrum as root biomass significantly 262 decreased in response to eCO 2 , which has also been reported for other plant species (Bader et 263 al., 2009;[START_REF] Kohler | Elevated CO 2 increases the effect of an 428 arbuscular mycorrhizal fungus and a plant-growth-promoting rhizobacterium on 429 structural stability of a semiarid agricultural soil under drought conditions[END_REF]. While root growth is widely assumed to be enhanced due to 264 the fertilising effect of eCO 2 , this is often negated by variations in nutrient availability and the 265 response may, in any case, decrease after several generations [START_REF] Van Noordwijk | 483 Global change and root function[END_REF]. 266 Moreover, plants that are 'inherently' slow growing (e.g. woody perennial crops) are 267 sometimes unable to modify growth rates and internal sinks to the same extent as faster 268 growing plants, so CO 2 induced responses are often less pronounced in such plants ( Van 269 Noordwijk et al., 1998). [START_REF] Bader | Fine root responses of mature deciduous forest 362 trees to free air carbon dioxide enrichment (FACE)[END_REF] reported that woody plants in their study showed 270 ca. 30% reduction in fine root biomass in response to eCO 2 , and suggested that lower water 271 requirements at eCO 2 may be responsible. It is unclear whether this could account for 272

F o r R e v i e w O n l y
reductions in root biomass reported in this study, but our findings provide further evidence 273 that eCO 2 does not necessarily promote root growth [START_REF] Rogers | Root to shoot ratio of crops as 456 influenced by CO 2[END_REF]. 274 275 Plant C and N concentrations were not statistically significantly affected by eCO 2 but root N 276 concentrations decreased by 8% at eCO 2 in control plants, which is similar to the average 277 generic decrease of 9% predicted by Cortrufo et al. (1998). Other studies have reported that 278 eCO 2 did not affect C and N concentrations in roots of woody plants [START_REF] Handa | No stimulation in root production in 399 response to 4 years of in situ CO 2 enrichment at the Swiss treeline[END_REF]279 Bader et al., 2009), so it seems plausible that C and N concentrations will not significantly 280 change in R. nigrum under eCO 2 scenarios. In any case, it seems unlikely that changes in C 281 and N were related to the reduced performance of weevils at eCO 2 . 282 283 Under both CO 2 conditions, weevil herbivory significantly reduced leaf C and increased root 284 C:N ratios. This most likely arose due to root herbivory causing a sink in the root system, 285 which diverted carbohydrates away from the foliage and impaired C fixation generally 286 [START_REF] Blossey | Belowground herbivory by insects: influence on plants and 378 aboveground herbivores[END_REF][START_REF] Johnson | Linking aboveground and belowground 416 herbivory[END_REF]. Similar negative effects of root herbivory 287 on C concentration have been reported for both roots (e.g. [START_REF] Hopkins | The relationship between 409 cabbage root fly (Delia radicum) larval feeding and the freeze-dried matter and sugar 410 content of Brassica roots[END_REF]Katovich et 288 al., 1999) and leaves (e.g. [START_REF] Murray | Impact of insect root herbivory on the growth and 451 nitrogen and carbon contents of white clover (Trifolium repens) seedlings[END_REF][START_REF] Dawson | Root feeding behaviour of Tipula 387 paludosa (Meig.) (Diptera : Tipulidae) on Lolium perenne L. and Trifolium repens L. 388[END_REF]. These findings contrast 289 with earlier assumptions that root herbivory generally causes increases in foliar C (e.g. 290 carbohydrates) through reduced water uptake and plant stress [START_REF] Masters | Plant mediated interactions between aboveground 444 and belowground insect herbivores[END_REF]. This 291 difference may be explained by the fact that leaf water concentrations were unaffected, 292 suggesting that root herbivory did not induce this type of nutritional stress response in leaves. 293

294

The concentrations of root phenolics increased in response to vine weevil herbivory at aCO 2 , 295 but the reverse was seen at eCO 2 . Increases in phenolic concentrations are a common anti-296 herbivore defence exploited by many plants [START_REF] Hartley | Plant chemistry and herbivory, or why the world is green[END_REF], and have been shown to 297

F o r R e v i e w O n l y
increase in response to root herbivore attack [START_REF] Kaplan | Constitutive and induced 424 defenses to herbivory in above-and belowground plant tissues[END_REF]. Indeed, there is growing 298 evidence that roots are highly responsive in terms of inducible defence mechanisms, 299 triggering synthesis of secondary metabolites in both leaves (e.g. phenolics) and the roots 300 (e.g. nicotine) [START_REF] Kaplan | Constitutive and induced 424 defenses to herbivory in above-and belowground plant tissues[END_REF][START_REF] Rasmann | In defense of roots: a research agenda for studying plant 454 resistance to belowground herbivory[END_REF]. This seems to be consistent 301 with the optimal defence theory [START_REF] Stamp | Out of the quagmire of plant defense hypotheses[END_REF], which suggests that plants invest more into 302 constitutive defences in those tissues that are regularly attacked, whereas they are more reliant 303 on induced defences when herbivore attack is more intermittent. By their nature, root 304 herbivores are generally aggregated in the soil and attacks on plant roots tend to be sporadic 305 and localised [START_REF] Brown | Insect herbivory below ground[END_REF] which would clearly make inducible defence a more 306 profitable strategy against root herbivores according to the optimal defence theory. Why eCO 2 307 resulted in altered defence responses is beyond the scope of this study, however, previous 308 work undertaken in Arabidopsis demonstrated that eCO 2 had significant effects on expression 309 of a number of transcripts involved in secondary metabolism, abiotic and biotic stress 310 responses, and cellular signalling [START_REF] Li | 435 Arabidopsis transcript and metabolite profiles: ecotype-specific responses to open-air 436 elevated [CO 2 ][END_REF]. It is therefore likely that signal 311 transduction and defence responses may be altered in R. nigrum at eCO 2 environment. 312

In this study, constitutive levels of phenolic compounds in roots that had not been attacked by 313 weevils were unaffected by eCO 2 , which is consistent with the predictions of Zvereva & 314 Kozlov (2006) for woody plant tissues. 315 316 While it seems likely that vine weevil larvae induced a defensive response in the roots of R. 317 nigrum, as has been reported elsewhere for spinach (Spinacia oleracea) roots (Schmelz et al., 318 1999), root phenolics were positively correlated with vine weevil population size and body 319 mass in this system. The defensive response following root damage therefore had no 320 detrimental effects on vine weevils at the concentrations found in this study. The positive 321 relationship between vine weevil performance and root phenolics may be partly explained by 322 the weak linkage between phenolic concentrations and root biomass (or another correlated 323 beneficial root feature) but it is also possible that phenolics could be beneficial for some 324 insects that are able to offset their negative effects. For instance, phenolics have been shown 325 to be nutritionally beneficial [START_REF] Bernays | Plant phenols utilized as nutrients by a phytophagous 369 insect[END_REF][START_REF] Bernays | Phenols as nutrients for a phytophagous 367 insect Anacridium melanorhodon[END_REF] and act as 326 phagostimulants [START_REF] Simmonds | Importance of flavonoids in insect-plant interactions: feeding and 467 oviposition[END_REF] for several insects. In particular, Bernays & Woodhead 327 (1992) suggest that insects could become adapted to using phenolics for cuticle sclerotization 328 to conserve amino nitrogen when feeding on woody plants, which are generally poorer in 329 terms of N concentration. A positive relationship between vine weevil performance and 330 phenolics has also been found in red raspberry (Rubus idaei L.) [START_REF] Clark | Linking aboveground and belowground insect herbivore interactions: a case 382 study with the vine weevil (Otiorhynchus sulcatus)[END_REF], which provides 331 further support for this observation. [START_REF] Zvereva | Consequences of simultaneous elevation of carbon dioxide 490 and temperature for plant-herbivore interactions: a metaanalysis[END_REF] suggested that constitutive 332 concentrations of phenolics in woody tissues are likely to decrease when elevated air 333 temperatures are combined with eCO 2 conditions, which may exacerbate the negative effects 334 of eCO 2 on vine weevils in terms of reduced phenolic concentrations but also mitigate the 335 effects through increased root growth at higher temperatures. 336 337 Conclusions 338

Given the strong positive correlation between root biomass and weevil population size (P < 339 0.001), it seems reasonable to assume that the impaired root growth seen at eCO 2 was related 340 to reduced weevil performance at eCO 2 . This is supported by studies between above-and 341 belowground herbivores, which have identified root biomass as the principal factor driving 342 root herbivore performance [START_REF] Moran | Interspecific competition between root-feeding and leaf-449 galling aphids mediated by host-plant resistance[END_REF][START_REF] Masters | Plant mediated interactions between aboveground 444 and belowground insect herbivores[END_REF], but see Soler et 343 al. (2007). Elevated CO 2 also reduced water concentration in roots, which was also closely 344 related to weevil performance, and may therefore have also contributed to the negative effects 345 of eCO 2 on weevils. Roots challenged by weevils under eCO 2 conditions had the lower 346 phenolic concentrations, a factor which was also associated with poorer performance. If 347 phenolics do promote vine weevil performance at the concentrations found here, rather than 348 act as an anti-feedant [START_REF] Bernays | Plant phenols utilized as nutrients by a phytophagous 369 insect[END_REF]

  Johnson 1* , Adam T. Barton 2 , Katherine E. Clark 1,3 , Peter J. Gregory 1

  to lower nutritional quality of the roots (increased C:N ratio, lower

(

  analysis of variance (ANOVA), with three complete blocks (runs) was used to 194 examine the effects of eCO 2 on weevil performance (population size and individual mass) and 195 for the effects of eCO 2 and weevils on plant responses (leaf and root dry mass, water, C and N 196 concentrations and root phenolics). The analysis had a hierarchical block structure whereby 197 CO 2 was applied at the main plot level (chamber) and, for plant responses, weevil presence 198 was applied at the sub-plot level (plant). Where necessary, data were transformed prior to 199 analysis as indicated in figure and table legends. Transformations were chosen to give 200 residual diagnostic plots which best fitted a normal distribution and showed least 201 heteroschedasticity[START_REF] Sokal | Biometry. 3rd edn[END_REF]. Analysis of covariance (ANCOVA) and 202 Spearman's rank correlation tests were used to ascertain any relationship between plant traits 203 and weevil performance at the two CO 2 conditions. All analysis was conducted in Genstat 204

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Effects of eCOeCO2 on vine weevil larvae performance; (a) number of larvae recovered per plant and (b) average larval mass. Mean values ± S.E. shown, (a) N = 30 (b) N = 29 as no larvae were recovered from one plant. Larval data log+1 transformed prior to analysis. 215x279mm (600 x 600 DPI)

Figure 4 .

 4 Figure 4. Relationship between root phenolic concentrations and (a) weevil mass and (b) weevil population size. Mean values shown. (a) N = 59 and (b) N = 60. Correlation analyses shown for each CO2 concentration and collectively. Line of best fit shown where P < 0.05. 209x296mm (600 x 600 DPI)

  can occur under eCO 2 conditions. A more 89 recent meta-analysis concluded that eCO 2 caused increased concentrations of phenolics in green plant 90 parts (at both ambient and elevated air temperatures), but did not affect constitutive concentrations in

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Table 1 .

 1 , then the failure of plants to mount a 349 'defensive' response at eCO 2 in this study may have actually been detrimental to weevils. 350 Summary of statistical analysis for plant responses to elevated CO 2 and weevil herbivory. Statistically significant effects indicated in bold where P < 0.05. Where appropriate, data was transformed ( 1 log+1, 2 arcsine square-root or 3 log, or 4 Box-Cox) prior to analysis. N = 30 in all cases. For CO 2 DF = 1,2; for weevils and the interaction between CO 2 and weevils DF = 1,112.

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Global Change Biology

Acknowledgements 352 We thank Sheena Lamond, Steven Gellatly, Lewis Fenton and Alison Vaughan for assistance 353 with this research, together with Gill Banks and Paul Walker for conducting plant chemical 354 analysis. The work was funded by the Scottish Government's Rural and Environment 355 Research and Analysis Directorate Workpackage 1.3 and a British Ecological Society SEP 356 grant 2327/2880. 357


Gabriel Yvon-Durocher
email: g.yvon-durocher@qmul.ac.uk

José M Montoya

Guy Woodward

J Iwan Jones

Trimmer
email: m.trimmer@qmul.ac.uk

Warming Increases the Proportion of Primary Production Emitted as Methane from Freshwater Mesocosms

Keywords: Global Warming, Methane, Carbon Cycle, Metabolic Theory, Primary Production, Ecosystem Respiration Global Warming, Methane, Carbon Cycle, Metabolic Theory, Primary Production, 23 Ecosystem Respiration. 24 25 26 27

Methane and carbon dioxide are the dominant gaseous end products of the remineralisation of organic carbon and also the two largest contributors to the anthropogenic greenhouse effect. We investigated whether warming altered the balance of methane efflux relative to primary production and ecosystem respiration in a freshwater mesocosm experiment. Whole ecosystem CH4 efflux was strongly related to temperature with an apparent activation energy of 0.85eV. Furthermore, CH4 efflux increased faster than ecosystem respiration or primary production with temperature, with all three processes having sequentially lower activation energies. Warming of 4°C increased the fraction of primary production effluxing as methane by 20% and the fraction of ecosystem respiration as methane by 9%, inline with the offset in their respective activation energies. Because methane is 21 times more potent as a greenhouse gas, relative to CO2, these results suggest freshwater ecosystems could drive a previously unknown positive feedback between warming and the carbon cycle.

production and ecosystem respiration in a freshwater mesocosm experiment. Whole ecosystem 32 CH 4 efflux was strongly related to temperature with an apparent activation energy of 0.85eV. 33 Furthermore, CH 4 efflux increased faster than ecosystem respiration or primary production with 34 temperature, with all three processes having sequentially lower activation energies. Warming of 35 4°C increased the fraction of primary production effluxing as methane by 20% and the fraction 36 of ecosystem respiration as methane by 9%, inline with the offset in their respective activation 37 energies. Because methane is 21 times more potent as a greenhouse gas, relative to CO 2 , these 38 results suggest freshwater ecosystems could drive a previously unknown positive feedback 39 between warming and the carbon cycle. The two most important gaseous end products of the remineralisation of organic carbon, carbon 52 dioxide (CO 2 ) and methane (CH 4 ), are also the two largest contributors to the anthropogenic 53 greenhouse effect (IPCC 2007). The net emission of greenhouse carbon gases from an ecosystem 54 is the balance between the CO 2 absorbed by the ecosystem by gross primary production (GPP) 55 and the carbon that is respired and released as CO 2 and/or CH 4 [START_REF] Whiting | Greenhouse carbon balance of wetlands: methane emission 518 versus carbon sequestration[END_REF]. 56

Further, the fraction of fixed carbon that is respired and released as either CO 2 or CH 4 may be 57 decisive for future global warming, as shifts in this balance will affect the greenhouse gas efflux 58 potential of ecosystems, because CH 4 has 21 times the radiative forcing potential of CO 2 over 59 periods of up to 20 years [START_REF] Rodhe | A comparison of the contribution of various gases to the greenhouse-effect[END_REF][START_REF] Lelieveld | Nato Advanced Research Workshop on Atmospheric 476 Methane : Sources, Sinks and Role in Global Change[END_REF][START_REF] Whiting | Greenhouse carbon balance of wetlands: methane emission 518 versus carbon sequestration[END_REF]. 60

Methanogenesis in freshwater ecosystems is the result of complex and often interrelated 61 biotic and abiotic processes (Christensen et al. 2003a). Methane is produced under strictly 62 anaerobic conditions during organic matter mineralisation but the net efflux of CH 4 from 63 ecosystems can be considerably reduced through oxidation by methanotrophs, which can 64 consume significant quantities of the CH 4 produced in the sediments of lakes (Kuivila et al. 65 1988) and wetlands [START_REF] Bartlett | Nato Advanced Research Workshop on Atmospheric Methane 425 : Sources, Sinks and Role in Global Change[END_REF][START_REF] Segers | Methane production and methane consumption: a review of processes 508 underlying wetland methane fluxes[END_REF]. Primary production by plants also 66 influences CH 4 production [START_REF] Joabsson | International Symposium on Polar Aspects of 458 Global Change[END_REF][START_REF] Joabsson | Vascular plant controls on methane emissions 460 from northern peatforming wetlands[END_REF], Christensen et al. 2003b), and this has 67 been attributed to the co-variability of organic carbon through root exudation (Chanton et al. 68 1995), the turnover of labile carbon, and/or litter production [START_REF] Joabsson | Vascular plant controls on methane emissions 460 from northern peatforming wetlands[END_REF], Christensen et 69 al. 2003b). These lines of evidence are supported by isotopic data which have shown that a large 70 fraction of the organic material that fuels methanogenesis in wetlands is derived from recently 71 synthesised carbon [START_REF] Chanton | Radiocarbon evidence for the substrates 433 supporting methane formation within northern minnesota peatlands[END_REF][START_REF] Joabsson | Vascular plant controls on methane emissions 460 from northern peatforming wetlands[END_REF]. Vascular plants can also enhance 72 emissions of CH 4 to the atmosphere via root aerenchyma that act as conduits across zones of 73 potential CH 4 oxidation in soils and sediments [START_REF] Kelker | The effect of clipping on methane emissions from Carex[END_REF][START_REF] King | Methane emission and transport by arctic sedges in 467 Alaska: Results of a vegetation removal experiment[END_REF]. 74 Clearly the mechanisms that influence CH 4 efflux are diverse, however, when all other 75 limiting factors (e.g. substrate limitation, water-table depth) are equal, temperature, via the 76 physiological stimulation of microbial metabolism, has been shown to exert strong control on 77 CH 4 efflux [START_REF] Schutz | Influence of soil-temperature on methane emission from 506 rice paddy fields[END_REF], Christensen et al. 2003a[START_REF] Gedney | Climate feedback from wetland methane emissions[END_REF]. Recently, 78 significant focus has been given to the temperature dependence of the biotic components of the 79 carbon cycle, and how the "metabolic balance" of ecosystems, that is the balance between the 80 gross sequestration and release of CO 2 , may respond to future global warming [START_REF] Allen | Linking the global carbon cycle to individual 420 metabolism[END_REF], 81 Lopez-Urrutia et al. 2006, Yvon-Durocher et al. in press). Recent evidence suggests that 82 autotrophic and heterotrophic metabolisms (e.g. photosynthesis and respiration) have different 83 temperature dependencies (or activation energies when depicted in an Arrhenius plot) at the 84 ecosystem level, such that respiration increases more rapidly with temperature than does 85 photosynthesis [START_REF] Allen | Linking the global carbon cycle to individual 420 metabolism[END_REF], Lopez-Urrutia et al. 2006, Yvon-Durocher et al. in press). We 86 have recently shown in an aquatic mesocosm experiment, that the differential temperature 87 dependence of these two processes reduced the ability of the warmed systems to sequester CO 2 88 because more of the carbon fixed by primary production was respired . The response of the greenhouse gas efflux potential of aquatic ecosystems to warming is 90 further complicated by considering the balance of CH 4 efflux in relation to carbon sequestration 91 and CO 2 emission. 92 A substantial body of work over the last two decades has established the strong 93 temperature dependence of methanogenesis in a wide range of ecosystems (i.e. from landfill sites 94 to high latitude wetlands) and from pure cultures of methanogens to whole ecosystem-level 95

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production [START_REF] Schutz | A 3 year continuous 502 record on the influence of daytime, season, and fertilizer treatment on methane emission 503 rates from an Italian rice paddy[END_REF][START_REF] Westermann | Temperature compensation in Methanosarcina 513 barkeri by modulation of hydrogen and acetate affinity[END_REF][START_REF] Conrad | Influence of temperature on energetics of hydrogen metabolism in 445 homoacetogenic, methanogenic and other anaerobic bacteria[END_REF], Schutz et al. 96 1990[START_REF] Walter | A process-based, climate-sensitive model to derive methane 510 emissions from natural wetlands: Application to five wetland sites, sensitivity to model 511 parameters, and climate[END_REF], Christensen et al. 2003a[START_REF] Gedney | Climate feedback from wetland methane emissions[END_REF]. Studies of the 97 temperature dependence of methanogenesis in pure cultures (under optimal conditions) have 98 revealed that its activation energy is typically higher than for other forms of metabolism, due to 99 the relatively large entropy change of the reaction [START_REF] Westermann | Temperature compensation in Methanosarcina 513 barkeri by modulation of hydrogen and acetate affinity[END_REF], Conrad & Wetter 100 1990[START_REF] Segers | Methane production and methane consumption: a review of processes 508 underlying wetland methane fluxes[END_REF]. Methanogenesis and potentially CH 4 efflux may, therefore, be especially 101 sensitive to increases in temperature which raises a number of important unanswered questions. 102

First, does the temperature dependence of CH 4 efflux at the ecosystem scale differ from that of 103 primary production and ecosystem respiration? Second, how will the balance between carbon 104 sequestration, ecosystem respiration and CH 4 efflux respond to warming? 105

Although our knowledge of CH 4 efflux and its regulation by temperature is extensive, it 106 is largely based on seasonal field surveys (e.g. in wetlands, soils, lakes) and laboratory 107 experiments (e.g. with peat monoliths and rice paddy-soil incubations) which cannot fully 108 address these unanswered questions. We sort to extend this knowledge using a controlled 109 freshwater mesocosm experiment where we compared the efflux of CH 4 with rates of gross 110 primary production (GPP) and whole ecosystem respiration (ER) in replicated mesocosms 111 maintained at either ambient temperature or ~ 4ºC above ambient, in line with warming scenarios 112 predicted for temperate latitudes by the end of the 21 st century (IPCC 2007). 113 The concentration of dissolved CH 4 was measured by removing a water sample (30 mL in a gas-133 tight syringe) and gently transferring it to a gas tight vial (12.5 mL Exetainers, Labco, High 134 Wycombe, UK), allowing it to overflow, fixing it with a bactericide (100 µL 50 % w/v ZnCl 2 ) 135 and sealing it. Samples were collected at hourly time intervals (in total 6 to 10 hours depending 136

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on the time of year) over a day for each replicate on alternate months for one year (April 2007to 137 April 2008, n = 1416 individual measurements). Upon return to the laboratory, a headspace (2 138 mL analytical grade helium) was introduced to the gas tight vial and the sample was shaken 139 vigorously for 0.5 minutes and then allowed to stand for a further 30 minutes to allow for 140 headspace equilibration, before analysis of the headspace concentration of CH 4 using a gas 141 chromatograph after [START_REF] Sanders | 499 Emission of methane from chalk streams has potential implications for agricultural 500 practices[END_REF]. Samples (50 µL) were withdrawn from the headspace 142 of the sample vials and injected into a gas-chromatograph fitted with a flame ionising detector 143 (GC/FID; Agilent Technologies, UK). Headspace concentrations of CH 4 were calculated from 144 peak areas calibrated against known standards (Scientific and Technical gases, Staffs, UK) and 145 the total amount of CH 4 in the gas tight vial (water plus headspace) was calculated using the 146 appropriate solubility coefficients [START_REF] Yamamoto | Solubility of methane in distilled water and 523 seawater[END_REF]. Finally, the 1416 individual 147 measurements were reduced in each case to give an average daily pool of dissolved CH 4 for each 148 pond (140 measures over the year for 70 heated and 70 ambient). 149 150

Methane Efflux 151

Measurements of the efflux of CH 4 were made simultaneously to those of dissolved CH 4 . A 152 single gas chamber was positioned at the water surface of each mesocosm on each sampling 153 occasion. The chambers were made of polycarbonate and enclosed a headspace (300 mL) of 154 ambient air at the air-water interface of the mesocosm (see figure S1 in supporting online 155 material). The lid of the gas chamber was equipped with a Teflon septum port, through which 156 samples of gas (1 mL) were removed using a gas-tight syringe (2 mL VICI gas tight syringe) 157 every 15 minutes for the first hour of the incubation, then hourly for up to 10 hours thereafter. 158

The samples were then transferred to water filled gas tight vials (3 mL, Exetainers; Labco, High 159 [START_REF] Odum | Primary production in flowing waters[END_REF][START_REF] Marzolf | Improvements to the diurnal upstream-482 downstream dissolved-oxygen change techniques for determining whole stream 483 metabolism in small streams[END_REF][START_REF] Mulholland | Inter-biome comparison of factors controlling 489 stream metabolism[END_REF]. This technique assumes that changes 176 in DO concentration over a diel cycle represent the metabolic activity (photosynthetic and 177 respiratory) of an aquatic ecosystem. YSI 600XLM multiparameter sondes equipped with 6562 178 rapid pulse™ dissolved oxygen sensors were deployed for 24 hours in each heated and unheated 179 treatment pair on each of the seven sampling occasions over the year. Measurements of DO and 180 temperature were taken every 15 minutes for 24 hours at the mid depth (0.25m) in the water 181 column of each pond. The daylight and night-time analysis periods were delimited as follows: 182

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9 the total analysis period was defined from the minimum O 2 concentration on the 1 st night and 183 extended for 24 hours to include the minimum O 2 concentration on the 2 nd night. Photosynthetic 184 dawn was identified as the minimum O 2 concentration after which all subsequent values were 185 greater than it. Photosynthetic dusk was defined as the maximum O 2 concentration after which 186 all subsequent values were lower [START_REF] Mulholland | Inter-biome comparison of factors controlling 489 stream metabolism[END_REF][START_REF] Bales | Automated routines for calculating whole stream metabolism. 422[END_REF]. The change in dissolved 187 oxygen (∆DO) over each 15 minute time interval was calculated as the difference in O 2 188 concentration between t 1 and t 2 (i.e., t 2 -t 1 ). Each ∆DO value was then assigned to a day or night-189 time category. The metabolic parameters GPP and ER were calculated, in turn, by numerical 190 integration according to: 191

day day R O GPP + ∆ = ∑ 2 (1) 192
where R day is day-time respiration. Since it is impossible to measure R day directly it was 193 estimated, in keeping with the literature, by extrapolating the mean night time respiration value 194 across the hours of daylight [START_REF] Cole | Persistence of net heterotrophy in lakes 442 during nutrient addition and food web manipulations[END_REF]. Current biogeochemical techniques cannot 195 discriminate between autotrophic and heterotrophic respiration at the ecosystem level 196 [START_REF] Mulholland | Inter-biome comparison of factors controlling 489 stream metabolism[END_REF]) and preclude the estimation of photorespiration (Marzolf et al. 1994). 197 Our measures of GPP using the DO change technique may, therefore, be slightly overestimated 198 given the inclusion of heterotrophic respiration in calculation of R day . ER was calculated as: 199 The rate of exchange of a gas between the surface water and the atmosphere is dependent 206 on two principal parameters: the concentration gradient of the gas between the water and the 207 atmosphere, and the gas transfer velocity, k. The gas transfer velocity is frequently modelled as a 208 function of wind speed, which controls the rate of gas transfer by determining turbulence in the 209 surface water [START_REF] Cole | Atmospheric exchange of carbon dioxide in a low-wind oligotrophic 440 lake measured by the addition of SF6[END_REF]. When wind speed drops below about 3 m s -1 , however, as 210 was the case in > 97 % of our measurements (see Yvon-Durocher et al. in press), gas transfer 211 becomes independent of wind speed, though it can remain substantial by being governed by 212 other physical (advective) processes besides wind [START_REF] Cole | Atmospheric exchange of carbon dioxide in a low-wind oligotrophic 440 lake measured by the addition of SF6[END_REF]. To determine whether 213 our experimental warming systematically altered the gas transfer velocity we estimated k from 214 our measurements of the efflux of CH 4 and dissolved CH 4 from: 215) /(

∑ ∆ + = night day O R ER
eq water C C f k - = (3) 216
where f is the measured efflux of CH 4 across the air-water interface, C water -C eq is the 217 concentration gradient of the gas in the water and the concentration in the water at equilibrium 218 with the atmosphere (C eq ). C eq was calculated using the equations of [START_REF] Yamamoto | Solubility of methane in distilled water and 523 seawater[END_REF] The concentration of CH 4 exhibited clear and near identical seasonal trends in the water of both 245 the heated and ambient mesocosms and, on average, over the year, was not significantly different 246 between treatments (Fig. 1a and Table 1). The concentration of CH 4 ranged from 0.04 µmol L -1 247 to 6.8 µmol L -1 , though the distribution of CH 4 concentration exhibited strong positive skew 248 (Shapiro Wilks test; W = 0.63; P < 0.005), such that 75% of all measurements were less than 249 0.66 µmol L -1 over the seasonal cycle. Using the 75th percentile for dissolved CH 4 as a 250 conservative estimate, and the average concentration of CH 4 that would be at equilibrium with 251 the atmosphere (~3.55 ×10 -3 µmol L -1 ), we estimated that the mesocosms were about 128 times 252 supersaturated with respect to CH 4 and the atmosphere. 253

Similarly, the rate of CH 4 efflux showed strong seasonal trends with peaks in early 254 summer and lowest rates in winter (Fig. 1b) and was strongly positively correlated (r = 0.94; 255 P<0.005) with the concentration of CH 4 in the water column. The rate of CH 4 efflux ranged from 256 0.35 to 7.02 µmol m -2 h -1 in the ambient mesocosms and from 0.96 to 8.14 µmol m -2 h -1 in the 257 warmed mesocosms. The distribution of CH 4 efflux between heated and ambient mesocosms was 258 also strongly positively skewed (Shapiro Wilks test; W = 0.74; P < 0.005), with 75% of 259 measurements falling below 3.6 µmol m -2 h -1 . Furthermore, the rate of CH 4 efflux was elevated 260 in the warmed mesocosms over parts of the seasonal cycle (April, August and February), and, on 261 average, the mean annual rate of CH 4 efflux was significantly greater in the warmed mesocosms 262 (Table 1). The gas transfer velocity, k, exhibited no clear seasonal variability (Fig. 2) and was not 263 significantly different between treatments (Fig. 2 and Table 1). 264

The rate of CH 4 efflux was strongly related to temperature (1/kT) (Table 2 and Fig. 3), 265 with an apparent activation energy in the order of 0.85 eV (95% confidence interval: 0.64 to 1.02 266 eV). In addition, CH 4 efflux was also related to GPP, though much more weakly (Table 2 and267 Fig. 4), with temperature explaining 43% of the variance in rate of CH 4 efflux, while GPP 268 explained only 23% (Fig. 2 and Fig. 4). 269 GPP and ER were also strongly related to temperature, as has been described previously 270 (Yvon-Durocher et al. in press), with apparent activation energies in the order of 0.45 eV (95% 271 confidence interval 0.38 to 0.53 eV) and 0.62 eV (95% confidence interval 0.55 to 0.69 eV) for 272 each, respectively. Importantly, here, the temperature dependence of CH 4 efflux was 273 significantly higher than that for either GPP or ER (Table 2) and, correspondingly, CH 4 efflux 274 increased more rapidly in response to warming than did either GPP or ER. 275 The balance between carbon absorption and CH 4 emission is given by the ratio of CH 4 276 efflux to GPP, which was found to be significantly elevated in the heated mesocosms, on 277 average, over the year (Table 1 andFig 5a). The mean annual ratio of CH 4 efflux to GPP was 278 elevated by 20 % in response to the ~4˚C experimental warming. Similarly, the ratio of CH 4 279 efflux to ER was significantly elevated in the heated mesocosms, on average, over the annual 280 cycle (Table 1 andFig µmol CH 4 m -2 h -1 ) were comparable to those measured in natural shallow lakes (Rudd & 288 Hamilton 1978[START_REF] Bastviken | Methane emissions from lakes: Dependence of 428 lake characteristics, two regional assessments, and a global estimate[END_REF], suggesting that our experimental scale was sufficient 289 enough to reproduce the complex biogeochemical cycling of carbon observed in natural 290

ecosystems. 291

Experimental warming might have artificially stimulated advective processes resulting in 292 elevated gas transfer at the air-water interface and a detailed consideration of this potential 293 artefact is fundamental before interpretation of our results. Our estimates of the gas transfer 294 velocity, based on detailed measurement of dissolved CH 4 and rates of CH 4 efflux, however, 295 suggest that this was not the case. If the gas transfer velocity was systematically enhanced by 296 artificial warming, we would have expected to observe considerable differences in the 297 concentration of dissolved CH 4 between treatments, but, again, this was not the case. At first 298 glance, the consistency in both the pool size of dissolved CH 4 and the estimated gas transfer 299 velocity between treatments appears at odds with the elevated efflux of CH 4 measured in the 300 warmed mesocosms. According to equation 3, the gas transfer velocity and the concentration 301 gradient (i.e. between the water and the atmosphere) drive the efflux of gas across the air-water 302 interface [START_REF] Cole | Atmospheric exchange of carbon dioxide in a low-wind oligotrophic 440 lake measured by the addition of SF6[END_REF]. This discrepancy can be explained when the relative magnitudes 303 of the respective processes and pool sizes are taken into consideration. Using the 75 th percentiles 304 for both CH 4 efflux and dissolved CH 4 (scaled to a whole mesocosm) of 11 µmol CH 4 305

F o r R e v i e w O n l y
mesocosm -1 h -1 and 662 µmol CH 4 mesocosm -1 , respectively, 75% of our measurements of the 306 efflux of CH 4 represented <1.7% of the total pool of dissolved CH 4 . The subtle differences 307 detected in the efflux of CH 4 between treatments would have been masked when analysing for 308 treatment effects at the level of the pool, because the overall magnitude of the pool size of 309 dissolved CH 4 was considerable relative to its minor diurnal variability, which drives the efflux. 310 Therefore, any error associated with the measurement of the CH 4 pool would likely overwhelm 311 the detection of any subtle statistical differences between treatments. This evidence suggests that 312 the physical influence of heating the mesocosms by ~4˚C had little discernable effect on 313 advective processes. We can therefore be confident that the biogeochemical patterns revealed by 314 our experiment are due to the biological consequences of warming on aquatic communities. 315

Our experimental results have implications for understanding the mechanisms controlling 316 CH 4 efflux from freshwater ecosystems, and how CH 4 dynamics in relation to carbon 317 sequestration rates might be affected by future global warming. Our experiment revealed that 318 temperature was the dominant driver of CH 4 efflux from our mesocosms. This result agrees with 319 other studies from a range of natural ecosystems, from soils to wetlands, which also highlight the 320 strong temperature dependence of CH 4 efflux [START_REF] Schutz | Influence of soil-temperature on methane emission from 506 rice paddy fields[END_REF][START_REF] Whiting | Greenhouse carbon balance of wetlands: methane emission 518 versus carbon sequestration[END_REF], 321 Christensen et al. 2003a[START_REF] Gedney | Climate feedback from wetland methane emissions[END_REF]. Because of the overriding influence of 322 temperature, the overall rates of CH 4 efflux were consistently elevated in the warmed mesocosms 323 relative to ambient over the course of our annual study, presumably reflecting the strong 324 physiological response to the temperature stimulation of methanogenesis. 325

To ascertain whether the strong temperature dependence of CH 4 efflux we observed 326 experimentally was due to the physiological stimulation of methanogenesis, we determined the 327 activation energy of methanogenesis in pure cultures at a range of temperatures, and under non-328

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limiting conditions, using previously published data (see S5 in supporting online information for 329 details of analysis and data collection). Our re-analysis of these data does indeed show that the 330 production of CH 4 in pure cultures has an equally strong temperature dependence (Fig. 5). 331

Interestingly, the temperature dependence of CH 4 efflux at the ecosystem level (E a = 0.85 eV, 332 95% confidence interval: 0.64 to 1.02 eV) was indistinguishable from that for methanogenesis in 333 pure culture (mean E a = 0.88 eV 95% confidence interval: 0.80 to 0.96 eV). This coherence 334 between organisational scales suggests that much of the potential complexity associated with 335 ecosystem level efflux of CH 4 might be reduced to the first principals of individual/cellular 336 kinetics. This result suggests that whole ecosystem metabolic fluxes can be scaled from the 337 individual to the ecosystem level, in line with predictions derived from the "metabolic theory of 338 ecology" [START_REF] Enquist | Scaling 448 metabolism from organisms to ecosystems[END_REF][START_REF] Allen | Linking the global carbon cycle to individual 420 metabolism[END_REF][START_REF] Lopez-Urrutia | Scaling the metabolic balance of 479 the oceans[END_REF]. Our study, therefore, 339 contributes to the growing body of evidence which suggests that metabolism is a fundamental 340 driver of the dynamics of ecological processes across multiple levels of organisation, by 341 demonstrating that CH 4 efflux at the ecosystem level appears to be constrained by the activation 342 energy of methanogenesis. Models derived from the metabolic theory of ecology might therefore 343 provide additional insight into the dynamics and temperature response of whole ecosystem CH 4 344 efflux in aquatic and other ecosystems. 345

As well as temperature, primary production has been shown to regulate the efflux of CH 4 346 to the atmosphere. Such regulation stems from the whole autotrophic assemblage providing 347 structural, labile carbon compounds in the form of dead biomass [START_REF] Whiting | Primary production control of methane emission from wetlands 516[END_REF], and 348 vascular plants producing root exudates in the form of organic acids [START_REF] Chanton | Radiocarbon evidence for the substrates 433 supporting methane formation within northern minnesota peatlands[END_REF], 349 Joabsson & Christensen 2001, Christensen et al. 2003b). Furthermore, rooted aquatic vascular 350 plants can act as conduits for the transport of CH 4 from the anaerobic zone of the sediment to the 351

F o r R e v i e w O n l y
atmosphere, bypassing the zones of potential CH 4 oxidation in the sediment and water column 352 [START_REF] Joabsson | Vascular plant controls on methane emissions 460 from northern peatforming wetlands[END_REF][START_REF] Joabsson | Methane emissions from wetlands and their relationship with 456 vascular plants: an Arctic example[END_REF]. In our experiment, however, the efflux of 353 CH 4 did not appear to be limited by substrates from GPP, as suggested by three lines of 354 evidence. Firstly, the weak correlation and gentle slope of the relationship between the efflux of 355 CH 4 and GPP indicated that the flux of CH 4 was relatively independent of the simultaneous rate 356 of photosynthesis and carbon fixation. Secondly, warming had no effect on the intercept of the 357 relationship between ln(CH 4 efflux) vs 1/kT between treatments (Table 2). If organic substrates 358 were limiting for CH 4 production and efflux, we would expect to see a lower intercept in the 359 warmed treatments because elevated physiological rates would be expected to reduce organic 360 substrates more rapidly, resulting in faster substrate limitation and thus a reduced intrinsic 361 capacity for methanogenesis. Thirdly, on average, the efflux of CH 4 represented a very small 362 fraction of GPP (mean annual value = 0.01%). If oxidation of methane production is assumed to 363 be 95% [START_REF] King | Distribution and rate of methane oxidation in the 464 sediments of the Florida Everglades[END_REF]), from our average annual CH 4 efflux measures (3 µmol m -2 h -1 ), we 364 estimate mean annual CH 4 production to be ~290 µmol m -2 h -1 which would represent only 10% 365 of the mean annual rate of GPP (2862 µmol m -2 h -1 ). Therefore, carbon sequestration and fixation 366 by photosynthesis is likely to exceed the demand of methanogenesis throughout the annual cycle. 367

The response of the greenhouse carbon gas balance of freshwater ecosystems to warming 368 could affect the strength of biotic feedbacks on a potentially global scale [START_REF] Woodwell | Biotic 521 feedbacks in the warming of the earth[END_REF]. 369

In our experiment, the fraction of carbon absorbed by GPP and subsequently remineralised via 370 the methanogenic pathway to efflux as CH 4 increased by 20% in response to the simulated global 371 warming scenarios projected for the end of the century. In addition, the efflux of CH 4 as a 372 proportion of ER was 9% greater in the warmed mesocosms. If, as aquatic ecosystems warm, 373 carbon remineralisation becomes increasingly dominated by methanogenesis this could result in 374 These patterns can be explained by the differential activation energies of the three metabolic 376 processes involved in the greenhouse carbon balance of ecosystems. 377

Here, and in previous research (Yvon-Durocher et al. in press), it has been demonstrated 378 that the three key ecosystem level carbon fluxes have progressively higher activation energies 379 (i.e., GPP = 0.45eV; ER = 0.62eV; CH 4 efflux = 0.85eV). The relative response of the 380 greenhouse carbon gas balance to warming observed here might be predictable from these 381 respective activation energies. To test this hypothesis, equations were derived from the metabolic 382 theory of ecology [START_REF] Brown | Toward a metabolic theory of 431 ecology[END_REF] to predict the response of the mean ratio of CH 4 efflux to 383 GPP over the year between heated and unheated treatments (R fixed H:U) which was given by: 384

( ) [ ] U T H kT U T H T Ep Ea U U H H fixed e p a p a U H R ) ( : - - = = (4) 385
where a H and a U are the allometric equations for the efflux of CH 4 in the heated and unheated 386 treatments respectively, while p H and p U are the allometric equations for GPP in heated and 387 unheated treatments. E a and E p are the activation energies for the efflux of CH 4 and GPP in our 388 experiment (0.85 and 0.45eV respectively), T H and T U are the mean annual temperatures (K) for 389 the heated and unheated treatments, respectively, and k is Boltzmann's constant (see S3 in 390 supporting online information for the full derivation of equation 4). Equation 4 predicts a 1.30 391 fold increase in R fixed H:U (range 1.18 to 1.38; based on the 95% confidence intervals of the 392 respective empirically measured activation energies),which is very close to our empirically 393 measured value of 1.20. Similarly, the response of the mean ratio for the efflux of CH 4 to ER 394 over the year between heated and unheated treatments (R emitted H:U) should be predicted by: 395

( ) [ ] U T H kT U T H T Er Ea U U H H emitted e r a r a U H R ) ( : - - = = (5) 396 F o r R e v i e w O n l y
where E a and E r are the empirically determined activation energies for the efflux of CH 4 and ER 397 (0.85 and 0.62eV respectively), respectively (see S4 in supporting online information for the full 398 derivation of equation 4). Using equation 5, we would expect a 1.16 fold increase in R emitted H:U 399 (range 0.17 to 1.24; based on the 95% confidence intervals of the respective empirically 400 measured activation energies). Our empirically measured value of R fixed H:U was 1.09, again very 401 close to our prediction. 402 Interestingly, equations 4 and 5 suggest that the relative offset of the carbon balance 403 between the ambient and warmed mesocosms can be predicted by the differences in activation 404 energies of metabolism and the degree of expected warming. This result is important because it 405 highlights the potential for a positive feedback between warming and the carbon cycle of 406 freshwater ecosystems, especially given the greater radiative forcing potential of CH 4 (Rodhe 407 1990[START_REF] Lelieveld | Nato Advanced Research Workshop on Atmospheric 476 Methane : Sources, Sinks and Role in Global Change[END_REF][START_REF] Whiting | Greenhouse carbon balance of wetlands: methane emission 518 versus carbon sequestration[END_REF]. Finally, accepting the caveats associated 408 with mesocosms, the close coherence between the activation energy of methanogenesis in pure 409 culture and that of whole system CH 4 efflux, suggests that much of the complexity of ecosystem 410 level fluxes can be reduced to produce simpler predictive models. and unheated treatments (black lines). The pool of dissolved methane exhibited strong seasonal trends and which were identical between treatments. Furthermore, the average annual pool of dissolved methane was identical between treatments (Table 1). (b) Differences in methane efflux, ln(ME) (±SE) between heated (red lines) and unheated (black lines) treatments. Methane efflux showed a strong seasonal pattern and was elevated, on average, over the annual cycle in warmed treatments reflecting its strong temperature dependence (Table 1). Fig. 2. Seasonal trends in the gas transfer velocity (k), between heated (red lines) and unheated (black lines) treatments. The gas transfer exhibited little seasonal variability and was not systematically affected by the heating of the mesocosms (Table 1). Data were natural log transformed for statistical analysis but presented here untransformed for ease of interpretation. [START_REF] Westermann | Temperature compensation in Methanosarcina 513 barkeri by modulation of hydrogen and acetate affinity[END_REF]. Data for b was reanalysed from data on rates of CH3COOH conversion to CH4 in Van den Berg et al., (1976), and data for c was reanalysed from data on rates of CH4 production in Huser et al., (1982). In each case, the slope of the temperature response equates to the activation energy of methanogenesis and agree very well with that derived in Fig. 3. 294x267mm (72 x 72 DPI)

  carried out between December 2005 and April 2008 at the Freshwater 116 Biological Association River Laboratory (2°10`W, 50°13`N) East Stoke, Dorset, UK. The 117 experiment consisted of twenty freshwater mesocosms (~1 m 3 , 0.5 m water depth): ten replicates 118 remained at ambient temperature, whilst the other ten were maintained at 3-5°C (mean 4°C) 119 above ambient. Warming was achieved by an electronic heating element at the base of the 120 mesocosm connected to a thermocouple which monitored the temperature in a given heated and 121 unheated treatment pair. Treatments were arranged in a randomised block design (5 blocks of 4 122 mesocosms) such that each block contained two replicates of each treatment. The mesocosms 123 were seeded in December 2005 with organic substrates and a suite of organisms [species list in 124 (Yvon-Durocher et al. in press)] to mimic the organismal composition and physical structure of 125 shallow lake ecosystems [e.g., after (McKee et al. 2003)]. The submerged macrophytes Elodea 126 canadensis Michaux, Myriophyllum spicatum L. and Ceratophyllum spicatum L. were added to 127 each pond in equal quantities (250g wet weight) and Chara contraria A. Braun ex Kutz 128 colonised all 20 ponds during the experiment. The biota was left to establish for ten months prior 129 to experimental warming, which commenced in September 2006

  ) through a two way valve with venting through a narrow bore needle. The gas-160 tight vials were then stored upside down prior to analysis. 161The concentration of CH 4 in the headspace of the sample was determined by gas 162 chromatography as described above. The efflux of CH 4 across the water-air interface was 163 calculated by regression analysis of the change in concentration of CH 4 in the chamber 164 headspace over time. Subsequently, one hour was used as an appropriate duration for accurately 165 estimating the flux of CH 4[START_REF] Lambert | Analytical Techniques for Measuring Fluxes of CO2 and CH4 472 from Hydroelectric Reservoirs and Natural Water Bodies[END_REF] (see figureS2in supporting online 166 material). As such, only data from the 1 st hour of the incubation were used to estimate the efflux 167 of CH 4 and we made a total of 140 measurements (70 heated and 70 ambient) over the annual 168 cycle. Regression slopes with a significance of P > 0.05 and/or an R-squared of below 0.9 were 169 considered non-significant and were excluded from further analyses (9 from the 140 were estimated simultaneously with the measurements of dissolved CH 4 and CH 4 174 efflux, by applying the well established single station dissolved oxygen (DO) change technique 175

  in the rate of gas transfer between the surface water and the 203 atmosphere due to physical forcing (e.g. advection) induced by the experimental treatment (e.g. 204 warming) may have biased the interpretation of our results. 205

  and 219 the measured mixing ratio for CH 4 in the air and temperature of the water on each occasion. 220221Statistical Analyses 222All data were checked for normality using the Shapiro Wilks test for normality and were natural 223 log transformed prior to statistical analysis where necessary. The activation energy of any 224 metabolism is given by the slope of the relationship of an Arrhenius plot between ln(x flux) and 225 1/kT, where k is Boltzmann's constant and T is absolute temperature (K). The temperature 226 dependence of ln(CH 4 efflux), ln(GPP) and ln(ER) (i.e. the activation energy) was determined by 227 ANCOVA. Furthermore, we used ANCOVA to test for statistical differences in the slopes and 228 relationships between treatments and months (i.e. n = 7 sampling occasions), 229 to identify the most parsimonious model for determining the activation energy (i.e. the 230 temperature dependence). Model comparison was carried out using the Akaike Information 231 Criterion (AIC). In the ANCOVA, temperature was delimited as a continuous variable and 232 defined as 1/kT. To account for temporal pseudo-replication in the statistical model pond identity 233 (n = 131) was nested within sampling occasion. ANCOVA computations were carried out in R 234 statistical software (R.[START_REF] Development | R Foundation for Statistical Computing[END_REF]. 235Categorical analyses (treating temperature as a fixed factor i.e. heated or unheated) of 236 CH 4 efflux, dissolved CH 4 pool, k gas transfer, CH 4 efflux /GPP and CH 4 efflux /ER was 237 conducted with restricted maximum likelihood methods using the lme (linear mixed-effects 238 model) function in R (R.[START_REF] Development | R Foundation for Statistical Computing[END_REF]). In the model treatment (heated or unheated) 239 was the fixed effect, and temporal pseudo-replication from repeated sampling of the mesocosms 240 over the year was accounted for by including mesocosm identity nested with sampling occasion 241 as random effects. The repeated measures model was used to test for overall statistical 242 differences between treatments in mean annual values of the above parameters. 243

  5b), with warming elevating the mean annual ratio of CH 4 efflux to ER by a compromise between the control and replication of 284 laboratory studies and the realism of descriptive field surveys but, despite their limitations, can 285 provide a fundamental tool for predicting how global change scenarios might affect ecosystem 286 level processes(Benton et al. 2007). Our measured rates of mesocosm CH 4 efflux (0.35 -8.14 287

  emitted to the atmosphere relative to CO 2 emission and carbon draw-down. 375

Fig. 1 .

 1 Fig. 1. (a) Differences in the pool of dissolved methane [ln(CH4)] (±SE) between heated (red lines)and unheated treatments (black lines). The pool of dissolved methane exhibited strong seasonal trends and which were identical between treatments. Furthermore, the average annual pool of dissolved methane was identical between treatments (Table1). (b) Differences in methane efflux, ln(ME) (±SE) between heated (red lines) and unheated (black lines) treatments. Methane efflux showed a strong seasonal pattern and was elevated, on average, over the annual cycle in warmed treatments reflecting its strong temperature dependence (Table1). 149x179mm (72 x 72 DPI)

  Fig. 1. (a) Differences in the pool of dissolved methane [ln(CH4)] (±SE) between heated (red lines)and unheated treatments (black lines). The pool of dissolved methane exhibited strong seasonal trends and which were identical between treatments. Furthermore, the average annual pool of dissolved methane was identical between treatments (Table1). (b) Differences in methane efflux, ln(ME) (±SE) between heated (red lines) and unheated (black lines) treatments. Methane efflux showed a strong seasonal pattern and was elevated, on average, over the annual cycle in warmed treatments reflecting its strong temperature dependence (Table1). 149x179mm (72 x 72 DPI)

Fig. 3 .Fig. 4 .Fig. 6 .

 346 Fig.2. Seasonal trends in the gas transfer velocity (k), between heated (red lines) and unheated (black lines) treatments. The gas transfer exhibited little seasonal variability and was not systematically affected by the heating of the mesocosms (Table1). Data were natural log transformed for statistical analysis but presented here untransformed for ease of interpretation.213x126mm (72 x 72 DPI)

Table 1 .

 1 Linear mixed effects model analysis. Analysing differences between heated and 531 unheated treatments in the annual means of methane efflux [ ln(CH 4 efflux)], the dissolve 532 methane pool [ln(CH 4 Pool)], the ratio of methane efflux to GPP [ln(CH 4 efflux)/ln(GPP)], and 533 the ratio of methane efflux to ER [ln(CH 4 efflux)/ln(ER)]. Significant P-values are given in bold. 534

	411
	412

Table 2 .

 2 Analysis of Co-Variance table for the relationships between ln(CH 4 efflux), ln(GPP) 537 and ln(ER) vs 1/kT. Significant P-values are given in bold.

	538
	539
	540
	541

Acknowledgements 413

We thank Brian Godfrey, Dan Perkins, and the Freshwater Biological Association for their help 414 with the experiment. Andrew P Allen and Ricard Solé discussed ideas and provided comments