You are on page 1of 9

SBB5265_proof 15 September 2012 1/9

Soil Biology & Biochemistry xxx (2012) 1e9

Contents lists available at SciVerse ScienceDirect

Soil Biology & Biochemistry


journal homepage: www.elsevier.com/locate/soilbio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 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

How does N deposition affect belowground heathland recovery following wildre?


Q3

E.R. Green a, **, R.J. Ellis b, c, S.R.M. Gadsdon a, d, A. Milcu c, S.A. Power a, e, *
a

Division of Biology, Imperial College London, Silwood Park, Ascot, Berkshire SL75 7PY, UK Specialist Scientic Services, Animal Health and Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, UK c NERC Centre for Population Biology, Imperial College London, Silwood Park, Ascot, Berkshire SL5 7PY, UK d City of London, Epping Forest Ofce, The Warren, Loughton, Essex IG10 4RW, UK e Hawkesbury Institute for the Environment, University of Western Sydney, Locked Bag 1797, Penrith NSW 2751, Australia
b

a r t i c l e i n f o
Article history: Received 29 September 2011 Received in revised form 22 August 2012 Accepted 23 August 2012 Available online xxx Keywords: Lowland heathland Wildre Nitrogen deposition Soil nutrient dynamics Microbial community composition Ericoid mycorrhizal fungi

a b s t r a c t
Climate change is predicted to increase the frequency and severity of summer res in the UK. However, the effects of re on ecosystem processes are likely to depend on pre-burn vegetation characteristics, soil chemistry and microbiology, all of which are known to be affected by changes in nitrogen (N) deposition. The occurrence of a severe summer re after eight years of experimental N additions at Thursley Common National Nature Reserve (UK) thus provided a unique opportunity to evaluate the impacts of a major perturbation on nutrient and microbial dynamics in soils of contrasting chemistry. Soil N and P concentrations were substantially reduced by re, although signicant pre-re treatment effects on soil N were still apparent immediately after the re. Soil nutrient availability, turnover rates and leaching losses were also increased by re. Concentrations of extractable soil N and leaching losses of Ca2, K, Cl and SO2 4 immediately after the re were signicantly higher in plots which had previously received N additions, demonstrating effects of N loading on post-re nutrient dynamics and economy. Effects of N addition on the activity of soil microbial populations were inuenced by soil moisture levels, with signicantly higher activity in N plots compared to controls when soil moisture levels were relatively high (>15%). Microbial community composition varied considerably between sampling dates, although clear N treatment differences were apparent, particularly for bacterial communities, both immediately after and one year on from the re. Persistent effects of wildre and N deposition on the stocks and availability of N, and microbial community dynamics, are likely to have longer-term consequences for the functioning of heathlands and other similar semi-natural ecosystems. 2012 Elsevier Ltd. All rights reserved.

1. Introduction Human activities, particularly those associated with agriculture, industry, transport and urban expansion have transformed the land surface and resulted in widespread changes to the structure and functioning of terrestrial ecosystems (Vitousek, 1997). Climate change is a strong driver of ecological change (Sala et al., 2000; Stocker et al., 2010), with predicted increases in temperature likely to affect the composition, distribution and functioning of ecosystems across the globe (Gitay et al., 2002). Changes in precipitation regimes are also predicted with, in particular, drier summers in mid

Q1

* Corresponding author. Division of Biology, Imperial College London, Silwood Park, Ascot, Berkshire SL75 7PY, UK. Tel.: 44 (0)207 5942318; fax: 44 (0)207 5942339. ** Corresponding author. Tel.: 44 (0)207 5942318; fax: 44 (0)207 5942339. E-mail address: s.power@imperial.ac.uk (S.A. Power). 0038-0717/$ e see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.soilbio.2012.08.025

latitude areas (Solomon et al., 2007). Such conditions are often associated with an increase in the frequency of wildre events (Dale et al., 2001) and there is indeed evidence linking a recent upsurge in the number and intensity of wildres to human inuence and climate change (Flannigan et al., 2000; Dale et al., 2001; Barcenas-Moreno et al., 2011). Fire has major impacts on the global carbon (C) cycle, converting organic C stores to CO2 (Cochrane, 2003; Jones and Cox, 2005; Kasischke et al., 2005; Randerson et al., 2005). In this way, re acts as a positive feedback on both climate change and the likelihood of ecologically damaging wildres (Borghesio, 2009). Resulting changes in temperature, soil moisture and organic matter quality will strongly inuence many important ecosystem processes including rates of decomposition and mineralisation (Swift and Heal, 1979; Schmidt et al., 2004). Anthropogenic disruption of the global nitrogen (N) cycle is also responsible for widespread change in the diversity and functioning of terrestrial ecosystems (Galloway et al., 2008; Gruber and

56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildre?, Soil Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025

SBB5265_proof 15 September 2012 2/9

E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9

111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175

Galloway, 2008; Bobbink et al., 2010). N is a limiting factor for plant growth in many semi-natural ecosystems, including forest, grassland and heathland, and many plant species associated with these systems are adapted to low levels of N (Bobbink et al., 1998; Aerts and Chapin, 2000). Indeed, 65e80% of Central European red list species are restricted to ecosystems with the lowest N status, since many of these plants can only compete in low nutrient situations (Bobbink et al., 2010). Responses to elevated N deposition include faster rates of nutrient cycling, species-specic changes in plant productivity and associated loss of diversity and N accumulation (Vitousek, 1997; Carroll et al., 2003; Galloway et al., 2008; Skogen et al., 2011). Interactions between N deposition and predicted increases in the frequency of summer drought and associated wildres are likely for several reasons. Lowland dry heaths are typically dominated by ericaceous dwarf-shrubs and, due to the large volume of fuel present, they tend to burn readily and produce relatively high temperature wildres (Tucker, 2003). N deposition has frequently been associated with increased biomass (e.g. Green, 2005; Carroll et al., 1999; Gordon et al., 1999; Power et al., 1998a,b) and the likelihood, severity, temperature and duration of a re may, therefore, be increased under elevated N deposition as a result of a greater fuel load. However, wildre also has the potential to remove a large proportion of accumulated N stores, potentially slowing or even reversing the process of nutrient build up associated with ecosystem N loading. Controlled burning is a management tool carried out under strict guidelines governing the season and intensity of burning, as described by Gimingham (1972). However, uncontrolled wildres tend to occur in the spring and summer and may burn intensely for a long time (Bullock and Webb, 1995). Flame temperatures between 225 and 1225  C have been recorded during wildres, although maximum temperatures are believed to reach as high as 2220  C (Dennison et al., 2006). The number and extent of heathland wildres is very dependent on weather conditions; in 2006 e one of the warmest summers on record (Prior and Beswick, 2007) e there were 923,000 such res recorded in the UK, an increase of 27% compared to the preceding year (McEvoy and Gamble, 2008). Whilst the devastating effects of wildre on vegetation and wildlife are immediately apparent, its impact on the belowground system, especially the microbial community and the ecological processes they control, are poorly understood. Several studies have shown that soil heating as a result of wildre decreases numbers of soil microorganisms and, in severe cases, can even temporarily sterilise the belowground environment (Neary et al., 1999; MataixSolera et al., 2009). The loss of nutrients (especially N) through combustion (Grdens et al., 2011) and production of charred N, a less accessible form of this element (Knicker, 2007), following re can result in low soil nutrient availability, slowing recovery of microbial populations. When populations do recover, changes in the soil chemical environment associated with heat and ash deposition e such as increased pH and soluble carbohydrate levels (Raison, 1979) e may make conditions more favourable for bacteria than for fungi. Therefore, not only will the soil environment be altered immediately by re, subsequent feedbacks are also likely as a result of changes in the microbial community (Raison, 1979). In this paper we present unique data from a long term heathland N addition experiment in southern England which experienced a severe, accidental summer re in 2006. This study aimed to determine the effects of wildre on soil nutrient status and the impacts of N addition on nutrient and microbial community dynamics following re. We hypothesise that: i) Wildre removes pre-existing treatment related differences in soil nutrient concentrations.

ii) N addition rapidly increases rates of nutrient availability and turnover, and modies microbial community composition following re. 2. Materials and methods 2.1. Site description and experimental design Thursley Common National Nature Reserve is an area of lowland heathland in Surrey, UK (Ordnance Survey Grid Reference SU 910404, Longitude 0 41.90 W, Latitude 519.30 N). The experimental area is a virtual monoculture of building phase Calluna (National Vegetation Classication type H2, Rodwell (1991), EUNIS site code UK930017, EUNIS (2011)) which was eight years old at the time of the wildre (Pic. S1). The soil is a podsol derived from Lower Greensand, comprising a thin (<1 cm) humus layer overlying a sandy mineral layer, with pure sand below 20 cm depth (Power et al., 1998a; Barker, 2001). Pre- and post-re soil physical and chemical properties are outlined in Table 1. In February 1998 a total of 16 plots (4 m 4 m) were set up in a factorial blocked design with 4 replicate blocks and 4 plots per block. Each plot is further sub divided into four sub-plots (2 2 m); management treatments were applied randomly to each of these 2 m 2 m sub-plots in February 1998 by Barker (2001) to provide a gradient of nutrient removal. As very few signicant management effects were detected, these are not considered here. Post-re sampling and analysis focused on two of these sub-plots which had previously been subjected to either a low intensity mow or a high temperature burn. From April 1998, half of the plots received increased N deposition (N: 30 kg N ha1 yr1) and the remaining plots served as controls (no extra N). Background deposition of N has been calculated at approximately 8e12 kg N ha1 yr1 following on site measurements of bulk deposition and gaseous concentrations of NH3 and NO2 (Power and Barker, 2003). Treatments, in the form of (NH4)2SO4, have been applied every two weeks, by adding 15 L N solution per 16 m2 plot on each occasion. Ammonium sulphate was used for comparability of results with an earlier experiment at the same site which started in 2 1989, when NH 4 co-deposition with SO4 occurred widely across the UK (NEGTAP, 2001). Control plots receive an articial rain 2 solution consisting of K, Mg2, Na, SO2 at concen4 , Cl , Ca trations found in rainfall measured at a nearby site (Uren, 1992), with additions representing 7% of the annual average rainfall at this site (Barker et al., 2004). Values for mean annual/growing season rainfall and temperature for the experimental period are presented in Table 2. The wildre occurred from 14the17th July 2006, after more than 8 years of N addition had taken place. The re resulted in the loss of all aboveground vegetation and the litter layer, as well as partial loss of the humus horizon in all experimental plots. The average ash layer depth was 2e3 mm, though this was largely washed away following a heavy rainfall event a month after the re. Treatment additions of N were suspended from JulyeOctober 2006, but resumed again after this time. The effect of re was determined by comparing nutrient stocks, availabilities and microbial activity with their pre-re values.
Table 1 Pre- and post-re soil chemical and physical properties at Thursley Common. Soil properties Total N mg kg1 Total P mg kg1 Bulk density g cm3 pH Loss on ignition (%) Pre-re 227.3 127.4 1.35 4.8 7.8 Post-re 66.2 79.4 1.40 5.0 1.8

176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildre?, Soil Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025

SBB5265_proof 15 September 2012 3/9

E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9

241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305

Table 2 Rainfall and temperature data for Thursley Common over the study period. Year Annual rainfall (mm) 690 740 589 850 925 Growing season (ApreSept) rainfall (mm) 203 307 239 335 443 Growing season (ApreSept) mean temp ( C) 14.4 14.0 13.8 14.7 14.1

2003 2004 2005 2006 2007

2.2. Soil chemistry and nutrient analyses Soil samples (0e10 cm) were collected from eight plots for Control and N treatments. Measurements included total N and phosphorus (P) concentrations (August 2006, October 2006 and February 2007), and extractable concentrations of nitrate, ammonium and phosphate (August 2006, October 2006, February 2007 and May 2007). Dried, ground soil samples were prepared for total N and P analyses using the Total Kjeldahl Nitrogen (TKN) method, based on the wet oxidation of organic material (Allen, 1989). 3 ml of sulphuric acid was added to 250 mg of soil, with a selenium catalyst, in order to convert organic N into the ammonium form. NO 3 and NH 4 ions were extracted from fresh soil samples using 1 M KCl (Allen, 1989). PO4 was also extracted from fresh soil samples, using Truogs reagent (Allen, 1989). All samples were analysed colorimetrically using a SKALAR San Flow segmented ow analyser (Skalar San Flow Autoanalyser, SKALAR, UK). Pre-re data for total (August 2003) and extractable (August 2004) soil nutrient concentrations were obtained using identical procedures. August data are presented to minimise the effect of seasonality when comparing pre-re conditions with those in August 2006, immediately after the re. Short term investigations into net mineralisation rates were carried out using in situ soil incubations over four month periods from MayeSeptember 2004 (pre-re) and MayeSeptember 2007 (post-re). On each occasion, paired soil samples were taken using PVC tubes (2.0 cm diameter, 10 cm length). One of each pair of tubes was taken for immediate analysis in the laboratory while the other was sealed and left in the eld for the incubation period. On return to the laboratory, 40 g of fresh soil was extracted using 250 ml 1 M KCl, and analysed for NH 4 and NO3 by colorimetric analysis. Net N mineralisation was taken as the increase in NH 4 eN plus NO3 eN, relative to concentrations in their initial paired samples (Van Vuuren and Van Der Eerden, 1992). Leachate samples were obtained using porous cup tension lysimeters installed to a depth of 45 cm. Pre-re data were collected monthly from April 1998 to May 1999 and from June to August 2003; post-re data were collected monthly from September 2006 until February 2007. Samples were stored at 5  C overnight and then NH 4 and NO3 concentrations were determined colorimetrically using a Skalar San Flow Autoanalyser (Skalar, UK). Subsamples from the collected leachate were also analysed for cation content using ion chromatography on a Dionex DX 500 HPLC/Ion Chromatography System (Thermo Scientic, USA). The pH of fresh soil samples (0e10 cm depth) was determined in August 2004 (prere), August 2006 and October 2006, using a 1:2 ratio of soil to deionised water and a 3 point calibrated Mettler Toledo MP230 pH meter (Mettler Toledo International, UK). 2.3. Soil microbial community analyses All microbial analyses were carried out on a single (0e10 cm) soil sample per plot. A sub-sample of soil (250 mg) was frozen

(18  C) within 12 h of sample collection for subsequent DNA extraction. Total dehydrogenase activity was determined on fresh soil (pre-re: August 2004, post-re: August 2006, October 2006, February 2007, May 2007, August 2007) as an indicator of overall microbial activity, using the method described by Ellis et al. (2001). One ml of 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT, SigmaeAldrich, UK) in 0.85% NaCl was added to 9 ml of the homogenized soil sample. This was shaken at 20  C for 24 h before formalin (0.5 ml) was added and the sample centrifuged at 5000 g for 20 min. The optical density (OD490) of the supernatant was determined and the pellet extracted with 10 ml methanol with 1% (v/v) Triton X-100 and shaken for 1 h at 20  C. The sample was then centrifuged at 5000 g for 20 min and the OD480 of the supernatant was determined. The extent of formazan production was then calculated according to standard conversion gures (Merlin et al., 1995). Molecular methods were used to assess total bacterial and fungal diversity, using primers targeted to the 16S and 18S ribosomal RNA genes, respectively (Muyzer et al., 1993, van Elsas et al., 2000). Post-re samples were collected in August 2006, October 2006, February 2007, May 2007 and August 2007 but no pre-re data were available. In addition, post-re diversity of 16S rRNA genes specic to ammonia-oxidizing bacteria within the b-proteobacteria (Kowalchuk et al., 1997), the functional genes required for N-xation, nifH (Lovell et al., 2000) and those associated with a key step in denitrication (nitrous oxide reduction), nosZ (Throbck et al., 2004) was quantied in October 2006, February 2007 and August 2007 for Control and N plots (Table S1). Furthermore, following recent evidence demonstrating the negative impacts of re on mycorrhizal fungi (Basitas et al., 2006) and the importance of ericoid mycorrhizae for growth of heather in restored lowland heath (Diaz et al., 2006) specic PCR primers were also developed to assess the presence and abundance of this important group of microorganisms following re. DNA was extracted from 250 mg of previously frozen soil, using Powersoil DNA isolation kits (MO BIO Laboratories Inc., USA). The PCR mix (50 ml/reaction) comprised 10 ml of each primer, 5 ml 1 Immobuffer, 2 mM MgCl2, 0.4 ml of 100 mM deoxynucleoside triphosphates (dNTP) mix, 1 unit of Immolase DNA polymerase (Bioline, London, UK) and 1 ml of extracted DNA. The cycle conditions used for each primer are described in the source article listed above for each gene. Amplied DNA fragments were separated using denaturing gradient gel electrophoresis (DGGE) as described in Ellis et al. (2003), using the INGENY phorU2 system (Netherlands). Samples were loaded onto 10% acrylamideebisacrylamide (37.5:1) gels with denaturing gradients from 30% to 60% (where 100% is 7 M urea and 40% [v/v.] deionized formamide) in 1 TAE electrophoresis buffer. Electrophoresis was performed at 100 V at 60  C for 16 h. Gels were then stained with SBYR Gold (Cambridge BioScience, UK) in 1 TAE for 30 min at room temperature and visualised under UV illumination. 2.4. Data analyses Mixed effects models, as implemented in the R Statistical package (lme function, R version 2.10.1; Pinheiro and Bates, 2009), were used to analyse the xed effects of N addition (N), re and their interaction, on soil chemistry and dehydrogenase activity. The random-effects of the initial (maximal) model, t by maximum likelihood, had a plot within block structure to account for temporal nesting and repeated measurements [R model: lme (y w Nitrogen*Fire*Time, random w1jBlock/plot), where time represents different sampling dates]. Following this approach (for each response variable) we performed two analyses, one comparing differences between the treatments pre-re and

306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildre?, Soil Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025

SBB5265_proof 15 September 2012 4/9

E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9

371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435

immediately after the re, whilst in the second analysis we compared the dynamics of the post-re recovery across several sampling dates. In order to explore the N addition impact on each sampling occasion a contrast (planned post-hoc test) analysis was performed to test for differences between Control and N plots. Soil moisture data were used as covariates in the model for extractable N and dehydrogenase activity measurements. Simplication of the maximal model was achieved by removing nonsignicant terms, comparing models using the Akaike information criterion (AIC) until reaching the minimum adequate model (Crawley, 2007). Digital images of DGGE gels were analysed using GelCompar II V5.10 software (Applied Maths, Belgium). Pairwise similarities in microbial community characteristics between samples were calculated using the dice similarity coefcient and allowing for 0.5% variation in band position. Graphical representations of similarity matrices were produced using UPGMA (unweighted pair group method using arithmetic averages). Throughout the paper, data are presented as mean SE and the threshold for statistical signicance is P 0.05. 3. Results 3.1. Soil nitrogen and phosphorus dynamics Immediately following the re (Aug 2006), total N and P concentrations for the 0e10 cm soil layer signicantly dropped below pre-re (Aug 2003) values [(N: F1,11 36.1, P < 0.001); (P: F1,12 5.51, P < 0.05)] (Fig. 1). However, earlier N treatmentrelated differences in soil N concentrations were still apparent immediately after the re (Aug 2006: F1,9 6.06, P < 0.05). Soil samples taken shortly after N additions resumed in October 2006 also had higher total N concentrations in N plots, relative to

Controls (post-re) (F1,10 7.01, P < 0.05). Effects of N additions on post-re total P concentrations were, however, not signicant (F1,10 0.35, P > 0.5). No discernable effects of either re or N treatments were found for extractable PO4 levels, although analyses showed that PO4 concentrations were higher when soil moisture levels were high (F1,20 18.29, P < 0.001). The temporal variation in extractable soil NH 4 and NO3 concentrations also reected the dynamic nature of moisture availability (Fig. 2). Post-re extractable NH 4 and NO3 concentrations were in fact higher than pre-burn (Aug 2004) values in Control and N treatments (NH 4 : F1,9 13.0, P < 0.005; NO3 : F1,9 200.4, P < 0.001). Pre-burn extractable N concentrations typically ranged from 0.2 to 0.6 mg kg1 during winter months, rising to 0.5e1.9 mg kg1 in the spring/autumn and with the lowest values (0.15e0.19 mg kg1) during the drier summer months (Green, 2005); post-re values were, therefore, high for the season (and soil moisture levels). Repeated measures analysis demonstrated a signicant overall effect of N addition on post-re extractable NH 4 concentrations (F1,21 12.9, P < 0.01), with signicant treatment effects seen individually on three of the four separate measurement occasions (Fig. 2a). Signicantly higher levels of extractable NO 3 were measured in N plots immediately after the re (Aug 2006) (F1,12 5.08, P < 0.05), however, levels of extractable NO 3 had returned to pre-re low values by the following spring (Fig. 2b).

Fig. 1. a) Pre- and post-re total soil content of a) nitrogen and b) phosphorus in relation to N treatment. Error bars represent 1 SEM. Signicance codes *** 0.001, ** 0.01, * 0.05, ns not signicant.

Fig. 2. Temporal and treatment-related patterns for KCl extractable a) NH 4 and b) NO 3 . Error bars represent 1 SEM. Signicance codes *** 0.001, ** 0.01, * 0.05, ns not signicant.

436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildre?, Soil Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025

SBB5265_proof 15 September 2012 5/9

E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9

501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565

N mineralisation rates following the re were not signicantly higher in N (4.4 g N m2) relative to Control (1.5 g N m2) plots. Values were, however, signicantly higher than pre-re mineralisation rates measured over a similar period in 2004 (0.16 g N m2 Control, 0.37 g N m2 N; F1,13 14.1, P < 0.01) (Fig. 3). Leaching losses of NO 3 and NH4 were generally small, and did not differ signicantly between Control and N plots (NH 4 F1,21 1.85, P > 0.05; NO 3 F1,21 0.81, P > 0.05). However, signicantly higher Ca (F1,21 6.00, P < 0.05), K (F1,21 23.4, P < 0.001) Cl (F1,21 8.77, P < 0.01) and SO2 4 (F1,21 11.5, P < 0.01) ion concentrations were present in leachate collected from N plots following the re (Sept 2006eFeb 2007), compared to Control plots. The biggest treatment effects were seen shortly after the re (Sept 2006), with Control/N plot values of 81/869 mg l1, 52/222 mg l1, 363/869 mg l1 and 54/ 194 mg l1 for Ca, K, Cl and SO2 4 , respectively, at this time. Soil pH was signicantly higher after the re than pre-re values recorded in August 2004 (control: 4.8, N: 4.3, Green, 2005); values in October 2006 increased by 0.2 unitse5.0 and 4.5 in Control and N plots, respectively (F1,10 29.6, P < 0.001). However, pre-re N treatment differences in pH remained highly signicant after the re (F1,10 17.3, P < 0.001). 3.2. Soil microbial communities Overall levels of microbial activity, as indicated by hydrogenase activity, appeared to be strongly inuenced by weather conditions preceding sampling; when post-re soil moisture levels were <15%, activity was generally low (Aug 06, Oct 06, May 07; <8 mM INT g1 h1) and there were no signicant effects of N at these times. Following relatively wetter weather (soil moisture > 15%), microbial activity increased, particularly in the N plots (Feb 07, Aug 07; >13 mM INT g1 h1) and N treatment effects were signicant (F1,9 8.53, P < 0.01) (Fig. 4). Interestingly, whilst measurements of soil microbial activity in August 2004 (pre-re) and August 2006 (immediately post-re) e both times when soil moisture levels and microbial activity were low e were not signicantly different, effects of N addition were signicant before (F1,9 32.5, P < 0.001), but not after the re. Assessment of post-re microbial molecular diversity showed effects of N treatment on various components of the soil microbial community. For total bacterial and fungal communities, cluster analysis and multidimensional scaling differentiated between samples from Control and N plots. Multidimensional scaling of bacterial communities from Aug 06, May 07 and Aug 07 showed that the variation between replicate samples from the same treatment taken at the same time was small compared to the

Fig. 4. Pre- and post-re soil dehydrogenase activity. Error bars represent 1 SEM. Signicance codes *** 0.001, ** 0.01, * 0.05, ns not signicant.

overall variation, but that there was overlap between different time/treatment groups (Fig. S1). Cluster analysis, however, revealed strong treatment differences between Control and N plots one year after the re, highlighting the persistence of treatment effects after a major disturbance event (Fig. 5). There was greater variability between sample dates than between N treatments for fungal communities (Fig. S2) although N treatment effects were apparent in the cluster analysis (Fig. 6) 12 months after the re occurred. Although populations of ammonia-oxidizing bacteria belonging to the b-proteobacteria were not particularly diverse they were highly variable, even within treatments, so denitive effects N addition could not be determined (results not shown). In contrast, there was little variation in the functional genes for N xation (nifH) and denitrication (nosZ) which were relatively stable over time and unaffected by N treatment (results not shown). The use of PCR primers specic for Rhizoscyphus ericae indicated that this

Fig. 3. Pre- (MayeSept 2004) and post-re (MayeSept 2007) net mineralisation rates. Error bars represent 1 SEM. Signicance codes *** 0.001, ** 0.01, * 0.05, ns not signicant.

Fig. 5. UPGMA cluster analysis of bacterial communities one year (Aug 2007) after the re, as assessed by PCR/DGGE of 16S ribosomal RNA genes.

566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildre?, Soil Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025

90

70

1 00

40

50

60

80

SBB5265_proof 15 September 2012 6/9

E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9

631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695

Fig. 6. UPGMA cluster analysis of fungal communities one year (Aug 2007) after the re, as assessed by PCR/DGGE of 16S ribosomal RNA genes.

mycorrhizal fungus was abundant in the soil of burnt areas one year after the re, although, again, no effects of N treatment were apparent.

4. Discussion 4.1. Changes in biogeochemistry The literature suggests that up to 95% of the N and 20e30% of the other principal plant nutrients in the standing vegetation and accumulated litter can be lost during heathland res (Neary et al., 1999; de Luis et al., 2003). In the current study, re resulted in a decrease in soil total N and P concentrations by an average of 72% and 42%, respectively, although pre-treatment differences in soil N concentrations were still apparent. The smaller proportional reduction in soil P stocks, compared to N, may reect the rapid incorporation of P deposited in ash (Christensen and Muller, 1975; Grove et al., 1986; Smithwick et al., 2005). In areas of moderate to high N deposition where N losses are quickly replaced, wildre may act to exacerbate P limitation in lowland heathland as replacement of P losses is a relatively slow process (Chapman et al., 1989). For example, Chapman (1967) suggest that following loss of soil organic matter at sites (such as Thursley Common) where the adsorption capacity is low, it may take c. 20 years for the lost P to be replaced. Phosphorus availability may, therefore, exercise control on the N economy of the system following wildre, particularly in areas of high N deposition (Aerts and Bobbink, 1999; Gordon et al., 2001; Turner et al., 2002). Under high rates of N deposition and concurrent P depletion (through re or controlled management removal of nutrients), soil conditions may also favour invasion and eventual dominance by species that are well adapted to P limitation (e.g. Molinia caerulea, Tomassen et al., 2003), with implications for the diversity, structure and conservation value of heathland ecosystems. Leaching losses of soil nutrients have been shown elsewhere to be high following a re if the soil has a low adsorption capacity (Robertson and Davies, 1965). Post-re losses of NH 4 and NO3 were, however, low in the current study as, in fact, they had been on all measurement occasions since the start of the experiment in 1998. An earlier 15N tracer study at the site demonstrated a very strong N immobilisation capacity in this system, with >75% of 15N inputs rapidly immobilised in the soil microbial biomass (Green, 2005). Whilst re undoubtedly affected the size and activity of microbial populations, their rapid recovery and high N sink strength may explain the very low leaching losses of N observed after the re. In contrast, however, not only were Ca2, K, Cl and SO2 4 concentrations elevated in the rst few months following the re e a likely consequence of ash deposition following combustion

of accumulated organic material (Raison, 1979) e they were also signicantly higher in N plots, demonstrating a negative effect of earlier N additions on the economy of these ions following a major perturbation. The high concentrations of extractable NH 4 present in N plots immediately after the re at Thursley may have contributed to a replacement of cations (Mg, Ca and K) at soil exchange sites (Brady and Weil, 1996) and thus account for the increased concentrations of Ca2 and K in leachate, relative to both control plot values (this study) and pre-re levels (C. Barker, unpublished data). Rainfall levels after the re were consistent with the long term seasonal average at the site (1998e2009, Meteorological Ofce, 2011) and are, therefore, unlikely to have resulted in unrepresentative dilution/concentration of lysimeter samples during the post-re analysis period. Niemeyer et al. (2005) also found increased leaching of Ca and K following a prescribed burn treatment, although these soil leaching losses were low compared to combustion losses from aboveground vegetation, and are likely to be replenished from precipitation within a few years (Allen, 1964; Robertson and Davies, 1965; Chapman et al., 1989). Typically, levels of available plant nutrients in a heathland system decline with time as they become immobilised in woody plant material (Tucker, 2003) and microbial biomass (Green, 2005), and are turned over only slowly. The immediate post-re increase in extractable NH 4 and NO3 concentrations found in this study in both Control and N plots is likely to be a consequence of the rapid oxidation of organic material and absence of a vegetation sink, as has been reported following wildres elsewhere (Allen, 1964; Kenworthy, 1964; Lloyd, 1971; Tucker, 2003). Signicant treatmentrelated differences in NH 4 concentrations were, however, in line with those found on multiple measurement occasions prior to the re, highlighting the consistency of response in extractable nutrient concentrations across seasons and years. Increased microbial activity after the re, compared to pre-re levels measured during the same season, imply high levels of oxidative activity in the soil (Taylor et al., 2002) and the retention of at least small amounts of soil organic matter following the wildre (Table 1). Although post-re mineralisation rates were not affected by N treatment, they were signicantly higher than pre-burn values (MayeSept 2004). Similar increases in mineralisation rates following wildres have been reported elsewhere for upland moorland (Pilkington et al., 2007), chaparral (Singh et al., 1991), tropical forest (Matson et al., 1987) and grassland (Hobbs and Schimel, 1984), indicating that more frequent res are likely to affect nutrient turnover and economy across a wide range of terrestrial systems. A review by Smithwick et al. (2005) evaluated 24 studies investigating the effects of various re types on forest ecosystems. They found that soil NH 4 and NO3 concentrations generally increased between 2 and 26 times following a re, with the pulse lasting less than two years. Conicting results were found with regard to mineralisation rates but, where increases were observed, they were attributed to a much reduced uptake of nutrients by aboveground vegetation, reduced microbial immobilisation (Klopatek et al., 1990), increased soil temperatures (Hobbs and Schimel, Q2 1984), increased anion exchange at depth (Matson et al., 1987) and higher pH levels in the soil (Allen, 1964; Christensen and Muller, 1975; Stevenson et al., 1996). A signicant increase in soil pH was observed in both the Control and N plots compared to pre-re levels and this may have contributed to the increase in post-re levels of microbial activity in the experimental plots. An increase in pH has been reported in many systems following wildre due to the addition of alkaline salts in ash deposits and the destruction of organic matter (DeBano et al., 1976; Giovannini et al., 1990; Forgeaard and Frenot, 1996). Overall the results of this study show

696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildre?, Soil Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025

1 00

50

60

90

70

80

SBB5265_proof 15 September 2012 7/9

E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9

761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825

that, even though nutrient stocks are undoubtedly reduced by re, nutrient availability may be increased in the short term. 4.2. Soil microbial communities Pre-re data collected at this site have shown clearly that N addition affects the structure and activity of soil microbial communities (Green, 2005). Post-re assessments revealed strong, persistent effects of N addition on the structure of bacterial and fungal communities. Soil microbial communities were seen to be highly dynamic, changing substantially between successive measurements, although with clear differences between Control and N plots evident on each measurement occasion. Wildre may actually make soil more favourable for bacteria (Raison, 1979; Alexander, 1977) due to an increase in pH and inputs of soluble carbohydrates. Renbuss et al. (1973) studied soil microbiology and nutrient changes following wildre which sterilised the soil in a forest ecosystem over a 15 month period. Bacterial recovery was reported to be swift, with populations increasing signicantly, to ten times pre-re levels within a month, although it took a year for bacterial community structure to return to pre-re compositions. In the current study, the compositions of both bacterial (Fig. S1) and fungal (Fig. S2) communities were highly dynamic in the year following re, with persistent differences in composition apparent between control and N plots. Measurements of microbial activity suggest that some of the temporal differences in community composition may have been linked to variations in soil moisture. Although the presence of functional genes for ammonia oxidation, N xation and nitrous oxide reduction, all of which are key steps in the N cycle, were not affected by N-treatment, it is probable that the relative abundance and activity of associated organisms are related to changes in soil chemistry. Whilst clear treatment-related differences in soil chemistry were apparent in August 2006, no N cycling-specic molecular data were available from this time. Given the relatively low overall levels of microbial activity in the nutrientpoor soils at this heathland site, any evidence of change would be difcult to detect using molecular methods three months on from a re. The detection of R. ericae in soil which was largely devoid of Calluna indicates that re-growth of the dominant vegetation would not have been limited by the availability of suitable mycorrhizal fungi. Microbial communities are the primary determinants of decomposition of organic matter and nutrient availability for plant uptake in terrestrial ecosystems, and play a critical role in ecosystem functioning (Schimel, 1995; Schimel and Gulledge, 1998; Cavigelli and Robertson, 2000). This study has shown that N deposition and wildre have the ability to alter the activity (and for N persistent changes in the structure) of microbial communities with associated changes in soil nutrient dynamics and economy which may, therefore, have long term consequences for the functioning of re-prone ecosystems. 5. Conclusion This study evaluates the consequences of two major global change phenomena for soil biodiversity and nutrient dynamics. Our rst hypothesis e that wildre would remove pre-existing treatment-related differences in soil nutrients e was not supported since, although re signicantly reduced soil N and P concentrations, pre-existing differences in soil N between control and Ntreated plots remained signicant immediately after the re. Temporal variation in nutrient availability and leaching was high, reecting patterns in microbial activity and climate. However, concentrations of extractable N and leaching losses of other ions were signicantly higher in N plots compared to controls,

supporting our second hypothesis. Whilst effects on specic microbial functional groups were not apparent, clear N-treatment and temporal differences in the activity and composition of the soil microbial community were found, also proving support for our second hypothesis. These ndings, combined with previous evidence of persistent, long term impacts of historical N loading at this site (Power et al., 2006), suggest that belowground effects of N and re may be highly persistent. This study clearly shows that, while severe summer res have the potential to remove a proportion of accumulated soil N stores, deposition-driven differences in nutrient availability and microbial community composition are quickly re-established and the long term consequences of elevated N inputs are, therefore, unlikely to be benecial at an ecosystem level. Acknowledgements This study was funded through an Urgency Grant from the UK Natural Environment Research Council (NE/E011063/1). We are grateful to the Site Wardens at Thursley Common, Simon Nobes and James Giles, for their support for this long term experiment, and to Kim Prior for assistance in both the eld and laboratory. We would also like to thank Dr Nathan Callaghan for comments on earlier versions on the manuscript and Dr David Ames for analysis of leachate chemistry. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2012.08.025. References
Aerts, R., Bobbink, R., 1999. The impact of atmospheric nitrogen deposition on vegetation processes in terrestrial, non-forest systems. In: Langan, S.J. (Ed.), The Impact of Nitrogen Deposition on Natural and Semi Natural Ecosystems. Kluwer Academic Press, The Netherlands. Aerts, R., Chapin, F.S., 2000. The mineral nutrition of wild plants revisited: a reevaluation of processes and patterns. Advances in Ecological Research 30, 1e67. Alexander, M., 1977. Introduction to Soil Microbiology. John Wiley & Sons, New York. Allen, S.E., 1964. Chemical aspects of heather burning. Journal of Applied Ecology 1, 347e367. Allen, S.E., 1989. Chemical Analysis of Ecological Materials. Blackwell Scientic Publications, Oxford. Barcenas-Moreno, G., Garcia-Orenes, F., Mataix-Solera, J., Mataix-Beneyto, J., Baath, E., 2011. Soil microbial recolonisation after a re in a Mediterranean forest. Biology and Fertility of Soils 47, 261e272. Barker, C.G., 2001. The Impact of Management of Heathland Response to Increased Nitrogen Deposition. PhD thesis, Imperial College, University of London. Barker, C.G., Power, S.A., Bell, J.N.B., Orme, C.D.L., 2004. Effects of habitat management on heathland response to atmospheric nitrogen deposition. Biological Conservation 120, 41e52. Basitas, B.A., Xu, Z., Cairney, J.W.G., 2006. Inuence of long-term repeated prescribed burning on mycelial communities of ectomycorrhizal fungi. New Phytologist 172, 149e158. Bobbink, R., Hicks, K., Galloway, J., Spranger, T., Alkemade, R., Ashmore, M., Bustamante, M., Cinderby, S., Davidson, E., Dentener, F., Emmett, B., Erisman, J.W., Fenn, M., Gilliam, F., Nordin, A., Pardo, L., De Vries, W., 2010. Global assessment of nitrogen deposition effects on terrestrial plant diversity: a synthesis. Ecological Applications 20, 30e59. Bobbink, R., Hornung, M., Roelofs, J.G.M., 1998. The effects of air-borne nitrogen pollutants on species diversity in natural and semi-natural European vegetation. Journal of Ecology 86, 717e738. Borghesio, L., 2009. Effects of re on the vegetation of a lowland heathland in north-western Italy. Plant Ecology 201, 723e731. Brady, N.C., Weil, R.R., 1996. The Nature and Properties of Soil. Prentice Hall, London, UK. Bullock, J.M., Webb, N.R., 1995. Responses to severe res in heathland mosaics in southern England. Biological Conservation 73, 207e214. Carroll, J.A., Caporn, S.J.M., Johnson, D., Morecroft, M.D., Lee, J.A., 2003. The interactions between plant growth, vegetation structure and soil processes in seminatural acidic and calcareous grasslands receiving long-term inputs of simulated pollutant nitrogen deposition. Environmental Pollution 121, 363e376.

826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildre?, Soil Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025

SBB5265_proof 15 September 2012 8/9

E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9 Kowalchuk, G.A., Stephen, J.R., de Boer, W., Prosser, J.I., Embley, T.M., Woldendorp, J.W., 1997. Analysis of ammonia-oxidizing bacteria of the b-subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplied 16S ribosomal DNA fragments. Applied and Environmental Microbiology 63, 1489e1497. Lloyd, P.S., 1971. Effects of re on chemical status of herbaceous communities of Derbyshire Dales. Journal of Ecology 59, 261e273. Lovell, C.R., Piceno, Y.M., Quattro, J.M., Bagwell, C.E., 2000. Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass, Spartina alterniora. Applied and Environmental Microbiology 66, 3814e3822. Mataix-Solera, J., Guerrero, C., Garcia-Orenes, F., Barcesnas, G.M., Torres, M.P., 2009. Forest re effects on soil microbiology. In: Cerda, A., Robichaud, P.R. (Eds.), Fire Effects on Soils and Restoration Strategies. Land Restoration and Management Series. Science Publishers, Eneld. Matson, P.A., Vitousek, P.M., Ewel, J.J., Mazzarino, M.J., Robertson, G.P., 1987. Nitrogen transformations following tropical forest felling and burning on a volcanic soil. Ecology 68, 491e502. McEvoy, P., Gamble, J., 2008. Fire Statistics: United Kingdom 2006. Crown Copyright: Communities and Local Government Publications, England. Merlin, G., Lissolo, T., Morel, V., Rossel, D., Tarradellas, J., 1995. Precautions for routine use of INT-reductase activity for measuring biological activities in soil and sediments. Environmental Toxicology and Water Quality 10, 185e192. Meteorological Ofce, 2011. Spring 2010 (Online). Available from: http://www. metofce.gov.uk/climate/uk/2010/spring.html (accessed 18.12.11.). Muyzer, G., de Waal, E.C., Uitterlinden, A.G., 1993. Proling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplied genes coding for 16S rRNA. Applied and Environmental Microbiology 59, 695e700. Neary, D.G., Klopatek, C.C., Debano, L.F., Folliott, P.F., 1999. Fire effects on belowground sustainability: a review and synthesis. Forest Ecology and Management 122, 51e71. NEGTAP, 2001. Transboundary Air Pollution, Acidication, Eutrophication and Ground Level Ozone in the UK. Report of the National Expert Group on Transboundary Air Pollution. Department of the Environment, Food and Rural Affairs, London, UK. Niemeyer, T., Niemeyer, M., Mohamed, A., Fottner, S., Hardtle, W., 2005. Impact of prescribed burning on the nutrient balance of heathlands with particular reference to nitrogen and phosphorus. Applied Vegetation Science 8, 183e192. Pilkington, M.G., Caporn, S.J.M., Carroll, J.A., Cresswell, N., Phoenix, G.K., Lee, J.A., Emmett, B.A., Sparks, T., 2007. Impacts of burning and increased nitrogen deposition on nitrogen pools and leaching in an upland moor. Journal of Ecology 95, 1195e1207. Pinheiro, J.C., Bates, D.M., 2009. Mixed-effects Models in S and S-PLUS. Springer Verlag. Power, S.A., Ashmore, M.R., Cousins, D.A., 1998a. Impacts and fate of experimentally enhanced nitrogen deposition on a British lowland heath. Environmental Pollution 102, 27e34. Power, S.A., Ashmore, M.R., Cousins, D.A., Sheppard, L.J., 1998b. Effects of nitrogen addition on the stress sensitivity of Calluna vulgaris. New Phytologist 138, 663e673. Power, S.A., Barker, C.G., 2003. Deposition measurements at Thursley Common heathland nature reserve. In: Achermean, B., Bobbink, R. (Eds.), Empirical Critical Loads for Nitrogen. Expert Workshop Proceedings, Berne, 11e13 November 2003. Swiss Agency for the Environment, Forests and Landscapes, Berne. Power, S.A., Green, E.R., Barker, C.G., Bell, N.B., Ashmore, M.R., 2006. Ecosystem recovery, heathland response to a reduction in N deposition. Global Change Biology 12, 1241e1252. Prior, J., Beswick, M., 2007. The record breaking heat and sunshine of July 2006. Weather 62, 174e182. Raison, R., 1979. Modication of the soil environment by vegetation res, with particular reference to nitrogen transformations: a review. Plant and Soil 51, 73e108. Randerson, J.T., van der Werf, G.R., Collatz, G.J., Giglio, L., Still, C.J., Kasibhatla, P., Miller, J.B., White, J.W.C., DeFries, R.S., Kasischke, E.S., 2005. Fire emissions from C-3 and C-4 vegetation and their inuence on interannual variability of atmospheric CO2 and delta(CO2)-C-13. Global Biogeochemical Cycles 19, GB2019. http://dx.doi.org/10.1029/2004gb002366. Renbuss, M.A., Chilvers, G.A., Pryor, L.D., 1973. Microbiology of an ashbed. Proceedings of the Linnean Society of New South Wales 97, 302e311. Robertson, R.A., Davies, G.E., 1965. Quantities of plant nutrients in heather ecosystems. Journal of Applied Ecology 2, 211e219. Rodwell, J.S., 1991. British Plant Communities. In: Woodlands and Scrub, vol. 1. Cambridge University Press, UK. Sala, O.E., Chapin III, F.S., Armesto, J.J., Berlow, R., Bloomeld, J., Dirzo, R., HuberSanwald, E., Huenneke, L.F., Jackson, R.B., Kinzig, A., Leemans, R., Lodge, D., Mooney, H.A., Oesterheld, M., Poff, N.L., Sykes, M.T., Walker, B.H., Walker, M., Wall, D.H., 2000. Biodiversity e global biodiversity scenarios for the year 2100. Science 287, 1770e1774. Schimel, J., 1995. Ecosystem consequences of microbial diversity and community structure. In: Chapin, F.S., Koerner, C. (Eds.), Arctic and Alpine Biodiversity: Patterns, Causes, and Ecosystem Consequences. Springer Verlag. Schimel, J.P., Gulledge, J., 1998. Microbial community structure and global trace gases. Global Change Biology 4, 745e758.

891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955

Carroll, J.A., Caporn, S.J.M., Cawley, L., Read, D.J., Lee, J.A., 1999. The effect of increased deposition of atmospheric nitrogen on Calluna vulgaris in upland Britain. New Phytologist 141, 423e431. Cavigelli, M.A., Robertson, G.P., 2000. The functional signicance of denitrier community composition in a terrestrial ecosystem. Ecology 81, 1402e1414. Chapman, S.B., 1967. Nutrient budgets for a dry heath ecosystem in the south of England. Journal of Ecology 55, 677e689. Chapman, S.B., Rose, R.J., Basanta, M., 1989. Phosphorus adsorption by soils from heathlands in southern England in relation to successional change. Journal of Applied Ecology 26, 673e680. Christensen, N.L., Muller, C.H., 1975. Effects of re on factors controlling plant growth in Adenostoma chaparral. Ecological Monographs 45, 29e55. Cochrane, M.A., 2003. Fire science for rainforests. Nature 421, 913e919. Crawley, M.J., 2007. The R Book. John Wiley & Sons Ltd, Chichester, UK. Dale, V.H., Joyce, L.A., McNulty, S., Neilson, R.P., Ayres, M.P., Flannigan, M.D., Hanson, P.J., Irland, L.C., Lugo, A.E., Peterson, C.J., Simberloff, D., Swanson, F.J., Stocks, B.J., Wotton, B.M., 2001. Climate change and forest disturbances. Bioscience 51, 723e734. de Luis, M., Gonzalez-Hidalgo, J.C., Raventos, J., 2003. Effects of re and torrential rainfall on erosion in a Mediterranean gorse community. Land Degradation & Development 14, 203e213. DeBano, L.F., Savage, S.M., Hamilton, D.A., 1976. The transfer of heat and hydrophobic substances during burning. Soil Science Society America Journal 40, 779e782. Dennison, P.E., Charoensiri, K., Roberts, D.A., Peterson, S.H., Green, R.O., 2006. Wildre temperature and land cover modelling using hyperspectral data. Remote Sensing of Environment 100, 212e222. Diaz, A., Green, I., Benvenuto, M., Tibbett, M., 2006. Are ericoid mycorrhizas a factor in the success of Calluna vulgaris heathland restoration? Restoration Ecology 14, 187e195. Ellis, R.J., Morgan, P., Weightman, A.J., Fry, J.C., 2003. Cultivation-dependent and -independent approaches for determining bacterial diversity in heavy-metalcontaminated soil. Applied and Environmental Microbiology 69, 3223e3230. Ellis, R.J., Neish, B., Trett, M.W., Best, J.G., Weightman, A.J., Morgan, P., Fry, J.C., 2001. Comparison of microbial and meiofaunal community analyses for determining impact of heavy metal contamination. Journal of Microbiological Methods 45, 171e185. EUNIS, 2011. Thursely Common. EUNIS Biodiversity Database (online) Available at: http://eunis.eea.europa.eu/sites/UK930017 (accessed 19.03.11.). Flannigan, M.D., Stocks, B.J., Wotton, B.M., 2000. Climate change and forest res. Science of the Total Environment 262, 221e229. Forgeaard, F., Frenot, Y., 1996. Effects of burning on heathland soil chemical properties: an experimental study on the effect of heating and ash deposits. Journal of Applied Ecology 33, 803e811. Galloway, J.N., Townsend, A.R., Erisman, J.W., Bekunda, M., Cai, Z., Freney, J.R., Martinelli, L.A., Seitzinger, S.P., Sutton, M.A., 2008. Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science 320, 889e892. Grdens, A.I., gren, G.I., Bird, J.A., Clarholm, M., Hallin, S., Ineson, P., Ktterer, T., Knicker, H., Nilsson, S.I., Nsholm, T., Ogle, S., Paustian, K., Persson, T., Stendahl, J., 2011. Knowledge gaps in soil carbon and nitrogen interactions e from molecular to global scale. Soil Biology and Biochemistry 43, 702e717. Gimingham, C.H., 1972. Ecology of Heathlands. Chapman and Hall, London. Giovannini, G., Lucchesi, S., Giachetti, M., 1990. Effects of heating on some chemical arameters related to soilfertility and plant growth. Soil Science 149, 344e350. Gitay, H., Surez, A., Watson, R. (Eds.), 2002. Climate Change and Biodiversity. IPPC Technical Paper V, Working Group II Technical Support Unit, Bern, Switerland. Gordon, C., Woodin, S.J., Alexander, I.J., Mullins, C.E., 1999. Effects of increased temperature, drought and nitrogen supply on two upland perennials of contrasting functional type: Calluna vulgaris and Pteridium aquilinum. New Phytologist 142, 243e258. Gordon, C., Wynn, J.M., Woodin, S.J., 2001. Impacts of increased nitrogen supply on high Arctic heath: the importance of bryophytes and phosphorus availability. New Phytologist 149, 461e471. Green, E.R., 2005. The Effect of Nitrogen Deposition on Lowland Heath Ecosystems. PhD thesis, Imperial College, University of London. Grove, T.S., Oconnell, A.M., Dimmock, G.M., 1986. Nutrient changes in surface soils after an intense re in Jarrah (Eucalyptus marginata donn ex sm) forest. Australian Journal of Ecology 11, 303e317. Gruber, N., Galloway, J.N., 2008. An Earth-system perspective of the global nitrogen cycle. Nature 451, 293e296. Hobbs, N.T., Schimel, D.S., 1984. Fire effects on nitrogen mineralization and xation in mountain shrub and grassland communities. Journal of Range Management 37, 402e405. Jones, C.D., Cox, P.M., 2005. On the signicance of atmospheric CO2 growth rate anomalies in 2002e2003. Geophysical Research Letters 32, L14816. http:// dx.doi.org/10.1029/2005GL023027. Kasischke, E.S., Hyer, E.J., Novelli, P.C., Bruhwiler, L.P., French, N.H.F., Sukhinin, A.I., Hewson, J.H., Stocks, B.J., 2005. Inuences of boreal re emissions on Northern Hemisphere atmospheric carbon and carbon monoxide. Global Biogeochemical Cycles 19, GB1012. http://dx.doi.org/10.1029/2004GB002300. Kenworthy, J.B., 1964. A Study if Changes in Plant and Soil Nutrients Associated with Moor Burning and Grazing. PhD thesis, University of St. Andrews, UK. Knicker, H., 2007. How does re affect the nature and stability of soil organic nitrogen and carbon? A review. Biogeochemistry 85, 91e118.

956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildre?, Soil Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025

SBB5265_proof 15 September 2012 9/9

E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9 Schmidt, I.K., Tietema, A., Williams, D., Gundersen, P., Beier, C., Emmett, B.A., Estiarte, M., 2004. Soil solution chemistry and element uxes in three European heathlands and their responses to warming and drought. Ecosystems 7, 638e649. Singh, R.S., Srivastava, S.C., Raghubanshi, A.S., Singh, J.S., Singh, S.P., 1991. MicrobialC, microbial-N and microbial-P in dry tropical savanna e effects of burning and grazing. Journal of Applied Ecology 28, 869e878. Skogen, K.A., Holsinger, K.E., Cardon, Z.G., 2011. Nitrogen deposition, competition and the decline of a regionally threatened legume, Desmodium cuspidatum. Oecologia 165, 261e269. Smithwick, E.A.H., Mack, M.C., Turner, M.G., Chapin, F.S., Zhu, J., Balser, T.C., 2005. Spatial heterogeneity and soil nitrogen dynamics in a burned black spruce forest stand: distinct controls at different scales. Biogeochemistry 76, 517e537. Solomon, S.D., Qin, M., Manning, Z., Chen, Z., Marquis, M., Averyt, K.B., Tignor, M., Miller, H.L. (Eds.), 2007. IPCC: Summary for Policymakers. Climate Change 2007: the Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge. Stevenson, A.C., Rhodes, A.N., Kirkpatrick, A.H., Macdonald, A.J., 1996. The Determination of Fire Histories and an Assessment of Their Effects on Moorland Soil. Scottish Natural Heritage, Edinburgh. Research, Survey and Monitoring Report No. 16. Stocker, T.F., Field, C.B., Qin, D. (Eds.), 2010. IPCC: Meeting report of the Intergovernmental Panel on Climate Change Expert Meeting on Detection and Attribution Related to Anthropogenic Climate Change. Cambridge University Press, Cambridge. Swift, M.J., Heal, O.W. (Eds.), 1979. Decomposition in Terrestrial Ecosystems. Blackwell Scientic Publications, Oxford.

1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041

Taylor, J.P., Wilson, B., Mills, M.S., Burns, R.G., 2002. Comparison of microbial numbers and enzyme activities in surface soils and subsoils using various techniques. Soil Biology and Biochemistry 34, 387e401. Throbck, I.N., Enwall, K., Javis, ., Hallin, S., 2004. Reassessing primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiology Ecology 49, 401e417. Tomassen, H.B.M., Smolders, A.J.P., Lamers, L.P.M., Roelofs, J.G.M., 2003. Stimulated growth of Betula pubescens and Molinia caerulea on ombrotrophic bogs: role of high levels of atmospheric nitrogen deposition. Journal of Ecology 91, 357e370. Tucker, G., 2003. Review of the Impacts of Heather and Grassland Burning in the Uplands on Soils, Hydrology and Biodiversity. English Nature, Document Number 550. Turner, B.L., Paphazy, M.J., Haygarth, P.M., Mckelvie, I.D., 2002. Inositol phosphates in the environment. Philosophical Transactions: Biological Sciences 357, 449e469. Uren, S.C., 1992. The Effects of Wet and Dry Deposited Ammonia on Calluna vulgaris. PhD thesis, Imperial College, University of London, UK. van Elsas, J.D., Duarte, G.F., Keijzer-Wolters, A., Smit, E., 2000. Analysis of the dynamics of fungal communities in soil via fungal-specic PCR of soil DNA followed by denaturing gradient gel electrophoresis. Journal of Microbiological Methods 43, 133e151. Van Vuuren, M.M.I., Van Der Eerden, L.J., 1992. Effects of three rates of atmospheric nitrogen deposition enriched with nitrogen-15 on litter decomposition in a heathland. Soil Biology and Biochemistry 24, 527e532. Vitousek, P.M., 1997. Human domination of Earths ecosystems. Science 278, 21.

1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildre?, Soil Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025