Chronic nitrogen additions suppress decomposition and sequester soil carbon in temperate forests

Transcription

1 Biogeochemistry (214) 121: DOI 1.17/s BIOGEOCHEMISTRY LETTERS Chronic nitrogen dditions suppress decomposition nd sequester soil cron in temperte forests S. D. Frey S. Ollinger K. Ndelhoffer R. Bowden E. Brzostek A. Burton B. A. Cldwell S. Crow C. L. Goodle A. S. Grndy A. Finzi M. G. Krmer K. Ljth J. LeMoine M. Mrtin W. H. McDowell R. Minoch J. J. Sdowsky P. H. Templer K. Wickings Received: 4 Ferury 214 / Accepted: 6 June 214 / Pulished online: 1 July 214 Ó Springer Interntionl Pulishing Switzerlnd 214 Astrct The terrestril iosphere sequesters up to third of nnul nthropogenic cron dioxide emissions, offsetting sustntil portion of greenhouse gs forcing of the climte system. Although numer of fctors re responsile for this terrestril cron sink, tmospheric nitrogen deposition contriutes y enhncing tree productivity nd promoting cron storge in tree iomss. Forest soils lso represent n importnt, ut understudied cron sink. Here, we exmine the contriution of trees versus soil to totl ecosystem cron storge in temperte forest nd Electronic supplementry mteril The online version of this rticle (doi:1.17/s ) contins supplementry mteril, which is ville to uthorized users. Responsile Editor: Mtthew Wllenstein S. D. Frey (&) S. Ollinger A. S. Grndy W. H. McDowell J. J. Sdowsky K. Wickings Deprtment of Nturl Resources nd the Environment, University of New Hmpshire, Durhm, NH 3824, USA e-mil: serit.frey@unh.edu S. Ollinger M. Mrtin Erth Systems Reserch Center, University of New Hmpshire, Durhm, NH 3824, USA K. Ndelhoffer J. LeMoine Deprtment of Ecology nd Evolutionry Biology, University of Michign, Ann Aror, MI 4819, USA R. Bowden Deprtment of Environmentl Science, Allegheny College, Medville, PA 16335, USA investigte the mechnisms y which soils ccumulte cron in response to two decdes of elevted nitrogen inputs. We find tht nitrogen-induced soil cron ccumultion is of equl or greter mgnitude to cron stored in trees, with the degree of response eing dependent on stnd type (hrdwood versus pine) nd level of N ddition. Nitrogen enrichment resulted in shift in orgnic mtter chemistry nd the microil community such tht unfertilized soils hd higher reltive undnce of fungi nd lipid, phenolic, nd N-ering compounds; wheres, N-mended plots were ssocited with reduced fungl iomss nd ctivity nd higher rtes of lignin ccumultion. We conclude tht soil cron ccumultion in response to N enrichment ws lrgely due to suppression of orgnic mtter decomposition rther thn enhnced cron inputs to soil vi litter fll nd root production. E. Brzostek Deprtment of Geogrphy, Indin University, Bloomington, IN 4745, USA A. Burton School of Forest Resources nd Environmentl Science, Michign Technologicl University, Houghton, MI 49931, USA B. A. Cldwell K. Ljth Deprtment of Forest Science, Oregon Stte University, Corvllis, OR 97331, USA S. Crow Deprtment of Nturl Resources nd Environmentl Mngement, University of Hwi i Māno, Honolulu, HI 96822, USA

2 36 Biogeochemistry (214) 121: Keywords Nitrogen deposition Soil cron Temperte forest Terrestril cron sink Introduction Of the estimted 8.7 Gt cron (C) relesed to the tmosphere nnully y humn ctivities (Boden et l. 212), only out hlf ccumultes, with the reminder eing sored y the ocens or tken up y the terrestril iosphere (Denmn et l. 27). The terrestril C sink represents s much s third of nnul CO 2 emissions (Myneni et l. 21; Deng nd Chen 211; Pn et l. 211), with disproportionte sink in temperte nd orel regions due to forest re-growth nd fire suppression in conjunction with CO 2 fertiliztion, climte wrming, lengthening growing sesons, nd tmospheric nitrogen (N) deposition (Schimel et l. 21). Nitrogen deposition in northern forests results in the indvertent fertiliztion of otherwise N-limited systems nd hs een estlished s key driver of net forest C storge (Sutton 28; de Vries et l. 29). Anthropogenic emissions of rective N to the tmosphere, primrily due to fossil fuel comustion nd griculture (Pinder et l. 212), nd the susequent deposition to Erth s surfce hve incresed nerly 2 % since the eginning of the industril revolution nd re projected to doule yet gin y 25 in mny prts of the world (Gllowy et l. 28). Nitrogen deposition in the sence of humn influence is typiclly\.5 kg N h -1 yer -1 ; however, in hevily populted nd industrilized regions, N deposition rtes rnge from 1 to 1 kg N h -1 yer -1 (Hyvönen et l. 27; de Vries et l. 29). C. L. Goodle Deprtment of Ecology nd Evolutionry Biology, Cornell University, Ithc, NY 14853, USA A. Finzi P. H. Templer Deprtment of Biology, Boston University, Boston, MA 2215, USA M. G. Krmer Soil nd Wter Science Deprtment, University of Florid, Ginesville, FL 32611, USA R. Minoch USDA Forest Service, Northern Reserch Sttion, Durhm, NH 3824, USA The C:N rtios of temperte forest woody iomss re high, such tht lrge mounts of C cn e fixed per unit N tken up y plnts. Some tree species exhiit incresed mortlity with exposure to high levels of N fertiliztion (Thoms et l. 21); however, growth enhncement ppers to e the generl response of forests to N deposition (Pinder et l. 212). Estimtes of N-induced tree C sequestrtion vry widely, ut severl recent dt syntheses suggest the most ecologiclly plusile upper limit is out 6 kg C kg -1 N (Hyvönen et l. 27; de Vries et l. 29; Thoms et l. 21), with the C uptke rte for given site eing highly dependent on tree species (Thoms et l. 21). Forest soils, which retin lrger nd more persistent pool of C thn tht contined in vegettion, represent n dditionl nd potentilly importnt reservoir for enhnced terrestril ecosystem C storge in response to N deposition (Pregitzer et l. 28; Zk et l. 28; Nve et l. 29; Jnssens et l. 21; Lovett et l. 213). A review of the literture shows growing consensus tht forest soils represent n importnt sink for dded N, resulting in enhnced C storge eyond wht is stored in tree iomss (summrized in Tle 1). It might e expected tht enhnced tree growth would fcilitte soil orgnic mtter ccumultion through incresed plnt litter nd root inputs. However, previous reserch suggests tht suppression of orgnic mtter decy my e the primry mechnism y which soils ccumulte C in response to long-term N dditions (Pregitzer et l. 28; Zk et l. 28; Jnssens et l. 21; Zk et l. 211). Despite long history of reserch on soil responses to enhnced N vilility, there hve een reltively few efforts to comprehensively exmine the reltive contriution of trees versus soils to net ecosystem C storge in conjunction with the concomitnt chnges in soil iogeochemistry tht could provide cler explntion for why soils ccumulte C in response to dded N (ut see Pregitzer et l. 28; Zk et l. 28; 211). Soil C pools re still the lest certin term of forest C udgets (Goodle et l. 22), nd etter understnding of N-induced soil C storge is criticl to ccurtely predict the net response of terrestril ecosystems to N deposition. In this study, we used long-term tree inventory nd litterfll dt coupled with mesurements of n extensive suite of soil properties to ssess the reltive importnce of tree versus soil C ccumultion for totl ecosystem C storge following 2 yers of

3 Biogeochemistry (214) 121: Tle 1 Cron sequestrtion in temperte forests per unit nitrogen dded Study loction Study durtion (yer) Nitrogen inputs (kg h -1 yer -1 ) Cron response (kg C kg -1 N) Trees Soils Totl Reference N. Americ & Europe (9 sites) Ndelhoffer et l. (1999) Europe (121 forest plots) c de Vries et l. (26) Sweden (orel forest) Hogerg et l. (26) d Finlnd, Sweden (15 sites) Hyvönen et l. (28) Northern US (4 hrdwood sites) Pregitzer et l. (28) Europe (4 forest plots) e Solerg et l. (29); Luhnn et l. (29) North-centrl US (24 tree species) * Thoms et l. (21) Glol nlysis (2 experiments) f Jnssens et l. (21) Northestern US (5 tree species) g 14 Lovett et l. (213) Northest US (hrdwood stnd) This study Northest US (hrdwood stnd) This study Northest US (pine stnd) This study Northest US (pine stnd) This study These estimtes of soil C storge were sed on the orgnic horizon nd some mount of minerl soil, though the depth of minerl soil smpling vried widely cross studies. The following studies reported minerl soil smpling s eing to depth of 12 cm (Lovett et l. 213), 2 cm (Hyvönen et l. 28), 4 cm (our study; entire soil profile smpled), nd 7 cm (Pregitzer et l. 28). Hogerg et l. (26) report smpling the orgnic horizon nd 5 15 nd depth of minerl soil. The depth of minerl soil smpling ws typiclly not specified in those studies where estimtes of soil C storge were sed on synthesis or met-nlysis lrge numer of sites or experiments (Ndelhoffer et l. 1999; de Vries et l. 26; Jnssens et l. 21) Estimtes sed on the short-term fte of 15 N trcer recovered fter 1 3 yers following N ddition c Derived from forest growth over 4 yers (196 2) under mient N deposition conditions (verge of 2.8 kg N h -1 yer -1 dditionl N deposition since 196) d As estimted y de Vries et l. (29) e Derived from forest growth over 7 yers (1993 2) under mient N deposition conditions f Vlue estimted from the reduction in heterotrophic soil respirtion in response to N fertiliztion g Averge from ll experimentl plots representing five different tree species experimentl N dditions to two temperte forest stnds (mixed hrdwood nd red pine) in Centrl Msschusetts, USA. We lso exmined fctors underlying the oserved soil response y ssessing the reltive importnce of enhnced C inputs to soil vi litterfll nd roots versus reduced C losses due to suppression of soil orgnic mtter decomposition. Mterils nd methods Experimentl site Our reserch ws conducted t the Chronic Nitrogen Amendment Experiment t the Hrvrd Forest Long- Term Ecologicl Reserch (LTER) site in Petershm, MA, USA. This site ws estlished in 1988 in two djcent forest stnds, one dominted y mture (*6 yer-old), even-ged red pine (Pinus resinos), the other y lck (Quercus velutin) nd red (Q. rur) ok mixed with lck irch (Betul lent), red mple (Acer rurum) nd Americn eech (Fgus grndifoli) (Mgill nd Aer 1998; Mgill et l. 24). The soils re Typic Dystrudepts of the Gloucester series (Peterjohn et l. 1994) with no significnt vrition mong N tretments in ph or concentrtions of the se ctions C nd Mg (Turlpti et l. 213). Men monthly tempertures rnge from 19 C in July to -5 C in Jnury nd verge nnul precipittion is 112 cm, distriuted reltively evenly throughout the yer. The current rte of mient N deposition t the site is *8 kgnh -1 per yer -1 (Ollinger et l. 1993).

4 38 Biogeochemistry (214) 121: Three m plots in ech forest stnd receive one of three N tretments: no N ddition (N; control); 5 kg N h -1 yer -1 (N5), or 15 kg N h -1 yer -1 (N15). The N fertilized plots re treted with n queous solution of mmonium nitrte pplied monthly during the growing seson. Ech tretment plot is divided into thirty-six m suplots with the outer suplots excluded from smpling to reduce edge effects. The N5 level is consistent with predictions for N deposition rtes in lrge prts of the world y 25 (Gllowy et l. 28). Even currently, some res in Western Europe see rtes of N deposition (totl wet nd dry) s high s 25 5 kg N h -1 yer -1 (Hollnd et l. 24), nd this is likely the cse in prts of Asi s well. Thus the N5 tretment is relevnt simultion of current nd future N deposition levels nd is consistent with other long-term N ddition experiments in the Northestern U.S. (Lovett nd Goodle 211; Lovett et l. 213) nd elsewhere (Cusck et l. 211). The high ddition rte (N15) ws not intended to e predictive, ut to push the system towrd sturtion nd to e spce-for-time sustitution to evlute underlying mechnisms/drivers in high N world (J. Aer, personl communiction). Smple collection nd nlysis Long-term dt from the site were otined from the Hrvrd Forest dt rchive ( hrvrd.edu/dt-rchive) nd included tree inventory, folir chemistry, nd litterfll iomss. Tree iomss ws determined from dimeter t rest height (DBH) dt collected every 3 yers since 1988 on ll trees greter thn 5 cm dimeter (Mgill et l. 24). Folir litterfll ws collected three times nnully since 1988 ccording to Mgill et l. (24). Fine roots (\2 mm-dimeter) were hnd-picked from orgnic horizon nd minerl soil smples collected in 28, 21, nd 211, dried t 6 C, nd weighed. Soil smples were collected in 28 from ech of six m suplots within ech tretment plot to complement the long-term oveground dt nd, specificlly, to ssess the effects of 2 yers of N dditions on soil C stocks nd ssocited iologicl nd chemicl properties. Orgnic horizon smples were collected in ech suplot y removing cm squre of the Oe nd O lyers to the depth of the minerl soil. Minerl soil smples were then collected in the sme loction in 1 cm increments to depth of 4 cm (ottom of soil profile) using gs-powered soil uger with 9 cm dimeter core. Smples were sieved (\2 mm) nd roots, rocks nd other deris[2 mm removed. Totl soil orgnic C nd N were determined y dry comustion on Costech 41 CHNS-O Anlyticl Comustion System. Bulk density, determined for ech smple, ws used to clculte volumetric soil C stocks (g C m -2 ). In situ soil respirtion nd temperture were mesured nine times over the growing seson (Apr 19, Jun 5 nd 26, Jul 13 nd 27, Aug 26, Sep 16, Oct 12 nd 3) using portle infrred gs nlyzer. Microil iomss nd community composition were determined on orgnic horizon smples (Oe nd O lyers) using phospholipid ftty cid (PLFA) nlysis (Guckert et l. 1985). Microil ctivity ws ssessed for the orgnic horizon nd top 1 cm of minerl soil y mesuring potentil extrcellulr enzyme ctivity. Proteolytic enzyme ctivity ws determined s the difference in mino cid concentrtions in susmples incuted for 4 h nd initil susmples tht were terminted immeditely (Brzostek et l. 211). Both incuted nd initil susmples were mended with sodium cette uffer (ph 5.), toluene to inhiit microil uptke, nd trichlorocetic cid solution to terminte the ssy. Orgnic mtter chemistry ws ssessed for the orgnic horizon (hrdwood stnd only) y determining the reltive percentges nd rtios of chemicl clsses determined using pyrolysis-gc/ms (Wickings et l. 211). Smples were pyrolyzed t 6 C nd products trnsferred to GC where compounds were seprted on 6 m cpillry column with strting temperture of 4 C followed y temperture rmp of 5 C min -1 to 27 C followed y finl rmp (3 C min -1 )to 3 C. Compounds were trnsferred to n ion trp mss spectrometer where they were ionized, detected vi electron multiplier, nd identified using compound lirry from the Ntionl Institute of Stndrds nd Technology (NIST). The dt were nlyzed using rnk nlysis of vrince (PROC RANK, PROC GLM, SAS Institute) with N ddition tretment (control, N5, N15) s the min fctor. A non-prmetric procedure ws selected ecuse the dt violted the ssumptions of normlity nd homogeneity of vrince for stndrd nlysis of vrince. The Ryn Einot Griel Welsch multiple rnge test ws used to determine significnt differences mong mens t P \.5. Seprte nlyses

5 Biogeochemistry (214) 121: Cron stocks (kg m -2 ) Cron stocks (kg m -2 ) Wood Litter were performed for ech stnd (hrdwood versus pine) nd soil horizon (orgnic versus minerl soil). In situ soil respirtion, mesured nine times over the growing seson, ws plotted s function of soil temperture, nd dummy vrile regression nlysis with slope nd intercept interction terms ws used to determine significnt differences mong tretments (P \.5). Nonmetric multidimensionl scling (NMDS) ws used to visulize differences in microil community composition nd soil orgnic mtter chemistry mong N ddition tretments. Results nd discussion N Addition (kg N h -1 yr -1 ) Fine roots Minerl soil Orgnic horizon Fig. 1 Totl C (kg m -2 ) stored in tree iomss, lef litter, fine roots (\2 mm), orgnic horizon, nd the minerl soil to depth of 4 cm in control nd N fertilized plots in the hrdwood () nd pine () stnds t the Chronic Nitrogen Amendment Experiment t the Hrvrd Forest Long-term Ecologicl Reserch (LTER) site. Vlues ove the rs indicte totl ecosystem C storge, ssuming 5 % C content for plnt components. Refer to Tle 2 nd Fig. 2 for within-tretment vriility for plnt nd soil C stocks, respectively Twenty yers of N fertiliztion resulted in significnt increse (38 %) to net loss (-5 %) of totl ecosystem C storge, with the response eing dependent on stnd type (hrdwood versus pine) nd N fertiliztion level (Fig. 1). The hrdwood stnd ccumulted 2 3 kg C kg -1 N dded over the 2 yer study (Tle 1), with more thn hlf (6 85 %) of this C storge due to n ccumultion of soil C (Fig. 2). At the lower N ddition rte (5 kg N h -1 yer -1 ), tree iomss in the hrdwood stnd incresed y 1 % compred to 33 % increse for soil C stored in the orgnic horizon (Fig. 2, Supplementl Tle S1). At the highest N ddition rte (15 kg N h -1 yer -1 ), hrdwood trees showed little dditionl response, incresing their totl iomss y only 3.7 % ove the lower N tretment. Soil C levels responded to much greter degree thn did trees in the higher N tretment, with soil C in the orgnic horizon incresing y 8 % over the control tretment. Additionlly, soil C stocks in the minerl soil (to 4 cm depth) significntly incresed under the highest N ddition rte, resulting in totl soil C ccumultion (orgnic horizon plus minerl soil) for this tretment of 25 kg C kg -1 N dded. The proportion of light frction (free prticulte) orgnic mtter in the top minerl soil lyer ( 1 cm) did not increse with N dditions (Supplementry Tle S2) nd is consistent with the lck of response of fine root iomss to N ddition (see elow), suggesting tht chemicl ssocition with minerl prticles ws the dominnt nd more persistent mechnism for the enhnced C storge t depth in the highest N tretment (s opposed to n increse in prticulte orgnic mtter). Increses in soil C in the pine stnd were lrgely offset y losses of tree iomss due to mortlity. We oserved n 8 16 % loss of totl stnding tree iomss nd n increse of 5 % in soil C in the orgnic horizon, resulting in slight net gin (1.4 %) to smll net loss (-5 %) t the lower versus higher N ddition rte, respectively. The negtive tree response oserved for pine is consistent with recent mesurements of forest inventory dt showing tht, of the 24 most common tree species in the Northest US, P. resinos (red pine) hd the lrgest negtive growth response to N deposition (Thoms et l. 21). In contrst to the hrdwood stnd, soil C ccumultion in the pine stnd ws only oserved in the orgnic horizon with no significnt chnge in minerl soil C levels. These C stock results demonstrte tht the soils t our site represent C sink of equl or greter mgnitude to tht of woody tree iomss following

6 31 Biogeochemistry (214) 121: two decdes of N enrichment. This finding is supported y 15 N trcer study t this site showing tht tree iomss ws reltively smll sink for dded 15 N, with most of the 15 N eing retined in the forest floor nd to lesser extent in the top 2 cm of minerl soil (Ndelhoffer et l. 1999, 24; Templer et l. 212). A review of pulished studies, where oth the tree nd soil response to mient or simulted N deposition in temperte forests hs een evluted (Tle 1), shows tht N-induced soil C ccumultion vries widely, rnging from 3 to 25 kg C kg -1 N dded nd representing 1 1 % of the totl C ccumultion oserved (trees? soils). The mount of soil C sequestered in response to N deposition represents the lnce etween C inputs (vi litterfll nd root iomss turnover/exudtion) nd C losses from litter nd soil orgnic mtter decomposition. Cron losses due to leching from the minerl soil re smll t our site (Currie et l. 1996; Evns et l. 28) nd erosionl losses re likely miniml. To ssess the reltive contriution of soil C inputs to the oserved soil C ccumultion t Hrvrd Forest, we nlyzed litterfll dt collected nnully over the 2 yer study nd exmined root dynmics in response to the long-term N dditions. Cumultive litterfll over the study durtion in the hrdwood stnd ws only 3 % higher in the N ddition plots thn in the control, nd verge nnul litterfll ws not significntly different mong tretments (Tle 2). Cumultive nd nnul litterfll in the pine stnd incresed significntly in response to N dditions (18 %), primrily due to high tree mortlity nd reduced needle longevity (Buer et l. 24). Fine root iomss (to 2 cm depth) ws not significntly different mong tretments in the hrdwood stnd (Tle 2), consistent with erlier estimtes of root iomss t this site (Mgill et l. 1997, 24). Nitrogen dditions in the pine stnd resulted in significnt reduction in root iomss t ll depths mesured. Root ctivity, ssessed y mesuring root respirtion, ws not higher in the N-mended plots (hrdwood or pine), nd root tissue N concentrtion ws generlly wek predictor of root respirtion in N-enriched soils (Supplementry Tle S3 nd Fig. S1). We lso did not oserve ny significnt differences in root production nd specific root length (Supplementry Tle S4). Thus, our reserch does not provide strong evidence for enhncement of root productivity or root respirtion in response to N dditions, finding similr to tht Soil C stocks (kg m -2 ) Soil C stocks (kg m -2 ) Orgnic horizon Minerl soil 1 g C / kg N* 25 g C / kg N** g C / kg N* 5g C / kg N* 5 15 Nitrogen ddition (kg h -1 yr -1 ) Fig. 2 Totl orgnic soil C (kg m -2 ) in the orgnic horizon (Oe? O) nd minerl soil to depth of 4 cm in the hrdwood () nd pine () stnds t the Chronic Nitrogen Amendment Experiment t the Hrvrd Forest LTER. Error rs represent ± one stndrd error for ech soil horizon. Different lower cse letters within soil horizon nd cross N tretments represent significnt difference t P B.5. Vlues ove the rs represent the mount of C ccumulted per unit of N dded over the 2 yer study. An * indictes significnt ccumultion of C with N ddition in the orgnic horizon only, while ** indictes significnt ccumultion in oth the orgnic nd minerl soil horizons (P B.5). The primry dt used for this nlysis re provided in Supplementry Tle S1 oserved t long-term N ddition study in Michign, USA (Pregitzer et l. 28; Zk et l. 28; Burton et l. 212) nd to results from glol met-nlysis demonstrting tht long-term N dditions did not increse fine root inputs to soil t orel, temperte, nd tropicl forest sites (Liu nd Grever 21). It hs een suggested tht the N-induced enhncement of oveground tree iomss my e due to shift in C lloction rther thn n increse in C ssimiltion, hypothesis tht is supported y studies showing tht N dditions often do not enhnce tree photosynthetic rtes or N use efficiency (Tlhelm et l. 211; Smithwick et l. 213). To summrize, our mesurements show tht N dditions did not enhnce C inputs

7 Biogeochemistry (214) 121: Tle 2 Totl nd live tree iomss, nnul litterfll mss, fine root mss, nd soil microil iomss for control nd nitrogenmended plots t the Chronic Nitrogen Amendment Experiment t the Hrvrd Forest Long-Term Ecologicl (LTER) site Ecosystem component Hrdwood Pine Nitrogen ddition (kg N h -1 yer -1 ) Nitrogen ddition (kg N h -1 yer -1 ) Vegettion (g m -2 ) Totl tree iomss* 2,218 22,262 23,87 24,735 22,657 2,694 Live tree iomss* 17,529 18,961 18,15 2,721 18,23 5,577 Annul litterfll 35 (6) 317 (1) 321 (12) 316 (14) 376 (19) 374 (26) Fine roots O horizon 261 (43) 363 (4) 39 (84) 127 (31) 82 (17) 41 (18) 1 cm minerl 227 (77) 23 (25) 295 (162) 386 (145) 69 (15) ND 1 2 cm minerl 11 (17) 1 (13) 168 (63) 82 (17) 65 (17) ND Microil iomss (nmol g -1 ) Bcteri Oe horizon 274 (31) 36 (55) 148 (23) 329 (16) 153 (19) 19 (13) O horizon 85 () 75 (16) 42 (17) 16 (3) 13 (29) 15 (3) Fungi Oe horizon 22 (23) 73 (8) 31 (9) 115 (28) 44 (8) 39 (5) O horizon 32 (2) 2 (3) 1 (5) 22 (6) 26 (8) \1 Vlues in prentheses re stndrd errors Vlues followed y different lowercse letter re significntly different t P B.5 ND no dt * Estimted for ech m plot sed on dimeter t rest height (DBH) dt collected every 3 yers since 1988 on ll trees greter thn 5 cm dimeter s descried y Mgill et l. (24) Fig. 3 The temperture response of in situ soil respirtion cross N ddition tretments in the hrdwood () nd pine () stnds Soil respirtion (µmol m -2 s -1 ) Control N5 N Soil temperture ( C) Soil temperture ( C) (oveground litterfll, root iomss, root production, nd root respirtion) to the hrdwood stnd. Nitrogen dditions did induce significntly higher litterfll in the pine stnd; however, this ws not due to enhnced tree productivity, ut rther to reduced tree vigor tht cused needles to drop nd which lso resulted in reduced root iomss. We conclude, sed on these results, tht n N-induced enhncement of plnt C inputs is not the primry mechnism underlying the ccumultion of soil C in our system. In contrst, we hve evidence tht the oserved C ccumultion is due primrily to suppression of orgnic mtter decomposition. In situ soil respirtion vried y stnd nd N tretment level, ut ws

8 312 Biogeochemistry (214) 121: Proteolysis (µg g -1 soil hr -1 ) Proteolysis (µg g -1 soil hr -1 ) Nitrogen ddition (kg h -1 yr -1 ) c Fig. 4 Microil ctivity s mesured y proteolytic enzyme ctivity (lg g -1 soil hr -1 ) for the orgnic horizon (Oe nd O lyers) nd the top 1 cm of minerl soil collected in the hrdwood () nd pine () stnds. Brs represent mens nd stndrd errors for ech soil horizon cross N ddition tretments. Different lower cse letters within soil horizon nd cross N tretments represent significnt difference t P B.5. Microil community composition of the orgnic horizon (c) ws ssessed y phospholipid ftty cid (PLFA) nlysis nd visulized using NMDS ordintion using the individul PLFA iomrkers listed in Supplementl Tles S9 nd S1. Arrows denote correltions etween ordintion scores nd individul microil mrkers consistently lower in the N ddition plots (Fig. 3). Using the tretment specific reltionship etween soil temperture nd respirtion, we estimte tht soil respirtion in the N-mended plots ws 7 22 % lower in the hrdwood stnd (P =.96) nd 1 16 % lower in the pine stnd (P =.54), results consistent with previous soil respirtion mesurements in these plots (Bowden et l. 24). Although our field soil respirtion mesurements included oth utotrophic nd heterotrophic respirtion components, the lck of difference in totl root iomss, root production, nd specific root respirtion in the hrdwood stnd suggests tht utotrophic respirtion ws lrgely unchnged y N dditions in this stnd nd tht the decline in totl respirtion in response to N fertiliztion ws the result of reduced heterotrophic respirtory flux. L incutions of root-free soils collected from these plots showed tht N ddition gretly suppressed heterotrophic respirtion (M. Weiss, unpulished dt), further supporting this conclusion. It is more difficult to ccurtely ssess the reltive contriution of utotrophic versus heterotrophic respirtion in the pine stnd where incresed tree mortlity hs resulted in n increse in needle inputs to the forest floor tht hve een offset y reduction in root iomss nd root respirtion. The suppression of in situ soil respirtion with N dditions coincided with significnt chnges in microil iomss, enzyme ctivity, nd community composition. Specificlly, N enrichment significntly lowered cteril nd fungl iomss (46 86 %; Tle 2) nd proteolytic enzyme ctivity (47 6 %; Fig. 4, ). The microil communities in the hrdwood nd pine stnds were distinct, with phospholipid ftty cid (PLFA) iomrkers from the hrdwood plots ordinting primrily long xis one nd the pine plots seprting to lesser degree long xis 2 of nonmetric multidimensionl scling (NMDS) ordintion (Fig. 4c). Nitrogen ddition in the hrdwood stnd ws positively ssocited with the cteril mrkers i15:, cy17:, 1me16:, nd 18:1x7c, nd negtively ssocited with the cteril mrker i17: nd the fungl mrker 18:1x9c. Oxidtive enzyme ctivity, mesured in the hrdwood stnd only, ws lso

9 Biogeochemistry (214) 121: significntly reduced y N ddition (42 87 %; Supplementry Tle S5). Previous work t our site hs demonstrted tht N dditions hve resulted in reduced rtes of lef litter decy (Mgill nd Aer 1998; Knorr et l. 25), microil growth (Kmle et l. 213), nd the ility of the microil community to utilize suite of C sustrtes typiclly found in soil (Frey et l. 24). Nitrogen dditions hve lso resulted in microil community whose growth is sustntilly limited y the vilility of redily ssimilted C sustrtes (Kmle et l. 213). Recent moleculr nlyses of the soil microil community t our site hve indicted significnt N-induced shifts in the composition nd diversity of oth the cteril (Turlpti et l. 213) nd fungl communities (E. Morrison nd S.D. Frey, unpul. dt). The lrgest N-induced responses in the microil community were oserved in the orgnic horizon of the hrdwood stnd where the gretest ccumultion of C occurred nd where mrked chnges in orgnic mtter chemistry were lso oserved (Fig. 5, ; Supplementry Tles S6 S8; Fig. S5). Nitrogen fertiliztion induced significnt reduction in the reltive undnce of lipids (53 57 %) nd N-ering compounds (19 33 %), wter-solule dissolved orgnic C (4 7 %; Supplementry Tle S7), nd polymine nd mino cid concentrtions (5 %; Supplementry Fig. S4). In contrst, the reltive undnce of lignin nd the lignin:phenol rtio were significntly higher under N ddition (Fig. 5). The lignin:phenol rtio represents n index of reclcitrnt orgnic C decomposition, nd significnt increse in this rtio indictes tht reltively more C ws present s unltered lignin in the fertilized plots, with less lignin trnsformtion resulting in lower liertion of phenolic compounds, of which lignin decy is mjor source. The iomss of soil fungi, the primry decomposers of lignin in temperte forests, ws positively correlted with the reltive undnce of lipids (r =.81; P =.21), phenols (r =.83; P =.6), nd N-ering compounds (r =.69; P =.39) nd negtively correlted with the lignin:phenol rtio (r =-.74; P =.23; Fig. 5c). To summrize, N enrichment hs resulted in shift in microil community composition nd orgnic mtter chemistry such tht the orgnic horizon of unfertilized soils hs greter reltive undnce of fungi nd Lignin:phenol c Lignin:phenol N Addition (kg N h -1 yr -1 ) R² = Fungl iomss (nmol g -1 ) Fig. 5 Soil chemistry nd microil community responses to N ddition in the orgnic horizon of the hrdwood stnd. Nonmetric multidimensionl scling (NMDS) ordintion of chemicl clsses determined using pyrolysis-gc/ms (finl stress =.485). Arrows denote significnt correltions (Person) etween ordintion scores nd orgnic mtter chemistry clsses or microil iomrkers (P \.5). Open tringles represent control plots nd closed tringles the N5 (grey symols) nd N15 tretment (lck symols), respectively. the lignin:phenol rtio s function of N ddition rte. Brs represent mens nd stndrd errors. Different lower cse letters cross N tretments represent significnt difference t P B.5. c the reltionship etween the soil fungl iomss nd lignin:phenol rtio. Refer to the Supplementl Mterils for the primry chemicl clss (Tle S6) nd microil iomrker dt (Tle S9)

10 314 Biogeochemistry (214) 121: lipid, phenolic, nd N-ering compounds; wheres, the N-mended plots re ssocited with reduced fungl iomss nd lignin ccumultion. The shift to greter dominnce y cteri in the N mended plots my explin the lrger mount of NO 3 - in soil solution ttriuted to nitrifiction, rther thn N pssing directly through from tmospheric deposition or fertilizer N inputs (Supplementry Fig. S4). Our results, sed on two decdes of experimentl N dditions, document tht long-term N fertiliztion of temperte forest hs resulted in n % increse in ecosystem C storge in hrdwood stnd, representing n ccumultion of 2 3 kg C kg -1 N dded. More thn hlf of this C storge ws ttriutle to n ccumultion of soil orgnic mtter, indicting tht the soil hs een more responsive to N dditions thn hs tree growth. In the pine stnd, loss of tree iomss due to high tree mortlity ws offset y significnt increse in orgnic horizon C, resulting in no net chnge in ecosystem C lnce. Soil C ccumultion in oth forest stnds, primrily in the orgnic horizon, ppers to e lrgely the result of suppressed microilly driven decomposition, rther thn to incresed litter or root inputs. A recent modeling exercise imed t simulting the effects of N dditions on soil C stocks t our site demonstrted tht model outcomes most closely mtched our oserved C stock vlues when orgnic mtter decomposition ws ggressively suppressed (Tonitto et l. 213), consistent with our experimentl results. Soil C responses to long-term N dditions hve yet to e incorported into glol-scle C lnce models (Whittinghill et l. 212), ut this is needed to ccurtely simulte future chnges in terrestril C storge in response to tmospheric N deposition (Thoms et l. 213, ). Acknowledgments We thnk Michelle Dy, Stephnie Juice, Meliss Knorr, Rich McLen, April Melvin, nd Mriss Weiss for ssistnce with smple collection nd nlysis. This work ws supported y the Ntionl Science Foundtion Longterm Ecologicl Reserch (LTER) Progrm. References Buer GA, Bzzz FA, Minoch R, Long S, Mgill A, Aer J, Berntson GM (24) Effects of chronic N dditions on tissue chemistry, photosynthetic cpcity, nd cron sequestrtion potentil of red pine (Pinus resinos Ait.) stnd in the NE United Sttes. For Ecol Mng 196: Boden TA, Mrlnd G, Andres RJ (212) Glol, regionl, nd ntionl fossil-fuel CO 2 emissions. Cron Dioxide Informtion Anlysis Center, Ok Ridge Ntionl Lortory, US Deprtment of Energy, Ok Ridge. doi:1.3334/ CDIAC/1_V212 Bowden RD, Dvidson E, Svge K, Ari C, Steudler P (24) Chronic nitrogen dditions reduce totl soil respirtion nd microil respirtion in temperte forest soils t the Hrvrd Forest. For Ecol Mng 196:43 56 Brzostek ER, Edwrd R, Finzi AC (211) Sustrte supply, fine roots, nd temperture control proteolytic enzyme ctivity in temperte forest soils. Ecology 92: Burton AJ, Jrvey JC, Jrvi MP, Zk DR, Pregitzer KS (212) Chronic N deposition lters root respirtion-tissue N reltionship in northern hrdwood forests. Glo Chng Biol 18: Currie WS, Aer JD, McDowell WH, Boone RD, Mgill AH (1996) Verticl trnsport of dissolved orgnic C nd N under long-term N mendments in pine nd hrdwood forests. Biogeochemistry 35: Cusck DF, Silver WL, Torn MS, McDowell WH (211) Effects of nitrogen dditions on ove- nd elowground cron dynmics in two tropicl forests. Biogeochemistry 14: de Vries W, Reinds GJ, Gundersen P, Ster H (26) The impct of nitrogen deposition on cron sequestrtion in Europen forests nd forest soils. Glo Chng Biol 12: de Vries W, Solerg S, Doertin M, Ster H, Luhnn D, vn Oijen M, Evns C, Gundersen P, Kros J, Wmelink GWW, Reinds GJ, Sutton MA (29) The impct of nitrogen deposition on cron sequestrtion y Europen forests nd hethlnds. For Ecol Mng 258: Deng F, Chen JM (211) Recent glol CO 2 flux inferred from tmospheric CO 2 oservtions nd its regionl nlyses. Biogeosciences 8: Denmn KL et l (27) In: Solomon S et l (eds) Climte chnge 27: the physicl science sis. Contriution of working group I to the fourth ssessment report of the intergovernmentl pnel on climte chnge. Cmridge University Press, Cmridge, pp Evns CD, Goodle CL, Cporn SJM, Dise NB, Emmett BA, Fernndez IJ, Field CD, Findly SEG, Lovett GM, Meesenurg H, Moldn F, Shepprd LJ (28) Does elevted nitrogen deposition or ecosystem recovery from cidifiction drive incresed dissolved orgnic cron loss from uplnd soil? A review of evidence from field nitrogen ddition experiments. Biogeochemistry 91:13 35 Frey SD, Knorr M, Prrent JL, Simpson RT (24) Chronic nitrogen enrichment ffects the structure nd function of the soil microil community in temperte hrdwood nd pine forests. For Ecol Mng 196: Gllowy JN, Townsend AR, Erismn JW, Bekund M, Ci Z, Freney JR, Mrtinelli LA, Seitzinger SP, Sutton MA (28) Trnsformtion of the nitrogen cycle: recent trends, questions, nd potentil solutions. Science 32: Goodle CL, Apps MJ, Birdsey RA, Field CB, Heth LS, Houghton RA, Jenkins JC, Kohlmier GH, Kurz W, Liu S, Nuurs GJ, Nilsson S, Shvidenko AZ (22) Forest

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