Glob Change Biol. 2019;00:1–11. wileyonlinelibrary.com/journal/gcb | 1© 2019 John Wiley & Sons Ltd
Received:29May2019 | Accepted:15August2019DOI: 10.1111/gcb.14823
P R I M A R Y R E S E A R C H A R T I C L E
Climate warming alters subsoil but not topsoil carbon dynamics in alpine grassland
Juan Jia1,2 | Zhenjiao Cao1,2 | Chengzhu Liu1,2 | Zhenhua Zhang3 | Li Lin4 | Yiyun Wang1,2 | Negar Haghipour5 | Lukas Wacker6 | Hongyan Bao7 | Thorston Dittmar8 | Myrna J. Simpson9 | Huan Yang10 | Thomas W. Crowther11 | Timothy I. Eglinton5 | Jin‐Sheng He4,12 | Xiaojuan Feng1,2
1StateKeyLaboratoryofVegetationandEnvironmentalChange,InstituteofBotany,ChineseAcademyofSciences,Beijing,China2CollegeofResourcesandEnvironment,UniversityofChineseAcademyofSciences,Beijing,China3KeyLaboratoryofAdaptationandEvolutionofPlateauBiota,NorthwestInstituteofPlateauBiology,ChineseAcademyofSciences,Xining,China4InstituteofEcology,CollegeofUrbanandEnvironmentalSciences,PekingUniversity,Beijing,China5GeologicalInstitute,ETHZürich,Zurich,Switzerland6LaboratoryofIonBeamPhysics,DepartmentofPhysics,ETHZürich,Zurich,Switzerland7StateKeyLaboratoryofMarineEnvironmentalScience,XiamenUniversity,Xiamen,China8ResearchGroupforMarineGeochemistry,InstituteforChemistryandBiologyoftheMarineEnvironment,CarlvonOssietzkyUniversityofOldenburg,Oldenburg,Germany9EnvironmentalNMRCentre,DepartmentofPhysicalandEnvironmentalSciences,UniversityofTorontoScarborough,Toronto,ON,Canada10StateKeyLaboratoryofBiogeologyandEnvironmentalGeology,ChinaUniversityofGeosciences,Wuhan,China11InstituteofIntegrativeBiology,ETHZürich,Zurich,Switzerland12StateKeyLaboratoryofGrasslandAgro‐ecosystems,CollegeofPastoralAgricultureScienceandTechnology,LanzhouUniversity,Lanzhou,China
CorrespondenceXiaojuanFeng,StateKeyLaboratoryofVegetationandEnvironmentalChange,InstituteofBotany,ChineseAcademyofSciences,Beijing100093,China.Email:[email protected]
Jin‐ShengHe,StateKeyLaboratoryofGrasslandAgro‐ecosystems,CollegeofPastoralAgricultureScienceandTechnology,LanzhouUniversity,Lanzhou,China.Email:[email protected]
Funding informationChineseNationalKeyDevelopmentProgramforBasicResearch,Grant/AwardNumber:2015CB954201and2017YFC0503902;InternationalPartnershipProgramofChineseAcademyofSciences,Grant/AwardNumber:151111KYSB20160014;NationalNaturalScienceFoundationofChina,Grant/AwardNumber:31370491,31630009,41422304 and 41773067
AbstractSubsoilcontainsmorethanhalfofsoilorganiccarbon(SOC)globallyandisconventionallyassumedtoberelativelyunresponsivetowarmingcomparedtothetopsoil.Here,weshowsubstantialchangesincarbonallocationanddynamicsofthesubsoilbutnottopsoilintheQinghai‐Tibetanalpinegrasslandsover5yearsofwarming.Specifically,warmingenhancedtheaccumulationofnewlysynthesized(14C‐enriched)carboninthesubsoilslow‐cyclingpool(silt‐clayfraction)butpromotedthedecompositionofplant‐derivedlignininthefast‐cyclingpool(macroaggregates).Thesechangesmirroredanaccumulationoflipidsandsug‐arsattheexpenseoflignininthewarmedbulksubsoil,likelyassociatedwithshortenedsoilfreezingperiodandadeepeningrootsystem.Aswarmingisaccompaniedbydeep‐eningrootsinawiderangeofecosystems,root‐drivenaccrualofslow‐cyclingpoolmayrepresentanimportantandoverlookedmechanismforapotentiallong‐termcarbonsinkatdepth.Moreover,giventhecontrastingsensitivityofSOCdynamicsatvarieddepths,warmingstudiesfocusingonlyonsurfacesoilsmayvastlymisrepresentshiftsinecosystemcarbonstorageunderclimatechange.
K E Y W O R D S
deepsoil,lignindecomposition,physicalfraction,radiocarbon,soilorganiccarbon,warming
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1 | INTRODUC TION
Theresponseofsoilorganiccarbon(SOC)cyclingtoglobalwarmingrepresents a critical feedback of terrestrial ecosystems to climatechange(Koven,Hugelius,Lawrence,&Wieder,2017;Melilloetal.,2017).Untilnow,mostresearchhasfocusedontopsoilcarbondy‐namics(Crowtheretal.,2016;Kovenetal.,2017).Incontrast,sub‐soil (i.e., residing>20cmbelowground)containingmorethanhalfofglobalSOCstocks(Rumpel,Chabbi,&Marschner,2012)remainspoorlyinvestigatedintermsofitsresponsetowarming.Thisknowl‐edge gap has emerged as one of the central uncertainties in ourunderstanding of terrestrial carbon storage under climate change(Ahrens&Reichstein,2017).
Subsoil carbon is conventionally assumed to be relatively sta‐bleandunresponsivetoairwarmingdueto its longturnovertimeand good insulation at depth (Harrison, Footen, & Strahm, 2011).However, according to the Intergovernmental Panel on ClimateChange(IPCC),subsoilsareprojectedtowarmatroughlythesamerateas surfacesoilsover thenextcentury (HicksPries,Castanha,Porras,&Torn,2017;IPCC,2013).Moreimportantly,emergingevi‐dencesuggeststhatsubsoils,havingvariedorganicmattersources,microbial communities, and substrate availability compared to thetopsoil (Rumpel et al., 2012), may show even stronger responseto warming‐induced shifts in microbial activities and functioning(Fontaineetal.,2007) inassociationwithalteredplantcommunitystructureanddistribution(Keuperetal.,2017;Liuetal.,2018).Forinstance,plantrootsareexpectedtogrowdeeperunderwarming‐inducedmoistureornutrientlimitation(Johnson,Rygiewicz,Tingey,&Phillips,2006;Liuetal.,2018;Wangetal.,2017),potentiallyin‐creasingrootinputs(includingrootlitterandexudates)andenhanc‐ingcarbonaccumulation in subsoils thatare relatively low inSOCcontent(Cotrufoetal.,2015).Alternatively,increasedfreshcarboninputand/orchangingsoilphysicalpropertiesmayleadtoshiftsinmicrobialcommunities(Chengetal.,2017)andacceleratethedegra‐dationofnativeSOC(Fontaineetal.,2007;Jiaetal.,2017).However,experimentalevidenceislackingtodemonstratehowtheabovepro‐cessesjointlyaffectSOCdynamicsinthesubsoil.Detectingchangesin subsoil carbon stock is particularlydifficult given the long resi‐dencetimeofSOC(Rumpeletal.,2012)andcounteractingeffectsofwarmingonvariouscarbonpools(Feng,Simpson,Wilson,Williams,&Simpson,2008).Hence,in‐depthinvestigationsintoSOCcompo‐sition and carbon allocation are essential to reveal changing SOCdynamicsandtoidentifySOCpoolsthataresensitivetowarming.
Deep soil carbon dynamics in high‐latitude and high‐altitudeecosystems warrant particular attention under warming becausethesesoilsstorethevastmajorityoftheglobalSOCpool(Tarnocaietal.,2009;Yangetal.,2008).Inaddition,thesecolderregionsareexperiencingahigher‐than‐averagewarming trend in recent years(Chen et al., 2013). On the world's highest and largest plateau,Qinghai‐TibetanPlateau(QTP),anaturalwarmingtrendinthepastdecadehasledtoasignificantincreaseofSOCinthesubsurfacesoil(10–30cm)whileshowingnoeffectonthetopmostsoil(0–10cm)in the alpine grasslands (Ding et al., 2017). These results stand in
contrastwithpreviousviewsthatwarmingmayinducelargecarbonlossesfromsoilswithahighSOCcontent(Crowtheretal.,2016),es‐peciallyinthehigh‐latitudeandhigh‐altitudeareas.IdentifyingthemechanismsgoverningthesedivergentresponsesofSOCtowarm‐ingatdifferentdepths is critical tominimizeuncertainty in futuresoilcarbonfeedbackprojections.
Here,weutilizeamanipulativesoilwarmingexperimentintheQTPalpinegrassland(Liuetal.,2018)tocomparewarmingeffectsonthecompositionandsourcingofSOCpoolsatdifferentdepths.Consistent with field observations over QTP alpine grasslandsfor the past decades, 5 years of continuous warming increasedgrasseswithdeeprootsanddecreasedsedgesandforbswithshal‐low roots in our experiment, resulting in elevated belowgroundnet primary productivity (BNPP) in the subsoil (30–50 cm; Liuetal.,2018).Comparedtoabovegroundbiomass(59.7g/m2),rootsaretheprimarycarboninputstotheQTPgrasslandsoils(330.5g/m2; Yang,Fang,Ji,&Han,2009).WithanenhancedBNPPinthesub‐soil,we anticipate a strongerwarming impacton subsoil carbondynamicscomparedtothetopsoil(0–10cm)intermsofnewcar‐bon accrual and labile‐carbon‐stimulated degradation of nativeSOC.Furthermore,thealteredplantcommunitycomposition(Liuetal.,2018)mayhavecascadingeffectsonthequantityandqual‐ityofplant‐derivedcarboninputsintosoilsandconsequentlythechemicalcompositionofsoilorganicmatter.Torevealthedetailedmechanisms,weuseradiocarbonanalysiscoupledwithsoil frac‐tionationtodisentangletheallocationofnewcarbonamongSOCpools of varied turnover times. Complementary molecular‐levelanalyses,thatis,biomarkers,nuclearmagneticresonance(NMR),and high‐resolution mass spectrometry, are further employedto investigate the fate and degradation of various plant‐ andmicrobial‐derived components in the soil. These state‐of‐the‐artanalysesofSOCcomponents,complementedbyinsitumonitoringofecosystemcarbonfluxes,helptodeliveradetailedunderstand‐ingofhowsoilcarbondynamicsrespondtowarminginthisalpinegrassland.
2 | MATERIAL S AND METHODS
2.1 | Experiment design and soil sampling
The field warming experiment is located at the Haibei AlpineGrasslandEcosystemResearchStation(101°19′E,37°36′N,3,215ma.s.l.).Thesitehasacontinentalmonsoonclimatewithameanannualtemperatureof−1.2°Candameanannualprecipitationof489mm.SoilsatthesiteareMat‐GryicCambisolwithaclayloamtextureandameanpHof8.01.Thesurfacelayersofthesoilareseasonallyfro‐zenwithfreezinginitiatingfromthetopinthelatefall.ThenativeplantcommunityisdominatedbyKobresia humilis,Carex przewalskii,Helictotrichon tibeticum, Stipa aliena, Saussurea pulchra, and S. pul‐chra.Afullfactorialdesignwithtwotemperaturelevels,thatis,con‐trolandyear‐roundwarming(W1;warmedby1.5–1.7°C),andthreeprecipitationlevels(ambient,+50%precipitation,and−50%precipi‐tation)weresetupinJuly2011(detailsinFigureS1).Eachtreatment
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hadsixrandomlydistributedplots(1.8m×2.2meach)onahomoge‐neouslandscape.Aswarmingratesarereportedtobesignificantlyhigher inwinter than the other seasons on theQTP (Chen et al.,2013), theexperimentwasexpanded to includeawinterwarming(W2) treatment inJanuary2012.Onanannualaveragebasis, sur‐face soil temperature in theW2treatment increasedby thesamemagnitudeasthatintheW1plots,butwithvariedscenariosofsea‐sonality(W2increasedby~3°Cduringlate‐Octobertolate‐Aprilandby0.5–1°Cfortherestoftheyear).Warmingplotswereheatedbyinfraredheatersinstalled1.6mabovegroundwhiledummyheaterswereinstalledabovethecontrolplots.Heatingstartedimmediatelyafterinstallationandsoiltemperaturewasmonitoredcontinuouslyatthedepthsof5,10,and20cm.
Threesoilcores(diameterof3cm,depthof70cm)wererandomlycollected fromeachplotof thecontrol andW1 treatments in July2011(beforewarming)andfromallcontrol,W1andW2plotsintheAugust of both2013 and2015 (after 3 and5 years ofW1warm‐ing,respectively).Soilswereseparatedintovariousdepthsandthosefromthesameplotandsamedepthwerehomogenized insitu.Forthisstudy,weselectedthetopsoil(0–10cm)andsubsoil(30–50cmfor2011;30–40cmfor2013and2015)fromfourplotsofeachtem‐peraturetreatments(i.e.,n=4).Afractionoffreshsoilswasstoredat−80°Cimmediatelyaftersamplingforglyceroldialkylglyceroltet‐raether(GDGT)analysis.Therestwaspassedthrough2mmsievesandfreeze‐driedimmediatelywithstonesandvisiblerootsremoved.
Assuming that the warming treatments did not change thechemicalcompositionofdominantspeciesduringourexperimentalperiod,wetookfouradditionalsoilcores(40cmindepth)fromadja‐centareasoutsidethecontrolplotsinAugust2015toanalyzeligninphenolsinplantroots.Rootmaterialswerepickedoutfromtheen‐tiresoilcores,immediatelywashedunderwaterandgroupedintosixspecies,namelygrasses(H. tibeticum and S. aliena),sedges(K. humilis and C. przewalskii),andforbs(S. pulchra and S. pulchra),whichtotallycontributedto~56%ofabovegroundnetprimaryproductivityatthesite.Allplantmaterialswerekeptat−20°C,freeze‐dried,andgroundintofinepowderbeforeanalysis.
2.2 | Ecosystem CO2 exchange measurement
To complement our SOCmeasurement related towarming effecton soil carbon stock changes, growing‐seasonnet ecosystemCO2 exchange (NEE) and ecosystem respiration (Reco) were measuredwithatransparentchamber (40cm×40cm×60cm)attachedtoaninfraredgasanalyzer(LI‐6400;LiCor)during2013and2015.WemeasuredNEEandReco2dayspermonthfromMaytoOctoberonsunny days during 9:00–11:00 (local time) for all treatments. Foreach measurement, six consecutive recordings of CO2 and watervaporconcentrationsweretakenat10s intervalsoveraperiodof60swithasmallfanrunningcontinuouslyinsidethechamberdur‐ingNEEmeasurement.ThefluxrateofCO2wascalculatedbasedontheslopeofthelinearregressionforthesixconcentrationrecordsinthetimeseries.RecowasmeasuredimmediatelyafterNEEmeas‐urementusingashadeclothtocoverthetransparentchamber.To
calculatedailyNEE,diurnalpatternsofNEEat2hr intervals (over24hr)weremonitoredandacalibrationcoefficientof0.17 (ratiosof daily averagevaluesbasedondiurnal patterns to valuesmeas‐uredduring9:00–11:00)wasused.Thecalibrateddailyvalueswerethenusedtoestimategrowing‐seasonNEE.Grossecosystempro‐ductivity(GEP)wascalculatedasthedifferencebetweenNEEandReco.PositiveandnegativevaluesofNEEindicateCO2releaseandfixation,respectively.Duringthenon‐growingseason(late‐Octoberto late‐April),diurnalchangesinRecoweresmall (Katoetal.,2006;Wangetal.,2014)andrespirationofnon‐growingseasonwasmeas‐ured once a month using the LI‐8100 Automated Soil CO2 FluxSystemwithLI‐8100‐103short‐termchamber(Li‐CorInc.).Recorep‐resentsNEEduringthenon‐growingseasonduetotheabsenceofphotosynthesis.
2.3 | Soil size fractionation and radiocarbon analysis
SoilswerefractionatedusingamethodmodifiedfromWilson,Rice,Rillig,Springer,andHartnett(2009)toexaminetheallocationofnewcarbonandbiomarkersamongmacroaggregates(>250µm),microag‐gregates(250–63µm),andsilt‐clayfractions(<63µm;detailsinTextS1.1).Theorganiccarbon(OC)and14Ccontentsofallsizefractionsandbulksoilweremeasuredafterremovalofinorganiccarbon(TextS1.1).Radiocarboncontentswere reportedas∆14C (‰)with fieldreplicates(n=4).AsthesubsoilshowedvaryingΔ14Cvaluesamongfractions(seeSection3.3),wefurtherestimatedtheturnovertimeofsubsoil(butnottopsoil)OCfractions.Soilfractionswereassumedto be in a steady state over themodeled period (~1 × 104 years)and their turnover timewas estimated based on∆14C accountingforbothradioactivedecayand incorporationofthebomb‐derived14Cproducedinthe1950sand1960s(Torn,Swanston,Castanha,&Trumbore,2009).
2.4 | Biomarker analyses
Arangeofbiomarkers(TableS1)wasselectedtoexaminechangesin thesourceanddegradationofSOCcomponents that representmajor plant and microbial contributions. Freeze‐dried bulk soils(<2mm)collectedbefore(2011)andafterwarming(2013and2015)aswellassoilsizefractionsin2015werefirstsolventextractedtoremoveextractablelipidsandthensequentiallysubjecttobasehy‐drolysisandcopperoxidationtoisolatehydrolysablelipids(includingsuberinandmicrobial‐derivedlipids)andligninphenols,respectively(Otto&Simpson,2007).Rootmaterialsofthesixplantspecieswerealsoanalyzedforligninphenols.Bulksoilscollectedfrom2015(butnotsizefractionsdueto limitedavailability)werefurtheranalyzedfor non‐cellulosic sugars (hereafter referred to as “sugars”; Eder,Spielvogel,Kӧlbl,Albert,&Kӧgel‐Knabner,2010)andcoreGDGTs(Tierney,Schouten,Pitcher,Hopmans,&SinningheDamsté,2012).Themacroaggregatesandsilt‐clayfractionsofthesubsoilsin2015weresolvent‐extractedusingdichloromethaneandmethanoltoiso‐latecoreGDGTsforcomparisonwiththebulksoil.DetailsregardingextractionprocedurescanbefoundinTextS1.2.
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BiomarkersofinterestwerequantifiedafterderivatizationonaTrace1310gaschromatographcoupledtoan ISQmassspectrom‐eter(MS;ThermoFisherScientific)oranAgilent1200liquidchro‐matographandtriplequadrupoleMSusinginternalstandards(TextS1.2). To better illustrate warming‐induced changes to biomarkerconcentrations after removing inter‐annual variabilities, relativechangesofmajormolecularcomponentswerecalculatedasthecon‐centrationoffsetbetweenwarming(W1andW2)andcontrolplotsrelativetotheconcentration inthecontrolplotsoftherespectivesamplingyearandexpressedaspercentages.
Otherthanligninphenols,hydroxyphenols,potentiallyderivedfrom proteins, tannins, and/or lignin degradation products in thesoil (Zaccone, Said‐Pullicino, Gigliotti, & Miano, 2008), were alsodetectedinsubstantialamountsintherootsofdominantplantspe‐ciesattheHaibeiStation(FigureS2).Inparticular,sedges(includingK. humilis and C. przewalskii)withshallowroots(<25cm,with95%oftherootbiomassdistributedintheupper10cm)weremostconcen‐tratedwithhydroxyphenolswhile grasses (includingS. aliena and H. tibeticum)withthedeepestroots(downto85cm;Liuetal.,2018)weremostenrichedwithligninphenolsintherootmass.Giventhehighvolumeofrootmass,bothhydroxyandligninphenolsarethere‐foreconsideredtomainlyderivefromplantsatoursite.
2.5 | Molecular characterization of bulk organic matter
Tofurthercharacterizewarming‐inducedchanges inorganicmat‐ter composition in the subsoil, water‐extractable organic matter(WEOM) and bulk soil were examined by Fourier transform‐ioncyclotronresonancemassspectrometry(FT‐ICRMS)andNMR,re‐spectively.Briefly,WEOMwasextractedfromfreeze‐dried,mixedsubsoilsusingdilutedHCl(0.1M),andconcentratedbysolid‐phaseextractionwith commercially availableBondElut cartridgeswithstyrene divinyl benzene polymer (PPL, Agilent; Text S1.3). FT‐ICRMS analysiswas performed on a solariX FT‐ICRMS (BrukerDaltonicGmBH) equippedwith an electrospray source and a 15TeslamagnetatOldenburgUniversity(TextS1.3).Identifiedcom‐poundswerealignedaccording to theiraromaticity index (AImod),ratios of oxygen‐to‐carbon and hydrogen‐to‐carbon in their for‐mula (Koch & Dittmar, 2006) and divided into five groups: (a)polycyclicaromatics, (b)polyphenols, (c)highlyunsaturatedcom‐pounds, includes lignindegradationproducts, (d)unsaturatedali‐phaticsandpeptides,and(e)saturatedcompounds,includinglipidsandcarbohydrates.
TheHCl‐extractedsoilresidueswerefurthertreatedwithhydro‐fluoricacid(10%)15timestoremoveparamagneticmaterialsandtoconcentrateSOC.The treated residuewas rinsed repeatedlywithdeionizedwater,freeze‐dried,andgroundintofinepowderforsolid‐state 13C NMR analysis. The 13C Cross Polarization/Magic AngleSpinningNMRspectrawereacquiredonaBrukerBioSpinAvanceIII500MHzNMRspectrometerwitha4mmprobeusingspinrateof13kHz,contacttimeof1ms,recycledelayof1s,acquisitiontimeof0.0135s,linebroadeningof75Hz,andwith2,048timedomain
points. Structures were represented by alkyl (0–45 ppm),O‐alkyl(45–110ppm),aromaticandphenolic(110–165ppm),andcarboxylicandcarbonyl(165–215ppm)carbon.Alkyl/O‐alkylratios,increasingwith increasingdegradation,werecalculatedbydividing theareasofthealkylandtheO‐alkylregionsofthespectra(Simpson,Otto,&Feng,2008).
2.6 | Statistical analysis
All statistical analyseswereperformedusingSPSS18.0 (SPSS).Homogeneity of variances and normal distribution of the dataweretestedbeforeapplyingparametricmethodsand logtrans‐formationwasperformedwherenecessary.Nonparametrictestswere conducted if normal distribution of the transformed dataor homogeneity of varianceswas not achieved. The t testwasused to examine differences in the concentrations of SOC andbiomarkers as well as acid‐to‐aldehyde (Ad/Al) ratios betweencontrol and W1 plots before warming. One‐way ANOVA wasperformedtotesttheeffectsofwarmingontheReco,GEP,NEE,SOCcontents,biomarkerconcentrations, and ratios in thebulksoil and size fractions. Two‐way ANOVAwas used to examinethemain and interactive effects ofwarming and soil size frac‐tions on SOC content, Δ14C, and biomarker concentrations in2015.Differenceswereconsideredtobesignificantatthelevelofp<.05.Principalcomponentanalysis(PCA)wasperformedforbiomarkers in soils of 2015 usingCANOCO4.5.Min–max nor‐malizationoforiginaldataintotherangeof[0,1]wasconductedbeforePCAanalysis.
3 | RESULTS
3.1 | Warming effects on ecosystem carbon fluxes and soil bulk properties
Comparedwiththecontrol,bothW1andW2treatmentssignificantlyincreasedsoiltemperatureatthedepthsof5,10,and20cmacross2013and2015(p<.05,FigureS3a–c).Soiltemperatureatthedepthof 30–40 cm was not monitored but estimated based on GDGTs(Weijers,Schouten,vandenDonker,Hopmans,&SinningheDamsté,2007),whichshowedanincreasingtrend(0.26±0.08,0.48±0.21,and0.69±0.14°Cforcontrol,W1,andW2,respectively;detailsaregivenintheSupportingInformation).Warmingincreasedthelengthof thawing period, decreased soil freezing depth (38.3, 35.4, and32.6cmforcontrol,W1,andW2,respectively)andthedurationoffreezing period (Lin,Wang, Zhang, &He, 2017). Soilmoisture de‐creasedatthedepthsof5,10,and20cmunderW1andat5cmunderW2comparedwiththecontrolinbothyears(p<.05;FigureS3d–f).
Ecosystem carbon fluxes were monitored in 2013 and 2015,exhibiting slight inter‐annual variabilities (Figure 1a) and yieldingsimilarresults(i.e.,NEEandReco)inthecontroltreatmentstothosemeasuredbyeddycovariancetechniques(Katoetal.,2006)orau‐tomated continuous measurement in the same grassland nearby(Wangetal.,2014).Comparedtothecontrol,GEPdidnotchange
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underwarminginthegrowingseason(late‐Apriltolate‐October)of2013 but increased inW1 and notW2 in 2015 (p < .05).Reco in‐creasedinbothW2andW1,relativetothecontrolduringthegrow‐ingseasonof2013and2015,respectively(p<.05).Respirationratesalsoincreasedunderwarming(exceptW1in2015)duringthenon‐growingseasonofbothyears(p<.05).Asaresult,NEEwasslightlylessnegative inW2than thecontrol in2013, indicating increasedcarbonrelease(insteadoffixation)underW2,whileNEEwassimilaracrossalltreatmentsin2015.Overall,theaboveresultssuggestthatwarminghadminimalimpactsoncarbonfluxesofthesealpinegrass‐landsduringthestudyperiod.
ChangesinNEEweretoosmalltobedetectedintheSOCstocksinthatSOCcontentsremainedsimilaramongalltreatmentsatbothdepthsbefore (forW1andcontrol in2011) andafterwarming (in2013and2015;p>.05;Figure1b).AsW2wasestablished1yearlater,initialsoilswerenotcollectedforcomparison.However,giventhehomogeneouslandscapeandvegetationcoverageatthestudysite,weassumesimilarSOCcontentsforthecontrolandW2plotsbeforewarming aswell. TheΔ14Cvaluesof SOC,beingmorede‐pletedinthesubsoil(−224±17‰;mean±SEM; n=4)thantopsoil(77±14‰)during2015,showednodifferenceamongtreatments,either(p>.05;Figure1c).
3.2 | Changing dynamics of major SOC molecular components under warming
Despite unaltered bulk SOC properties, considerable changeswere observed inmajor plant‐ andmicrobial‐derivedmolecularcomponents.Importantly,theseeffectsweredetectedinthesub‐soilonly,withnochangesinthetopsoil.Specifically,biomarkersofvariousoriginshadcomparableabundancesinthecontrolandW1plotsatbothdepthsatthestartoftheexperiment(p>.05;Figure2).Neitherwarmingtreatmentaffectedbiomarkerabun‐dancesinthetopsoilin2013or2015(p>.05).However,subsoilsinbothW1andW2plotsdisplayedadecreaseofplant‐derivedphenols (Figure 2a) relative to the control in 2013 and 2015(p<.05)despiteenhancedgrowthofdeepgrassroots(Liuetal.,2018) that are relatively abundant in lignin (Figure S2). In con‐trast, microbial‐derived hydrolysable lipids (referred to as “mi‐crobial lipids”hereafter)andsugarsofbothmicrobialandplantoriginsincreasedintheW1plotsin2015(p<.05).ThelattertwogroupsofbiomarkersalsoshowedanincreasingtrendrelativetothecontrolintheW2subsoilsin2015,butthedifferencewasnotstatistically significant, probably due to large spatial heteroge‐neitythatobscuredtheseeffects.GDGTsderivedfromarchaealandbacterialmembranelipidsalsoincreasedintheW2subsoilsin2015(p<.05).Bycomparison,root‐derivedsuberinlipidsre‐mainedsimilaramongtreatments(p>.05).
Changes in the entire SOC molecular profile were identifiedusingPCAofbiomarkersinthewarmedandcontrolsoils,whichex‐plained78.2%and73.7%ofvarianceinthetop‐andsubsoilof2015,respectively(Figure3).Whilethetopsoilsamplesdidnotclusterac‐cordingtotreatments,subsoilsfrombothwarmingtreatmentswereseparated from those under controlwith phenolsweighing in theoppositedirectionofsugarsandGDGTsonthex‐axisandmicrobiallipidsandsuberinweighingonthey‐axis.Theseresultscollectivelysuggestanaccumulationoflipidandsugarcomponentsderivedfromboth plants andmicrobes at the expense of lignin in thewarmedsubsoil.
Enhanced lignindegradation in thewarmed subsoilwas fur‐ther evidenced by the Ad/Al ratios of vanillyl (V) and syringyl(S) phenols, which typically increasewith elevated degradation(Otto&Simpson,2006).Beforethewarmingtreatmentinitiatedin2011,bothratioswerehigherinthetop‐thansubsoils(p<.05;FigureS4a),butbecamesimilarbetweendepthsin2015(p>.05;FigureS4b), indicatingconvergenceof ligninoxidationstatebe‐tweendepthsunderwarming.Moreover,incontrasttothecom‐monobservationofincreasingAd/Alratioswithsoildepths(Otto&Simpson,2006), the lower (Ad/Al)S ratio in thesubsoilat thissiteindicatedarelativelylowoxidationstageofligninlikelyduetosubstrateand/ortemperaturelimitationsatdepth.Bycompar‐ison, thehigher (Ad/Al)V ratio in the top, relative to subsoils of2011,canalsobepartlyattributedto the influenceof theshal‐low‐rooted, dominant vegetation (K. humilis) with a very high(Ad/Al)Vratioinitsroots(FigureS2d).
F I G U R E 1 Changesinecosystemcarbonfluxes(a),soilorganiccarboncontent(SOC,b),and∆14CofSOC(c)underwarming.Reco,ecosystemrespiration;NEE,netecosystemCO2exchange;GEP,grossecosystemproductivity;growingseasonrefertolate‐Apriltolate‐Octoberofeachyear.Negativeandpositivevaluesindicatecarbonuptakeandrelease,respectively.Meanvaluesareshownwithstandarderror(n=3fora;n=4forbandc).W1andW2representtheyear‐roundwarmingandwinterwarming,respectively.Lowercaselettersindicatedifferentlevelsamongdifferenttreatments(p<.05)
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3.3 | Shifting carbon allocation in soil physical fractions
Different size fractions had similar OC and 14C contents in thetopsoilin2015(p>.05),buttheOCcontentsandΔ14Cvaluesde‐creasedwith decreasing size of fractions in the subsoil (p < .05;Figure4a,b). In thecontrol subsoil, themacroaggregatesand silt‐clay fractions had a turnover time of ~1,500 and ~3,100 years,respectively,confirmingtheslow‐cyclingnatureofOCassociatedwith fineparticles.Warmingdidnot change themassproportionofsizefractionsateitherdepth(p>.05;FigureS5a).TheOCand14Ccontentsdidnotchangeforanytopsoilfractionunderwarming,either.However,warminghadsignificanteffectsonthesubsoilsilt‐clayfractionbyincreasingtheOCcontentofW2(by~56%)andin‐creasingtheΔ14CvaluesofW1relativetothecontrol(p<.05).Thesilt‐clay fraction ofW2 subsoils also showed an increasing trendin Δ14C, but the changewas not significant due to a high spatialheterogeneity (p> .05).Theseresultssuggestanaccumulationofrecentlysynthesizedcarbon in the fine fractionofsubsoilsunderwarming.Warmingdidnotproduceinteractiveeffectswithfraction
sizeonthemassproportion,OC,or14Ccontentsofsoilfractionsateitherdepth(p>.05).
Phenolsandlipidswerefurtherexaminedinvarioussoilfractionstoexplaintheirchangesinthebulksoil(Figure4;FiguresS5andS6).Inthetopsoil,neitherconcentrationsnorproportionsofbiomarkerswereaffectedbywarminginanyfraction.Bycontrast,suberincon‐centrationsincreasedintheW2relativetocontrolandW1subsoilswithall fractionsconsidered (p= .00)whileGDGTconcentrationsdecreasedinthesilt‐clayfractionofW2thancontrolandW1sub‐soils(p<.05).Warmingdecreasedphenolsinthesubsoilmacroag‐gregates,withsignificantlylowervaluesintheW2thancontrolplots(p<.05).Asaresult,warmingdecreasedthepercentageofphenolsinthemacroaggregatesofW2subsoilscomparedwithcontrol(p<.05)withoutaffectingtheallocationofotherbiomarkers(FigureS6).
Lignin Ad/Al ratios did not differ among soil size fractionsorwarming treatment in the topsoil in 2015 (p > .05; Figure S7).However, macroaggregates had lower (Ad/Al)S ratios relative toothersoil fractions inthecontrolsubsoils (p< .05).Moreover, theW1treatment increasedthe (Ad/Al)S ratio inthemacroaggregatesbutdecreasedthe(Ad/Al)Vratiointhesilt‐clayfractionofsubsoils
F I G U R E 2 Warmingeffectsontheconcentrationofmajorsoilmolecularcomponents.(a)Phenols,includingligninandp‐hydroxyphenols;(b)suberin;(c)microbiallipids:microbial‐derivedhydrolysablelipids;(d)glyceroldialkylglyceroltetraethers(GDGTs;explainedinTableS1);(e)sugars.Percentagechangesarecalculatedastheconcentrationoffsetbetweenthewarmedandcontrolplotsrelativetotheconcentrationinthecontrolplotsoftherespectivesamplingyear.Meanvaluesareshownwithstandarderror(n=4).Blueandredarrowsindicatesignificantdecreaseandincrease,respectively(p<.05).W1andW2representtheyear‐roundwarmingandwinterwarming,respectively.RawconcentrationdataarelistedinTableS2
F I G U R E 3 Principalcomponentanalysisofbiomarkersinthetopsoil(a)andsubsoil(b)in2015.Theresultsareexpressedasabiplot,wherethedistanceanddirectionfromtheaxiscenterhavethesamemeaningforsoilsamplesandbiomarkervariables.Numbersinparenthesisrepresentdatavariationsexplainedbythefirsttwoprincipalcomponents(PC).BiomarkersaredefinedinTableS1.W1andW2refertotheyear‐roundwarmingandwinterwarming,respectively
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relativetothecontrol(p<.05).AsimilaralbeitnonsignificantpatternwasobservedfortheW2subsoil,indicatingincreasedligninoxida‐tioninthemacroaggregatesandadecreasedoxidationstageinthefinefractionofsubsoilslikelyduetofreshlignininputs.
3.4 | Composition of bulk organic matter in the subsoil
Warming‐inducedchangestobulkorganicmattercompositionwerefurther investigated for the subsoil in 2015. Unsaturated aliphat‐icsandpeptides (e.g.,proteins)weremoreabundant intheWEOMfrombothwarmedsubsoils(especiallyinW1)relativetothecontrol(Figure5).Meanwhile,polycyclicaromatics,polyphenols,andhighlyunsaturatedcompoundswerelessabundant(especiallyinW2),sug‐gestingadecreaseofsolublearomaticcompounds(includingtanninsand lignindegradationproducts) underwarming.These results areconsistentwiththedecreaseofphenolicbiomarkersandincreaseoflipidsinthebulksubsoil.Bycomparison,solid‐state13CNMRanalysisofbulkSOCshowedincreasedratiosofalkyl/O‐alkylinthesubsoilsofW1(1.09)andW2(1.11)comparedwithcontrol(0.96;TableS3),againsuggestingenhanceddegradationofsubsoilSOCunderwarm‐ing,despitenegligiblechangesinthetopsoil.
4 | DISCUSSION
Five years ofwarming (forW1) did not alter SOC concentrationsin the studied alpine grassland due to relatively small changes inNEE comparedwith soil carbon stocks. However, given the com‐plex composition and varying turnover of SOC pools, shifts incarbon allocation and dynamics among various soil fractionsmay
F I G U R E 4 Changesinbulkpropertiesandbiomarkerconcentrationsofsoilsizefractionsunderwarmingin2015.Meanvaluesareshownwithstandarderror(n=4)fororganiccarbon(OC)content(a),Δ14Cvalues(b),andconcentrationsofphenols(c)andsuberin(d).Lowercaselettersindicatedifferentlevelsamongthecontrol,year‐roundwarming(W1),andwinterwarming(W2)treatmentsinthesamesizefraction(p<.05).ValuesshownforT,S,and T×Srepresentthepvaluesgeneratedfromtwo‐wayANOVAtestsfortheeffectsoftemperature(warmingtreatment),sizefraction,andinteractionbetweenthetwo,respectively
F I G U R E 5 Relativechangesinthemoleculesidentifiedinthewater‐extractableorganicmatterofsubsoilsin2015.RedandbluedotsrepresentproducedandremovedmoleculesidentifiedbyFouriertransform‐ioncyclotronresonancemassspectrometry(FT‐ICRMS)insamplesofyear‐roundwarming(W1;a)andwinterwarming(W2;b)relativetothecontrol,respectively.Boxeswithdifferentcolorsinthediagramcorrespondtomajorclassesofcompounds.Moleculargroupswereassignedtomolecularformulasaccordingtotheiraromaticityindex(AImod),hydrogen‐to‐carbon(H/C),andoxygen‐to‐carbon(O/C)
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impartcontrastingwarmingfeedbacksinthelongterm,eventhoughchanges inbulksoilcarbonstocksarenotdetectedat thecurrent(initialwarming) stage.Using a combination of sensitivemeasure‐ments on SOC characteristics, we reveal divergent responses ofdifferentSOCcomponents,providingevidenceforshiftingcarbondynamics (reflected inbothmolecularand14Ccomposition) in thesubsoil.Thesechangesmayindicatefutureshiftsinthecarbonstor‐agepotentialofsoilsunderwarming.Importantly,noneoftheseef‐fectsweredetectedinthetopsoil,highlightingthatthevastsubsoilcarbonstocksmaybethemostsensitivecomponentsofSOCpoolsinthisstudyregion(Figures2and3).
In contrast to lignin accrual in the macroaggregates of a tall‐grassprairiesoilunderelevatedlitter inputs (Cotrufoetal.,2015),plant‐derivedphenolsdecreasedinthewarmedsubsoils(especiallyinmacroaggregates) relative to thecontrol (Figure2a)despiteen‐hancedinputsoflignin‐enrichedgrassroots(FigureS2).Ourresultspointtowarming‐enhancedlignindegradationatdepthinthisalpinegrassland.Thisfindingiscorroboratedbytherelativechangeoflig‐ninAd/Al ratioswithprogressivewarming (FigureS4), in linewithincreased alkyl/O‐alkyl ratios (Feng et al., 2008; Pengerud et al.,2017)inthewarmedsubsoilsanddecreaseofsolublearomaticsde‐rivedfromligninandtannin,etc.,intheWEOM(Figure5;TableS3).Thedecreaseofphenolsinbulksubsoilswaslargelycausedbytheirdeclining concentrations and proportions in the macroaggregates(Figure 4c; Figure S6a),which are enrichedwith fungal communi‐ties(andhyphae)astheprimarylignindegrader(Gleixner,Czimczik,Kramer,Lühker,&Schmidt,2001)andpoor inmineralsconferring
physiochemical protection for carbon substrates (Wilson et al.,2009).Enhancedligninoxidationinthemacroaggregatesisalsoevi‐dencedbythehigher(Ad/Al)Sratiointhewarmedthancontrolsub‐soilfractions(FigureS7a).
Theabovechangescollectivelysuggest that lignindegradationismoresensitivetowarminginthesub‐thantopsoilofthisalpinegrassland. This may be linked to several factors. First, underde‐graded lignin in the subsoil (indicated by lignin Ad/Al ratios) maybemoresusceptibletodegradationinthewarmedsoilswithanex‐tendedperiodofmicrobialactivity(Linetal.,2017)andapotentiallyincreasedsizeofenzymepools(Alvarezetal.,2018),highlightingthevulnerabilityof relatively fresh ligninpartially “cryo‐locked” in thedeephorizonsofalpinesoils.Second,microbialdegradationofmil‐lennia‐oldSOCwasfoundtobefueledbyincreasedinputsoflabilecarbonviarootpenetrationtothedeeplayersofatemperategrass‐land (Shahzad et al., 2018). Similarity, lignin decomposition couldhavebeenstimulatedinouralpinesubsoilduetowarming‐inducedrootdeepening.Thisexplanationissupportedbyastrongercomet‐abolicdecayofligninintheglucose‐amendedincubationofsubsoilsfromthewarmingcomparedtocontrolplots(Jiaetal.,2017).Third,microbialgenesinvolvedindegradingcomplexorganicssuchaslig‐ninareshowntoincreaseunderwarmingintundrasoils(Sistlaetal.,2013;Xueetal.,2016),andmicrobialcommunitiescanshiftinfavoroffungithatdominatelignindecomposition(Gleixneretal.,2001).InaQTPalpinemeadow,warminghasbeenreportedtoinducemi‐crobial community shifts toward bacteria at 0–10 cm but towardfungiinthedeeperlayers,highlightingthatfunctionalshiftsinsoil
F I G U R E 6 Conceptualfigureshowingcontrastsinsoilcarbondynamicsinresponsetowarmingatvarieddepthsinthealpinegrassland.Warming‐inducedvegetationshifts(Liuetal.,2018)areobservedtocausedeeperrootdistributionandelevatedBNPPinthesubsoil,potentiallyincreasinginputofrootexudates.Thesechangesleadtonewcarbonaccumulationinthesilt‐clayfraction,increasedconcentrationsofsugarsandlipidsaswellasenhanceddegradationoflignininthesubsoillikelyduetoelevatedprimingeffect(Jiaetal.,2017).TheaboveshiftsstandincontrasttotheunalteredBNPPandsoilorganiccarbon(SOC)dynamicsinthetopsoil,underscoringthehighsensitivityofdeepSOCcyclingtowarminginthealpinegrassland
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communitiescouldunderpinthesesubsoilchangesinSOCdynamics(10–50cm;Zhangetal.,2015).
In contrast to phenols, root‐ andmicrobe‐derived biomarkerssuggestthatbulkSOCreceivedincreasedinputsoflipidsandsug‐ars frombothplantsandmicrobes in thewarmedsubsoil. Inpar‐ticular,accompaniedbyanincreasedgrassrootredistributionintothedeeper soil (Liu et al., 2018), the silt‐clay fractionofwarmedsubsoilsshowedan increaseofOCcontent (Figure4a)andanac‐cumulationofnewlysynthesizedcarbon(Figure4b)aswellasun‐degraded (fresh) lignin indicated by lower (Ad/Al)V ratios relativetothecontrol (FigureS7b).Theseresultsagreewithan increasedsilt‐clayfractioninawarmedsubarcticgrassland(Poeplau,Katterer,Leblans,&Sigurdsson,2017)andwith favorablesequestrationofnew carbon in the fine‐sized soil particles under elevated rootgrowth(Desjardins,Barros,Sarrazin,Girardin,&Mariotti,2004).Itis also notable that in contrast to their increase in the bulk sub‐soil (Figure2d), archaea‐andbacteria‐derivedGDGTsdeclined inthesilt‐clayfractionofW2relativetocontrolsubsoils(FigureS5c),likelyduetodilutionbyfreshplant‐derivedOC.Therefore,newlysequestered carbon in the fine fraction appeared to be mainlyplant(root)derived.Takentogether,ourresultsindicatethatwhilewarming‐enhanceddegradation(ofphenols)mainlyoccurredinthefast‐cycling pool (i.e., macroaggregates), sequestration of newlysynthesizedcarbonwaselevatedintheslow‐cyclingpool(i.e.,silt‐clayfraction;Figure6).Asfine‐sizedormineral‐associatedfractionis considered to be responsible for the long‐term stabilization ofSOC (Cotrufoet al., 2015), the shiftingallocationof carbon fromfast‐toslow‐cyclingpoolsmaybenefitlong‐termSOCstorageandexplain the reported increase of subsurface carbon stocks withwarmingontheQTP(Dingetal.,2017).
Insummary,ourstudyprovidescompellingevidenceforshiftingsoilcarbondynamicsontheQTPunderwarming (Figure6).Giventhattheseeffectswereobservedinthesubsoilandnottopsoil,ourresultshighlightthehighsensitivityofdeepSOCcyclingtowarminginalpinegrasslands(Dorrepaaletal.,2009;HicksPriesetal.,2017).Untilnow,studieshavefocusedprimarilyonthewarmingresponseoftopsoil.OurresultssuggestthatthesestudiesmaymisrepresentorunderestimatetotalchangesinSOCdynamicsorterrestrialcar‐bon cycling under warming. The specific ecological mechanismsidentified can help to provide insights into the vulnerability ofhigh‐altitude and high‐latitude regions under warming.Moreover,aswarmingisreportedtoinducedeeperrootdistributioninawiderangeofecosystems(Arndal,Tolver,Larsen,Beier,&Schmidt,2018;Johnson et al., 2006; Keuper et al., 2017; Leppälammi‐Kujansuuetal.,2013;Limetal.,2018;Liuetal.,2018;Shietal.,2017;Wangetal.,2017),theobservedroot‐drivenaccelerationofsubsoilcarboncyclingandnewcarbonsequestrationmayalsooccurinnon‐alpinesystems. It is important to assesswhether,where, andwhennewcarbonaccrualinthefine‐sizedfractionmayoutweighthedecayofnativeSOCatdepth.Itisalsonecessarytoexamineiftheoutcomeisrelatedtothenatureoforganicmatterpreserved(suchasthefresh‐nessof lignin components) and/or the statusofnutrient availabil‐ity in the soil, given thatbothmicrobial activity andplant growth
are strongly regulated by nutrient cycling under global changes(Fontaineetal.,2011;Perveenetal.,2014).Giventhetremendouscarbonstorageinsubsoils,addressingthesequestionswillbecriticaltoimproveconfidenceinfutureprojectionsofsoilcarbondynamicsunderclimatechange.Thiscouldalsohelpus to identifypotential“hotspots”fornewcarbonsequestrationversusoldcarbonpreser‐vationunderwarming.
ACKNOWLEDG EMENTS
This studywas supported financiallyby theChineseNationalKeyDevelopment Program for Basic Research (2017YFC0503902,2015CB954201),theNationalNaturalScienceFoundationofChina(41422304,31630009,31370491,41773067),andtheInternationalPartnership Program of Chinese Academy of Sciences (Grant no.151111KYSB20160014). J. Jia thanks China Scholarship Councilfor supportinghervisit toETHZürich.Wearegrateful toXinyingZhang,TingLiu,andZhiyuanMaforhelpinsamplinganddataanaly‐ses.Thedatausedare listed inthetables,figures,andSupportingInformationofthepaper.
CONFLIC T OF INTERE S T
Theauthorshavenocompetingintereststhatmightbeperceivedtoinfluencetheresultsanddiscussionreportedinthispaper.
AUTHOR CONTRIBUTIONS
X.F.andJ.J.designedthestudy.J.S.H.,L.L.,andZ.Z.designedandcarriedoutthewarmingexperiment.J.J.,Z.C.,C.L.,andY.W.carriedout sample analyseswith help fromN.H., L.W., andT.I.E. for 14C,H.B.andT.D.forFT‐ICRMS,M.J.S.forNMR,andH.Y.forGDGTs.J.J.,X.F.,andT.W.C.wrotethepaperwithinputsfromallco‐authors.
ORCID
Myrna J. Simpson https://orcid.org/0000‐0002‐8084‐411X
Xiaojuan Feng https://orcid.org/0000‐0002‐0443‐0628
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SUPPORTING INFORMATION
Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.
How to cite this article:JiaJ,CaoZ,LiuC,etal.Climatewarmingalterssubsoilbutnottopsoilcarbondynamicsinalpinegrassland.Glob Change Biol. 2019;00:1–11. https://doi.org/10.1111/gcb.14823
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