C Stocks in Forest Floor and Mineral Soil of Two Mediterranean Beech Forests

20 Pages·2016·3.11 MB·English

Article
C Stocks in Forest Floor and Mineral Soil of
Two Mediterranean Beech Forests
AnnaDeMarco1,AntoniettaFioretto2,MariaGiordano1,MicheleInnangi2,CristinaMenta3,
StefaniaPapa2andAmaliaVirzoDeSanto1,*
1 DepartmentofBiology,ComplessoUniversitariodiMonteSant’Angelo,ViaCintia,21,80126Napoli,Italy;
[email protected](A.D.M.);[email protected](M.G.)
2 DepartmentofEnvironmental,BiologicalandPharmaceuticalSciencesandTechnologies,
SecondUniversityofNaples,ViaVivaldi,43,81100Caserta,Italy;antonietta.fi[email protected](A.F.);
[email protected](M.I.);[email protected](S.P.)
3 DepartmentofLifeSciences,UniversityofParma,ViaFarini,90,43124Parma,Italy;[email protected]
* Correspondence:[email protected];Tel.:[phone]or+39-348-353-5925
AcademicEditor:BjörnBerg
Received:19May2016;Accepted:16August2016;Published:22August2016
Abstract: ThisstudyfocusesontwoMediterraneanbeechforestslocatedinnorthernandsouthern
Italy and therefore subjected to different environmental conditions. The research goal was to
understandCstorageintheforestfloorandmineralsoilandthemajordeterminants. Relativetothe
northernforest(NF),thesouthernforest(SF)wasfoundtoproducehigheramountsoflitterfall(4.3vs.
2.5Mg·ha−1)andtostorelessCintheforestfloor(~8vs. ~12Mg·ha−1)butmoreCinthemineral
soil(~148vs. ~72Mg·ha−1). Newly-shedlitterofNFhadlowerP(0.4vs. 0.6mg·g−1)buthigherN
concentration(13vs. 10mg·g−1)thanSF.DespiteitslowerMnconcentration(0.06vs. 0.18mg·g−1),
SFlitterproducesaMn-richerhumus(0.32vs. 0.16mg·g−1)thatislessstable. Thedatasuggestthat
decompositionintheNFforestfloorislimitedbytheshortergrowingseason(178daysvs. 238days)
andthehigherNconcentrationsinnewlyshedlitterandforestfloor. DifferencesinCstockinthe
mineralsoilreflectdifferencesinecosystemproductivityandlong-termorganic-matteraccumulation.
TheverticalgradientofsolubleandmicrobialfractionsinthesoilprofileofSFwasconsistentwitha
fasterturnoveroforganicmatterintheforestfloorandgreaterCaccumulationinmineralsoilrelative
toNF.Withreferencetoregional-scaleestimatesfromItalianNationalForestInventorydata,theC
stockinthemineralsoilandthebasalareaofItalianbeechforestswerefoundtobesignificantly
related,whereasCstockintheforestfloorandCstockinthemineralsoilwerenot.
Keywords: litter fall; forest floor; mineral soil; N; P and Mn; GDD (growing degree days); INFI
(ItalianNationalForestInventory)
1. Introduction
Forests play a key role in mitigating climate change as they may act as carbon sinks [1].
AsubstantialportionofEarthbiospherecarbon(C)isstoredinforestabovegroundandbelowground
biomass,deadwoodandsoil,thelastbeingthemajorcarbonstock[2].Factorssuchastreespecies[3–5]
andclimate[6],influencethesizeoftheforestsoilCstocks. Commonbeech(FagussylvaticaL.)forests
are found in Europe from temperate to mountainous Mediterranean areas with a wide range of
climaticconditionsandsoiltypes,andareofutmostimportanceforCsequestrationonacontinental
scale. InItaly,beechisthemostcommontreespeciesandisfirst-rankintermsofgrowingstock[7].
The considerable diversity of climatic conditions and soil types in the Italian territory may result
indifferencesinbeechlitterchemicalcomposition,whichinturnarelikelytoaffectorganic-matter
dynamicsandsoilCstocks. ThesizeoforganicCstockinforestsoilistheresultofthebalancebetween
twomainprocesses: theannualinputofCwithlitterfallandCoutputthroughdecomposition.
Forests2016,7,181;doi:10.3390/f7080181 www.mdpi.com/journal/forests
Forests2016,7,181 2of20
Litterdecompositionprimarilydependsonlitterchemicalcomposition,interactionswithsoil
biotacommunity,andsiteproperties[8]. Litterconcentrationofessentialnutrients,suchasnitrogen
(N)andphosphorus(P),andlitterstoichiometry(C:nutrientratios)havebeenusedaslitterdecay
predictors. The initial litter N concentration has been found to be negatively related to the limit
value,i.e.,thestageatwhichdecompositionvirtuallystopsorproceedsveryslowly,thusleadingto
humusaccumulationandCsequestration. InPinuslitters,thelimitvalueispositivelyrelatedtoinitial
Mnconcentration,whichthereforemaybeusedtopredicttheaccumulationofhumus[9,10]. Mnis
importantforlitterdecompositionmainlybecausefungiuseMnperoxidaseforlignindegradation[11].
ThecausalrelationshipbetweenMnconcentrationandlitterdecayhasrecentlybeendemonstrated
in a litter bag study using beech litter experimentally enriched in Mn [12]. Litter quality affects
not only the rate of litter mass loss, but also nutrient dynamics. During litter decomposition, the
nutrientsaretakenupbythemicroorganismsandonlyaftermicrobialdemandhasbeenmetisthe
surplusreleasedandmadeavailabletoplants. NandPreleasesarepositivelyrelatedtotheinitial
concentrationoftheseelementsandthereforenegativelyrelatedtolitterC:NandC:P,respectively[13].
Climate[14,15]andsitecharacteristics[16]mayinducewithin-speciesdifferencesinlitterchemical
compositionandhenceinlitterdecayandthesizeofsoilorganicCstocks. Acrossawideclimatic
gradient,NandPconcentrationsofnewly-shedpineneedlehavebeenfoundtobesignificantlyand
positivelyrelatedtothemeanannualtemperature(MAT),whileMnconcentrationshowedasignificant
negativeexponentialrelationshipswithMAT[17]. Theconcentrationofsomenutrients,e.g.,Mnand
Ca, inlitteralsovarieswithsoilpH,whichinfluencestheirmobility[8]. Caccumulationinsoilis
affectedbyforestageandproductivity. Asaparameterthatintegratesthenumberandsizeoftrees,
basalareaisconsideredagoodpredictorofabovegroundbiomassC[18]. Accordingly,soilCinItalian
forestsissignificantlycorrelatedwithabovegroundCstocksandwithbasalarea[4].
Inthelastfewyears,severalstudieshaveinvestigatedsizeandtypesofsoilcarbonpoolsinbeech
forestsofcentralEurope[3,19]includingsolubleandmicrobialfractions,whicharebothpivotaltosoil
organic-matterformationandaccumulation[20–22]. Verylittleinformationispresentlyavailablefor
beechforestsoftheMediterraneanregion[23],inspiteofthefactthatthisisoneoftheworldareas
mostdramaticallythreatenedbyclimatechange[24].
Italianbeechforestsarefoundat900–1900maltitudeandaredistributedfromtheAlpstoSicily
across two biogeographical regions, the Central-European and the Mediterranean, separated by a
boundary running along the Apennines from Northern Liguria to Southern Emilia-Romagna [25].
MeanannualtemperaturesarelowerintheMiddle-EuropeanthanintheMediterraneanbiogeographic
region(9–13◦Cvs. 14–18◦C).Beechforestscoverapproximately1,000,000hectares,arepresentin20of
Italy’s21administrativeregionsand,accordingtotheItalianNationalForestInventory(INFI),storeat
country-scale195.3Mgofcarbonha−1,ofwhich4.5isinlitterand96.6isinsoil[4]. SoilCstockper
hectareishigherinbeechforestsintheMediterraneanthanintheCentral-Europeanbiogeographic
region(about115vs. 80Mg·ha−1)[26]. EstimatesofregionalCstocksinItalianbeechforestsareinthe
rangeof2.1–9.3Mg·ha−1 forlitter,1.2–26.5Mg·ha−1 fororganichorizons,and29.5–171.6Mg·ha−1for
mineralsoiluptothefixed30-cmdepth. Suchlargerangesarelikelytoreflectmajorenvironmental
constraintsoverforestproductivity,litterquality,litterdecompositionandCaccumulationinsoil.
ThispaperpresentsadetailedstudyofsoilCandNstocks,solubleCandNfractions,andthe
microbial-biomassCfractionsintheforestfloor-mineralsoilcontinuumoftwobeechforestswith
relevantdifferencesinclimaticandedaphicconditions,locatedinnorthernandsouthernApennines
(Italy),respectively. WealsofocusedontheamountoflitterfallanditsN,PandMncontents. Nitrogen
andParethemostcommonlimitingnutrientsinforestecosystems,[8]. Phosphorusmainlyaffectsthe
earlyphaseoflitterdecomposition,whichisdominatedbyfast-growingmicroorganismswithhigh
demandsforP[27]. NitrogenandMnareknowntocontrollate-stagesoflitterdecomposition[8,12]
andmaycontributetoregulatetheprimarysequestrationofCintheorganiclayer[28]. Theprimary
goalofourworkwastounderstandCstorageattheshortandlongtimescaleinanorthernanda
southernItalianbeechforest;thus,weinvestigateddifferencesbetweenthetwoforestsforCandN
Forests2016,7,181 3of20
stocksinforestfloorandinthemineralsoilandtheputativemajordriversoflitterdecompositionand
organic-matterdynamics. Asecondgoalwastocomparetheresultsofourstudywiththepattern,
ifany, ofCdistributionintheorganiclayersandmineralsoilofbeechforestsofthewholeItalian
territory and their relationship with basal area. To do this, we used the regional-scale estimates
reportedbyINFI.
2. MaterialsandMethods
2.1. StudySites
The two study sites had similar stand age and management history. They were located in
mountainareasofhighenvironmentalrelevance: (a)“GuadinePradaccio”BiogeneticNaturalReserve
(Corniglio,PR,Italy)onTosco-EmilianoApennines(henceforthreferredtoasnorthernforest,NF)and
(b)Laceno,aninternalpartofRegionalNaturalReserveofMontiPicentini(BagnoliIrpino,AV,Italy),
CampaniaApennines(henceforthreferredtoassouthernforest,SF).Thestudysites,withasurface
areaofabout3000m2,werecoveredby75-year-oldcoppicedbeechtrees(FagussylvaticaL.)andhad
neverbeenaffectedbyfire,grazingorclear-cuttingduringthistimespan(informationprovidedbythe
staffofthetwoNaturalReservesandAuthor’scheckofcharcoalremains). Basalarea(BA;m2·ha−1),
definedasthesumofcross-sectionalareasatbreastheight(1.3m)ofalltreesinastand,wasobtained
bymeasuringthestemdiameteratbreastheight(DBH)onthetreesstandingin10plotsof100m2each.
ThesoilwasanUmbricLeptosolsinNFandaHaplicAndosolinSF(FAOSoilClassificationSystem).
Data about climatic conditions were obtained from the Meteorological Station of Lagdei
(Pradaccio) for NF and the Meteorological Station of Piano Laceno for SF. Temperature data were
adjustedforaltituderelativetothelocationofmeteorologicalstations.Thelengthofthegrowingseason
wascalculatedasgrowingdegreedays(GDD)i.e.,thenumberofdayswithminimumtemperature
over5◦C[29].
2.2. LitterFallCollection
Litterfallfromtreeswascollectedintwoconsecutiveyears(2010and2011)using9circularlitter
traps,eachwithasamplingareaof0.5m2,placedunderthecanopyatabout1mabovetheground.
Thecollectorwasaloosely-hangingpolyesternetwithameshsizeof5mm×8mm. Thismethod
underestimatescertainlitterfractionssuchaslargebranchesandbark[30].Littercollectionwascarried
outeverytwoweeksduringtheperiodoflitterfall. Thelitterwasdriedat75◦Cuntilconstantweight
andweighedinordertocalculatetheannuallitterinputperunitarea. Samplesoftheleaffraction
collectedinthetraps(newly-shedleaves)wereusedtodeterminethelitterchemicalcompositionas
describedbelow.
2.3. ForestFloorLitterandMineralSoilSampling
Samples of the forest floor litter and underlying mineral soil were collected in autumn 2010
at the peak of litter fall and in Spring 2011 with the recovery of vegetative growth so to include
temporalvariationsoftheinvestigatedparameters. Litterandsoilweresampledinsixplotsrandomly
selected within the study areas and spaced at least 10 m apart from each other in order to avoid
spatialautocorrelation[31]. Ineachplot,forestfloorlitterwascollectedusinga20cm×20cmsteel
frame. ThelitterfromtheOLlayer,consistingofplantresidueseasilydiscerniblebynakedeye,was
manuallyseparatedfromstronglyfragmentedandamorphousmaterialbelongingtotheOF+OH
layer. ThesamplesofOLandOF+OHlayerswereweighedanddriedat75◦Ctoconstantweight.
Ineachsamplingplot,thesoilunderlyingtheforestfloorwascollectedbymeansofasteelcore,
4.8cmindiameter,fromthe0to40cmlayer;thesoilcoresweredividedintofoursections,0–5,6–15,
16–30 and 31–40 cm, that were processed separately for chemical analyses after sieving through a
2-mmmesh.
Freshsamplesofforestfloorandmineralsoillayerswerestoredat4◦Candusedwithin24hto
determinefungalbiomass,microbialbiomassandpotentialmineralization.
Forests2016,7,181 4of20
2.4. LitterandSoilAnalyses
LitterandsoilpHwasmeasuredbythepotentiometricmethodindistilledwater(1.0/2.5,litteror
soil/water). Thewatercontentwasmeasuredgravimetricallybyoven-dryingofsamplesat75◦Cto
constantweight. CationExchangeCapacity(CEC)wasdeterminedonthefinefractionaccordingto
theprotocolbyMinisterodellePoliticheAgricoleeForestali,Milan,Italy,2000[32]. Soiltexturewas
determinedmeasuringparticlesizedistributionbygravitationalliquidsedimentationusingafixed
positionpipetteapparatus(protocolbyMinisterodellePoliticheAgricoleeForestali)[32].
Thechemicalcompositionoflitterandmineralsoilwasdeterminedonoven-dried(75◦C)samples
powderedbyaFritschPulverisette(type00.502,Oberstein,Germany)equippedwithagatemortarand
ballmill. Theorganicmatterconcentration(OM)wasdeterminedasdifferencebetweentheresidual
andtheinitialmassofthesampleafterheatingat550◦Cfor2h. OrganicCandNweredetermined
by gas chromatography (CNS analyzer—Thermo Finnigan, Flash 112 Series EA Strumentazione,
Milan,Italy). Water-solubleorganicmatterwasextractedfromthelitterandmineralsoilaccording
toGarcìaetal. (modified)[33]. Oven-driedsamples(0.5gforlitterand20gforsoil)weresoaked
indistilledwater(70mLand200mLrespectively)andincubatedfor24hunderstirring(Universal
Table-Shaker709,130rpm)withtwosonications. Thewaterextractswerecentrifugedat5000rpmfor
15minandfiltered(WhatmanØ15µm). TheconcentrationsofsolubleCandNwereevaluatedbygas
chromatography(CNSanalyzer—ThermoFinnigan,Flash112SeriesEAStrumentazione,Milan,Italy)
afterlyophilisationofwaterextracts.
TocalculatethesizeofCandNstocksineachlayer(OLandOF+OH)oftheforestfloor,Cand
Ncontentpergramofsamplewasmultipliedbythemassofmaterialperunitarea. Asthewhole
massofmaterialhadbeendriedatroomtemperature,acorrectionfactorforconvertingairdriedto
water-freedrymasswascalculatedusingsubsamplesdriedtoconstantweightat75◦C.Thestocksof
CandNinmineralsoilwerecalculatedfromCandNcontentpergramofsamplemultipliedbythe
bulkdensityofthecorrespondingsoillayer. Bulkdensitywascalculatedasmass/volumeafterdrying
at75◦Csamplescollectedwithacorerofknownvolume.
MnandPconcentrationsweredeterminedafterdrymineralizationinamufflefurnaceat480◦C
for16h. Asheswererehydratedwith1mL1:3HNO dilutedwith9mLdouble-distilledwater,filtered
3
andthenanalyzedbyinductively-coupledplasmaatomicemissionspectrometry(ICP-AES)without
ultrasonicnebulization[34].
2.5. DeterminationofActiveFungalMycelium,MicrobialCandPotentialMineralization
Thehyphallengthofmicrofungiinforestfloorandmineralsoilwasdeterminedbytheintersection
methodofOlson[35]. Eachsample(1g)wassuspendedin100mLof60mMphosphatebufferatpH
7.5andhomogenizedat6000rpmfor2min.Aliquots(0.5mL)ofsuspensionwerecollectedandfiltered
undervacuumonnitrocellulosefilterwithaporesizeof0.45µm. Membranefilterswereprepared
accordingtoSundmanandSivelä[36]andtreatedwithafluoresceindiacetatesolutiontodetectactive
fungalmycelium.Thevalueswerereportedasmgdryfungalbiomassg−1organicmatter,assumingan
averagehyphalcrosssectionof9.3µm2,adensityof1.1g·mL−1andadrymassof15%[37]. FungalC
was calculated from active-mycelium mass on the basis of mean fungal values of C/N ratio [38]
and N content [39]. Microbial carbon (Cmic) was evaluated by the method of substrate-induced
respiration(SIR)accordingtoAndersonandDomsch[40]basedonthemeasurementofCO evolution
2
fromlitter/soil(25◦C,55%ofwater-holdingcapacity)inresponsetoadditionofglucose,aneasily
mineralizablesubstrate. Themagnitudeofrespiratoryresponsewasconvertedtomgofmicrobial
biomasscarbonusingtheconversionfactorintroducedbySparling[41]. Potentialmineralizationof
litterandsoilsampleswasestimatedasCO evolutionafteradding3mLdistilledwaterto3gof
2
sample[42]. CO evolutionwasmeasuredafterincubation(25◦C,55%ofwater-holdingcapacity)in
2
tightcontainersfor10daysbyNaOHtrappingfollowedbytwo-phasetitrationwithHCl[43].
Forests2016,7,181 5of20
2.6. Statistics
Differencesbetweensitesforchemicalcompositionofnewly-shedleavesandforCstockswere
assessedbyt-test(overallsignificancelevel=0.05). Two-wayANOVAwasappliedtogetadistinct
viewoftheinfluenceofsiteandseasononthevariabilityofsolublefractionsandmicrobialparameters.
Pairwisemultiplecomparisonswereperformedbymeansofthepost-hocHolm-Šídákmethod(overall
significancelevel=0.05). Simplelinearregressionanalyseswereperformedtoevaluaterelationships
amongCstocksoforganicandminerallayersandbasalareaofItalianbeechforestsusingregional
scaleestimatesfromtheItalianNFI(NationalInventoryofForestsandforestCarbonpools—INFC).
Suchdataareavailableonthewebsite“inventarioforestale.org”. Datawerecheckedfornormalityand
heteroscedasticityandwhennecessarylog-transformed.
ThestatisticalanalyseswereperformedusingSystat_SigmaPlot_12.2software(JandelScientific,
SanJose,CA,USA).
3. Results
3.1. SiteFeatures
Thetwositesdifferedformeanannualtemperatureaswellasformeantemperatureofthecoldest
andthewarmestmonth,thatwerebothloweratthenorthernsite(Table1). Thelengthofthegrowing
seasonwas178and238daysforthenorthernandsouthernsite,respectively(Table1). Asexpected,
soilpHwashigheronthesouthernsitewhichcoincidedwiththelargercationexchangecapacity
(CEC;Table1).
Table 1. Location, climate and soil features of two 75-year-old beech forests in the Apennines
mountains(Italy).
NorthernForest SouthernForest
Latitude/Longitude 43◦23(cid:48)N;10◦01(cid:48)E 40◦48(cid:48)N;15◦07(cid:48)E
Altitude(metersabovesealevel) 1350 1150
*Meanannualtemperature(MAT) 6.0◦C 8.7◦C
*Meantemperatureofthewarmestmonth(TM) 15.4◦C 17.4◦C
*Meantemperatureofthecoldestmonth(Tm) −2.7◦C 0.3◦C
*Meanannualprecipitation(MAP) 2900mm 2300mm
*Maximummonthlyprecipitation(PM) 415mm 430mm
*Minimummonthlyprecipitation(Pm) 42mm 31mm
Numberofgrowingdegreedays 178 238
Arenaceous,Lithological Calcareouscoveredby
Soilparentalsubstrate
formation:Macigno pyroclasticmaterial
Soil0–15cmdepth
Texture(%):
Sand 34.2 29.7
Silt 40.4 47.4
Clay 25.4 22.8
watercontent(%d.w.) 72.4±10.7 78.2±2.6
bulkdensity(g·cm−3) 1.06±0.06 0.99±0.05
organicmattercontent(%) 20.2±1.0 22.8±1.2
CEC(cmol·kg−1) 29.4±3.6 35.7±2.8
pH(H2O) 3.9±0.1 5.6±0.1
N(mg·g−1d.w.) 6.52±1.75 9.38±0.58
P(mg·g−1d.w.) 0.04±0.007 0.16±0.01
C/N 14.7±2.1 12.7±0.7
C/P 3742±348 1118±247
*Observationperiod2008–2013.DataforthesouthernforestfromMeteorologicalStationofPianoLacenoand
forthenorthernforestfromMeteorologicalStationofLagdei(Pradaccio)locatedat1110mand1256ma.s.l.,
respectively.Temperaturedataadjustedforaltitudeandtheactuallocationofmeteorologicalstations.Thevalues
forsoilparametersaremean±standarderror;n=36(6samplingplots,2samplingseasons,3replicateanalyses).
Forests2016,7,181 6of20
3.2. StandFeatures
Thebasalarea(BA)inthenorthernforestwas75%ofthevaluemeasuredforthesouthernforest;
thetreedensitywashigherandmeandiameteratbreastheightwassmallerinthenorthernforest
(Table2).
InlinewithlowerBAvalues,theannuallitterinputtothegroundinthenorthernforestwasonly
58%thatofthesouthernforest. Inbothcases,theleavesaccountedfornearly90%ofthewholelitter
massreachingtheforestfloor(Table2).Incontrast,themassofforestfloorlitterwasquitesimilarinthe
southernandthenorthernforests,i.e.,21.18vs. 23.89Mg·ha−1(Table2). Theratioorganic-matterstock
toorganic-matterinputwas4.9and9.6,respectively,inthesouthernandnorthernforest(Table2).
3.3. Nutrients
Newly-shed leaves from the southern forest had higher P content but were poorer in N and
Mnrelativetothenorthernforest,withmeanconcentrationsdifferingbyafactorof1.6,1.3and2.3,
respectively,forP,N,andMn(Table2,Figure1).
Table2. Stand,litterfallandforestfloorfeaturesoftwo75-year-oldbeechforestsintheApennines
mountains(Italy).
NorthernForest SouthernForest
Slope(degree) 13.98◦ 12.13◦
Treedensity(number·ha−1) 1100±55 800±40
Diameteratbreastheight(cm) 25.4±1.3 34.4±2.2
Basalarea(m2·ha−1) 55.7 74.3
Litterinput(Mg·ha−1)(n=9) 2.50±0.18 4.31±0.43
Forestfloorlitterstock(Mg·ha−1) 23.89±2.12 21.18±2.69
Ratiolitterstocktolitterinput 9.6 4.9
Nutrientcontentofnewlyshedleaves(mg·g−1)(n=9)
C 472.0±1.45 466.7±0.90
N 13.06±0.05 10.17±0.22
C/N 36 46
P 0.40±0.06 0.62±0.06
Mn 0.14±0.005 0.06±0.001
Nutrientfluxeswithlitterfall(kg·ha−1)
N 32.65 43.83
P 1.00 2.67
Mn 0.35 0.26
Mainfeaturesofforestfloorlitter(OLandOF+OH)*
pH 5.82±0.10 6.18±0.03
(H2O)
watercontent(%d.w.) 191.4±13.0 224.7±21.3
OM(mg·g−1d.w.) 910.1±15.4 728.1±30.9
N(mg·g−1d.w.) 21.21±1.42 15.68±1.27
P(mg·g−1d.w.) 0.18±0.01 0.38±0.03
Mn(mgg−1d.w.) 0.16±0.02 0.25±0.03
C/N 24 32
C/P 2925 1328
C/Mn 3690 2184
C/Ca 53 26
*Thevaluesforsoilparametersaremean±SE;n=36(6samplingplots,2samplingseasons,3replicate
analyses)foreachofthetwolayers.
Forests2016,7,181 7of20
Forests 2016, 7, 181  7 of 21 

Figure 1. Nutrient concentrations in newly‐shed leaves, floor litter (OL and OF + OH layers) and 
Figure1. Nutrientconcentrationsinnewly-shedleaves,floorlitter(OLandOF+OHlayers)and
mineral soil at different depths in a northern and a southern beech forest. Values are means ± standard 
mineralsoilatdifferentdepthsinanorthernandasouthernbeechforest.Valuesaremeans±standard
error; n = 36 (6 sampling plots, 2 sampling seasons, 3 replicate analyses). Different lower‐case letters 
error;nin=di3ca6te(6 sisganmifipcalinntg dpiffleortesn,c2ess abmetwpleienng ssiteeass (ot‐ntess,t3; orveeprlailcla stiegnainfiaclaynscees l)e.vDeli f=f e0.r0e5n).t lower-caseletters
indicatesignificantdifferencesbetweensites(t-test;overallsignificancelevel=0.05).
Forest floor in the southern forest had higher P and lower N concentrations relative to the 
northern counterpart (Figure 1). In both forests, the N content increased whereas the P content 
Fodreecsrteaflsoeodr firnomth ethseo uOtLh etron OfoFr e+s tOhHa dlahyiegrh, ei.re.P, wanitdh lporwoegrreNssicnogn clietntetrr adteiocanys r(Feilgautirvee 1t)o. tDhieffneroerntht ern
countepraptaterrtn(sF iogf uvraeri1a)t.ioInn obfo Mthnf coornecsetsn,trthateioNn wcoenrete fnotunindc rine atsheed twwoh feorreeasstst. hIne tPhec osnoutetnhetrdne fcorreeasst,e Mdnfr om
theOLcotoncOenFtr+atOioHn lianycreera,sie.ed. ,5w tiitmhepsr ofrgormes snienwglyli‐tstheerdd elecaavye(sF, itghurorueg1h). tDheif fOerLe ntot pthaett eOrFn s+o OfHva rlaiayteiro, nof
Mncowncheenretraas tiito rnemwaeinreedf oviurtnudalilny uthnechtawnogefdo irne sthtse. nIonrtthheernso fourtehset r(Fnigfourree s2t),. MC/nnuctorinencet nrattriaot wioans hinigchreear sed
5timesfromnewly-shedleaves,throughtheOLtotheOF+OHlayer,whereasitremainedvirtually
unchangedinthenorthernforest(Figure2). C/nutrientratiowashigherinthenorthernsite,with
theexceptionofC/Nthatwashigherinthesouthernsite(Table2). Inthesouthernforest,NandP
Forests2016,7,181 8of20
Forests 2016, 7, 181  8 of 21 
fluxesiwn tihthe nliottrethrfearnll s(ilteea, fwliitthte trhme eaxscsep×tiolintt oerf Cn/uNtr tiheantt wcoasn cheignhterar tiino nth)ew soeurethlearrng seirte( f(aTcatboler s21).. 3Ina tnhde 2.7,
southern forest, N and P fluxes with litterfall (leaf litter mass × litter nutrient concentration) were 
respectively)whereasMnreturnwassmaller(factor0.7)(Table2). Mineralsoilofthesouthernforest
larger (factors 1.3 and 2.7, respectively) whereas Mn return was smaller (factor 0.7) (Table 2). Mineral 
hadhigherpH,CEC,NandPconcentrations,andlowerC/nutrientratios,whereastheOMandwater
soil of the southern forest had higher pH, CEC, N and P concentrations, and lower C/nutrient ratios, 
contentweresimilarinthetwoforests(Table1).
whereas the OM and water content were similar in the two forests (Table 1). 
Inbothsites,CandNconcentrationstypicallydeclinedwithsoildepth(Figure1).Incontrastwith
In both sites, C and N concentrations typically declined with soil depth (Figure 1). In contrast 
forestfloor,soilNconcentrationwashigheratthesouthernsite(Tables1and2;Figure1). Asexpected,
with forest floor, soil N concentration was higher at the southern site (Tables 1 and 2; Figure 1). As 
theC/eNxpreactteiod,d tehcel Cin/eNd rwatiioth ddecelpintehdi nwibtho tdhepsitthe sin( Sbuotphp slietems e(SnutaprpyleMmeantetarriayl MsFaitgeruiarles SF1ig).ure S1). 

FigureF2ig.uMren 2c. oMnnc econntrcaetnitornatsioinnsn inew nelyw-lsyh‐sehdedle laevaevsesa annddfl floooorr lliitttteerr iinn aa nnoortrhtehrenr nanadn ad saoustohuetrhne brenecbhe ech
forest.foVreasltu. eVsalfuoers nfoerw nelyw-lsyh‐eshdedli tltitetrera arree mmeeaannss ± ±stasntdaanrdda errdroer;r nro =r ;9.n V=alu9e.s Vfoarl fuoeressft oflroofor raerset mfleoaonrs are
± standard error; n = 36 (6 sampling plots, 2 sampling seasons, 3 replicate analyses); different lower‐
means±standarderror;n=36(6samplingplots,2samplingseasons,3replicateanalyses);different
case letters indicate significant differences between sites (t‐test; overall significance level = 0.05). 
lower-caselettersindicatesignificantdifferencesbetweensites(t-test;overallsignificancelevel=0.05).
3.4. Stocks of Organic C and N, Water Soluble C and N, Microbial and Fungal C 
3.4. StocksofOrganicCandN,WaterSolubleCandN,MicrobialandFungalC
The organic C and N pools in the forest floor of SF were smaller than in the northern forest 
T(hFeigourrgesa 3n iacndC 4a, nanddN Tapbloeo 2l)s. in the forest floor of SF were smaller than in the northern forest
(Figures3aAnltdho4u,gahn dC Taanbdl eN2 c)o.ncentrations in mineral soil were lower than in forest floor (Figure 1), the 
Amltihnoeruagl hsoCil satnordedN mcuocnhc leanrgtrear taiobnsosluinte maminoeurnatls soof iCl wanedre Nl o(Fwigeurrtehs a3n ainnd f4o)r deuste fltoo oitrs (gFreigatuerre  1),
thickness. The organic C stock in the 0–40 cm mineral layer was one order of magnitude higher than 
themineralsoilstoredmuchlargerabsoluteamountsofCandN(Figures3and4)duetoitsgreater
in forest floor (Figure 4). In contrast with the pattern observed for forest floor, the stocks of C and N 
thickness. TheorganicCstockinthe0–40cmminerallayerwasoneorderofmagnitudehigherthanin
in the mineral soil were higher in the southern forest (Figures 3 and 4). 
forestfloor(Figure4). Incontrastwiththepatternobservedforforestfloor,thestocksofCandNin
The concentrations of water‐soluble C and N per gram litter/soil were one order of magnitude 
themineralsoilwerehigherinthesouthernforest(Figures3and4).
higher in the forest floor than in the mineral soil (Supplementary Materials Figure S2). When 
Tehxepcreosnsceden atsr aat iforancstioonf wofa ttoetra-ls Col uanbdle NC, haonwdeNverp, ewragtrear‐msolluitbteler /Cs aonildw Ne rine othnee foorredset rflooform aangdn iint ude
higherthine tmhienfeorarel sstoflil ohoardt hmaonrei nsitmheilamr ivnaeluraels s(oFiilg(uSrue p5p). lTemhee snotlaurbyleM fraatcetriioanlss oFfi gCu arendS 2N). iWn fhoerneset xflporoers sed
asafrwacetrieo nhigohfetro itna lSCF tahnand iNn N,hFo, wwiethv tehr,e wexacteeprt-isoonl oufb sloeluCblaen Nd fNracitniotnh ien fsoprreinstg fl(Foiogruraen 5d). iInn cthonetrmasint, eral
soilhatdhem froarcetiosinms oilfa wrvataelru‐seoslu(Fbilge uCr ean5d) .NT ihne msoinluerballe sofrila wcteioren hsiogfheCr ainn NdFN thiannf oinr eSsFt (flFoigourrew 5e)r. eInh bigothhe rin
forests the soluble fractions increased from OL to OF + OH in the forest floor and from the 0 to 5 cm 
SFthaninNF,withtheexceptionofsolubleNfractioninspring(Figure5). Incontrast,thefractions
layer to deeper layers in mineral soil (Figure 5). Significant differences between seasons for soluble C 
ofwater-solubleCandNinmineralsoilwerehigherinNFthaninSF(Figure5). Inbothforeststhe
fraction were found only in the mineral soil of NF. The soluble N fraction was significantly higher in 
solublefractionsincreasedfromOLtoOF+OHintheforestfloorandfromthe0to5cmlayerto
spring than in autumn both in forest floor and in mineral soil of the northern forest. In the forest floor 
deeperlayersinmineralsoil(Figure5). SignificantdifferencesbetweenseasonsforsolubleCfraction
of SF, soluble N decreased significantly from autumn to spring, while in mineral soil no significant 
werefoundonlyinthemineralsoilofNF.ThesolubleNfractionwassignificantlyhigherinspring
change was observed. 
thaninautumnbothinforestfloorandinmineralsoilofthenorthernforest. IntheforestfloorofSF,
solubleNdecreasedsignificantlyfromautumntospring,whileinmineralsoilnosignificantchange
wasobserved.
Forests2016,7,181 9of20
Forests 2016, 7, 181  9 of 21 

Figure3.CFiagnudre N3. Cp aonodl sNi npotohlse inc othnet cinonutuinmuufmo rfoersetstfl folooorr ‐-mmininerearla sloisl ooifl ao nfoarthneornrt ahnedr an saountdhearns boeuetchh ernbeech
forest. Valufoersesat.r Veamlueesa anrse m±easntas n± dstaarnddaerrdr eorrr;orn; n= =3 366 ((66 ssaammpplinlign pgloptsl,o 2t ss,am2pslainmg pselainsognss, e3a rseopnlicsa,t3e replicate
analyses). Different lower‐case letters indicate significant differences between sites (t‐test; overall 
analyses). Differentlower-caselettersindicatesignificantdifferencesbetweensites(t-test; overall
significance level = 0.05). 
significancelevel=0.05).
Forests 2016, 7, 181  10 of 21 

FigureF4i.gurCe 4s. tCo cstkosckisn inf foorreesstt flfloooro arnadn mdinmerainl seorial lins ao inloritnhearn naondrt ah esorunthaenrnd beaecsho fuotrhesetr. nC sbtoecekcsh  forest.
in the mineral soil 0–30 cm depth are reported for comparison with INFI data. Values are means ± 
Cstocksinthemineralsoil0–30cmdeptharereportedforcomparisonwithINFIdata. Valuesare
standard error; n = 36 (6 sampling plots, 2 sampling seasons, 3 replicate analyses). Different lower‐
means±standarderror;n=36(6samplingplots,2samplingseasons,3replicateanalyses).Different
case letters indicate significant differences between sites (t test; overall significance level = 0.05). 
lower-caselettersindicatesignificantdifferencesbetweensites(ttest;overallsignificancelevel=0.05).
8 8
Northern Autumn Spring
C) Southern C)
nic  6 6 nic 
a a 2 a
g g
r r
O O
%  4 2 a 4 % 
C ( 1 2 2 C (
Soluble  2 1 b a 1a 1 1 2a a a b 2 Soluble 
b b b b b
0 0
OL OF+OH 0‐5 cm 6‐15 cm16‐30 cm31‐40 cm OL OF+OH 0‐5 cm 6‐15 cm16‐30 cm31‐40 cm
25 25
Autumn Spring
1
N) 20 a 20 N)
al  1 2 a 2 al 
Tot 15 a a a 15 Tot
%  b % 
N ( N (
e  10 b 2 a 10 e 
ubl 1 b 2 b b ubl
ol 5 1 2 5 ol
S a S
1
b b
0 0
OL OF+OH 0‐5 cm 6‐15 cm 6‐30 cm 1‐40 cm OL OF+OH 0‐5 cm 6‐15 cm 6‐30 cm 1‐40 cm
1 3 1 3

Figure 5. Soluble C and N fractions % of total C and N in the continuum forest floor ‐mineral soil of a 
northern and a southern beech forest. Values are means ± standard error; n = 18 samples (6 sampling 
plots, 3 replicate analyses). Different lower‐case letters indicate significant differences between sites;
Forests 2016, 7, 181  10 of 21 

Figure 4. C stocks in forest floor and mineral soil in a northern and a southern beech forest. C stocks 
in the mineral soil 0–30 cm depth are reported for comparison with INFI data. Values are means ± 
Forests2016,7,181standard error; n = 36 (6 sampling plots, 2 sampling seasons, 3 replicate analyses). Different lower‐ 10of20
case letters indicate significant differences between sites (t test; overall significance level = 0.05). 
8 8
Northern Autumn Spring
C) Southern C)
nic  6 6 nic 
a a 2 a
g g
Or Or
%  4 2 a 4 % 
C ( 1 2 2 C (
Soluble  2 1 b a 1a 1 1 2a a a b 2 Soluble 
b b b b b
0 0
OL OF+OH 0‐5 cm 6‐15 cm16‐30 cm31‐40 cm OL OF+OH 0‐5 cm 6‐15 cm16‐30 cm31‐40 cm
25 25
Autumn Spring
1
N) 20 a 20 N)
al  1 2 a 2 al 
% Tot 15 a b a a 15 % Tot
N ( N (
e  10 b 2 a 10 e 
ubl 1 b 2 b b ubl
ol 5 1 2 5 ol
S a S
1
b b
0 0
OL OF+OH 0‐5 cm 6‐15 cm16‐30 cm31‐40 cm OL OF+OH 0‐5 cm 6‐15 cm16‐30 cm31‐40 cm

Figure5. FSiogulureb 5le. SColuabnled C Nandfr Na cfrtaioctniosns% % ooff ttootatal Cl Canda nNd inN thein cotnhtienucuomn tfionreustu fmloofr o‐mreinsetrflalo sooirl o-fm a ineralsoil
northern and a southern beech forest. Values are means ± standard error; n = 18 samples (6 sampling 
of a northern and a southern beech forest. Values are means ± standard error; n = 18 samples
plots, 3 replicate analyses). Different lower‐case letters indicate significant differences between sites; 
(6samplingplots,3replicateanalyses). Differentlower-caselettersindicatesignificantdifferences
betweensites; differencesbetweenseasonsareindicatedbydifferentnumbers(two-wayANOVA
followedbypost-hocHolm-Šídákmethod;overallsignificancelevel=0.05).
SimilarconcentrationsofmicrobialandfungalC(SupplementaryMaterialsFigureS3)werefound
inforestfloorandinmineralsoil,withapeakinthetopminerallayer(0–5cm). Incontrast,when
expressedasafractionoftotalorganicC,bothfungalandmicrobialCsignificantlyincreasedfrom
forestfloortomineralsoilatbothsites;fungalandmicrobialCfractionsofforestfloorandtopmineral
layerssignificantlyincreasedfromautumntospringatbothsites(Figure6). Differencesbetweensites
wererecordedonlyforthemineralsoil,whichinthenorthernforesthadhigheramountsofmicrobial
andfungalCrelativetothesoutherncounterpart(Figure6). However,fungalCincreasedsignificantly
fromautumntospringintheforestfloorandinthetopmineralsoilofNF;inspringfungalCinthe
forestfloorwashigherinNFthaninSF(Figure6).
Potentialmineralizationwassimilarinthetwoforestsanddeclinedfromforestfloortomineral
soil (Figure 7). The metabolic quotient was always slightly lower in spring than in autumn and a
significantdifferencewasfoundinautumnbetweenthetwoforestsinmineralsoil(Figure7).