Effects of anabolic and catabolic nutrients on woody plant encroachment after long-term

24 Pages·2017·8.95 MB·English

RESEARCHARTICLE
Effects of anabolic and catabolic nutrients on
woody plant encroachment after long-term
experimental fertilization in a South African
savanna
AnthonyJ.Mills1*,AntoniV.Milewski2,DirkSnyman3,JorrieJ.Jordaan4
1 DepartmentofSoilScience,StellenboschUniversity,Matieland,SouthAfrica,2 PercyFitzPatrickInstitute,
DST/NRFCentreofExcellence,UniversityofCapeTown,Rondebosch,SouthAfrica,3 C4EcoSolutions,
Tokai,SouthAfrica,4 UniversityofLimpopo,Sovenga,SouthAfrica
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*[email protected]
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Abstract
Thecausesoftheworldwideproblemofencroachmentofwoodyplantsintograssyvegeta-
tionareelusive.Theeffectsofsoilnutrientsoncompetitionbetweenherbaceousandwoody
OPENACCESS plantsinvariouslandscapesareparticularlypoorlyunderstood.Along-termexperimentof
60plotsinaSouthAfricansavanna,comprisingannualapplicationsofammoniumsulphate
Citation:MillsAJ,MilewskiAV,SnymanD,
JordaanJJ(2017)Effectsofanabolicandcatabolic (146–1166kgha-1yr-1)andsuperphosphate(233–466kgha-1yr-1)overthreedecades,and
nutrientsonwoodyplantencroachmentafterlong- subsequentpassiveprotectionoveranotherthreedecades,duringwhichindigenoustrees
termexperimentalfertilizationinaSouthAfrican
encroachedondifferentplotstoextremelyvariabledegrees,providedanopportunityto
savanna.PLoSONE12(6):e0179848.https://doi.
org/10.1371/journal.pone.0179848 investigaterelationshipsbetweensoilpropertiesandwoodyencroachment.Alltopsoils
wereanalysedforpH,acidity,EC,water-dispersibleclay,Na,Mg,K,Ca,P,S,C,N,NH ,
Editor:DafengHui,TennesseeStateUniversity, 4
UNITEDSTATES NO3,B,Mn,CuandZn.Applicationsofammoniumsulphate(AS),butnotsuperphosphate
(SP),greatlyconstrainedtreeabundancerelativetocontrolplots.Differencesbetweencon-
Received:January3,2017
trolplotsandplotsthathadreceivedmaximalASapplicationwereparticularlymarked(16.3
Accepted:June4,2017
±5.7versus1.2±0.8treesperplot).SoilpropertiesmostaffectedbyASapplications
Published:June29,2017 includedpH(H O)(controltomaximalASapplication:6.4±0.1to5.1±0.2),pH(KCl)(5.5±
2
Copyright:©2017Millsetal.Thisisanopen 0.2to4.0±0.1),acidity(0.7±0.1to2.6±0.3cmolkg-1),acidsaturation(8±2to40±5%),
accessarticledistributedunderthetermsofthe Mg(386±25to143±15mgkg-1),Ca(1022±180to322±14mgkg-1),Mn(314±11to118
CreativeCommonsAttributionLicense,which
±9mgkg-1),Cu(3.6±0.3to2.3±0.2mgkg-1)andZn(6.6±0.4to3.7±0.4mgkg-1).Mag-
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal nesium,B,MnandCuwereidentifiedusingprincipalcomponentanalysis,boundaryline
authorandsourcearecredited. analysisandKruskal-Wallisranksumtestsasthenutrientsmostlikelytobeaffectingtree
DataAvailabilityStatement:Allrelevantdataare abundance.TheratioMn/Cuwasmostrelatedtotreeabundanceacrosstheexperiment,
withinthepaperanditsSupportingInformation supportingthehypothesisthatcompetitionbetweenherbaceousandwoodyplantsdepends
files.
ontheavailabilityofanabolicrelativetocatabolicnutrients.Thesefindings,basedonmore
Funding:WegratefullyacknowledgetheSouth thansixdecadesofexperimentation,mayhaveglobalsignificanceforthetheoreticalunder-
AfricanGovernment’sDepartmentof
standingofchangesinvegetationstructureandthusthepracticalcontrolofinvasivewoody
EnvironmentalAffairs,NaturalResources
plants.
ManagementProgramme,andtheNational
ResearchFoundation(grantnumber
FA2005040700027)forfundingthisresearch.The
fundershadnoroleinstudydesign,datacollection
PLOSONE|https://doi.org/10.1371/journal.pone.0179848 June29,2017 1/24
Constraintsonwoodyencroachmentafterfertilization:Effectsofanabolicvscatabolicnutrients
andanalysis,decisiontopublish,orpreparationof Introduction
themanuscript.
Theencroachmentofwoodyplantsintorangelandsisagrowingproblemworldwide[1,2,3].
Competinginterests:Theauthorshavedeclared
Long-termcontrolofsuchchangesdependsonanunderstandingoftheultimatereasonsfor
thatnocompetinginterestsexist.TheauthorDirk
whyestablishmentoftreesisconstrainedingrasslandbiomesandwithinthegrassymatrixin
Snymanholdsacommercialaffiliation,being
savannabiomes.Suchanunderstandingofvegetationstructurehasremainedelusive.Although
employedbyC4EcoSolutions.Thisdoesnotalter
ouradherencetoPLOSONEpoliciesonsharing theliteratureonthetreelessnessofgrasslandshasemphasisedtheadverseeffectsofdrought,
dataandmaterials. fireandfrostonwoodyplants[4,5,6,7],thesefactorsarelikelytobemechanismsratherthan
causesunderlyingthenaturalcompetitivepowerofgrassesandtheconsequentsuppressionor
exclusionofeventhosetaxaoftreesadaptedtosuchadversities[8].
Notwithstandingtheeffectsoffactorssuchaswateravailability,firefrequencyandintensity
ofherbivory,theavailabilityofcertainnutrientelementsislikelytobeoneultimatecauseof
thedominanceoftreesoverherbaceousplantsinaparticularlandscapeorregion[9,10].The
waysinwhichvariousnutrientsinsoilsaffectthecompetitionbetweenherbaceousandwoody
plantsremain,however,unclear.Aparticularconundrumisthatmanyoftheworld’sgrass-
lands,shrublandsandsavannasarewarmandwetenoughtosupportclosedforests[11,12,
13].Severalauthorshaveproposedthatnutrientdeficienciesconstraintheestablishmentof
treesinthesesystems[9,14,15,16,17,18,19,20,21,22,23].Yet,eventhemostnutrient-poor
savannasandshrublandsappeartohavesufficientconcentrationsofnutrientsfortheestab-
lishmentofforests[24].AsBond[24]notes:“Theapparentfailureoflownutrientstocksto
explainthemissingforestsonnutrient-poorsoilsemphasisestheneedfornewideasonhow
nutrients,aloneorincombinationwithotherfactorssuchasfire,influencevegetationstruc-
ture”.Bond[24]alsourgesresearchersto“...takeafreshlookathownutrientsinfluencevege-
tationstructure”.
Alittle-knownagronomicapproachistouseratiosofelements–suchasCa/Mg–todeter-
minetheamountofaparticularnutrientrequiredtomaximiseherbaceousortreecroppro-
duction[25].Thisisbecausetherearesynergisticandantagonisticeffectsamongnutrient
elementsthataffecttheiruptakebyrootsandtheirmetabolicrolesoncetheyenterlivingcells.
Thepotentialeffectsofratiosofvariousnutrientsonvegetationstructureinnaturalecosys-
temshave,however,beenneglectedintheliterature.Thisisaccordinglyoneapproachwhich
wouldoffera‘freshlookathownutrientsinfluencevegetationstructure’.Recently,Milewski
andMills[8]providedatheoreticalframework(hereafterreferredtoastheAnabolic/Catabolic
Theory)forwhyratiosofanabolicnutrients(e.g.MnandMg)tocatabolicnutrients(e.g.Cu
andZn)wouldplayastrongroleincompetitionbetweenherbaceousplantsandtreeseedlings.
Thetheoryholdsthatherbaceousplantsarecapableofcompetitivelyexcludingtreesonsites
wherethesupplyofcatabolicnutrientsmeetsthedemandcreatedbythephotosyntheticpoten-
tialofthelocalenvironment.ArecentstudyingrasslandsandwoodlandsinNewSouth
Wales,Australia,corroboratedthetheory,findingthatthesoilsundergrasslandswerericher
innutrients,particularlycatabolicnutrients,thanthoseunderadjacentwoodlands[26].
Inthispaper,wereportontheeffectsofalong-termexperimentof60plotsatthe
TowoombaAgriculturalDevelopmentCentreinaSouthAfricansavanna,namelySpringbokv-
lakteThornveld[27],ontheabundanceoftreesandarangeofsoilproperties.Thetreatments
entailedvariousapplicationsofammoniumsulphate(146–1166kgASha-1yr-1;Table1)and
superphosphate(233–466kgSPha-1yr-1;Table1)aswellasharvestingofhayeachyearfor
threedecadesovertheperiod1949to1981,followedbyafurtherthreedecadesofpassivepro-
tection.Duringtheperiodofpassiveprotectiontherewasextremelyvariablewoodyencroach-
mentacrosstheexperiment,withsomeplotsbecomingdenselywoodedandothersresisting
invasion.Thetreespeciesencroachingthemostwasthefrost-anddrought-tolerantVachellia
karroo,anacaciaknownasoneofthemostimportantwoodyinvadersofgrasslandsinSouth
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Constraintsonwoodyencroachmentafterfertilization:Effectsofanabolicvscatabolicnutrients
Table1. ExperimentaltreatmentsatTowoombaovertheperiod1949to1981.
Treatment AS SP Treatment AS SP Treatment AS SP
AS SP 0 0 AS SP 0 233 AS SP 0 466
0 0 0 1 0 2
AS SP 146 0 AS SP 146 233 AS SP 146 466
1 0 1 1 1 2
AS SP 291 0 AS SP 291 233 AS SP 291 466
2 0 2 1 2 2
AS SP 583 0 AS SP 583 233 AS SP 583 466
3 0 3 1 3 2
AS SP 1166 0 AS SP 1166 233 AS SP 1166 466
4 0 4 1 4 2
AS=ammoniumsulphate(kgha-1yr-1);SP=superphosphate(kgha-1yr-1)
https://doi.org/10.1371/journal.pone.0179848.t001
Africa[28,29].TheoriginaldesignoftheTowoombaexperimentwasbasedonanattemptto
enhancenaturalgrazingbymeansoffertilizationsolelywithN,S,PandCa.However,thefer-
tilizationtreatmentshavefortuitouslyandindirectlyprovidedaninvaluableopportunityto
determinetheeffectsofarangeofsoilpropertiesontheabundanceofwoodyplantsinthis
savanna.Theperiodofpassiveprotectionintheexperimenthappenedtocoincidewiththe
first-recordedworldwidewaveofwoodyencroachmentinsavannasandformerlytreeless
grasslands[30,31,32,33].Itthusalsofortuitouslyprovidesaninsightintoacurrentglobal
problem.
Insummary,theTowoombaexperimentcomprisedtwophases,eachspanningapproxi-
matelythreedecades.Thefirstphaseentailedintensivefertilization,harvestingofgrass,and
protectionfromlivestock.Thesecondphase–duringwhichwoodyplantencroachmenttook
placeacrosstheexperiment–entailedprotectionfromlivestock.Fireonlyoccurredtwiceon
thesiteinthesixdecadesoftheexperiment.Ourcentralquestionofcauseandeffectwas
‘whichsoilproperties,atthescaleoftheentirecommunityofplants,areultimatelyresponsible
forthevirtualfailureoftreestoestablishincertainplotsinthesecondphaseoftheTowoomba
experiment?’Inordertoanswerthis,wehavefocussedonvariouseffectsonsoilcomposition
notoriginallyenvisagedinthedesignoftheexperiment.Theresultisananalysisthatlinkssoil
propertieswithtreeabundancesfromwithinanexperimentthatspannedmorethansix
decadesinanAfricansavanna.
Methods
Studyareaandexperimentalsite
TheTowoombaAgriculturalDevelopmentCentre(28o21’E,24o54’S)islocatednearBela-Bela,
LimpopoProvince,SouthAfrica(Fig1).ThisfacilitywasestablishedbytheformerTransvaal
ProvincialAdministrationin1935.TheTowoombalong-termfertilizationexperimentwhich
weanalysedconsistsofasplit-plotdesignwith15treatmentsincludingcontrols,totalling60
plotseachmeasuring6.4x9.1m[34].Attheoutset,a5x3factorialdesignwaslaidoutinran-
domisedblockswithfourreplications–arrangedasfourrowsintherectangularexperimental
site(S1Table).AlthoughtheTowoombaexperimenthassince1995beenmaintainedona
reducedbudget,theoriginalplotshavebeenpreservedandprotectedtothisday(Fig2).
ThestudyareaisrepresentativeofthesouthernSpringbokFlats,aplainwithsoilsderived
fromigneousandsedimentaryrockscoverednaturallybylowopensavannaatthetimewhen
Europeanfarmersarrivedaround1870[27].Theexperimentalsite(120x35m)atTowoomba
islevelwithnegligibletopographyandhasfreely-drained,base-rich,red-yellow,apedalsoil
with30–35%clayinthetopsoil,probablyderivedfromdoleriteswithanalluvialinfluence
[37].Thesearerhodic,eutric,haplicferralsols(correspondingtotheHuttonform,Stellafam-
ilyintheSouthAfricanterminology,[38].Vertisols(darkcrackingclays),derivedfrombasalt
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Constraintsonwoodyencroachmentafterfertilization:Effectsofanabolicvscatabolicnutrients
Fig1.LocationofTowoombaAgriculturalDevelopmentCentreinthesouthernSpringbokFlatsofLimpopoProvinceinSouthAfrica.Thismap
wasgeneratedbasedondatasetsfromthefollowingsources:[35,36].
https://doi.org/10.1371/journal.pone.0179848.g001
[39],occurimmediatelyadjacenttotheexperimentalsitebutnotwithintheexperimental
plots.
Thestudyareaismesicwithmeanannualprecipitation(concentratedinsummer)of629
mmandfrequentfrost,butnosnow,inthedrywinter.Temperaturevariesfromameanmaxi-
mumof29˚Candameanminimumof16˚CinDecembertoameanmaximumof21˚Cand
meanminimumof4˚CinJuly[40].
Althoughthenaturalsavannainthestudyareacanprovidefairlyvaluablegrazingfor
domesticlivestockthroughouttheyear,therainfallissufficientinmostyearstosupportarable
cropping.TherangelandontheSpringbokFlatsisofmixedvalue,withsomespeciesofgrasses
remainingattractiveasforageforungulatesinthedrywinter,whilethedormanttissuesofoth-
ersofferonlystrawinsufficienttomaintainthebodymassofthegrazers[27].
Overthelastseveraldecades,theremainingunploughedrangelandsinthestudyareahave
beendegradedbywidespreadencroachmentofindigenouswoodyplantsintheabsenceofany
erosion[41].Giventhisregionalincreaseintheabundanceoftrees,auniqueopportunitywas
providedbythefactthatthedegreeofwoodyencroachmentintheexperimentalsitehascome
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Constraintsonwoodyencroachmentafterfertilization:Effectsofanabolicvscatabolicnutrients
Fig2. (a)SatelliteimageshowingtheapproximatepositionoftheexperimentalplotsatTowoomba
AgriculturalDevelopmentCentre,SouthAfrica;and(b)aphotographtakeninSeptember2014oftwo
experimentalplots.Blackbordersin(a)depicttheplotsshownin(b).Foregroundin(b):aplotwithnowoody
encroachmentthatreceivedthemaximumapplicationsofAS(1166kgha-1yr-1)andSP(466kgha-1yr-1).
Backgroundin(b):aplotwithintensewoodyencroachmentbyVachelliakarroothatreceivedminor
applicationsofAS(146kgha-1yr-1)andmoderateapplicationsofSP(233kgha-1yr-1).[Depictedistheauthor
AntoniMilewskiwhoconfirmedconsentforpublication].
https://doi.org/10.1371/journal.pone.0179848.g002
toshowconsiderablevariationaccordingtotheformerlevelsoffertilizationamongthe60
plots(Fig2).
Thetreesandshrubs(hereaftercollectivelyreferredtoastrees)whichhaveencroachedto
thegreatestextentontheexperimentalsiteatTowoombaareV.karrooandtheconfamilial
Dichrostachyscinerea,bothofwhicharecapableofsymbioticfixationofNinrootnodules
[29](S2Table).Otherplantfamiliescontributingtowoodyencroachmentinthestudyarea,
butincapableofN-fixation,includee.g.Anacardiaceae(Searsialancea,S.leptodictya,S.pyr-
oides),Tiliaceae(Grewiaflavescens),Combretaceae(Combretumimberbe),Ehretiaceae(Ehretia
rigida)andEbenaceae(Diospyroslycioides).
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Constraintsonwoodyencroachmentafterfertilization:Effectsofanabolicvscatabolicnutrients
Thecommonperennialgrassesintheexperimentalsitehaveatussockformandthereisa
negligibleincidenceoflawn-formingspecies(S1Text).Allgrasseshavegrownspontaneously
withintheexperimentalsite,withnonebeingdeliberatelyplanted.Themainspeciesatthe
startoftheexperiment–Themedatriandra,CymbopogonpospischilliandHeteropogoncontor-
tus–immediatelygavewaytoothers,theswardincertainexperimentalplotschangingtempo-
rarilyfromperennialtussockstoannualsleavingbaregroundinthedryseason[42].Fifteen
yearsafterthestartoftheexperiment,Louw[42]foundthefollowingshiftsinthefloristic
compositionofperennialgrassesintheplots:withmaximalapplicationofAS,Panicummaxi-
mum,Urochloamosambicensis,EragrostisrigidiorandBrachiariaserrataincreasedinabun-
dance;withmaximalapplicationofSP,Hyparrheniahirta,P.maximum,U.mosambicensisand
Sporobolusstapfianusincreasedinabundance;andwithmaximalapplicationofbothASand
SP,therewasa60-foldincrease[42]intheabundanceofU.mosambicensis,afavouritefoodof
bovinelivestock[43].Anytendencytowoodyencroachmentduringthethreedecadesoftreat-
mentwasnotapparentbecauseofannualcuttingofallbiomassaboveaheightof5cm.
ApplicationsofASandSPintheexperimentalsiteshowedtoxiceffectsonseveralspeciesof
grasses[34],butboostedtheoverallproductionofhayasfollows:thesmallestharvests(control
treatments)averaged1.4tha-1overthreedecadesandthegreatestharvestsaveraged5.3tha-1
byvirtueofmaximalapplicationsofSP(466kgha-1)andnext-to-maximalapplicationsofAS
(583kgha-1).Anaverageof5.2tha-1ofhaywasharvestedinthetreatmentwithmaximal
applicationofbothASandSP(S3Table).Thistreatmentlaterexperiencedtheleastwoody
encroachmentinthewholeexperiment.Thestatusofwoodyplantsinthestudyareaoverthe
fullcourseoftheTowoombaexperimentcanbesummarisedasfollows.
1. Before1949,theincidenceoftreesinthesouthernSpringbokFlatsisunrecorded,butthey
wereprobablyscarceinwhatwastobecometheexperimentalsite.
2. Duringtheperiod1949to1981,allexperimentalplotsweremaintainedfreeoftreesby
repeatedcuttingofbiomass.
3. Duringtheperiod1982to1985,someeffortsweremadetocleartreesthatstartedto
encroach(mainlybycoppicing)ontheexperimentalsite,buthaywasnolongerharvested.
4. Duringtheperiod1986tothepresent,theexperimentalsitewaspassivelyprotectedwith
strictlimitationsongrazingandburning.Onlytwowildfires(2010and2012)occurred.
Treeswerethereforefreetoestablishandgrowamongthedensecoverofperennialgrasses
intheexperimentalplots.
Treesalreadyoccurredallaroundtheexperimentalsiteinunfertilisedsavannaby1985.
However,woodyencroachmenthasbeensovariablethatinsomeplotstreeabundancenow
exceedsthatobservedinmostoftheremainingrangelandontheSpringbokFlats,whereasin
otherstreeabundanceisnegligibleandthusprobablylessthanthatfoundbythefirstEuro-
peanfarmersaround1870.Weassumedthatseedsfromtreessurroundingtheexperimental
sitewouldhavedispersedacrossallplotsandthatvariationinthesuccessoftreesinindividual
plotsreflectedsoilconditionsandcompetitionwithgrassratherthananylackofpropagules.
AlthoughtheoriginalintentionoftheTowoombaexperimentwassimplytofertilisewith
N,S,PandCa,thetreatmentsinevitablyaffectedothernutrientsviacomposites,additivesand
contaminantsinthefertiliserformulas,depletioninharvestedhay,andchangestoavailability
ofcertainnutrientsbyvirtueofchangesinpH.TheonlyfertiliserseverappliedwereASand
SP(aspertreatmentspresentedinTable1).TheSPfertiliseravailablefortheexperimental
treatmentduringthemiddleofthetwentiethcenturywasabouttwo-foldpoorerinPthanthat
availabletoday,andwasnotyetdeliberatelyenrichedwithZn.TheoriginofthisoriginalSP
PLOSONE|https://doi.org/10.1371/journal.pone.0179848 June29,2017 6/24
Constraintsonwoodyencroachmentafterfertilization:Effectsofanabolicvscatabolicnutrients
wouldhavebeenrockphosphateminedinMoroccoandlaterlocallyinSouthAfrica(Phala-
borwaand/orLangebaan).Atthattime,thefertiliserproducedfromsuchrockphosphates
containedsomeMgandimpuritiesincludingvarioustracemetals[44].
Samplingandlaboratorymethods
AttheheightofthedryseasoninlateSeptember2014,compositesamplesofthesoilsurface–
i.e.pedoderm[45]–weretakenfromalltreatmentplotstoadepthof2cmusingaglassjar.
Thepedodermwassampledbecausethisisthelayerofsoilinwhichcompetitionbetween
grassesandtreeseedlingsislikelytobeatitsmostintensei.e.shortlyaftergerminationofthe
treeseed.Glasswasusedtopreventanycontaminationofthesoilsamplewithtraceelements
frommetallicsamplingdevices.Eachcompositesamplecomprised6–8subsamplesofsoil
takenrandomlyfromwithineachplot.Areasundertreecrownswereavoided,andanysurface
leaflitterwasremovedpriortosampling.Soilsampleswereair-driedandsievedto<2mm.
PermissiontocollectsoilsampleswasgrantedbytheLimpopoDepartmentofAgriculture.
Awiderangeofanalyseswasundertakenonthecompositesamplestomeasurenutrient
concentrationsandotherpropertiesofthesoils.pH(H O)andelectricalconductivity(EC)
2
weredeterminedusingsoil/distilledwaterratiosof1:5and1:2.5,respectively.pH(KCl)was
determinedina1MKClsolutionusingasoil/KClsolutionratioof1:2.5[46].Exchangeable
aciditywasdeterminedusing0.5mollitre-1potassiumsulfateandanalysedbymeansofvolu-
metrictitration[46].Acidsaturationwasthencalculatedusingtheformula:Acidsaturation=
(Exchangeableacidity(cid:4)ECEC)×100;whereECEC=Ca+Mg+K+Na+exchangeableacid-
ity.Water-dispersibleclay(WDC)wasdeterminedusingthemethoddescribedinSetaand
Karathanasis[47].InductivelyCoupledPlasmaMassSpectrometry(ICP-MS)wasusedtoana-
lyse:(i)Na,Mg,KandCa(extractedin1%citricacid)[48];(ii)P(extractedin1%citricacid
[49];(iii)S(extractedincalciumphosphate[50];(iv)B(extractedinhotwater[51];and(v)
Mn,ZnandCu(extractedin0.02Mdi-ammoniumEDTA)[52,53].Cwasdeterminedusing
theWalkley-Blackmethod[54,55].TotalNwasdeterminedusingtheDumasCombustion
method[56,57].Ammoniumandnitratewereextractedusing1moldm-3potassiumchloride
andanalysedusingthecalorimetricmethod[46].
Intermsoftoxicityeffectsintheexperiment,wenotethatlong-termapplicationofSPcan
potentiallycontaminatesoilswithheavymetals,particularlyAs,CdandPb[44].Althoughwe
didnotanalyseforthesetoxicelements,wenotethatanyadverseeffectsbyAs,CdandPb
wouldhavelittleexplanatorypowerintermsofwoodyencroachmentbecauseapplicationof
SPdidnotsignificantlyaffecttheeventualabundanceoftrees.
ThenumberandheightoftreesineachexperimentalplotwasrecordedbetweenNovember
2011andJanuary2012.Wereferthroughoutthispapertonumbersofindividualtreesperplot
as‘treeabundance’.Aspreviouslymentioned,theexperimentalsitehaslargelybeenprotected
fromfiredespitethecessationofexperimentaltreatments.Onlytwoaccidentalfireshave
occurred(duringthedryseasonsof2010and2012),withnonoticeableeffectsontree
abundance.
Dataanalysis
StatisticaltestsconductedonthedatacollectedincludedFriedmantests[58],Kruskal-Wallis
one-wayanalysesofvariancebyranks[59],Pearsoncorrelationcoefficients,principalcompo-
nentanalyses(PCA),boundarylineanalyses,andKruskal-Wallisranksumtests.Alldatawere
analysedusingthestatisticalsoftwareRversion3.3.0[60].Non-parametricstatisticalmethods
wereusedforanalysingtheresultsoftherandomisedblockdesignexperimentbecauseofthe
relativelysmallnumberofreplicatesi.e.fourplotspertreatment.WhereFriedmantesting
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Constraintsonwoodyencroachmentafterfertilization:Effectsofanabolicvscatabolicnutrients
showedoverallsignificantdifferencesacrosstreatments(p<0.05),Kruskal-Wallisone-way
analysesofvariancebyrankswereusedtoinvestigatewhethertheASand/orSPtreatments
hadsignificanteffectsonallmeasuredvariables.PrincipalcomponentanalysesusingRpack-
age“ade4”[61]wereusedtodepictintwodimensionalspacetherelationshipbetweentree
abundanceandallmeasuredsoilproperties.Boundarylineanalyses[62,63]wereundertaken
onallscatterplotsoftreeabundanceversussoilpropertiesaswellasall91possiblenutrient
ratios.Thelikelihoodofaparticularboundarylinepatternbeingaresultofchancewascalcu-
latedbyrunning100,000iterationsofrandomiseddataforeachscatterplot(S2Text).Lastly,
Kruskal-Wallisranksumtests,aswellasmultiplecomparisonsrunwiththeRfunctionnpar-
comp(usingTukeyspecialcontrastandalogittransformation),wereusedtodeterminewhich
soilpropertieschangedsignificantly(p<0.05)acrossfourdifferentcategoriesoftreeabun-
dance,namely0–1,2–4,4–8and>8treesperplot.
Results
Ammoniumsulphateapplicationsovertheperiod1949to1981hadmarkedeffectsonsubse-
quentwoodyencroachment:thegreaterthefertilizationwithAS,themoreconstrainedwas
woodyencroachmentintermsofbothtreeabundance(numbersofindividuals)andcumula-
tiveheightsofthesametrees(Fig3andS5Table;p<0.001).Controlplotshadamaximumof
30andanaverageof16.3(±5.7)treesperplotcomparedwithamaximumof3andanaverage
of1.3(±0.8)treesperplotinplotsfertilisedwith1166kgha-1ofAS(Fig3andS8Table).By
contrast,fertilizationwithSPdidnothaveasignificanteffectontreeabundance(Fig3andS6
Table).
Treeabundancewasstronglycorrelatedwiththecumulativetreeheightinthesameplots
(Pearsoncorrelationcoefficientof0.92,S7Table);ofthesetwovariables,treeabundancegen-
erallyshowedthestrongerrelationshipswithsoilpropertiesandwethereforeconcentratedon
thisvariable.Thewoodyfloravariedlittleamongexperimentalplots–beingdominatedbyV.
karrooupto5.3mhigh(S2Table).
Fig3.AbundanceandcumulativeheightoftreesaccordingtoexperimentaltreatmentatTowoombain2011/12.Box-plotsshowmedians(darkbar),
upperandlowerquartiles(topsandbottomsofboxes),andranges(upperandlowerwhiskers).AS=ammoniumsulphate;SP=superphosphate.pvalues
showtheresultsoftheKruskal-WallistestingforindividualeffectsofASandSPafterFriedmantestingfordifferencesacrossalltreatments.
https://doi.org/10.1371/journal.pone.0179848.g003
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Constraintsonwoodyencroachmentafterfertilization:Effectsofanabolicvscatabolicnutrients
Theresultsofexperimentaltreatmentsonsoilpropertiesshowedstatisticallysignificant
effects(p<0.05,FriedmanandKruskal-Wallistesting)ofASapplicationonpH,acidity,acid
saturation,Mg,Ca,Mn,CuandZn(Fig4;Table2andS8Table).Comparisonsbetweencon-
trolplotswiththosereceivingthemaximalASapplicationshowedareductioninpH(H O)
2
(6.4±0.1to5.1±0.2),areductioninpH(KCl)(5.5±0.2to4.0±0.1),anincreaseinacidity
(0.7±0.1to2.6±0.3cmolkg-1;263%increase),anincreaseinacidsaturation(8±2to
40±5%;432%),anddepletionofthefollowingnutrients:Mg(386±25to143±15mgkg-1;
63%reduction),Ca(1022±180to322±14mgkg-1;69%),Mn(314±11to118±9mgkg-1;
62%),Cu(3.6±0.3to2.3±0.2mgkg-1;36%),Zn(6.6±0.4to3.7±0.4mgkg-1;43%),respec-
tively(S9Table).Bycontrast,comparisonsbetweencontrolplotswiththosereceivingthemax-
imalSPapplicationshowedstatisticallysignificanteffectsonCa(1022±180to1395±95;36%
increase),P(14±1.5to124±3.0;820%)andZn(6.6±0.4to7.7±0.9;17%)(Table2andS9
Table).Notably,therewasnoeffectofASorSPapplicationonK,S,N,NH ,NO orB.
4 3
Teststatistics(Hvalues)andpvaluesfromnon-parametricKruskal-Wallisone-wayanaly-
sesofvariancebyranksfollowedbyFriedmantests.
Soilpropertiesshowingstrongcorrelations(i.e.>0.8)included:MgwithCa(0.84);Mg
withMn(0.84);CawithMn(0.80);CawithZn(0.81);BwithC(0.84);CuwithMn(0.88);EC
withB(0.80);MnwithpH(KCl)(0.84);CawithpH(KCl)(0.83);MgwithpH(KCl)(0.88);and
acidsaturationwithMg(-0.82)(S7Table).InaPCAshowingseparationofellipseswithtree
abundancesof0–3individualtreesperplotfromthosewithfourormore(Fig5),centroidsof
ellipseswereseparatedfromeachotherwithrespecttobothcomponentsinthedirectionof
pH,acidity,acidsaturation,MnandCu.
Soilpropertiesandnutrientratiosthatshowedstrongrelationshipstotreeabundancein
bothboundarylineanalysesandKruskal-WallisranksumtestsincludedpH(KCl),soilacidity,
acidsaturation,B,Mn/Cu,Mg/CuandCa/P(Figs6and7andS3Fig).OnlytheratioMn/Cu
hadanupperboundarylinedepictingastrongconstraintontreeabundancewithadecrease
oftheratioaswellasalowerboundarylineshowingapredictableincreaseintreeabundance
astheratioincreasedtowardsitsmaximalvalue.
Allrawdatausedinthisstudyisprovidedasanonlinefile(S1RawData).
Discussion
Applicationofammoniumsulphate(AS)overthreedecadesinthesavannaecosystemofthe
SpringbokFlatsgreatlyconstrainedwoodyencroachmentoverthesubsequentthreedecades.
Bycontrast,applicationofsuperphosphate(SP)–despiteboostingtheconcentrationsofPin
soilsbymorethananorderofmagnitudeandwiththeeffectstillevidentthreedecadesafter
thelastapplicationofSP–hadnoeffectontreeabundance(Figs2and3).
AlthoughthemainpurposeofASapplicationwastofertilisewithN,andalthoughNis
regardedasoneofthemostimportantnutrientsforplants[64,65],ouranalysesshowedthat
thiselementpersehadnegligibleeffectsonwoodyencroachment.Thesoilpropertiessignifi-
cantlyaffectedbyASapplication,andthereforepossiblyconstrainingtreeabundanceacross
thesite,wereratherpH,soilacidity,acidsaturation,Mg,Ca,Mn,CuandZn(Fig4).Such
effectsofASonsoilpropertiesmaybeexpectedbecausethisformoffertiliserisknowntoacid-
ifysoilswithconcomitantlossesofcationsvialeaching[66,67].Furthermore,majorreduc-
tionsinpH,MgandCaasaresultofASapplicationintheTowoombaexperimenthadalready
beenreportedbyDonaldsonetal.[34].
Whereacidificationwasextreme–e.g.plotswithpH(KCl)lessthan4andacidsaturation
greaterthan40%–Altoxicityislikelytohaveaffectedplantgrowth.Itisconceivablethattree
seedlingsintheTowoombaexperimentweremoresusceptiblethangrassestoAltoxicityand
PLOSONE|https://doi.org/10.1371/journal.pone.0179848 June29,2017 9/24
Constraintsonwoodyencroachmentafterfertilization:Effectsofanabolicvscatabolicnutrients
Fig4.ConcentrationsofsoilpropertiesaccordingtoexperimentaltreatmentatTowoombain2014.Box-plotsshowmedians(darkbar),upperand
lowerquartiles(topsandbottomsofboxes),andranges(upperandlowerwhiskers).AS=ammoniumsulphate;SP=superphosphate.pvaluesshowthe
resultsoftheKruskal-WallistestingforindividualeffectsofASandSPafterFriedmantestingfordifferencesacrossalltreatments.
https://doi.org/10.1371/journal.pone.0179848.g004
thatsomeoftheconstraintonwoodyencroachmentevidentinFigs6and7isattributableto
Altoxicity.However,boundarylineanalyses(Figs6and7)andKruskal-Wallisranksumtests
showedmarkeddeclinesintreeabundanceacrossrangesinpH(KCl)(e.g.4.5–6)andacidsat-
uration(e.g.5–25%)inwhichAltoxicityisimplausible.Therearethereforelikelytobefactors
beyondacidificationthatunderminedthecompetitivepoweroftreesatTowoomba.
Boundarylineanalysesandcategorisationsofthedataaccordingtotreeabundanceallowed
ustodistinguishtwocategoriesofvariables,namelythoseensuringconstraintoftreeabun-
danceandthoseensuringpromotionoftreeabundance.Inadditiontosoilacidityandacid
saturation,Ca/P,Mn/CuandMg/Cuwereassociatedwithconstraint,whileMn/CuandB
PLOSONE|https://doi.org/10.1371/journal.pone.0179848 June29,2017 10/24