Segregating the Effects of Seed Traits and Common Ancestry of Hardwood Trees on Eastern Gray ...

16 Pages·2015·0.57 MB·English

RESEARCHARTICLE
Segregating the Effects of Seed Traits and
Common Ancestry of Hardwood Trees on
Eastern Gray Squirrel Foraging Decisions
MekalaSundaram1*,JannaR.Willoughby1,NathanaelI.Lichti1,MichaelA.Steele2,Robert
K.Swihart1
1 DepartmentofForestryandNaturalResources,PurdueUniversity,WestLafayette,Indiana,UnitedStates
ofAmerica,2 DepartmentofBiology,WilkesUniversity,Wilkes-Barre,Pennsylvania,UnitedStatesof
America
* [email protected]
Abstract
Theevolutionofspecificseedtraitsinscatter-hoardedtreespeciesoftenhasbeenattrib-
utedtogranivoreforagingbehavior.However,thedegreetowhichforaginginvestments
andseedtraitscorrelatewithphylogeneticrelationshipsamongtreesremainsunexplored.
OPENACCESS
Wepresentedseedsof23differenthardwoodtreespecies(familiesBetulaceae,Fagaceae,
Citation:SundaramM,WilloughbyJR,LichtiNI,
Juglandaceae)toeasterngraysquirrels(Sciuruscarolinensis),andmeasuredthetimeand
SteeleMA,SwihartRK(2015)Segregatingthe
distancetravelledbysquirrelsthatconsumedorcachedeachseed.Weestimated11physi-
EffectsofSeedTraitsandCommonAncestryof
HardwoodTreesonEasternGraySquirrelForaging calandchemicalseedtraitsforeachspecies,andthephylogeneticrelationshipsbetween
Decisions.PLoSONE10(6):e0130942.doi:10.1371/ the23hardwoodtrees.Variancepartitioningrevealedthatconsiderablevariationinforaging
journal.pone.0130942 investmentwasattributabletoseedtraitsalone(27–73%),andcombinedeffectsofseed
Editor:HepingCao,USDA-ARS,UNITEDSTATES traitsandphylogenyofhardwoodtrees(5–55%).AphylogeneticPCA(pPCA)onseedtraits
Received:February19,2015 andtreephylogenyresultedin2“global”axesoftraitsthatwerephylogeneticallyautocorre-
latedatthefamilyandgenuslevelandathird“local”axisinwhichtraitswerenotphyloge-
Accepted:May27,2015
neticallyautocorrelated.Collectively,theseaxesexplained30–76%ofthevariationin
Published:June25,2015
squirrelforaginginvestments.ThefirstglobalpPCAaxis,whichproducedlargescoresfor
Copyright:©2015Sundarametal.Thisisanopen
seedspecieswiththinshells,lowlipidandhighcarbohydratecontent,wasnegatively
accessarticledistributedunderthetermsofthe
relatedtotimetoconsumeandcacheseedsandtraveldistancetocache.Thesecond
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany globalpPCAaxis,whichproducedlargescoresforseedswithhighprotein,lowtanninand
medium,providedtheoriginalauthorandsourceare lowdormancylevels,wasanimportantpredictorofconsumptiontimeonly.ThelocalpPCA
credited.
axisprimarilyreflectedkernelmass.Althoughitexplainedonly12%ofthevariationintrait
DataAvailabilityStatement:Allrelevantdataare spaceandwasnotautocorrelatedamongphylogeneticclades,thelocalaxiswasrelatedto
withinthepaperanditsSupportingInformationfiles.
allfoursquirrelforaginginvestments.Squirrelforagingbehaviorsareinfluencedbyacombi-
Funding:ThisstudywasfundedbyaFredM.van nationofphylogeneticallyconservedandmoreevolutionarilylabileseedtraitsthatisconsis-
EckscholarshipawardedtoMSbytheFredM.van
tentwithaweakormorediffusecoevolutionaryrelationshipbetweenrodentsandhardwood
EckFoundationforPurdueUniversity,andtheJohn
treesratherthanadirectcoevolutionaryrelationship.
S.WrightEndowment.Thefundershadnorolein
studydesign,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
PLOSONE|DOI:10.1371/journal.pone.0130942 June25,2015 1/16
Phylogenetic&EcologicalDeterminantsofGraySquirrelForaging
Introduction
Scatter-hoardingrodentsinfluenceseedlingestablishmentinmanycommunitiesbyactingas
seeddispersalagentsandseedpredators[1,2].Whereasmostseedsharvestedbyrodentsare
ultimatelyconsumed[1,3],manyrodentsenhancetheprobabilityofgerminationandestab-
lishmentbycachinginsuitablemicrosites[4]andthensubsequentlyfailingtorecoverapor-
tionoftheseseeds[1,5].Mostrodentscantherefore,easilyshiftfrommutualismtoseed
predation,andthisconditionalnatureofmutualismbetweenseed-bearingtreesandscatter-
hoardersisadelicateone[1]thatoftenfollowsfromtherodents’responsestoseedcharacteris-
tics[2,6].
Fromtheperspectiveofarodent,seedpreferenceandseedhandlinginvolveasequenceof
behavioraldecisions.Ateachstepintheprocess,scatter-hoarderspresumablyevaluatecosts
andbenefitsassociatedwithalternativestomaximizeuseofresources[7,8].Experimentswith
artificialandnaturalseedssuggestthatseeddispersalandhandlingbehaviorisrelatedtothe
perceivedvalueofaseed,orthebenefitsofattractivetraitsdiscountedbythecostsleviedby
defensiveseedtraits[8,9].Whilenumerousstudieshavefocusedontherelationshipbetween
seedtraitsandfate,fewerhaveexaminedtheinfluenceofseedtraitsonthespecificbehavioral
decisionsofthescatterhoarder[10,11].Fine-scaleassessmentsoftheseedhandlingprocess
provideevidencethatrodentsevaluatetheconditionofseeds(e.g.bypawmanipulationin
Sciurusniger),whichmaybepredictedbytraitsindicatingseedqualityorcondition[11].
Fromtheperspectiveofatree,someseedtraitsmayimprovetheprobabilityofbeingcached
orconsumed,therebyinfluencingfitness[12].Creatinganattractiveseed(e.g.,withahigh
caloricvalue)incomparisontothatofcompetitorsisbeneficialbecausesuchseedsmaybedis-
persedlongerdistances[13,14,15]reducingdensity-dependentmortalityfromseedlingcom-
petitorsorseedpredation[16–18].Ontheotherhand,aseedwithdefensivetraitssuchasa
hardshellmaybebeneficialbecausesuchseedsincreasehandlingcostsandinduceseedcach-
ingbehaviorstherebyincreasingseedsurvival[10,19–22].SeedsofhardwoodtreesinChina,
forexample,showtrade-offswithrespecttoinvestmentinseedtraitsthatlikelyinfluencehan-
dlingcostsandseeddispersal[12].
Observationsofseedhandlingandseedchemistryprovideabundantevidenceforreciprocal
evolutionaryeffectsbetweenrodentsandwoodyplants[23].Intreesquirrels,suitesofbehav-
ioraladaptationsandmorphologicaladaptationshaveresultedfromselectivepressuresassoci-
atedwithseedmorphologyandchemistry[23].Forexample,gradientsoftannin
concentrationshavebeenobservedinoakkernels,whichimpartphysiologicalandmetabolic
coststorodentsandresultinportionsofkernelsbeingrejected[24–27].Inresponse,some
rodentshaveadaptedtodetoxifytannins[28,29],andmanycacheacornswithhightannin
concentrationsmorefrequently,possiblytoavoidordelaythecostsassociatedwithingesting
tannins[30,31].Intrees,changesinseedchemistryandmorphologyhaveevolvedinresponse
topredationanddispersalpressuresexertedbytreesquirrels[23].Forinstance,whiteoak
seedsshownodormancyandmultiple-seededacornspossiblytominimizelengthofexposure
topredationandescapemortalityfromembryo-excisionbehaviorsbyeasterngraysquirrels
[31,32].Thusfarallapproachestorodent-treecoevolutionhavecollectedsomecombinationof
detailedmorphological,physiological,behavioral,andbiogeographicdata[23].Whilethese
studiessuggestthatcoevolutionaryinteractionsexistbetweengranivorousrodentsandhard-
woodtrees,thestrengthofthisinteractionisvariable[23].
Coevolutionaryrelationshipsbetweenplantsandseeddispersershavebeendescribedacrossa
continuum,rangingfromstrong,pairwiseinteractionstoweak,diffuseinteractions.Pairwise
coevolutionoccursbetweenspecificspeciesandleadstostrongselectivepressuresontraits[33].
Recentobservationsofacorn(Quercusspp.)embryoexcisionbehaviorshownbynaïvesquirrels
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Phylogenetic&EcologicalDeterminantsofGraySquirrelForaging
(Sciuridaespp.;[31,32])isanexampleofabehavioraladaptationandsuggestsastrongandper-
hapspairwisecoevolutionaryrelationshipbetweenoaksandsquirrels[31,34].Incontrast,weak
selectivepressuresduetodisparitiesinevolutionaryratesofwoodyspeciesandanimaldispers-
ers,unpredictabilityofconditionsforseedgermination,andaplethoraofotherfactorsleadsto
diffuseorweakcoevolution[35,36].
Tobetterunderstandthenatureofcoevolutionaryinteractionsbetweenhardwoodtree
seedsandsquirrels,weusedacombinationofphylogeneticandecologicalmethodstodeter-
minehowseedcachingandseedconsumptionbehaviorsofeasterngraysquirrels(Sciuruscar-
olinensis)variedasafunctionofseedtraitsandphylogeneticrelationshipsof23hardwood
treesspecies.Specifically,wehypothesizedthatgraysquirrelswouldinvestmoretimeand
travellongerdistancestoconsumeandcacheseedswithstrongphysicaldefensesandhigh
nutrientvalues.Inaddition,wepredictedthatconspecificinterferencewouldberelatedto
physicaldefensesofaseedandnutrientvalueofaseed.Weincorporatedphylogeneticrelation-
shipsofhardwoodtreesintoouranalysestoevaluatetherelativeproportionofgraysquirrel
seedhandlingbehaviorsthatisinfluencedbycommonancestryofhardwoodtreespeciesand
todeterminewhichphylogeneticallyrelatedseedtraitsinfluencehandling.Wehypothesized
thatifstrongcoevolutionaryrelationshipsoccurbetweengraysquirrelsandhardwoodtreespe-
cies,mostofthevariationingraysquirrelbehaviorwouldbeexplainedbyseedtraitswith
strongphylogeneticsignals.However,weaker,morediffusecoevolutionaryrelationships
wouldbeexplainedbyamixofseedtraitswithastrongphylogeneticsignalandthosetraits
withnophylogeneticsignal.Finally,ifnocoevolutionaryrelationshipsoccur,phylogenetically
conservedtraitsshouldexplainasmallportionofthevariationingraysquirrelbehavior.
MaterialsandMethods
Seedhandlingmetrics
Free-rangingeasterngraysquirrelsonthecampusofPurdueUniversitywerepresentedwitha
sequenceofseedtypesinpseudo-randomorderbetween1October2011and15February2012.
Seedswereobtainedfromcommercialseedcompanies(F.W.SchumacherCo.,EastSandwich,
MAandSheffield’sseedCo.,Locke,NY)andfrombeneathtreesoncampus.Seedswerestored
at4°C,separatedbyspeciesinplasticcontainers.Seedpresentationswereconductedbytossing
aseedtoasquirrelfrom1–4m.Ifthesquirrelretrievedtheseed,theobservervideorecorded
theeventuntiltheseedwasconsumedorcached.Whentheseedwasconsumed,theobserver
recoveredtheremainsoftheseedkernelandshellbysiftingthroughthelitterandsoil.Thedis-
tanceasquirrelmovedtocacheorconsumeaseedwasdeterminedbyretracingthepathofthe
squirrel,markingthefinaldestination,andthenmeasuringthestraight-linedistancebetween
thepointofencounteroftheseedandthefinallocation.Ifthedistancetravelledwastoogreat
tobeaccuratelyestimatedwithatapemeasure,aGPS(GarminModelNo.72)wasusedto
obtaincoordinates.Straight-linedistancewasthencalculatedinArcGIS10(EnvironmentalSys-
temsResearchInstitute2010).Asecondseedwaspresentedtoasquirrelonlyafterthefirstseed
washandled,andtheprocessrepeateduntilsquirrelsnolongerrecoveredseeds.Presentations
ofthesameseedtypeswerespatiallyseparatedbyselectinglocationsthatwere>100mapart
toincreasethelikelihoodthatdatawerecollectedfromdifferentsquirrels.Exceptionswereper-
mittedonlyiftwodifferentsquirrelscouldberecordedsimultaneously.Theprotocolincluded
non-intrusiveobservationwithnohindrancetotheanimals,andtherefore,wedidnotseek
additionalapprovalfromPurdueAnimalCareandUseCommittee(PACUC).
Foragingtrialvideoswerecollectedfor23seedtypesfromthetreefamiliesBetulaceae,Faga-
ceae,andJuglandaceae(S1Table).Thetimetakentoconsumeorcacheeachseedwascalcu-
latedfromthevideos.Timerequiredtoconsumeandcacheaseedwasdefinedasthetime
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Phylogenetic&EcologicalDeterminantsofGraySquirrelForaging
neededtoconsumeandcompletelyburyaseedrespectively,anddidnotincludetraveltime.
Wequantifiedthenumberofrecordedtrialsperseedtypeinwhichthefocalsquirrelwas
chasedbyaconspecificwhilehandlingaseed,whichwedefineasconspecificinterference[37].
Seedtraits
Thevalueofeachseedtraitwasmeasuredfor3seedsforeachofthe23seedtypesunlessspeci-
fiedotherwise(S2Table).Percentageofmoistureinseedswascalculatedbydryingsamplesat
103°Cfor3days.Percentageoftannicacidequivalentsinnutmeatwasdeterminedbyperform-
ingradialdiffusionassays[38,39].Thevaluewasexpressedastannicacidequivalentsoccurring
in100gramsofdrynutmeat.Energycontentofthenutmeatwasobtainedinabombcalorime-
ter(PARR1262bombcalorimeter,ParrInstrumentCo.,Moline,IL)usingbenzoicacidasa
calibrationstandard.Thevaluewasadjustedtoobtaincaloriespergramofdryweight.Seed
hardnesswasestimatedasthepeakload(kilograms)requiredtobreakaseed’sshellandpierce
thenutmeat.TestingwasdonewithaMTS/Sintechcomputerizedtestingmachine(MTSCor-
poration,EdenPrairie,MN)usingacrossheadfashionedaftertheskullofaneasterngray
squirrelanddesignedtomimicasquirrel’sincisoraction(S1Fig).Onlytheupperincisors
wereusedtoobtainhardnessestimates.Peakloadwithin5mmcrossheaddisplacementwas
sufficienttobreaktheseedshellofmostseedsandwasusedasanestimateofseedhardness.
Onlybutternut(Juglanscinerea)seedshellswerenotpiercedwiththe5mmcrosshead;there-
fore,10mmdisplacementwasemployedforthisseed.Seedshellthicknesswasdeterminedby
visualizingthecrosssectionofseedshellswithastereoscopicmicroscopeattachedtoaNikon
ImagingSystemat4.91micrometersperpixel.Proximateanalysiswasperformedfollowing
AssociationofOfficialAnalyticalChemists(AOAC)protocolstoestimatepercentageofcrude
proteins(Kjeldahl,AOACOfficialMethod984.13A-D,2006),fats(etherextraction,AOAC
OfficialMethod920.39A,2006)andcarbohydrates(differencemethod,FAO2003)innut-
meat.Wecombinedkernelsfrommultipleseedstoobtainatleast10gofdriedmaterial,and
duplicatesampleswereusedinproximateanalysesforeachseedtype.Finally,weusednumber
ofdaysofcoldstratificationrequiredbeforegerminationinachamberasanindexofdormancy
periodofseedtypes.Estimatesofcoldstratificationdayswereobtainedfromtheliterature
[40–42],andaverageswerecomputedandusedwheneverarangeofdayswasprovided.
Molecularandphylogeneticmethods
Weconductedaphylogeneticanalysistoestimateandincorporateevolutionaryrelationships
betweenhardwoodtreespeciesintoanalysesinthisstudy.Toachievethisgoal,wefirst
extractedDNAfromplanttissueusingtwomethods.Forthemajorityofspecies,weextracted
DNAfromleaftissueusingaphenol-chloroformprotocol[43],modifiedbygrindingfreshleaf
tissuewith500mgPVPPinliquidnitrogen[44].WeusedthePowerPlantProDNAIsolation
Kit(MoBioLaboratories,Inc;Carlsbad,CA)toextractDNAfromCaryatomentosaleaftissue
andembryosremovedfromtheseedsofQuercusprinusandQuercusbicolor.
Tocreateaphylogenyforour23hardwoodtreespecies,weusedsequencedatafromtwo
chloroplastgenes-ribulose-bisphosphatecarboxylaseoxygenaselargesubunit(rbcL)and
maturaseK(matK)aswellaportionofthenucleargenomethatincludedtheinternaltran-
scribedspacer1,5.8SrRNAgene,andtheinternaltranscribedspacer2(ITS).Whenpossible,
weobtainedrelevantsequencesfromGenbank(S6Table).Fortheremainingspecies,we
sequencedeachsampleusingpolymerasechainreaction(PCR)onanEppendorfMastercycler
(Eppendorf,Westbury,NewYork).Initialamplificationwasperformedin20μLreactionscon-
tainingapproximately40ngtemplateDNA,1unitofNEBTaqpolymerase,0.3lMofeach
primer,1.5mMMgCl2,10mMTris-HCl,50mMKCl,0.5mg/mlBSA,and0.2mMofeach
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dNTP.PCRparametersincludedaninitialdenaturingtemperatureof95°Cfor30secondsfol-
lowedby30cyclesof95°Cfor30secondsdenaturing,annealingtemperatureof53°CforrbcL
andmatK[45]and58°CfortheITSgene(primersITS5andITS4;[46]),and72°Cfor1minute
elongation,withafinalelongationtemperatureof72°Cfor5minutes.Wecleanedtheresulting
PCRproductwiththeMinElutePCRPurificationKit(Qiagen,Valencia,California).Sanger
sequencingreactionswereperformedat10μLandcontained50ngoftemplateandBigDye
3.1.WeusedanABIPrism3730XLsequencer(AppliedBiosystems,FosterCity,CA,USA)and
trimmedsequencesinSequencher4.7(GeneCodesCorp.,AnnArbor,MI).
WealignedthesequencesforeachgeneusingM-coffeewebserverhostedbytheCentrefor
GenomicRegulation[47,48].Wetestedforcongruenceofspeciespairwisedistancematrices
derivedforthe3alignedgenesinthisstudybycalculatingKendall’sWstatisticofconcordance
asimplementedbythefunction‘CADM’withinthepackage‘ape’inR[49].Afterrejectingthe
nullhypothesisofincongruencebetweendistancematrices,weconcatenatedsequencesofthe
3genesforfurtheranalyses.Thefunction‘modelTest’inthepackage‘phangorn’wasusedto
selectthebestnucleotidesubstitutionmodelfortheconcatenatedsequences[50].ModelSYM
(symmetricalmodel;[51])+G(gammadistributedratevariation)wasselectedbasedonBIC
values.
Wegeneratedaphylogenyforour23speciesusingtheprogramBEAST[52],andprepared
ourinputfileusingBEAuti[53].Weusedarelaxedlognormalmolecularclock[54],theYule
speciationmodel[55,56],andRubusoccidentalisasanoutgroup.Weran5independentruns
of50millionsteps,thinningtoevery1000trees.Weconfirmedconvergenceofeachrunwith
Tracer,andfilteredthefirst10%eachgroupofrunsusingTreeAnnotator.
Statisticalanalyses
WeestimatedpairwiseSpearmanrank-correlationsbetweentheseedhandlingmetricsandall
seed-traitvaluesinthisstudy.WealsocomputedMoran’sImetricofphylogeneticsignal[57]
forallseedtraitsandevaluatedsignificancebypermutingseedtraitvaluesacrosstipsofthe
phylogeny1000times.Forallfurtherstatisticalanalyses,weincludedthefollowingseedtraits
inourseedtraitmatrix:percentlipids,percentcarbohydrates,percentproteins,caloricconcen-
tration,dormancyperiod,loghardness,logshellthickness,loginteractionofhardnessand
thickness,logkernelmass,logshellmass,andlogtanninconcentration.Naturallogtransfor-
mationwasusedforseedtraitswithskeweddistributions.Beforelog-transformingtannin
concentrations,alowvalueof0.35%TAEwasassignedtothe3seedswith0%TAEtoavoid
undefinednumbers.
Weincorporatedthephylogenyofhardwoodtreesintoourstatisticalanalysesusingtwo
methods—variancepartitioninganalysesandphylogeneticPCA.Topartitionthevariationin
eachseedhandlingmetricbetweenseedtraitsandphylogeneticrelationships,weusedphyloge-
neticeigenvectorsandapartialregressionanalysisimplementedintheRpackage‘PVR’[58].
ThePVRmethodconvertsamatrixofdouble-centeredphylogeneticdistancesintoeigenvec-
tors,whicharethenusedaspredictorstoexplainvariationinatraitofinterest.Thefirstfew
eigenvectors,whichcapturemostofthevariationinthedistancematrix,representdifferences
betweencladesattherootofthephylogeny.Subsequenteigenvectorscapturevariationamong
taxaorgroupsclosertothetips.Selectionofalleigenvectorsaspredictorsinregressionsisnot
necessaryandmayresultinlargeorinflatedR2values.Incontrast,selectionoftoofeweigen-
vectorscanresultinresidualautocorrelationbecauseallofthephylogeneticdependenceisnot
captured.Forouranalyses,weselectedphylogeneticeigenvectorsthatminimizeresidualauto-
correlationasestimatedbyMoran’sI[59,60].ThePVRmethodthenusesthephylogenetic
eigenvectorsandenvironmentalvariables(i.e.seedtraits)aspredictorsinapartialregression
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Phylogenetic&EcologicalDeterminantsofGraySquirrelForaging
analysestopartitionthevariationinatrait.WeusedthePVRmethodtoobtainthevariation
ineachseedhandlingmetric(handlingtimeanddistancestravelledpriortohandlingfor
cachedandconsumedseeds)explainedbyseedtraitsalone,selectedphylogeneticeigenvectors
alone,informationsharedbetweenseedtraitsandselectedphylogeneticeigenvectors,and
finallyunexplainedfactors.
Next,weidentifiedlinearcombinationsofseedtraitsthatexhibitautocorrelationswiththe
hardwoodtreecladesobservedinourphylogeny.Weperformedaphylogeneticprincipalcom-
ponentsanalysis(pPCA)usingtheAbouheifmethod(equivalenttoMoran’sImetric)ofcom-
putingphylogeneticsignals[61].ThepPCAgroupsseedtraitsintopossible‘global’and‘local’
structuresgivenacandidatephylogenyandtraitsofinterest.The‘global’structuresconstitute
pPCaxesthatarepositivelycorrelatedtocladesattherootofthephylogeny.Largepositive
eigenvaluesrepresentaxesshowinglargevarianceandpositivephylogeneticautocorrelationor
Moran’sI.Incontrast,‘local’structuresconstitutepPCaxesandtraitswithnegativephyloge-
neticautocorrelationindicativeoftraitsthataredifferentbetweenrelatedtaxa.Largenegative
eigenvaluesrepresentaxesexplainingalargevarianceandaxeswithanegativeMoran’sI.
Together,theglobalandlocalpPCaxescansuggestspecificlifehistorystrategiesadoptedby
thetaxa.WeperformedapPCAonourseedtraitmatrixusingthefunction‘ppca’inthepack-
age‘adephylo’[62].Toensurethatinconsistenciesinthephylogenywerenotdrivingthe
observedrelationships,wecollapsedsubcladesintogroupsandperformedthepPCAasecond
time.Thecollapsedcladesinourphylogenyincluded-walnuts(Juglans),hickories(Carya),
whiteoakgroup(QuercussectionQuercus),redoakgroup(QuercussectionLobatae),and
chestnuts(Castanea).Inaddition,thetreecontainedthefollowingsingletontaxa—hazelnut
(Corylusamericana),beechnut(Fagusgrandifolia)andtanoak(Notholithocarpusdensiflorus).
WeretainedthefirstthreepPCaxes(2globaland1localasdeterminedbythesignofthe
eigenvalue)explainingthehighestproportionofvariation.WetestedourselectedpPCaxesfor
significantpositiveandnegativephylogeneticautocorrelationbyperformingAbouheif’stest
onthePCscoresusingthe‘abouheif.moran’function.Finally,weregressedthe4squirrelseed
handlingmetricsin4differentmultiplelinearregressionmodelsagainstour2globaland1
localpPCaxes.Plotsofresidualsindicatedunequalvariancesanddeparturesfromnormality,
soweevaluatedsignificanceofpredictorsbypermutingtheresponsevariable1000timesand
estimatingthenulldistributionofcoefficients.Finally,weperformedapost-hocPoisson
regressiontopredictnumberoftrialswhereaconspecificinterferedwithseedhandlingusing
our3pPCaxesaspredictors.Toevaluateifinterferencecausedanincreaseinhandlingtime,
weperformedapairedt-testcomparingaveragetimesspenthandlingaseedtypewheninter-
ferencewasnotobservedtowheninterferencewasnotobservedpoolingacrossseedtypes.All
analyseswereperformedinR3.1.0.
Results
Werecorded272foragingtrials,whichincluded5–7seed-consumptionand4–6seed-caching
trialsperseedtype(meanof5.04seed-consumptionand5.52seed-cachingtrials/seedtype).
Fortanoak(N.densiflorus)4cachingtrialswererecorded.Forblackwalnut(J.nigra),thedis-
tancetravelledtoconsumeaseedwascomputedfor4ofthe5consumptiontrials.Kernels
weretypicallyconsumedorcached(S1Table).
PairwiseSpearmanrankcorrelationsbetweenseedtraitsofthe23seedtypesrevealedsev-
eralpatternsofcorrelationbetweengroupsofseedtraits(S3Table).Nutrientandperishabil-
ity-relatedvariablessuchaspercentageofproteins,carbohydratesandlipidsinthekernel,
caloricconcentration,anddormancyperiodwerepositivelycorrelated.Physicaldefensivetraits
includinghardness,shellthickness,hardnessandthicknessinteraction,anddrymassofshell
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werealsopositivelycorrelated.Inaddition,lipidconcentrationwaspositivelycorrelatedto
shellthicknessandhardness.Tanninconcentrationofkernelswascorrelatedtoproteincon-
centrationanddormancyperiodonly(S3Table).
WesuccessfullyextractedDNAfromplanttissuefor12taxaforwhichsequenceswerenot
availableinGenBank(S6Table).Theconcatenateddatasetcontained1864sites,outofwhich
332wereparsimonyinformative.TopologyoftheBayesiantree(Fig1)issupportedbyother
existingphylogeniesandcurrenttaxonomicclassificationofthehardwoodtreesspecies[63–
67].Inaddition,posteriorprobabilitiesassociatedwiththecladeswereusually>0.9.Weak
nodesupportoccurredonlyatthetipsofthephylogenywithinthewhiteoakgroup(Quercus
sectionQuercus),andthesplitofshagbarkandpignuthickory(CaryaglabraandCaryaovata,
Fig1).
Afterdeconstructingourphylogenyintoeigenvectors,weperformedvariancepartitioning
toestimatetheproportionofsquirrelseedhandlinginvestmentsexplainedbyseedtraitsand
phylogeny(Table1).Seedtraitvariationaloneexplainedbetween27and73%ofthevariation
intheseedhandlingbehaviorofsquirrels.Phylogenyindepdentofthemeasuredtraits
explained<6%ofallseedhandlingbehaviors.Fortimerequiredtoconsumeaseedanddis-
tancetravelledtocacheaseed,thevariationexplainedbythecombinationofseedtraitswitha
phylogeneticstructurewas44%and56%,respectively.Unexplainedvariationwasrelatively
high(21%and32%)fordistancemovedtoconsumeaseedandtimerequiredtocacheaseed.
The11seedattributesof23seedtypes(Fig2,S4Table)werereducedtothreephylogenetic
principalcomponents(pPC),cumulativelyexplaining83.3%ofthetotalvariation.Weinter-
pretedthefirstglobalpPCaxisasfamily-leveldifferencesbetweenseeds.Thisaxisaccounted
for63.5%ofthetotalvariation.Percentageofcarbohydratesloadedhighlyandcontrastedwith
Fig1.Bayesianmaximumcladecredibilitytreefor23hardwoodtreespeciesfromthefamilies
Fagaceae,JuglandaceaeandBetulaceae,usingRubusoccidentalisasanoutgroup.Treeinferredfrom
rbcL,matKandITSgenesequences.Posteriornodesupportindicatedbynodelabels.
doi:10.1371/journal.pone.0130942.g001
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Table1. Variancepartitioningresultsforeachsquirrelseedhandlingbehavior,includingtimetoconsume,distancetravelledtoconsume,timeto
cache,anddistancetravelledtocacheaseed.
Responsevariable Seedtraitsalone Combinedtraits&phylogeny Phylogenyalone Unexplained
Timetoconsume 0.502 0.440 0.006 0.052
Distancetravelledtoconsume 0.731 0.055 0.000 0.214
Timetocache 0.409 0.213 0.058 0.320
Distancetravelledtocache 0.274 0.556 0.004 0.156
Valuesareproportionofvariation(R2)ofeachbehaviorexplainedbyseedtraitinformationalone,combinedinformationbetweenseedtraitand
phylogeny,phylogeneticinformationalone,andunexplainedsourcesofvariation.
doi:10.1371/journal.pone.0130942.t001
percentageoflipidsandshellthickness.ScoresfrompPC1werephylogeneticallyautocorre-
lated(I=0.83,p=0.002),anddifferentiatedlipid-rich,thickshellseedsofJuglandaceaeand
Betulaceaefromcarbohydrate-richFagaceae(Fig2).ThesecondglobalpPCaxisexplained
8.4%ofthevariation,andreflectedgenusandsection-leveldifferencesinseeds.Theaxisposi-
tivelyloadedproteinconcentration,andnegativelyloadedtanninconcentrationanddormancy
period.ScoresfrompPC2axiswerealsophylogeneticallyautocorrelated(I=0.36,p=0.004)
andseparatedhighproteinJuglansseedsfromCaryaseedsandhighproteinCastaneaseeds
fromlowdormancyQuercussectionQuercusseeds(Fig2).Finally,thethirdpPCaxisreflected
kernelsizeofseedsandexplained12.4%ofthetotalvariation.Thisaxispositivelyloadedkernel
andshellmassandnegativelyloadedenergyvalueofseed.ScoresfrompPC3werenotsignifi-
cantlyphylogeneticallyautocorrelated(I=-0.12,p=0.694,Fig2).ThepPCaxesdidnot
changesubstantiallyafterreducingthephylogenytoonlywell-establishedclades.
TimerequiredtoconsumeaseedwaspredictedbythetwoglobalpPCaxesandthelocal
pPCaxis(R2=0.76,Table2).Thedistancemovedtoconsumeaseedwaspositivelypredicted
predominantlybythelocalpPCaxisandmarginallybythefirstglobalpPCaxis(R2=0.30,
Table2).TimerequiredtocacheaseedwasnegativelypredictedbythefirstglobalpPCaxis
andpositivelytothelocalpPCaxis(R2=0.43,Table2).Distancemovedtocacheaseedwas
correlatednegativelywiththefirstglobalpPCaxisandpositivelywiththelocalpPCaxis
(R2=0.76,Table2).
Competitiveinterferencewasobservedin10seedtypes(range0–3trialsperseedtype)and
wasobservedin3seed-cachingvideosand11seed-consumptionvideos.Numberoftrialsin
whichconspecificinterferencewasobservedwasnotpredictedbyanyofthepPCaxes.The
meanhandling(consumptionand/orcaching)timedidnotdifferwithinaseedtypebetween
trialsinwhichinterferencewasobservedandtrialswhereinterferencewasnotobserved
(Pairedt-testt=0.22,df=10,P=0.95).
Discussion
Ourresultsprovideevidencethatforaginginvestmentsareinfluencedbyamixtureofseed
traitsthatarephylogeneticallyautocorrelated(massofshell,hardnessofshell,shellthickness,
lipidconcentrations,andcarbohydrateconcentrations)andthosethatarenot(kernelsizeand
tanninconcentrations,seeTable2,S5Table).Wesuggestthattheseresultssupporttheexis-
tenceofadiffusecoevolutionaryrelationshipbetweeneasterngraysquirrelsandhardwood
treeseeds.
Byincorporatingphylogeneticinformation,wefoundthatgraysquirrelforaginginvest-
mentsareinfluencedtodifferentdegreesbyseedtraitinformation,phylogeneticrelatednessof
hardwoodtreesandunexplainedsourcesofvariation.Distancemovedtoconsumeaseedand
timetocacheaseedshowedarelativelyhighdegreeofunexplainedvariationinthevariance
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Phylogenetic&EcologicalDeterminantsofGraySquirrelForaging
Fig2.Biplotsofseedtraitsandscoresof23hardwoodtreespeciesobtainedfromaphylogenetic
principalcomponentsanalysis(pPCA).Distributionofhardwoodtreespeciesacrossthefirst2‘global’
pPCaxes(A).Distributionoftreespeciesacrossthefirst‘global’andthird‘local’pPCaxes(B).Separationof
speciesbelongingtofamiliesJuglandaceaeandBetulaceae(symbols:squareanduprighttriangle)from
Fagaceae(symbols:circlesandinvertedtriangle)isobservedacrossphylogeneticallyautocorrelatedpPC1.
SeparationofJuglans(grayfilledsquare)fromCarya(blackfilledsquare)andseparationofCastanea(black
filledcircle)fromQuercussectionQuercus(opencircle)andQuercussectionLobatae(grayfilledcircle)is
seenacrosspPC2.Thethirdaxisisnotsignificantlyphylogeneticallyautocorrelatedandspeciesarenot
differentiatedbytaxonomiccladesacrosspPC3.BiplotsalsoincludeNotholithocarpusdensiflorus(inverted
graytriangle)andFagusgrandifolia(invertedopentriangle).Biplotarrowsplottedonlyforseedtraitswith
loadingsgreaterthan75thpercentileofabsoluteloadings(pPCloadingsand75thpercentilecutoffinS4
Table).
doi:10.1371/journal.pone.0130942.g002
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Phylogenetic&EcologicalDeterminantsofGraySquirrelForaging
Table2. Regressionsofsquirrelforagingbehavior(timetoconsume,distancetravelledtoconsume,timetocache,anddistancetravelledto
cacheaseed)against3phylogeneticPCaxes(pPC1,pPC2,pPC3).
Responsevariable Predictor Slope t-statistic p-value
Timetoconsumeaseed Intercept 9.216 7.039 0.000
pPC1 -3.112 -6.208 0.000
pPC2 4.196 3.039 0.006
pPC3 2.843 2.388 0.033
Distancemovedtoconsumeaseed Intercept 6.048 5.341 0.072
pPC1 -0.736 -1.699 0.090
pPC2 -0.007 -0.006 0.994
pPC3 2.460 2.389 0.039
Timetocacheaseed Intercept 2.090 10.044 0.039
pPC1 -0.220 -2.864 0.016
pPC2 0.130 0.614 0.544
pPC3 0.507 2.133 0.047
Distancemovedtocacheaseed Intercept 26.283 12.400 0.000
pPC1 -5.263 -6.757 0.000
pPC2 0.437 0.204 0.840
pPC3 8.742 3.621 0.002
Estimatesofslope,t-statisticandp-valueareprovidedforeachpredictor.Boldfacedepictsvaluesofp<0.05;italicsdepict0.05(cid:1)p<0.10.
doi:10.1371/journal.pone.0130942.t002
partitioninganalyses,suggestingthatthesemetricsmaybeinfluencedtoarelativelygreater
degreebyvariablesnotmeasuredinthisstudy.Adifferentpatternofvariancepartitioningwas
observedfortimerequiredtoconsumeaseedanddistancetocacheaseed,bothofwhichare
behaviorsrelatedtocachingdecisionsandseedsurvival.Specifically,seedswithlargehandling
timesmaybecachedmoreoften,increasingthepossibilityofgerminationandestablishment,
andseedscachedatlongdistancesmayescapedensity-dependentsourcesofmortality
[16,17,18,68].Thesebehaviorswereexplainedtoalargeextentbythesharedinformationin
seedtraitsandphylogeny(Table1),suggestingpossiblecoevolutionbetweenhardwoodtrees
andeasterngraysquirrels.
Coevolutionresultsfrominteractionsthatoccuroverlongtimescalesandshouldextendto
ancestralstatesexistingpriortodifferentiationofgenera[36].OurpPCAmethodsallowedus
todeterminewhichseedtraitvariantscorrespondtofamilyandgenus-leveldifferentiations
amongour23hardwoodtreespecies.ByregressingsquirrelforaginginvestmentsagainstpPC
axes,weindirectlydeterminedifgraysquirrelsaresensitivetofamilyorgenus-levelseedtrait
variations.
Family-levelseedtraitdifferencesweremoreimportantinpredictinggraysquirrelbehavior
thangenus-orsection-leveldifferences.Theearliestsplitobservedinourhardwoodtreephy-
logenycorrespondstotheseparationofJuglandaceaeandBetulaceaefromFagaceae(Fig1),
whichcoincideswithtwodifferentstrategiesofmakingseeds(assuggestedbytheglobalpPC1
axis)—lipid-richandthickshelledseedsinJuglandaceaeandBetulaceae,asopposedtothin-
shelledbutcarbohydrate-richFagaceaeseeds(Fig2).Specifically,squirrelstravelfartherto
cacheandinvestmoretimecaching/consuminglipid-richandthick-shelledJuglandaceaeand
Betulaceaeseeds(Table2),potentiallyimprovingseedsurvivalandfitnessofthesetrees.The
secondpPCaxis,describingdifferencesinseedtraitsbetweengeneraandsectionclades,was
alsoincludedasapredictorinourregressionanalysesbutpositivelyexplainedonlytimeto
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