Biotic and Abiotic Drivers of Sap Flux in Mature Green Ash Trees

17 Pages·2017·1 MB·English

by  Flower, Charles

Article
Biotic and Abiotic Drivers of Sap Flux in Mature
Green Ash Trees (Fraxinus pennsylvanica)
Experiencing Varying Levels of Emerald Ash Borer
(Agrilus planipennis) Infestation
CharlesE.Flower1,2,* ID,DouglasJ.Lynch2,3,KathleenS.Knight1and
MiquelA.Gonzalez-Meler2 ID
1 USDAForestService,NorthernResearchStation,359MainRd.,Delaware,OH43015,USA;
[email protected]
2 DepartmentofBiologicalSciences,UniversityofIllinoisatChicago,845W.TaylorSt.,Chicago,
IL43015,USA;[email protected](D.J.L.);[email protected](M.A.G.-M.)
3 LI-CORBiosciences,4421SuperiorStreet,Lincoln,NE68504,USA
* Correspondence:charlesfl[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:16April2018;Accepted:24May2018;Published:28May2018
Abstract: Whiletherelationshipbetweenabioticdriversofsapfluxarewellestablished, therole
of biotic disturbances on sap flux remain understudied. The invasion of the emerald ash borer
(AgrilusplanipennisFairmaire,EAB)intoNorthAmericainthe1990srepresentsasignificantthreat
to ash trees (Fraxinus spp.), which are a substantial component of temperate forests. Serpentine
feeding galleries excavated by EAB larvae in the cambial and phloem tissue are linked to rapid
treemortality. ToassesshowvaryinglevelsofEABinfestationimpacttheplantwaterstatusand
stresslevelsofmaturegreenash(FraxinuspennsylvanicaMarshall)trees,wecombinedtree-levelsap
flux measurements with leaf-level gas exchange, isotopes, morphology and labile carbohydrate
measurements. Results show sap flux and whole tree water use are reduced by as much as
80% as EAB damage increases. Heavily EAB impacted trees exhibited reduced leaf area and
leaf mass, but maintained constant levels of specific leaf area relative to lightly EAB-impacted
trees. Alteredfoliargasexchange(reducedlightsaturatedassimilation,internalCO concentrations)
2
pairedwithdepletedfoliarδ13CvaluesofheavilyEABimpactedtreespointtochronicwaterstressat
thecanopylevel,indicativeofxylemdamage. Reducedphotosyntheticratesintreesmoreimpacted
by EAB likely contributed to the lack of nonstructural carbohydrate (soluble sugars and starch)
accumulation in leaf tissue, further supporting the notion that EAB damages not only phloem,
butxylemtissueaswell,resultinginreducedwateravailability. Thesefindingscanbeincorporated
intomodelingeffortstountanglepostdisturbanceshiftsinecosystemhydrology.
Keywords: emeraldashborer(Agrilusplanipennis); invasivespecies; Fraxinus; forestdisturbance;
sapflux;treewateruse;thermaldissipationprobe
1. Introduction
Patternsofsapfluxintreesareusedinestimatesofwhole-treewateruse,tree-leveltranspiration,
andareevenscaledtoecosystemtranspiration,assuchprecisemeasurementsofwaterexchangesare
essentialforcoupledbiosphere-atmospheremodels. Thevariablesdrivingwaterfluxesthroughthe
soil-plantcontinuumaregenerallywellunderstood[1–4].However,theimpactsofbioticfactorssuchas
treeboringforestpestsonhostwaterusearelargelyunderstudied(butsee[5]),despitetheconsiderable
impacts they have on forest systems [6]. Abiotic variables including light levels, vapor pressure
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Forests2018,9,301 2of17
deficit (VPD), and soil moisture have long been shown to drive sap flux, generally with higher
irradiance,higherVPD,andlowersoilmoisturedrivinghigherratesofsapflux[2,7,8]. Fromabiotic
standpoint, substantial variability has been shown to occur among species [9–11], between trees
of the same species which differ in size [2], and based on tree canopy position (i.e., dominant,
codominant or suppressed) [12]. Several studies have demonstrated reduced sap flux and water
use associated with the disease progression and susceptibility of host trees to fungal pathogens
(e.g.,[13–16])aswellasforestresponsetodefoliation(e.g.,[17,18]). Todate,wecouldidentifyonly
asinglestudydescribestree-levelsapfluxdeclinesfollowingatreeboringbeetleattack(i.e.,mountain
pinebeetle(DendroctonouspoderosaeHoplins)infestationoflodgepolepine(PinuscontortaDouglas)[5].
When assessing the impacts of biotic forest disturbances on tree sap fluxes however, potentially
uniquecharacteristicsofagivendisturbanceagentmayresultinavarietyofresponsesnecessitating
furtherinvestigation.
Emerald ash borer (Agrilus planipennis Fairmaire, EAB) is a tree-boring beetle native to Asia,
which was inadvertently imported into the United States in the 1990s [19–23]. Emerald ash
borers feed almost exclusively on trees in the genus Fraxinus [24,25], but see [26] whose
constituentspeciesarewidelydistributedacrossxeric(e.g.,F.AmericanaL.,F.quadrangulataMichx.),
mesic(e.g.,F.pennsylvanicaMarshall)andhydric(e.g.,F.nigraMarshall,F.profundaBush)forestsin
thecontinentalUS[27–29]andsouthernCanada[30]. InforestsoftheGreatLakesRegionoftheU.S.,
ashoccupiesanecologicallyimportantnicheinriparianandwetlandsystemsandaccountsfor~5–9%
ofabovegroundlivecarbonmass[28]. Unlikemostnativetreeboringbeetleswhichtypicallyattack
treesexperiencingphysiologicalstress,initsinvadedrangeEABattackshealthyandstressedtrees,
withpreferencefordamagedtrees[31]. EABcausesprogressivecanopydecline[32]thatculminates
withashtreemortalityin2–5yearsandalmostcompleteashtreemortalityattheforestlevelwithin
6years[33]. ItisgenerallyrecognizedthatEABlarvaecauserapidtreemortalityviatheirfeedingin
thecambialandphloemtissue,whichcreatesserpentinegalleriesthatseversaptransportbetween
shootsandroots[22,34,35]. Whilesimulatedgirdlingstudieshavebeenconductedtoassessthedecline
ofblackashinwetlands(F.nigra)[36],theeffectsofEABontree-levelwateruptakeandsapfluxremain
undocumented. Thus,directevidenceofthephysiologicalmechanismsalteredbyEABinfestation
arelacking. ChangesinwaterrelationscausedbyEABwillnotonlyacceleratetherateoftreemortality,
butalso mayhavelocal consequencesforsurface hydrologyand theenergy balanceofassociated
ecosystems[6,37].
Forest pests that damage cambial tissue can impact tree water use and canopy processes by
at least three non-exclusive mechanisms. First, forest pests can impact tree water use via direct
feedback mechanisms that regulate photosynthesis [38]. Specifically, leaf stomatal closure may be
caused by direct loss of turgor pressure in guard cells associated with changes in the hydraulic
conductance along the soil-leaf continuum [39–41] or by increased concentrations of free abscisic
acid [42]. Second, the sink limitation imposed by the forest pest’s girdling behavior may result in
theaccumulationofphotosynthateinsourceleaves[43]causingdownregulationofphotosynthesis,
photoinhibitionandrelatedeffects[44]. Third,viapersistentandchronicdirectmechanicaldamageto
vasculartissueoveraperiodofyears,impactedtreesmayexhibitreducedleafareaatbothcanopy
andsingleleaflevels,resultinginconcomitantreductionsinwateruse[45,46]. Understandingthe
underlyingmechanismsinvolvedinashtreeresponsestoEABwillfacilitateabetterunderstanding
oftheassociatedhydrologicalresponsestoforestpests[36]. StudyingtheseeffectsonEABimpacted
forestsisofparticularimportancebecauseourunderstandingofthehydrologicalresponsestoforest
pestdisturbancespredominantlyrelylargelyongirdlingstudiesorinoculationsoffungalpathogens,
whose damage symptom progressions differ at the tree level. Specifically, sap flux measurements
studyingmortalityfromfungalpathogeninoculationscanoccurinasfewas1year[14,15],girdlingin
asquicklyas2years[47],andEABinducedmortalityfrom2–5years[33].
The objective of this study was to examine the impacts of the invasive EAB on ash foliar
morphologyandtreewaterusewithinanashdominatedforestincentralOhio,U.S.Theeven-aged
Forests2018,9,301 3of17
forest stand utilized in this study is comprised of mature overstory green ash (F. pennsylvanica
Marshall) trees displaying all stages of EAB infestation [35], thus providing a unique opportunity
to investigate biotic (i.e., EAB) and abiotic drivers of tree water use. Using Granier type sap flux
probes (described by Lu et al. [8]), we measured sap flux density as a proxy for direct water use
inninegreenashwhichcomprisedthefullrangeofEABimpactedconditions. Wealsomeasured
thebulkleafcarbon(C)isotopiccomposition,δ13C ,ofleaftissuefromthefocaltreesasaproxy
leaf
for relative changes in intrinsic water use efficiency (the ratio of water used per carbon gain at
theleaflevel)[48]. Foliarnonstructuralcarbohydrateconcentrationsweremeasuredtoassesssink
limitations[43]. Finally,weconductedleaflevelgasexchangemeasurementsofcanopyleavesand
collected leaf tissue from the canopy of ash trees across a gradient of EAB induced mortality to
assessmorphologicalcharacteristics(surfacearea(SA),drymass(DM),andspecificleafarea(SLA)).
TheseparametersareusedtoelucidatetheplanthydrologicalconsequencesofEABinfestationandto
assesstheroleofwateronmortalityofinfectedash.
2. MaterialsandMethods
2.1. SiteDescriptionandTreeSelection
Thisstudywasconductedin2010withina35-yearold(asdetermineddendrochronologically)
abandoned tree plantation established on an agricultural field in central Ohio located at the
USDA Forest Service Northern Research Station (40◦ 23(cid:48) N, 83◦ 02(cid:48) W) previously described
by Floweretal.[49]. Following abandonment ~30 years prior to the study, succession occurred
unimpeded. Theforestischaracterizedbysiltyloamsoils,isatanelevationof290mabovesealevel,
anditsaverageannualprecipitationfrom2006–2010was92.04cm±3.71cm. Theforestcanopyis
dominatedbyF.pennsylvanica(greenash)andF.americanaL.(whiteash),whichrepresent~80%oftree
basalarea,aswellasUlmusamericanaL.(Americanelm),andTiliaamericanaL.(basswood).
Greenashtreeswereselectedforinclusioninthesapfluxstudybyfirstassessingthediameterat
breastheight(DBH,~1.37mfromground)ofeachtreeintheforeststandandifanashtree,ratingthe
ash canopy condition (see below for description of AC classes). Additionally, the height of each
treewasmeasuredpriortothestudywithahypsometer(VertexIV;Haglöf,Sweden). Ninetreesof
comparableDBHwererandomlyselectedwithintheplotacrosstherangeofACclasses(seeTable1
formoredetailsonstudytrees). Investigativesapfluxstudieshavebeensuccessfullyconductedwith
lowreplicationwhentheeffectsizesforthevariablesofinterestarelarge[13,50]. Onlyco-dominant
overstoryashtreeswereselectedforsapfluxmeasurements,sincecanopypositionhasbeenshown
toimpactsapflowrates[51]. Selectedtreeswerelocated>10mfromtheforestedgetominimizethe
microclimaticvariabilityanddifferencesinturbulenceandmomentumfluxesnearforestedgeswhich
canimpactcanopylevelboundaryconditions,transpirationandsapfluxrates[52–55]. Measurements
ofsolarradiationandVPDwerederivedfromameteorologicalstationlocatedattheUSDAFSstation
<0.5kmfromthestudysite.
Table1.CharacteristicsoftheexperimentalFraxinuspensylvanicasampledforsapfluxdensity.
%EABGallery
TreeNo. AC DBHa(cm) TreeHeight(m) Avg.DailySapFlux(kg/day±se) Leafδ13C
Cover
1 1 18.8 11.9 13.69±0.33 −28.12 0
2 1 22.4 13.2 13.13±0.10 −28.11 5
3 1 17.8 11.2 13.01±0.31 −27.39 5
4 2 20.1 12.1 4.54±0.30 −26.97 10
5 2 19.8 12.3 3.78±0.28 −25.94 17.5
6 3 19.3 11.6 3.74±0.16 −26.49 25
7 4 17.0 10.5 2.20±0.15 −25.91 45
8 4 18.2 11.7 1.17±0.22 −25.94 50
9 4 18.8 11.5 1.48±0.10 −25.92 55
aDiameteratbreastheight.
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Each live co-dominant ash tree within the interior of the stand was evaluated for EAB
infestationusingavisualassessmentofcanopyhealthcalledashcanopycondition(AC).Thisvisual
assessment has been shown to exhibit a significant relationship with tree-level EAB densities
(larvae/adult)andEABgallerycoverandthusitrepresentsasolidvisualproxyforEABinfestation
levels[35,56]. Briefly,ashcanopyconditionisgradedona1(healthy)to5(dead)scale:(1)healthy/full
canopy—ahealthyashcanopyisfullandexhibitsnodefoliation;(2)thinningcanopy—slightreduction
inleafmass,alltopbranchesexposedtosunlighthaveleaves;(3)dieback—canopyisthinningand
sometopbranchesexposedtosunlightaredefoliated,lowerbrancheswhichexhibitnaturalthinning
arenotconsidered;(4)>50%dieback—thecanopyhaslessthan50%oftheleavesofaclass1treeand/or
overhalfofthetopbranchesaredefoliated;and(5)deadcanopy—noleavesremaininthecanopy
portionofthetree,regardlessofepicormicsprouts[57]. Canopyconditionwasdeterminedvisually
inJuneafterleafexpansionwascomplete. Additionally,EABlarvalgallerycoverwasquantifiedon
22August2018followingterminationofsapfluxmeasurementsontwodebarked10×10cmwindows
(onthenorthandsouthsideofeachtree)at1.5mheightasdescribedbyFloweretal.[35].
2.2. SapfluxMeasurements
Granier [58] type systems consisting of thermal dissipation probes (TDP, manufactured by
PlantSensors,Nakara,Australia)weredeployedtomeasuresapfluxinnineashtreesalongagradient
ofEABinfestation. EachsystemisapairofTDPprobes(each2mmindiameter)insertedradially
intothesapwoodoftheboletoadepthof2cmandseparatedvertically,withtheupperprobeheated
andthelowerprobeanunheatedreference. TwoTDPsystemsweredeployedpertreeataheight
of 1.37 m, one on the north-facing side and one on the south facing-side of each tree to account
for flux variability along the stem circumference. In all trees, the depth of the sapwood exceeded
thedepthoftheTDP.Theheatedandreferenceprobeswereplaced~10cmapart(sothereference
measurements are not affected by the heating) and the heating power was adjusted to 0.2 W and
inducedamaximumtemperaturedifferenceof8–10◦CunderzerofluxconditionsasdescribedbyDo
andRocheteau[59]. Inordertoreducetheeffectofnaturaltemperaturegradientswhichcanimpact
measurementaccuracy,weutilizedaheating/coolingcycleof15min/15minasdescribedbyDoand
Rocheteau[59]. Additionally,theTDPsystemsweresurroundedbyclosedcellfoamandwrappedin
areflectiveheatshield(afoilcoatedbubblesheet~0.6minheight,wrappedaroundtheTDPsystem
andtree)tominimizetemperaturegradientsassociatedwithdirectradiation. Siliconecaulkingwas
usedatthetopandbottomoftheheatshieldtoexcluderainandstemflowfrominterferingwiththe
TDPsystems. Sapfluxprobesweredeployedon17June2010anddatawasrecordedthrough1August,
because of interference with the sap flux probes from raccoons we will only report results from
beforethisinterference(17–20June). Temperaturedifferentialsweremeasuredevery5minbetween
theheatedandreferencethermocouplejunctionsoneachprobeand30minmeanswererecorded
on a Campbell CR10 datalogger (Campbell Scientific, Logan, UT, USA). Temperature differentials
betweenprobeswereinfluencedbysapfluxdensitysurroundingtheheatedprobe. Sapfluxdensity
(SFD,ms−1)foreachsensorwascalculatedaccordingtoGranier[12,58]usingthefollowingequations:
SFD=αKβ, (1)
whereβ=1.231andα=119×10−6ms−1[59]. Thefluxindex(K,adimensionlessvalue)isdefinedas:
K=(∆T −∆T )/∆T , (2)
0 u u
where∆T isthemaximumtemperaturedifferenceobtainedunderzerofluxconditionsand∆T isthe
0 u
measuredtemperaturedifferenceatagivenfluxdensity. Itwasassumedthatnosapfluxoccurredat
pre-dawn. Treelevelwateruse(TWU,kgh−1)wascalculatedas:
TWU=SFD×S , (3)
A
Forests2018,9,301 5of17
where S is the cross-sectional area of the sapwood at the level of the heating probe [12].
A
Thecross-sectionalareaofsapwoodwasestimatedvisually,bywoodcolorasdescribedbyMeadows
andHodges[60],fromincrementcoresremovedfollowingsapfluxmeasurements. Dailytreelevel
wateruse(TWU ,kg−1 day−1)wascalculatedasthe24-hdailysumofTWU.Insummary,assap
D
flowspasttheunheated(lower)thermocouple,itrecordstheambientsaptemperatureandtheupper
heatedneedleiscooled. Rapidsapfluxresultsinlowdifferencesinthethermocoupletemperaturesas
theupper(heated)thermocoupleiscooled.
2.3. LeafTissueSampling
InlateJune2010,leaftissuewasexcisedfromtheuppersunexposedcanopyofeachfocal(sap
flux)ashtreeduringmid-dayusingashotgun. Collectedleaftissuefromeachexperimentalashtree
was immediately oven-dried at 60 ◦C for ~48 h to constant mass to remove any moisture prior to
isotopicanalysis. Thebulkleafcarbonisotopiccomposition,δ13C,representsanunbiasedintegrator
of a plant water relations over the longevity of the leaf tissue and can be used as a surrogate for
intrinsicwateruseefficiency. Theintrinsicwateruseefficiencyisnon-linearlyproportionaltothe
differencebetweenambientCO concentrations(C )andinternalleafCO concentrations(C)[48].
2 a 2 i
Thus,underconstantC ,andconstantδ13CofatmosphericCO ,δ13Creflectsarelativeandintegrated
a 2
measureofintrinsicwateruseefficiencyamongtreesofdifferentAC.
On11–12July2013,atleast3replicateleafsampleswerecollectedfromtheuppersunexposed
canopy of 20 ash trees across a gradient of AC classes to assess differences in leaf morphology.
Leaftissuewascollectedfrom09:00–10:30andthesurfaceareadeterminedimmediatelyfollowing
collectionusingaLI-COR3100leafareameter(LI-COR,Lincoln,NE,USA).Sampleswerethendried
inaforced-airconvectionovenat60◦Cfor48horuntilnochangeinmasswasdetectedandanalyzed
for%carbon(%C),%nitrogen(%N),andδ13Casdescribedbelow. Replicateswereaveragedpriorto
statisticalanalyses.
2.4. FoliarGas-ExchangeMeasurements
During June 2015, sun-exposed branches were collected between 09:30–11:30 from the upper
canopy of green ash trees across the AC gradient (n = 12 trees). Branches were re-cut under
deionizedwatertoreduceembolismandgas-exchangemeasurementswereconductedimmediately
thereafter. An open-system portable IRGA (LI-6400 equipped with the 6400-02 LED light source
attachment,LI-COR,Inc.,Lincoln,NE,USA)wasusedtomeasurethelightsaturatedphotosynthetic
rate(A @1600PAR;µmolCO m2s−1),internalCO concentrations(C;ppmCO ),andstomatal
sat 2 2 i 2
conductance(gs;mmolm−2s−1)for3fullyexpandedleaves/branch.Beforeeachmeasurementtheleaf
chamberwascheckedforleaksandstabilityinflow(500µmols−1),CO (400ppm),andH O(<80%).
2 2
2.5. ElementalandIsotopicAnalysisofLeafTissue
Driedplanttissuewasfinelygroundusingaballmillpriortoanalysis. Thecarboncontentof
leaftissuewasmadeviaflashcombustionusingaCostechElementalAnalyzer(Valencia,CA,USA)
withazero-blankautosamplerandelectronicactuatortoeliminateaircontaminationinthesamples.
StableisotopeanalysesofCwereconductedonthesamesampleusinganIRMS(isotoperatiomass
spectrometer, Finnigan Deltaplus XL, Bremen, Germany) operated in continuous flow mode and
calibratedtothe13CViennaPeeDeeBelemnite(VPDB)scaleusingUSGS40withaprecisionof0.05 .
Elementalandsolidinternationalandsecondaryisotopestandardswereusedforinstrumentcalibratio(cid:104)n
andsampleconversiontoδvalues.Isotopicvaluesarereportedindeltanotationrelativetothestandard
VPDBforcarbon,(δ =1000((R /R )−1),R=13C/12C).Allmeasurementsweredone
sample sample standard
atthestableisotopelaboratoryattheUniversityofIllinoisatChicago.
Forests2018,9,301 6of17
2.6. PlantTissueNon-StructuralCarbohydrateAnalysis
In2010,leaftissue(aspreviouslydescribed)fromsapfluxtreeswasanalyzedfornonstructural
carbohydrates (starch and soluble sugars; NSC). Briefly, we quantified soluble sugars and starch
in woody tissues using standard methods [61]. Leaf tissue was dried in a forced convection
oven immediately following collection until no mass loss and ground with a ball mill for 3 min.
Solublesugars(sucrose,glucose,andfructose)wereextractedfrom25mgoftissuewith80%ethanol
(5mL)at80◦Cfor5min. Extractswerecentrifugedandthesupernatantspooled;a2mLaliquotwas
removedanddriedusingavacuumevaporator. Driedextractwasresuspendedwith3mLdeionized
waterand40mgpolyvinylpolypyrrolidoneandspundownusingacentrifuge. A0.5mLaliquotwas
colorimetricallyassayedforsolublesugars[61]andmodifiedforuseondeciduoustissuesaccording
toCurtisetal.[62]. Solublesugarrecoverywas>95%.
Starchwasquantifiedfromthesolublesugarextractedtissuepelletsbyresuspensionusing1mL
of 0.2 N KOH and incubated at 80 ◦C for 25 min. KOH was neutralized by adding 0.2 mL of 1 N
aceticacid. Starchwashydrolyzedtoglucosewithα-amyloglucosidasesolution(pH7.05)at55◦Cfor
1.5handassayedaccordingtoJonesetal.[61]. Starchrecoverywas>95%. Thetotalnonstructural
carbohydrateconcentration(TNC)wasthesumofsolublesugarsandstarch.
2.7. StatisticalAnalysis
The effect of AC on ash tree sap flux density rates (SFD) were investigated using a repeated
measuresanalysisofvariance(RMANOVA)test,withthehourlySFDmeasurementsastherepeated
measure. SapfluxdensityrateswerecomparedbetweenACusingTukey’sHSD,α=0.05. Becauseof
lackofreplication,treesinAC2and3werepooledpriortoanalysisandthusrepresentamoderate
levelofEABdecline. Separately,wetestedtheroleofACandabioticfactorsonSFDusingANOVA,
inwhichACwastreatedasamaineffectandtheenvironmentalparameters(radiationandVPD)were
treatedascovariates. Additionally,theeffectofAConaverageTWU weretestedusingANOVA,
D
dailytreelevelwaterusewascomparedbetweenACusingTukey’sHSD,α=0.05. Therelationship
betweendailysapfluxratesandthecarbonisotopiccompositionofleaftissue(δ13C )wastested
Leaf
usingSpearman’sRankCorrelationAnalysis,duetothenon-linearrelationshipbetweendailysapflux
ratesandthecarbonisotopiccompositionofleaftissue(δ13C ). Thistestprovidesadistributionfree
Leaf
testofindependencebetweenthevariables. Theeffectofashconditionclasses(AC)onmorphological
leaf measurements (surface area, dry mass, specific leaf area), gas exchange parameters (A , g ,
sat s
andC)andleafcarbonisotopiccompositionwereinvestigatedusingindependentanalysisofvariance
i
(ANOVA)tests. VariableswerecomparedbetweenACclassesusingTukey’sHSD,α=0.05. Theeffect
ofAConleafstarch,solublesugar,andTNCconcentrationswasassessedwithANOVA,treesinAC2
and3werepooledpriortoanalysis. AllstatisticalanalyseswereconductedusingSYSTATstatistical
software(v13,SYSTAT2007).
3. Results
3.1. SapFluxDensityRatesbyAshCanopyConditionClassesandAbioticDrivers
Sapfluxdensitiesrangedfrom0.00to1.49ms−1andexhibitedsignificantvariabilityattributed
toashcanopyconditionclasses(ANOVA;F =566.3,p<0.001). Sapfluxdensitiesinhealthytrees
(2624)
(AC1;0.583m3 m−2s−1 ±0.045SE)weresignificantlyhigherthanthoseofmoderately(AC2or3;
0.144m3 m−2 s−1 ± 0.011SE)andheavily(AC4; 0.112m3 m−2 s−1 ± 0.009SE)infestedashtrees
(Tukey’sHSDp<0.001forallACvalues;Figure1). Sapfluxdensityalsoexhibitedsignificanttemporal
variabilityacrossthesamplingperiod(ANOVA;F =105.018,p<0.001). Finally,weobserved
(103,624)
asignificantdifferenceintheinteractionterm(AC×time),highlightingincreasedresponsivenessin
sapfluxinthehealthytreesovertime(Figure1;ANOVA;F =36.251,p<0.001).
(206,624)
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AC 1
1.4 AC 2/3
AC 4
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168 169 170 171 172
Day of Year

ForestsF 2i0g1u8r, e9 , 1x. FSOaRp PflEuExR dReEnVsIiEtyW ( S FD) of Fraxinus pennsylvanica trees during 26–30 June 2010 (AC1 = low7 of 17
Figure1. Sapfluxdensity(SFD)ofFraxinuspennsylvanicatreesduring26–30June2010(AC1=low
infestation levels, AC 2 & 3 moderate infestation levels, and AC4 = high infestation levels). Error bars
infestationledveenlost,eA ±1C SE2. &3moderateinfestationlevels,andAC4=highinfestationlevels).Errorbars
AC 1
denote±1SE. 1.4 AC 2/3
Although substantial vaArCia 4t ion existed between AC classes and over time, SFD exhibited
considerable diurn1a.2l fluctuations within trees generally peaking during mid-day when the
Althougehvapsourabtsivtea dn1et)miaalndv waarsi agrteiaotnest e(Fxigisutree d1; Tbabeltew 2)e. Tehne SAFDC wcasl amsosset sstraonngdly dorviveern btyi minceo,mSinFgD exhibited
solar radiation- hand 1V.0PD (Table 2). Higher order interactions of AC*VPD and AC*radiation were also
considerablediurnalflu2ctuationswithintreesgenerallypeakingduringmid-daywhentheevaporative
significant, ind-micati0n.g8 the differential impacts that the abiotic factors have on the SFD of trees across
demandwasdgirffeearetnets At(CF c3ilgaussrees (1T;aTbaleb 2l)e. S2p)e.cTifihcaellSy,F SDFDw waass mlesoss rtessptroonsnivgel ytod inrcivreeansedb yraidniactoiomn ains gEAsBo larradiation
m
and VPD (Tainbdleuc2ed) .caHnoipg(yh deer0c.l6ionre dmearniifnestteedr a(Fcitgiuorne 2s).o f AC*VPD and AC*radiation were also significant,
D
indicatingthedifferentiaFlim0.4pactsthattheabioticfactorshaveontheSFDoftreesacrossdifferentAC
S
classes(Table2). Specifically,SFDwaslessresponsivetoincreasedradiationasEABinducedcanopy
0.2
declinemanifested(Figure2).
0.0
Table2.Analysisofvarianc1e6s8tatisticsfo1r6t9hemaineff1e7c0tsofashca1n7o1pyconditi1o7n2class,vaporpressure
deficit(VPD),andradiationonashtreesapfluxD(SaFyD o)f dYeenasrityrates.

Figure 1. Sap flux density (SFD) of Fraxinus pennsylvanica trees during 26–30 June 2010 (AC1 = low
Souirncfeestation levels, AC 2T &y p3 meoIdIeIraSteS infestationd lfevels, and ACM4 =S high infestatioFn- Rlevaetliso). Error bars p-Value
denote ±1 SE.
AC 0.215 2 0.108 7.34 0.001
VPD 0.904 1 0.904 61.65 <0.001
Although substantial variation existed between AC classes and over time, SFD exhibited
Radiation 11.867 1 11.867 809.37 <0.001
considerable diurnal fluctuations within trees generally peaking during mid-day when the
ACev×apoVraPtDive demand was grea0t.e4s2t 6(Figure 1; Tabl2e 2). The SFD0 .w21as3 most strongl1y4 d .5ri3ven by incomi<ng0 .001
AC×solRara draidaitaitoionn and VPD (Tab7le.4 24).1 Higher order 2interactions 3of.7 A2C1*VPD and A2C5*3r.a7d6iation were al<so0 .001
Figure 2. Half hourly sap flux density (SFD) of Fraxinus trees in relation to solar radiation (W/m2)
AC×VPsiDgn×ificRanatd, iinadtiiocanting the diff1e.r5e6n2tial impacts th2at the abiotic0 f.7ac8t1ors have on t5h3e. 2S8F4D of trees acro<s0s .001
during 26–30 June 2010 (AC1 = low, AC2/3 = moderate, AC 4 = high infestation levels).
dEifrferroernt AC classes (Table 21)3. .S5p6e2cifically, SFD92 w5as less res0p.0o1ns5ive to increased radiation as EAB
i3n.d2.u Dceadil yc aSnaop pFylu dxe cline manifested (Figure 2).
Reduced tree level SFD resulted in significant differences in daily sap flux rates (TWUD) between
lightly and heavily infested trees (Figure 3, ANOVA; F (2,6) = 554.014; p < 0.001). Values of TWUD
tended to be greater for trees which were lightly or not infested with EAB (AC 1 = 13.27 kg day−1 ±
0.25 SE) relative to moderately (AC 2 & 3 = 4.02 kg day−1 ± 0.31 SE) and heavily infested trees (1.62 kg

Figure 2. Half hourly sap flux density (SFD) of Fraxinus trees in relation to solar radiation (W/m2)
Figure2. Halfhourlysapfluxdensity(SFD)ofFraxinustreesinrelationtosolarradiation(W/m2)
during 26–30 June 2010 (AC1 = low, AC2/3 = moderate, AC 4 = high infestation levels).
during26–30June2010(AC1=low,AC2/3=moderate,AC4=highinfestationlevels).
3.2. Daily Sap Flux
Reduced tree level SFD resulted in significant differences in daily sap flux rates (TWUD) between
lightly and heavily infested trees (Figure 3, ANOVA; F (2,6) = 554.014; p < 0.001). Values of TWUD
tended to be greater for trees which were lightly or not infested with EAB (AC 1 = 13.27 kg day−1 ±
0.25 SE) relative to moderately (AC 2 & 3 = 4.02 kg day−1 ± 0.31 SE) and heavily infested trees (1.62 kg
Forests2018,9,301 8of17
3.2. DailySapFlux
ReducedtreelevelSFDresultedinsignificantdifferencesindailysapfluxrates(TWU )between
D
lightlyandheavilyinfestedtrees(Figure3, ANOVA;F =554.014; p<0.001). ValuesofTWU
(2,6) D
tendedtobegreaterfortreeswhichwerelightlyornotinfestedwithEAB(AC1=13.27kgday−1
± 0.25SE)relativetomoderately(AC2&3=4.02kgday−1 ± 0.31SE)andheavilyinfestedtrees
Forests 2018, 9, x FOR PEER REVIEW 8 of 17
(1.62kgday−1 ±0.37SE,Figure3). Furthermore,aSpearman’srankcorrelationrevealedthatTWU
D
wasnegatidvaeyl−y1 ±c o0r.3r7e lSaEt,e Fdigwuriet h3)t. hFeurδt1h3eCrmore, (ar S=pe−arm0.a9n3’7s 2r,anpk< co0r.r0e5la)t.ion revealed that TWUD was
Leaf s
negatively correlated with the δ13CLeaf (rs = −0.9372, p < 0.05).
Forests 2018, 9, x FOR PEER REV1I6EW 8 of 17
14 a
day−1 ± 0.37 SE, Figure 3). Furthermore, a Spearman’s rank correlation revealed that TWUD was
negatively correlatedy) with1 2the δ13CLeaf (rs = −0.9372, p < 0.05).
a
d 1106
g/
(kD 184 a
WUy) 162 b
Tda 140
g/ c
k 28
(D
U 06
W 1 2 anbd 3 4
T
4 Ash Canopy Conditionc
2
Figure 3. Mean daily sap flux (TWUD) across trees of different ash condition classes. Letters represent
Figure3.Meandailysapflux(TWU )acrosstreesofdifferentashconditionclasses.Lettersrepresent
significance levels determ0ined bDy Tukey’s HSD pairwise comparisons (p < 0.05), error bars denote ±1
significancSeE.l evelsdeterminedbyTukey’s1HSDpa2ir awndi 3secompa4risons(p<0.05),errorbarsdenote
±1SE. Ash Canopy Condition
3.3. Leaf Traits
Figure 3. Mean daily sap flux (TWUD) across trees of different ash condition classes. Letters represent
3.3. LeafTraitsIsnigdniivfiicdaunacel lleevaef lss udreftaecrme ianreeda bvya Triuekde yfr’so HmS 7D. 1p atoir w41is.e2 ccomm2p aanridso wnsa (sp s<i g0.n0i5f)i,c earnrotlry b laarrsg deern ionte h ±e1a lthy
trees S(AE.C 1 and AC 2) relative to trees exhibiting EAB induced canopy decline (AC 3 and AC4) (Figure
Indiv4idAu; AalNlOeVafAs; uF r(3f,1a6)c =e 1a0r.7e3a1,v pa <r i0e.0d01f)r. oLmeaf7 d.1ryt moa4s1s .w2acsm al2soa snigdniwficaasntslyig lanrigfierc ainn htleyaltlhayr gtreeresi nhealthy
(3A.3C. L 1e aafn Tdr aAitCs 2) relative to trees exhibiting canopy decline (AC 3 and AC4) (Figure 4B; ANOVA; F
trees (AC 1 and AC 2) relative to trees exhibiting EAB induced canopy decline (AC 3 and AC4)
(Figure4A(;3,1A6) =NI 9nO.d02Viv6Ai,d p;u =Fa l0 .l0e0a1f )s. u=Arsf1a sc0ue.c 7ah3r, e1lae, avpf ad<rriey0d m. 0far0os1sm )e .x7Lh.1ie btaiotf e4dd1 .ar2 y lcinmme2aa ras npsdow switaiavsse s arilegslnaoitfiisocinagsnnhtliiypfi lwcaarigtnhet rll eyianf l hsauerargflaethcryei nhealthy
area (Figure S1; (A3,d16j )r2 = 0.98). Because of the conserved relationship between leaf dry mass and leaf
trees (AC 1 and AC 2) relative to trees exhibiting EAB induced canopy decline (AC 3 and AC4) (Figure
trees(AC1suarnfadceA arCea2, )nor esligantiivficeatnot dtrifefeersenecxehs iwbeitrien ogbscearvneodp iyn tdhee cslpineceifi(cA leCaf3 araena d(dAataC n4o)t (pFriegsuenrteed4;B p; ANOVA;
4A; ANOVA; F (3,16) = 10.731, p < 0.001). Leaf dry mass was also significantly larger in healthy trees
F =9.=0 206.4,7p2).= 0.001). As such, leaf dry mass exhibited a linear positive relationship with leaf
(3,16) (AC 1 and AC 2) relative to trees exhibiting canopy decline (AC 3 and AC4) (Figure 4B; ANOVA; F
surfaceare(a3,16()F =i 9g.u02r6e, pS 1= ;0A.00d1j).r A2s= su0c.9h8, l)e.AaBf dercya umasaesso efxthhibeitceod na slienrevaer dporseitliavtei orenlasthioinpshbiept wwiethe nlealef asufrdfarcye massand
leafsurfacearaear e(Fai,gnuore sSi1g; nAidfij cra2 n= t0.d98i3)f0.f eBreecanucsees owf theer ecoonbasesrevrevde rdelaintiotnhsehispp beectwifiecenl eleaaff adrreya m(adssa taandn loetafp resented;
p=0.472).surface area, no signific2)ant differences were observed in the specific leaf area (data not presented; p
= 0.472). m 20
c
A ( b b
S 10 A a
30
a
2)
m 20 B a
A (c 0.3 a b b
M (g)S 01.20
D b b
0.1 B a
0.3 a
M (g) 0.2 AC 1 AC 2 AC 3 AC 4
Figure 4. Leaf surfaceD area (SA) (A) and leaf dry mass (DM) (bB) in relatibon to ash tree canopy condition
0.1
class (AC). Significance levels depict results of Tukeys HSD pairwise comparisons (p < 0.05).
AC 1 AC 2 AC 3 AC 4
Figure 4. Leaf surface area (SA) (A) and leaf dry mass (DM) (B) in relation to ash tree canopy condition
Figure4.Leafsurfacearea(SA)(A)andleafdrymass(DM)(B)inrelationtoashtreecanopycondition
class (AC). Significance levels depict results of Tukeys HSD pairwise comparisons (p < 0.05).
class(A C).SignificancelevelsdepictresultsofTukeysHSDpairwisecomparisons(p<0.05).
Forests2018,9,301 9of17
3.4. FoliarChemistry
Forests 2018, 9, x FOR PEER REVIEW 9 of 17
Leafcarbonisotopicsignaturesexhibitedsignificantenrichmentalongwithprogressivecanopy
decline(Fi3g.u4.r Feol5iaAr ;CAheNmiOstrVy A;F =9.08,p<0.001),whilenosignificantdifferenceswereobservedin
(3,16)
thefoliarnitroLgeeanf ciasrobotonp isioctospigicn saigtnuarteusre(sF eixghuibriete5dB s;igAniNficOanVt Aen;riFch(m3,1e6n)t =alo2n.1g5 w2i,thp p=ro0g.r1e3ss4i)v.e Bcaunlokpyle afcarbon
contentgedneecrlianlel y(Fiignucrree 5aAs;e AdNaOsVaAsh; Fc (a3,1n6) o= p9y.08c, opn <d 0i.0ti0o1n), winhcilree naos esisg.nTifihcaenct adrifbfeornenccoesn wteenret oobfselervaevde sinAC1
wassignifiinc athnet floylilaor wniterorgtehna insottohpaict soigfntarteuersesi (nFiAguCre 25Ba; nAdNOAVCA;4 F ((3P,16o) =s t2h.1o5c2, Tp u= k0.e1y34’)s. BHuSlkD leapf <car0b.o0n5 ;datanot
content generally increased as ash canopy condition increases. The carbon content of leaves in AC 1
shown). BulkleafnitrogencontentalsoexhibitedvariabilityacrossACclasses,althoughdifferences
was significantly lower than that of trees in AC 2 and AC 4 (Posthoc Tukey’s HSD p < 0.05; data not
werelesspsrhoonwonu). nBcuelkd l,eaanf nditAroCgen1 cwonatsenotn allysod eixfhfiebrietendt vtahraianbiAlitCy a2craonssd AAC Ccl3as(sPeso, satlhthoocugThu dkieffye’rsenHceSsD p<0.05;
datanotshwoewre nle)s.sH priognhoufonclieadr, abnudl kACN 1 awnads olonwly dbiuffelkrenCt tihnanth AeCf o2 laianrd tAisCs3u (ePoosfthtorce eTsukineyA’s CHS1Dl ap r<g elyledto
0.05; data not shown). High foliar bulk N and low bulk C in the foliar tissue of trees in AC 1 largely
thesignificantdifferencesinleafC:NratiosbetweenACclasses(Figure5C;ANOVA;F =6.241,
(3,16)
led to the significant differences in leaf C:N ratios between AC classes (Figure 5C; ANOVA; F (3,16) =
p=0.005).
6.241, p = 0.005).

Figure 5. Relationship between green ash foliar 13C and ash canopy decline (A), foliar 15N (B), and
Figure 5. Relationship between green ash foliar 13C and ash canopy decline (A), foliar 15N (B),
foliar carbon to nitrogen ratio (C). Significance levels depict results of Tukeys HSD pairwise
and foliarccomarpbaorinsontos (pn <it 0r.o0g5)e. n ratio (C). Significance levels depict results of Tukeys HSD pairwise
comparisons(p<0.05).
Soluble sugar concentrations ranged from 13 to 51 mg g−1 and starch concentrations ranged from
1.2 to 22 mg g−1 (see Table 3 for means). In general, foliar soluble sugar concentrations were greater
Solubtlheans ustgaracrh ccoonncecnetnrattriaontiso (nTasbrlea 3n)g. Nedo sfirgonimfica1n3t dtoiffe5r1enmcesg ing s−o1luabnled susgtaarr (cph = 0c.o13n)c, setnartcrha t(pio =n s ranged
from1.2to0.32621)m, org TgN−C1 c(osneceenTtraabtiolens3 (pf o= r0.m66)e wanerse) .obIsnergveedn beertawl,eefno lAiaCr clsaossluesb. lesugarconcentrationswere
greater than starch concentrations (Table 3). No significant differences in soluble sugar (p = 0.13),
Table 3. Foliar soluble sugar, starch concentrations, and total nonstructural carbohydrates
starch(p=0.361),orTNCconcentrations(p=0.66)wereobservedbetweenACclasses.
(TNC) (mg/g) between ash canopy condition classes. Values represent mean and (SE). No
significant differences across AC classes were observed between soluble sugars, starch, or
Table 3. FToNliCar cosnocluenbtlreatsioungsa wr,erseta rrecvheacleodn vciean otnraet-wioanys ,AaNnOdVtAo.t al nonstructural carbohydrates (TNC)
(mg/g) between ash canAoCp y conSdoliutibolne Sculagsasres s. ValuSteasrcrhe presentTmNeCa n and (SE). No significant
differencesacrossACclas1s eswereo3b8s.0e r(0v.e9d) between4.s9o (l1u.5b)l esuga4r2s.9, s(2ta.3r)c h,orTNCconcentrations
werer evealedviaone-wayANOVA.
AC SolubleSugars Starch TNC
1 38.0(0.9) 4.9(1.5) 42.9(2.3)
2/3 46.1(4.0) 4.7(2.3) 50.8(2.1)
4 28.3(7.9) 12.0(5.9) 40.3(13.8)
Forests 2018, 9, x FOR PEER REVIEW 10 of 17
Forests2018,9,301 10of17
2/3 46.1 (4.0) 4.7 (2.3) 50.8 (2.1)
4 28.3 (7.9) 12.0 (5.9) 40.3 (13.8)
3.5. FoliarGasExchange
3.5. Foliar Gas Exchange
Foliar gas exchange parameters were tightly linked to EAB induced canopy deterioration.
Foliar gas exchange parameters were tightly linked to EAB induced canopy deterioration.
Specifically,A declinedprogressivelyfromamaximumof14.5µmolm2s−1(±0.39)to6.69(±0.35)as
Specificaslalyt, Asat declined progressively from a maximum of 14.5 μmol m2 s−1 (±0.39) to 6.69 (±0.35) as
ashtreesbecameincreasinglyimpactedbyEAB(Figure6A).Additionally,internalCO concentrations
ash trees became increasingly impacted by EAB (Figure 6A). Additionally, internal CO2 co2ncentrations
(Ci)in(cCrei)a insecrdeainselde ainv leesavoefsa osfh ascha ncaonpoipeisese xepxpeerrieienncciinngg ssiiggnnifiificacnatn EtAEBA dBadmaamgea (gFeig(uFrieg 6uBr)e. 6B).
16
A. a
14
-1s) 12 b
2
m 10
ol c
m 8
µ c
( at 6
As 4
2
0
B.
c
250 b bc
a
200
Ci150
100
50
0
1 2 3 4
Ash Canopy Condition
Figure 6. Mean light saturated photosynthetic rate (Asat; at 1600 PAR; panel (A)) and internal leaf CO2
Figure6. Meanlightsaturatedphotosyntheticrate(Asat;at1600PAR;panel(A))andinternalleaf
concentration (Ci; panel (B)) across foliage from ash trees of different ash condition classes. Letters
CO concentration (Ci; panel (B)) across foliage from ash trees of different ash condition classes.
2 represent significance levels determined by Tukey’s HSD pairwise comparisons (p < 0.05), error bars
LettersrepresentsignificancelevelsdeterminedbyTukey’sHSDpairwisecomparisons(p<0.05),
denote ±1 SE.
errorbarsdenote±1SE.
4. Discussion
4. Discussion
The results presented herein indicate that EAB larvae damage ash tree cambial tissue resulting in
Tdhreasrteiscu clhtasnpgreess teon pteladnth weraetienr rineldatiicoantse ltehaadtinEgA toB clharrovnaiec dwaamtera gstereasssh. Atrse EeAcBa msybmiapltotimss pureogreressuslitoinn gin
drasticincchreaansgeeds, StoFDp lwanats wreadtuecrerde,l aintihoibnistinlega dAisnat gantod crhedrounciincgw leaatef rsiszter easnsd. AmsasEsA. IBncsryemaspintgo mEApBr olagrrveasls ion
feeding gallery cover or emergence holes within the stem have previously been linked to shifts in leaf
increased,SFDwasreduced,inhibitingA andreducingleafsizeandmass. IncreasingEABlarval
sat
and canopy level traits [24,32,35]. Although the experiment presented herein was not designed to
feedinggallerycoveroremergenceholeswithinthestemhavepreviouslybeenlinkedtoshiftsin
investigate progressive canopy thinning as a mechanism by which ash trees can re-assimilate nutrients
leafandcanopyleveltraits[24,32,35]. Althoughtheexperimentpresentedhereinwasnotdesigned
for later use as a means to cope with stress [45], our results lend support to this notion. Specifically, EAB
to investigate progressive canopy thinning as a mechanism by which ash trees can re-assimilate
damage resulted in significant reductions in leaf morphological characteristics (SA and DM; Figure
nutrie4nat,sb)f,o cronlasitseternut sweitahs leaafm reesapnosnsteos tcoo wpeatewr istthressst,r eysets S[L4A5] w, oasu crornesseurvltesd l(eFnigdurseu Sp1p).o Trhtet roedthucistionnost ion.
Specifiinc aSlAly ,anEdA dBry dmaamssa sgeeenr iens uthlitse sdtuidny swigernei ficocmanptarraebdleu tcot tihoonsse ionf Ploesasfenm eot rapl. h[6o3l]o. gHiocwalevcehra, rthaec tleacrkis tics
(SAanodf aD SMLA; Freisgpuornese4 aas,b E)A, Bco inmspiastcetendt twreeitsh colenatrfasrtess tphoe nfisnedsintgos wofa Ptoesrsesntr eets asl,. y[6e3t] wSLhAo owbsaesrvceodn as 5e–rved
(Figur1e0%S1 i)n.crTeahsee irne dthuec StLioAn osf iBnetuSlAa aannd dPodpurylusm asassoscisaeteedn winitht whiest asntudd dyryw treearetmceonmts.p Tahrea δb1l3eC tiosottohpoisc e of
enrichment of leaf tissue as ash trees progressed from healthy (AC 1) to heavily EAB impacted (AC 4)
Possenetal.[63]. However,thelackofaSLAresponseasEABimpactedtreescontraststhefindingsof
further supports the notion of EAB-induced chronic water stress in ash trees of this forest (Figure 5A)
Possenetal.[63]whoobserveda5–10%increaseintheSLAofBetulaandPopulusassociatedwithwet
anddrytreatments. Theδ13Cisotopicenrichmentofleaftissueasashtreesprogressedfromhealthy

(AC1)toheavilyEABimpacted(AC4)furthersupportsthenotionofEAB-inducedchronicwater
stressinashtreesofthisforest(Figure5A)andisconsistentwiththepreviousreportsofshiftsinfoliar
carbonisotopiccompositionwithdifferencesinplantwateruseefficiency[64,65].
At the tree-level, sap flux rates were impacted by both biotic (EAB larvae) and abiotic (solar
radiationandVPD)drivers(Table2,Figure1). ThediurnalpatternsofSFDofhealthytreesreported