Population genetics of purple saxifrage (Saxifraga oppositifolia) in the high Arctic archipelago of

14 Pages·2015·0.64 MB·English

Research Article
Composition of fungal soil communities varies with plant
D
o
w
abundance and geographic origin nlo
a
d
e
d
Vanessa Reininger1,2,3*, Laura B. Martinez-Garcia1,4, Laura Sanderson1 and Pedro M. Antunes1 fro
m
1 DepartmentofBiology,AlgomaUniversity,SaultSte.Marie,ON,P6A2G4Canada h
2 Presentaddress:Agroscope,InstituteforPlantProductionSciencesIPS,Schloss1,8820Wa¨denswil,Switzerland ttp
s
3 Presentaddress:ETHZurich,InstituteofIntegrativeBiology,ForestPathologyandDendrology,Universita¨tstrasse16,8092Zurich, ://a
Switzerland ca
4 Presentaddress:DepartmentofSoilQuality,WageningenUniversity,6700AA,Wageningen,TheNetherlands de
m
Received:27May2015;Revised:30July2015;Accepted:1September2015;Published:14September2015 ic.o
u
AssociateEditor:JeanH.Burns p
.c
o
Citation:ReiningerV,Martinez-GarciaLB,SandersonL,AntunesPM.2015.Compositionoffungalsoilcommunitiesvarieswithplant m
abundanceandgeographicorigin.AoBPLANTS7:plv110;doi:10.1093/aobpla/plv110 /a
o
b
p
la
dArbivsetrsraofcptl.anInttceormacmtiounnsitoyfsbtreuloctwugreroiunnindvfaudnegdalgcroamssmlanudnsit.ieHsowwietvheer,xfioetilcdasnudrvneaytsivlienkpilnagntpslapnetciceosmmmayunbietyimstpruocrttaunret /article
withbelowgroundfungalcommunitiesaremissing.Weinvestigatedwhetheraselectednumberofabundantandrela- -ab
s
tivelyrareplants,eithernativeorexotic,fromanold-fieldsiteassociatewithdifferentfungalcommunities.Wealso tra
c
assessedwhethertheseplantsshoweddifferentsymbioticrelationshipswithsoilbiotathroughtheirroots.Wechar- t/d
acterizedtheplantcommunityandcollectedrootstoinvestigatefungalcommunitiesusing454pyrosequencingand oi/1
assessedarbuscular mycorrhizalcolonizationandenemy-inducedlesions.Differencesinfungalcommunitieswere 0.1
consideredbasedontheassessmentofa-andbdiversitydependingonplant‘abundance’and‘origin’.Plantabun- 09
3
danceandorigindeterminedthefungalcommunity.Fungalrichnesswashigherfornativeabundantasopposedto /a
o
relativelyrarenativeplantspecies.However,thiswasnotobservedforexoticsofcontrastingabundance.Regardlessof bp
la
theirorigin,bdiversitywashigherforrarethanforabundantspecies.Abundantexoticsinthecommunity,whichhap- /p
pentobegrasses,weretheleastmycorrhizalwhereasrarenativesweremostsusceptibletoenemyattack.Ourresults lv1
1
suggestthatcomparedwithexotics,therelativeabundanceofremnantnativeplantspeciesinourold-fieldsiteisstill 0/1
linkedtothestructureofbelowgroundfungalcommunities.Incontrast,exoticspeciesmayactasadisturbingagent 80
3
contributingtowardsthehomogenizationofsoilfungalcommunities,potentiallychangingfeedbackinteractions. 79
1
b
Keywords: Arbuscularmycorrhizalfungi;communityecology;adiversity;bdiversity;454pyrosequencing. y g
u
e
s
t o
Introduction n
communityassemblythroughtestingplant–plantcom- 0
9
Oneofthemajorgoalsinecologyistounderstandwhy petitiveinteractionsandplant–abioticinteractionssuch A
p
some plant species in natural communities are more asnutrientandlightavailability(Tilman1985;Thompson ril 2
abundantthanothers(Hubbell2001;Chave2004;McGill 1987;WilbyandBrown2001;Milleretal.2005;Dybzinski 01
9
etal.2007;Dumbrelletal.2010).Manystudiesfocusing andTilman2007;Beveretal.2012).Thereisincreasing
on plantcommunityecologyhaveprovidedinsightinto evidencethatbelowgroundplant–microbeinteractions
*Correspondingauthor’se-mailaddress:[email protected]
PublishedbyOxfordUniversityPressonbehalfoftheAnnalsofBotanyCompany.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/
licenses/by/4.0/),whichpermitsunrestrictedreuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 1
Reiningeretal.—Surveyofassociatedfungalcommunitiesfromanold-field
areimportanttoexplainplantcommunitydynamics(Bever 2002). However, (cid:2)50% of the studies do not support
etal.1997,2010,2012;ReynoldsandHaubensak2009). the Enemy Release Hypothesis (Agrawal et al. 2005;
Many studies have already been conducted focusing on Jeschkeetal.2012).Thesecontrastinghypotheseshigh-
belowground microbes and their interactions with the lighttheuncertaintysurroundingmicrobialcommunities
hostplant (Johnsonetal.1997;JumpponenandTrappe associatingwithnativeandexoticplantspeciesinnatural
1998; Reynolds et al. 2003; Bonfante and Anca 2009; communitiesandhowalterationsimposedbyexoticsmay
Compant et al. 2010; Porras-Alfaro and Bayman 2011; affect the soil biota community and by extension, the
Reininger et al. 2012; Reininger and Sieber 2012, 2013). entireplantcommunity. D
o
However,littleisknownabouttheimpactofplantcommu- Next-GenerationSequencingtechnologiessuchas454 wn
lo
nitystructureonthebelowgroundsoilmicrobialcommu- pyrosequencing using universal primers provide an a
d
nity(Klironomos2003;Reynoldsetal.2003;Bagchietal. opportunity to survey the soil microbial diversity asso- ed
2010; Bever et al. 2010, 2012) especially with regard to ciatedwithplantspeciesinnaturalcommunities(Bue´e fro
m
plant communities which are progressively invaded by etal.2009;CostaandAntunes2012).Thesesurveyscon- h
exotic plant species (Levine 2000; Klironomos 2002). tributetoimproveourunderstandingofthebiodiversityof ttp
s
AccordingtotheWildSpeciesReportissuedbytheGovern- microorganisms that co-exist and interact with plant ://a
c
ment of Canada, extant natural plant communities in rootsacrossspatialscalesandpossiblyinfluenceplants’ ad
e
Canadanowincludealargeportionofexoticspecies,com- relative abundance and the dynamics between native m
ic
prising24%ofallthevascularplants(WildSpecies2010, andexoticplantspecies.Specifically,fungiarekeydrivers .o
u
http://publications.gc.ca/collections/collection_2011/ec/ of primary productivityand plant communitystructure p.c
o
CW70-7-2010-eng.pdf,November2014).Duetoestablish- due to their diverse roles as mycorrhizal fungi, endo- m
mentofexoticplantspecies,plantcommunitystructure phytes,decomposersandpathogens.Theydirectlyinter- /ao
b
canchangeandconsequently,interactionswithsoilmicro- actwithplantrootsresultinginfunctionaloutcomesthat pla
bialcommunitiesmaychangeaccordingly.Bycomparing range from mutualism to parasitism (Schulz and Boyle /a
theassociationsbetweenco-occurringnativeandexotic 2005;SinclairandHoward2005;SmithandRead2008). rticle
plant species and their respective belowground fungal Betterknowledgeoffungalcommunitystructurewithin -ab
s
communities,wemaybebetterabletounderstandand plantrootsamongdifferentplantspecies is afirststep tra
c
predictplantcommunitydynamicsininvadedecosystems. towardsunderstandinghowfungiinfluenceplantcom- t/d
o
AccordingtoKlironomos(2002),plantspeciesrelative munitydynamics. i/1
abundance may partially be explained by interactions Wesurveyedthefungaldiversitypresentintherootsof 0.1
0
betweenplantsandsoilmicrobes.Plant–soilfeedbacks fournativeandfourexoticplantspeciesfromanatural 9
3
aredefinedastheeffectthatplantspeciesexertonsoil old-field plant community. Within each of these two /a
o
b
biotic and abiotic factors, which in turn can feedback groups,twospecieswerehighlyabundantinthecommu- p
la
intogrowthandfitnesseffectsonsubsequentconspecific nityandtheothertwowererelativelyrare.aDiversitywas /p
lv
or heterospecific plants (Van der Putten et al. 2013). In usedtomeasurethefungaldiversityineachplantspecies 1
1
0
native plants, feedbacks are subjected to co-evolution whereasbdiversitywasameasureofvariationinfungal /1
8
betweensymbionts.Exoticspeciesinvadinganewrange composition among groups (abundant versus rare and 0
3
7
willalsoestablish interactionswiththesoil biotabutby native versus exotic) (Anderson et al. 2011). We also 9
1
virtueofthehostplant’snoveltyinthesystem,thoseinter- determinedlevelsofarbuscularmycorrhizalcolonization b
y
g
actionsmaynotyethaveexperiencedco-evolutionarypro- andlesionsinflictedbyenemiestoplantroots. u
e
cesses.Asaresult,exoticsmayassociateindiscriminately Wepredictedthatfungalcommunitiesassociatingwith st o
with soil microbial communities and function as a non- plantrootsdifferdependingonplantspecies‘abundance’ n
0
hostforsomeplantpathogens,i.e.theyshowasymptom- and ‘origin’. More specifically, abundant natives would 9 A
lBeosyslein2fe0c0t5io).nT(hNeu¨srenbfuenrggearlasnymdbLiiopnkats,2a0c0c5u;mScuhlautliznganind arasrseoncaiattiveeswaitshthmeyoareresmimoirlearofftuenngsaulrrcooumndmeudnbiytiecosntshpaen- pril 20
1
exotic species without necessarily causing population cificsthatarelikelytoharborthesamefungalcommunity. 9
declines, may in turn negatively affect native plants to However,exoticswereexpectedtoassociatewithamore
whichtheyarepathogenic(RoyandHandley2012;Flory variablefungalcommunityastheyarenotyetrestricted
andClay2013;VanderPuttenetal.2013). toaparticular(i.e.co-evolved)fungalcommunity.Abun-
Exotic plants are assumed to leave their enemies dant and rare exotic plant species were expected to
behindintheirhomegeographicrange(EnemyRelease show the same variable pattern as they have not had
Hypothesis)andthiscouldexplainthedifferentialinfeed- the same amount of time to interact with the below-
backintheirnewrangerelativetonatives,whicharemore groundfungalcommunitiesandestablishspecies-specific
affected by a negative feedback (Keane and Crawley relationshipsincomparisontothenatives.
2 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015
Reiningeretal.—Surveyofassociatedfungalcommunitiesfromanold-field
Methods zone.Onehundredplotswereselectedrandomlyforassess-
mentofplantspeciesrichnessandabundance.Speciesrich-
Studysiteandplantcommunitydiversity
nesswasassessedinthesecondweekofeachmonthfrom
The study site, our long-term ecological research site MaytoAugust2012.Plotswerethoroughlyexaminedandall
(LTERS), is a 100×100m flat homogeneous (i.e. plant speciespresentwererecorded.Percentoccurrenceofspe-
coverandedaphicconditions)oldagriculturalfieldatthe cieswascalculated by counting the numberof plots (i.e.
Ontario Ministry of Natural Resources Research (OMNR) the100randomlyassignedplots)containingeachspecies.
Arboretum in Sault Ste. Marie, Ontario (46832′34.1′′N, Thisprovidedanestimateofspecies’abundance(i.e.percent D
o
84827′29.4′′W).Thefieldsitehasnotbeencultivatedfor occurrence)inourold-fieldsite(Fig.1). wn
six decades and has not been mowed since 2009. The Thesurveycomprisedfourexoticandfournativeplant loa
d
soil is a silty loam and soil analysis conducted in 2011 species(Table1).Ineachcategory,i.e.exoticandnative, ed
were as follows (n¼6): 22.6+1.06% clay, 53.1+ twospecieswereabundantandtwowererelativelyrare fro
m
0.27% silt, 24.4+1.07% sand, 0.13+0.001% total N, (termedabundantandrarehereon)atthisold-fieldsite h
1.81+0.02%totalC,11.4+0.07mgkg21Brayavailable (Fig.1).Thespecieswerechosenaccordingtotheirnat- ttp
s
P,360.7+2.55mgkg21Ca,45.0+0.23mgkg21K,38.9+ ural relative abundance at the old-field site and, as ://a
c
0.32MgandpH¼5.74+0.01.Thefieldwasdividedinto such, the lack of a balanced phylogenetic design was ad
e
twohundredandeighty-nine1×1mplotsona17×17 unavoidable. Exotic grasses dominate the site and we m
ic
plotgrid.Eachplotwassurroundedbya1mwidebuffer selected two of the most dominant for our exotic .o
u
p
.c
o
m
/a
o
b
p
la
/a
rtic
le
-a
b
s
tra
c
t/d
o
i/1
0
.1
0
9
3
/a
o
b
p
la
/p
lv
1
1
0
/1
8
0
3
7
9
1
b
y
g
u
e
s
t o
n
0
9
A
p
ril 2
0
1
9
Figure1. Plantcommunityattheold-fieldsiteinSaultSte.Marie,ON,Canada.Relativeabundancewascalculatedasthenumberofplotsin
whicheachspecieswaspresentoutof100plots(1×1m).Blackbarsandspeciesnameswithanasteriskindicatethespeciesusedinthisstudy.
E,exoticorigin;N,nativeorigin.
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 3
Reiningeretal.—Surveyofassociatedfungalcommunitiesfromanold-field
Table1. PlantspeciesfromourLTERSinvestigatedinthisstudy. at2808Candfreeze-driedfor4days(LabconcoFreeZone
2.5,USA).Toverifypropersurfacesterilizationthree0.5-cm
Nativerare Nativeabundant Exoticrare Exotic
rootpiecesofeachindividualplantwereexcisedandsur-
abundant
facesterilizedasdescribedaboveandincubatedatroom
..................................................................................
R.hirta S.canadensis H.perforatum B.inermis temperatureonpotatodextroseagarplates.Surfacester-
ilizationoftheplantrootsystemswassuccessfulasfungi
S.lanceolatum P.arundinacea P.recta D.glomerata
started growing out of the roots onlyafter several days
at208C.Thismeans organismsattachedtothesurface D
o
abundantcategory (i.e. Dactylisglomerata and Bromus wereexcludedbutfungiwithintherootswerestillactively wnlo
growing. a
inermis).Forthenative-abundantcategoryweselected d
DNAwasextractedusingtheDNeasy96PlantKit(Qia- ed
themostabundantnativesatthesite(i.e.Solidagocana- gen, Toronto, ON, Canada) according to the manufac- fro
densis and Phalaris arundinacea). Rare (i.e. relatively m
rarer) specieswerechosenbasedon thepresenceofat tpuurreifir’esdpursoitnogcothl.eTGheenoexmtricacDtNeAd CDlNeaAnw&aCsocnlceeannteradtoarnTMd http
least four replicates in the field. Potentilla recta and Kit (ZymoResearch,USA).AfterDNApurification,thesix s://a
Hypericumperforatumweretheselectedrareexoticspe- c
cies and Symphyotrichum lanceolatum and Rudbeckia technicalreplicatescomingfromonesinglerootsystem ade
were pooled together leading to 32 samples in total m
hirtatherarenatives.Ourmaingoalwasnottocontrol (eight plant species×four replicates). The amount of ic.o
for phylogenetic relatedness or functional group but u
insteadtosurveyand compareplantspeciesaccording DNAwasmeasuredusingaBioTekTM EpochTM Microplate p.c
o
Spectrophotometer(Winooski,VT,USA). m
totheirrelativeabundanceinthiscommunity.Oureight PrimersfITS7asaforward(5′-GTGARTCATCGAATCTTTG-3′) /ao
selectedspeciesrepresent24%ofallthespeciesinthe b
community. Across the entire old-field site four indivi- andITS4asareverse(5′-TCCTCCGCTTATTGATATGC-3′)were pla
chosen to sequence samples using 454 pyrosequencing /a
dpvioudtauelasnlptpieaerlrsspppleaacntiietasslpwveaecrriieeasbs)ailwimtyep,rleseesdtasfmoorpfrloreeodptsilniaccanltodestseo(pmornoinexiimimnidiztiey- (sIphercmifiacrkfuentgaall.2p0ri1m2e).rWfITeSs7pbeceicfiacuaslleywcheowseerteheinnteerweshteigdhilny rticle-abs
to each other in a smaller area wherever possible. To amplifyingtheentirefungalcommunitypresentwithinthe trac
account for this possible spatial variation we included plantroots.IncombinationwithITS4thispairspecifically t/d
o
thefactor‘replicate’inourstatisticalmodel.Thechoice amplifiestheITS2regionandproducesshorteramplicons i/1
offourreplicatesisconsistentwithotherstudiesinvesti- compared with the ITS1f/ITS4 primer pair. We expected 0.10
that as we sequenced an entire fungal community any 9
gating belowground fungal community interactions 3
bias resulting from different sequence lengths would be /a
with plants(Johnsonet al.2004;Mart´ınez-Garc´ıaet al. ob
minimizedbyamplifyingtheshorterITS2regioninstead p
2011). la
oftheentireinternaltranscribedspacer(ITS)region.Accord- /p
lv
ing to a BLASTN search, our selected primer pair should 1
1
454Pyrosequencingoffungalcommunities match68%oftheGlomeromycota(Ihrmarketal.2012). 0/1
8
Todeterminethefungalcommunityintheselectedspe- SamplesweresenttoGenomeQue´bec(Montre´al,Canada) 0
3
7
cies,plantswerecollectedincludingtheirwhole-rootsys- andpurifiedanddiluted1:4usingaMultiScreenPCRfiltra- 9
1
tem using a spade within 2 days in the second half of tionplate(Millipore,Canada).PCRwithprimersfITS7and b
y
August 2013. They were placed into Ziploc bags and ITS4wasconductedin25mLreactionvolumecontaining gu
e
cooledimmediately inacold boxandlaterat48Cuntil 2.5mL purified DNA, 2.5mL 10× buffer, 1.5mM MgCl2, st o
furtherprocessing.Plantswerestoredat48Cnolonger 0.2mMdNTPs,0.4mMofeachprimerand5UmL21HotStar- n 0
than24h.Rootsystemswerewashedinthelaboratory TaqDNAPolymerase(Qiagen)asfinalconcentrations.PCR 9 A
wanityhrcoooldt tfraapgwmaetnetrstothraetmwoveerethneotsociloannndecttoedseptoartahtee rautn9n6in8gCfcoorn1d5itimoninsfwoleloreweadsbfoyll3o5wcsy:cilneistiaolfddeennaattuurraattiioonn pril 20
1
mainsystemandwhichcouldbelongtoadifferentplant. at968Cfor30s,annealingat528Cfor30s,extensionat 9
DNAwasextractedfromsixtechnicalreplicatesperindi- 728C for 1min and a final extension step at 728C for
vidualplanttoensurehavingarepresentativeamountof 10min.Samplesweretaggedusing10-bpmultiplexidenti-
DNA.Foreachtechnicalreplicatetwenty-one0.5-cm-long fiers(MIDTags)inaseparatebarcodingPCRreaction.The
rootpieceswereexcised.Forverythickroots,theamount tagged,equimolarampliconsweresequencedonaRoche
ofrootmaterialwasadjustedaccordinglytoavoidover- 454GSFLXinstrumentusingtheFLX+chemistryatGenome
loadingtheDNA extraction column. Rootswere surface Que´bec.Atotalof327867singlereadswereproducedina
sterilizedfor30sin30%hydrogenperoxideandthereac- one-fourthregionofa454plate.Sequencedataweresub-
tionwasstoppedusing70%EtOH.Theywerethenfrozen mittedtoNCBI(accessionno.SRP043133).
4 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015
Reiningeretal.—Surveyofassociatedfungalcommunitiesfromanold-field
Symbioticrelationships effect of plant abundance and origin was calculated by
measuringtheeffectofeachfactorafteraccountingfor
To investigate symbiotic (i.e. functional) relationships
betweenmutualisticandantagonisticbelowgroundbiota thevarianceexplainedbytheotherfactor(varianceparti-
andourtargetspecies,plantswerecollectedatourold- tioning (Schmid et al. 2002)). This was done after first
field site in mid-August 2012, with the exception of accountingforourfourreplicates(i.e.spatialvariability)
R.hirta,whichcouldnotbefoundonthatoccasion.Four and we also took into consideration the two different
individualplantswerecollectedforeachspeciesusinga plantfunctionalgroups(i.e.monocotyledonsanddicotyle-
spade(exceptforH.perforatumforwhichwecouldonly dons).Wefirstranamodelincluding‘plantspecies’asa Do
samplethreeindividuals).Inthelaboratory,rootsamples thirdfactorand,asexpected,plantspecieswereasignifi- wn
lo
werecarefullywashedanddividedintotwosub-samples. cantpredictoroffungalcommunitycomposition.Bythis ad
Onesub-samplewasusedtoquantifythepercentageof factor, 42% of the variance was explained. However, ed
root colonizationbyarbuscularmycorrhizalfungi (AMF) since our main goal was to survey general patterns of fro
m
usingthe gridline intersect method byMcGonigle et al. fungaldiversityratherthanspecies-specificeffects,we h
(1990). The roots werestained using 0.03% Chlorazol- considered species as ‘plant functional group’ thereby ttps
blackEandaminimumof100intersectionsperreplicate gainingdegreesoffreedominourtwofactormodel. ://a
c
wereexamined.Theothersub-samplewasusedtoquan- Tocalculatethevarianceexplainedbythefactor‘abun- ad
e
dance’(fullmodelincludinginteraction)weusedthefol- m
tifytheincidenceofdiseaseandenemyattackbyvisually ic
estimatingdamage(i.e.thenumberoflesionsperroot) lowingformula: .ou
p
caused by pathogens, parasites and herbivores. This Dissimilaritymatrix(cid:2)replicate+ plant functionalgroup .co
methodisidenticaltotherootstainingandquantification m
method used for AMF,excludingthe stainingstep.This +origin+abundance+origin:abundance /ao
b
approachprovidesaconsistentestimate ofplanttoler- (1) pla
/a
a20n0c3e;Sacghaninitszetrpeattahl.o2g0e1n1s).aFnordbootthheArMeFnaenmdileessio(Mniqtcuhaenl-l Tocalculatethevarianceexplainedbythefactor‘origin’ rticle
tification,H.perforatumsampleshadtoundergoanadd- (fullmodelincludinginteraction)weusedthefollowing -ab
s
itionalbleachingstepusingalkalinehydrogenperoxideto formula: tra
c
removerootpigmentationtoallowvisualization. t/d
Dissimilaritymatrix(cid:2)replicate+ plant functionalgroup o
i/1
+abundance+origin+abundance:origin 0.1
Bioinformaticsandstatisticalanalysis (2) 09
3
BioinformaticswereperformedatGenomeQue´becusing /a
o
b
Qiime(version1.7.0)thepipelinefordataprocessing.In Theplantfunctionalgroupwaskeptinthemodelsinceit p
la
total, 327867 readsweregenerated,assessed fortheir explained9.21%ofthetotalvariance.Bykeepingit,the /p
lv
qualityand clustered.Reads witha Phred qualityscore variancefromthisfactorcouldbedeductedfromourvari- 1
1
≥30, ,3 undetermined bases and ,4 bases having a ablesofinterest(‘abundance’and‘origin’).Asdissimilar- 0/1
8
Phredqualityscore ,20passedthequalitycontrolstep. ity measure, Bray–Curtis was used to account for the 0
3
7
Allreadswerecuttoafinallengthof220bases.Reads composition of the fungal communities as well as to 9
1
wereclusteredat100%ID,followedby99%andfinally exclude joint absences. a Diversity wastested foreach b
y
g
by97%.Chimeraclusterswereremovedduringthisstep. sampleusingthefunction‘rarefy’fromtheveganpack- u
e
SequenceswereblastedagainsttheITS2databaseofthe age (Oksanen et al. 2013) in R (R CoreTeam 2013) and st o
UNITEITSdatabase(Ko˜ljalgetal.2013),trimmedtothe an ANOVA was run to test for significant differences n 0
ITS2region.AnOperationalTaxonomicUnits(OTU)table dependingonthefactors‘abundance’and‘origin’.Adis- 9 A
wraaresfigeednteora1t0e0d0rreepardessepnetrinsgamfupnlgei.TohnelyQaiinmdespaimpeplliensew(veerre- t(Oankscaenemnaettriaxl.w20a1s3c)rineaRte(RdCuorseinTgeatmhe20v1e3g)atnoepsatcimkaagtee pril 20
1
sion1.7.0)wasusedtocalculatetherarefractioncurves. b diversity using the function ‘betadisper’ and to draw 9
Afterrarefaction,singletonsanddoubletonswereremoved principlecoordinatesanalysis(PCoA)plots.
andtheresultingOTUtablewasusedforfurtheranalysis. AnotherANOVAwasruntotestwhetherAMFrootcol-
To determine whether fungal communities differed onization and lesions varied according to ‘abundance’
betweenthefactors‘abundance’(i.e.rareandabundant and‘origin’.Tomeettherequirementsofthestatistical
plantspecies)and‘origin’(i.e.nativeandexoticplantspe- tests,variableswerearcsinetransformed.Meansamong
cies),aPermutationalMANOVAwasconductedusingfunc- treatmentswerecomparedusingTukey’shonestlysignifi-
tion‘adonis’fromtheveganpackage(Oksanenetal.2013) cant difference (HSD) test (P,0.05). These statistical
implementedinthesoftwareR(RCoreTeam2013).The analyses were performed with the software StatSoft,
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 5
Reiningeretal.—Surveyofassociatedfungalcommunitiesfromanold-field
Inc.(2013),STATISTICA(dataanalysissoftwaresystem), Table2. Taxonomicfungalgroupsdetectedintherootsofallplant
version12.www.statsoft.com. speciesinthefilteredandrarefiedOTUtableexcludingsingletons
anddoubletons.
Taxonomicgroup No.of OTUs No.of Reads
Results
OTUs in% reads in%
..................................................................................
Fungalcommunitypatterns Ascomycetes 138 84 26754 95.5
Wedetected164fungalOTUsacrossallsamples.Thephy- Helotiales 57 41.3 17964 67.1 D
o
lum Ascomycota comprised 138 OTUs (84% of fungal Chaetothyriales 25 18.1 1291 4.8 wn
lo
OTUs) whereas Basidiomycota only comprised 23 OTUs a
Pleosporales 17 12.3 1522 5.7 d
(14%offungalOTUs),theremainingthreeOTUs(1.8%of ed
fungal OTUs) could only be assigned to Kingdom Fungi Capnodiales 9 6.5 1522 5.7 fro
m
(Table 2). We were unable to recover AM fungi as we Sordariales 7 5.1 809 3 h
expectedbasedonourprimerchoice.Fungalrichnessof Hypocreales 3 2.2 129 0.5 ttps
rarefiedandnon-rarefiedsamplesforeachplantspecies Xylariales 2 1.4 195 0.7 ://a
c
isprovidedinTable3.ThetwoplantspeciesH.perforatum ad
Chaetosphaeriales 2 1.4 121 0.5 e
and P. arundinacea had the highest number of fungal m
OTUs(Table3)andtheHelotialeswasthemostcommon Dothideales 2 1.4 50 0.2 ic.o
u
fungalorderacrossallplantspecies(Fig.2andSupporting Hysteriales 1 0.7 52 0.2 p.c
o
Information—TableS1fordetailedtaxonomicresults). Basidiomycetes 23 14 1258 4.5 m
Permutational MANOVA showed significant fungal Agaricales 6 26.1 198 15.7 /aob
Rco2m¼m0u.1n4it0y,dPis¼sim0.0ila0r1it)ieasnfdor‘o‘aribguinn’d(aFnce’¼(F14,2.599¼0,5.R129¼4, Auriculariales 5 21.7 102 8.1 pla/a
0.120, P¼0.001) after accounting fo1r,2b9oth the spatial Sebacinales 3 13 630 50.1 rticle
variation (factor ‘replicate’) in our field site (F3,29¼ Cantharellales 2 8.7 285 22.7 -ab
s
1.322 with R2¼0.103, P¼0.161 (1) and, R2¼0.103, Polyporales 1 4.3 8 0.6 tra
c
P¼0.140 (2)) and our two plant functional groups Undetermined(fungi) 3 1.8 3134 11.2 t/d
o
(monocotyledons and dicotyledons) which explained i/1
9.21% of the total variance (F1,29¼3.756 with R2¼ 0.1
0.092,P¼0.005(1)andR2¼0.092,P¼0.002(2)).How- 09
3
ever,theinteractionof‘abundance’and‘origin’wasnot Table 3. Fungal richness per plant species for non-rarefied and /a
o
significant(F ¼1.235,R2¼0.034,P¼0.255)ineither rarefiedsamples. bp
1,29 la
ofthetwomodelsandtherefore,wecontinuedwiththe /p
Plantspecies Non-rarefied Rarefied lv
analysisusingthereducedmodels(i.e.byexcludingthe .................................................................................. 11
interaction but keeping the factor ‘replicate’) (Table 4). R.hirta 45.26127 27.25000 0/1
8
ThePCoAplotswereinconcertwiththeresultsfromthe S.lanceolatum 78.96880 49.00000 03
7
permutationalMANOVApointingtowardsthestructuring S.canadensis 75.55551 44.33333 91
offungalcommunitiesasafunctionof‘abundance’and b
P.arundinacea 107.10158 64.75000 y g
‘origin’(Fig.3).Figure3Cshowsthatour‘plantfunctional u
groups’didnotsharethesamefungalcommunitiesbut H.perforatum 108.59286 55.25000 est o
those were rather dependent on origin except for P.recta 63.01892 40.33333 n 0
H.perforatumandS.canadensis. B.inermis 69.51156 43.50000 9 A
affaecDtievedrsbiytyt,hi.ee.ftahcetnoursm‘abbeuronfdOaTnUces’p(eFr1s,2a6m¼p1le.1,w08a,sPno¼t D.glomerata 64.02056 39.25000 pril 201
0.302)or‘origin’(F ¼0.163,P¼0.690)buttheirinter- 9
1,26
actionwassignificant(F ¼6.908,P¼0.014)(Fig.4A).
1,26 native plants, fungal community composition among
Fungalbdiversityvariedsignificantlydependingonplant
exoticswasmoresimilar(Fig.3B).
species’relative‘abundance’(F ¼5.77,P¼0.017)but
1,28
notontheir‘origin’(Fig.4B).
Symbioticrelationshippatterns
Overall, abundant plant species tended to associate
with a more similar fungal community compared with Species‘abundance’aswellastheinteractionbetween
rareplants(Fig.3A).Thiswasalsosupportedbybdiversity ‘abundance’ and ‘origin’ significantly determined AMF
(Fig.4B).Inaddition,PCoAindicatedthatcomparedwith colonization by hyphae in our selected plant groups
6 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015
Reiningeretal.—Surveyofassociatedfungalcommunitiesfromanold-field
Incontrast,hyphalcolonizationdidnotvarysignificantly
withplantrelativeabundancefornativeplants(Fig.5A).
Arbuscularcolonizationshowedthesametrendsbutthe
resultswerenotstatisticallysignificantduetoahighvari-
ancefortherareexoticspecies,whichwereunderrepre-
sented(Fig.5B).Wefoundasignificanteffectof‘origin’as
well as‘origin’בabundance’ for vesicularcolonization
(F ¼7.663, P¼0.011 and F ¼6.937, P¼0.015, D
1,23 1,23 o
respectively).However,thiswasincontrastwithhyphal wn
lo
colonization. The rare native species had the greatest a
d
numberofvesiclesandrareexoticstheleast(Fig.5C). ed
‘Origin’wasasignificantfactordeterminingthesuscep- fro
m
tibility of plant species to enemy attack belowground h
(F ¼17.811,P≤0.001),anddespitethelowerreplica- ttp
1,22 s
tion of rare plants due to the absence of R. hirta, we ://a
c
detected a marginally significant effect of ‘abundance’ ad
e
(F ¼4.148,P¼0.054).Inaddition,therewasamargin- m
1,22 ic
allysignificanteffectfortheinteraction(F1,22¼3.273,P¼ .ou
0.084).Rarenativeswerethemostsusceptiblegroupto p.c
o
enemy attack whereas for exotics, their lesion number m
was the same regardless of their relative abundance in /ao
b
thecommunity(Fig.5D). pla
/a
rtic
Discussion le-a
b
s
Figure2. Rarifiedabundanceoffungalsequencereadsdiscrimi- Themainobjectiveofthisstudywastoinvestigatepatterns tra
nated by fungal order for each plant species. ‘Undetermined’ connectedwithplantandbelowgroundfungalcommunity ct/d
meansOTUsnotdeterminedtothelevel‘Order’butatleasttothe o
kPAin,gPd.oamrunodfiafunnagcie.aR;HH,PR,.Hh.iprtear;foSrLa,tSu.mla;nPcRe,oPl.arteucmta;;SBCI,,BS..icnaenramdies;nDsiGs;, aspsseecmiesbloyf.Mcoonretrasspteincgificreallalyt,ivweeadbeutnedrmaninceedanwdhegtehoegrrapplahnict i/10.1
0
D.glomerata. origininanaturalold-fieldcommunitytypicalofeastern 93
NorthAmericaassociateddifferentlywiththebelowground /a
o
b
fungalcommunity.Ourmaingoalwastoprimarilysurvey p
Table 4. Factors and their interaction referring to community la
plants based on their relative abundance. As such, the /p
dissimilarityandresultingfromthePermutationalMANOVAforthe lv
responsevariables‘abundance’and‘origin’;*Significantata¼0.05. lackofabalancedphylogeneticdesignwasanunavoidable 11
0
featureofthestudysystem.Ourchoiceofplantspeciespro- /1
F-value R2 P-value videsinsightintohownaturalplantdiversitymaycontrib- 80
.................................................................................. 3
7
Calculatingfactor‘abundance’ utetoinfluencethebelowgroundfungalcommunityofa 91
typicalold-fieldgrasslandineasternNorthAmerica.Never- b
Abundance 5.194 0.14 0.001* y g
theless, in addition to plant abundance (abundant and u
Replicate 1.322 0.103 0.161 e
rare) and plant origin (native and exotic) we accounted st o
Plantfunctionalgroup 3.756 0.092 0.005* fortheproportionofthevarianceexplainedbyplantfunc- n 0
Abundance×origin 1.235 0.034 0.255 tional groups (monocotyledons and dicotyledons). Our 9 A
Calculatingfactor‘origin’ rceosmulmtsuwnietireescaosnssoisctieantetdwditihffeoruerntplyredwiictthionplathnattsfpuencgieasl pril 20
Origin 4.59 0.12 0.001* 1
dependingontheplants’relative‘abundance’and‘origin’. 9
Replicate 1.322 0.103 0.14
Plantspecies‘abundance’wasoneofthetwofactors
Plantfunctionalgroup 3.756 0.092 0.002* influencingfungalcommunitiesinplantrootssuggesting
Abundance×origin 1.235 0.034 0.255 thatsoilcommunitiesmayalsoplayaroleindetermining
therelativeabundanceofourplantspeciesinthecom-
munity.Thisisconsistentwithreportsemphasizingthe
(F ¼11.548, P¼0.002 and F ¼6.972, P¼0.015, importanceofsoilmicrobialcommunitiesinstructuring
1,23 1,23
respectively).Rareexoticplantshadthelargestamount plantcommunities(Klironomos2002;Beveretal.2010;
of AMF colonization and abundant exotics the least. Pendergastetal.2013).
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 7
Reiningeretal.—Surveyofassociatedfungalcommunitiesfromanold-field
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/a
o
b
p
la
/a
rtic
le
-a
b
s
tra
c
t/d
o
i/1
Figure3. Principalcoordinateanalysisplotsdiscriminatingbetweenbelowgroundroot-associatedfungalcommunitiesaccordingto(A)abun- 0
.1
dance,(B)originand(C)plantspecies.In(AandC)exoticplantspeciesarelabeledwithastarandnativespecieswithtrianglesandin(B)abun- 0
9
dantplantspeciesarelabeledwithatriangleandrarespecieswithastar.Confidenceinterval¼80%for(AandB)and75%for(C).RH,R.hirta; 3
/a
SL,S.lanceolatum;SC,S.canadensis;PA,P.arundianacea;HP,H.perforatum;PR,P.recta;BI,B.inermis;DG,D.glomerata. o
b
p
la
/p
lv
Partlyconsistentwithourprediction,wefoundbdiver- species occupya greater numberof microsites to avoid 1
1
0
sitytobesignificantlyhigherforrarethanforabundant competitionastheyarepresumablyratherweakcompeti- /1
8
plantspecies,indicatingthatfungalcommunitiesamong torscomparedwithabundantplantspecies.Duetothese 0
3
7
abundant plant species were more similar compared differentenvironmentsrareplantscannotbeveryselective 9
1
withthosepresentinrareplantspecies(Fig.4B).Thisresult inchoosingtheirfungalcommunities.Thisinturnmight b
y
wasadditionallysupportedbythePCoAplot(Fig.3A).How- influencetheirbdiversitywhichwasratherhighcompared gu
e
ever, the interaction between ‘abundance’ and ‘origin’ withabundantspecies.Sincerarityincommunitiesmaybe st o
wasnot significant contrary to our predictionthat pat- drivenbynegativefeedback(Klironomos2002)basedon n
0
ternsoffungal communitydiversitywere connectedto ourresultsitistemptingtospeculatethatrarenativespe- 9 A
nboifitchapnltalnytdaibffuenrednatncbeetawnedeonrigeixno.tbicDaivnedrsnitaytwivaessnpoetcsieigs-. c(Bieesvemraeytaasl.s2o0c1ia2t;eVwanithdepruPtauttitveenlyemtaolr.e20a1n3ta).gTohnisiswticasfufnugr-i pril 20
1
bDiversityofrareplantsmaybeinfluencedbythefungal thersupportedbyourrootlesiondata,showingthatroot 9
communityofneighboringplantspecieswhicharelikely enemiesaffectedmostlyrarenativeplants(Fig.5B).Add-
to be composed of different plant species. Conversely, itionally,anegativefeedbackhasbeenshowntobepreva-
abundantplantsaremoreoftensurroundedbyconspeci- lent in grasslands maintaining a high plant diversity
ficsandtherefore,shareamoresimilarfungalcommu- (Kulmatiskietal.2008).
nitythanrare plant species.Alternatively,our fourrare Fungalrichness(adiversity)inabundantnativeplants
species (Table 1) may be more selectivein their fungal washigherthaninrarenativesbutforexoticswedidnot
associations,explainingthehigheroverallbdiversitybut observe a significant difference (Fig. 4A). Perhaps such
lower a diversity. Another scenario could be that rare higher fungal diversity in the roots of native plants
8 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015
Reiningeretal.—Surveyofassociatedfungalcommunitiesfromanold-field
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/a
o
b
p
la
/a
rtic
le
-a
b
s
tra
c
t/d
Figure4. a(A)andb(B)diversityoffungalcommunitiesforthefactors‘abundance’and‘origin’inplantrootsfromanaturalold-fieldcommu- o
nity.aDiversityshowedasignificantinteractionbetween‘abundance’and‘origin’(P¼0.014).Standarderrorsareindicatedbyerrorbars.Means i/10
inabarsharingthesameletterarenotsignificantlydifferentaccordingtoTukey’sHSDatasignificancelevelofP≤0.05. .1
0
9
3
/a
o
b
couldbeimportanttoexplaintheirabundance.Ecosys- toassociatewithaspecificbelowgroundmicrobialcommu- p
la
tems and communities may be considered to be more nity. Despite the importance of fungi as drivers of plant /p
lv
stable if biodiversity is higher since productive species communitystructure(VanderHeijdenetal.1998;Reynolds 1
1
0
cancompensateforotherspeciesthatmaynotperform etal.2003)onlyveryfewstudieshavecomparedthefungal /1
8
aswell(NaeemandLi1997).Thismayalsobelinkedto communitiesassociatingwithexoticsversusnativesina 0
3
7
the success of individual plant species. For instance, naturalcommunity(Bongardetal.2013).Consistentwith 9
1
highergenotypicdiversityofroot-associateddarkseptate our results, Nelson and Karp (2013) found that distinct b
y
g
endophyticfungihasbeenshowntoincreasetheirhost communities of pathogenic fungi associate with native u
e
plants’ growthrate (Reiningeret al.2012). Perhapsthe andexotichaplotypesofPhragmitesaustralis,suggesting st o
microbialcommunitiesassociatedwithabundantnative that exotic plants mayassociate with a different fungal n
0
plant species may be even more conducive to positive communitythannativespecies. 9 A
feeAdsbparcekdi(cVtaend,dfuenrgPaultctoemnemtuanl.it2ie0s1a3s)s.ociatingwithexotic beOlounrgminogletocuthlaerAmsectohmoydcoolotag,icwahlaicphpirsotahcehladregteescttecdlafduenogfi pril 20
1
speciesweredifferentfromfungalcommunitiesassociat- fungi(Kirketal.2001;Wangetal.2006),andBasidiomy- 9
ingwithnativespecies.However,wefoundnodifferences cota.Morespecifically,themostrepresentedorderacross
in a- and b diversity between abundant exotic and rare allplantspeciesregardlessoftheir‘abundance’and‘origin’
exoticspecies,andexoticandnativespecies,respectively. weretheHelotialesbelongingtotheAscomycota(Fig.2).
Thissuggeststhattherelativeabundanceofexoticsinthe Thisverydiverseandlargefungalorderincludes (cid:2)2300
communitymaybeindependentfromtheirfungalcommu- specieswhichtakeseveralecologicalfunctionalrolesran-
nities.Sinceexoticspeciesmayhaveescapedfromtheir gingfromericoidmycorrhiza,endophytes,saprobesand
co-evolvedsoilenemies(KeaneandCrawley2002;Agrawal pathogens(WebsterandWeber2007).Therefore,itisnot
etal.2005),theymightbeuncoupledfromtherequirement surprisingthatwefoundsomanyOTUsbelongingtothis
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 9
Reiningeretal.—Surveyofassociatedfungalcommunitiesfromanold-field
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/a
o
b
p
la
/a
rtic
le
-a
b
s
tra
c
t/d
o
i/1
0
.1
0
9
Figure5. Arbuscularmycorrhizalfungalcolonizationbyhyphae(A),arbuscules(B)andvesicles(C)andincidenceoflesions(D)intherootsof 3
/a
nativeandexoticplantsofcontrastingrelativeabundanceinaruderalnaturalplantcommunity.Meansinabarsharingthesameletterarenot o
b
significantlydifferentaccordingtoTukey’sHSDatasignificancelevelofP≤0.05. pla
/p
lv
1
1
0
largeanddiversefungalorder.SinceOTUswerenotdeter- ITSandLSUregions.Furthermore,duetothefactthatin /1
8
minedtospecieslevel,wewereunabletomakeanystate- ourstudytheprimersweredegenerate,thereisanincreas- 0
3
7
mentabouttheirparticularfunctioninourold-fieldsite. ingchanceofgettingunspecificresultsbyrunningtoomany 9
1
TheITSregionhasbeenshownahighdegreeofsuccess PCRcycles (Ihrmarket al. 2012). This primer bias can be b
y
forthebroaddetectionoffungi(Bue´eetal.2009)andwas expectedasaresultofahighnumberof PCRcycles(35) gu
e
recentlyproposedtoserveastheprimaryfungalbarcode andthelowGCcontentoftheGlomeromycota(Stockinger st o
marker(Schochetal.2012).Eventhoughtheprimerpair etal.2010;Gianinazzi-Pearsonetal.2012;Kauserudetal. n
0
fITS7/ITS4hasbeentestedpositivelytomatchGlomero- 2012;PintoandRaskin2012).Nevertheless,alloursamples 9 A
tmhyocuogthaA(IMhrFmwaerkreecteartl.ai2n0ly12p)r,ewseentfaiinledthteorodootssoofevoeunr wcaeprteurteretahteedfuellqbureaalldytahnodftehveenfutnhgoaulgKhinwgdeowme,roeuurn4a5b4lepytro- pril 20
1
plantspecies(Fig.5A).Thisisincontrastwith aBLASTN osequencing approach was able to consistently detect a 9
searchatNCBIwithfITS7,whichmatched68%oftheGlo- broaddiversityoftaxaamongourplantgroups.Thisisin
meromycota5.8Ssequences(Ihrmarketal.2012).Even accordancewithIhrmarketal.(2012)asourprimersare
thoughweselectedthisprimerpairtakingintoconsider- supposedtobetterpreservethequantitativecomposition
ation the shorter sequence length, it is known that the of the template between-sample differences in fungal
choice of ITS could bias against certain groups such as communitycomposition. Wealsoneedto considerthat
EarlyDivergingLineages,AscomycotaandGlomeromycota fungalcommunitycompositionshiftsseasonally(Thoms
(seeSchochetal.2012).Forthelattergroup,Stockinger and Gleixner 2013;Vorˇ´ısˇkova´ et al.2014).The sampling
et al. (2010) proposed a larger barcode, combining the was undertaken mid-summer to ensure that fungal
10 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015