an improved method for utilizing high-throughput amplicon ... · 1 an improved method for utilizing...

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Animprovedmethodforutilizinghigh-throughputampliconsequencingtodeterminethe1

dietsofinsectivorousanimals2

3

Authors4

MichelleA.Jusino1++*,MarkT.Banik1++,JonathanM.Palmer1,AmyK.Wray2,3,LeiXiao4,Emma5

Pelton3,5,JesseR.Barber6,AkitoY.Kawahara4,ClaudioGratton3,M.ZachariahPeery2,and6

DanielL.Lindner1*7

8++indicatessharedfirstauthorshipbasedonequalcontributions9

*indicatescorrespondingauthors10

11

Affiliations12

(1) UnitedStatesForestService,NorthernResearchStation,CenterforForestMycology13

Research,OneGiffordPinchotDrive,Madison,Wisconsin,USA14

(2) UniversityofWisconsin-Madison,DepartmentofForestandWildlifeEcology,Madison,15

Wisconsin,USA16

(3) UniversityofWisconsin-Madison,DepartmentofEntomology,Madison,Wisconsin,USA17

(4) McGuireCenterforLepidopteraandBiodiversity,FloridaMuseumofNaturalHistory,18

UniversityofFlorida,Gainesville,USA19

(5) TheXercesSocietyforInvertebrateConservation,Portland,Oregon,USA20

(6) DepartmentofBiologicalSciences,GraduatePrograminEcology,Evolutionand21

Behavior,BoiseStateUniversity,Boise,Idaho,USA22

23

*MichelleA.Jusino:USFSNRSCFMR,OneGiffordPinchotDrive,Madison,WI,5372624

[email protected],[email protected]

*DanielL.Lindner:USFSNRSCFMR,OneGiffordPinchotDrive,Madison,WI,5372626

[email protected],[email protected]

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Runningtitle:ImprovedHTSofinsectivorediets 29

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3184v1 | CC BY 4.0 Open Access | rec: 24 Aug 2017, publ: 24 Aug 2017

2

Abstract30

DNAanalysisofpredatorfecesusinghigh-throughputampliconsequencing(HTS)31

enhancesourunderstandingofpredator-preyinteractions.However,conclusionsdrawnfrom32

thistechniqueareconstrainedbybiasesthatoccurinmultiplestepsoftheHTSworkflow.To33

bettercharacterizeinsectivorousanimaldiets,weusedDNAfromadiversesetofarthropodsto34

assessPCRbiasesofcommonlyusedandnovelprimerpairsforthemitochondrialgene,35

cytochromeoxidaseCsubunit1(CO1).WecompareddiversityrecoveredfromHTSofbat36

guanosamplesusingacommonlyusedprimerpair“ZBJ”toresultsusingthenovelprimerpair37

“ANML”.Toparameterizeourbioinformaticspipeline,wecreatedanarthropodmock38

communityconsistingofsingle-copy(cloned)CO1sequences.Toexaminebiasesassociated39

withbothPCRandHTS,mockcommunitymemberswerecombinedinequimolaramountsboth40

pre-andpost-PCR.Wevalidatedoursystemusingguanofrombatsfedknowndietsandusing41

compositesamplesofmorphologicallyidentifiedinsectscollectedinpitfalltraps.InPCRtests,42

theANMLprimerpairamplified58of59arthropodtaxa(98%)whereasZBJamplified24of5943

taxa(41%).Furthermore,inanHTScomparisonoffield-collectedsamples,theANMLprimers44

detectednearlyfour-foldmorearthropodtaxathantheZBJprimers.Theadditionalarthropods45

detectedincludemedicallyandeconomicallyrelevantinsectgroupssuchasmosquitoes.46

ResultsrevealedbiasesatboththePCRandsequencinglevels,demonstratingthepitfalls47

associatedwithusingHTSreadnumbersasproxiesforabundance.Theuseofanarthropod48

mockcommunityallowedforimprovedbioinformaticspipelineparameterization.49

50

51

Keywords:arthropodmockcommunity,batguano,dietaryanalysis,insectivore,next-52

generationsequencing,NGS53

54


3

Introduction55

High-throughputampliconsequencing(HTS)hasbecomethepreferredmethodforrapid56

molecularidentificationofmembersofmixedecologicalcommunities.HTSisnowalso57

increasinglyusedtoidentifythearthropoddietarycomponentsofawidetaxonomicrangeof58

animalsincludingmammals(Busscheetal.2016;Clareetal.2014a;Clareetal.2014b;Mallott59

etal.2015;Rydelletal.2016;Vesterinenetal.2016),birds(Crisol-Martínezetal.2016;Jedlicka60

etal.2016;Trevellineetal.2016),reptiles(Kartzinel&Pringle2015),fish(Harms-Tuohyetal.61

2016),andarthropods(Krehenwinkeletal.2016).IdentificationoftheDNAofdietary62

componentsisaccomplishedby“metabarcoding”,whichinvolvesextractingDNAfromfecal63

samples,amplifyingoneormorebarcodingloci,preparingDNAlibraries,andfinallysequencing,64

bioinformatics,anddataanalysis.Eachofthesestepsinvolvesdecisionsandassumptionsthat65

significantlyaffectresults.Forexample,biasesareunavoidablewhenamplifyingenvironmental66

DNAwithPCR-basedmethods(Brooksetal.2015)andcarefulconsiderationshouldbe67

exercisedwhenselectingaprimerpairforHTS.Thus,whileDNAmetabarcodingisapowerful68

toolforstudyingtrophicinteractions,conclusionsshouldtakeintoaccounttheshortcomings69

andparametersofthetechniques(e.g.:Brooksetal.2015;D’Amoreetal.2016;Lindahletal.70

2013;Nguyenetal.2015;Pompanonetal.2012).71

ThemitochondrialcytochromeoxidaseCsubunit1locus(CO1)isthemostfrequently72

usedbarcodinglocusforidentifyingawiderangeoftaxonomicgroups,includingarthropods.73

BecauseCO1hasthemostextensivereferencelibraryforarthropods(BOLDsystems,74

Ratnasignham&Hebert,2007),itisthemostcommonlyusedlocusfordietarystudiesof75

insectivorousanimals(Clarkeetal.2014).TheentireCO1barcodingregionisabout658base76

pairs(bp)andcurrentlytoolongtobeusedwithmostHTSplatforms.Thereforeitisnecessary77

tosequenceshorterregionsoftheCO1locus,whichhasprovenchallengingduetoalackof78

conservedprimingsiteswithintheCO1region(Deagleetal.2014).Therefore,novelprimer79

pairsshouldbetestedagainstasmanyexpectedtargetDNAsequencesaspossible.80

Zealeetal.(2011)developedtheZBJ-ArtF1c/ZBJ-ArtR2c(hereafterZBJ)primerpairfor81

detectingarthropodpreyDNAinbatguanobyamplifyinga157bpfragmentoftheCO1region.82

Intheinitialstudy,whichemployedcloningandsequencingratherthanHTS,theZBJprimers83


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amplified37taxafrom13arthropodorders,butdidnotamplifybatCO1DNA.TheZBJprimers84

weredesignedtotargetashortfragmentinordertoamplifythepresumablydegradedDNA85

presentinguanoandcoincidentallythelengthoftheamplicongeneratediscompatiblewith86

manyHTSplatforms.Subsequently,numerousresearchershaveemployedtheZBJprimersin87

HTSstudiesthatanalyzedietsofinsectivorousanimals,includingbats(Busscheetal.2016;88

Clareetal.2014a;Clareetal.2014b;Rydelletal.2016;Vesterinenetal.2016)andbirds89

(Crisol-Martínezetal.2016;Jedlickaetal.2016;Trevellineetal.2016).AlthoughtheZBJ90

primershavebeenwidelyutilized,thereareindicationsthattheyhaveanarrowtaxonomic91

range(Brandon-Mongetal.2015;Clarkeetal.2014;Mallottetal.2015).92

TheassumptionsandparameterscommonlyemployedinHTSenvironmentalDNA93

analyseshavealargeimpactontheoperationaltaxonomicunits(OTUs)thatarerecovered.94

Bioinformaticsclusteringalgorithmscaninfluenceapparentdiversitywithinasample,oran95

entirelibraryofsamples,andtrimmingandfilteringparameterscanimpacttheresulting96

communitycomposition(Deagleetal.2013).Avalidationorcontrolisneededtoparameterize97

bioinformaticspipelines;therefore,theuseofmockcommunitiesaspositivecontrolsinHTSis98

increasinglybecomingcommon,especiallyamongresearcherswhoworkwithfungaland99

bacterialcommunities(Bokulich&Mills2013;Bokulichetal.2013;Nguyenetal.2015).Mock100

communitiescanbeusedtoexaminebiases,startingatthesamplingstepandendingatthe101

bioinformaticsandcommunityanalysissteps.102

Hereweusedareferenceinsectcommunitytoidentifyspecificamplificationbiases103

associatedwiththreecommonlyusedprimerpairs,includingZBJ,andtwonovelprimerpairs,104

LCO1-1490/CO1-CFMRa(hereafterANML)andLCO1490/CO1-CFMRb(hereafterCFMRb),for105

theCO1region(Table1).Tofurthertestprimers,wecomparedHTSresultsfromtheZBJ106

primerstoournovelANMLprimerpairusingfield-collectedbatguanosamples.Wedesigned107

anarthropodmockcommunitybasedonsingle-copy(cloned)mitochondrialCO1sequences,108

whichcanserveasastandardinHTSsequencingandtohelpparameterizeabioinformatics109

pipeline.Finally,wevalidatedtheaccuracyofoursystemofnovelprimers,themock110

communitycontrol,andourbioinformaticspipelinebyusingguanofrombatsfedknowninsect111

dietsandcompositesamplesofmorphologicallyidentifiedarthropodsfrompitfalltraps.112


5

MethodsandMaterials113

Testingofprimerpairsagainstknowninsectsamples114

DNAwasextractedfrom67arthropodtaxa(Table2)followingtheprotocolinLindner&115

Banik(2009)withmodificationsforinsects(SupportingInformation,AppendixS1).Briefly,DNA116

wasextractedfromexcisedlegmusclesoflargerinsectsor,forsmallerinsects,thethoraxwas117

puncturedandtheentireinsectwasusedforextraction.Legmusclesandsmallinsectswith118

puncturedthoraxeswereplacedin100μLoffilteredcelllysissolution(CLS;Lindner&Banik119

2009)andfrozenat-20°C,andtheextractionproceeded.FollowingDNAextraction,the120

effectivenessofthefollowingfiveprimerpairsinamplifyingthe67purifiedDNAswas121

evaluated:LCO1490/HCO2198(Folmeretal.1994;Hebertetal.2003;hereafterCO1L/H),ZBJ-122

ArtF1c/ZBJ-ArtR2c(Zealeetal.2011;ZBJ),LCO1-1490/CO1-CFMRa(ANML),LCO1490/CO1-123

CFMRb(CFMRb),andLepF1/mLepR(Hebertetal.2004;Smithetal.2006;hereafterLEP).The124

CO1-CFMRaandCO1-CFMRbprimersdesignedforthisstudywerederivedfromtheZBJ-ArtR2c125

primerandhadsequencesof5’-GGWACTAATCAATTTCCAAATCC-3’and5’-126

GGNACTAATCAATTHCCAAATCC-3’,respectively.TheCO1-CFMRaandCO1-CFMRbprimingsites127

arelocatedintheCO1geneapproximately180bpawayfromtheLCO1490primingsite128

(SupportingInformation,FigureS1).AlistoftheprimersusedispresentedinTable1.129

AmplificationoftheextractedDNAusingallprimerpairs,exceptZBJ,usedthefollowing130

reagentvolumesper15LμLreaction:7.88μLDNA-freemoleculargradewater,3μLGreen131

GoTaq5xbuffer(Promega),0.12μLof20mg/mLBSA,0.3μLof10μMdNTPs,0.3μLofeach10132

μMprimer,0.1uLof5u/μLGoTaqpolymerase(Promega),and3μLofextractedarthropod133

templateDNA.TheZBJprimerpairwasusedwithtwodifferentreagentregimes.One,termed134

themodifiedprotocol,wasthesameasaboveexcept1.0μLofeach10μMprimerwasadded135

andthesecondregimewasthatdescribedbyoriginalauthors(Zealeetal.2011).The136

thermocyclerparametersfortheCO1L/H,ANML,andCFMRbprimerpairswerethosedescribed137

byHebertetal.(2003)withonemodification:thefinalextensionat72°Cwasincreasedfrom5138

to7minutes.TheLepfF-1/mLepR-5amplificationparameterswerethoseofSmithetal.(2006),139

whiletheZBJprimerpairamplificationparameterswerethosedescribedbyZealeetal.(2011).140

Followingamplification,3μLofproductwasrunina2%agarosegelfor20minutesat110V,141


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stainedwithethidiumbromideandvisualizedusingUVlight.Presenceorabsenceofbandswas142

recordedforeachprimerpairandDNAcombination.Toprovidereferencesequencesforeach143

speciestheLCO1490/HCO2198PCRproductswereSangersequencedwithABIPrismBigDye144

(AppliedBiosystems)sequencingfollowingthemethodofLindner&Banik(2009).Theresulting145

sequencesweresubjectedtoanNCBIBLASTsearchtoconfirmtheidentitiesoftheinsect146

speciesoforigin.147

HTSoffield-collectedguanosamplesusingtwodifferentprimerpairs148

ThearthropodDNApresentinthreefield-collectedbatguanosampleswasanalyzed149

usingtheANMLandZBJprimerpairs.DNAwasextractedfromthreeMyotislucifugusguano150

samplesfromthreedifferentlocationsinsouthernWisconsin(allcollectionswereapprovedby151

theWisconsinDepartmentofNaturalResources).Onesamplecontaining100mgofguano,152

approximately10pellets,wasextractedfromeachsiteusingQIAampDNAstoolMiniKit153

followingtheprocedureinAppendixS2oftheSupportingInformation.TheDNAwasthen154

amplifiedusingprimersmodifiedformetabarcodingbyaddinganIonTorrentXpresstrP1155

adaptersequenceonthereverseprimerandbarcodesequenceandIonTorrentXpressA156

adaptersequenceoneachforwardprimer(seeSupportingInformation,TableS1forbarcoded157

primersequences).AmplificationconditionsfortheANMLpairfollowedtheprotocolusedfor158

theprimerpairtestandconditionsfortheZBJpairfollowedthemodifiedprotocolforZBJ159

describedintheprimerpairtest.Followingamplification,eachoftheuniquelybarcodedPCR160

productswaspurifiedviasizeselectingE-GelCloneWellGels(Invitrogen)atapproximately161

180bp.Thesize-selectedproductswerethenquantifiedonanInvitrogenQubit2.0Fluorometer162

andbroughttoaconcentrationof2000pMusingDNA-free,moleculargradewater.Wethen163

combinedtheproductsinequalamountstoproducethesequencinglibrary.Thelibrarywas164

dilutedto13pMpriortotemplatingontoionsphereparticles(ISPs)withtheIonOneTouch2165

system(LifeTechnologies)andaPGMHi-QOT2templatingkit(ThermoFisher#A27739),166

accordingtothemanufacturer'srecommendations.ThetemplatedISPswerethenpurifiedand167

thetemplatedDNAwassequencedusingtheIonTorrentPersonalGenomeMachine(PGM;168

ThermoFisher)withtheIonPGMHi-QSequencingKit(ThermoFisher#A25592)accordingtothe169

manufacturer'sprotocolfor400bpsequencing.170


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Bioinformatics171

HTSdatawereprocessedusingthe‘DADA2’methodviatheAMPtkpipeline172

(https://github.com/nextgenusfs/amptk).Briefly,theAMPtkpipelineprocesses(de-173

multiplexes)HTSampliconsequencingreadsby:1)identifyingavalidbarcodeindexineach174

read,2)identifyingforwardandreverseprimersequences,3)trimmingbarcodeandprimer175

sequences,4)renamingthereadbasedonbarcodeindex,and5)trimming/paddingthereads176

toasetlength.TheDADA2algorithm(Callahanetal.2016)isanalternativetowidelyused177

sequence-clusteringalgorithms(e.g.,UPARSE,UCLUST,nearest-neighbor,SWARM,etc.)and178

functionsto“denoise”HTSsequencingreads.DADA2hasbeenshowntobeveryaccurateand179

issensitivetosinglebasepairdifferencesbetweensequences(Callahanetal2016).AMPtk180

implementsamodifiedDADA2algorithmthatproducesthestandard“inferredsequences”181

outputofDADA2aswellasclustersthe“inferredsequences”intobiologicallyrelevantOTUs182

usingtheUCLUST(Edgar2010)algorithmemployedinVSEARCH(Rognesetal.2016).The183

resultingAMPtkOTUtablescanbefilteredbasedonspike-inmockcommunities(described184

below).TaxonomyformtCO1isassignedinAMPtkusingacombinationofglobalsequence185

alignment,UTAX(http://www.drive5.com/usearch/manual/utax_algo.html),andSINTAX(Edgar186

2016)usingaCO1referencedatabase.ThecurrentCO1databasedistributedwithAMPtkwas187

derivedfromcollatingsequencesfromrepresentativebarcodeindexnumbers(BIN)from188

chordatesandarthropodsintheBarcodeofLifev4database(BOLD;Ratnasingham&Hebert189

2007)andisavailableat:190

https://github.com/nextgenusfs/amptk/blob/master/docs/reference_databases.md.191

Developmentandtestingofanarthropodmockcommunity192

ToproduceamockcommunitytoserveasacontrolforHTSdataanalysis,43ofthe193

arthropodtaxausedintheprimerpairtestwerechosenascandidates(Table2).DNAfromeach194

arthropodwasamplifiedusingLCO1490/HCO2198primersasdescribedpreviously.Toremove195

intragenomicvariation(Songetal.2008),theresultingampliconswereclonedintoE.coliusing196

thePromegapGem-Tvectorsystemfollowingthemanufacturer’sinstructionswiththe197

modificationsusedbyLindner&Banik(2009).Threeclonesofeacharthropodtaxonwere198

subsequentlySangersequencedtoverifythepresenceoftheCO1insertsequence.Twoofthe199


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clonedarthropodsproducedclonedsequencevariants,andthesevariants(3total)werealso200

includedinthemockcommunity,bringingourmockcommunitytotalto46.Plasmidswere201

purifiedusingstandardalkalinelysisandtheresultantDNAwasthenquantifiedonan202

InvitrogenQubit2.0Fluorometerandbroughttoaconcentrationof1500pMusingDNA-free,203

moleculargradewater.Plasmidswerethendiluted1:20usingDNA-freemoleculargradewater204

andindividuallyamplifiedusingtheionANMLprimerswiththesamebarcode.Theindividual205

PCRproductswerethenvisualizedona2%agarosegel,cleanedandsizeselectedat≥150bp206

usingZymoResearchSelect-A-SizeDNAClean&Concentratorspincolumns,quantifiedand207

equilibratedto2000pMasdescribedpreviously,andsubsequentlycombinedinequal208

amounts.Thisampliconmixtureisreferredtoasour“post-PCRcombinedmockcommunity”,209

whichservesasacontroltovalidatesequencingefficiencyofeachmockmember.Tomeasure210

initialPCRbiasandtoparameterizeourbioinformaticspipeline,wealsocreated“apre-PCR211

combinedmockcommunity”bycombiningour1500pMplasmidsinequalamounts.Thepre-212

PCRcombinedmockcommunitywasthendilutedtoa1:8000concentrationpriorto213

amplificationwithANMLbarcodedprimers.TheresultingbarcodedPCRproductwasthen214

visualized,sizeselected,quantified,andbroughtto2000pMasdescribedbefore.Theresulting215

barcodedPCRproductswerethenpreparedandsequencedonanIonTorrentPGManddata216

werebioinformaticallyprocessedasdescribedabove.217

Testingofknownmixedsampleswithmockcommunityandourpipeline218

TotestpreyDNArecoveryfrombatguano,twobats,oneEptesicusfuscusandone219

Lasiuruscinerus,werefedknowndietsofGalleriamellonella,TenebriomolitorandAntheraea220

polyphemusaloneandincombination(Table4).Thebatswerefedeachknowndietforone221

day,andguanopelletswerecollectedduringthefollowing24hours(approvedbyBoiseState222

UniversityInstitutionalAnimalCareandUseCommittee006-AC14-018).Weanalyzedthree223

knowndietcombinationsfromtheE.fuscusindividualandtwoknowndietcombinationsfrom224

theL.cinerusindividual.DNAwasextractedfromguanosamplesusingQiagenQIAampmini225

Stoolkits,followingthemodifiedprotocoldescribedinZealeetal.(2011).DNAfromtheknown226

dietsampleswasamplifiedwithbarcodedANMLprimers,andtheresultingPCRproductswere227


9

thenvisualized,sizeselectedat≥150bpusingZymoResearchSelect-A-SizeDNAcleanand228

concentratorspincolumns,quantified,andbroughtto2000pMasdescribedbefore.229

Totesttheeffectivenessofthemethodoncomplexinsectcommunities,fivesamples230

frompitfalltrapsfromtheSnakeRiverBirdsofPreyConservationAreainKuna,Idahowere231

analyzed.Eachpitfalltrapconsistedofaglassjarcontainingpropyleneglycol.Trapswereleft232

outsidefor2-3days,atwhichpointthecontentsofthetrapswererinsedwith100%ethanol233

andsubsequentlytransferredtoglassvialscontaining100%ethanolforstorageatroom234

temperature.AlltrapsamplesweresenttotheFloridaMuseumofNaturalHistoryin235

Gainesville,Floridaforvisualidentificationtoarthropodfamilyandlong-termstorageat-20°C.236

Initialidentitiesofthearthropodspresentintheinsecttrapsampleswereobtainedusing237

traditionalmorphologicalkeys,andmostwereidentifiedbyeyetothefamilylevel,withthe238

followingexceptions:allspringtailswereidentifiedtoorder(Collembola),centipedeswere239

identifiedtoclass(Chilopoda)andmiteswereidentifiedtosubclass(Acari).240

ThesamplesweresenttotheUnitedStatesForestService,NorthernResearchStation,241

CenterforForestMycologyResearchinMadison,Wisconsin,wheretheywereprocessedfor242

molecularanalysis.ArthropodsfromthetrapsampleswererinsedinDNA-freemoleculargrade243

waterandpreparedforDNAextractionintwoways:(1)theexcisedlegmusclesoflarger244

arthropods,andsmallerarthropodswithopenthoraxeswerecombinedandsubmersedinCLS245

andvortexed(dissectedsample),or(2)theintactarthropodswereaddedto15mLCLSand246

maceratedwithasterilepestleandvortexed(maceratedsample).DNAextractionfollowed247

detailsdescribedinAppendixS1oftheSupportingInformation;metabarcodingPCR,andHTS248

thenproceededaspreviouslydescribed.Datawerebioinformaticallyprocessedasdescribed249

before.250

Results251

Testingofprimerpairsagainstknowninsectsamples252

Fifty-eightofthe59taxa(98%)amplifiedwiththeANML(LCO1490/CO1-CFMRa)and253

CFMRb(LCO1490/CO1-CFMRb)primerpairs,withbothpairsfailingtoamplifythesamecarabid254

beetle(Table2).Fifty-twoof59taxa(89%)amplifiedwiththeCO1L/Hprimerpairand48of59255

(81%)amplifiedwiththeLEPprimerpair;theLEPpairamplified100%oftheLepidopteransand256


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Dipteranstested(Table2).TheZBJprimerpairamplified24ofthe59(41%)taxatestedwiththe257

Zealeetal.(2011)protocoland27ofthe59(46%)taxausingourmodifiedprotocol.TheZBJ258

primerpairsuccessfullyamplifiedatleastonerepresentativefromeacharthropodordertested259

(Table2).260

HTSoffield-collectedguanosamplesusingtwodifferentprimerpairs261

BoththeZBJandtheANMLprimersproducedanamplificationproductfromthethree262

Myotislucifugusguanosamples.Forbothprimersetscombined,atotalof64OTUs(Table3)263

weredetected,ofwhich59couldbeidentifiedtothefamilylevel,representing10orders264

comprisedof28families.TheANMLprimersdetected56OTUsandtheZBJprimersdetected15265

OTUs.Sevenofthe64totalOTUsweredetectedwithbothsetsofprimers,49weredetected266

onlywiththeANMLprimerswhile8wereonlydetectedwiththeZBJprimers.Representatives267

fromalltenordersand26familieswererecoveredusingtheANMLprimerpair,whiletheZBJ268

pairrecoveredrepresentativesfromthreeordersandeightfamilies.Themostoftendetected269

familywasthedipteranmidgefamilyChironomidae,with27OTUs,24ofwhichweredetected270

bytheANMLprimersand6byZBJ.Thesecondmostoftendetectedfamilyweremosquitoes271

(Family:Culicidae),with5OTUsdetectedbyANMLbutonlyonebyZBJ.Allbutoneofthe272

remainderofthefamilieswererepresentedbyonlyoneOTUeach.273

Developmentandtestingofanarthropodmockcommunity274

Theindividualplasmidcomponentsofourpost-PCRcombinedmockcommunity275

generatedreadcountsthatrangedfrom3740to4;themeanwas2119andstandarddeviation276

+/-799,with89%(41outof46)yieldinggreaterthan1500reads(Figure1;supplementaltable277

1).Allmockmembersinthepost-PCRcombinedcommunitywererecovered,although3278

generatedfinalreadcountsbelow100(range4to12).Incontrast,individualmembersofour279

pre-PCRcombinedmockcommunitygeneratedreadcountsthatrangedfrom10,577to0witha280

meanof2174andstandarddeviationof+/-2238,with54%(25of46)yieldingmorethan1500281

reads.Twoofourmockmembersdidnotgenerateanysequencesinthepre-PCRcombined282

communityandanadditional4generatedfinalreadcountsbelow100(range2to39).283

Testingofknownmixedsampleswithmockcommunityandourpipeline284


11

TheresultsoftheknowndietHTSsamplesaresummarizedinTable4.WedetectedDNA285

fromalloftheexpecteddietarycomponentsinall5oftheknowndietsamplestested.286

Additionally,wedetectedDNAfromtwopossibleaccidentaldietarycomponents(Empria287

takeuchiiandAgrotisipsilon)inbigbrownbat(Eptesicusfuscus)dietsamplesthatincluded288

bothGalleriaandTenebrioasdietarycomponents.WealsodetectedDNAfromaparasitoid289

wasp(Family:Ichneumonidae)in3of4(75%)dietsamplesthatincludedGallerialarvae.Finally,290

wedetectedbigbrownbat(E.fuscus)DNAin2ofthe3samplesfrombigbrownbats,and291

hoarybat(L.cinereus)DNAinboth(2of2)ofthesamplesfromhoarybats(Table4).Thesedata292

wereprocessedbioinformaticallywithDADA2,withandwithout97%clusteringappliedtothe293

inferredsequencetablethatresultedfromtheDADA2output.Withoutclustering,weobtained294

oneinferredsequenceforG.mellonella,Antheraeapolyphemus,E.takeuchii,A.ipsilonandE.295

fuscus,butobtained11inferredsequencesforTenebriomolitor,7fromIchneumonidae,and3296

forL.cinereus.Afterclusteringat97%,wemaintainedtheOTUnumberforalltaxathathadone297

OTUbeforeclustering,andobtained2OTUsforT.molitor,1OTUforIchneumonidae,and2298

OTUsforL.cinereus.299

Theresultsofthepitfalltrapsamplesaresummarizedbasedonpresenceorabsenceof300

familiesinTable5.Thereappearstobenosignificanteffectofthemethodinwhichthe301

communitieswereextracted(dissectedsamplesormaceratedsamples)ontheefficiencyof302

taxonrecovery.Overall,in5samples37familiesidentifiedusingconventionalmorphological303

methodswerealsorecoveredwithHTS,whileafurther18familiesmorphologicallyidentified304

werenotrecoveredwithHTSand16familieswereonlyrecoveredwithHTS.Ofthe18families305

missedbyHTS,9wereprobablyaresultofeitheramorphologicalorsequence306

misidentification,withtheremaining9mostlikelylostthroughsystembias.307

Discussion308

Throughanamplificationtestof5primerpairsagainstataxonomicallydiverse309

communityofarthropods,wedemonstratedthatourANMLandCFMRbprimerpairsamplified310

moretaxathanpreviouslydescribedprimerpairs(CO1L/H,ZBJ,andLEP)inastandardPCR.311

Throughadirectcomparisonoffield-collectedguanosamplessubjectedtoHTSwithtwoprimer312

pairs,ANMLandZBJ,wedemonstratedthattheANMLprimerpairamplifiedsubstantiallymore313


12

taxathantheZBJprimers,thecommonlyusedprimerpairforHTSstudiesexaminingthediets314

ofinsectivorousanimals.Whenweusedbothpairsonthesameenvironmentalsamples,the315

ANMLpairyieldedalmostfourtimesasmanyarthropodtaxathantheZBJpair.Wealso316

detectedchiropteran(bat)sequencesinfecalsamplesfrombatsusingtheANMLprimerpair,317

althoughthenumberofchiropteranDNAsequencesandOTUswasinsignificantcomparedto318

theoverallnumberofsequencesgenerated.Thus,theamplificationofchiropteranDNAdidnot319

significantlyimpacttherecoveryofarthropodDNA,afeaturethathelpsconfirmtheidentityof320

thebattargetspecies,aswellastheirdietarycomponents.ItislikelythattheCO1regionof321

othervertebratescouldalsobeamplifiedbytheANMLprimers,thushelpingtoconfirmthe322

identityoftheconsumerinarangeofsystems(e.g.othermammalspecies,reptiles,323

amphibians,andbirds).BecausetheyproducelongerPCRproducts,theANMLprimers(180bp324

product)alsoallowforbettertaxondelineationcomparedtotheZBJprimers(157bpproduct).325

Improveddetectionofpestspecies326

Insectivorousanimalsarevaluedasprovidersofpestcontrol;however,thetotal327

economicvalueofthisecosystemserviceisdifficulttoestimate(Boylesetal.2011;Clevelandet328

al.2006;Maine&Boyles2015;Williams-Guillénetal.2016).Determiningthefullvalueis329

dependentonthereliabledetectionofthepestspeciespresentinthedietsofinsectivorous330

animals.HTScanbeapowerfultoolforhelpingtobuildtheempiricalbasisnecessaryto331

estimateecosystemservices,butthesuccessofthisapproachdependsinpartonprimer332

efficacy.Basedonouranalyses,theANMLprimersareamajormethodologicalimprovement333

overexistingprimers,allowingforthedetectionofgreaterarthropoddiversityinthe334

environmentalsampleswetested,includingagreaterdiversityofknownpestssuchas335

mosquitoes(Family:Culicidae).Theprevalenceofmosquitoesisusuallyverylowinother336

molecularstudiesofbatguanothatrelyupontheZBJprimers(Clareetal.2014a;Clareetal.337

2014b;Gonsalvesetal.2013;Rolfeetal.2014),andsomehavegoneasfarastosaythat338

mosquitoesarenotimportantpreyitemsforbats(Fenton2012).Specifically,inourguano339

samples,theZBJpairwasonlyabletodetectAedesvexans,whiletheANMLpairdetectedA.340

vexansplusfourotherCulicidaespeciesinthesamesamples.Thus,theANMLprimersallowfor341


13

betterestimationoftheecosystemservicesofbats,andperhapsotherinsectivores,as342

predatorsofmosquitoesandothereconomicallyimportantpestspecies.343

Single-copyarthropodmockcommunity,sourcesofunexpectedvariationandsomesolutions344

WhilesomeauthorshavenotedthatHTSdataareunreliableasasourcetomeasure345

communitymemberabundance(Piñoletal.2015),manyHTSstudiesofenvironmentalsamples346

continuetouseabundancemetricsbasedonreadnumbers.Totestthevalidityofreadnumber347

asanestimateofrelativeabundance,wecombinedpre-andpost-PCRmockcommunitiesin348

equimolaramountspriortosequencing.Wepredictedthatiftheapproachisvalid,read349

numbersshouldbeequalacrosstaxa.Instead,eventhougheachmemberofthemock350

communityamplifiedwellinindividualPCRs,weobservedalargevariationinreadnumbersfor351

thepre-PCRcombinedmockcommunity,withsomemembersbeingabsent.Incontrast,the352

post-PCRcombinedmockwasfarlessvariable(Figure1).TheinitialPCRintroducedalarge353

amountoftaxonomicbiasbypreferentiallyamplifyingsometaxa,asinferredfromthe354

differenceinvariabilityinreadnumbersbetweenthepostandpre-PCRmixesofourarthropod355

mockcommunity.Sequencingitselfalsointroducedbiasresultingindifferencesinread356

numbersbetweenthemockmembersthatwerecombinedpost-PCR.Someofthevariationin357

readnumbersamongmockcommunitymemberswasprobablyinducedbymismatchesinthe358

primingsite,giventhatsomememberspossessingthreeormoreprimermismatches.While359

thisnumberofmismatchesdidnotinhibitamplificationinindividualPCRs,inacompetitive360

mixedPCRthemismatchescouldresultinanamplificationbias.Differencesinreadnumbers361

canalsobeattributedtosequencecharacteristicssuchashomopolymerregionsandGC362

content.Ourmockcommunitydatademonstratedthatusingreadnumbersasproxiesfor363

abundanceinenvironmentalsamplesisproblematic,especiallyincomplexsamples.364

Becauseourarthropodmockcommunityconsistsofsingle-copyclonedplasmids,we365

expectedtofindonlyoneOTUpermockmember,allowingtheconclusiveidentificationof366

spuriousorchimericsequencesgeneratedduringthesequencingprocess.Someofthese367

chimerasaretheresultofsimplebinningerrorsandothersaretruechimeras(i.e.,hybrid368

sequencesasaresultofPCRandsequencingerror).Acriticalcomponentofchimerafilteringis369

havingacurateddatabaseofreferencesequences.Weinitiallyattemptedtouseallavailable370


14

CO1sequencesinBOLD,butencounteredmanyinconsistencies;thus,wemanuallycurateda371

subsetofthosesequencestouseforreferencesequences.Thiscuratedreferencedatabaseis372

availableat373

https://github.com/nextgenusfs/amptk/blob/master/docs/reference_databases.md.As374

additionalwell-documentedsequencesareaddedtothedatabase,theabilitytoidentify375

chimericsequenceswillcontinuetoimprove,thusenhancingtheaccuracyofOTUidentification376

inHTSofCO1.377

Withouttheuseofamockcommunity,finalOTUcountsmaybegreatlyinflated378

becauseitisdifficulttoidentifyspuriousOTUs.SpuriousOTUsmayarisefromPCR-or379

sequencing-basedchimeraformationaswellaserrorsgeneratedbyclusteringalgorithms.380

Usingawidelyusedclusteringalgorithm(UPARSE;Edgar2013)andfine-tunedfiltering381

parameters,ourinitialOTUestimateforour46membersinglecopymockcommunitywas70,382

andthusinflatedbyatleast52%bythegenerationofspuriousOTUs.Throughmanual383

inspectionofthesequences,mostofthespuriousOTUsinthemockcommunitywerePCR-384

basedchimerasthatpassedthechimerafilterandwerenotobservedinanyothersample.385

Usingourmockcommunityasareference,wewereabletoassesstheefficacyofanalternative386

OTUpickingalgorithm,DADA2(Callahanetal.2016).UsingtheDADA2algorithmfollowedby387

97%UCLUSTclustering,wewereabletoreducethenumberofOTUsinourpre-PCRcombined388

mockcommunityfrom70to43.Thismethodisstillimperfect,asoneoftheOTUswas389

attributedtosequencingerrorandonewasachimera,thusreducingthefinalnumberto42.390

Twoofourmockmemberswerelostbecausetheydidnotsequencewell,andanadditional391

twowereintra-individualvariantsofothermockmembers(HarmoniaaxyridisandPhalangium392

opilio),andclusteredwiththeir“sibling”sequencesafterUCLUSTwasappliedtotheDADA2393

output.WhenweusedthecuratedreferencedatabaseforchimerafilteringwithUCHIMEin394

combinationwiththeDADA2algorithm,wewereabletoremoveallbutonespuriousOTUfrom395

ourmockcommunity,demonstratingthatclusteringalgorithmscanbefine-tunedtominimize396

spuriousOTUgenerationwiththeuseofsingle-copymockcommunities.397

Estimatesoftaxonomicrichnessmayalsobeinflatedbyintragenomicvariabilityin398

barcodingregions.Intragenomicvariabilityisknowninsomeofthemostcommonlyused399


15

barcodingregions,suchasthefungalITSregion(Lindner&Banik2011;Lindneretal.2013;400

Schochetal.2012),aswellasthemitochondrialCO1region(Songetal.2008).Therefore,401

intragenomicvariabilitycouldbeacommonissuewithmanyotherloci.Thepresenceofthis402

individual-levelvariationcanleadtotheinflationoftaxonnumbersbecauseintragenomic403

variantsareoftenmisclassifiedasseparateOTUs(Lindner&Banik2011;Songetal.2008).Two404

conditionsthatcancausethisapparentvariationintheCO1locus,specifically,are405

heteroplasmyandthepresenceofnuclearmitochondrialpseudogenes(numts),whichare406

piecesofmitochondrialDNAthathavebeenincorporatedintothegenome(Songetal.2008).407

Wedetectedintra-individualvariationintheCO1regioninHarmoniaaxyridisandPhalangium408

opilioviastandardcloningandsequencing,eventhoughalimitednumberofcloneswere409

sequenced(i.e.,twosequencevariantsweredetectedbysequencingonlythreeclonesfrom410

eachoftheseindividuals).Basedontheseobservationsfromaverylimitedsamplingof3clones411

perindividual,itseemslikelythatindividualsharbormanysuchvariantsandthatindividual-412

levelvariabilitycouldsignificantlyinflatediversityestimatesinHTSoftheCO1region.TheH.413

axyridisvariantsonlydifferedby2.1%(14of658bp)buttheP.opiliovariantsweremorethan414

threepercentdifferent(3.5%,or23of659bp).Manyofthesedifferencesoccurredinthe415

fragmentamplifiedbytheANMLprimersandthustraditionalclusteringwouldhaveidentified416

themasdistinctOTUs.BothvariantsofH.axyridisandP.opiliowereincludedinourarthropod417

mockcommunitytodetermineifourbioinformaticspipelinewouldbinthesequencevariants418

fromthesameindividualintoseparateinferredsequences.WhenweappliedtheDADA2419

algorithmwithoutclustering,thevariantsseparatedintoseparateOTUs.Afterweapplied97%420

clusteringtotheresultingDADA2inferredsequences,thevariantsweobservedinoursingle421

copymockcommunitybinnedtogether.Theuseofsingle-copyclonedplasmidDNAformock422

communitymembersiscrucialbecauseitremovescrypticsourcesofbiologicalvariationthat423

mightotherwiseoccurwithinthemockcommunity.424

ValidationoftheANMLprimerpairandmockcommunity425

WefurthervalidatedourprimersandHTSsystemusingtwotypesofsampleswith426

knowncomposition:(1)guanosamplesfrombatsfedknowndietsand(2)samplesfrominsect427

trapsthatwereidentifiedbymorphology.Fromtheguanosamples,werecoveredalltaxa428


16

includedintheknowndietsandalsorecoveredadditionalOTUs(Table4).TheinitialtotalOTU429

estimateforourfiveknowndietsampleswas42basedonUPARSEclustering,31inferred430

sequencesbasedonDADA2withoutclustering,and10OTUsbasedonDADA2with97%431

clustering.MuchofthetaxonomicreductionintheknowndietsamplesafterusingDADA2with432

clusteringcanbeattributedtosequencevariantsoftwotaxa,Tenebriomolitorand433

Ichneumonidaesp.Thesetwotaxayieldedupto11and7inferredsequencespersamplewith434

theDADA2algorithm,respectively,before97%clusteringwasapplied.However,after435

clusteringwasapplied,theyyieldeduptotwoOTUspersample.Thedegreetowhichthese436

variantsrepresentintra-individualsequencevariation,orvariantsamongindividuals,cannotbe437

determinedhere,butoffersaninterestingtopicforfutureinvestigation.Theestimatewith438

DADA2withclusteringismuchclosertotheexpectedrichnessof5OTUsthanotherestimates.439

SeveralOTUsdetectedfromtheknowndietsampleswereunexpected,butprobablyreal440

componentsofthebatdiet.TwooftheseOTUs,E.takeuchiiandAgrotisipsilon,arelikely441

contaminantsinthedietarycomponentsbecausetheirlarvalformsmayhavebeenmixedinto442

theG.mellonellalarvaethatcomprisedthediet.Wealsodetectedanichneumonidparasitoid443

wasp,whichwasperhapsparasitizingoneormoreoftheinsectsinthediet.Theunexpected444

taxacouldhavebeenanticipatedbysequencingasubsampleoftheknowndietarycomponents445

priortofeeding.446

HTSsuccessfullyrecoveredthemajorityofarthropodspresentinmixedsamplesfrom447

pitfalltraps(Table5).Aftertakingintoaccountprobablemorphologicalidentificationerrors,448

approximately80%ofthetaxaidentifiedbymorphologywerealsoidentifiedviaHTS.Those449

taxamissedbyHTSmayhavebeenmissedduetobiasesinthemolecularpipelinesuchasPCR450

biasesthataroseinthesecomplexcommunities,orperhapsthesetaxarequiremorespecific451

primers.TherewerealsotaxathatweredetectedwithHTSbutmissedbymorphological452

identification.Theseadditionaltaxamayhavebeenmayhavebeenconsumedbyorotherwise453

associatedwiththearthropodscollectedinthetraps,misidentifiedduringthemorphological454

identification,ormaybeDNAcontaminationofthetrapsorothercollectionequipment.455

Conclusion456


17

WedemonstratedthattheANMLprimerpairdetectsagreaternumberofarthropod457

taxathanotherfrequentlyusedCO1primerpairs.TheuseofHTSreadnumbersasameasure458

ofabundanceinenvironmentalsamplesisproblematicduetobiasesintroducedduringboth459

PCRandHTS.Thesebiasesmaybepartiallyalleviatedinthefuturebynon-PCRbased460

techniquessuchasshotgunmetagenomicsandtargetcapturetechniques.However,shotgun461

metagenomicsarecurrentlyfarmoreexpensivethanampliconsequencingandmaybecost-462

prohibitivetomostresearchers,andtargetcapturehasnotyetbeenthoroughlyevaluatedfor463

communitycharacterizationofenvironmentalsamples.Failingtouseappropriatepositive464

controlsforamplicon-basedstudiescanleadtoover-estimationofdiversity,andthe465

persistenceof“nonsensetaxa”.Thus,mockcommunitycontrolsarenecessarytoparameterize466

downstreambioinformatics,especiallyfordiversityandcommunitystructurerelatedquestions467

andweadvocatefortheinclusionofaspike-inmockcontrolineveryHTSrun.468

469

Acknowledgements470

FundingforthisworkwasprovidedbytheUSForestService,NorthernResearchStation471

andtheAgriculturalExperimentStationattheUniversityofWisconsin–MadisonviaHatch472

FormulaFunds,andNSFIOS-1121739toAYKandIOS-1121807toJRB.Wesincerelythank473

JamesSkeltonforinsightfuldiscussionthatimprovedpreviousversionsofthismanuscript,J.474

PaulWhiteandHeatherKaarakkaoftheWisconsinDepartmentofNaturalResourcesfor475

assistancewithcoordinatingfieldcollectionofguanosamples,MarceloFerreiradeMeloand476

ErinGreenforassistancewitharthropoddissections,andEliCintoMeijaandMitchLevenhagen477

forinsectcollectionfrompitfalltraps.478

479

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600


23

TablesandFigures601

602

Table1:Sequencesandreferences,andprimerpairnamesfortheprimerstestedagainst603

knownarthropodsamples.604

605

Primername Primersequence Reference PairnameLCO1490 5’-GGTCAACAAATCATAAAGATATTGG-3’ Folmeretal.1994 CO1L/HHCO2198 5’-TAAACTTCAGGGTGACCAAAAAATCA-3’ Folmeretal.2003 LEPF1 5’-ATTCAACCAATCATAAAGATATTGG-3’ Hebertetal.2004 LEPmLEPR 5’-CTTGTTCCAGCTCCATTTT-3’ Smithetal.2006 ZBJ-ArtF1c 5’-AGATATTGGAACWTTATATTTTATTTTTGG-3’ Zealeetal.2011 ZBJZBJ-ArtR2c 5’-WACTAATCAATTWCCAAATCCTCC-3’ Zealeetal.2011 LCO1490 5’-GGTCAACAAATCATAAAGATATTGG-3’ Folmeretal.1994 ANMLCO1-CFMRa 5’-GGWACTAATCAATTTCCAAATCC-3’ Thisstudy LCO1490 5’-GGTCAACAAATCATAAAGATATTGG-3’ Folmeretal.1994 CFMRbCO1-CFMRb 5’-GGNACTAATCAATTHCCAAATCC-3’ Thisstudy


24

Table2:Resultsfromtestingthe5primerpairslistedinTable1onknowninsectsamples.Shadingandavalue606

of1indicateamplification;noshadingandavalueofzeroindicatenoamplification.Amplificationwas607

attemptedonavarietyofDNAconcentrationsforeachtemplateDNAsamplebeforeassigningavalueofzero608

(noamplification).ArthropodmockcommunitymembersareindicatedwiththesuperscriptIM.Anasterisk609

indicatesthattwodifferentclonedsequencevariantsofanindividualwereaddedtothearthropodmock610

community.**Indicatesthatthreedifferentclonedsequencevariantsofanindividualwereaddedtothe611

arthropodmockcommunity.612

Order Family Identity

ANML

CFMRb

CO1L/H

ZBJZealeetal.2011protocol

ZBJmodifiedprotocol LEP

Blattodea Blattidae PeriplanetafuliginosaIM 1 1 1 1 1 1Blattodea Ectobiidae SupellalongipalpaIM 1 1 1 0 0 1Coleoptera Cantharidae Chauliognathuspennsylvanicus 1 1 1 1 1 1Coleoptera Carabidae Carabidaesp. 0 0 0 0 0 0Coleoptera Cerambycidae Tetraopessp. 1 1 1 1 1 1Coleoptera Chrysomelidae PariafragariaeIM 1 1 1 1 1 1Coleoptera Coccinellidae HarmoniaaxyridisIM** 1 1 1 0 0 0Coleoptera Hydrophilidae Hydrophilidaesp.IM 1 1 1 0 1 1Coleoptera Meloidae Epicautasp. 1 1 1 0 0 1Coleoptera Scarabaeidae EuphoriafulgidaIM 1 1 1 0 0 1Coleoptera Tenebrionidae Tenebriomolitor 1 1 1 1 0 1Coleoptera unk.Coleoptera Polyphagasp.IM 1 1 1 0 0 1Dermaptera Forficulidae Forficulidaesp. 1 1 0 0 0 1Diptera Anthomyiidae DeliaplaturaIM 1 1 1 1 1 1Diptera Bombyliidae LepidophoraluteaIM 1 1 1 1 1 1Diptera Chironomidae Dicrotendipessp.IM 1 1 0 0 0 1Diptera Chironomidae Procladiussp.IM 1 1 1 1 1 1Diptera Culicidae AedesAlbopictusIM 1 1 1 0 0 1Diptera Culicidae AedesvexansIM 1 1 1 1 1 1Diptera Leptoceridae Oecetisinconspicua 1 1 1 0 0 1Diptera Tipulidae NephrotomaferrugineaIM 1 1 1 0 1 1Ephemeroptera Ephemeridae Hexagenialimbata1 1 1 1 0 0 1Ephemeroptera Ephemeridae Hexagenialimbata2IM 1 1 1 0 0 1Ephemeroptera Heptageniidae LeucrocutamaculipennisIM 1 1 1 1 1 1Ephemeroptera unk.Ephemeropotera Ephemeropterasp. 1 1 0 0 0 0Hemiptera Aphididae AphishelianthiIM 1 1 1 0 0 0Hemiptera Cicadellidae Osbornellusauronitens 1 1 1 0 0 0Hemiptera Cicadidae Cicadidaesp. 1 1 1 0 1 1Hemiptera Corixidae Corixidaesp. 1 1 0 0 0 1Hemiptera Corixidae SigaraalternataIM 1 1 1 1 1 1Hemiptera Pentatomidae AcrosternumhilareIM 1 1 1 0 0 0


25

Hymenoptera Apidae ApismelliferaIM 1 1 1 0 0 0Hymenoptera Crabonidae Spheciusconvallis 1 1 1 0 0 1Hymenoptera Eucharitidae Eucharitidaesp.IM 1 1 1 1 1 1Hymenoptera Formicidae Formicafusca 1 1 1 0 0 0Hymenoptera Formicidae Formicasp.IM 1 1 1 0 0 0Hymenoptera Tenthredinidae EmpriatakeuchiiIM 1 1 1 1 1 0Lepidoptera Crambidae CrambusagitatellusIM 1 1 1 1 0 1Lepidoptera Crambidae ElophilaobliteralisIM 1 1 1 0 1 1Lepidoptera Crambidae UdearubigalisIM 1 1 1 1 1 1Lepidoptera Depressariidae DepressariapastinacellaIM 1 1 1 1 1 1Lepidoptera Erebidae HypenascabraIM 1 1 1 1 1 1Lepidoptera Erebidae HyphantriacuneaIM 1 1 1 0 0 1Lepidoptera Erebidae IdiaaemulaIM 1 1 1 1 1 1Lepidoptera Erebidae ReniafactiosalisIM 1 1 1 1 1 1Lepidoptera Geometridae HaematopisgratariaIM 1 1 1 1 1 1Lepidoptera Noctuidae AgrotisipsilonIM 1 1 1 1 1 1Lepidoptera Tortricidae ChoristoneurarosaceanaIM 1 1 1 1 1 1Neuroptera Chrysopidae ChrysopaoculataIM 1 1 1 0 0 1Neuroptera Mantispidae Mantispidaesp.IM 1 1 1 0 1 1Opiliones Phalangiidae PhalangiumopilioIM* 1 1 1 0 0 1Orthoptera Acrididae MelanoplusfemurrubrumIM 1 1 1 0 0 1Orthoptera Tettigoniidae ScudderiacurvicaudaIM 1 1 1 0 0 1Orthoptera Tettigoniidae Tettigoniidaesp.IM 1 1 1 1 1 1Trichoptera Hydropsychidae Potamyiaflava 1 1 0 0 0 1Trichoptera Hydroptilidae Orthotrichiasp.IM 1 1 1 0 0 1Trichoptera Leptoceridae CeracleamaculataIM 1 1 1 1 1 1Trichoptera Leptoceridae LeptocerusamericanusIM 1 1 1 0 0 1Trichoptera unk.Trichoptera Trichopterasp. 1 1 0 0 0 0

Negativecontrol 0 0 0 0 0 0

Total 58 58 52 24 27 48

%Total 98.31 98.31 88.14 40.68 45.76 81.36

613


26

Table3:OperationalTaxonomicUnits(OTUs)recoveredusinghigh-throughputampliconsequencing(HTS)and614

eithertheANMLprimersortheZBJprimerson3field-collectedguanosamples.Numbers(0-3)and615

representativeshadingindicatethenumberofguanosampleseachOTUwasdetectedinforeachprimerpair.616

617

ANML ZBJ Phylum Class Order Family Genus species1 0 Arthropoda Arachnida Araneae Theridiidae Theridion Theridionfrondeum2 0 Arthropoda Arachnida Trombidiformes Limnesiidae Limnesia Limnesiasp.1 0 Arthropoda Arachnida Trombidiformes

Trombidiformessp.

1 0 Arthropoda Arachnida

Arachnidasp.1 0 Arthropoda Insecta Coleoptera Coccinellidae Harmonia Harmoniasp.2 0 Arthropoda Insecta Coleoptera Elateridae Melanotus Melanotussimilis1 0 Arthropoda Insecta Coleoptera Hydrophilidae Helocombus Helocombusbifidus1 0 Arthropoda Insecta Coleoptera Scarabaeidae

Scarabaeidaesp.

1 0 Arthropoda Insecta Coleoptera Tenebrionidae Tenebrio Tenebriosp.1 0 Arthropoda Insecta Coleoptera

Coleopterasp.

1 0 Arthropoda Insecta Diptera Bibionidae Bibio Bibiosp.1 0 Arthropoda Insecta Diptera Ceratopogonidae Bezzia Bezziasp.0 2 Arthropoda Insecta Diptera Chaoboridae Chaoborus Chaoboruspunctipennis1 1 Arthropoda Insecta Diptera Chironomidae Ablabesmyia Ablabesmyiaamericana1 0 Arthropoda Insecta Diptera Chironomidae Ablabesmyia Ablabesmyiaannulata1 0 Arthropoda Insecta Diptera Chironomidae Ablabesmyia Ablabesmyiasp.11 0 Arthropoda Insecta Diptera Chironomidae Ablabesmyia Ablabesmyiasp.23 0 Arthropoda Insecta Diptera Chironomidae Chironomus Chironomusplumosus1 1 Arthropoda Insecta Diptera Chironomidae Chironomus Chironomussp.10 2 Arthropoda Insecta Diptera Chironomidae Chironomus Chironomussp.21 0 Arthropoda Insecta Diptera Chironomidae Coelotanypus Coelotanypussp.1 0 Arthropoda Insecta Diptera Chironomidae Conchapelopia Conchapelopiasp.1 0 Arthropoda Insecta Diptera Chironomidae Cryptochironomus Cryptochironomussp.12 0 Arthropoda Insecta Diptera Chironomidae Cryptochironomus Cryptochironomussp.22 0 Arthropoda Insecta Diptera Chironomidae Dicrotendipes Dicrotendipestritomus2 1 Arthropoda Insecta Diptera Chironomidae Parachironomus Parachironomussp.11 0 Arthropoda Insecta Diptera Chironomidae Parachironomus Parachironomussp.21 0 Arthropoda Insecta Diptera Chironomidae Parachironomus Parachironomussp.32 0 Arthropoda Insecta Diptera Chironomidae Polypedilum Polypedilumsp.11 0 Arthropoda Insecta Diptera Chironomidae Polypedilum Polypedilumsp.22 2 Arthropoda Insecta Diptera Chironomidae Procladius Procladiussp.11 0 Arthropoda Insecta Diptera Chironomidae Procladius Procladiussp.21 0 Arthropoda Insecta Diptera Chironomidae Procladius Procladiussp.30 1 Arthropoda Insecta Diptera Chironomidae Procladius Procladiussp.41 0 Arthropoda Insecta Diptera Chironomidae Xenochironomus Xenochironomussp.3 0 Arthropoda Insecta Diptera Chironomidae

Chironomidaesp.1

1 0 Arthropoda Insecta Diptera Chironomidae

Chironomidaesp.22 0 Arthropoda Insecta Diptera Chironomidae

Chironomidaesp.3


Chironomidaesp.4


27


Chironomidaesp.51 0 Arthropoda Insecta Diptera Culicidae Aedes Aedesabserratus1 0 Arthropoda Insecta Diptera Culicidae Aedes Aedesexcrucians1 0 Arthropoda Insecta Diptera Culicidae Aedes Aedesprovocans1 1 Arthropoda Insecta Diptera Culicidae Aedes Aedesvexans1 0 Arthropoda Insecta Diptera Culicidae Culiseta Culisetamelanura1 0 Arthropoda Insecta Diptera Hybotidae Platypalpus Platypalpussp.0 1 Arthropoda Insecta Diptera Limoniidae Shannonomyia Shannonomyialenta1 0 Arthropoda Insecta Diptera Psychodidae Psychoda Psychodaalternata1 1 Arthropoda Insecta Diptera Tachinidae

Tachinidaesp.

1 0 Arthropoda Insecta Diptera Tipulidae Nephrotoma Nephrotomaferruginea0 1 Arthropoda Insecta Diptera Tipulidae Tipula Tipulakennicotti1 1 Arthropoda Insecta Ephemeroptera Caenidae Caenis Caenisyoungi1 0 Arthropoda Insecta Ephemeroptera Palingeniidae Pentagenia Pentageniavittigera1 0 Arthropoda Insecta Ephemeroptera Siphlonuridae Siphlonurus Siphlonurustypicus1 0 Arthropoda Insecta Hemiptera Corixidae Trichocorixa Trichocorixaborealis1 0 Arthropoda Insecta Hemiptera Miridae Lygus Lyguslineolaris0 1 Arthropoda Insecta Lepidoptera Blastobasidae Blastobasis Blastobasisglandulella1 0 Arthropoda Insecta Lepidoptera Depressariidae Antaeotricha Antaeotrichaleucillana

1 0 Arthropoda Insecta Lepidoptera Tortricidae ArgyrotaeniaArgyrotaeniapinatubana

1 0 Arthropoda Insecta Lepidoptera

Lepidopterasp.1 0 Arthropoda Insecta Megaloptera Corydalidae Chauliodes Chauliodesrastricornis1 0 Arthropoda Insecta Trichoptera Hydroptilidae Oxyethira Oxyethiraserrata0 2 Arthropoda Insecta

Insectasp.

3 0 Chordata Mammalia Chiroptera Vespertilionidae Myotis Myotislucifugus 618


28

Table4:NumberofOTUsidentifiedfrombatsfedknowninsectdiets,brokendownbyexpecteddietarycomponents,possible619

accidentaldietarycomponents,andbatDNA.Blanksarezeros.DADA2isdatafromDADA2,withoutclustering.DADA297%clusteris620

datafromDADA2with97%clusteringappliedtotheOTUtable.Shadedcellsaredietarycomponentsthatwereexpected(i.e.known621

tobefedtothebat).EPFU1,EPFU2,andEPFU3arefromBigBrownBats(Eptesicusfuscus),andLACI1andLACI2arefromHoaryBats622

(Lasiuruscinereus).623

624

Expecteddietarycomponents Possibleaccidentaldietarycomponents BatDNA

Galleriamellonella

Tenebriomolitor

Antheraeapolyphemus

Empriatakeuchii

Agrotisipsilon Ichneumonidaesp.

Eptesicusfuscus

Lasiuruscinereus

DADA2 DADA297%

cluster

DADA2 DADA297%

cluster

DADA2 DADA297%

cluster

DADA2 DADA297%

cluster

DADA2 DADA297%

cluster

DADA2 DADA297%

cluster

DADA2

DADA297%

cluster

DADA2 DADA297%

clusterEPFU1 1 1 8 1 1 1 1 1 1 1 EPFU2 1 1 1 1 1 1 1 1 1 1 7 1 1 1 EPFU3 1 1 1 1 LACI1 1 1 7 1 3 2LACI2 11 2 3 2


29

Table5:ComparisonofmorphologicalandHTSfamily-levelidentificationsofarthropods625

collectedfrom5pitfalltraps.Arthropodsfromtraps1and4weredissectedpre-extraction,and626

arthropodsfromtraps2,3,and5weremaceratedpre-extraction.A“+”indicatespresenceofa627

family.628

629

Trap1 Trap2 Trap3 Trap4 Trap5

Key NGS Key NGS Key NGS Key NGS Key NGS

TotalTaxa 7 10 17 13 17 19 4 5 10 7

Class Order/subclass Family

Insecta Blattodea Ectobiidae + Insecta Coleoptera Carabidae + + Insecta Coleoptera Elateridae + Insecta Coleoptera Melyridae + + + +

InsectaColeoptera

Ptinidae/

+

Anobiidae

Insecta Coleoptera Scarabaeidae + Insecta Coleoptera Silphidae + + Insecta Coleoptera Tenebrionidae + + Entognatha Collembola + + Insecta Diptera Anthomyiidae + + + + Insecta Diptera Bombyliidae + +

Insecta Diptera Calliphoridae + + + Insecta Diptera Cecidomyiidae + Insecta Diptera Culicidae + Insecta Diptera Dipterasp. + Insecta Diptera Heleomyzidae + Insecta Diptera Phoridae + + + Insecta Diptera Scathophagidae + + Insecta Diptera Sciaridae + Insecta Diptera Syrphidae + +

Insecta Hemiptera Aphididae + + + +

Insecta Hemiptera Cicadidae + + Insecta Hemiptera Cicadellidae + + + + + Insecta Hemiptera Geocoridae + Insecta Hemiptera Miridae + + Insecta Hemiptera Pentatomidae + + Insecta Hemiptera Psyllidae + + Insecta Hymenoptera Formicidae + + + + + + + +

Insecta Hymenoptera Braconidae +


30

Insecta Hymenoptera Ceraphronidae + Insecta Hymenoptera Chalcidoidea + Insecta Hymenoptera Crabronidae + Insecta Hymenoptera Dryinidae + Insecta Hymenoptera Halictidae + +

Insecta Hymenoptera Hymenopterasp. + Insecta Hymenoptera Ichneumonidae + + + Insecta Hymenoptera Pompilidae + + Insecta Lepidoptera Gelechiidae + + Insecta Lepidoptera Tortricidae + +

Insecta Neuroptera Chrysopidae + Insecta Orthoptera Acrididae + Insecta Orthoptera Tettigoniidae + Insecta Thysanoptera Thripidae + Insecta Thysanoptera Hydroptillidae + Arachnida Acari + + + + + + + + Arachnida Araneae Araneaesp. + Arachnida Araneae Gnaphosidae + + + + Arachnida Araneae Lycosidae + + Arachnida Araneae Pisauridae + Arachnida Araneae Salticidae + + + + Arachnida Araneae Thomisidae + Chilopoda + +

630


31

Figure1:Heatmapofthehigh-throughputampliconsequencingreadnumbersofthe631

arthropodmockcommunity,equilibratedandcombinedbothpre-andpost-PCR.Thepost-PCR632

combinedmockcommunitywasfarmoreevenandrepresentativeoftheequalamountsof633

DNAaddedforeachmockmemberthanthepre-PCRcombinedmockcommunity.634

635

636


32

DataAccessibility637

ThecorrespondingdataforthispaperwasdepositedintheNCBISRA(SRAstudySRP102878;638

BioProjectPRJNA380665),andbarcodedprimerinformationisprovidedinthesupplemental639

information.640

641

AuthorContributions642

MAJ,MTB,andJMPwrotethepaper;MAJ,MTB,JMP,andDLLdesignedresearch;MAJ,MTB,643

JMP,AKW,andEPperformedresearch;AYK,LX,JRB,CG,andMZPcontributedsamples;MAJ,644

MTB,JMPanalyzedthedata;andMAJ,MTB,JMP,AKW,JRB,AYK,CG,MZP,andDLLedited645

draftsofthepaper.646

647

SupportingInformation648

AppendixS1–DNAextractiondetailsfortheCLSextractionforarthropods649

AppendixS2–DNAextractiondetailsforthemodifiedbatguanoextraction650

TableS1–SequencesforIonbarcodedprimers651

FigureS1–MitochondrialcytochromeoxidaseCsubunit1locus(CO1)primermap652


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