Towards a DNA Barcode Reference Database for Spiders and Harvestmen of Germany

24 Pages·2016·2.22 MB·English

RESEARCHARTICLE
Towards a DNA Barcode Reference Database
for Spiders and Harvestmen of Germany
JonasJ.Astrin1*,HubertHöfer2*,JörgSpelda3*,JoachimHolstein4*,SteffenBayer2,
LarsHendrich3,BernhardA.Huber1,Karl-HinrichKielhorn5,Hans-JoachimKrammer1,
MartinLemke6,JuanCarlosMonje4,JérômeMorinière3,BjörnRulik1,MaltePetersen1,
HannahJanssen1,ChristophMuster7
1 ZFMK:ZoologischesForschungsmuseumAlexanderKoenig,Bonn,Germany,2 SMNK:Staatliches
MuseumfürNaturkundeKarlsruhe,Karlsruhe,Germany,3 ZSM:ZoologischeStaatssammlungMünchen,
München,Germany,4 SMNS:StaatlichesMuseumfürNaturkundeStuttgart,Stuttgart,Germany,5 Karl-
a11111 HinrichKielhorn,Berlin,Germany,6 MartinLemke,Lübeck,Germany,7 ZoologischesInstitutundMuseum,
UniversitätGreifswald,Greifswald,Germany
*[email protected](JJA);[email protected](HH);[email protected](JS);joachim.
[email protected](JH)
Abstract
OPENACCESS
Citation:AstrinJJ,HöferH,SpeldaJ,HolsteinJ, AspartoftheGermanBarcodeofLifecampaign,over3500arachnidspecimenshavebeen
BayerS,HendrichL,etal.(2016)TowardsaDNA
collectedandanalyzed:ca.3300Araneaeand200Opiliones,belongingtoalmost600spe-
BarcodeReferenceDatabaseforSpidersand
cies(median:4individuals/species).Thiscoversabout60%ofthespiderfaunaandmore
HarvestmenofGermany.PLoSONE11(9):
e0162624.doi:10.1371/journal.pone.0162624 than70%oftheharvestmenfaunarecordedforGermany.Theoverwhelmingmajorityof
speciescouldbereadilyidentifiedthroughDNAbarcoding:mediandistancesbetweenclos-
Editor:MatjaKuntner,ScientificResearchCentreof
theSlovenianAcademyofSciencesandArt, estspecieslayaround9%inspidersand13%inharvestmen,whilein95%ofthecases,
SLOVENIA intraspecificdistanceswerebelow2.5%and8%respectively,withintraspecificmediansat
Received:June3,2016 0.3%and0.2%.However, almost20spiderspecies,mostnotablyinthefamilyLycosidae,
couldnotbeseparatedthroughDNAbarcoding(althoughmanyofthempresentdiscrete
Accepted:August25,2016
morphologicaldifferences).Conspicuouslyhighinterspecificdistanceswerefoundineven
Published:September28,2016
morecases,hintingatcrypticspeciesinsomeinstances.Anewprogramispresented:DiS-
Copyright:©2016Astrinetal.Thisisanopen
tatscalculatesthestatisticsneededtomeetDNAbarcodereleasecriteria.Furthermore,
accessarticledistributedunderthetermsofthe
newgenericCOIprimersusefulforawiderangeoftaxa(alsootherthanarachnids)are
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany introduced.
medium,providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles.
Sequencesareappendedasalignment,theirtrace
Introduction
filesareavailablefromBOLD(http://www.
boldsystems.org/).TheDiStatsscriptisavailable
Long-termmonitoringofbiodiversityisoneofthemostimportantchallengesinconservation
throughGitHub(https://github.com/mptrsen/distats)
biology.To evaluatetheconservationstatusandanthropogenicimpactofhabitats,sufficient
andthroughtheZFMKhomepage(www.zfmk.de/en/
research/research-centres-and-groups/distats). knowledgeonspeciescompositionofnaturalenvironmentsisneededonaregionallevel.For
manyifnotmostinvertebratetaxa,wearestillfarfromachievingthisgoal.Onepromising
Funding:TheGBOLprojectisfinancedbythe
approachtomeetthischallengeisDNAbarcoding[1],atechniquethatusestheeasytohomol-
GermanFederalMinistryofEducationandResearch
(BMBF#01LI1101).Partofthesequencescontributed ogize,well-quantifiable,discretetaxonomiccharacterscontainedinDNAsequencedatafor
byZSMresultedfromfundingbytheBavarianState standardized,rapid,andrelativelycheapspeciesidentification.DNAbarcodingdependson
PLOSONE|DOI:10.1371/journal.pone.0162624 September28,2016 1/24
DNABarcodingofSpidersandHarvestmeninGermany
MinistryofEducationandCulture,Scienceandthe lowlevelsofintraspecificvariationcoupledwithmarkedgeneticdifferentiationbetweenspe-
Arts(BarcodingFaunaBavarica,BFB).The cies(the'barcodinggap',investigatedinspidersin[2–4]).
sequencingworkofZSMwassupported,inpart,by
Withmorethan45,800describedspecies[5],spidersareamongthemostdiverseanimal
fundingfromtheGovernmentofCanadatoGenome
orders[6].Theyareabundantinallterrestrialhabitats.Asubiquitouspredators,theyoccupya
CanadathroughtheOntarioGenomicsInstitute,while
theOntarioMinistryofResearchandInnovationand keypositioninfoodwebs.Manyspeciesshowpreferencesforspecifichabitatstructuresor
NSERCsupporteddevelopmentoftheBOLD environmentalfactors,e.g.temperature,humidity,shading[7],whichturnsthemintopotential
informaticsplatform.Thefundershadnoroleinstudy indicators[8].Easytoobserveanddocument,spidersareseenasamodelgroupforecological
design,datacollectionandanalysis,decisionto
studies[9,10].
publish,orpreparationofthemanuscript.
ThespiderfaunaofGermany,comprisingapproximately1000species[11],iswellknown,
CompetingInterests:Theauthorshavedeclared andchecklistsandredlistsofendangeredspecieshavebeenpublishedforGermanyandmost
thatnocompetinginterestsexist.
ofitsfederalstates(see[11]andreferencestherein).The'ArachnologischeGesellschafte.V.'
(www.arages.de)offersregularlyupdatedoccurrencemaps,basedonasteadilygrowingdata-
base.Therefore,spidersareregularlyusedinhabitatassessments,biodiversityinventories,and
ecologicalstudies(e.g.[12–18]).Spidersareparticularlypromisingasindicatorsofsustainable
forestmanagement[19],habitatstructure[20],successionalstages[21,22],orconservation
value[23,24].Therehavebeenseveralattemptstoclassifyspidersaccordingtotheirhabitator
nichepreferencesinGermanyorCentralEurope[7,25–30]andtousethesedatatoclassify
habitatsorassesshabitatqualitybyidentifyingtheproportionofrare,endangered,stenotopic,
orcharacterspecies(e.g.[18,31,32]).IdentificationofGermanspidersisfacilitatedbythe
onlinekeysforspidersofEuropeatwww.araneae.unibe.ch[33].However,morphologicaliden-
tificationtospecieslevelrequiresadultspecimensinmostinstances.About80–200spiderspe-
ciescanoccurinanear-naturalhabitatinGermany,ofwhichonlyasmallfractioncanbe
directlyrecordedandidentifiedinthefield(pers.obs.,H.Höfer,C.Muster).Foranecolog-
icallymeaningfulassessmentoraclosetocompleteinventory,muchmoretimeneedstobe
investedtocapture,process(oftenmeaningdissectionofsexualorgans)andidentifythe
(adult)spiders,requiringconsiderableexpertise.Regularly,severalspecimensremainthathave
tobecheckedbythefewavailabletaxonomicspecialistswithsufficientknowledgeonmorpho-
logicalvariabilityintherespectivespeciesandwithaccesstoreferencecollections.
Withsome6500speciesworldwide,harvestmen(Opiliones)constitutethethird-largest
orderofarachnids[34].Currently,52specieshavebeenrecordedfromGermany[35].The
omnivorousharvestmenconstitutearegularcomponentofterrestrialfaunas,withhighestden-
sitiesindampandshadedhabitats[36].Theiruseinappliedandecologicalstudiesisexplained
bytheexistenceofbothstenotopicspecieswithstrictmicrohabitatrequirements(andoften
limitedgeographicranges)andinvasivespeciesthatexhibitimmensecolonizationpotential
[37,38].DeterminationofmostGermantaxaisreliablyachievableusingtheworkof[39].
However,recentstudieshaverevealedhighlevelsofcrypticdiversityinCentralEurope[40–
42],suggestingapromisingperspectiveforDNAbarcodinginthistaxon.
TheuseofmitochondrialCOIbarcodes[43]fromanextensivereferencedatabaseofspider
andharvestmenspecieswillaidnon-specialistsinthedeterminationofthesegroups.Species
thathavehithertobeenproblematicorevenimpossibletoidentifymorphologically–eitherin
generalorforaparticularsex–maybereliablydiscriminated.EventhoughnotfrequentinGer-
many, therearestillmanyspiderspeciesinwhichoneofthe(dimorphic)sexesisstill
unknown,andbarcodingcanprovidethelinkbetweensexes(demonstratede.g.in[44]).More-
over,disputedinstancesofsynonymymayberesolved[45].Notleast,aconsiderableadvantage
ofbarcodingisthepossibilitytoidentifyjuvenilespecimens[3,46–49].Thiswillnotonlymake
inventoriesmorecomplete,butwillalsoallowspecies-levelinclusionofjuvenilesintoecologi-
calanalyses.Tapping intothisrichmaterialresourcewillallowstudyingmoreecologicalques-
tionswithoutthenecessityforexhaustiveandexpensivesampling.Afuturebroadapplication
ofroutineDNAbarcodinginspidersisfacilitatedthroughmass-trapping,sincesomeofthe
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DNABarcodingofSpidersandHarvestmeninGermany
samplingsolutionsemployedintrapspreserveDNAwellenoughforbarcoding[50,51].The
methodfurtherholdsthepotentialtorevealcrypticspeciesortoidentifycaseswheremorpho-
logicalplasticitymayhavebeenover-interpreted.Barcodingmaythusactasacatalystfor
alphataxonomy[52].Whileintrogressionevents[53],retentionofancestralpolymorphisms
[49],nuclearmitochondrialpseudo-genes[54]orendosymbionts(Wolbachia bacteriaetc.)
[55,56]allposepotentialproblemstoDNAbarcodingapproaches,agrowingnumberofstud-
iesshowthegeneralfeasibilityofDNAbarcodingforarachnids[2–4,46,47,57–66].
TheGermanBarcodeofLife(GBOL)campaignisimplementedbyanationalnetworkofca.
20biodiversityresearchinstitutionsandmorethan200taxonspecialists[67].Itpursuesthe
goaltoestablishaDNAbarcodelibraryofasmanyanimal,fungalandplantspeciesaspossible
thatoccurinGermany.Theprojectaimsatcollecting,ifpossible,tenspecimensperspecies,
fromlocationsasdistinctaspossiblethroughoutthecountryinordertocapturegeneticvari-
ability.Somespecieswithwiderrangesmayalsoincludespecimenscollectedinneighboring
countries.
NaturalhistorycollectionsconstitutethecoreinfrastructureofGBOL,takingintoaccount
thatbarcodingprojectsproduceavaluablelegacyofvouchers(morphologicalspecimensand
molecularsamplesalike)whichbecomerelevantinsubsequentstudiesduetothehighquality
oftheunderlyingtaxonomicassignmentsandgranularityofthemetadata.Thesevouchers
formthephysicalfoundationthatfuturemonitoringprojectswillbebasedon,warrantingcon-
tinuoustestability,validation,andcoherentexpansionofthebarcodingreferencedatabase–
ideallyforcenturiestocome.
WithintheGBOLconsortium,arachnidshavereceivedwideattention,asnolessthanfour
GBOLinstitutesandtheirrespectiveexternalarachnologistpartnerscollaborateintensivelyon
compilinganationalmolecularinventoryofspidersandharvestmen:StaatlichesMuseumfür
NaturkundeKarlsruhe(SMNK),StaatlichesMuseumfürNaturkundeStuttgart(SMNS),Zool-
ogischesForschungsmuseumAlexanderKoenig(ZFMK),andZoologischeStaatssammlung
München(ZSM).AcariarebeinginvestigatedinanotherGBOLsubproject(bySenckenberg
MuseumfürNaturkundeGörlitz,SMNG).
Forspiders,country-focused,taxonomicallybroadbarcodingdatasetshavesofarbeenpub-
lishedforCanada[61](1018speciescovered),forSloveniaandforSwitzerland[66](together
298species)and,inapilotproject,fortheNetherlands[68](31species)(foralistofongoing
Europeanprojects,seehttp://www.araneae.unibe.ch/barcoding/content/15/Barcoding-of-
European-spiders).Thepresentstudycontributesthefirstdatasetofaspiderbarcodingcam-
paignforGermanyandthefirstdatasetworldwideofthiskindforharvestmen.
MaterialsandMethods
Sampling
Forthisstudy,3537arachnidspecimens,3339Araneaeand198Opiliones,weresampledfrom
Germany(91%ofthematerial)andneighboringcountries.WithinGermany,24%ofthespeci-
menswerecollectedinBaden-Württembergand13%inSchleswig-Holstein.MostotherGer-
manstateswererepresentedby6–10%oftheGermanspecimenseach.Thuringia(0.1%),
Hesse(1%)andRhineland-Palatinate(2%)werelesswellrepresented,aswerethecity-states
andthecomparativelysmallSaarland(1%).Fig1illustratesthesamplingpattern.
SMNKandexternalpartnersareresponsibleforandcontributed14%ofthespecimens,
SMNSandpartners10%,ZFMKandpartners56%,ZSMandpartners20%.
To date598morphologicalarachnidspecies(561spp.inspidersvs.37inharvestmen)in
269genera(246vs.23)and50families(44vs.6)couldbeintegrated.Settingthisintorelation
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DNABarcodingofSpidersandHarvestmeninGermany
Fig1.Geographicsamplingofarachnidspecimensunderlyingthepresentstudy.ImageproducedusingGPSVisualizer
(www.gpsvisualizer.com).
doi:10.1371/journal.pone.0162624.g001
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DNABarcodingofSpidersandHarvestmeninGermany
Fig2.Numberofspeciessampledperfamily(excludingfamiliesrepresentedonlyby1or2speciesinthisstudy).Numbersabovebarsare
percentagesshowingspeciescoverageforGermany,asderivedfromthechecklistsmentionedinthetext.Familynamesprefixedwith"O."belongto
Opiliones,allothersarespiderfamilies.
doi:10.1371/journal.pone.0162624.g002
withtheGermanchecklists[11,35],speciescoverageis57%forspidersand71%forharvest-
men.SpeciesnumbersareplottedforthemorefrequentfamiliesinFig2.
Thespeciesinthedatasetwererepresentedby6individualsonaverage(median:4).Almost
19%,i.e.112species,were'singletons'.With48specimens,Pardosalugubriswasthespecies
withmostindividuals;allotherspecieswererepresentedby30orfewerindividuals(seeFig3).
Mostindividuals(98%)werecollectedspecificallyforGBOLbetweentheyears2011and
2015.Theoldestspecimenprocessedinthisstudywascollectedin2003.
Collectingwasmostlydonebyhand,andmostspecimenswerekilledandpreserveddirectly
in96%or100%ethanol.7%ofthespecimenswereinitiallypreservedin70%water-dilutedeth-
anoland8%werecollectedinpropyleneglycol.Thelatterwasusedascapturefluidinpitfall
traps;soonafteridentification,tissueforDNAextractionwastransferredtoabsoluteethanol.
Allmaterialusedinthisstudyispropertyofthefederalstatesoftheinvolvedinstitutions.
Materialacquiredbytheseinstitutionsisonlyacceptedafteracheckthatitwascollectedin
compliancewithnationalandinternationallaws,regulationsandconventionsandthatthe
materialisfreefromthirdpartyrights.Furthermore,inordertobecomecertifiedasaGBOL
collector,itisrequiredtoaccepttheproject'sgeneraltermsandconditions,whichdemand
abidingbytheregulationsoftheConventiononBiologicalDiversityandnationallegislations.
Fieldworkpermitswereissuedbythefollowingauthorities:BayerischesStaatsministeriumfür
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DNABarcodingofSpidersandHarvestmeninGermany
Fig3.Numberofspecimenssampledperspecies.19%ofthespecieswere'singletons',whilethemedianrepresentationlayat4individualsper
species.
doi:10.1371/journal.pone.0162624.g003
UmweltundGesundheit,München;RegierungspräsidiumStuttgart;Regierungspräsidium
Karlsruhe;Struktur-undGenehmigungsdirektionKoblenz;KreisverwaltungRhein-Sieg-Kreis,
AmtfürNatur-undLandschaftsschutz;AmtfürUmwelt,Verbraucherschutz undLokale
Agenda,UntereLandschaftsbehörde,Bonn;NationalparkamtMüritz,Hohenzieritz;Biosphär-
enreservatsverwaltungNiedersächsischeElbtalaue,Hitzacker;NationalparkforstamtEifel,
Schleiden-Gemünd;AmtfürdasBiosphärenreservatSüdost-Rügen,Putbus;Landesamtfür
Umwelt,NaturschutzundGeologieMecklenburg-Vorpommern,Güstrow;Landesamtfür
Landwirtschaft,UmweltundländlicheRäumeSchleswig-Holstein,Flintbek;LandratKreis
HerzogtumLauenburg;LandratKreisRendsburg-Eckernförde,FachdiensteuntereNat-
urschutzbehörde.Thepermitscoverstateforests,publiclandandprotectedareasaswellasthe
fivespeciesofArachnidaprotectedinGermany:Arctosacinerea,Dolomedesfimbriatus,Dolo-
medesplantarius,EresuscinnabarinusandPhilaeuschrysops.
Altogether,over100collectorscontributedmaterial.Fielddataforallanalyzedspecimens
canbeaccessedinS1Table.Juvenilespecimensanalyzedbelongtotaxathatareeasilyidentifi-
ablealsoinjuvenilestage(e.g.basedoncoloration)orforwhichproblematic('look-alike')con-
genersdonotoccurinthestudyarea.Juvenilesthatclusteredconspicuouslyinthetreewere
removedfromthedataset.
Allmorphologicalspecimenvouchersandalsomolecularvouchers(DNAandoftentissue)
aredepositedatandareavailablefromthefollowingfourGermanpubliccollections(perma-
nentrepositories):StaatlichesMuseumfürNaturkundeKarlsruhe(SMNK),Staatliches
MuseumfürNaturkundeStuttgart(SMNS),ZoologischesForschungsmuseumAlexanderKoe-
nig(ZFMK),Bonn,ZoologischeStaatssammlungMünchen(ZSM).Allvouchernumbersare
giveninS1Table.ThevoucherIDsinS1Table aswellasthenamesinthetreesincludethe
institutionalcode,sothattheassociationofagivensampletooneoftheGBOLpartnerinsti-
tutescanbeeasilyestablished.
SequencedataareavailableonBOLD[69]viaDOIdx.doi.org/10.5883/DS-GBOLARA,and
onGenBank.Specimendatawillalsobeaccessible,alongsidespecimenimages,throughthe
GBOLportal(www.bolgermany.de).
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DNABarcodingofSpidersandHarvestmeninGermany
Molecularmethods
Analyseswereperformedmostlyinthreeseparatelaboratories:ZFMK,SMNS,andCanadian
CentreforDNABarcoding(CCDB)inGuelph.SMNSandZFMKusedtheirownfacilities(up
tothepointofsequencing),SMNKsampleswereprocessedatbothSMNSandZFMK,ZSM
samplesatCCDB.
Total genomicDNAwasusuallyisolatedfromlegs.Inverysmallspecimens(especially
manyLinyphiidae)atZFMKandSMNS,DNAwasextractednon-destructivelyfromwhole
specimenswhichwererecoveredafterlysis(cf.[70]).
AtZSM,singlelegswereremovedfromeachspecimenandsentin96welllysisplatesto
CCDBforstandardizedDNAextraction,PCRamplificationandbidirectionalSangersequenc-
ing.CCDBlabprotocolsareavailableunderwww.ccdb.ca/resources.php.
AtZFMKandSMNS,silica-basedmethodswereemployedtoextractDNA.SMNSfollowed
theprotocolby[71]withPallAcroPrepTM96filterplates(PallCorporation,PortWashington,
NY, USA),whileaQiagen(Hilden,Germany)BioSprint96magneticbeadextractorandcorre-
spondingkitswereusedatZFMK.
Polymerasechainreactionforthe5'partofthemitochondrialcytochromecoxidasesubunit
1(COI)genewascarriedout,atZFMK,intotalreactionmixesof20μl,including2μlofundi-
lutedDNAtemplate,0.8μlofeachprimer(10pmol/μl),andstandardamountsofthereagents
providedwiththe'MultiplexPCR'kitfromQiagen(Hilden,Germany).AtSMNS,PCRreac-
tionsof25μlvolumecontained4μlDNA,5unitsofTaq KAPA extrapolymerase(KAPA-
BIOYSYTEMS,Boston,USA),10μlof5xKAPA Taq extrabuffer,3μlofMgCl (25mM;both
2
solutionsprovidedbythemanufacturer),1μlofeachprimer(10pmol/μl),and1μlof10mM
dNTPmix(Bioline,Luckenwalde,Germany).
ThePCRprimersused(alsoforsequencing)aregiveninTable 1.
Thermalcyclingwasperformed,atZFMK,onAppliedBiosystems2720ThermalCyclers
(LifeTechnologies,Carlsbad,CA,USA),usingaPCRprogramwithtwocyclesets,asa
Table1. Listofprimersusedforamplificationandsequencingofthe5'partofthemitochondrialCOIgene.
Primername Sequence Publication Usedat
LCO1490 5'-GGTCAACAAATCATAAAGATATTGG Folmeretal.1994 SMNS,CCDBforZSM,ZFMK
HCO2198 5'-TAAACTTCAGGGTGACCAAAAAATCA Folmeretal.1994 SMNS,CCDBforZSM,ZFMK
LepF1 5'-ATTCAACCAATCATAAAGATATTGG Hebertetal.2004 CCDBforZSM
LepR1 5'-TAAACTTCTGGATGTCCAAAAAATCA Hebertetal.2004 CCDBforZSM
C_LepFolF cocktailofLepF1andLCO1490 www.boldystem.org/index.php/Public_Primer_PrimerSearch CCDBforZSM
C_LepFolR cocktailofLepR1andHCO2198 www.boldystem.org/index.php/Public_Primer_PrimerSearch CCDBforZSM
LCO1490-JJ 5'-CHACWAAYCATAAAGATATYGG Astrin&Stüben2008 ZFMK
HCO2198-JJ 5'-AWACTTCVGGRTGVCCAAARAATCA Astrin&Stüben2008 ZFMK
LCO1490-JJ2 5'-CHACWAAYCAYAARGAYATYGG new ZFMK
HCO2198-JJ2 5'-ANACTTCNGGRTGNCCAAARAATCA new ZFMK
LCO1490-JJ4a 5'-CNACNAAYCAYARRGAYATYGG new ZFMK
HCO2198-JJ4a 5'-AIACYTCNGGRTGICCAAARAATC new ZFMK
LCO1490-JJ4 5'-CIACIAAYCAYAARGAYATYGG new ZFMK
HCO2198-JJ4 5'-ANACTTCNGGRTGNCCAAARAATC new ZFMK
Species—alsomanyothersthanarachnids—withstronglymodified bindingsitescouldusuallybesuccessfullyamplifie datZFMKwithasetofnewly
(manually)designed,highlydegenerateprimers(mostoftenusingcombinationLCO1490-JJ2&HCO2198-JJ2).ThecombinationLCO1490-JJand
HCO2198-JJconstitutesthestandardsetofprimersusedatZFMK.ThestandardsetofprimersusedatCCDBforZSMwasthecombinationC_LepFolF
andC_LepFolR,acocktailconsistingoftheprimerslistedabove.
doi:10.1371/journal.pone.0162624.t001
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DNABarcodingofSpidersandHarvestmeninGermany
combinationofa'touchdown'anda'step-up'routine:firstcycleset(15repeats):35sdenatur-
ationat94°C,90sannealingat55°C(−1°Cpercycle)and90sextensionat72°C.Secondcycle
set(25repeats):35sdenaturationat94°C,90sannealingat45°C,and90sextensionat72°C.
AtSMNS,PCRamplificationwascarriedoutinaLabcyclerbySensoQuest(Göttingen,Ger-
many).PCRconditionswere:35cyclesof60sdenaturationat93°C,90sannealingat50°Cand
60sextensionat72°C.
PCRproductsweresubsequentlysentforbidirectionalSangersequencingtovariouscom-
panies:ZFMKtoBGI(HongKong,China)andMacrogen(Amsterdam,Netherlands),SMNS
toLGCGenomics(Berlin,Germany)in2013andfrom2014ontoGATCBiotech(Konstanz,
Germany).
DNAsequencealignmentwasperformedusingparallelizedMAFFTver.7.123[72].PAUP(cid:3)
ver.4.0b10[73]wasusedforp-distancetransformationsandforevaluatingbasecomposition
andinformationcontent.Statisticalparsimonynetworks[74]werecalculatedwiththeTCS
algorithm[75]inPopART(http://popart.otago.ac.nz).Statisticalevaluationofthedatawas
performedusingSPSS,R(boxplots),SpeciesIdentifierver.1.7.7–3[76](extractionof'splits'
and'lumps'),andthePerlscriptDiStats(intraspecificdistances,individualizeddataonclosest
speciespairsandonmostdistantcongeners).DiStatshasbeendevelopedforthisstudyandis
available,includingdocumentation,underGitHub(https://github.com/mptrsen/distats)and
throughtheZFMKhomepage(www.zfmk.de/en/research/research-centres-and-groups/
distats).ThescriptusesFASTA asinputformat,calculatesp-distancesorK2Pdistancesand
canbeparallelizedinordertoprocesslargedatasets.Itcanproducetwooutputfiles:atable
withstatisticsforeachspeciesandoptionallyalsothematrixofallpairwisedistancesinthe
dataset.Foranalignmentcontaining1000COIbarcodesequences,theanalysiswilltake
around6minuteswhenusingasinglethread(ona3.4GHzprocessor).DiStatshasanalgorith-
miccomplexity(O)ofapproximatelyO(n2),whichmeansthatruntimeincreasesexponen-
tiallywiththenumberofinputsequences(n).UsingmultipleCPUthreadsreducestherun
timebyafactorof1/c,wherecisthenumberofthreads.
PAUP wasalsousedforreconstructionofapheneticneighbor-joining(NJ;[77])treeasa
quickmolecularidentificationcheck.PhylogeneticreconstructionsusingMaximumLikelihood
(ML;[78])wereperformedwithRAxMLver.7.3.0[79].EvolutionarymodelselectionfortheML
analysiswasimplemented,usinghierarchicallikelihoodratiotesting,inModelGeneratorver.
0.85[80]andindicatedGTR+I+Γasthebest-fittingmodel[81].TheCOIdatasetwasparti-
tionedtotreat3rdcodonpositionsseparatelyfrom1stand2ndpositions.Theanalysiswasrun
for1milliongenerationsandincluded1000bootstrapreplicates.FortreerootingpurposesinNJ
andMLanalyses,wechoseamitesequencefromBOLDasoutgroup(seeS1Alignment).
Results
AverageCOIsequencelengthforthe3537sequenceswas650bp.To accommodatemany
slightlyshortersequences,whileavoidinggeneticdistanceartifacts,alignmentlengthwassetto
653bp.Sequencesshorterthan500bpwereexcludedfromtheanalysis.Theshortestincluded
sequencewascomposedof509residues.
Thedatasetcomprised2099distincthaplotypes,meaningthat1438sequenceswerenon-
unique.
Amongnucleotides,therewasacompositionalbiastowardsAT: 67.5%,whichiscloseto
levelspreviouslyreportedforspiders(e.g.[4,82,83]).Indetail,overallbasecompositionwas:A
25.3,C13.3,G19.2,T42.2%.
Altogether,5,572,791pairwisedistanceswerecomputedforspiders;ofthese,17,867were
intraspecificdistances.ForOpiliones,therewere19,503pairwisedistancesofwhich896were
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DNABarcodingofSpidersandHarvestmeninGermany
Table2. Estimatorsusedtocharacterizegeneticdistancestructureinthedataset.
[%] intraspecific interspecific
median mean range 95thperc. median mean range 5thperc.
Araneae 0.3 0.7 0.0–10.1 2.5 17.5 17.4 0*-28.2 13.6
Opiliones 0.2 1.3 0.0–8.9 8.1 19.3 19.4 7.0–30.1 13.6
Aran.K2P 0.3 0.7 0.0–11 2.6 20.0 20.0 0.0–35.6 15.0
Opil.K2P 0.2 1.3 0.0–9.5 8.7 22.6 22.7 7.5–38.6 15.1
Theuppertworowsindicateuncorrecteddistancesforspidersandharvestmen,respectively,whilethethirdandfourthrowsgiveK2Pdistances(asrequired
forabarcodedatarelease).Medianandmeandistancesaregivenforbothintraspecific andinterspecific distances,alongwiththerangebetweenthe
smallestandlargestobservationintherespectivedatacategory.*:Therewerecasesofsharedhaplotypesamongspecies,seetext.
doi:10.1371/journal.pone.0162624.t002
distancesbetweenconspecificspecimens.Table2givesanoverviewofintra-andinterspecificdis-
tances,separatedbyorder.Table3summarizesdistancesforallclosestspeciespairsandforthe
mostdistantcongenericpairs;S2andS3Tablesillustratetheseinmoredetail,givingindividual
statisticsbytaxon.S2Table(spiders)andS3Table(harvestmen)furthermoreindicateintraspecific
distancerangesandcentraltendenciesforallanalyzedspecies.S4Tableindividuallyliststhehigh-
estintraspecificdistancesinthedataset,whileS5Tablegivesthelowestinterspecificdistances.
Therangecoveredbyintraspecific(p-)distanceswassimilarforspiders(0–10%)andhar-
vestmen(0–9%),withanarithmeticmeanof0.7%inspidersand1.3%inharvestmen.The
influenceofhighoutlierswasstrongerinthecomparativelysmallharvestmandataset(median
at0.2%vs.meanat1.3%),causedmostlybythedeepsplitswithinMitopusmorio(Fabricius,
1779)andPhalangiumopilioLinnaeus,1758(bothdiscussedbelow),butalsoinNemastoma
lugubre(Müller,1776)(seeS4Table).
Theinterspecificdistancerangevariedforthetwoarachnidorders:0.0–28%inspiders,
7–30%inharvestmen.Infourcases,haplotypesweresharedamongnominalspiderspecies
(seeS5Table,alldiscussedbelow):EnoplognathalatimanaHippa&Oksala,1982/E.ovata
(Clerck,1757);Pardosalugubris(Walckenaer, 1802)/P.saltansTöpfer-Hofmann, 2000;Tibel-
lusmaritimus(Menge,1875)/T.oblongus(Walckenaer, 1802);Xysticusaudax(Schrank,
1803)/X.cristatus(Clerck,1757).Interspecificarithmeticmeanswere17%forspiders,19%
forharvestmen.
Table3. Statisticsforclosestspeciespairsandmostdistantcongenericpairs.
[%] closestspeciespairs mostdistantcongenerpairs
range median(ofalldist.forspeciespairs) range median(oflargestdistances)
Araneae 0.0*-20.1(22.0) 9.2 1.8–20.2 11.8
Opiliones 7.0–21.8(22.8) 13.8 12.6–18.8 14.9
AraneaeK2P 0.0–21.0(28.6) 9.9 1.9–23.8 12.8
OpilionesK2P 7.5–24.0(29.0) 15.5 13.7–22.0 16.5
Theuppertworowsindicateuncorrecteddistancesforspidersandharvestmen,respectively,whilethethirdandfourthrowsgiveK2Pdistances(asrequired
forabarcodedatarelease).Therangefortheclosestspeciespairsindicatestheminimumandmaximumamongallsmallestpairwisedistancesbetween
closestspeciespairs.Ifaclosestspeciespairisrepresentedbyseveralindividuals,theremaybelargerdistancesaswell:themaximalclosestspecies
distanceintherespectivedatasetisindicatedinparentheses.Whilefortheclosestspeciespairsnoclassific atorial(genus)backgroundinformationwas
used,thelasttwocolumnsinthistableorientthemselvesatdistancesfromrepresentativeswithinthesamegenus(butdifferentspecies).Therangeforthe
mostdistantcongenerpairsextractstheextremesamongthesemaximalpairwisedistancesbetweenfarthestcongenericspeciespairs.Informationonthe
individualclosestspeciespairsandontherespectivemostdistantcongenericspeciespairsisgiveninS2TableforspidersandS3Tableforharvestmen.
*Therewerecasesofsharedhaplotypesamongspecies,seetext.
doi:10.1371/journal.pone.0162624.t003
PLOSONE|DOI:10.1371/journal.pone.0162624 September28,2016 9/24
DNABarcodingofSpidersandHarvestmeninGermany
Fig4.Boxplotofp-distancesfortheorderAraneae.Sortedbydistancecategory:betweenspecimensofdifferentgenera(allogeneric),between
specimensbelongingtodifferentspecies,buttothesamegenus(congeneric),andbetweenspecimensthatbelongtothesamespecies(intraspecific).
Boxesindicateinterquartilerange(IQR:betweenupper[Q3]andlower[Q1]quartile).Blackbarsdesignatemedians,whiskersindicatevalueswithin
1.5×IQRbeneathQ1or1.5×aboveQ3.Circlesdepictoutliers(aboveorbelow1.5×IQR).
doi:10.1371/journal.pone.0162624.g004
Meanintraspecificdistancesvariedconsiderablyamongfamilies.Forthebest-represented
spiderfamilies,theselaybetween0.4%(Agelenidae,Lycosidae,Philodromidae)and1.0%(Tet-
ragnathidae).Clubionidae,LinyphiidaeandTheridiidaehadmeanintraspecificdistancesof
0.6%,Gnaphosidae0.7%,Araneidae0.8%,SalticidaeandThomisidae0.9%.
Table 2andtheboxplots(Figs4and5)indicatethatauniversalbarcodinggapisabsent
fromthedataset.However,mostofthespeciesseparatewell;whenignoringthe5%most
extremeoutliers(ahypotheticalscenarionotsurpassingtheusualsignificancethreshold),the
barcodinggapforharvestmenwouldspan5.5%andforspiderseven11%(seeTable 2).The
mediandistanceforclosestspeciespairswas9%inspidersand13%inharvestmen.
ApheneticreconstructionusingNeighborJoining(NJ;S1Fig)andaphylogeneticrecon-
structionusingMaximumLikelihood(ML;S2Fig)deliveredtreesinwhichthespeciesover-
whelminglyformedmonophyleticclusters(butseediscussion–severalofthecaseswith
conspicuousdistanceswerealsorecoveredasparaphyleticandpolyphyletic).MLbootstrap
analysispredominantlyindicatedveryhighsupportforspecies-levelnodes,butdidusuallynot
allowmuchinsightintodeepertreetopology.
Discussion
ManypreviousstudiesusingCOIhaveshownthatspeciesdifferentiationviaDNAbarcoding
isgenerallyfeasibleandpromisinginarachnids(mostlyspiders:e.g.[2–4,46,47,57–66]–butsee
PLOSONE|DOI:10.1371/journal.pone.0162624 September28,2016 10/24