throughput sequencing offers insight into mechanisms of resource partitioning in cryptic bat species

15 Pages·2013·0.63 MB·English

High-throughput sequencing offers insight into mechanisms
of resource partitioning in cryptic bat species
OrlyRazgour1,ElizabethL.Clare1,MattR.K.Zeale1,JuliaHanmer2,IdaBærholmSchnell3,
MortenRasmussen3,ThomasP.Gilbert3 &GarethJones1
1SchoolofBiologicalSciences,UniversityofBristol,BristolBS81UG,UK
2BatConservationTrust,QuadrantHouse,250KenningtonLane,LondonSE115RD,UK
3CentreforGeoGenetics,NaturalHistoryMuseumofDenmark,UniversityofCopenhagen,1350Copenhagen,Denmark
Keywords Abstract
Diet,interspecificcompetition,molecular
scatology,nextgenerationsequencing, Sympatriccrypticspecies,characterizedbylowmorphologicaldifferentiation,pose
Plecotus. a challenge to understanding the role of interspecific competition in structuring
ecologicalcommunities.Weusedtraditional(morphological)andnovelmolecular
Correspondence methodsofdietanalysistostudythedietoftwocrypticbatspeciesthataresympatric
OrlyRazgour,SchoolofBiologicalSciences,
insouthernEngland(PlecotusaustriacusandP.auritus)(Fig.1).UsingRocheFLX
UniversityofBristol,WoodlandRoad,Bristol
454(Roche,Basel,CH)high-throughputsequencing(HTS)anduniquelytagged
BS81UG,UK.Tel:[phone];
generic arthropod primers, we identified 142 prey Molecular Operational Taxo-
Fax:[phone];
E-mail:[email protected] nomicUnits(MOTUs)inthedietofthecrypticbats,60%ofwhichwereassigned
toalikelyspeciesorgenus.Thefindingsfromthemolecularstudysupportedthe
FundedbytheHon.VincentWeirthroughthe resultsofmicroscopicanalysesinshowingthatthedietsofbothspeciesweredom-
BatConservationTrust,UK,andfromthe inatedbylepidopterans.However,HTSprovidedasufficientlyhighresolutionof
DanishNationalResearchFoundation
prey identification to determine fine-scale differences in resource use. Although
GeoGeneticsgrant(MTPG).ELCwasfunded
bothbatspeciesappearedtohaveageneralistdiet,eared-mothsfrom thefamily
throughaNaturalSciencesandEngineering
Noctuidaewerethemainpreyconsumed.Interspecificnicheoverlapwasgreater
ResearchCouncilofCanadapost-doctoral
fellowship. thanexpectedbychance(Ojk=0.72,P<0.001)duetooverlapintheconsumption
ofthemorecommonpreyspecies.Yet,habitatassociationsofnongeneralistprey
Received:08September2011;Accepted:13 species found in the diets corresponded to those of their respective bat predator
September2011 (grasslands for P. austriacus, and woodland for P. auritus). Overlap in common
dietaryresourceusecombinedwithdifferentialspecialistpreyhabitatassociations
EcologyandEvolution2011;1(4):556–570
suggests that habitat partitioning is the primary mechanism of coexistence. The
performance of HTS is discussed in relation to previous methods of molecular
doi:10.1002/ece3.49
andmorphologicaldietanalysis.Byenablingspecies-levelidentificationofdietary
components, the application of DNA sequencing to diet analysis allows a more
comprehensivecomparisonofthedietofsympatriccrypticspecies,andtherefore
canbeanimportanttoolfordeterminingfine-scalemechanismsofcoexistence.
titionforlimitingresources(Chesson2000).Thereisample
Introduction
evidencetosupportbothecologicalcharacterdisplacement
Interspecificcompetitionisanimportantmechanismstruc- (species occupying different niches in sympatry; reviewed
turing ecological communities (Schoener 1983), outweigh- in Dayan and Simberloff 2005) and limits to the similarity
ing the effect of positive mutualistic interactions (Alexan- ofcoexistingcompetitors(e.g.Kingstonetal.2000).More-
drouetal.2011),andpromotingincreasedspeciationrates over resource partitioning has been identified as a mecha-
(Thierryetal.2011).Stablecoexistenceinthefaceofinter- nismthatfacilitatescoexistenceinavarietyofanimalcom-
specificcompetitionistheresultofecologicalnichedifferen- munities (reviewed in Schoener 1974). Nevertheless, some
tiation,wherebyphenotypiccharacterdivergencepromotes studieshaveshownthatecologicallysimilarspeciescanco-
differentialresourceuse,thusreducingtheeffectofcompe- exist through neutral processes that promote phenotypic
556 (cid:2)c 2011TheAuthors.EcologyandEvolutionpublishedbyBlackwellPublishingLtd.
ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution-NonCommercialLicense,whichpermits
use,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycitedandisnotusedforcommercial
purposes.
O.Razgouretal. MolecularDietAnalysisofCrypticSpecies
character convergence rather than divergence (Leibold and sis(e.g.,fecalorstomachcontentanalysis),polymerasechain
McPeek2006). reaction(PCR)amplificationandDNAsequencingofprey
Traditional views of niche-based species coexistence are remainsfrompredatorfecalsamplesallowsidentificationto
challenged by the presence of sympatric, morphologically the species level of both soft- and hard-bodied consumed
similar,butgeneticallyisolated,species(crypticspecies)that prey(Symondson2002).DespitethehighpotentialofDNA
do not appear to differ sufficiently in their morphology to sequencingtoaididentificationatfinerresolutionthanthe
allow niche differentiation (Wellborn and Cothran 2007). ordinallevelforimportantinsectprey,suchasLepidoptera
However, morphological similarity may not be a sufficient (Whitakeretal.2009),thistechniquehasonlyrecentlybeen
indication of ecological similarity, and even small differ- applied to study the diet of insectivorous bats. However,
ences can influence access to resources (Saunders and Bar- molecular diet studies to date have focused on individual
clay 1992). Cryptic species appear to be common in bats species (Clare et al. 2009, 2011b; Zeale et al. 2011) or on
(order Chiroptera) (Clare 2011; Clare et al. 2011a), and in sympatricspeciesthatarenotcloserelatives(Bohmannetal.
particular among insectivorous bats, because differences in 2011),ratherthanoncrypticspecies,wherefinerresolution
their sensory abilities, in the form of ultrasonic echoloca- ofpreytypesmaybefundamentaltodeciphermechanisms
tion calls, are not readily distinguished by humans. More- ofresourcepartitioning.
over,echolocationgivesbatsanaddeddimensionforniche We applied traditional (morphological) and novel next
separationthatmaybelessconservedinitsevolutionthan generation sequencing approaches to study the diet of two
morphology per se (Jones 1997), and therefore may pro- sympatriccrypticsisterspeciesinsouthernEngland,thegray
moteecologicaldivergencepotentiallyleadingtospeciation andbrownlong-earedbats,Plecotusaustriacus andP.auri-
(KingstonandRossiter2004).Becauseforaginghabitatuse tus.Plecotusaustriacus(Fig.1)isoneoftherarestmammals
andpreyselectionininsectivorousbatsarecloselylinkedto inBritain,withaprebreedingpopulationestimatedat1000
wingmorphology(AldridgeandRauthenback1987;Norberg individuals,andisrestrictedtosouthernEngland,whileP.au-
andRayner1987)andecholocationcallstructure(Jonesand ritusismorecommonandwidespreadthroughouttheBritish
Rydell2003),evenmoderatedifferencesinthesefeaturesmay Isles(Harrisetal.1995).Thelong-earedbatgenuscontains
allowspeciestopartitionresources. at least 19 distinct cryptic species, nearly all of which were
Despite their morphological similarity, sympatric cryp- onlyidentifiedinthepastdecadebasedonmolecularstudies
ticbatspeciesmayshowpronouncedspatialsegregationof (Spitzenbergeret al. 2006;Mayer et al. 2007). Thoughrec-
primaryforaginghabitats.Forexample,partitioningoffor- ognized as separate species in the 1960s (Corbet 1964), P.
aginghabitat,intheabsenceofapparentdifferencesinwing austriacusandP.auritusshowhighoverlapinmostmorpho-
morphology(Jones1997),wasidentifiedamongtherecently logical characteristics (Ashrafi et al. 2010), including their
separated cryptic Pipistrellus species, whereby P. pygmaeus wing morphology (Sevcik 2003) and echolocation call pa-
selects riparian habitats, while P. pipistrellus prefers a wide rameters(RussoandJones2002).Significantdifferencesin
range of habitats including deciduous woodlands and pas- theshapeofthebaculum(Dietzetal.2009)maypartlyac-
ture(Davidson–Wattsetal.2006;NichollsandRacey2006). countforreproductiveisolationbetweenthesecrypticspecies
Differencesinecholocationcallstructurecanfacilitateniche (PattersonandThaeler1982).Thetwobatsaresympatricin
differentiationinsympatricbatspeciesbyaffectingthedetec- southern England and parts of Europe. However, while P.
tiondistancesofpreyofvarioussizes(KingstonandRossiter austriacusisprimarilyasouthernEuropeanspecies,P.auri-
2004),aswellascontributingtoforagingperformanceun- tusisabundantincentralandnorthernEurope,butconfined
derdifferentlevelsofhabitatclutter(SiemersandSchnitzler tomountainousareaswithcoolerclimatesinsouthernEu-
2004).Forbatsforaginginthesamehabitat,sensoryadap- rope(Spitzenbergeretal.2006).Similaritiesinmorphology
tationsmayallowaccesstodifferentprey,thusreducingre- andecholocationcallssuggestthatthesespeciesshareseveral
sourcecompetition(SiemersandSwift2006).However,the niche dimensions, and therefore studying their patterns of
roleofinterspecificcompetitionindrivingtrophicresource resourceusemayrevealimportantfine-scalemechanismsof
partitioninginbatsisstilldebated(Husar1976;Arlettazetal. coexistence(Tokeshi1999).
1997;SchoemanandJacobs2011). Our objectives were to assess the ability of PCR-coupled
Theadventoffinerresolutionmethodsofdietanalysismay nextgeneration(RocheFLX,Roche,Basel,CH)sequencing
offeraclearerpictureoftrophicresourcepartitioningamong toidentifyfine-scaledifferencesintrophicecologyfromfe-
coexisting species. Indeed some studies that failed to iden- calsamples,andcomparetheperformanceofthistechnique
tifyresourcepartitioningamongmorphologicallysimilarbat withpreviousmolecular(PCR-coupledcloningandfragment
species suggest that finer scale differences in microhabitat targeting)andnongeneticmethodsofdietanalysis.Wehy-
use or finer resolution diet analysis may reduce the extent pothesizedthatdespiteshowingfewphenotypicdifferences,
ofresourceoverlap(JacobsandBarclay2009;Schoemanand crypticspeciesareabletocoexistduetodifferencesintheir
Jacobs2011).Contrarytotraditionalmethodsofdietanaly- ecology.Therefore,wepredictedthat(1)coexistenceamong
(cid:2)c 2011TheAuthors.EcologyandEvolutionpublishedbyBlackwellPublishingLtd. 557
MolecularDietAnalysisofCrypticSpecies O.Razgouretal.
of30min.Feceswereimmediatelystoredin100%ethanol
inindividualtubes,andfrozenwithin12hofcollection.For
themorphologicaldietanalysis,feceswerecollectedoncea
monthbetweenAprilandOctober2009fromseedcollection
traysplacedonthefloorofaP.austriacusmaternitycolony
inDevon(50◦3(cid:4)N,3◦3(cid:4)W).
DNAextractionandPCRamplification
DNAwasextractedfromfecesusingtheQIAampDNAstool
extraction kit (Qiagen, Valencia, CA) following the modi-
fications described in Zeale et al. (2011). A short (157 bp
excludingprimer)regionofthecytochromec oxidasesub-
unitI(COI)mitochondrialDNAgenewassubsequentlyPCR
amplifiedfromeachDNAextract.Weusedgenericarthropod
primerswithawidetaxonomiccoveragethatincludes13in-
sectandarachnidorderscommonlyfoundininsectivorous
bat diets (ZBJ-ArtF1c and ZBJ-ArtR2c; Zeale et al. 2011).
Theprimersweremodifiedinto5(cid:4)-tagged“fusionprimers,”
inordertoenableRocheFLXsequencingofpoolsoftheam-
plicons,andsubsequentbioinformaticsortingintooriginal
PCR(Binladenetal.2007).Post-PCRamplification,theam-
pliconswerepurified,quantifiedbyreal-timePCR(qPCR)
(Meyeretal.2007),andthenpooledbyspeciesatequimo-
lar ratio. Each pool of amplicons was deep sequenced on
one-eighthofaRocheGS-FLXplatformusingTitaniumse-
quencingchemistry.PCRreactionconditions,cycleprogram,
andsequencingproceduresfollowedBohmannetal.(2011)
andareprovidedinSupportingInformation2.
AnalysisofFLXsequencingdata
Weusedaconservativeapproachdiscardingsequencesthat
didnotmatchprimersexactly,fragmentsthatweretooshort,
andallsingletonsequences(uniquesequencesthatwereonly
Figure1. (a)Thegreylong-earedbat,Plecotusaustriacus,and(b)the
brownlong-earedbat,Plecotusauritus. presentonce)tominimizetheeffectofsequencingerrors.Se-
quenceswerealignedusingtheClustalWalgorithminBioEdit
P.austriacusandP.auritusismediatedviafine-scalemecha- (T. Hall, http://www.mbio.ncsu.edu/bioedit/bioedit.html),
nismsofresourcepartitioning;(2)theextentofdietaryniche and the nucleotide and amino acid alignmentwas checked
overlapanddietselectivityareaffectedbyvariationinprey byhandwithreferencetoknownarthropodsequences.All
availability;and(3)thedietofthetwobatspeciescorresponds sequences were collapsed into Molecular Operational Tax-
topatternsofforaginghabitatselection. onomic Units (MOTUs), approximating species, using the
softwarejMOTU(Jonesetal.2011,https://www.nematodes.
Methods org/bioinformatics/jMOTU/). This approach is useful for
describing niche breadth and overlap when identification
Samplecollection
of sequences is limited by an incomplete reference dataset
Wecollectedfecalsamplesdirectlyfrom30P.austriacus(rep- (Clare et al. 2011b). The MOTU assignment was tested at
resentingapproximately3%oftheprebreedingBritishpop- aseriesofthresholdscorrespondingto0–10%sequencedi-
ulation[Harrisetal.1995])and30P.auritusbatscaughtat vergence and the resulting assignments were graphed. For
sitesinsouthernEngland(Devon,IsleofWight,andSomer- furtheranalysis,weselectedathreshold(2%)basedonthe
set).TheP.austriacussampleswerecollectedbetweenApril infliction point of the graph of distribution of MOTUs re-
2009andOctober2010,andtheP.auritussamplesbetween covered,thatis,thepercentofsequencedifferenceswherethe
June and October 2010 (Supporting Information 1). Bats graphreachedanasymptoteandnewMOTUswerenotbe-
were caughtunder license eitherin roosts, bat boxes, or in ingrecognizedbutexistingMOTUshadnotbeencollapsed.
woodlands,andheldinseparateholdingbagsforamaximum Thisvalueisbiologicallymeaningfulandiswellwithinex-
558 (cid:2)c 2011TheAuthors.EcologyandEvolutionpublishedbyBlackwellPublishingLtd.
O.Razgouretal. MolecularDietAnalysisofCrypticSpecies
pectedvariationatthespecieslevelinsurveyedinsects(see WeusedPianka’s(1973)measureofnicheoverlap(Equa-
Clare et al. 2011b for a discussion of MOTU divergences). tion 3) to quantify dietary resource overlap at the MOTU
Representative sequences from each MOTU (the three levelbetweenthetwobatspeciesandwithineachbatspecies
mostcommonsequenceswhenidenticalsequencesarecol- between the two main collection sites (Devon and Isle of
lapsed into haplotypes) were compared to reference se- Wight). Null models were used to test whether the extent
quences using the Barcode of Life Data System (BOLD, of niche overlap is greater than expected by chance, and
www.barcodinglife.org)identificationengine.Weusedacon- determine the effect of season and sex on dietary resource
servativethresholdof>98.5%sequencesimilarityforspecies use.Wegenerate10,000simulatedmatricesofrandomized
identificationbasedonmeansequencedivergencevalueses- MOTUdietcomposition,usingthesoftwareEcoSim(version
timatedbyZealeetal.(2011)fortheamplifiedCOIregion. 7;http://grayentsminger.com/ecosim.htm)withRandomisa-
PhylogeneticapproachesemployedbyClareetal.(2009, tionAlgorithm3,andcomparedobservedandrandomlysim-
2011b)tocomplimentdistance-basedspeciesidentification ulatedextentsofnicheoverlap.Bonferronicorrectionswere
were not possible due to the short length of the sequences appliedtoretainsignificancevalueatP <0.05,resultingin
(157bp).Moreover,shortsequencelengthmeantthatsome significancelevelsetatP<0.017.
MOTUsgave>98.5%matchtomorethanonespecies,some (cid:2)n
of which belonged to different genera or even families. To p p
ij ik
overcome these obstacles, we used the following criteria to
createthreeidentificationconfidencelevels: Ojk = (cid:3)(cid:4)(cid:4)(cid:2)ni (cid:2)n , (3).
(cid:5)
1. Solid match (>98.5%) to one species—species-level as- pi2j pi2k
signment,ormatch(>98.5%)tomorethanonespecies,all i i
belongingtothesamegenus—genus-levelassignment. where P is the proportion that resource i is of the total
ij
2. Match(>98.5%)tomorethanonespeciesbelongingto resourcesusedbyspeciesj;P istheproportionthatresource
ik
different genera, only one of which was a U.K. species— iisofthetotalresourcesusedbyspeciesk;andnisthetotal
species-levelassignmenttoU.K.species. numberofresourcestates(totalnumberofMOTUs).
3. Match(>98%)toseveralspeciesofdifferentgenerawithin Dietary prey composition was compared within each
thesamefamilyortoreferencesequencesonlyidentifiedto bat species between seasons using nonparametric tests. We
thefamily-level—familylevelassignment. compared the proportion of Lepidoptera MOTUs within
eachfecalsamplebetweenthespring(April–May),summer
Dietanalysis
(June–August),andautumn(Sep–Oct)forP.austriacus,and
ThedietofP.austriacusandP.aurituswasquantifiedbased between the summer and autumn for P. auritus. Statistical
onthepercentfrequencyofoccurrence(%FO)ofpreyorders analyseswerecarriedoutinPASWStatistics18.
(thenumberoffecescontaininganorderdividedbythetotal Arthropodpreyhabitatassociationsandpresenceinsouth-
occurrencesofallorders).Allotheranalyseswerecarriedout ernEnglandweredeterminedbasedonpreviouslypublished
attheMOTU(species)level. information (Heath and Emmet 1983; Waring et al. 2003;
Theextentofdietaryspecializationanddiversitywasde- Chinery 2005; Manley 2008; Chandler 2010). Arthropod
terminedattheMOTUlevelusingthestandardizedLevins’ species commonly found in a range of habitat types were
measureofnichebreadth(Equation1)andShannon’sdiver- classifiedashabitatgeneralists,whilespeciesthatwereexclu-
sityindex(Equation2). sivelyassociatedwitheithergrasslandandopenhabitatsor
woodlandhabitatswereclassifiedasgrasslandandwoodland
1
B = (cid:2) . (1) specialists,respectively.
p2
i
B −1 Morphologicaldietanalysis
Standardizedas: B =
A n−1 Primergenerality
where B is Levins’s measure, p is the proportion of fecal
i Inordertotestforprimeramplificationbiasesagainstcertain
samplesinwhichMOTUiwasfound,andnisthenumber
prey orders, we used traditional microscopic fecal analysis
ofpossibleMOTUsinthediet.
methods (Whitaker et al. 2009) to determine prey compo-
(cid:2)s sition in all fecal samples analyzed genetically. We looked
H(cid:4) =− piLn(pi), (2) for the presence of prey orders that were absent from the
i=1 molecularstudyandfordifferencesinpreyordercomposi-
wherep istheproportionoffecalsamplesinwhichMOTU tionintermsofpercentfrequencyofoccurrence(%FO)in
i
iwasfound. diet.
(cid:2)c 2011TheAuthors.EcologyandEvolutionpublishedbyBlackwellPublishingLtd. 559
MolecularDietAnalysisofCrypticSpecies O.Razgouretal.
Traditionalmorphologicaldietanalysis Dietcompositionandspecialization
Wecomparedtheresultsofthehigh-throughputsequencing ThedietofP.austriacuscontained90MOTUs,whilethatof
(HTS)analysistoapreviousmicroscopicfecalanalysisstudy P. auritus contained 68. Analysis carried out at the MOTU
of the diet of P. auritus from Somerset, southern England level showed that both bat species had relatively narrow
(Hollyfield1993).However,becausetherewerenoprevious niches (Levins’ measure: B = 0.21, for both species) but
A
studies of the diet of P. austriacus in Britain, we analyzed highdietarydiversity(Shannondiversityindex:P.austriacus
fecescollectedfromamaternitycolonyinDevon,usingtra- H=4.1,P.auritusH=3.9).Weidentifiedsixpreyordersin
ditionalmicroscopicfecalanalysismethods(Shieletal.1997; thedietofP.austriacus (Lepidoptera,Diptera,Neuroptera,
Whitaker et al. 2009). We randomly selected 30 droppings Hemiptera,Trichoptera,andIsopoda)andfiveordersinthe
foranalysisfromeachmonthlycollection,andteasedthem dietofP.auritus(Lepidoptera,Diptera,Coleoptera,Araneae,
apartunderadissectionmicroscope,pickingoutallrecog- and Isopoda). The diet of both bat species comprised pri-
nizablearthropodfragmentsforidentificationwithreference marilyofpreyfromtheordersLepidopteraandDiptera,with
slidesandidentificationguides(Shieletal.1997;M.James Lepidopteraaccountingfor66.7%ofthedietofP.austriacus
pers.comm.).ThedietsofP.austriacus andP.auritus were and64.7%ofthedietofP.auritus (Fig.2).Thesameprey
quantifiedbasedonpercentfrequencyofoccurrenceofprey orderswereidentifiedatsimilarproportionsinthemorpho-
orders(%FO). logicalanalysisofpreyremainsingeneticallyanalyzedfecal
samples, with the exception of Coleoptera, which was only
foundinthemorphologicalanalysisoftheP.austriacussam-
ples. However, it is important to note that Coleoptera was
only identified in one P. austriacus fecal sample, and com-
Results
prisedlessthan5%ofthevolumeofthatsample.
Themajorityofthelepidopteranspeciesidentifiedinthe
Of the 30 fecal samples collected for each bat species, 28
diet of both bat species were eared-moths of the family
of the P. austriacus (93%) and 24 of the P. auritus extrac-
Noctuidae (proportion of Lepidoptera MOTUs: P. ausitra-
tions (80%) produced PCR amplicons, and were therefore
cus71%,P.auritus83.3%;Fig.3).SpeciesofthefamilyTip-
included in subsequent analyses. The sequencing run gen-
ulidae were the most common dipterans identified in the
erated 100,508 and 73,606 sequences containing correctly
diet of P. austriacus (76%), while species of the dipteran
barcodedprimersfortheP.austriacusandP.auritussamples,
family Muscidae were more common in the diet of P. au-
respectively.Subsequentfilteringofthedatatoreturnonly
ritus (65%). The most common prey species in the diet of
high-quality,identifiablearthropodsequencesandremoval
P.austriacuswereN.pronuba,Autographagamma,Hepialus
ofsingletonsequencesreducedtheseto91,476and63,715,
sp.,Apameamonoglypha,andTipulaoleracea,whilethemost
respectively.
commonpreyspeciesinthedietofP.aurituswereN.pronuba,
Thehigh-qualityarthropodsequenceswerecollapsedinto
Xestiaxanthographa,Hepialussylvina,andPolieteslardarius
142 MOTUs (using a 2% sequence divergence threshold),
(Table1).
belongingtothreespeciesofarachnids,twospeciesofCrus-
Thedietcompositionattheordinallevelofbothbatspecies
tacea, and at least six orders of insects. We identified 60%
differedbetweentheseasons.Plecotusaustriacus consumed
of the MOTUs to the likely species or genus (69% were
higherproportionofLepidopterainsummerthaninspring
solidmatches—confidencelevel1,while31%matchedmore
(Kruskal–Wallistest:H=7.72,N=14,8,P=0.002;Fig.4),
than one species, only one of which was a U.K. species—
while P. auritus consumed more Lepidoptera in summer
confidence level 2), and 16% to the family level (Table 1).
than in autumn (Mann–Whitney test: U = 33, N = 10,
TheremainingMOTUs(24%)couldnotbeidentifiedconfi-
14,P=0.014;SupportingInformation3).
dentlybasedonourclassificationschemeandweretherefore
leftasunknown.InLepidoptera,wherethereferencedataset
isrelativelycomplete,59discreteMOTUswereidentifiedas Dietarynicheoverlap
52separatespecies;suggestingthatusingcurrenttaxonomic
We found little evidence for dietary resource partitioning
classificationinBOLD,MOTUsslightlyoverestimatedspecies
amongthesecrypticbatspecies.Interspecificnicheoverlap,
richness(by12%).
measured based on MOTUs, was significantly higher than
ThemajorityofMOTUs(80%)wereconsumedbyasingle expected by chance (O = 0.72, P < 0.001) and higher
jk
individual, though the most common prey species in the than intraspecific niche overlap (P. austriacus: O = 0.63,
jk
diet of both species, Noctua pronuba, was consumed by 19 P = 0.009; P. auritus: O = 0.31, not significant) (Fig. 5).
jk
P. austriacus and 13 P. auritus bats. Number of prey taxa
Nicheoverlapbetweenbatspecieswasgreaterinthesummer
±
(MOTUs)perdroppingrangedbetween1and17(means (O =0.62,P =0.012)thantheautumn(O =0.48,not
± ± jk jk
SD:P.austriacus,5.3 4;P.auritus,4.2 3).
significant).
560 (cid:2)c 2011TheAuthors.EcologyandEvolutionpublishedbyBlackwellPublishingLtd.
O.Razgouretal. MolecularDietAnalysisofCrypticSpecies
Table1. Listofarthropodpreyidentifiedinthefecesof28Plecotusaustriacusand24P.auritusfromsouthernEngland,includingthenumberof
individualsfromeachbatspeciesthatconsumedthepreytaxa.ConfidencelevelsarebasedontheBOLDidentificationsystem,wherebyconfidence
level1=solidmatchtoonespeciesorgenus(>98.5%);level2=matchtomorethanonespecies(>98.5%),onlyoneofwhichwasaU.K.species;
andlevel3=match>98%toseveralspeciesofdifferentgenera,ortoreferencesequencesonlyidentifiedtothefamilylevel.
Order Family Species P.austriacus P.auritus Confidencelevel
Lepidoptera Aractiidae Unknown 2 0 3
Diaphoramendica 1 0 1
Seirarctiasp. 1 0 1
Spilosomaluteum 3 0 1
Crambidae Agriphilatristella 2 0 1
Chrysoteuchiaculmella 2 0 2
Crambusperlella 1 0 2
Elachistidae Depressariadaucella 1 0 1
Semioscopissp. 2 0 1
Gelechiidae Carpatolechiadecorella 1 2 1
Geometridae Gymnoscelisrufifasciata 1 0 2
Hepialidae Hepialussp. 6 2 2
Hepialussylvina 6 3 1
Noctuidae Unknown 6 3 3
Acronictaalni 0 1 1
Agrocholalitura 0 1 1
Agrocholalota 0 1 1
Agrocholalychnidis 0 1 2
Agrocholamacilenta 0 1 1
Agrotisexclamationis 4 2 1
Agrotisipsilon 2 0 2
Agrotissegetum 1 0 1
Agrotisputa 1 0 1
Allophyesoxyacanthae 1 2 2
Apameacrenata 0 1 1
Apameaepomidion 0 1 1
Apameamonoglypha 6 0 2
Apameasp. 1 0 1
Autographagamma 11 2 1
Charanycatrigrammica 1 0 2
Conistrasp. 0 1 2
Diarsiasp. 0 2 2
Hoplodrinaambigua 1 0 1
Hydraeciamicacea 1 0 2
Hydraeciasp. 1 0 1
Hypenaproboscidalis 0 1 1
Lithophanehepatica 1 0 2
Mythimnaalbipuncta 2 0 1
Mythimnapallens 0 1 2
Noctuacomes 2 0 2
Noctuapronuba 19 13 1
Noctuasp. 1 1 2
Ochropleurasp. 0 1 2
Oligiasp. 0 1 1
Omphaloscelislunosa 1 1 1
Orthosiagothica 1 0 2
Phlogophorameticulosa 2 0 1
Scoliopteryxlibatrix 1 0 1
Tholeracespitis 0 1 1
Tholeradecimalis 2 0 1
Xestiaxanthographa 2 6 2
Oecophoridae Diurneafagella 0 1 2
Endrosissarcitrella 0 1 2
(continued)
(cid:2)c 2011TheAuthors.EcologyandEvolutionpublishedbyBlackwellPublishingLtd. 561
MolecularDietAnalysisofCrypticSpecies O.Razgouretal.
Table1. Continued
Order Family Species P.austriacus P.auritus Confidencelevel
Diptera Anisopodidae Unknown 0 1 3
Calliphoridae Unknown 0 1 3
Polleniasp. 0 1 2
Muscidae Unknown 0 2 3
Eudasyphoracyanella 1 2 1
Eudasyphorasp. 0 1 1
Helinaimpuncta 1 0 1
Morelliasimplex 0 1 1
Phaoniasubventa 1 3 1
Polieteslardarius 0 3 1
Scathophagidae Scathophagastercoraria 1 1 1
Syrphidae Platycheirussp. 0 2 2
Tachinidae Leskiasp. 1 4 1
Tipulidae Unknown 1 0 3
Tipulaoleracea 8 1 1
Tipulasp. 1 0 1
Neuroptera Chrysopidae Unknown 2 0 3
Coleoptera Dermestidae Anthrenusfuscus 0 1 1
Hemiptera Miridae Unknown 3 0 3
Phytocoristiliae 1 0 1
Trichoptera Limnephilidae Stenophylaxsp. 1 0 2
Isopoda Porcellionidae Porcellioscaber 1 1 1
Porcelliosp. 0 1 1
Araneae Anyphaenidae Unknown 0 1 3
Anyphaenaaccentuata 0 1 1
Tetragnathidae Metellinasegmentata 0 1 1
Preyhabitatassociations intheproportionofpreyorderswerenotstatisticallysignifi-
cant(Pairedt-tests,Arcsintransformation:P=0.98).Similar
The majority of the prey MOTUs identified to the species
totheresultsofthemoleculardietanalysis,theproportion
levelwerehabitatgeneralists.Generalistpreyspeciestended
of Lepidoptera in the diet differed between the study sea-
tobeconsumedbybothbatspeciesandformedthemajority
sons,beingsignificantlylowerinspring(Kruskal–Wallistest
of their diet (P. austriacus 65%, P. auritus 68%). Of the 25
withArcsintransformation:H =47.7,df=2,P <0.001),
preyMOTUswithspecifichabitatassociations,themajority
while the proportion of Diptera was highest in the spring
ofspeciesthattheliteratureconsideredasfoundexclusively
(Fig.4).
inwoodlands(67%)wereconsumedbyP.auritus,whilethe
A comparison with a previous morphological study of
majorityofspeciesassociatedwithmoreopengrasslandhabi-
the diet of P. auritus (data adapted from Hollyfield 1993,
tats(75%)wereconsumedbyP.austriacus.Patternsofprey
N =240)showedsimilarproportionsofLepidoptera(68%)
selectionbyhabitatwerenotrandom(Chisquare:χ2=13.1,
and Diptera (20%) but higher proportions of Coleoptera
df=1,P<0.01).
(6%)thanthoseidentifiedinourmolecularstudy(Fig.2).
The morphological analysis revealed four additional prey
Concordancebetweenmorphologicaland
orders absent from our study, Neuroptera, Trichoptera,
molecularanalysis
Hymenoptera, and Plecoptera, though all contributed
Amorphologicalanalysisof170P.austriacus fecalsamples together to less than 1.6% of the diet. As in the diet of P.
resultedintheidentificationofsixpreyorders,themostcom- austriacus,therewerenosignificantdifferencesintheresults
monofwhichwasLepidoptera(62%),followedbyDiptera of the molecular and microscopic diet analysis (P = 0.59).
(32%). The morphological analysis identified two prey or- NicheoverlapbetweenthedietofP.austriacusandP.auritus
ders that were not recorded in the molecular diet analysis, at the ordinal level, based on the result of the morpholog-
Coleoptera and Hymenoptera, but instead did not identify icaldietstudies,wasveryhighandsignificant(O =0.95,
jk
anyTrichopteraorIsopoda(Fig.2).Nevertheless,differences P=0.01).
562 (cid:2)c 2011TheAuthors.EcologyandEvolutionpublishedbyBlackwellPublishingLtd.
O.Razgouretal. MolecularDietAnalysisofCrypticSpecies
Figure2. PiechartsshowingthedietcompositionattheordinallevelofthetwobatspeciesinsouthernEnglandbasedonmolecular([A]Plecotus
austriacus[N=28];[B]P.auritus[N=24])andmorphologicaldietanalyses([C]P.austriacusfromtheDevoncolony[N=170];[D]P.auritusadapted
fromHollyfield1993[N=240]).Theproportionofpreyordersinthedietispresentedaspercentfrequencyofoccurrence(%FO).
Discussion patternsof resource partitioningamongcompeting species
withalevelofdetailnotpreviouslypossible(Valentinietal.
Useofhigh-throughputDNAsequencing
2009a).Thisstudyisthefirstapplicationofmoleculartech-
tostudyinterspecificinteractions
niquestostudyinterspecificcompetitionbetweensympatric
High-throughput DNA sequencing technology has the po- crypticspeciesthatareexpectedtoshowhighdietaryover-
tential to increase the scope of dietary studies by revealing lap due to strong morphological similarities (Findley and
(cid:2)c 2011TheAuthors.EcologyandEvolutionpublishedbyBlackwellPublishingLtd. 563
MolecularDietAnalysisofCrypticSpecies O.Razgouretal.
Figure3. TheproportionofLepidopterafamiliesidentifiedinthediet
ofP.austriacus(blackbars,N=28)andP.auritus(graybars,N=24)in
southernEngland.
Black 1983). Unlike previous DNA sequencing techniques,
whichreliedoneithercloning(e.g.,Deagleetal.2005;Zeale
et al. 2011) or the selection of prey fragments (Clare et al.
2009,2011b),high-throughputDNAsequencingexploitsen-
tiresamplesanddoesnotrelyonsubsampling,andtherefore
evenveryraredietaryitemscanpotentiallybedetected(Dea-
gleetal.2009).Moreover,theuseofuniquelytaggedprimers
(Binladenetal.2007)makesHTSmoreeconomicalbyen-
ablingthepoolingofmanysamples(upto30fecalsamples
per sequencing lane in our study) together while retaining
theabilitytoascribepreysequencestoanindividualbatcon-
sumer,thusallowingtheidentificationofseasonalandsexual
dietaryandnicheoverlappatterns.Yet,duetodifferencesin
primer binding, prey digestibility, and amount of DNA in Figure4. Seasonalvariationintheproportionofpreyordersidentified
inthedietofP.austriacusinsouthernEnglandbasedon(A)themolec-
preytissue,neithermethodofmoleculardietanalysistodate
ularstudy(Spring:N=8,Summer:N=14,Autumn:N=6)and(B)
canprovideanaccuratequantitativemeasureofdietcompo-
themorphologicalstudy(Spring:N=60,Summer:N=80,Autumn:
sitionwithinanyonesample(Siposetal.2007;Deagleetal. N=30).Dietcompositionispresentedaspercentfrequencyofoccur-
2010). rence(%FO).
Thenumberofpotentialpreyitems(MOTUs)identifiedin
thedietofthetwobatspeciesinourstudy(P.austriacus[90],
P. auritus [68]) did not exceed those identified in previous The application of molecular techniques did not result
molecularstudiesofthedietofinsectivorousbats(Clareetal. intheidentificationofmorelepidopteranpreyspeciesthan
2009—127preyspeciesinthedietofLasiurusborealis;Zeale previous studies of culled prey remains. Bauerova (1982)
2011—89preyspeciesinBarbastellabarbastellus).However, recorded 140 species of Lepidoptera in the diet of P. aus-
the number of MOTUs per fecal sample in our study was triacus in central Europe, while Robinson (1990) found 34
muchhigher(weidentifiedamaximumof17preyMOTUs species of Lepidoptera in the diet of P. auritus in England.
per dropping in P. austriacus and 16 in P. auritus, versus However,becauseDNAsequencingtechniquesarelesslikely
a maximum of seven and nine prey species in Clare et al. tobebiasedtowardlargehard-bodiedprey(Whitakeretal.
(2009)andZeale(2011),respectively),suggestingthatHTS 2009),wewereabletosuccessfullyrecordavarietyofsmaller
hasgreaterpotentialtorevealraredietarycomponentsand andsofterbodiedpreyitemsofordersoverlookedinprevious
specieswithlessvisibleremainsinthefeces. studies.
564 (cid:2)c 2011TheAuthors.EcologyandEvolutionpublishedbyBlackwellPublishingLtd.
O.Razgouretal. MolecularDietAnalysisofCrypticSpecies
AssuggestedbyZealeetal.(2011),theprimerssuccessfully
amplifiedDNAfromawiderangeofarthropodorders,in-
cludingacrustaceanorder(Isopoda)neverbeforerecorded
inthedietsofthetwobatspecies.Zealeetal.(2011)fedan
individualP.auritus anexperimentallymanipulateddietof
Diptera,Coleoptera,andLepidoptera,andfoundcloseagree-
mentbetweenitemsrecordedinthedietbymolecularanalysis
andthefooditemsfedtothebat6–24hpreviously,suggest-
ingthattheprimersdonotappeartosufferfromsignificant
amplificationbiases.Microscopicanalysisconfirmedthediet
compositionresultsconcludedfromgeneticdata.Although
previousmorphologicalstudiesfoundhigherproportionsof
Coleopterainthedietofbothbatspecies,ourmicroscopic
analysisofgeneticallyanalyzedfecalsamplesconfirmedthe
Figure 5. Extent of between (interspecific) and within species niche lowproportionofColeopterainoursamplesandtheabsence
overlapamongP.austriacusandP.auritusinsouthernEnglandbased
ofotherpreyorders.Hence,theprimersappeartoprovide
onPianka’smeasureofnicheoverlap,includingthesignificanceofniche
overlaprelativetorandomsimulations(**P<0.001;*P<0.01).Within goodcoverageofthemainpreyorderscommonlyfoundin
thedietofinsectivorousbats.
speciesnicheoverlapwastestedbycomparingthedietofDevonandIsle
ofWightcoloniesseparatelyforeachbatspecies(P.austriacus:Devon Molecularapproacheswereabletoovercomemanyofthe
N=19,IsleofWightN=9;P.auritus:N=10,7). biasesassociatedwithtraditionalbatdietanalysistechniques
(reviewedinWhitakeretal.2009),whileprovidinganaccu-
Strong overall agreement between the diet composition rateestimateofdietcompositionandmorefine-scaleextent
and seasonal patterns of diet composition of the two bat ofnicheoverlap(seeTable2forcomparisonoftheperfor-
species identified in our HTS study and our own and pre- manceofthevariousdietanalysismethods).Yet,molecular
vious (Hollyfield [1993] for P. auritus) morphological diet diet analysis is still constrained by an incomplete reference
studiessupportsthevalidityofthemoleculardietcomposi- sequencesdatabase,thoughthisproblemwasatleastpartly
tionestimationsattheordinallevel.Moreover,thefactthat overcomeinourstudythroughtheuseofMOTUsasasurro-
similarresultswereobtainedfrommorphologicalstudiescar- gateforpreyspeciesidentification.Highcorrespondencebe-
riedouton>170fecalsamplessuggeststhatoursamplesize tweenMOTUsandlepidopteranspecies,forwhichtheBOLD
wassufficientforrepresentingdifferencesinthedietsofthe referencedatasetisrelativelycomplete,suggeststhatMOTUs
twobatspecies. areaneffectivemethodofdietassessment.However,ourre-
Theshortsequencelength(157bp)andlackofcomplete sultssuggestthattheymayoverestimatepreyspeciesrichness
overlapbetweenthecoverageoftheprimersusedinourstudy by12%duetotheproportionofrepeatedspeciesidentifica-
andavailableCOIreferencesequencesinBOLD(only∼130- tion in our study. This overestimation can be attributed to
bp overlap) meant that not all MOTUs could be identified intraspecificpolymorphismintheamplifiedregion(Valen-
to the species level, and we were not able to use phyloge- tini et al. 2009b), though it can also be an artifact of the
netictreestoconfirmidentification.Inaddition,manyofthe incompletereferencedatasetortaxonomicambiguityamong
sequences showed 100% match to more than one species somepreyspecies.
distributed globally, some of which belonged to different
genera, thus forcing us to create a three-tiered confidence
Trophicecologyrelativeto
systemandtoidentifysomespeciesbasedonwhetherthey
resourceavailability
are found in the United Kingdom. Nevertheless, because a
highproportionofpreyDNAfragmentsrecoveredfromfe- Insectivorousbatsaresometimesperceivedasopportunistic
ces are degraded and relatively short (Deagle et al. 2006), foragers(Kunz1974),consumingpreybasedontheiravail-
short amplicons may provide a more accurate estimate of abilitybothintheenvironment(Swiftetal.1985)andtothe
dietcomposition,includingalsopreyofhigherdigestibility, batitself,giventheconstraintsofitssensoryandflightabili-
andaremorelikelytoidentifyraredietarycomponents.As ties(Fenton1990;SiemersandSchnitzler2004).Clareetal.
such,shortampliconscanovercomeproblemsencountered (2011b)showsthatthesamepredatorcanberegardedasa
byClareetal.(2011b)oflowamplificationsuccessandhigh generalist,basedonthenumberofpreyMOTUsidentifiedin
contamination by nonprey DNA. The source material may thediet,andasaspecialistwhenthedietisconsideredphylo-
thusdictatethechoiceofprimerlength,atrade-offbetween genetically,ifallthepreyspeciesidentifiedinthedietbelong
lengthofampliconforidentificationandtheimpactofDNA tothesameorder.Inourstudy,dietselectivityattheordinal,
degradation. andevenfamilylevel,appearstobehigh.Lepidoptera,and
(cid:2)c 2011TheAuthors.EcologyandEvolutionpublishedbyBlackwellPublishingLtd. 565