Innovative Bayesian and Parsimony Phylogeny of Dung Beetles

86 Pages·2015·4.88 MB·English

RESEARCHARTICLE
Innovative Bayesian and Parsimony
Phylogeny of Dung Beetles (Coleoptera,
Scarabaeidae, Scarabaeinae) Enhanced by
Ontology-Based Partitioning of
Morphological Characters
SergeiTarasov1*,FrançoisGénier2
1 DepartmentofResearchandCollections,NationalCenterforBiosystematics,NaturalHistoryMuseum
UniversityofOslo,P.O.Box1172BlindernNO-0318,Oslo,Norway,2 ColeopteraSection,Canadian
NationalCollectionofInsects,ArachnidsandNematodes,AgricultureandAgri-FoodCanada,960Carling
Avenue,Ottawa,K1A0C6,Ontario,Canada
* [email protected]
OPENACCESS
Abstract
Citation:TarasovS,GénierF(2015)Innovative
BayesianandParsimonyPhylogenyofDungBeetles
Scarabaeinedungbeetlesarethedominantdungfeedinggroupofinsectsandarewidely
(Coleoptera,Scarabaeidae,Scarabaeinae)Enhanced
byOntology-BasedPartitioningofMorphological usedasmodelorganismsinconservation,ecologyanddevelopmentalbiology.Duetothe
Characters.PLoSONE10(3):e0116671. conflictsamong13recentlypublishedphylogeniesdealingwiththehigher-levelrelation-
doi:10.1371/journal.pone.0116671
shipsofdungbeetles,thephylogenyofthislineageremainslargelyunresolved.Inthis
AcademicEditor:BenJMans,Onderstepoort study,weconductrigorousphylogeneticanalysesofdungbeetles,basedonanunprece-
VeterinaryInstitute,SOUTHAFRICA
dentedtaxonsample(110taxa)anddetailedinvestigationofmorphology(205characters).
Received:May3,2014 Weprovidethedescriptionofmorphologyandthoroughlyillustratetheusedcharacters.
Accepted:December11,2014 Alongwithparsimony,traditionallyusedintheanalysisofmorphologicaldata,wealso
applytheBayesianmethodwithanovelapproachthatusesanatomyontologyformatrix
Published:March17,2015
partitioning.Thisapproachallowsforheterogeneityinevolutionaryratesamongcharacters
Copyright:©2015Tarasov,Génier.Thisisanopen
fromdifferentanatomicalregions.Anatomyontologygeneratesanumberofparameter-par-
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits titionschemeswhichwecompareusingBayesfactor.Wealsotesttheeffectofinclusionof
unrestricteduse,distribution,andreproductioninany autapomorphiesinthemorphologicalanalysis,whichhithertohasnotbeenexamined.Gen-
medium,providedtheoriginalauthorandsourceare
erally,schemeswithmoreparameterswerefavoredintheBayesiancomparisonsuggest-
credited.
ingthatcharacterslocatedondifferentbodyregionsevolveatdifferentratesandthat
DataAvailabilityStatement:Allrelevantdataare
partitioningofthedatamatrixusinganatomyontologyisreasonable;however,treesfrom
withinthepaperanditsSupportingInformationfiles.
AdditionalfilesareavailablefromMorphoBank(http:// theparsimonyandalltheBayesiananalyseswerequiteconsistent.Thehypothesizedphy-
www.morphobank.orgproject1157). logenyrevealsmanynovelcladesandprovidesadditionalsupportforsomecladesrecov-
Funding:VisitsofSergeiTarasovtotheMNHNin eredinpreviousanalyses.Ourresultsprovideasolidbasisforanewclassificationofdung
ParisreceivedsupportfromtheSYNTHESYSgrant beetles,inwhichthetaxonomiclimitsofthetribesDichotomiini,DeltochiliniandCopriniare
(http://www.synthesys.info).Thefundershadnorole
restrictedandmanynewtribesmustbedescribed.Basedontheconsistencyofthephylog-
instudydesign,datacollectionandanalysis,decision
enywithbiogeography,wespeculatethatdungbeetlesmayhaveoriginatedintheMesozo-
topublish,orpreparationofthemanuscript.
iccontrarytothetraditionalviewpointingtoaCenozoicorigin.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
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Morphology-BasedPhylogenyofScarabaeinae
Introduction
DungbeetlesofthesubfamilyScarabaeinaeareawell-knowngroupofinsectsthankstotheir
exploitationofanimalfeces,abehavioraltraitwithaglobalimpactonEarth’secosystems.
Withmorethan6200speciesin267generaandwithanestimated30–50%ofthespeciesstill
undescribed(FG,personaldatabase),dungbeetlesexhibitdiversitycomparabletotheAves(ca.
9.800species[1]),aswellasbeingthesecondmostcitedsubfamilyofbeetlesonGoogleScholar
(Fig.1).Dungbeetleshavebeenshowntoplayamajorroleinnutrientrecycling,bioturbation,
enhancementofplantgrowth,secondaryseeddispersal,parasitesuppressionanddispersal,fly
control,trophicregulationandpollination[2].Throughthesevariousmechanisms,theypro-
videecosystemservicesvaluedat$380millionannuallyforthecattleindustryintheUSalone
[3].
Theirassociationwithephemeralfoodresourcescoupledwithinterestingandcomplex
feeding,breedingandnestingbehaviorsmakedungbeetlesamodelforvariousecologicalstud-
ies[4].Dungbeetleshavebeenselectedasindicatorsforbiodiversityinventoryandmonitoring
duetotheirglobaldistribution,highabundance,easeofcapturecombinedwithtightconnec-
tionstospecificsoilandvegetationtypes;theirsensitivitytocommunitydisturbanceallowsas-
sessmentofhumanimpactaroundtheglobe[4,5].Dungbeetles,amongstfewother
invertebratetaxa,wereselectedfortheIUCNRedListIndexprogram[6,7].Thescarabaeine
genusOnthophagus,oneofthemostspecies-richgenerainanimalkingdom,inwhichhorns
exhibitfascinatingphenotypicdiversity,hasemergedasamodelsysteminevolutionarydevel-
opmentalbiologyandecologicaldevelopment[4,8].
Duetotheextensivebiologicalknowledgeacquiredaboutthemandtheiruniquebiological
traits,dungbeetlesstandoutamongstotherinvertebrates,asorganismsidealforunraveling
generalpatternsofnature.Dobzhansky’sfamous"Nothinginbiologymakessenseexceptin
thelightofevolution"[9]applieswellascomparativedatainbiologycanbeonlyinterpretedif
Fig1.Phylogenetictimelineandcitationchart.Onthetop:phylogenetictimelineillustratinghistoryofphylogeneticstudiesindungbeetles.Descriptionof
thephylogeneticstudiesisprovidedinTable1.Leftonthebottom:citationchartofColeopterasubfamilies.ThepopularityofeachColeopterasubfamilyis
basedonthenumberofcitationsonGoogleScholar.Thediagramshowsthe16mostpopularColeopterasubfamilieswhosenumberofcitationsexceed
1500.ThecitationswereobtainedbyqueryingarecentlyupdatedlistofallColeopterasubfamilies(providedbyAlfredNewton,FieldMuseum,Chicago,IL)in
GoogleScholarusingRscript(availableuponrequest).Scarabaeinaeemergesasthesecondtopsubfamily,with4740citations.Numbersabovecolumns
indicatethenumberofcitations.
doi:10.1371/journal.pone.0116671.g001
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Morphology-BasedPhylogenyofScarabaeinae
Table1.ThekeyphylogeneticstudiesinthesubfamilyScarabaeinae. Seealsophylogenetictime-line
inFig.1.
# Year Authors Description
1 2013 Mlamboetal.[29] MOLECULAR:48species,COI,16S,28Sd3;majorityofAfrican
DeltochiliniandDichotomiini,5Madagascanand1Orientaltaxon
2 2011 Baietal.[23] MORPHOLOGY:81species119characters;Chinesetaxa
3 2010 SoleandScholtz MOLECULAR:35species,COI,16S,28Sd2,28d3,CAD;majorityof
[28] Africangenera
4 2010 Wirtaetal.[27] MOLECULAR:155species,COI,16S,18S,28S,COI;focusedon
MadagascanDeltochilinibutalsoincludesmanyotherScarabaeinae
taxa
5 2008 Wirtaetal.[26] MOLECULAR:44species,COI,18S,28S,COI,Cytb,16S;focusedon
MadagascantribeHelictopleurinibutalsoincludesothergeneraof
Scarabaeinae
6 2007 Monaghanetal. MOLECULAR:224species,COI,16S,28S;globalcoverageof
[25] Scarabaeinae
7 2007 Vaz-de-Mello[22] MORPHOLOGY:12species,20characters;someNeotropicalgenera
ofDichotomiiniandDeltochilini
8 2007 Vaz-de-Mello[11] MORPHOLOGY:87taxa,297characters;globalcoverageof
Scarabaeinae
9 2006 Ocampoand MOLECULAR:45species,18S,28Sd2,28d3,variousScarabaeinae
Hawks[24] genera
10 2004 Philipsetal.[20] MORPHOLOGY:48species,199characters;47generafromvarious
biogeographicregions
11 2002 Villalbaetal.[18] MOLECULAR:33species,COI,COII;Iberianspecies
12 1998 Montreuil[17] MORPHOLOGY:29genera,42characters;someDichotomiiniand
Deltochilini
13 1983 Zunino[19] MORPHOLOGY:18generafromvariousbiogeographicregions;
intuitivecladisticapproach
doi:10.1371/journal.pone.0116671.t001
weknowthephylogenyoftheorganismsunderstudy.Dungbeetles’all-aroundpopularityin
thelasttwodecadeshasmadethemthesubjectof13keyphylogeneticstudiesaimingtoresolve
theirevolutionaryhistoryusingbothmolecularandmorphologicalcharacters(Fig.1,Table1).
Despitesuchintensescrutiny,thephylogenyofdungbeetlesremainslargelyunresolved.Cur-
rentphylogeniesoftenresolveonlyveryshallowdivergencesbetweencloselyrelatedgenera
andlargelydisagreewitheachotherconcerningdeeperrelationships(seenextchapterforde-
tails).Asaresult,westilldonothaveafirmbasisforcomparativeevolutionaryandecological
researchandagoodnaturalclassificationforthisimportantgroup.
Usuallymolecularphylogeniesdealingwithdungbeetlesrelyonalimitednumberofmito-
chondrial(bothprotein-codingandrDNA)andnuclearrDNAmarkers.Protein-codingmito-
chondrialmarkers,affectedbyfasterevolution,arelesssuitableforuncoveringdeeper
divergences,whilerDNAsequencesposealignmentproblemsandchallengemodelingoftheir
evolution[10].AlthoughmitochondrialandrDNAgenesaregoodcandidatesforresolving
shallowdivergences,theyseemtoincludenoiseandlessinformationforelucidatingrelation-
shipsatahigherlevel[10].Morphologicalphylogeniesaresensitivetotaxonsamplingthatcan
significantlyaffecttheirresults.Onaverage,morphologicalstudiesindungbeetlesuseupto50
terminals;thereisonlyonestudycomprising80taxaandarepresentativesampleofthegenera
[11].Inaddition,morphologicalphylogenies,toacertainextent,lackathoroughinvestigation
ofmorphologythatwouldincludetotalexaminationoftheirinternalandexternalstructuresas
wellasgenitalia.Suchin-depthinvestigationiscriticalasmorphologicalphylogeniesstrictly
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dependonhomologyassessmentandincorporationofallpossiblephylogeneticinformation
preservedinthephenotype.
Inthispaper,weaimatareconstructionofdungbeetlephylogenybasedonanunprece-
dentedanalysisintermsoftaxonsampleanddepthofmorphologicalinvestigation.
Inthecurrenteraofphylogenomics,ourchoiceofamorphologicalapproachisbasedupon
twoconsiderationsrenderingmorphologyirreplaceable.First,evenbigmulti-genestudiesmay
providebiasedestimatesandperformworsethanmorphology[12].Thus,morphologyisanal-
ternativesourceofdataimportantfortheassessmentofphylogeneticaccuracy.Second,mor-
phologyisessentialtothedevelopmentofanewrobustnaturalclassificationfordungbeetles,
whichrequiresmorphologicalcharactersfordiagnosisandidentificationoftaxa.
Thepresentstudyhasseveralkeyadvantagesoverpreviousmorphologicalstudies.Itcovers
allbiogeographicandmorphologicaldiversityandincorporatesthemajorityofputativeline-
ages,andsupragenericcategoriesconsideredphylogeneticallyquestionable.Weperformedthe
mostdetailedinvestigationofendo-andexoskeletonstructuresindungbeetlesusingadissect-
ingtechniquedescribedinthematerialsandmethodssection.Wespeciallyfocusonstudying
theinternalscleritesofmalegenitalia.Thesestructuresrecentlyhavebeenshowntobephylo-
geneticallyinformative[13],althoughremainingpoorlystudiedastheirpreparationandexam-
inationisrathercomplex.Weprovidethefirstlargescaleassessmentofhomologyforinternal
scleritesandtheirdetailedillustration.Ourthoroughexaminationofmorphologyrevealed
manynovelcharacters,whichareinformativeforuncoveringphylogeneticrelationshipsin
dungbeetles.
Complementarytotheparsimonyapproachprevailinginphylogeneticinferenceusingmor-
phology,wealsouseaBayesianmethod.PreviousstudiesapplyingBayesianmethodologyto
morphologyrevealedthat,althoughresultsoftheBayesianandparsimonyanalysesaresimilar,
theBayesianapproachmightinferinterestingpatternsneglectedbyparsimony[14–16].Bayes-
ianinferenceisbasedonsubstitutionmodels,accountsforrateheterogeneityandcanincorpo-
rateautapomorphiccharacters,ignoredbyparsimony.Bypartitioningthedataset,model-
basedmethodsallowdifferentpartitionstoevolveatdifferentevolutionaryrates.Suchanap-
proachseemsbiologicallymorerealisticthanparsimony,andthereforecanbeinterestingfor
phylogeneticinference.However,thepartitioningofthemorphologicaldatasets,unlikethe
molecularones,isnotstraightforward.Here,weuseanatomicontologyi.e.,anatomicalrela-
tionshipsamongcharactersforpartitioning.Thisapproachassignscharactersinpartition
giventheiranatomicallocationonthebeetlebody,ontheassumptionthatcharactersofthe
sameanatomicalregionundergosimilarevolutionarydynamics.Thedatasetcanbepartitioned
inmultiplewaysthat,alsoallowstestinghypothesesaboutcharacterevolution.WeuseBayes
factortoelucidatethebestpartitioningschemeforphylogeneticinferenceandanswerthefol-
lowingbiologicalquestions.Ispartitioningofthemorphologicaldatasetmeaningful?Docharac-
tersonthesameanatomicalregionevolveatsimilarrates?And,howcanautapomorphic
charactersaffecttreetopology?
Howmanypatternsareoutthere:areviewofScarabaeinaephylogenies
ThecurrentclassificationsplitsthesubfamilyScarabaeinaeinto12tribesandapproximately
267genera.MorethanhalfofthegeneraareclassifiedinthetribesDeltochilini(formerly
Canthonini,103genera)andDichotomiini(43genera).Allphylogeneticstudiessupportthe
polyphylyofthesetribes,whichresultsinaconstantshufflingofgeneraamongDeltochilini,
Dichotomiiniandtheothertribes.Ofallthetribes,Deltochilini,Dichotomiini,Coprini,and
Onthophaginihaveaglobaldistribution.Despitethat,thetribesDeltochilini,Dichotomiinido
notshareanygeneraamongtheNewWorld,OldWorldandAustralasianRegions.Thetribes
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Morphology-BasedPhylogenyofScarabaeinae
Gymnopleurini,ScarabaeiniandOnitiniareendemictotheOldWorld,whileEucraniini,Eur-
ysterniniandPhanaeiniareendemictotheNewWorld.Twotribes,OniticelliniandSisyphini,
occurinboththeOldandNewWorld,buttheirNewWorldpresencelikelyrepresentsrather
recentdispersal.Interestingly,theAustralasianRegion,knownforitshighgenericendemism,
lacksanycurrentlyrecognizedendemicdungbeetletribes[4].
Thefirststudyofdungbeetlephylogeny,basedonanintuitivephylogeneticapproachand
illustratedbyatreedrawnbyhand,datesbackthreedecades[35].Then,afterafifteenyearshi-
atus,Montreuil[17]publishedthefirstmorphologicalphylogenybasedonparsimony,witha
datasetcomprising29generamostlyfromthetribesCoprini,DeltochiliniandDichotomiini
andusing42characters.Fouryearslater,thefirstmolecularphylogeny[18]usingtwogenere-
gions(COIandCOII)andfocusingonIberiandungbeetleswaspublished.Thispublication
startedanongoingandstillincreasinginterestinmolecularandmorphologicalinvestigationof
dungbeetlesresultinginasteadyflowofpublicationseveryyearortwo.Uptodate,7morphol-
ogy-based[11,17,19–23]and6molecular-basedstudies[18,24–29]relevanttohigher-level
phylogenyofScarabaeinaehavebeenpublished.Asummaryofthesepublications(Table1and
Fig.1)ispresentedasahistoricaltimelinethatcanbefoundathttp://embed.verite.co/timeline/
?source=0Aqe3bAUelOltdG9ac1VEdmczbXdUSGdwSWU5QVF2T2c.Although,thesephy-
logeniespursuedifferentgoalsanddifferintaxonomiccontentandbiogeographiccoverageas
wellasthesetofmolecularormorphologicalmarkersused,wecanconductcomparisonsbe-
tweenthem,duetothesignificanttaxonoverlap.
Thelargeststudiesintermsofbiogeographiccoverageandtaxonsamplearethemorpho-
logicalphylogenyofVaz-de-Mello[11](87species,297characters)andthemolecularphyloge-
nyofMonaghanetal.[25](224species,3generegions).Someoftheotherphylogeniesfocus
mainlyonspecificbiogeographicregions;e.g.,[24]Neotropicaltaxa;[28,29]Deltochiliniand
DichotomiinigenerafromAfricaandMadagascar[26,27].
Molecularphylogeniesuseanalmostuniformsetof2–6generegionswhichrepresentmito-
chondrialprotein-codinggene(s)andnuclearand/ormitochondrialrDNAgenes.Asingle
broad-scalestudyattemptedincorporatingnuclearprotein-codingmarkers[28].Nuclear
markerswereshowntobehighlyinformativeforasmaller-scalephylogenydealingwithtaxa
ofOnthophagini[30].
Thesubstantialdisagreementbetweenthe13keyphylogeniescomplicatesinductionof
sharedpatterns,asalmosteverysinglestudyhasitsuniquetree.Here,wehighlightthemost
criticalcasesandsummarizepatternssharedbetweentwoormorephylogeniesorthosewith
interestingbiogeographicalcorrelations.Eachanalysisisbriefonpurposeinordertodemon-
stratethedramaticincongruenceoftheresults.Scholtzetal.[4]provideanexcellentin-depth
reviewofexistingphylogenies.
AllphylogeniessupportmonophylyofScarabaeinae.Themajorityofphylogeniesdonot
challengethemonophylyof8(Onitini,Sisyphini,Gymnopleurini,Scarabaeini,Eurysternini,
Oniticellini,Phanaeini,Eucraniini)outof12scarabaeinetribes,whiletwotribesDichotomiini
andDeltochilini,emergeashighlypolyphyletic.Despitethesesimilarities,substantialdiscrep-
anciesappearintherelationshipsbetweenmonophyletictribes(Fig.2)andgeneraofthepoly-
phyleticDichotomiiniandDeltochilini.
Toexemplify,twomolecularphylogenies[24,25]recoverthetribeOniticellininestedwith-
inthetribeOnthophagini,anotherphylogenybasedonmorphologyrecoverspolytomycom-
prisingOnthophaginiandOniticellini[24],whiletheothermorphologicalphylogeny[11]
recoversOniticelliniasabasallineageontheScarabaeinaetree,onlyremotelyrelatedto
Onthophagini(Fig.2).Atthesametime,thereisalsoalackofconsensusonthemonophylyof
thetribeOnthophaginithatemergespolyphyleticorparaphyleticinbroad-scalemolecular
phylogenies[24,25],orcomesupmonophyleticinonemorphologicalphylogeny[13].
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Fig2.Phylogeneticpatternsrevealedbypreviousphylogenies.Thetreesillustraterelationships,amongtribesOnthophagini,Oniticellini,Onitini,
Sisyphini,Eurysternini,Deltochiliniandsomegenera.Interruptedlineindicatesremoterelationshipbetweencladesi.e.,itomitsirrelevanttaxa/clades
branchingbetweenthecladesitjoins.
doi:10.1371/journal.pone.0116671.g002
ThephylogeneticpositionsofthetribesGymnopleuriniandEurysterninihavebeenincon-
stantfluxamongphylogeniesastheysupportdifferentplacementsofthesetwotribesasdem-
onstratedinFig.2.
ThestudieswithsufficienttaxonsamplingtotestthemonophylyofCoprini,uncoverits
polyphyleticnature:twomorphologicalphylogenies[17,20]separateCopriniintotwoline-
ages,whileamolecularandanothermorphologicalphylogenyseparateCopriniintothreeline-
ages[11,25].Additionally,allfourphylogeniesrevealingpolyphylycontradicteachotherin
theaffiliationofseparateCoprinilineages.
Twomolecularphylogenies[24,25]supportmonophylyofNeotropicaltribesPhanaeini+
EucraniiniandtheirsisterrelationshiptotheNeotropicalgenusDichotomiusanditscloserela-
tives.Conversely,twomorphologicalphylogeniesarenotconsistentwiththispattern,and
placeEucraniiniassistertoCircellium+Scarabaeini[20]orrecoveracladecomposedofOni-
tini+OnthophaginiwithinthecladePhanaeini+Eucraniini[11].
ManyphylogeniesareconsistentinthepositionofthegeneraSarophorus,Frankenbergerius,
Coptorhina,Dicranocara,Odontolomaandallieswhichemergeasbasalornearlybasallineages
[11,25,28,29];howeverrelationshipsbetweenthesegeneradifferamongthosestudies.
Twomorphological[11,20]andmolecularphylogenies[25]confirmthesisterrelationship
betweenCircellium(orCircellium+Ateuchus)andthetribeScarabaeini.However,twoother
molecularphylogeniesplaceCircelliumaseithersisterofPedaria[28]orbelongingtoaclade
consistingofthegeneraJanssensantus+Tanzanolus[29].
ThemolecularphylogenyofMonaghanetal.[25]recoversaninterestingcladecomprising
almostallAustralasianendemicgenerawithNewZealand,NewCaledonia,andoneAfrican
genusPedarianestedwithin.OnlytwoAustralasiangenera,BoletoscapterandMonoplistes,not
fallingintheAustralasianclade,breakthemonophylyofAustralasianendemicgenerainthat
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phylogeny.ThatstudyalsorecoversmonophylyforNewCaledoniangenera.Suchconsistency
relatedtogeographymakesthisphylogeneticpatternquitemeaningfulbiogeographically,al-
thoughnotsupportedbyanyotherphylogeneticstudy.
TheevolutionaryscenarioforthemajorityofMadagascantaxaisalsocontradictory.Ac-
cordingto[27],DeltochiliniincludesthreeseparatelineagesonMadagascar:Arachnodes+
Epilissus,Epactoides,andNanos+Apotolamprus.TheEpactoideslineageissistertotheOrien-
talgenusOchicanthon,withwhomtheyformacladesistertotheAustralasiangenusMono-
plistes;thisAustralasian—Oriental—Madagascanclade,inturn,issistertoanother
MadagascanlineagecomprisingNanos+Apotolamprus.However,thelatterbiggercladeis
weaklysupportedinthatanalysis(posteriorprobability0.54).Thesistergrouptothelineage
Arachnodes+Epilissuswasnotinferredinthatstudyasthislineageendsinapolytomywith
manyothertaxausedinthestudy.SimultaneousanalysisofMadagascanandAfrotropicaltaxa
[29]corroboratesthedivisionofMadagascanscarabaeines[27]intothreelineages,and,addi-
tionally,placestheEastern-ArcmountaingenusMadaphacosomawithintheEpactoides+
Ochicanthonlineage.However,thelargestmolecularphylogeny[25]mergesallthreeMadagas-
canlineagesalongwithOchicanthonandMonoplistesinoneclade,andatthesametimeplaces
EpilissussplendidusFairmaire,1889andArachnodessp.assistertotheNeotropicalgenus
Trichilium.
MaterialsandMethods
Outlineofthemethodologicalprocedure
Toconstructacharactermatrixforthephylogeneticanalyses,weperformeddetailedexamina-
tionofendo-andexoskeletonmorphology(205characters)fortheselecteddungbeetletaxa
(110species).Tomakeourstatementsofmorphologicalcharactersclearandusableinfurther
researchwedocumentthembyprovidingillustrationsofalmostallcharactersused(seecharac-
terreportandillustrations).
Toinferphylogeneticrelationshipsweransixparsimonyanalyses(parsimonyanalysessec-
tion).Inadditiontothem,weran184Bayesiananalysesusingdifferentschemesofcharacter
matrixpartitioningtoinferphylogenetictreesandbestpartitioningscheme(Bayesiananalyses
section).Thepartitioningofthematrixisbasedonourinnovativeapproachthatusesanatomy
ontologytoguidethepartitioning(anatomyontologyconstructionsection).
Illustrationsandabbreviations
PhotosweretakenwithCanonEOS500DdigitalcameraattachedtoaLeicaMZ16microscope.
Aedeagi,endophallicscleritesandsomeskeletalstructureswerephotographedinanalcohol-
basedhandsanitizercomprisingadensegelthatwasusedtofixthepositionofstructuresfor
photography.ThecolorschemesoftheendophallicscleritesweredrawninAdobeIllustrator.
Thetechnicalnotesontheillustrationsareprovidedincharacterreportsection.Abbreviations
usedinthetextandfiguresaredetailedinTable2.
Taxonselection
Theentiredatamatrixcomprises110taxa,4ofwhichcomprisetheoutgroup.Outgrouptaxa
belongtothesubfamiliesAphodiinae:tribeAphodiini(AphodiuserraticusandPodotenusfulvi-
ventris)andtribeAulonocnemini(Manjarivolosp.andAulonocnemiscrassecostataafricana)
(S1Table).AphodiinaeareconventionallyconsideredthesistergrouptoScarabaeinae.This
factgainssupportfromvariousmolecularandmorphologicalphylogenies[31–33].Theyshare
44synapomorphiesinmorphology-basedphylogenyofthefamilyScarabaeidae[34].However,
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Table2.Abbreviationsusedinthetext.
? indicatesscleriteswithunclearhomology
AcS accessorysclerites
AIS additionalinferiorsclerite
AMS additionalmedialscleriteoflamellacopulatrix
AS additionalsclerite
ADP anteriordepressionofpropleura
ARP anteriorridgeofpropleuron
A axialsclerite
BSG basalscleriteofgalea
BS basalscleriteofinternalsac
BSSg basalscleritespiculumgastrale
BSc basalsemicircularsclerite
BFP basisternalfurcaofprothorax
Blp basolateralparamerite
Blp basolateralplate
BShA borderdelimitingareaofheadshieldedbypronotum
Br bristle
Cav cavity
CRP clypealrectangularpattern
CxDp coxaldepression
DASG dorsalarticularscleriteofgalea
DPT dorsalprocessoftentorium
ExL externallobe
Eye eye
FLP* fronto-lateralperipheralscleritemarkedwith*isnotconsideredtobehomologousto
FLP
FLP,FLP1,FLP2 fronto-lateralperipheralsclerite,fronto-lateralperipheralsclerite1(outer),fronto-lateral
peripheralsclerite2(inner).
Gl glossa
GlFl glossalflap
Gr groove
InLM incisorlobeofmandibles
IAS1,IAS2 inferioraccessorysclerite1and2respectively
IL inferiorleftlobeoflamellacopulatrix
ILb inferiorlobe
IP inferiorportion
IR inferiorrightlobeoflamellacopulatrix
ICP internalcarinaofpronotum
InL internallobe
InS internalsack
LC lamellacopulatrix
LlS laterallabialsclerite
LP lateralprocess
MP medialperipheralsclerite
MsArRp mesofurcalarm,rearprocess
MtSc metascutellum
MtEp metepisternum
PIS parameralinferiorside
PSS parameralsuperiorside
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Morphology-BasedPhylogenyofScarabaeinae
PhFS phallobasefrontalside
PhRS phallobaserearside
PhG pheromonegland
pit pit
Pit pitofdifferentorigin
PRP posteriorridgeofpropleuron
Pr projection
PsnSt prosternalsternellum
Ri ridge
X scleriteofunknownhomology
SclP sclerotizedplateofparameralinferiorside
SclR sclerotizedroof
ScP scutellumplate
ScAP scutellum,apicalprocess
setae setae
TS spur-shapedlobeofA+?SAcomplex
SA(SA1,SA2, subaxialsclerites(subaxialsclerite1,subaxialsclerite2,subaxialsclerite3).
SA3)
SL superiorleftlobeoflamellacopulatrix
SLb superiorlobe
SR superiorrightlobeoflamellacopulatrix
SRP superiorrightperipheralsclerite
S suture
Sw swell
TFP trochantofemoralpit
Tub tubercle
VPCAM ventralprocessofclypealanteriormargin
VsSt ventralsurfaceofabdominalsternite
WScP weaklysclerotizedpart
doi:10.1371/journal.pone.0116671.t002
sinceAphodiinaestilllackanythoroughphylogeneticanalysis,thereisnorobustevidencethat
couldsupportmonophylyofthisgroup.Giventhehighmorphologicalheterogeneityoftaxa
withinAphodiinaeanditsnon-phylogenybasedsystematics,thereisahighprobabilitythat
Aphodiinaemightbeparaphyletic(seealsoPhilips[31]).Therefore,forthepurposeofthecur-
rentanalysisweselectedrepresentativesofAphodiinaesensustricto(i.e.,tribeAphodiini)and
sensulato(i.e.,tribeAulonocnemini)toaccountforphylogeneticuncertainty.Wespecifically
selectedthetribeAulonocnemini,whichisconsideredasubfamilybysomeauthors,sincethe
speciesofthistribeshareanumberofcharacteristicscommonforScarabaeinaeandusuallyab-
sentinotherAphodiinae.Thesecharacteristicsare:metatibiawithoneapicalspur,anterior
portionofhypomerondepressed,protibiatruncatedapically.Thetaxonomicallydifferentout-
grouptaxawereincludedfortheirpotentiallyinformativeinputinresolvingbasalrelationships
inScarabaeinae.
Theingrouptaxaweresampledtocovertaxonomic,biogeographic,andmorphologicaldi-
versity.Theybelongtoall12tribesandto101generaofthesubfamilywhichrepresents37%of
thetotalgenericdiversity(Scarabaeinaecurrentlytotalsca.258–267generaaccordingtoinfor-
mationcompiledfromScholtzetal.[4]andFGpersonaldatabase).Thesamplingoftaxaacross
taxonomiccategorieswasbiasedonpurpose.Accordingtotheavailablephylogeniesthemono-
phylyofthemajorityofthetribesisalmostentirelyundoubted,exceptfortwotribes
PLOSONE|DOI:10.1371/journal.pone.0116671 March17,2015 9/86
Morphology-BasedPhylogenyofScarabaeinae
Deltochilini(formerlyCanthonini)andDichotomiiniwhicharealwaysshowntobepolyphy-
letic(seereviewofphylogeniessection).Together,thesetwotribescomprise146generaand
arethemostgenera-richinthesubfamily.Therefore,inthepresentstudy,weselectedafew
representativesfromthetribeswithsupportedmonophylyandconcentratedgreatestfocuson
thetribesDeltochiliniandDichotomiinicoveringalmostallputativephylogeneticlineages.In
thispaperthetribalclassificationforgenerafollowsthemostrecentlistofgeneraprovidedby
Scholtzetal.[4]withsometaxonomicmodificationintroducedbyVaz-de-Mello[35].
Materialdeposition
ThedepositionofmaterialforeachtaxonusedintheanalysesissummarizedinS1Table.The
codonsusedinthetableareasfollows:
(cid:129) CASCCaliforniaAcademyofSciences,SanFrancisco(N.Penny,D.Kavanaugh)
(cid:129) CMNCCanadianMuseumofNature,Ottawa(F.Génier)
(cid:129) CNCICanadianNationalCollectionofInsects,ArachnidsandNematodes,Ottawa(V.Gre-
bennikovandB.Gill)
(cid:129) cSTSergeiTarasovprivatecollection
(cid:129) FMNHFieldMuseumofNaturalHistory,Chicago(M.ThayerandJ.Boone)
(cid:129) MNHNMuseumnationald’Histoirenaturelle,Paris,France(O.MontreuilandA.
Mantilleri)
(cid:129) TMSADitsongNationalMuseumofNaturalHistory(formerlyTransvaalMuseum),Pretoria
(R.Müller)
(cid:129) UPSAUniversityofPretoria,Insectcollection(C.DeschodtandC.Scholtz)
(cid:129) ZMUCNaturalHistoryMuseumofDenmark(A.Solodovnikov)
Examinationofspecimensandmorphology
Specimenswereeitherdry-pinnedoralcohol-preserved.Atleastonespecimenperspecieswas
entirelydissected.ThenumberofmalesandfemalesdissectedperspeciesisgiveninS1Table.
Inadditiontothedissectedspecimens,onaverage,weexamined1–3dry-pinnedspecimens
perspeciestostudyexternalmorphologyanddissectaedeagi.
Thedissectionprocessinvolvedthefollowingphases.Mainbodyparts(usuallyhead,pro-
thorax,pterothorax,legs,wings,elytra,abdomen,genitalia)ofalcohol-preservedspecimens
wereseparatedusingforceps/scalpelandplacedin10%KOHforseveralhoursorovernight;
wingsweredirectlyplacedindistilledwater.Dryspecimensweresoftenedinwarmdistilled
waterpriortoseparationoftheirbodyparts.AfterKOHtreatment,thespecimenswererinsed
indistilledwater;aedeagusandspiculumgastralewereplacedinglycerinforstudyandperma-
nentstorage;internalsacwasseparatedfromtheaedeagus.Theremainingtissueonthebody
partswasremovedbywashingunderafinestreamofwater.Darkandstronglypigmented
bodyparts,inwhichstructuraldetailsweredifficulttoobserveunderlightmicroscope,were
bleachedwith3%hydrogenperoxidecontainingonedropofammoniumhydroxide.Thetime
requiredforbleachingrangedfromfewminutestofewhoursdependingonthespecimensize
anddesiredresult.Afterbleaching,thebodypartswererinsedindistilledwater,andalong
withtheremainingdissectedelementsincludingwings,placedinabsoluteethanolforafew
minutes;afterwhichtheywerepreservedinglycerinforstudyandpermanentstorage.We
PLOSONE|DOI:10.1371/journal.pone.0116671 March17,2015 10/86