Genes Suggest Ancestral Colour Polymorphisms Are Shared across Morphologically Cryptic

26 Pages·2015·2.31 MB·English

RESEARCHARTICLE
Genes Suggest Ancestral Colour
Polymorphisms Are Shared across
Morphologically Cryptic Species in Arctic
Bumblebees
PaulH.Williams1*,AlexandrM.Byvaltsev2,BjörnCederberg3,MikhailV.Berezin4,
FrodeØdegaard5,ClausRasmussen6,LeifL.Richardson7,JiaxingHuang8,Cory
S.Sheffield9,SuzanneT.Williams1
1 DepartmentofLifeSciences,TheNaturalHistoryMuseum,London,UnitedKingdom,2 Departmentof
GeneralBiologyandEcology,NovosibirskStateUniversity,Novosibirsk,Russia,3 SwedishSpecies
InformationCentre,SwedishUniversityofAgriculturalSciences,Uppsala,Sweden,4 Entomological
Department(Insectarium),TheMoscowZoo,Moscow,Russia,5 NorwegianInstituteforNatureResearch,
Trondheim,Norway,6 DepartmentofBioscience,AarhusUniversity,Aarhus,Denmark,7 GundInstitutefor
EcologicalEconomics,UniversityofVermont,Burlington,Vermont,UnitedStatesofAmerica,8 Key
LaboratoryforInsect-PollinatorBiologyoftheMinistryofAgriculture,InstituteofApiculturalResearch,
ChineseAcademyofAgriculturalSciences,Beijing,China,9 RoyalSaskatchewanMuseum,Regina,
OPENACCESS
Saskatchewan,Canada
Citation:WilliamsPH,ByvaltsevAM,CederbergB,
*[email protected]
BerezinMV,ØdegaardF,RasmussenC,etal.(2015)
GenesSuggestAncestralColourPolymorphismsAre
SharedacrossMorphologicallyCrypticSpeciesin
ArcticBumblebees.PLoSONE10(12):e0144544. Abstract
doi:10.1371/journal.pone.0144544
Editor:ZuogangPeng,SOUTHWESTUNIVERSITY, Ourgraspofbiodiversityisfine-tunedthroughtheprocessofrevisionarytaxonomy.Ifspe-
CHINA ciesdoexistinnatureandcanbediscoveredwithavailabletechniques,thenweexpect
Received:June18,2015 theserevisionstoconvergeonbroadlysharedinterpretationsofspecies.Butfortheprimar-
ilyarcticbumblebeesofthesubgenusAlpinobombusofthegenusBombus,revisionsby
Accepted:November19,2015
someofthemostexperiencedspecialistsareunusualforbumblebeesinthattheyhaveall
Published:December10,2015
reacheddifferentconclusionsonthenumberofspeciespresent.Recentrevisionsbasedon
Copyright:©2015Williamsetal.Thisisanopen
skeletalmorphologyhaveconcludedthattherearefromfourtosixspecies,whilevariation
accessarticledistributedunderthetermsofthe
incolourpatternofthehairraisedquestionsastowhetheratleastsevenspeciesmightbe
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany present.Evenmorespeciesaresupportedifweaccepttherecentmoveawayfromviewing
medium,providedtheoriginalauthorandsourceare speciesasmorphotypestoviewingtheminsteadasevolutionarilyindependentlineages
credited.
(EILs)usingdatafromgenes.EILsarerecognisedhereinpracticefromthegenecoales-
DataAvailabilityStatement:Allsequencesare centsthatprovidedirectevidencefortheirevolutionaryindependence.Weshowfromfitting
availablefromtheonlinegene-sequencedatabases:
bothgeneralmixedYule/coalescent(GMYC)modelsandPoisson-tree-process(PTP)mod-
GenBank(ncbi.nlm.nih.gov/genbank/)andBOLD
elstodataforthemitochondrialCOIgenethatthereissupportforninespeciesinthesubge-
(boldsystems.org).Adetailedlistofaccession
numbersforthesequencenumbersdepositedin nusAlpinobombus.ExaminationofthemoreslowlyevolvingnuclearPEPCKgeneshows
thesedatabasesisgiveninthesupporting furthersupportforapreviouslyunrecognisedtaxonasanewspeciesinnorthwesternNorth
information(S2File)inthemanuscript.
America.Thethreepairsofthemostmorphologicallysimilarsisterspeciesareseparated
Funding:Thedatabaseofspecies’distributionsin allopatricallyandpreventedfrominterbreedingbyoceans.Wealsofindthatmostofthe
Russiawascreatedwiththefinancialsupportfroma
speciesshowmultiplesharedcolourpatterns,givingtheappearanceofmimicryamong
GrantofthePresidentoftheRussianFederationfor
youngRussianscientists(grantMK-6176.2015.4)(to partsofthedifferentspecies.However,reconstructingancestralcolour-patternstates
AB).DNAbarcodesforsomeofthematerialwere
PLOSONE|DOI:10.1371/journal.pone.0144544 December10,2015 1/26
CrypticArcticDiversity
fundedbytheSaskatchewanMinistryofAgriculture showsthatspeciationislikelytohavecutacrosswidespreadancestralpolymorphisms,
andtheCanada-SaskatchewanGrowingForward2 withoutorlargelywithoutconvergence.IntheparticularcaseofAlpinobombus,morphologi-
bi-lateralagreement(grant20130112)(toCS).Work
cal,colour-pattern,andgeneticgroupsshowlittleagreement,whichmayhelptoexplainthe
inGreenlandwassupportedbytheCarlsberg
Foundation(grants2009-01-0534and2011-01-0463) lackofagreementamongprevioustaxonomicrevisions.
(toCR).DNAbarcodesforsomeoftheNorwegian
materialwerefundedbytheResearchCouncilof
Norway(grant266134/F50)andtheNorwegian
BiodiversityInformationCentre(grant70184209).
TheNorwegianpartoftheprojectwasfundedbythe Introduction
TaxonomyInitiativeoftheNorwegianBiodiversity
Recentlyauthorshavesoughttoreducesubjectivityinthepracticeofspeciesdiscoveryor
InformationCentre(grant70184228)(toFO).DNA
sequencingforsomeofthematerialwasfundedby ‘delimitation’[1]bycomparingtheresultsfromapplyingmultiplecriteriainanexplicitlyinte-
theAgriculturalScienceandTechnologyInnovation grativetaxonomy[2].Thisisasearchforcongruentresultsfrommultiplecriteriaascorrobora-
Program,China(grantCAAS-ASTIP-2015-IAR)(to tion,eventhoughthedifferentsetsofcharactersemployed(fromdifferentdisciplines)may
JH).Partofthedatabaseofspecies’distributionsin
havedifferentevolutionaryconstraintsrequiringdifferentevolutionaryexplanations[2].
NorthAmericawascreatedwithfinancialsupport
Indeed,notallstudiesusingdifferentcriteriacanbeexpectedtoagree,becauserelationships
fromgrantsfromtheNationalScienceFoundationto
JohnAscher,DouglasYanega,andJeromeRozen betweencriteriaandspeciationeventsdiffer[1,3,4].Ithasevenbeenacknowledgedthatthose
(grantsNSF-DBI0956388andNSF-DBI0956340)(to criteriathatrelatemostcloselytospeciationcouldformaminoritypatterninsomecases[2].
LR).Thefundingagencieshadnoroleinthestudy Butforsomearcticbumblebees,taxonomicrevisionsbyexperiencedspecialistshaveshown
design,datacollectionandanalysis,decisionto
unusuallypooragreementonthenumberofspecies,wesuggestinpartbecauseofpooragree-
publish,orpreparationofthemanuscript.Theother
mentamongresultsfromapplyingdifferentcriteria.
authorsreceivednospecificfundingforthiswork.
Bumblebees(genusBombusLatreille)havebeendescribedasmorphologicallyhomoge-
CompetingInterests:Theauthorshavethe
neouscomparedwithotherbees[5].Itisthereforeunsurprisingthatbumblebeespecieswere
followinginterests:workinGreenlandwassupported
diagnosedinitiallyintermsofthestrikingcolourpatternsofthehaironthedorsumofthe
bytheCarlsbergFoundation(grants2009-01-0534
body[6].Butthen,morethanacenturyago,itwasrealisedthatthegroupssupportedbyskele-
and2011-01-0463).Therearenopatents,productsin
developmentormarketedproductstodeclare.This talmorphologyareactuallymoreclearlycircumscribed,andthatwithinthesemorphological
doesnotaltertheauthors'adherencetoallthePLOS species,bumblebeesarehighlyvariableincolourpatterns[7–10].Thisissupportedbyrecent
ONEpoliciesonsharingdataandmaterials,as geneticstudies[11–14],reaffirmingthelong-heldviewthatbumblebeecolourpatternsdonot
detailedonlineintheguideforauthors.
necessarilydiagnosespecies[15].Detailedstudiesofbumblebeevariationhaveledtothereali-
sationthatspeciescanbecrypticinbothmorphologyandcolourpattern[16,17],nowcon-
firmedbygeneticstudies[18,19].
Particularlyintriguingistheobservationthatbumblebeecolourpatternsareoftenimpres-
sivelysimilaramongspecies[8,20–26].Inmanycases,asinglespeciesmayshowdifferentcol-
ourpatternsindifferentgeographicalregions,ineachofwhichtheymayresembleclosely
otherspeciesasmembersofregionalcolour-patterngroups[24,27].Severalpossibleexplana-
tionshavebeenproposed(reviewedin[26,28]),includingMüllerianmimicry[22,29].Recent
advancesinunderstandingevolutionaryrelationshipshavebeguntoclarifythechronologyof
theevolutionofsomeoftheseresemblances[30],animportantpre-requisitetodiscerning
causes[31].Incasesofregionalcolour-patternresemblance,thesimilarspeciesareoftenonly
distantlyrelated,sothatthesimilarityhasbeeninterpretedastheresultofevolutionaryconver-
gence[26].Weexploreanotherpossibleexplanationfortheevolutionofcolour-patternresem-
blance,asanalternativetoconvergence:thatinsomesituationsapolymorphismwithinan
ancestralspeciesmayhavebeeninheritedinparallelbyseveraldescendentspecies.
ThebumblebeesofthesubgenusAlpinobombusSkorikovareasmallgroupofcloselyrelated
species[32].Previoustaxonomicrevisions(includingglobalrevisionsandbroaderregional
faunalrevisionswithglobalreferencing)ofAlpinobombusbysomeofthemostexperienced
bumblebeespecialistshavereliedonbothmorphology(especiallythemorevariablecharacters
ofthemalepenisvalve,volsellaandgonostylus,andofthefemaleoculo-malarareaandhind
tibia)andonthecolourpatternsofthehairandhavealldifferedintheirconclusionsonthe
numberofspeciespresent(Table1).Severalrecentfaunallistshaveacceptedasvalidjustthe
PLOSONE|DOI:10.1371/journal.pone.0144544 December10,2015 2/26
CrypticArcticDiversity
fivemoreclearlymorphologically-diagnosablespecies(interpretedinabroadsense,fromthe
shapeofthefemalehead,malegenitalia,etc.):B.alpinus,B.polaris,B.balteatus,B.neoboreus,
andB.hyperboreus[10,33–35].Butvariationincolourpatternpromptedsomeauthorsto
questionwhetheratleasttwomorespeciesmightbeaddedtothislist:B.pyrrhopygus(but
usingthejuniorsynonymsB.diabolicusFrieseandB.alpiniformisRichardsforbeeswiththe
Scandinaviancolourpatternofthisspecies)andB.kirbiellus,eventhoughbothlackedclearly
discretediagnosticmorphologicalcharacters[36–38].ThecolourpatternsofAlpinobombus
specieshavebeendescribedasbeingbothvariablewithinspecieswhileshowingclosesimilari-
tiesamongspecies[36,37,39–43].Thesespeciesareunusualevenamongbumblebees[10,44]
foroccupyingextremearcticoralpinehabitats[40,43,45].Therefore,atleastinthefarnorth,
theyco-occurwithfewornootherbumblebeespecies[36,37,43,46],whichsimplifiesthesys-
temforanalysisifmimicrywereinvolved.
WeusesequencesforpartsoftheCOIandPEPCKgenesfromfreshlycollectedspecimens
andfrommuseumspecimenstorecognisetheevolutionarilyindependentlineagesofthesub-
genusAlpinobombusasspeciesandtoestimatethephylogeneticrelationshipsamongthese
species.Wethentestwhetherthecolour-patternpolymorphismsaremorelikelyeithertobe
ancestral,ortohavearisenmorerecentlyandconvergentlywithinthedaughterspecies.From
ouranalysesweconclude:(1)thatcoalescentsfortheCOIgenesupportnearlytwiceasmany
species(ninecf.five)ashavebeenrecognisedfromskeletalmorphologyandthatthreeofthese
species,includingonetaxonnotrecognisedpreviously,arealsosupportedbyPEPCKpolymor-
phisms;and(2)thatsimilarityincolourpatternamongpartsofthesespeciesislikelytohave
arisenfrombroadlyconservedancestralpolymorphisms,notfromconvergence.Thework
reportedherebuildsonasurveyofthemorphologyandcolourpatternsoftheNorthAmerican
Alpinobombus[42]andispartofarevisionarystudyofAlpinobombusbumblebeesworld-wide,
whichwilltreattypespecimensandnomenclaturalissuesseparately.
Table1. ListsandnumbersofSpeciesoftheSubgenusAlpinobombusfromprevioustaxonomicrevisions.a
Richards1931[39] Skorikov1937[43] Milliron1973[36] Løken1973[37] Thisstudy
alpinus alpinus alpinus alpinus alpinus
pyrrhopygus
arcticus arcticus polaris arcticus polaris
kincaidii kincaidi
balteatus balteatus balteatus balteatus balteatus
tristis
kirbyellus kirbiellus
neoboreus neoboreus ? neoboreus
strenuus strenuus strenuus ?
unnamed
natvigi
hyperboreus hyperboreus hyperboreus hyperboreus hyperboreus
Total 7 8 6 4 9
Splitb 2 3 0 0 -
Lumpedb 3 3 2 3 -
aSpeciesnamesandspellingsarethoseusedintheoriginalpublications.TherevisionbyLøkendidnotcovertheNewWorldfaunaandsodidnottreatB.
neoboreus/B.strenuus.
bCountsofsplitandlumpedspeciescomparedtothisstudyexcludethe‘unnamed’taxonthatwetreat(conservatively)asunsampledpreviously.
Authorsofthetaxonnames:alpinus(Linnaeus),arcticusKirby,balteatusDahlbom,hyperboreusSchönherr,kincaidiiCockerell,kirbiellusDahlbom,natvigi
Richards,neoboreusSladen,polarisCurtis,pyrrhopygusFriese,strenuusCresson,tristisFriese.
doi:10.1371/journal.pone.0144544.t001
PLOSONE|DOI:10.1371/journal.pone.0144544 December10,2015 3/26
CrypticArcticDiversity
MaterialsandMethods
Ethicsstatement
Nohumansorvertebrateanimalswereusedinthisresearch.Fieldpermits:(Russia)Bumblebees
ofthesubgenusAlpinobombusarenotincludedinanyRedListsandarenotlistedunderthelaw
‘Onenvironmentalprotection’inRussia.Noneoftheareaswherefieldworkwascarriedoutare
private.OnlyMeduzaBayinWestTaimyrisapartoftheGreatArcticReserve,wherethe
researchwasconductedwiththepermissionoftheReservemanagement.Specimensfromthe
researchcollectionofNovosibirskpassedtheStateveterinarycontrolbeforeexportonloan.
(Norway)NopermitswereneededforcollectingoftheNorwegiandata.
(Sweden)NoneofthebeeswerecollectedwithinNationalParksorNatureReserves.They
arenotsubjecttoanycollectionrestrictionsandthereforepermitsarenotrequired.Atpresent
thereisnolegislationorrestrictionsthatregulatestheexportofgeneticsamplesfromSweden.
ThereisnootherrestrictiononthetransferofscientificmaterialofthissortbetweenEUmem-
berstates.
(Denmark/Greenland)TheGreenlandEcosystemMonitoringCoordinationGroupatthe
NationalEnvironmentalResearchInstitute,AarhusUniversity,approvedourresearchpropos-
alsforaccessandresearchactivitiesinboth2010and2011.
(Canada)Nopermitswererequiredforcollectionsitesvisited.
(USA)CollectionsfromtheRockyMountainBiologicalLaboratoryinColoradoweremade
accordingtoanagreementbetweentheRMBLandtheUnitedStatesForestService.
WebringtogethersamplesoftaxaofthesubgenusAlpinobombusfromthroughouttheir
globaldistributions.Taxonomicandgeographicalbreadthisessentialinataxonomicrevision
forachievingarepresentativesampleinordertoassessthefullrangeofpatternsofvariation
andrelationship[47,48].Thisisespeciallyimportantforreducingthebiasthatmightother-
wiseresultifsamplingwererestrictedtojustoneortwoisolatedregionsorweretoexclude
majorlineages.Tocoverallvariation,wemadeapreliminarysurveyofspecimensfrom
museumcollections(Fig1;seethesupportinginformation).
TorecognizespeciesofthesubgenusAlpinobombus,weacceptthebroadlyheldconceptof
speciesasevolutionarilyindependentlineages(EILs)[1].Torecognizethesespeciesinpractice,
weusestatisticaltestsdesignedtoidentifygenecoalescents,whichprovidedirectevidencefor
EILs[49].Coalescencemethodsaresuggestedtobeespeciallyusefulcomparedtolessdirect
indicators(includingcriteriarelatedtomaterecognitionandreproductiveisolation)because
theycandetectlineageseparationeveninitsearlystages[1,3].Weuseoneofthefastestevolv-
ing,mosthighlyvariablemitochondrialgenes,codingforCOI(cytochromeoxidasesubunitI).
Thisisparticularlyappropriatefordetectingspeciesbecauseofitsrapidcoalescencewithinlin-
eages[3,50].Fromamongrecentlycollectedspecimensshowingthemostobviousmorpholog-
icalandcolourvariation,wesequencedthe‘barcode’regionoftheCOIgene(Fig1).
Mostoftheextraction,amplification,andsequencingworkwasdoneattheBiodiversity
InstituteofOntario,Guelph,usingstandardprotocols[51]andprimers[52].Aminorityof
specimenswereprocessedattheInstituteofApiculturalResearch,Beijing.Becausethegeo-
graphicrangeoccupiedbyAlpinobombusislargeandmanyareasaredifficulttoaccess,mito-
chondrialDNAwithmanycopiespercellhastheadvantageofbeingmoreeasilyextracted,so
thatevenpinnedmuseumspecimenscanbesampled.Although70%ofAlpinobombusspeci-
menssurveyedareknowntobelessthanacenturyold,only8.6%arelessthan10yearsold.A
problemwitholdermuseumspecimensisthatthesequencesobtainedareoftennotoffull
length[53].SequencealignmentsweremadeusingtheClustalWfunctioninMEGA(version
6.06,accessed2014:megasoftware.net[54])andaminoacidtranslationswerecheckedwith
EMBOSSTranseq(accessed2014:ebi.ac.uk/Tools/st/emboss_transeq/).
PLOSONE|DOI:10.1371/journal.pone.0144544 December10,2015 4/26
CrypticArcticDiversity
Fig1.GlobalSamplingoftheSubgenusAlpinobombus.Greyspotsshowsamplesitesforthe4345
specimensexamined(Alpinobombusspeciesareunknownfromthetropicsorfromthesouthern
hemisphere;collectionslistedinTableAinS1File);blackspotsshowthesitesforthe173sampleswithCOI
barcodes;andquestionmarksshowthesitesfordoubtfulrecords(specimenssupposedlyfromsitesthat
combinelowlatitudewithlowelevation).Polarprojection,NorthPole(starred)atthecentreofthemap.
TerrestrialnationalboundariesandtheArcticCircleareshownasnarrowgreylines.ImagecreatedinArcGIS
usingWorld_Shaded_ReliefbasemapwhichisCopyright2014ESRI.
doi:10.1371/journal.pone.0144544.g001
Tocheckforpossiblemismatchesbetweentreesfordifferentgenes,andhenceforpossible
mismatchesbetweengenetreesandspeciestrees[49,55],wealsosequencedthenucleargene
PEPCK(phosphoenolpyruvatecarboxykinase;accessionnumbersforsequenceslistedin
TableAinS2File,obtainedusingprimersfrom[32]).Thisgenehasbeenpopularforstudies
ofbumblebees[11,30,32].
Torecognisespecies,single-thresholdgeneralmixedYule/coalescent(GMYC)modelsseek
atransitionexpectedinthetree-branchingratesbetweeninterspecificbranchesandintraspe-
cificbranches[56–61].GMYCanalysisrequiresanultrametricestimateofthephylogenetic
treethatincludesonlyuniquehaplotypes[59,62](T.Barraclough,pers.comm.)inorderto
avoidspuriousinflationofestimatesofterminalbranchingrates.Aftersortingthesequences
bydecreasinglength,thelongestexamplesofeachuniquehaplotypearerecognisedusingCol-
lapse(version1.2,accessed2011:softpedia.com/get/Science-CAD/Collapse.shtml),which
ignoressiteswithmissingdata.Asoutgroupsforrootingthetreewiththisfastgene,weinclude
speciesfromcloselyrelatedbumblebeesubgenera[32]:B.(Cullumanobombus)rufocinctus
Cresson,B.(Pyrobombus)vagansSmith,B.(Bombus)ignitusSmith,B.(Bombus)terrestris
(Linnaeus),andB.(Bombus)cryptarum(Fabricius).Thebestnucleotide-substitutionmodel
forCOIaccordingtotheBayesianinformationcriterion(BIC)obtainedfromMEGAisthe
generaltime-reversiblemodelwithagamma-frequencydistributionofchangesamongsites
(GTR+Γ).BEAST(version1.8.0,accessed2013:beast.bio.ed.ac.uk)wasusedforBayesiananal-
ysisofmultiplemodeltrees[63].Theclockmodelwassettotheuncorrelatedlognormal
(relaxedclock),thetree-speciationpriorwassettoaconstant-sizecoalescentprocess(consis-
tentwiththenullhypothesisthatthereisasinglespeciesinthedata),andthechainlengthfor
theMarkov-chainMonteCarlo(MCMC)algorithmwassettotwobilliongenerations,with
samplingofthetreesevery200,000generations.ThesampleofresultingtreesfromtheMCMC
PLOSONE|DOI:10.1371/journal.pone.0144544 December10,2015 5/26
CrypticArcticDiversity
algorithmwasexaminedusingTracer(version1.6.0,accessed2013[63]),whichshowedthat
stationarityhadbeenachievedwithinthefirst1%ofthetotalMCMCgenerations.Thelarge
numberofMCMCgenerationswasneededwithourdatatoincreaseeffectivesamplesizes.A
maximumclade-credibilitytreewasobtainedfromthepostburn-intreesampleafterrejecting
thefirst1%ofsampledtreesusingTreeAnnotator(version1.8.0,asforBEAST).Afterremoval
oftheoutgroups,theGMYCmodelwasappliedtothetreeusingtheSPLITSlibrary(version
1.0–11,accessed2011:r-forge.r-project.org/projects/splits/)runningontheRplatform(ver-
sion2.12.1,accessed2011:www.r-project.org).Theoldestavailablename[64]isappliedto
eachcoalescentgroupasaprospectivespecies.Multiplerunsofallanalysesweremadeandthe
stabilityoftheresultschecked.
TochecktherobustnessoftheGMYCresultsbytakingintoaccounttheuncertaintyinesti-
matesofphylogeny,weappliedaBayesianGMYCanalysis[59]usingthebGMYClibrary(ver-
sion1.0.2,accessed2015:sites.google.com/site/noahmreid/home/software)ontheRplatform.
bGMYCwasappliedtothelast100treesfromthesampleof10,000treesfromtheBEASTrun
oftwobillionMCMCgenerations(asamplerepresentingthelast20milliontrees)thathad
beencheckedpreviouslywithTracerforstationarity.bGMYCwasrunwiththedefaultsettings
(MCMC50,000generations,burn-in40,000,thinning100).
Asanothercheckonthenumberofspeciesrecognised,weapplyanotherrelatedapproach
fordiscoveringspeciesdesignedforusewithsingle-locusdata,lookingforchangesinthenum-
bersofnucleotidechangesalongbranchesfrombetween-towithin-speciesrelationships,
basedonPoisson-treeprocesses(PTP:[65]).PTPanalysisislessdemandingoftreeinforma-
tionthantheGMYCapproachandthereissomeevidencethatitcanperformbetter[65].We
applytheBayesianimplementationonthebPTPserver(accessed2015:species.h-its.org)toa
metricmaximum-clade-credibilitytreefromCOIbarcodesobtainedwithMrBayes(version
3.1.2,accessed2011:[66]),usingthesamedata,outgroups,andnucleotide-substitutionmodel,
fourchains(temperature0.2),and100milliongenerationsoftheMCMCalgorithmwitha1%
burn-inselectedusingTracer.WeusedthedefaultbPTPoptionsafterremovingoutgroups
fromtherootedtree.
ThevariationincolourpatternsforthesubgenusAlpinobombusincludesmanysubtledif-
ferences[42],whicharesimilarinthetwosexes.Inallcasescolourpatternsrefertothecolour
ofthehaironvariousbodyregions,nottothecolourofthebodysurface,whichisbrownor
nearlyblackthroughout.Generally,weobservethatthepresenceofnon-blackhaironthedif-
ferentbodysegmentsisnotindependent,butappearstobeconditionallydependentamong
segments[8](e.g.therearenoAlpinobombusbeeswiththehaironmetasomaltergum2black
andontergum3yellow,orontergum4orangeandontergum5black,althoughinbothcases
thereverseconditionsarecommon).Thereforeitshouldbereasonabletobeginbycoding
bumblebeecolourpatternsforthepresenceorabsenceofareasofpalehairandthenlateradd-
ingcharactersasmodifiersfortheextentofthepalehair[26,67].Asafirstapproximation,we
assumethatAlpinobombuscolourpatternscanbereducedtoarepresentationastwoprincipal
characters,eachwithtwostates.Character(1):whethertherearetransverseyellowbandsin
thehaironthebody,usuallyanteriorlyandoftenposteriorlyonthethoracicdorsumand/oron
metasomalterga1–2(stateB,banded:minimallywithanobviousyellowbandonmetasomal
tergum2,varyingfrompalestrawyellowtodarkerorangebrown),orwhetherthehairinthese
areasisblackornearlyblack(stateU,unbanded).Character(2):whetherthereisobviouspale
hair(mostoftenredororange,butsometimesyelloworwhite)onthe‘tail’,consistingofmeta-
somalterga4–6(stateP,pale:minimallycoveringtergum5or6),orwhetherthehairofthetail
isblackornearlyblack(stateD,dark).Themostwidespreadcolourpatternbothamongall
bumblebees[26]andamongthesister-grouptothesubgenusAlpinobombus,thesubgenus
Bombussensustricto[19],hasboththeyellowbandsandapaletail.Replacementofthepale
PLOSONE|DOI:10.1371/journal.pone.0144544 December10,2015 6/26
CrypticArcticDiversity
bandsandthepaletailwithblackcanalsobepolymorphicwithinsomeBombuss.str.species,
althoughthisisrelativelyrare(occurringin3/17and1/17speciesforthetwocharactersrespec-
tively).Colourcharacterswerescoredforasampleof1646Alpinobombusspecimensofboth
sexesfromacrossthedistributionrange.Toreconstructthehistoryofcolour-patterncharac-
ters,weestimatethespeciestreeusingBEASTlinkedtreesfromCOIandPEPCKexonand
intronsequences.Thebest(unlinked)nucleotide-substitutionmodelsaccordingtotheMEGA
BICareHKYforboththePEPCKexonandintrons,thetree-speciationpriorwassettoa
birth-deathprocessappropriateforamulti-speciestree,andfromexaminationoftheresults
withTracer,theMCMCalgorithmwassettorunfor1.5billiongenerations.Outgroupsand
othersettingswereasforthefirstanalysis.NofossilsofAlpinobombusspeciesareknownfor
datingpartsofthetree,sothephylogenywascalibratedwithadatefromamolecularstudy:
Hines[68]estimatedtheageofdivergencebetweenthesubgenusAlpinobombusandthesubge-
nusBombuss.str.tobe13Ma.Mesquite(version3.02,accessed2015:mesquiteproject.org
[69])wasusedtoreconstructancestralcharactersstatesonthistree.Weusedparsimonyanaly-
sisbecausewewantedtoretaininformationonalternativeexplanationsratherthanjustthe
mostlikely.Wecodedpolymorphicpopulationsasathirdintermediateorderedstate,allowing
statechangesineitherdirection([70];W.Maddison,pers.comm.).
Results
Wefound46uniquehaplotypesamong173COI-barcodesamplesofthesubgenusAlpinobom-
bus(accessionnumberslistedinTableAinS2File).TheseCOIdataareinformationrich,with
92/658nucleotidesitesphylogeneticallyinformative.TheGMYCmodelsshowasignificant
changeinbranchingratethroughtimeintheCOIbarcodetree(likelihoodratio14.03between
theGMYCmultiple-speciesmodelandthenullmodelthatthereisasinglespeciesinthe
group,p=0.0028).Thisthreshold(at-0.0041substitutionspernucleotide)leadsustorecog-
niseninecandidatesforprospectivespecies(witha95%confidenceintervalof8–11species)
withinAlpinobombusasinFig2,whichalsoshowsthateachofthesespeciesisstronglysup-
portedasamonophyleticgroup.
ThebGMYCanalysisshowedstationarityandahighmodalcoalescent/Yuleratioconsistent
withsuccess.TheposteriorprobabilitydistributionisshownagainstthefirstsampletreeinFig3
toprovidea‘heat’mapoftheprobabilitiesthatthehaplotypesareconspecific.Adoptingathresh-
oldprobabilityof0.5fromtheposteriormeanfromtheanalysis(correspondingtoamoderate
position,midwaybetweena‘splitter’anda‘lumper’)recognisesninespecieswithinthesubgenus
Alpinobombusonthediagonal(unsurprisingly,thespecieswithmorehaplotypeshavemorestruc-
turewithinthem).ThesearethesameninespeciesasareidentifiedintheGMYCanalysisinFig2.
TheBayesianPTPsolutionwiththemostsupport(Fig4)recognisesthesamenineprospec-
tivespecies(witharangeof7–30species),althoughtheindividualBayesiansupportvaluesfor
thespeciesarenothigh(0.48–0.79).
Asexpected,wefoundmuchlessvariationinthePEPCKgeneamongspeciesofthesubge-
nusAlpinobombus.Only4/903nucleotidesitesinoursequencesareuniquelydiagnosticfor
species,withuniquePEPCKnucleotidechangessupportingthespeciesB.alpinus(baseposi-
tion351),B.neoboreus(bp755),andtwofortheunnamedspecies(bp360,533)(Table2).
AdditionalPEPCKnucleotidedifferencesgivefurthersupportforthedistinctionbetweenthe
speciespairB.neoboreus/unnamed(bp769).TheestimateofphylogenyforAlpinobombus
speciesfromthecombinedgenedataisshowninFig5.Thisshowsstrongsupportforall
groupsexceptforthepositionofB.alpinus,whichremainsuncertain.
TheprincipalcolourpatternsrecordedforthesubgenusAlpinobombusshowthreeofthe
fourpossiblecombinationsofthetwostatesforthetwocharacterswesurvey(Table3,Fig6):
PLOSONE|DOI:10.1371/journal.pone.0144544 December10,2015 7/26
CrypticArcticDiversity
Fig2.RecognisingSpeciesoftheSubgenusAlpinobombuswithGMYC.The173sequencesreducedto46uniqueCOI-barcodehaplotypesona
BEASTultrametricgenetree(outgroupsnotshown).Thesinglethreshold(T=-0.0041)fromtheGMYCmodelisshownbytheverticalgreybarandthe
intersectinglineagesareinterpretedassubtendingnineprospectivespecies(blackspotsshowthecoalescentnodeforeachspecies).Thetreeisthe
Bayesianultrametricmaximum-clade-credibilitytreefromasampleoftreesafter1%burn-infrom2billiongenerationsoftheMCMCalgorithminBEAST.
ValuesnexttothenodesareBayesianposteriorprobabilitiesshowingbranchsupport.Eachhaplotypeisrepresentedbyoneofthelongestsample
sequences,labelledwithaspeciesname,acodethatconsistsofanidentifierfromtheprojectdatabaseand(afterthehyphen)fromtheBOLDdatabase,
followedbyitsgeographicorigin.
doi:10.1371/journal.pone.0144544.g002
PLOSONE|DOI:10.1371/journal.pone.0144544 December10,2015 8/26
CrypticArcticDiversity
Fig3.RecognisingSpeciesoftheSubgenusAlpinobombuswithBayesianGMYC.The173sequencesreducedto46uniqueCOI-barcodehaplotypes
ononeBEASTultrametricgenetree(outgroupsnotshown).Theposteriorprobabilitydistribution(right,colourscalefarright)isplottedagainstasampletree
fromBEAST(left)toprovidea‘heat’mapoftheprobabilitiesthathaplotypesareconspecificbybGMYC.Blackspotsshowthecoalescentnodeforeach
speciesfromFig2.
doi:10.1371/journal.pone.0144544.g003
UP,unbandedpaletails;BP,bandedpaletails,andBD,bandeddarktails.Thefourthstate,
entirelyblack(UD),isnotrecordedhere,althoughonespecimenofB.balteatusfromKara-
ginskiyIsland(Kamchatka,Russia)comesclose,withonlyanarrowandincompleteyellow
bandonmetasomaltergum2.Fig7showsthedistributionsofthethreeprincipalcolourpat-
terns.TheUPcolourpatternisthemostrestrictedgeographicallyandisconcentratedprimar-
ilyinEurope;UPalsooccursinisolatedpocketsinRussiaandinNorthAmericabutis
unrecordedfromGreenland.Incontrast,theBPandBDpatternsco-occurbroadly,bothtaxo-
nomically(Table3)andgeographically(Fig7).ThefrequencyoftheBDcolourpatternrelative
toBPcolourpatternmayincreasetowardsthenorth(Fig7).Nonetheless,thethreeprincipal
colour-patterngroupsareeachsharedbythreeofthenineprospectivespecies(Table3;B.
PLOSONE|DOI:10.1371/journal.pone.0144544 December10,2015 9/26
CrypticArcticDiversity
Fig4.RecognisingSpeciesoftheSubgenusAlpinobombuswithBayesianPTP.The46uniqueCOI-barcodehaplotypesasinFig2onaMrBayes
metricgenetreewiththeBayesianPTPsolutionwithhighestsupport,showingnineprospectivespecies(blackspotsshowthecoalescentnodeforeach
species;outgroupsnotshown).Thetreeisthemaximum-clade-credibilitytreeafter1%burn-infrom100milliongenerationsoftheMCMCalgorithmin
MrBayes.ValuesabovethenodesarePTPBayesiansupportvaluesthatalldaughterhaplotypesbelongtoasinglespeciespopulation;valuesbelowthe
nodesareBayesianposteriorprobabilitiesshowingbranchsupport.HaplotypeselectionandlabelsasinFig2.Thescalebarforbranchlengthsshows0.02
substitutionsperbaseposition.
doi:10.1371/journal.pone.0144544.g004
pyrrhopygus,B.polaris,andB.balteatus),aswellasbeingrecordedfromboththeOldWorld
andNewWorldregions,aswidespreadpolymorphisms.Whilethereareoftensmalldifferences
betweenthemostfrequentpatternsforeachspecies,nonethelessinallcasesitispossibleto
findspecimensthatresembleotherspeciessocloselythatdiagnosisbycolourpatternisnot
alwayspossible.
PLOSONE|DOI:10.1371/journal.pone.0144544 December10,2015 10/26