Table Of ContentOdonatologica37(2):101-109 JuneI.2008
A phylogenyof Celithemis inferredfrom
mitochondrialandnuclearDNAsequence data
and morphology
(Anisoptera: Libellulidae)
G.M.Baskinger¹,J.L.Ware¹,D.D.Kornell¹+, M.L.May¹* andK.M. Kjer²
1DepartmentofEntomology,RutgersUniversity,NewBrunswick, NJ08901,United States
2 DepartmentofEcologyEvolution and Natural Resources,RutgersUniversity,New Brunswick,
NJ08901,UnitedStates
ReceivedNovember 9,2007/ReviewedandAcceptedDecember 14,2007
Thedragonfly genus Celithemis consists of8 spp.,someofthem commonand
brightlycolored,thatareconfined largelytoeasternNorthAmerica.Severalspp.have
beenusedinbehavioral,ecological,andmorphologicalstudies,buttheirintrageneric
phylogenyisunclear.Inthispaperisprovidedaphylogenybasedonmorphologyand
ondata frommitochondrial andnuclear DNAsequences ofmultipleindividuals of
eachspecies.Thegenus appearstobemonophyletic,withonenested speciespair(C.
amanda+C. martha)receivingstrongbootstrapsupportbybothparsimony ormaxi-
mum-likelihood criteriaaswellashighBayesianposteriorprobability.Asecondgroup
(C.bertha,C.elisa,C. ornataand C.fasciata) iswell-supportedin Bayesiananalysis
butonly weaklyby parsimonyand maximum-likelihood bootstrapvalues. C. verna
and C.eponinaareprobablybasaltoboththesegroups,buttheirrelationshiptoeach
other is unclear. Allindividuals assignedtoaspecies recognizedonmorphological
-groundswererecovered asmonophyletic.Theproblematictaxa,C. monomalaenaand
C. bertha leonora,areshown definitivelytobesynonyms ofC.fasciataandC.bertha,
respectively.
INTRODUCTION
Aside from the inherentinterestof theirevolutionary history, Odonata have
been important in research onfreshwatersystemsas wellas behavioralecology,
sexual selection, and insect physiology and functionalmorphology. Thegenus
Celithemis, in particular, hasbeenused in studiesof functionalmorphology of
*Direct reprintrequeststothisauthor
+Presentaddress: 12418N.E. 137thPlace,Kirkland,WA98034,United States
102 G.M.Baskinger,J.L.Ware, D.D.Kornell,M.L. May& K.M.Kjer
thepenis(MILLER. 1981),reproductive behavior(MILLER, 1982),competition
(COWLEY & JOHNSON, 1992), energybalance (BENKE& BENKE, 1975,
BENKE, 1976),and predation pressure (GRESENSet al., 1982). Understand-
ing relationships amongtaxa isessentialforcomparative assessment ofadaptive
similaritiesanddifferencesinbehavioralecology. Thusestablishmentofareliable
phylogeny isaprerequisiteforanydeep understandingofselectionandcharacter
evolutioninCelithemis, as inany groupoforganisms.
HAGEN (1861) establishedthegenus Celithemisto includeLibellula eponina
Drury, Celithemissuperbum sp. n. and the fossil species, C. cellulosasp. n. Ce-
lithemissuperbum was moved to the monotypic genus,Pseudoleon Kirby, 1881,
and C. cellulosais clearly distantfrom our current conceptof Celithemis(see
GARRISON etal., 2006.for afulldiagnosis) byvirtueof having themidribof
theanalloop stronglybent,andhaving manymorecells intheforewing triangle
and supratriangle and manymore antenodalcrossveins than in extant species.
Thusthesetwo species are notconsideredfurther.
Celithemishas not beenrevised since WILLIAMSON (1922), and no mod-
ern analysis ofthe species boundariesorrelationships hasbeenattempted. The
namedtaxa.C. monomalaenaWilliamson, 1910,and C. berthaleonoraWestfall,
C1952, haverecently beenconsideredsynonymsofC. fascia.ta Kirby, 1889,and
b. bertha Williamson, 1922, respectively, but unequivocal datasupporting syn-
onymy havenotbeenpublished. KORNELL (2003)investigated phylogeny and
species distinctions using molecularand morphological data, but several gaps
andinconsistenciesinherdatahaveencouraged thisre-examination.
Our principal objective hereis to deriveawellsupported phylogeny resolving
therelationships amongspecies ofCelithemis.Sincemorphological featureslead
to littleresolution, we combinethis withsequencedatafromthemitochondrial
protein coding genecytochrome oxidase I(COI)andfromthe D2regionofthe
nuclear28S rDNA gene.We also provide datasupporting thesynonymy of C.
monomalaenaand C.berthaleonora.
MATERIALAND METHODS
MOLECULARTECHNIQUES — Twenty-eightspecimensformolecularanalysis,includingrep-
resentatives ofallputativespeciesand subspeciesofCelithemisandthree outgroupspecies, Cordulia
shurtleffi, Libellula pulchella,and Leucorrhinia glacialis,werefieldcollected ortakenfromthecollec-
tions ofMLMand RutgersUniversity.Tissuewasobtainedbyexcisingthoracicmuscle orremoving
aleg,which wasthen placedinanEppendorftube. Extractionproceduresfollowed manufacturer’s
recommendations (QIAGEN, DNeasyTissuehandbook).
AftertheDNAwasextracted,polymerasechainreaction (PCR)wasused foramplificationofa
COI fragmentfrom the mtDNA and the D2 regionof thelargesubunit (28S)nuclear ribosomal
codingregion.Weused theCl73IFandCI14RprimersofArtisset al.(2001)forCOIamplification
andtheD20DUPandD2DnBprimers(5TGCTTGAGAGTGCAGCCCAA3’and 5’CCTT-
GGTCCGTGTTTCAAGAC3’).WeamplifiedtheDNAwith Ipieach ofupanddown primer,
1to 5pioftemplateDNA, 12.5piMaster Mixsolution,and 5.5to 9.5piddH.O.
Phylogeny ofCelithemis 103
AmplifiedDNA wasseparatedona 1.5%low meltingagarose gel(FMC Bioproducts),thenpu-
rified usingQIAquick PCR Purification Kit Protocol (QIAGEN) accordingtothe manufacturers
instructions. The purifiedproductwassequencedin bothdirectionsusingBigDyeTerminatorsCy-
cleSequencingKit (AppliedBiosystems) and anAB1 3100capillary sequencer. COIsequences were
alignedusingClustal(THOMPSON, 1997)then,toassurehomology,translatedtothecorrespond-
ingproteinsequenceinMacClade (MADDISON& MADDISON, 1992).D2 sequencealignments
weremadeusingClustal,andtheresultingfileswerethen alignedmanuallyin MicrosoftWordusing
the structural methodsdescribedin KJER etal.(1994).KJER (1995),KJER etal.,(2007)andsec-
ondarystructuremodels based onGUTELL et al.(1993).
D2 sequences onlywereprovidedforLeucorrhinia orientalis (specimenobtained fromDr Elena
Malikova)and a Leucorrhinia sp sequence obtained fromGenBank (Acc. # AY859583)).COIse-
quenceswerescoredasmissingdata forthese twospecies.
Thirty-sixmorphologicalcharacters,including3larvalcharacters,werescoredforallputativetaxa
ofCelithemis and forthe3outgroupspecies. Alladult characters were observed directly,mostlyus-
ingaWild™stereomicroscope.CharactersofthesecondarygenitaliawereexaminedwithanHitachi
S510scanningelectronmicroscopeaftersputtercoatingwith gold-palladium.Dataonlarval char-
actersweretakenfromNEEDHAMetat. (2000).
PHYLOGENETIC ANALYSIS - Each datapartitionwas analyzedindividuallyand in com-
bination. Thepartitionedanalyses werenotseriouslyincongruent,althoughthey differed in their
level ofresolution. MorphologyandD2 gavepoorresolution,while theCOItreewaswellresolved.
Theanalysesofthecombined dataarepresentedhereafter.Amaximum parsimonyheuristic search
of 1,000random replicates wasconducted usingPAUP— 4.0bl0(SWOFFORD, 1999)withTBR
branchswapping.Toassessbranchsupport,400bootstrappseudoreplicates(FELSENSTEIN,1985)
wereperformedusing 10random addition searches perpseudoreplicate.Modeltest 3.06(POSADA
&CRANDALL, 1998)wasused to obtainanevolutionarymodelforeachmolecular datasetinthe
Bayesian and maximum likelihood analyses; in eachcasethe GTR+1+Twas estimated tobe the
best model formolecular data,with mk model used formorphology.Usingtheprogram MrBayes
(HUELSENBECK,2000),Bayesiananalysis(4MCMCchains: 1cold,3hot)wasrunonthecom-
bined data for2,873,500generations;the first 100,000werediscardedasburn-in.
MORPHOMETRICANALYSIS - WILLIAMSON(1910)distinguishedC. monomalaenafrom
C.fasciatabased onsize,wingcoloration,and severalvenational characters.Numerous authors(e.g.
NEEDHAM etal., 2000)have doubted thevalidityof C. monomalaem, and CARLE (1982)indi-
catedthatthesupposeddifferenceswereeitherclinalorshoweddiscordantvariation.Nonetheless,no
quantitativeevaluation ofthesupposeddistinctionshasbeenpublished.Wemeasured tothenearest
0.5mm orcounted therelevantcharacters(see Results)in 59-66specimensfromtheNational Mu-
seumofNatural History (NMNH),theFlorida State Collection of Arthropods(FSCA),and the
collection ofMLM,usingaWildstereomicroscopewhennecessary. Datawereplottedasafunction
oflatitude andevaluated bylinear regressiontoassessgeographicvariation ofeach character.
RESULTS
Figure 1 presentsastrictconsensusofthefourmostparsimonioustrees(length
1065).Celithemisisrecoveredas monophyletic withgood support.Allnamedtaxa
arerecoveredexcept thatthetwo C. amandaare ina polytomywith C. martha.
Note also that C.fasciata and C. monomelaenaare mutually polyphyletic and
C. b.berthaisparaphyletic relativeto C. berthaleonora. C. vernaand C. eponina
formapolytomy withanapparentcladethatincludesall otherspecies (but this
cladeissupported with very low bootstrap support). Thestronglysupported C.
104 G.M. Baskinger,J.L.Ware,D.D.Kornell,M.L. May& K.M.Kjer
amandaandC. marthacladeis apparentlybasalinthepectinate arrangementof
the remaining species.
Figure 2 is themajorityruleconsensustreeobtainedby Bayesian analysis. Ce-
lithemisvernaissisterto theremainingspecies, followedsuccessively by C.eponina
andthe C. amanda+ C.marthacladeandfinally apolytomy comprising C. ber-
tha, C. ornata + C. elisa,and C.fasciata. Posteriorprobabilities provide strong
support forthe monophyly ofCelithemisandfor thatofmost of thespecies ex-
Fig. 1. Strict consensusof fourmostparsimonioustrees of length1065 based oncombined data
fromCOI, D2and morphology.Numbers abovethebranches indicatebootstrapvalues > 50%. The
Cnamesinquotations,“leonora”and“monomalaena”,areregardedassy.nonyms ofC. berthaand
fasciata,respectively,but areretained inthetree toindicatespecimensthatexhibit characteristicsof
those supposedtaxa. Superscriptsindicatespecimensforwhich sequence data wereobtained from
E.Malikova (I)orfromGenBank (2).
PhylogenyofCelithemis 105
cept C. amanda, whichis, however, recoveredas monophyletic. C.fasciata and
C. monomelaenaare againmutually polyphyletic, and C. b. leonoraformsa clade
with C. b. bertha. Species groupings are at leastmoderately wellsupported ex-
cept fortheterminalpolytomyandwithinthelatter, thepossible cladeincluding
C. ornata and C. elisa.Itis worthnoting thatBayesian analysis oftheCOIdata
alone (which was the mostinformativedatapartition used) recovered C. verna,
C. eponina, andC.amanda+C.marthainabasalpolytomybutgavefairlystrong
support(93% posterior probability)to the C. ornata + C. elisaclade.
Morphometricanalyses ofspecimens oftheC.fasciata/C. monomalaenaclade
showgraduallatitudinaldinesinhindwinglength,numberof cellsbetweenveins
A andA in thehindwing, numberof hindwing marginalcellsbasalto theanal
2
Fig.2.Majorityruleconsensusof11,096treesobtainedforcombinedCOI,D2,andmorphologydata
fromBayesiananalysis. Posteriorprobabilities>50%areshown abovethecorrespondingbranches.
SeeFig. 1forexplanationofquotationmarksandsuperscripts.
106 G.M.Baskinger,J.L.Ware,D.D. Kornell,M.L.May& K.M.Kjer
loop(Fig. 3A),andextentoftheforewingnodalspot; hindwinglengthalso tends
to increaseclinally fromeast to west(p =0.09,not shown). Charactersclaimed
by WILLIAMSON(1910)to distinguishthetaxa donot varyconcordantly from
northtosouth(Fig. 3B),although specimens fromnorthof38N dotendonaver-
agetohavefewerforewingtriangle andposttrigonal cellsandfewerbasalparanal
cells(see NEEDHAM etal., 2000). Theproportion ofspecimens withyellow at
thebaseofthehindwing, supposedly characteristicofnominateC.fasciata, was
not quantified becauseof variationinitsintensityandextent,but, although this
colorpatternwas clearly morefrequent insouthern specimens, individualswith
completely clearhindwings occurredas farsouthas 32.3°N(GA, CrawfordCo.)
andwithdistinctyellowbasalareas as fara42.2°N(NY, Chataqua Co.).
DISCUSSION
All 8 generally acknowledged species appear tobe valid, monophyletic taxa,
withthepossible exceptionofC. martha, whichcouldbeajuniorsynonymof C.
amanda; this pair is quitesimilaringeneral appearanceandmorphology except
forthe strongdarkening of mature malesof C. martha.We do not advocatea
taxonomicchange, however,pending amorethorough study ofthesetwo nomi-
nal taxa.
Wesuggestthattheputativetaxa C.monomalaenaandC. berthaleonoraherel-
egated tosynonymy withCfasciata and C. bertha, respectively. Inthefirst case,
individualsidentifiedas C.monomalaenaonmorphological groundsare mingled
haphazardly ina cladewithtypical C.fasciata (Figs 1-2).Furthermore,Figure 3
provides no morphological basisfordistinguishing thesetaxaevenas subspecies.
Inthecaseof C.b. leonoraand C. h. bertha, recognition of“leonora”leavesthe
nominotypical formparaphyletic (Fig. 1).Asalready described by WESTFALL
(1952), the subterminal dark wing spot distinguishing “leonora”varies from
about0.5 cm indiameterto vanishingly small, sothat thesupposed subspecies
is doubtfully diagnosable. Moreover, the “leonora”form is not geographically
distinctby virtueofbeingrestricted to theFlorida panhandle butalso occurs at
leastinSouthCarolina,from whichour sequenced specimen originated. Thusit
isgeographically broadly coextensivewithC. b. berthaandhencedoesnotfitthe
usualdefinitionofa subspecies (e.g. MAYR, 1963).
Noformalmorphology-based phylogeny hasbeenproposed nor, to ourknowl-
edge, hasany otherdiscussion ofrelationships withinthe genusappeared since
WILLIAMSON’S(1922) very brieftreatment. One might assume a closerela-
tionship amongC. elisa, C. eponina, andC.fasciata basedonwingcoloration,as
Williamsondid,oramong C. eponina, C.fasciata, C. ornata,and C. vernabased
onlarvalfeatures(NEEDHAM etal.,2000),especially theunusualeyemorphol-
ogy ofC. verna, C.fasciata, andC. ornata,butneitherofthesegroupings issup-
portedhere.Rather, elaboratelypatterned wingsandconicallarvaleyesandlong
Phylogeny ofCelithemis 107
abdominalspines arelikely tohaveevolvedindependently multipletimes.Astrict
consensus tree(not shown) basedon morphology aloneandanalyzed usingthe
maximumparsimony criterion showedlittleresolutionwithinCelithemisexcept
thatitgrouped C.amandaand C. martha,basedonthoracic colorandtheshape
ofthecornuofthe penis, andC. elisa, C. eponina,andC.fasciatabasedon wing
coloration.
Ourhypothesis isnot withoutweakness.Theparsimony tree(Fig. 1),although
well-resolved, provides minimalsupportforinterspecificrelationships. TheBaye-
Fig. 3:(A)Relation ofcell countsandhindwinglength,suggestedbyWILLIAMSON(1910)todif-
ferentiate C.fasciataand C monomelaena,asafunction oflatitude. Lines represent linear regres-
sionoftherespectivemeasurementsonlatitude. Eachquantitydeclined significantly(p<0.001)but
smoothlywith latitude,indicatingaN-Sdine;measurementsofthetransverse extentofthe forew-
ingnodal spotgavesimilar results(notshown;p <0.01).— (B) Proportionofcell counts,suggest-
edby Williamson tocharacterize thesupposednorthern species.C. monomelaena,asafunction of
latitudinal range.Ineach casethehighestproportionoccursatthehighestrangebutwith secondary
peaks atthe lowest range and with minima discordant,at 30-34°N(marginalcells andpostrigonal
cells)or 34-38°N(cells intriangle).
108 G.M.Baskinger,J.L.Ware,D.D. Komell,ML. May&K.M. Kjer
siantree(Fig. 2), however, giveshigh posterior probabilities formostbranches,
aCnd the two trees are concordantinmost respects. There.lative positions of
eponina andthe C. amanda+ C. marthaclade, and the relationships among C.
bertha, C. ornata, C. elisaand C.fasciataare uncertain,butinotherrespectswe
thinkthephylogenies havenumerous commonfeaturesthatcanbeaccepted with
someconfidence.
Wehopethisstudywillencourageadditionalcomparative investigations ofthis
interesting genus.Tomentiononly twointriguingpossibilities:(1)sinceextensive
wingmaculationator beyond the nodusappearsto haveevolvedindependently
two or threetimes, studiesoftheeffectofthis characteronintra-or interspecific
signaling couldbeproductive; (2) similarly, extremely conicallarvaleyes seemto
haveevolvedindependently two or threetimes, so the adaptive advantages and
physiological consequencesofthis unusualmorphology areopento investigation.
Although thegenusisasmallone, itmaywellprovide numerousotheropportu-
nitiesforcomparisons ofbehavioral, morphological, andecological properties.
ACKOWLEDGEMENTS
WearegratefultoBOBBEHRSTOCK,DANADENSON, DANJOHNSON,STEVEKROTZER,
ELENA MALIKOVAand LYNNSMITHforsupplyingspecimensforDNA analysis and toO.S.
FLINTand DAVID FORTH(NMNH) andBILL MAUFFRAY (FSCA)forallowingustoexam-
inespecimensintheircare.SupportedbyNSFGrantDEB-0423834with REUSupplementtoGMB;
NJAESPublication D-08-0S190-04-07,supportedby Stateand U.S.HatchActFunds.
REFERENCES
ARTISS,T,T.SCHULTZ, DA.POLHEMUS&C. SIMON,2001. Molecularphylogeneticanalysis
oftheodonate genus Libellula (Odonata:Libellulidae)basedonmitochondrial cytochrome
oxidase I and 16SrRNA sequence data.Mol. Phylog.Evol. 18:348-361.
BENKE,A.C., 1976. Dragonflyproductionandprey turnover. Ecology57: 915-927,
BENKE,A.C.&S.S.BENKE, 1975.Comparativedynamicsandlifehistories ofcoexistingdragon-
flypopulations.Ecology56: 302-317.
CARLE,F., 1982. Acontribution totheknowledgeofOdonata,pt 2.PhD Diss., VirginiaPolytech.
Inst.St.Univ., Blacksburg,VA.
CROWLEY, PH. & DM. JOHNSON, 1992. Variability and stability ofadragonflyassemblage.
Oecologia90: 260-269.
DRURY, D., 1773. Illustrationsofnaturalhistory,Vol. 2.White,London
FELSENSTEIN, J., 1985.Confidence limitsonphylogenies:anapproachusingthebootstrap. Evo-
lution39:783-791.
GARRISON, R.W,N. VON ELLENR1EDER & J.A. LOUTON, 2006. Dragonflygeneraof the
New World. An illustratedand annotatedkey tothe Anisoptera.John HopkinsUniv. Press,
Baltimore.
GENSENS,S.E.,M L.COTHRA& J.H.THORP, 1982.Theinfluence oftemperatureonthefunc-
tionalresponse ofthe dragonflyCelithemis fasciata(Odonata:Libellulidae).Oecologia53:
281-284.
GUTTELL,R.R., M.WGRAY & M.N.SCHINARE, 1993.Acompilationoflargesubunit (23S
PhylogenyofCelithemis 109
and 23S-like)ribosomal RNAstructures. NucleicAcidsRes. 21: 3055-3074,
HAGEN, H.A., 1861.SynopsisoftheNeuropteraofNorthAmerica,withalistoftheSouthAmeri-
canspecies. Smithson, misc. Colins4: 1-347.
HUELSENBECK, J.P.,2000. MrBayes: Bayesianinferenceofphytogeny.Distributedbytheauthor.
DeptBiol.,Univ. Rochester.
KJER, K.M., 1995. Use ofrRNA secondarystructure inphylogeneticstudiestoidentifyhomolo-
gouspositionsand exampleofalignmentand datapresentationfromthefrogs.Mol. Phylog.
Evol. 5:314-330.
KJER, K.M.,J.J.GILLESPIE&K.A.OBER,2007. Opinionsonmultiplesequencealignment,and
anempirical comparisonofrepeatabilityand accuracy between POYand structural align-
ments.Syst. Biol. 56: 133-146.
KORNELL, D., 2003.PhytogenyofCelithemis Hagen(Libellulidea:Odonata):aclaudisticanalysis.
M.S.Thesis,RutgersUniv.,NewBrunswick,NJ.
MADDISON,W.P.&D.R.MADD1SON, 1992. MacClade version3:Analysisofphytogenyandchar-
acter evolution.Sinauer Associates,Sunderland /MA.
MAYR,E., 1963. Animalspeciesandevolution. BelknapPress,Cambridge,MA.
MILLER,P.L., 1981.FunctionalmorphologyofthepenisofCelithemiseponina(Drury)(Odonata;
Libellulidae).Odonalologica10:293-300.
MILLER, P.L., 1982. Observation on thereproductivebehaviour of Celithemis eponina(Drury)
(Odonata:Libeliulidae)inFlorida. Ent.mon. Mag. 117: 209-212.
NEEDHAM, J.G.,M.J.WESTFALL& M.L.MAY,2000.DragonfliesofNorth America[Revised
edn],Scient. Publishers,Gainesville,FL.
POSADA, D.&K.A.CRANDALL, 1998.Modeltest: testingthemodelofDNAsubstitution. Bio-
informatics 14:817-818.
QIAGEN,2002. QIAquickPCR Purification KitProtocol. QIAquickSpinHandbook,p. 18.
QIAGEN, 2004. Protocol: purification oftotal DNA from animal tissues. DNeasy Tissue Hand-
book, pp. 18-20.
SWOFFOLD, D.L., 1999. PAUP-.PhylogeneticAnalysis UsingParsimony (andothermethods).
Version4.Sinauer Associates, Sunderland,MA.
THOMPSON, J.D.,T.J. GIBSON, F. PLEVNIAK,F. JEANMOUGIN&D.G.HIGGINS, 1997.
TheClustalXwindows interface; flexiblestrategiesformultiplesequencealignmentaidedby
qualityanalysistools. NucleicAcidsRes. 24: 4876-4882.
WESTFALL,M.J.,1952. CelithemisberthainFlorida,with adescriptionofanewsubspecies(Odo-
nata).FlaEnt. 35: 109-116.
WILLIAMSON,E.B., 1910. AnewspeciesofCelithemis (orderOdonata).OhioNaturalist 10: 153-
160.
WILLIAMSON,E.B., 1922.Notes onCelithemis with descriptionoftwonewspecies(Odonata).
Occ.Pap.Mus. Zool., Univ. Mich. 108: 1-22.
