Research Article Molecular Population Structure of Junonia ...downloads.hindawi.com/journals/psyche/2014/897596.pdf · evarete .Neild[ ]alsodesignatednewtypesfor J.evarete and J

Research ArticleMolecular Population Structure of Junonia Butterflies fromFrench Guiana Guadeloupe and Martinique

Amber P Gemmell Tanja E Borchers and Jeffrey M Marcus

Department of Biological Sciences University of Manitoba Winnipeg MB Canada R3T 2N2

Correspondence should be addressed to Jeffrey M Marcus marcusccumanitobaca

Received 29 May 2014 Revised 20 August 2014 Accepted 15 September 2014 Published 12 October 2014

Academic Editor Martin H Villet

Copyright copy 2014 Amber P Gemmell et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Up to 9 described species of Junonia butterflies occur in the Americas but authorities disagree due to species similaritiesgeographical and seasonal variability and possible hybridization In dispute is whether Caribbean Junonia are conspecific withSouth American species Cytochrome oxidase I (COI) barcodes wingless (wg) sequences and Randomly Amplified Fingerprints(RAF)were studied to reveal Junonia population structure in FrenchGuiana GuadeloupeMartinique andArgentina Phylogeneticanalysis of COI recovered 2 haplotype groups but most Junonia species can have either haplotype so COI barcodes are ambiguousAnalysis of nuclear wingless alleles revealed geographic patterns but did not identify Junonia species Nuclear RAF genotypingdistinguished 11 populations of Junonia arranged into 3 clusters Gene flow occurs within clusters but is limited between clustersOne cluster included all Argentinian samples Two clusters included samples from French Guiana Martinique and Guadeloupeand appear to be divided by larval host plant use (Lamiales versus Scrophulariales) Many Junonia taxa were distributed acrosspopulations possibly reflecting patterns of genetic exchange We had difficulty distinguishing between the Caribbean forms Jzonalis and J neildi but we demonstrate that Caribbean Junonia are genetically distinct from South American J evarete and Jgenoveva supporting the taxonomic hypothesis that they are heterospecific

1 Introduction

Buckeye butterflies genus Junonia (Nymphalidae) are animportant model system for experimental research in theLepidoptera [1 2] Junonia species have been widely usedto study the evolution and development of butterfly wingcolour patterns [2ndash9] Experimental tools to manipulate geneexpression developed in Junonia are broadly applicable acrossthe Lepidoptera [10ndash14] Junonia has also been used in studiesof insect endocrinology [15ndash17] and has been an importantsystem for examining the evolution of larval host plantpreference and tolerance to host plant toxins [18ndash21]

Junonia butterflies are found throughout theOld andNewWorld tropics In the Western Hemisphere forms of Junoniaoccur from southern Canada to Tierra del Fuego [22ndash24]and have a complicated taxonomic history In 1775 Cramer[25] identified and described two similar species of JunoniaJ evarete and J genoveva from Suriname a Dutch colony onthe north coast of South AmericaThe species were described

according to the standards of the time (without designatedtype specimens) and the descriptions were accompanied byhand-tinted plates that reproduced the colours from theoriginal watercolour drawings of specimens of each form(republished in [26])

In the 20th century there was considerable disagreementin the scientific community about whether Cramerrsquos twospecies were truly distinct [27 28] or whether all of thespecimens belonged to J evarete [29ndash33] This is a resultof the geographical [29 34] and seasonal [35] variabilityof Junonia and the fact that some Junonia forms closelyresemble one another [36 37] In addition different Junoniaforms share identical karyotypes (119873 = 31) [26 38] andare capable of hybridization and the production of fer-tile offspring [39ndash41] further complicating the process ofassigning names to Junonia specimens These features makeapplying the biological species concept [42] phylogeneticspecies concept [43] or morphospecies concept [44] verydifficult in Junonia Operationally we use the isolation species

Hindawi Publishing CorporationPsycheVolume 2014 Article ID 897596 21 pageshttpdxdoiorg1011552014897596

2 Psyche

concept that defines species as systems of populations suchthat genetic exchange between these systems is limited orprevented by one ormore reproductive isolatingmechanisms[45 46] Identifying and understanding the reproductiveisolating mechanisms operating in Junonia will be of greatimportance in clarifying Junonia taxonomy

Authorities who favoured the two-species hypothesis inJunonia called the larger form J genoveva and the smallerform J evarete In 1985 Turner and Parnell [26] using spec-imens from Jamaica and Florida USA verified the existenceof two Junonia species in both regions However afterconsulting Cramerrsquos hand-tinted plates and comparing themto specimens from Jamaica and Florida Turner and Parnell[26] switched the names so the larger species was now Jevarete and the smaller species was J genoveva Neild [22]using specimens fromVenezuela (geographicallymuch closerto the type locality of Suriname) also confirmed the existenceof two species However Neild [22] unsatisfiedwithCramerrsquos[25] published plates (copies of which differ fromone anotherdue to variation among the watercolourists who tinted themand differences in how the plates aged) consulted Cramerrsquosoriginal watercolours and Junonia specimens from manylocalities in South America Using this reference materialNeild [22] reversed Turner and Parnell [26] so that the largerspecies was again J genoveva and the smaller species was Jevarete Neild [22] also designated new types for J evarete andJ genoveva to facilitate future taxonomic work

Recently L Brevignon and C Brevignon [23 47 48]identified 5 Junonia species (J evarete J genoveva J wahl-bergi J litoralis and J divaricata) from French Guiana Thisrepresents the most diverse assemblage of Junonia in theNewWorld There are two forms of Junonia known from theCaribbean Islands ldquozonalisrdquo and ldquoneildirdquo which were initial-ly recognized as subspecies of mainland J evarete and Jgenoveva respectively [49] and later as two distinct speciesJ zonalis and J neildi [23] In recognizing J neildi and Jzonalis as distinct species L Brevignon and C Brevignon[23] restricted the use of the species epithets J evarete and Jgenoveva to mainland Central and South American forms Ifit were confirmed that the Caribbean forms are actually dis-tinct species with respect to Junonia from the mainland thiswould explain some of the widespread difficulty of assigningappropriate taxonomic names to specimens from FloridaJamaica and elsewhere in the West Indies Finally there aretwo additional Junonia species J coenia fromNorth Americaand J vestina from the Andes mountains of South Americafor a current total of up to 9 species of NewWorld Junonia

The first molecular phylogenetic approaches to under-standing the relationships among the species discussed hereestablished that the New World fauna appears to be mono-phyletic and that the various New World forms are indeedin the genus Junonia [50 51] (some authorities had pre-viously placed these species in the related genus Precis)[52 53] Unfortunately these early studies which incor-porated data from both mitochondrial and nuclear locihad limited taxon sampling including data from only 3New World species [50 51] More recent studies of themolecular phylogeny of New World Junonia [23 54 55]which have better taxon sampling have focused entirely on

the mitochondrial cytochrome oxidase I (COI) locus whichis widely used as a barcoding locus for animal taxa [56 57]Based on mitochondrial haplotype sequences the relation-ships amongmanyNewWorld Junonia species are ambiguousand most species are not reciprocally monophyletic [23 2454] The degree to which recent divergences retained poly-morphisms andor hybridization events contribute to thesepatterns in Junonia is unknown because only mitochondrialmarkers were considered What is apparent is that thereare two very divergent COI haplotype groups (4 sequencedivergence between them) present in New World JunoniaGroup A which predominates in South America and is alsopresent in the Caribbean and Group B which predominatesin North and Central America but which also occurs in theCaribbean and South America [24 54] Sequences belongingto each of these haplotype groups can occur in differentindividuals of the same species at the same locality [24]

The most successful study to date to distinguish betweenNew World Junonia taxa using molecular markers employeda combination of mitochondrial and nuclear markers toexamine populations of Junonia in Buenos Aires ArgentinaBorchers and Marcus [24] used DNA sequences from thenuclear wingless gene and anonymous nuclear loci identifiedby Randomly Amplified Fingerprinting (RAF) (a techniqueused to assess genetic diversity within populations [58ndash60]and gene flow between populations [61]) in addition tosequences from the mitochondrial COI gene They identified3 distinct populations of Junonia from Buenos Aires onepopulation with dark-coloured wings referred to as J evareteflirtea [62] which Borchers and Marcus [24] suggested maycorrespond to J wahlbergi and 2 light-coloured populationsthat correspond to J genoveva hilaris and either a geneticallydisparate population of J genoveva hilaris or an undescribedcryptic Junonia species However the relationship of theseArgentinian forms with J evarete and J genoveva fromSuriname and French Guiana is not known so we refer tothem as J ldquoflirteardquo and J ldquohilarisrdquo

In the current study we extend the genetic tools that wereemployed by Borchers and Marcus [24] to Junonia popula-tions from French Guiana and the French Antilles in orderto study the distinctiveness of the named taxa within andbetween these two localities This will allow an explicit test ofthe 7-species taxonomic hypothesis (2 species in the FrenchAntilles plus 5 species in French Guiana) of L Brevignonand C Brevignon [23] and also detect possible hybridizationevents between named forms By using a common set ofmarkers we will also be able to compare these populations topreviously studied Junonia from Argentina [24]

2 Materials and Methods

21 Specimens and DNA Preparation A total of 104 Junoniaspecimens were collected from the wild as adults rearedfrom wild-collected larvae or reared from eggs laid by wild-collected adults and frozen at minus20∘C (Table 1) DNA was iso-lated from legs removed from each specimen Some samples(42 specimens) were prepared by the Canadian Centre forDNA Barcoding at the University of Guelph as previouslydescribed [23] The remaining samples (62 specimens) were

Psyche 3

Table 1 Number of Junonia specimens included in this study eitherentirely processed in our laboratory or extracted at the University ofGuelph and sent to us for further study

Species and localityDNA extracted

in ourlaboratory

DNA extracted byGuelph and wholegenome amplifiedby our laboratory

J coenia Florida USA 0 2J divaricata French Guiana 0 5J evarete French Guiana 0 6J genoveva French Guiana 32 6J litoralis French Guiana 8 4J neildi Guadeloupe 6 2J neildiMartinique 2 3J wahlbergi French Guiana 0 10J zonalis Guadeloupe 7 2J zonalis Martinique 7 2

processed in our laboratory using the Qiagen DNEasy Bloodand Tissue kit (Qiagen Dusseldorf Germany) as previouslydescribed [24] except that the extractions were performed ina Qiagen QIAcube instrument using the standard instrumentprotocol for purification of total DNA from animal tissueExtracted DNA was stored at minus20∘C

Only 10 120583L aliquots of DNA were available for the 42Junonia specimens processed at the University of Guelphwhich was insufficient for the number of experiments wewished to conduct To produce additional template wholegenome amplification using Illustra Genomiphi V2 (GEHealth Care Life Sciences Pittsburgh PA USA) protocolwas performed as follows 1 120583L of DNA template and 9 120583Lof sample buffer were incubated at 95∘C for 3min cooled to4∘C mixed with 9 120583L of reaction buffer and 1 120583L of enzymeincubated at 30∘C for 90 minutes and then 65∘C for 10minutes and cooled to 4∘C Deionized distilled water wasused as the template for a Genomiphi amplification negativecontrol Genomiphied samples were stored at minus20∘C

22 Mitochondrial Cytochrome Oxidase I Protocol Cyto-chrome oxidase I (COI) PCR products were generated using aseminested two-step amplification with LCO1490 and Nancyprimers followed by a reamplification with LCO1490 andHCO2198 (Table 2) [63 64] Quick-Load Taq 2X Mastermix(New England Biolabs Ipswich MA USA) was used inPCR reactions with total volumes of 25120583L Amplificationprotocols were run on a BioRad MyCycler or S1000 ThermalCycler (BioRad Hercules California USA) for these and allother PCR amplifications unless otherwise specified LCO14-90Nancy PCR reaction conditions were 95∘C for 5 minutes40 cycles of 95∘C for 1 minute 46∘C for 1 minute 72∘C for 15minutes and a final 5-minute extension at 72∘C before beingplaced on a 4∘C hold LCO1490HCO2198 PCR reactionconditions were 95∘C for 5 minutes 35 cycles of 94∘C for 1minute 46∘C for 1 minute 72∘C for 15 minutes and a final5-minute extension at 72∘C before being placed on a 4∘Chold PCR reactions were evaluated by gel electrophoresis

(1 agarose in TAE buffer 78 V for 1 hour visualized withethidium bromide)

Samples that failed to amplify with LCO1490 and HCO-2198 were reamplified with M13-uniminibarF1 (miniCOIF)andM13-uniminibarR1 (miniCOIR) (Table 2) [65] MiniCOIPCR reaction conditions were 95∘C for 2 minutes 5 cycles of95∘C for 1 minute 46∘C for 1 minute 72∘C for 30 seconds35 cycles of 95∘C for 1 minute 53∘C for 1 minute 72∘Cfor 30 seconds and a final 5-minute extension at 72∘Cbefore being placed on a 4∘C hold Further reactions werecarried out to obtain overlapping PCR products that couldbe assembled as contigs to obtain additional sequence dataAdditional primerswere designed to bind to invariant regionsof the Junonia COI gene (miniCOIF2 and miniCOIR2 in onereaction and either miniCOIF3 and HCO2198 or miniCOIF2and HCO2198 (Table 2) in a second reaction) to selectivelyamplify required sequences Reaction conditions for theseprimers were the same as the miniCOI protocol describedabove

23 Nuclear Wingless Protocol Wingless PCR products weregenerated using lepwg1 and lepwg2 primers (Table 2) [66]Wingless PCR reaction conditions were 94∘C for 5 minutes40 cycles of 94∘C for 1 minute 46∘C for 1 minute 72∘Cfor 2 minutes and a final 10-minute extension at 72∘Cbefore being placed on a 4∘C hold While these primerstypically work well in Junonia [24] the samples analyzedhere failed to produce detectable products likely due to poorpreservation of nuclear DNAThese PCR reactions were usedas the template for PCR reamplification with miniwgF andminiwgR (Table 2) which we designed to bracket the mostinformative interval of the Junonia wingless coding sequence(Table 3) Mini-wingless reaction conditions were 95∘C for 5minutes 40 cycles of 95∘C for 1 minute 57∘C for 1 minute72∘C for 1 minutes and a final 5-minute extension at 72∘Cbefore being placed on a 4∘C hold

24 Sequencing Correctly sized PCR products weresequenced as previously described [24] Products weresequenced in both directions usually with the same primersthat generated the products When the miniwgR primerproduced poor quality sequences samples were reamplifiedwith miniwgF and T7-miniwgR and sequenced using T7primer (Table 2) Sequencing reactions were analyzed onan ABI 3730xl automated sequencer and edited using Se-quencher 46 software [67] Sequences were trimmed to theappropriate size (Table 3) and aligned in CLUSTALW [68]

25 Randomly Amplified Fingerprinting Protocol RandomlyAmplified Fingerprinting (RAF) was used to gather a largemultilocus data set [60] Amplifications were carried outusing single fluorescently labelled primers that act as both for-ward and reverse primers A product is produced only if theprimers bind in the correct orientation and close enough toone another for amplificationThe 3 RAF primers each cova-lently bound to a 6-FAM fluorescent molecule (IntegratedDNA Technologies Iowa City Iowa USA) used in theseamplifications were RP2 (51015840-6-FAMATGAAGGGGTT-31015840)

4 Psyche

Table 2 Primer sequences used in cytochrome oxidase I (COI) and wingless (wg) PCR reactions

Primer name SequenceCOI

Nancy 51015840CCCGGTAAAATTAAAATATAAACTTC31015840

LCO1490 51015840GGTCAACAAATCATAAAGATATTGG31015840

HCO2198 51015840TAAACTTCAGGGTGACC AAAAAATCA31015840

M13-uniminibarF1 51015840GTAAAACGACGGCCAGTGGAAAATCATAATGAAGGCATGAGC31015840

M13-uniminibarR1 51015840GGAAACAGCTATGACCATGTCCACTAATCACAARGATATTGGTAC31015840

miniCOIF2 51015840ATACTATTGTTACAGCCTCATGC31015840

miniCOIR2 51015840TGTTGTAATAAAATTAATAGCTCC31015840

miniCOIF3 51015840CCCCACTTTCATCTAATATTGC31015840

wglepwg1 51015840GARTGYAARTGYCAYGGYATGTCTGG31015840

lepwg2 51015840ACTNCGCRCACCATGGAATGTRCA31015840

miniwgF 51015840ATCGCGGGTCATGATGCCTAATACG31015840

miniwgR 51015840GTTCTTTTCGCAGAAACCCGGTGAAC31015840

T7-miniwgR 51015840TAATACGACTCACTATAGGGGTTCTTTTCGCAGAAACCCGGTGAAC31015840

Table 3 Expected sequence length of trimmed PCR products(primers removed) for each primer pair

Primer pair Trimmed sequence length(base pairs)

LCO1490Nancy 725LCO1490HCO2198 658M13-uniminibarF1M13-uniminibarR1 153mCOIF2mCOIR2 292mCOIF2HCO2198 520mCOIF3HCO2198 295Lepwg1Lepwg2 402miniwgFminiwgR 137

RP4 (51015840-6-FAMTGCTGGTTCCC-31015840) and RP6 (51015840-6-FAMTGCTGGTTTCC-31015840) [59] Amplifications were per-formed in triplicate alongwith positive and negative (distilleddeionized water) controls for a total of 954 RAF amplifica-tions Reaction volumes of 10120583L were used Samples wererun in a BioRadMyCyclerThermocycler under the followingreaction conditions 95∘C for 5 minutes 30 cycles of 94∘Cfor 30 seconds 57∘C for 1 minute 56∘C for 1 minute 55∘Cfor 1 minute 54∘C for 1 minute 53∘C for 1 minute and afinal 5-minute extension at 72∘C before being placed on a4∘C hold Reactions were shipped at room temperature to theBiotechnology Core Facility at Western Kentucky University(Bowling Green Kentucky USA) 10 120583L HiDye formamideand 1 120583L RX-500 GeneScan Size Standard (Applied Biosys-tems Carlsbad California USA) were added to each PCRtube upon receipt The solution was then vortexed for 1-2seconds and placed in a microcentrifuge at 13000 rpm for 30seconds at room temperature Samples were placed into indi-vidual wells on a sequencing plate and incubated at 95∘C for

4 minutes in a thermocycler Following 3ndash5 minutes on icesamples were loaded into an ABI 3130 automated sequencer(Applied Biosystems) which was fitted with a 50 cm capillaryfilled with Pop-7 sequencing polymer for fragment analysis

26 Mitochondrial Cytochrome Oxidase I Analysis A subsetof the samples in the current study was used in a previousbarcoding study performed in another laboratory [23] Toensure that there was no confusion or contamination of DNAsamples during transfer we resequencedCOI from 17 samples(of 42 transferred) that had been previously sequenced In allcases identical sequences were obtained by our laboratory aspreviously reported [23]COI sequence alignments were con-verted to NEXUS format for phylogenetic analysis using sev-eral different reconstruction methods (distance parsimonyand likelihood) that rely on vastly different assumptionsabout sequence evolution each ofwhich recovered essentiallythe same tree For the sake of brevity we will only presentthe maximum likelihood analysis (HKY model 10 replicateheuristic searches with random number seeds tree bisectionand reconnection branch swapping algorithm) [69] Otherpreviously published Junonia COI sequences were includedin this phylogenetic analysis [23 24 51 57 70ndash74] We alsoconducted a maximum likelihood bootstrap analysis of thisdataset (500 fast addition replicates collapsing all notes withfrequency less than 50)The aligned COI FASTA sequencesgenerated by this study along with 22 previously publishedArgentinian Junonia COI sequences [24] were analyzedusing Arlequin 35 [75] We employed an AMOVA analysiswith the following settings 1000 permutations determiningthe minimum spanning network (MSN) among haplotypescomputing distance matrix and pair-wise difference witha gamma value of 0 The minimum spanning tree out-put from AMOVA was put into HapSTAR-07 [76] whichdisplays the haplotype network in graphical form Sinceanalysis in Arlequin requires all sequences to be of the same

Psyche 5

length the analysis was first conducted using all samplesthat amplified using LCO1490HCO2198 (Figure 2) and thenrepeated after trimming all sequences to the length of mini-COIF2HCO2198 (Figure 3) Additional adjustments to thenetwork were made using Canvas X (ACD Systems SeattleWashington USA) such as scaling the population circles toreflect sample size and adding pie charts to reflect the RAFpopulation assignment or geographical location and species

27 Nuclear Wingless Analysis For the Junonia species se-quenced in this study and Argentinian Junonia wingless se-quences from a prior study [24] individuals heterozygousfor single nucleotide polymorphisms (SNPs) in the codingsequence were identified using sequencing chromatogramsand CLUSTALW alignments For each polymorphism thegenotype of each individual was entered into PHASE 211[77] and analyzed using the default settings PHASE uses theMarkov Chain-Monte Carlo method to group coinheritedSNPs in order to determine the most probablewingless allelespresent in each individual The most likely alleles identifiedin PHASE were assigned to each individual and the datawas then entered into GENEPOP 4010 [78] GENEPOPwas used to test for genetic differentiation (Exact G test[79]) by determining if the alleles from each subpopulationwere drawn from the same distribution GENEPOP settingsused for testing all populations were a demorisation of10000 10000 batches and 10000 iterations per batch FinallyStructure 233 [80] was used to analyze the wingless datasince unlike GENEPOP [78] Structure does not requirethe a priori assignment of individuals to specific subpop-ulations Population structure exhibited by wingless alleleswas analyzed using Structure 233 [80] with settings forcodominant alleles a 10000 step burn-in and 1 millionMarkov Chain-Monte Carlo Method replicates Ten replicatestructure searches tested each of 15 different populationmodels with 1 to 15 subpopulations among the 88 winglesssequences The maximum log likelihood (ln119875(119863)) for the10 replicate searches for each population model was usedto calculate the posterior probability (119875(119870 = 119899)) of eachpopulation model Haplotype networks of wingless alleleswere constructed in the same manner as COI except thatPHASE output identifying the most likely wingless genotypeswas formatted for input into Arlequin 35 [75]

28 Randomly Amplified Fingerprinting Analysis Fragmentanalysis sample runs were combined with previously studiedArgentinian Junonia [24] and analyzed using GENEMAP-PER version 37 software (Applied Biosystems) An allelicbin size of 3 base pairs was selected in order to detect poly-morphic alleles without introducing excessive noise into theanalysis associatedwith small differences in run time betweensamples The resulting GENEMAPPER genotypic classifi-cations were exported to an Excel spreadsheet (MicrosoftRedmondWashington USA) for further analysis Bands thatappeared in negative control amplifications (of deionizeddistilledwater with noDNAadded)were considered artefactsand removed from further analysis for all samples Withinthe 3 replicate RAF fragment runs for each primer from

an individual butterfly allele-calling for the presence orabsence of the dominant allele at each RAF locus was basedon a majority rule determination (at least 2 of the 3 runshad to show the allele for it to be scored as present) Eachlocus was coded in binary with 0 indicating the absence of anallele and 1 indicating the presence of the allele Such binarydata was analyzed using Structure 233 software [80] with thesame settings as described previously for the wingless dataexcept that in the case of the RAF data set only dominantalleles could be scored A total of 50 replicate searches werecarried out on each of 119870 = 1ndash15 populations first includingonly the samples genotyped in this study (primarily fromFrench Guiana and the Caribbean) and then again includingthe 22 Argentinian specimens genotyped in a previous study[24]

Allele frequencies for each RAF locus were calculatedfor each population identified in Structure and formattedfor input for the CONTML application of PHYLIP 35[81] as implemented in EMBOSS Explorer [82] CONTMLuses a rigorous maximum likelihood algorithm to estimatephylogenies based on allele frequencies In this model alldivergence between populations is assumed to be due togenetic drift in the absence of newmutations [83] CONTMLtrees were exported in NEXUS format and rendered inEvolView [84] for interpretation A parallel analysis wasconducted in CONTML for the RAF data set and the allelefrequency data obtained for COI and wingless

3 Results

31 Mitochondrial Cytochrome Oxidase I Results New COIDNA sequences generated by this project were depositedin Genbank (67 accessions numbers KJ469059ndashKJ469126)with the exception of specimens with only COI minibar-code sequence fragments [65] which were submitted tothe DNA Databank of Japan (DDBJ 5 accessions AB9353-41ndashAB935345) Full COI barcode sequences covering theinterval between LCO1490 and HCO2198 were recov-ered from 65 specimens (17 reported previously [23] and48 new sequences) 15 of which required assembling 3sequence contigs to obtain the 658 bp sequence Partial bar-code sequences were obtained from miniCOIFR sequencesassembled into contigs with miniCOIF2R2 sequences (2specimens) miniCOIFR sequences assembled into con-tigs with miniCOIF3HCO2198 sequences (2 specimens)and miniCOIF2HCO2198 sequences alone (16 specimens)Overall some COI sequence was recovered from 90 of the104 specimens

Analysis of theCOI sequences produced amaximum like-lihood phylogenetic tree (Figure 1) As previously reported[24 54] there are two distinct mitochondrial haplotypegroups in NewWorld Junonia Haplotype group A is found inSouth American and Caribbean specimens while haplotypegroup B includes many North American Central Americanand Caribbean specimens as well as some South Americanspecimens A few forms of Junonia appear to be associatedwith only one haplotype group (groupA the SouthAmericanforms J ldquoflirteardquo and J vestina group B the North Americanforms J coenia and J ldquonigrosuffusardquo) All other Junonia species

6 Psyche

0005 substitutionssite

J divaricata French Guiana (LCB13)J wahlbergi French Guiana (LCB25)

J evarete Brazil (NW1513)J genoveva French Guiana (LCB9)J neildi Martinique (LCB37)

J evarete Brazil (NW1552)J divaricata French Guiana (LCB15)

J neildi Guadeloupe (UK419)J zonalis Martinique(LCB157)J zonalis Martinique (LCB158)J zonalis Martinique (LCB161)J zonalis Guadeloupe (LCB165)J neildi Guadeloupe (LCB174)J neildi Guadeloupe (LCB176)J neildi Guadeloupe (LCB178)J zonalis Martinique (LCB162)

J zonalis swifti Guadeloupe (NW136 17)J neildi Guadeloupe (UK418)

J litoralis French Guiana (LCB320)LCB179LCB181LCB182

LCB185LCB186

LCB184

LCB196LCB197

LCB190

LCB203LCB204

J genoveva French Guiana (LCB191)J genoveva French Guiana (LCB207)

J genoveva French Guiana

J genoveva French Guiana (UK414)Junonia sp French Guiana (UK416)

LCB201

LCB187

LCB199LCB198

LCB180

J divaricata French Guiana (LCB14)

LCB189 J genoveva French Guiana

J evarete Brazil (NW12620)J evarete Brazil (NW12930)


J litoralis French Guiana (LCB321)



J zonalis Martinique (LCB163)

LCB195

LCB192LCB193

LCB208LCB200

LCB194

LCB209


J zonalis Martinique (LCB160)J evarete French Guiana (UK415)J evarete Brazil (NW362)J wahlbergi French Guiana (LCB22)

LCB1LCB2LCB4LCB5LCB20LCB18LCB19LCB21LCB23LCB26


J zonalis Martinique (LCB31)J litoralis French Guiana (LCB41)J neildi Guadeloupe (LCB33)

J vestina Peru (NN07)

CIAD10 B12CIAD10 B13CIAD10 B16CIAD10 B18CIAD10 B21CIAD10 B19

J evareteSonora MX

J evarete nigrosuffusa Sonora MX (CIAD10 B32)J evarete nigrosuffusa Sonora MX (CIAD10 B35)J evarete Sonora MX (CIAD10 B23)J evarete Sonora MX (CIAD10 B11)J evarete nigrosuffusa Sonora MX (CIAD10 B34)J evarete Costa Rica (05 SRNP 58220)

05 SRNP 3118405 SRNP 58208

05 SRNP 5799905 SRNP 31186

03 SRNP 2806905 SRNP 5800105 SRNP 5799805 SRNP 32756

00 SRNP 3055

05 SRNP 5799595 SRNP 6024

J evareteCosta Rica

J evarete Costa Rica (03 SRNP 28074)J coenia coenia TN USA (NW8513)J evarete Costa Rica (03 SRNP 28071)

J evarete Sonora MX (CIAD10 B28)J evarete Costa Rica (05 SRNP 58241)

CIAD10 B27CIAD10 B17CIAD10 B37CIAD10 B20CIAD10 B10CIAD10 B01CIAD10 B22

J zonalis Guadeloupe (LCB170)J neildi Martinique (LCB172)J neildi Guadeloupe (LCB175)

J neildi Guadeloupe (LCB173)J zonalis Guadeloupe (LCB169)

J neildi Guadeloupe (LCB177)J neildi Martinique (NW13616)

J evarete Morelos MX (JM610)Jcoenia coenia MA USA (TDWG0126)J evarete nigrosuffusa Sonora MX (CIAD10B25)J coenia coenia KY USA (Bio17517)J evarete nigrosuffusa Sonora MX (CIAD10B26)J evarete nigrosuffusa Sonora MX (CIAD10B36)J zonalis Guadeloupe (LCB166)J evarete nigrosuffusa Sonora MX (CIAD10B30)J evarete nigrosuffusa Sonora MX (CIAD10B24)J evarete nigrosuffusa Sonora MX (CIAD10B33)

J evarete nigrosuffusa Sonora MX (CIAD10B31)J coenia coenia NC USA (DNAATBI0816)

J evarete Costa Rica (04SRNP15110)J coenia coenia FL USA (LCB27)

J coenia coenia NC USA (DNAATBI0802)

J zonalis Guadeloupe (LCB167)J evarete Morelos MX (NW1627)

J zonalis Guadeloupe (LCB164)J coenia coenia UT USA (NW3818)

J evarete Costa Rica (05SRNP58293)J evarete Costa Rica (05SRNP57796)

05 SRNP 3118703 SRNP 2807605 SRNP 5822105 SRNP 5800905 SRNP 58257


CIAD10 B04CIAD10 B05


CIAD10 B07


J coenia griseaCalifornia USA

J evareteCosta Rica

J neildi Guadeloupe (LCB34)J evarete Panama (YBBCI12765)J genoveva French Guiana (LCB8)J wahlbergi French Guiana (LCB24)J wahlbergi French Guiana (LCB95)

J genoveva French Guiana (LCB7)J divaricata French Guiana (LCB16)

J litoralis French Guiana (LCB38)J coenia coenia FL USA (LCB28)

J zonalis Martinique (LCB32)J zonalis Guadeloupe (LCB29)

J neildi Martinique (LCB36)


J hierta China (huangshangyan1)

J villida Australia (NW991)

J natalica Zimbabwe (NW8311)

J terea Captive Reared (NW6815)J terea Uganda (NW833)J terea Tanzania (NW8212)

J terea Tanzania (NW8214)J gregorii Uganda (NW832)


J lemonias India (SEC19LP05)

J oenone Captive Reared (NW681)J hierta Zimbabwe (NW8812)

J hierta Indonesia (NW806)

J orithya China (NW8811)J orithya China (EF683678)

J orithya Malaysia (NW291)J orithya India (SEC03BP05)

J westermanni Uganda (NW8211)J westermanni Uganda (NW834)

J sophia Uganda (NW8310)J westermanni Central African Republic (DQ385855)

J orithya Uganda (NW8811)J lemonias Thailand (NW814)

J lemonias Bangladesh (NW10115)J lemonias Taiwan (NW979)

J lemonias China (sheyan4)J lemonias Taiwan (NW9710)

J almana almana China (MYJD001)

J almana Malaysia (NW293)J almana Vietnam (NW1318)J almana Vietnam (NW1317)

J erigone Papa New Guinea (NW812)J hedonia Australia (NW332)J hedonia Australia (NW785)

J atlites Captive Reared (NW784)J atlites Malaysia (NW296)J atlites Uganda (NW1319)J atlites China (bowenyan1)

J atlites Indonesia (NW982)J iphita Captive Reared (NW6817)

J iphita China (gouchiyan2)J iphita India (SEC05C05)

J cymodoce Zambia (NW1148)

J artaxia Zambia (NW11411)J touhilimasa Zambia (NW9515)

J ansorgei Uganda (NW1391)

Hypolimnas deois Indonesia (NW808)Yoma sabina Australia (NW1322)

J evarete French Guiana

J wahlbergi French Guiana

J evarete Sonora MX


J hierta Zimbabwe (NW8810)J hierta Bangladesh (NW10116)

J natalica Captive Reared (NW6813)

J sp affin ldquohialrisrdquo Argentina (ARG10)

J ldquoflirteardquo Argentina (ARG17)


J ldquoflirteardquo Argentina (ARG19)J ldquoflirteardquo Light Phenotype Argentina (ARG18)



J ldquohilarisrdquo Argentina (ARG1)J ldquohilarisrdquo Argentina (ARG5)J ldquohilarisrdquo Argentina (ARG6)J sp affin ldquohilarisrdquo Argentina (ARG7)J sp affin ldquohilarisrdquo Argentina(ARG8)

J ldquohilarisrdquo Argentina (ARG3)

J sp affin ldquohilarisrdquo Argentina (ARG11)J ldquoflirteardquo Argentina (ARG20)J ldquoflirteardquo Argentina (ARG21)J ldquoflirteardquo Argentina (ARG22)



J sp affin ldquohilarisrdquo Argentina (ARG9)J sp affinldquohilarisrdquo Argentina (ARG12)

J sp affin ldquohilarisrdquo Argentina (ARG4)

Haplotype group Asouth America and

Caribbean

Haplotype group Bentire western

hemisphere

All J ldquoflirteardquoand J vestina

All J coeniaand J evarete nigrosuffusa

51

73

63

91

88

64

53

56

55

9198

71

80

94

96

100

88

86

98

9699

5295

959377

97

74

76

100

56

80

9356

98

98

98

72

73

Figure 1 Phylogenetic tree depicting the two distinct mitochondrial COI haplotype groups found among New World Junonia Group Ahaplotypes occur primarily in South America and the Caribbean Group B haplotypes occur throughout the Western Hemisphere but aremost common in North and Central America Most Junonia species include individuals with both haplotypes Only J ldquoflirteardquo and J vestinapossess exclusively group A haplotypes while only J coenia and J evarete nigrosuffusa possess exclusively group B haplotypes In this figurethe entire haplotype Group B clade has been translated horizontally so that the tree fits better on the page The dotted portion of the lineconnecting this clade to the tree is not included in calculations of sequence distances

Psyche 7

Haplotypegroup A

Haplotypegroup B

ARG 4

ARG 9LCB 008LCB 024LCB 095

LCB 177 LCB 164LCB 170LCB 172LCB 175

LCB 027

LCB 166LCB 169 LCB 167

LCB 192LCB 193LCB 194LCB 195LCB 200LCB 208LCB 209

LCB 160

LCB 162 LCB 163

LCB 188



LCB 001LCB 002LCB 004LCB 005LCB 018LCB 019LCB 020LCB 021LCB 023LCB 026


LCB 207

LCB 191

LCB 204

LCB 159

LCB 320

ARG 18ARG 19

ARG 16

ARG 17

ARG 2

ARG 3ARG 1

ARG 14

ARG 10

ARG 13

ARG 15

ARG 5 ARG 20ARG 6ARG 7ARG 8ARG 11ARG 21ARG 22

LCB 013LCB 025

LCB 173

ARG 12

J neildi MartiniqueJ zonalis GuadeloupeJ neildi Guadeloupe

J genoveva French GuianaJ litoralis French GuianaJ wahlbergi French Guiana

J evarete French GuianaJ divaricata French GuianaJ zonalis Martinique

J coenia USAJ ldquoflirteardquo ArgentinaJ ldquohilarisrdquo Argentina

(a)

RAF population 1RAF population 2ARAF population 2B

RAF population 3ARAF population 3BRAF population 3C

RAF population 4RAF population 5RAF population 6A

RAF population 6BRAF population 6C

Haplotypegroup A

Haplotypegroup B

LCB 008LCB 024LCB 095

LCB 170 LCB 177 LCB 164LCB 172LCB 175

LCB 027LCB 166

LCB 169

LCB 173

LCB 167

LCB 192

LCB 320

LCB 162

LCB 160

LCB 193LCB 194LCB 195LCB 200LCB 208LCB 209

LCB 157

LCB 188

LCB 163



LCB 179LCB 191LCB 181LCB 182LCB 184LCB 185LCB 186LCB 190


LCB 196LCB 197LCB 203LCB 204

LCB 159

LCB 207

LCB 025LCB 013

ARG 17

ARG 2

ARG 3ARG 1ARG 16

ARG 15

ARG 13

ARG 14

ARG 10


ARG 18ARG 19

ARG 12

ARG 4

ARG 9

(b)

Figure 2 Haplotype networks generated using complete barcode fragment mitochondrial COI haplotypes Circles are scaled to represent thenumber of individuals that contain a specific COI haplotype Divisions and colours of circles in (a) reflect geography and species associatedwith each COI haplotype French Guianan J genoveva is orange French Guianan J litoralis is yellow French Guianan J wahlbergi is redFrench Guianan J evarete is pale orange French Guianan J divaricata is pale yellow American J coenia is grey Martiniquan J zonalis ispurple Martiniquan J neildi is pink Guadeloupean J zonalis is dark blue Guadeloupean J neildi is light blue Argentinian J ldquohilarisrdquo is lightgreen and Argentinian J ldquoflirteardquo is dark green Colours of circles in (b) reflect the RAF population associated with each COI haplotypePopulation 1 is yellow Population 2A is dark blue Population 2B is light blue Population 3A is dark purple Population 3B is light purplePopulation 3C is pink Population 4 is red Population 5 is orange Population 6A is light green Population 6B ismediumgreen andPopulation6C is dark green All 11 RAF populations are represented in group A haplotypes Group B haplotypes are comprised of RAF Populations 2A2B 3C 5 and 6B

8 Psyche

Haplotypegroup A

Haplotypegroup B





LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 017 LCB 163 LCB 162

LCB 188



LCB 209


LCB 179LCB 181LCB 182LCB 184LCB 185LCB 186LCB 190LCB 191LCB 196LCB 197LCB 203LCB 204LCB 207

LCB 001LCB 002LCB 004LCB 005LCB 018LCB 019LCB 020LCB 021LCB 022LCB 023LCB 026

LCB 159

LCB 160

LCB 320

LCB 033LCB 041

LCB 031

LCB 007LCB 029

LCB 032

LCB 036

LCB 037LCB 009

LCB 025LCB 013


LCB 172LCB 175

ARG 4

ARG 9ARG 16 ARG 18

ARG 14

ARG 1

ARG 2

ARG 10

ARG 13

ARG 15

ARG 5ARG 6ARG 7ARG 8ARG 11ARG 21ARG 22

ARG 20

ARG 3

ARG 19

ARG 12

ARG 17

(a)





Haplotypegroup A

Haplotypegroup B

LCB 007

LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 031

LCB 041LCB 033

LCB 017

LCB 320LCB 163

LCB 188

LCB 013

ARG 17

LCB 025

LCB 009LCB 037

LCB 029

LCB 032







LCB 209LCB 179LCB 181LCB 182LCB 184LCB 185LCB 186LCB 190LCB 191LCB 196LCB 197LCB 203LCB 204LCB 207LCB 001


LCB 159

LCB 160

LCB 162

ARG 4

ARG 9

ARG 12

ARG 16 ARG 18

ARG 2

ARG 5ARG 6ARG 7

ARG 11ARG 21ARG 22

ARG 10

ARG 13ARG 15

ARG 8

ARG 14

ARG 20

ARG 1

ARG 3

ARG 19

(b)

Figure 3 Haplotype networks constructed from those samples with partial mCOIF2HCO fragment COI sequences The network in (a)reflects geography and species while the network in (b) reflects RAF population assignmentThe key to the colours is the same as in Figure 2

Psyche 9

(J divaricata J evarete J genoveva J ldquohilarisrdquo J litoralis Jneildi J wahlbergi and J zonalis) were found to include indi-viduals with haplotypes in both group A and group B COIcoding sequences from the two haplotype groups containno internal stop codons no insertions or deletions and few(and generally conservative) nonsynonymous substitutionsand show little evidence of heterozygosity (no double bandsin PCR products very few double peaks in sequencing readsto indicate heterozygous sites within PCR products) suggest-ing that these are not pseudogenes or nuclear copies ofmitochondrial DNAs but true allelic alternatives

Junonia litoralis fromFrenchGuiana and J neildi from theCaribbean both of which feed on black mangrove (Avicenniagerminans) as larvae include individuals with mitochondriafrom both COI haplotype groups Junonia genoveva fromFrench Guiana which has often been considered conspecificwith J neildi from the Caribbean also includes individualsthat carry haplotypes in groups A and B Junonia evarete fromFrench Guiana have exclusively group A COI haplotypesbut J zonalis from the Caribbeanwhich has sometimes beenconsidered conspecific with J evarete includes individualsfrom both haplotype groups In specimens fromGuadelouperegardless of species group A haplotypes are relatively rare(515 33 type A) Specimens from Martinique which iscloser to the South American mainland than Guadeloupeprimarily have group A haplotypes (912 75 type A) Spec-imens from French Guiana regardless of species primarilyhave group A haplotypes (4956 875 type A) as do speci-mens from Argentina (1922 86 type A)

Analysis of the haplotype networks produced from COIsequences revealed the same general pattern regardless ofwhether 86full-length 658 bp barcode sequences (Figure 2)or 102 partial 520 bp barcode sequences (Figure 3) wereanalyzed Haplotype groups A and B are clearly delineatedwith 11 nucleotide changes between the genotypes fromgroups A (J zonalis specimen LCB164) and B (J litoralis spec-imen LCB320) that are most similar to each other The twonetworks differ primarily with respect to how some group Agenotypes are connected to LCB320 a specimen of J litoralislocated near the center of haplotype group A There are alsosome minor rearrangements among the genotypes in haplo-type group B between the two networks There is a stronggeographic signal in the COI haplotype network with spec-imens from the same place often sharing identical or similargenotypesMany genotypes are found only in FrenchGuianathe French Antilles or Argentina and there is only one groupB genotype that can be found in all 3 localities (Figure 3(a))

The haplotype networks also reveal that almost all Jwahlbergi and almost all J evarete from French Guiana sharea single groupA genotype that is rare in other Junonia speciesSimilarly the vastmajority of J genoveva fromFrenchGuianapossess one of 3 disparate genotypes within COI haplotypegroup A (Figures 2(a) and 3(a)) that are rare in all otherJunonia species Most of the remaining J genoveva specimenshave sequences that are one nucleotide removed from one ofthe three haplotype A genotypes or carry a genotype fromhaplotype groupB Formost other species of Junonia there areno abundant COI genotypes that are diagnostic for particularspecies

32 NuclearWingless Results Short 137 bpwingless sequenceswere recovered from 66 of the 104 specimensThe newly gen-erated sequences were submitted to DDBJ (accession num-bers AB935346ndashAB935395 andAB936758ndashAB936773)Thesesequences were analyzed in combination with 22 winglesssequences fromArgentinian Junonia specimens [24] Follow-ing CLUSTALW alignments of the sequences of the mini-wingless PCR products with the Argentinian wingless prod-ucts 22 single nucleotide polymorphisms (SNPs)were identi-fied in this highly variable regionThese sites were confirmedin the chromatograms by the presence of a double peakwhich indicates heterozygosity Of the 22 SNPs 21 are binarySNPs and 1 SNP contains 3 alternate nucleotides Analysisof the SNPs in PHASE 211 [77] identified a total of 35wingless alleles The most probable allelic combinations foreach Junonia specimen were identified in PHASE and thenassigned to each individual for entry into GENEPOP 4010[78]

GENEPOPwas first used to test for genetic differentiationamong all populations Separating Junonia populations solelyby geography (119875 = 000017) or species (119875 = 00000) andby both geography and species (119875 = 00000) suggests sig-nificant genetic differentiation and distinct wingless alleledistributions associated with these two factors However sep-arating individual Junonia specimens using mitochondrialCOI haplotype alone to define populations shows no sta-tistically significant distinct distribution of wingless allelesassociated with mitochondrial haplotypes (119875 = 0776) How-ever when specimens were categorized by geography andCOI haplotype (119875 = 000014) species and COI haplotype(119875 = 00000) or geography species and COI haplotype (119875 =00000) the wingless alleles again appear to be drawn fromsignificantly different distributions among subpopulationslikely due to the extremely strong influence of geography andspecies on the distribution of wingless alleles

Thewinglessdatawas also analyzed in Structure 233 [80]The results of the Structure analysis testing for 1 to 15 sub-populations (Table 4) showed that themodelwith the highestposterior probability (119875(119870 = 119899)) based on the wingless allelicdata is 119870 = 1 (all samples belonging to a single population)Haplotype networks of wingless alleles show that one allele(a9) that is common in J genoveva populations from FrenchGuiana (50) is much rarer in Junonia from bothMartiniqueand Guadeloupe (15) (Figure 4) A second Junonia winglessallele (a7) is fairly common in both French Guianan (38)and French Antillean (54) populations

33 Randomly Amplified Fingerprinting Results RAF frag-ments for the RP2 RP4 and RP6 primers were recoveredfrom all 104 Junonia specimens in this study and 22 Argen-tinian Junonia from our prior work [24] RAF producesfragments of several different sizes from amplification witha given primer 43 RP2 loci 61 RP4 loci and 18 RP6 loci wereidentified for a total of 122 variable RAF loci (Table 5) Struc-ture 233 software [80] was used to test for 1 to 15 subpop-ulations among the French Guianan and Caribbean Junoniabutterflies The results of this analysis (Table 6) showed thatthe model with the highest posterior probability (119875(119870 = 119899) =1) is 119870 = 8 (samples belonging to 8 separate populations)

10 Psyche





LCB 183LCB 183LCB 203LCB 204LCB 027

LCB 019LCB 027

LCB 019 LCB 158LCB 179ARG 3

LCB 026ARG 2LC B014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005

ARG 9ARG 10ARG 13ARG 14ARG 14ARG 21LCB 172LCB 185

ARG 15ARG 17ARG 22LCB 192 ARG 21

LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095

LCB 157 LCB 175 LCB 002LCB 157 LCB 175 LCB 002LCB 158 LCB 177 LCB 014LCB 159 LCB 177 LCB 026LCB 160 LCB 178 ARG 1LCB 160 LCB 178 ARG 5LCB 161 LCB 180 ARG 10LCB 161 LCB 181 ARG 20LCB 162 LCB 187LCB 162 LCB 187LCB 163 LCB 188LCB 164 LCB 191LCB 164 LCB 195LCB 168 LCB 196LCB 169 LCB 197LCB 170 LCB 200LCB 170 LCB 209LCB 173 LCB 210LCB 173 LCB 322LCB 174 LCB 322LCB 174 LCB 326

ARG 6ARG 8ARG 12ARG 15

ARG 2

a3

a7

a21 a20

a22

a31

a27

a4

a14

a11

a18a17

a24

a15

a9a19

a13a28

a10

a23

a16

a2

a26

a34

a35

a12

a29

a33

a30

a32

a25

a8

a7

a5

a9

a6

a1

LBC 001 LBC 189 LBC 210LBC 001 LBC 189 LBC 208

LBC 007 LBC 190 LBC 326LBC 021 LBC 191 ARG 1LBC 024 LBC 192 ARG 3LBC 025 LBC 194 ARG 4LBC 159 LBC 194 ARG 4LBC 163 LBC 195 ARG 6LBC 166 LBC 197 ARG 7LBC 166 LBC 198 ARG 8LBC 167 LBC 198 ARG 9LBC 167 LBC 199 ARG 11LBC 171 LBC 199 ARG 12LBC 171 LBC 200 ARG 13LBC 172 LBC 201 ARG 16LBC 179 LBC 201 ARG 18LBC 180 LBC 202 ARG 19LBC 181 LBC 202 ARG 20LBC 182 LBC 203 ARG 22LBC 186 LBC 204LBC 188 LBC 208

(a)


LCB 019LCB 027


LCB 026

ARG 2LCB 014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005


ARG 15ARG 17ARG 22LCB1 92 ARG 21

LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095






LCB 001 LCB 189 LCB 208LCB 001 LCB 189 LCB 210LCB 007 LCB 190 LCB 326LCB 021 LCB 191 ARG 1LCB 024 LCB 192 ARG 3LCB 025 LCB 194 ARG 4LCB 159 LCB 194 ARG 4LCB 163 LCB 195 ARG 6LCB 166 LCB 197 ARG 7LCB 166 LCB 198 ARG 8LCB 167 LCB 198 ARG 9LCB 167 LCB 199 ARG 11LCB 171 LCB 199 ARG 12LCB 171 LCB 200 ARG 13LCB 172 LCB 201 ARG 16LCB 179 LCB 201 ARG 18LCB 180 LCB 202 ARG 19LCB 181 LCB 202 ARG 20LCB 182 LCB 203 ARG 22LCB 186 LCB 204LCB 188 LCB 208


ARG 2

a21

a7

a3

a20

a22

a31

a27

a4

a1

a6

a5a8

a7 a9

a23

a16

a2

a12

a10

a29

a33

a30

a32

a13

a28

a9

a19

a15

a24

a17a18

a11

a14

a25

a35

a34

a26

(b)

Figure 4 Haplotype networks generated usingwingless alleles Circles are scaled to represent the number of individuals that contain a specificwingless allele with the exception of alleles a7 and a9 (if scaled proportionately would be 50 and 61 times larger resp than shown) Coloursfor both (a) and (b) are as described in Figure 2 (a) Divisions and colours of circles reflect geography and species associated with eachwinglessallele Allele a9 is much rarer in the Caribbean populations than it is in the mainland populations 50 of a9 allele is comprised of J genovevaThe majority of wingless alleles found in Argentinian Junonia are allele a9 or its derivatives (b) Divisions and colours of circles reflect theRAF populations associated with each wingless allele All 11 RAF populations carry the most common allele a9 Individuals from 10 of the 11RAF populations carry the other common allele a7

Psyche 11

Table 4 Inferring 119870 the number of populations testing for 1ndash15subpopulations in STRUCTURE for Argentinian French GuiananGuadeloupean and Martiniquan Junonia wingless data

119870 (number ofpopulations in model)

Medianln[119875(119863)]

PR (119870 = 119899) (posteriorprobability of the model)

1 minus4270 09722 minus4654 00213 minus5603 1587119864 minus 06

4 minus5368 1664119864 minus 05

5 minus13683 1287119864 minus 41

6 minus13559 4426119864 minus 41

7 minus17316 2136119864 minus 57

8 minus13003 1156119864 minus 38

9 minus14933 4797119864 minus 47

10 minus6183 4806119864 minus 09

11 minus5965 4251119864 minus 08

12 minus5559 2465119864 minus 06

13 minus5393 1296119864 minus 05

14 minus4928 000115 minus4782 0006

Curiously when this analysis was repeated with the inclusionof Junonia samples from Argentina the model with the high-est posterior probability (119875(119870 = 119899) = 1) was119870 = 6 Howeverthe Structure analysis did not distribute the Argentiniansamples among the populations previously established forFrench Guiana and the Caribbean Instead all of the Junoniafrom Argentina were assigned to 1 population while thesamples from French Guiana and the French Antilles wereredistributed among 5 populations This was very curiousbecause in our prior study the Argentinian Junonia whenanalyzed by themselves using Structure were divided into 3populations [24]The ability of Structure to detect populationsubdivision is reduced when sample sizes are very small butthe software is far more sensitive to insufficient numbersof variable markers [85] This study employs more variableRAF markers (122 loci) than our earlier study (51 loci) butthe additional loci are fixed in Argentinian Junonia [24]When the Argentinian data set is analyzed in isolation bothsets of RAF loci show the model with the highest posteriorprobability (119875(119870 = 119899) = 1) is 119870 = 3 (samples belongingto 3 separate populations) as expected Populations thatwere unchanged in composition whether or not Argentiniansamples and were included in the analysis are populations 14 and 5 (Figure 5) Structure identifies major discontinuitiesin population structure most readily When both major andminor discontinuities exist in the same data set the minordiscontinuities can be missed because Structure employs aheuristic search algorithm that explores the solution spacerather than calculating an exact solution [80] In the absenceofmajor discontinuities the algorithmmore readily identifiesminor discontinuities For populations that fused or dividedbetween analyses with and without Argentinian samples wereanalyzed each group of affected specimens in Structure sep-arately from all other specimens and we used the population

Table 5 Fragment sizes in base pairs of RAF amplification pro-ducts for RP2 RP4 and RP6 primer

RP2 (bp) RP4 (bp) RP6 (bp)35 37 3539 42 3841 46 4743 50 5047 56 6149 58 6351 62 7254 65 7356 68 7758 71 8260 79 8564 82 8966 85 9368 87 14772 89 14975 93 19076 95 19379 99 19780 10084 10386 11287 14188 14590 14992 15295 15598 158100 161107 164135 168136 175148 179150 196158 201160 202162 209182 223200 228228 231233 234234 237240 240241 247

249258261277280287289

12 Psyche

Table 5 Continued

RP2 (bp) RP4 (bp) RP6 (bp)291342345363369371389391394440483

assignments from these separate analyses as the definitivegroup definitions Populations defined in this way are indi-cated by a shared number followed by letters (eg 2A and 2B)

Overall there are 11 populations established by Structureanalysis of RAF alleles (Figure 5(a)) RAF Population 1(yellow) primarily includes specimens of J litoralis but 2 Jzonalis 1 J genoveva and 1 J evarete specimens also showgenetic influence from this populationPopulation 2A (darkblue) includes all specimens of J wahlbergi and J divaricataand all but one of the J evarete specimens Population 2A alsoincludes individuals from all of the other FrenchGuianan andFrench Antillean Junonia species Most of the specimens inPopulation 2B (light blue) also show genetic influence fromPopulation 2A but there are 2 Caribbean J zonalis specimenswhose primary genetic influence is population 2B Population3A (dark purple) is comprised of J genoveva and J zonalisspecimens Populations 3B (light purple) and 3C (pink)both include J genoveva J neildi and J zonalis specimensPopulation 4 (red) contains only J genoveva specimensPopulation 5 (orange) is primarily composed of J genovevaspecimens although there is 1 J zonalis specimen that alsoshows influence from this population Populations 6A-C arethe same as the 3 Argentinian Junonia populations previouslydescribed [24]

Finally of particular interest is the distribution of speci-mens showing the genetic influence ofmore than one popula-tion The populations exist in 3 distinct clusters with somegenetic exchange within each cluster but little apparentgenetic exchange between clusters (Figure 5(a)) Populations1 3A 3B 3C 4 and 5 belong to one such cluster with Popula-tion 3C as the ldquohubrdquo population showing genetic exchangewith all of the other ldquosatelliterdquo populations which havevarying amounts of (often very limited) genetic exchangewith one another It is interesting to note that the 3C hubpopulation is almost exclusively composed of specimens fromthe French Antilles Specimens of J zonalis and J neildi fromthe Carib-bean and J genoveva and J litoralis from FrenchGuiana comprise the remainder of this cluster (along with 1specimen of J evarete) In this cluster many individuals haveRAF genotypes with influence from more than 1 populationsuggesting possible past or current genetic exchange amongthese populations A second cluster includes Populations 2A

Table 6 Inferring 119870 the number of populations testing for 1ndash15 subpopulations in STRUCTURE for the Argentinian FrenchGuianan Guadeloupean and Martiniquan Junonia RAF data

119870 (number of populations inmodel)

Medianln[119875(119863)]

PR (119870 = 119899) (posteriorprobability of the

model)Without Argentinian Junonia

1 minus23598 9598119864 minus 47

2 minus17872 7078119864 minus 22

3 minus14720 3458119864 minus 08

4 minus14308 2129119864 minus 06


8 minus13003 09959 minus14933 4130119864 minus 09

10 minus15254 1659119864 minus 10

11 minus14613 1008119864 minus 07

12 minus16062 5136119864 minus 14

13 minus15026 1630119864 minus 09

14 minus15784 8279119864 minus 13

15 minus157205 1562119864 minus 12

With Argentinian Junonia1 minus38419 3286119864 minus 78

2 minus30045 7665119864 minus 42

3 minus23734 1963119864 minus 14

4 minus20828 00825 minus20797 01126 minus20627 06117 minus21473 00008 minus20828 00829 minus29936 2291119864 minus 41

10 minus31386 1156119864 minus 47

11 minus34271 3398119864 minus 60

12 minus25891 8416119864 minus 24


15 minus27082 5658119864 minus 29

and 2B which show extensive genetic exchange betweenthem but limited genetic exchange with all of the other pop-ulations we have identifiedThe 2A-2B cluster shows a stronggenetic influence from J evarete J wahlbergi and J divaricatafrom French Guiana although it also includes specimensfrom the other 2 French Guianan species specimens of Jneildi and J zonalis from the Caribbean and J coenia fromFlorida A third cluster includes Populations 6A 6B and 6Cand contains only Argentinian specimens Like the 1-3A-3B-3C-4-5 cluster one population (6B) has genetic exchangewiththe other two populations but there may be little or no directgene flow between 6A and 6C [24]

Psyche 13





6 Junonia litoralis1 Junonia genoveva1 Junonia evarete

6 Junonia genoveva9 Junonia wahlbergi4 Junonia evarete5 Junonia divaricata1 Junonia zonalis2 Junonia neildi1 Junonia coenia

2 Junonia genoveva1 Junonia zonalis 4 Junonia genoveva

4 Junonia neildi1 Junonia zonalis

8 Junonia genoveva

4 Junonia genoveva

4 Junonia ldquohilarisrdquo

3 Junonia sp affinldquohilarisrdquo

2 Junonia zonalis

2 Junonia genoveva

1 Junonia genoveva

1 Junonia zonalis

1 Junonia neildi

1 Junonia genoveva1 Junonia zonalis

2 Junonia zonalis2 Junonia neildi3 Junonia zonalis

1 Junonia neildi3 Junonia genoveva

6 Junonia genoveva1 Junonia wahlbergi4 Junonia zonalis3 Junonia neildi1 Junonia coenia6 Junonia litoralis1 Junonia evarete

2 Junonia ldquoflirteardquo2 Junonia ldquohilarisrdquo



7 Junonia ldquoflirteardquo1 Junonia ldquoflirteardquo LightPhen

2 Junonia zonalis

(a)

POP 3A

POP 3BPOP 3C

POP 5POP 6A

POP 6B

POP 6CPOP 2A

POP 1POP 2B

POP 4

00163

000450006 00242

00172004750043

001350052100549

00414

00142001

00474

001

(b)

Figure 5 Summary of population genetic analysis of RAF nuclear genotyping (a) Venn diagram illustrating gene flow occurring betweenpopulationsThe 11 Junonia populations are distributed into 3 clusters which show extensive genetic exchange within a cluster but little or nogenetic exchange between clusters (b) CONTML analysis of the RAF allele frequency data for populations identified in Structure Clustersidentified in (a) tend to be adjacent to one another on the tree which shows a very strong influence on larval host plant use Most of theindividuals in RAF Populations 2A 2B 6A 6B and 6C are from species that utilize larval host plants in the order Scrophulariales Virtuallyall of the Junonia in the remaining RAF populations are from species that use larval host plants in the order Lamiales

14 Psyche

CONTML analysis of the RAF allele frequency data forpopulations identified in Structure produced an arbitrarilyrooted tree that shows much of the same clustering that wasapparent from other analyses of this data set (Figure 5(b))Populations 2A and 2B which containmany Caribbean spec-imens are sister groups on the tree while Populations 6A 6Band 6C which consist of only Argentinian specimens arise asan unresolved polytomy from a single common ancestorTheremaining populations which make up the 1-3A-3B-3C-4-5cluster are also adjacent to one another on theCONTML treealthough they do not form a monophyletic group CONTMLanalysis of the RAF allele frequency data combined withCOI and wingless allele frequency data produces a tree ofidentical topology with minuscule changes in branch length(not shown)

4 Discussion

We hoped that using a set of molecular tools that had workedwell to distinguish between forms of Junonia from Argentina[24] would allow us to unambiguously distinguish betweenforms from the French Antilles and French Guiana Unfortu-nately this is not entirely the caseCytochrome oxidase I (COI)barcodes are clearly unreliable for distinguishing amongmostNew World forms of Junonia because individuals of mostnamed species contain haplotypes from either of the twomain haplotype groups (A and B Figure 1) This is consistentwith the findings of prior studies that have found to be identi-cal haplotypes in Junonia [23 24 54] CertainCOI haplotypesare found primarily in particular geographic regions butmost are found in more than one species in that region andthus are not diagnostic (Figures 2 and 3)There are examplesof significant apparently intraspecific mitochondrial haplo-type divergence in other species and are typically associatedeither with the existence of cryptic species (each with dif-ferent haplotypes) [86 87] or with hybridization followedby introgression of mitochondria [88 89] The presence ofcryptic species is usually corroborated by the demonstrationof either consistent phenotypic differentiation between cryp-tospecies population subdivision based on nuclear markersbetween cryptospecies or both Neither this study nor pre-vious studies of Junonia have been able to provide corrobo-ration supporting the existence of cryptic species associatedwith COI haplotype diversity [23 24 54] so we feel weight ofthe evidence ismore consistentwith a history of hybridizationand introgression Except for certain special cases in whichhybridization between forms of Junonia is apparently absentfrom a region [55] COI barcoding should probably be aban-doned as amethod for distinguishing taxa amongNewWorldJunonia At the same time the segregation of haplotypes Aand B separated by approximately 4 sequence divergencein Junonia populations across much of the Western Hemi-sphere is a phenomenon worth studying in its own right

There appears to be a north-south gradient across theCaribbean with respect to the frequency of COI haplotypeswith the typeA haplotype ranging from0 inNorthAmerica[54] to 87 in the South American mainland (this studyand [24])There is a long-standing hypothesis that CaribbeanJunonia are a ring species [29 34] which is defined as a group

of species or subspecies that exhibit a ring-like distributionsuch that the forms at the extreme ends of the range overlap[42] Gene flow occurs through intermediate forms in themiddle of the ring but in a classic ring species forms foundin the overlapping region of the ring do not interbreed [9091] Western Cuba and the nearby Island of Pines were iden-tified as the possible region of overlap for the putative Junoniaring species since several phenotypically distinct forms ofJunonia coexist there [34 92] Our results suggest that ifthere is a region of secondary contact between two ends of aCaribbean Junonia ring species then hybridization betweenthe terminal forms appears to be occurring and the zone ofoverlap is not limited to CubaMore extensivemapping of thedistribution of the haplotypes in combination with phyloge-netic analysis of additional mitochondrial sequence data mayprovide insights into the origin of these haplotypes (currentlyunknown [51]) and what evolutionary forces may be con-tributing to their continued presence in Junonia populations

Alleles from the nuclear locus wingless which were help-ful in distinguishing between Junonia from Argentina [24]were less effective in this study This is probably due at leastin part to the small 137 bp sequence fragment ofwingless thatwe were able to recover from Caribbean and French GuiananJunonia (versus 402 bp previously recovered for ArgentinianJunonia [24])While thewingless fragment that was recoveredis the most variable portion of the New World Junoniawingless coding sequence (Figure 4) potentially informativesequence variation elsewhere in the gene was not available forus to study It would be highly desirable to obtain additionalspecimens of Junonia from FrenchGuiana and the Caribbeanwith better-preserved nuclear DNA so that larger portions ofthe wingless gene could be analyzed Another factor that maymakewingless sequences less useful in FrenchGuiana and theCaribbean is the large number of forms of Junonia coexistingand possibly interbreeding in this region [23] If winglesscoding sequences or sequences closely linked to winglessare adaptive for example contributing to colour patternphenotypes under selection [8 93] such sequences may besubject to introgression after hybridization events [94 95]As such the evolutionary history of the introgressed regionof the genome may not be representative of the evolutionaryhistory of the organism as a whole [96] Finally winglesssignalling may contribute to the development of colourpattern phenotypes that are used as fieldmarks for identifyingspecies of Junonia [8] most notably the prominent palemedian stripe on the ventral hind wing of some species [9798] This connection between developmental processes andspecies-specific phenotypes in combination with transgenictechniques [10ndash12 14] may permit us to identify the specificmutations responsible for phenotypic evolution in Junoniaand to characterize the molecular mechanisms responsiblefor colour pattern diversity

In the past Randomly Amplified Fingerprint (RAF)genotyping was extremely effective at distinguishing betweenforms of Junonia from Argentina [24] In this study thesepopulations remained distinguishable from one another(Populations 6A 6B and 6C) and were genetically distinctfrom populations sampled from other regions (Figure 5) Infact the Argentinian populations are all more genetically

Psyche 15

similar to one another than to any populations in FrenchGuiana or the FrenchAntilles Based onwing colour patternsArgentinian Junoniahave been conventionally referred to as Jgenoveva hilaris (C amp R Felder) the light buckeye butterflyand J evarete flirtea (Fabricius) the dark buckeye butterfly[62] However Borchers and Marcus [24] identified 3 ge-netically distinct Argentinian populations based on RAFgenotypes After consulting a key for the Junonia of FrenchGuiana that relies on colour patterns andmorphological traits[23] Borchers and Marcus [24] suggested that the two light-coloured Argentinian populations correspond to J genovevaand either a genetically disparate population of the samespecies or an undescribed cryptic species while the dark-coloured population corresponds to J wahlbergi The resultsof this study suggest that there is no close affinity betweenthe Argentinian forms and J genoveva or J wahlbergi fromFrench Guiana so the tentatively assigned scientific namesshould be revised We refer to the 2 light-coloured forms as Jldquohilarisrdquo and J sp affin ldquohilarisrdquo and the dark-coloured formas J ldquoflirteardquo

The samples from French Guiana and the Caribbean aredivided into two major clusters (Figure 5) The first cluster iscomposed of RAF Populations 1 3A 3B 3C 4 and 5 Overallthis cluster appears to be strongly associated with forms ofJunonia whose larval host plants are in order Lamiales Jlitoralis from French Guiana and J neildi from the Caribbean(both use larval host blackmangroveAvicennia germinans) Jgenoveva fromFrenchGuiana (larval hostHyptis atrorubens)and J zonalis from the Caribbean (larval hosts Stachytarphetajamaicensis S urticifolia and Lippia nodiflora) [23 99]Since all Junonia from the Caribbean feed on plants in theLamiales this cluster contains the bulk (1930) of Caribbeanspecimens in our analysis In contrast the population clusterthat includes Populations 2A and 2B contains the bulk ofspecimens from species whose larval host plants are in theorder Scrophulariales J coenia J divaricata J evarete andJ wahlbergi Junonia divaricata and J evarete use Utriculariahispida as their larval host while J wahlbergi and J evareteuseAgalinis hispidula [23] Junonia coenia from North Americafeeds on a wide variety of larval hosts in the order Scro-phulariales including several Agalinis species [18] Sharinglarval host plants may facilitate habitat overlap among extantforms and the cooccurrence of organisms is a necessaryprecondition for interspecific hybridization and gene flow totake place The overall congruency between the populationclustering based on RAF genotyping and host plant usesupports the hypothesis of L Brevignon and C Brevignon[23] that host plant use defines twomajor lineageswithinNewWorld Junonia However it should also be noted that thereare exceptions to this congruence 1 specimen of J evareteclustered with the Lamiales feeders while 12 specimens ofJ genoveva 5 specimens of J zonalis and 5 specimens of Jneildi clustered with the Scrophulariales feeders (Figure 5)This might be interpreted as evidence for some gene flowbetween these two lineages

Some species of Junonia were not readily distinguishablefrom one another in the Structure analysis of the RAF dataJunonia coenia J divaricata J evarete and J wahlbergi areall associated with the same RAF population cluster (2A-2B)

We suspect that our inability to distinguish between theseforms is due at least in part to artefact because thesefour species were represented by the smallest number ofindividuals in the RAF Structure analysis (between 2 and10 individuals sampled depending on the species Table 1)Thus the statistical power of the algorithm is poor for thesespecies [80] With additional sampling of these forms a morerobust analysis of population structure for these Junoniaspecies will be possible While we were not able to reliablyseparate J zonalis and J neildi from each other we are ableto conclude based on the available data that these Caribbeanformsappear to be genetically differentiated from the taxa thatoccur in French Guiana with the vast majority of Caribbeanspecimens assigned to Populations 2B and 3C (Figure 5)Thissupports the taxonomic hypothesis of L Brevignon and CBrevignon [23] which elevated these taxa to full speciesIt also explains why it has been so challenging to applytaxonomic names based on South American types to formsfound in the Caribbean [22 26ndash28]

The other pattern emerging from the analysis of RAFgenotypes is for individuals of one species to be spread acrossmultiple RAF populations (Figure 5) Junonia litoralis fromFrench Guiana has several individuals assigned to the sameRAF population (Population 1) but that population alsoincludes individuals from at least two other species Further-more other J litoralis are assigned to a different populationcluster (Populations 2A and 2B) In the most extreme caseof distribution among RAF populations J genoveva fromFrenchGuiana has individuals assigned to 6 RAF populations(spread across two major population clusters with apparentlylittle gene flow between the clusters) J zonalis from theFrench Antilles is similarly distributed across 4 populationswhile J neildi is assigned to at least 2 populations

This is similar to what has been observed previouslyfor the light buckeye Junonia ldquohilarisrdquo from Argentina [24]and has been replicated in the current analysis of RAF datawhich divided this species into two separate populations(Populations 6A and 6B) Previously we suggested that thepresence of two very phenotypically similar but geneticallydistinct populations of Junonia existing simultaneously inBuenos Aires Argentina may be due to mass migrations ofindividuals from geographically disparate areas [24] Massmigration of Junonia is a phenomenon that has been doc-umented in Argentina and elsewhere in the New World[100 101] However mass migration of Junonia has not beenobserved in French Guiana (C Brevignon pers com) andmass migration followed by hybridization between formswould tend to homogenize the genotypes of the interactingpopulations over time While some of the named Junoniataxa from French Guiana and the French Antilles have beendescribed relatively recently [23 47ndash49] all of these formshave been observed in the region for decades (C Brevignonpers com) and in some cases for centuries [25 102]

Many different forms of New World Junonia tested inlab crosses are interfertile and produce viable fertile hybrids[39ndash41] but many of these interfertile forms are separatedgeographically or by habitat preference (see below) andwouldhave limited contact in the wild Preliminary attempts atsome interspecific pairings of sympatric Junonia from French

16 Psyche

Guiana have been unsuccessful (C Brevignon pers com)Wolbachia bacterial infections can prevent otherwise genet-ically compatible insects from producing viable offspringblocking gene flow [103] perhaps contributing to the patternwe see in Junonia Several species of Junonia have beentested for Wolbachia (including J evarete from Panama)but thus far infections have only been detected in Asian Jalmana [104 105] Junonia species have characteristic malegenitalia [23 24] but there is no evidence of male-femalegenitalic incompatibility as has been reported in some pairsof snail and moth sister species [106 107] Also Junoniaspecies have been observed to engage in courtship flights withheterospecifics in the wild [108] and wild-caught individualsthat appear to be of hybrid origin have been identifiedbased on their RAF genotypes [24] (Figure 5) This suggeststhat if mass migration were sufficiently common to bringindividuals from geographically distinct populations intocontact at a specific site it would very quickly eliminate mostof the genetic population structure in Junonia at that localityunless there is assortative mating between forms

In addition to explaining patterns of genetic overlapamong Junonia RAF populations we have to explain thepossible subdivision of some Junonia species across multiplepopulations (eg J genoveva in French Guiana with 6 dif-ferent RAF subpopulations Figure 5) Frequently individualsfrom one named taxon collected from a single locality wereassigned to different RAF subpopulations An alternativecause of the complex genetic population structure found insome forms of Junonia is that these species which are definedon the basis of morphology and colour patterns may includeraces that are specializing on different larval host plantsHost plant specialization is a widespread mechanism forpopulation differentiation causing rapid evolution of adaptivetraits for feeding on new hosts and for assortative matingto maintain favourable combinations of traits [109ndash113] Insome cases it has been suggested that this has been a driverfor reproductive isolation and incipient speciation in manyinsects [113ndash117]

Most New World Junonia are currently known to onlyfeed on a single species of larval host plant in the wild [23 4997] However J coenia feeds on many alternative hosts [18]Under artificial conditions many varieties of Junonia larvaecan be reared on alternate host plants or on artificial dietscontaining alternate host plant leaves ([40 118] and JeffreyM Marcus pers observation) When presented with severalalternative hosts female J coenia choose to oviposit on thesame primary host plant used by the wild population fromwhich the female was derived [21 119] If additional larvalhost plants for South American forms of Junonia exist thismay explain some of the extensive population structure seenin some species such as J genoveva NewWorld Junonia hostplants contain iridoid glycoside secondary compounds thepresence of which may be a necessary precondition for useas a host by Junonia [120] This may help identify possibleadditional larval host plants for South American forms ofJunonia

A variety of mechanisms permit assortative mating andallow genetically distinct but reproductively compatible pop-ulations of species to persist in the same habitat Habitat

partitioning allows individuals from different populations touse different portions of available habitat thereby makingit less likely that they will interact and mate [121 122] Insome North American habitats where multiple Junonia taxaoccur one of the authors (Jeffrey M Marcus) has observeddifferences in habitat use In mangrove swamps along thecoast of Florida USA most J coenia males appear to patrolmating territories in clearings without trees or other verticalhabitat structure In contrast males of a form that resemblesJ neildi (the forms have yet to be compared genetically)and whose larvae feed on black mangrove trees (Avicenniagerminans) tolerate more vertical structure and establishmating territories in close proximity to their larval hostplants Similarly in coastal dune habitats in south TexasUSA J coenia males establish mating territories on theforeshore between the sand dunes and the water line A fewmeters away males of the darkly pigmented J ldquonigrosuffusardquo(taxonomic affinities uncertain) appear to establish matingterritories in the interdune and slack areas between thesand dunes It is not clear whether these North AmericanJunonia habitat preferences are due to preferences for abioticconditions of the microhabitat itself or whether the presenceor relative abundance of preferred larval host plants foreach form in the favoured microhabitats is the driver ofhabitat preference [123] In French Guiana the presenceof different Junonia taxa appears to be closely tied to theabundance and phenology of larval host plants [23]Whetherthere are similar patterns of microhabitat subdivision amongcooccurring Junonia species elsewhere is unknown

A second mechanism for assortative mating is for differ-ent forms to become reproductively active at different timesreproducing in different years [124] at different times of theyear [125] or at different times of the day [107 126] Thisreduces the likelihood of interspecific mating and permitsthe continued coexistence of allochronic species There areno known diurnal differences in habitat usage among formsof Junonia but there are seasonal differences that maycontribute to an allochronic mechanism for persistence InFrench Guiana the foliage of Junonia larval host plants in theLamiales is persistent while the foliage of larval hosts in theScrophulariales deteriorates quickly during the dry seasonThe flight times of adults of J divaricata J evarete and Jwahlbergi coincide temporally with each other and with thepresence of larval hosts in the Scrophulariales while the flighttimes of J genoveva J litoralis J neildi and J zonalis are lessseasonally restricted [23] This difference in phenology maycontribute to the continued distinctiveness of the two majorJunonia lineages (clusters of RAF populations) in FrenchGuiana and the French Antilles (Figure 5) but do not presentan obvious mechanism for maintaining distinctive forms orspecies within a cluster

A further possible mechanism may be differences in theamount or chemical composition of the pheromone or com-bination of pheromones used in the mating systems ofdifferent strains or species which may allow individuals toestablish a preference for other members of their own species[127] Pheromonesmay differ because of intrinsic genetic dif-ferences between strains [128] or because of differences in theavailability of pheromone precursors in the host plants used

Psyche 17

by different strains [129] Unfortunately nothing is currentlyknown about Junonia pheromone use or composition Othercharacteristics of mating systems may also contribute to theassortativemating between strains or species including vocal-izations displays of colour and physical interactions betweensexes [130 131]Of particular interest in this regard are severalcharacteristics of the courtship flights that are known todiffer among the North American and Caribbean forms ofJunonia [26 108] Variation in courtship flight patterns inother Junonia forms has not yet been documented There arealso differences in the colour patterns of NewWorld Junoniaspecies [22 23 97] but any roles that these colour pattern dif-ferences play in the mating systems are also undocumented

Operationally we use the isolation species concept thatdefines species as systems of populations such that geneexchange between these systems is limited or prevented byone or more reproductive isolating mechanisms [45 46]

5 Conclusions

While the molecular tools employed here cannot yet distin-guish between all named forms of Junonia we are gettingmuch closer to having a set of reliable molecular markersfor defining groups of populations within which geneticexchange is extensive and between which genetic exchangeis limited providing a means by which we can begin to dis-tinguish between species While COI barcodes are of limitedutility nuclear wingless sequences and RAF genotyping areeffective at identifying some individual species of Junoniaand have been very helpful in examining the relationshipsof Junonia forms from different geographic regions Usingthese tools we have determined that in spite of phenotypicsimilarities Junonia from the FrenchAntilles FrenchGuianaand Argentina are genetically distinct from one another andthat different species likely occur in each region Junonia pop-ulations also appear to cluster according to larval host plantuse supporting the hypothesis that there are two Junonialineages one which feeds primarily on plants in the orderScrophulariales and the other which uses larval host plantsin the order Lamiales The rapid growth in our knowledge ofthe natural and evolutionary history of New World Junoniain combination with the powerful experimental tools thatare available for use in these organisms shows much promisein making this group an excellent model for the studyof processes of speciation host plant adaptation and theevolution and development of colour pattern phenotypes

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authorsrsquo sincere thanks are due to Christian Brevignonfor providing the Junonia samples analyzed in this paper andfor his extensive and insightful comments on an earlier draftof this paper The authors also benefitted greatly from thehelpful comments ofMartin Villet NiklasWahlberg and two

anonymous reviewers Thanks are due to Roohollah AbbasiKahlia Beaudette Ashley Haverstick Moiz Kapasi BonnieMcCullagh Jacob Miller Melissa Peters and Stephanie Roz-bacher for assistance in the laboratory and for helpful com-ments during the preparation of this paperTheywould like tothank Margaret Docker for the use of the gel imager Thanksare due to Rodolphe Rougerie Evgeny Zakharov and SureshNaik for their assistance in obtaining access to the JunoniaDNA extractions processed at the Canadian Centre for DNABarcoding at the University of Guelph Finally they thankNaomi Rowland from the Biotechnology Core Facility ofWestern Kentucky University and the staff of the UniversityCore DNA Services Lab of the University of Calgary for pro-cessing of RAF and sequencing samples respectively Supportfor this project was provided by a University of ManitobaUndergraduate Research Award and an NSERC-USRA (toAmber P Gemmell) in addition to an NSERC DiscoveryGrant RGPIN386337-2011 and a Canada Foundation forInnovation Award (to Jeffrey M Marcus)

References

[1] R I Vane-Wright andW J Tennent ldquoColour and size variationin Junonia villida (Lepidoptera Nymphalidae) subspecies orphenotypic plasticityrdquo Systematics and Biodiversity vol 9 no4 pp 289ndash305 2011

[2] J M Marcus ldquoJumping genes and AFLP maps transforminglepidopteran color pattern geneticsrdquo Evolution amp Developmentvol 7 no 2 pp 108ndash114 2005

[3] S B Carroll J Gates D N Keys et al ldquoPattern formation andeyespot determination in butterfly wingsrdquo Science vol 265 no5168 pp 109ndash114 1994

[4] J M Otaki ldquoArtificially induced changes of butterfly wingcolour patterns dynamic signal interactions in eyespot devel-opmentrdquo Scientific Reports vol 1 article 111 2011

[5] U Kodandaramaiah ldquoEyespot evolution phylogenetic insightsfrom Junonia and related butterfly genera (NymphalidaeJunoniini)rdquo Evolution and Development vol 11 no 5 pp 489ndash497 2009

[6] P B Koch R Merk R Reinhardt and P Weber ldquoLocalizationof ecdysone receptor protein during colour pattern formation inwings of the butterfly Precis coenia (Lepidoptera Nymphalidae)and co-expression with Distal-less proteinrdquoDevelopment Genesand Evolution vol 212 no 12 pp 571ndash584 2003

[7] H F Nijhout ldquoMolecular and physiological basis of colorpattern formationrdquo Advances in Insect Physiology vol 38 pp219ndash265 2010

[8] A Martin and R D Reed ldquoWingless and aristaless2 define adevelopmental ground plan formoth and butterflywing patternevolutionrdquo Molecular Biology and Evolution vol 27 no 12 pp2864ndash2878 2010

[9] U Kodandaramaiah P Lindenfors and B S Tullberg ldquoDeflec-tive and intimidating eyespots a comparative study of eyespotsize and position in Junonia butterfliesrdquo Ecology amp Evolutionvol 3 no 13 pp 4518ndash4524 2013

[10] K Beaudette T M Hughes and J MMarcus ldquoImproved injec-tion needles facilitate germline transformation of the buckeyebutterfly Junonia coeniardquo BioTechniques vol 56 no 3 pp 142ndash144 2014

[11] B Dhungel Y Ohno R Matayoshi and J M Otaki ldquoBac-ulovirus-mediated gene transfer in butterfly wings in vivo

18 Psyche

an efficient expression system with an anti-gp64 antibodyrdquoBMC Biotechnology vol 13 article no 27 2013

[12] D L Lewis and C R Brunetti ldquoEctopic transgene expressionin butterfly imaginal wing discs using vaccinia virusrdquo BioTech-niques vol 40 no 1 pp 48ndash54 2006

[13] M S Serfas and S B Carroll ldquoPharmacologic approaches tobutterfly wing patterning Sulfated polysaccharides mimic orantagonize cold shock and alter the interpretation of gradientsof positional informationrdquo Developmental Biology vol 287 no2 pp 416ndash424 2005

[14] D L LewisM A Decamillis C R Brunetti et al ldquoEctopic geneexpression and homeotic transformations in arthropods usingrecombinant Sindbis virusesrdquo Current Biology vol 9 no 22 pp1279ndash1287 1999

[15] A L Miner A J Rosenberg and H Frederik Nijhout ldquoControlof growth and differentiation of the wing imaginal disk ofPrecis coenia (Lepidoptera Nymphalidae)rdquo Journal of InsectPhysiology vol 46 no 3 pp 251ndash258 2000

[16] C Kremen and H F Nijhout ldquoControl of pupal commitmentin the imaginal disks of Precis coenia (Lepidoptera Nymphali-dae)rdquo Journal of Insect Physiology vol 44 no 3-4 pp 287ndash2961998

[17] H F Nijhout and L W Grunert ldquoBombyxin is a growthfactor for wing imaginal disks in lepidopterardquo Proceedings of theNational Academy of Sciences of theUnited States of America vol99 no 24 pp 15446ndash15450 2002

[18] A Knerl and M D Bowers ldquoIncorporation of an introducedweed into the diet of a native butterfly consequences forpreference performance and chemical defenserdquo Journal ofChemical Ecology vol 39 no 10 pp 1313ndash1321 2013

[19] L A Richards E C Lampert M D Bowers C D Dodson AM Smilanich and L A Dyer ldquoSynergistic effects of iridoid gly-cosides on the survival development and immune response ofa specialist caterpillar Junonia coenia (Nymphalidae)rdquo Journalof Chemical Ecology vol 38 no 10 pp 1276ndash1284 2012

[20] M D Bowers and S K Collinge ldquoFate of iridoid glycosides indifferent life stages of the Buckeye Junonia coenia (LepidopteraNymphalidae)rdquo Journal of Chemical Ecology vol 18 no 6 pp817ndash831 1992

[21] M D Camara ldquoA recent host range expansion in JunoniacoeniaHubner (Nymphalidae) oviposition preference survivalgrowth and chemical defenserdquo Evolution vol 51 no 3 pp 873ndash884 1997

[22] A F E NeildThe Butterflies of Venezuela Part 2 NymphalidaeII (Acraeinae Libytheinae Nymphalinae Ithomiinae Morphi-nae) Meridian London UK 2008

[23] L Brevignon and C Brevignon ldquoNouvelles observations sur legenre Junonia en Guyane Fran120591aise (Lepidoptera Nymphali-dae) (Seconde partie)rdquo Lepidopteres de Guyane vol 7 pp 8ndash352012

[24] T E Borchers and J M Marcus ldquoGenetic population structureof buckeye butterflies (Junonia) from Argentinardquo SystematicEntomology vol 39 no 2 pp 242ndash255 2014

[25] P Cramer Papillon Exotiques des Trois Parties du Monde LrsquoAsieLrsquoAfrique et LrsquoAmerique vol 1 S J Baalde Amsterdam TheNetherlands B Wild Utrecht The Netherlands 1775

[26] TW Turner and J R Parnell ldquoThe identification of two speciesof Junonia Hubner (Lepidoptera Nymphalidae) J evarete andJ genoveva in Jamaicardquo Journal of Research on the Lepidopteravol 24 pp 142ndash153 1985

[27] W P Comstock ldquoInsects of Puerto Rico and the Virgin Islandsrdquoin Scientific Survey of Puerto Rico and the Virgin Islands vol 12pp 421ndash622TheNewYorkAcademy of Sciences NewYorkNYUSA 1944

[28] N D Riley A Field Guide to the Butterflies of the West IndiesNew York Times Book New York NY USA 1975

[29] W T M Forbes ldquoVariation in Junonia lavinia (LepidopteraNymphalidae)rdquo Journal of the New York Entomological Societyvol 36 no 4 pp 306ndash321 1928

[30] E G Munroe The Genus Junonia in the West Indies (Lepi-doptera Nymphalidae) vol 1498 of American Museum Novi-tates American Museum of Natural History 1951

[31] F M Brown and B Heineman Jamaica and Its Butterflies E WClassey London UK 1972

[32] A Seitz Die Gross Schmetterlinge der Erde Alfred KernenStuttgart Germany 1914

[33] B DAbrera Butterflies of the Neotropicale Region Part IVNymphalidae (Conc) amp Satyridae Black Rock Hill HouseVictoria Australia 1987

[34] C L Remington ldquoGenetical differences in solutions of the crisesof hybridization and competition in early sympatryrdquoBolletino diZoologia vol 52 pp 21ndash43 1985

[35] D B Rountree and H F Nijhout ldquoHormonal control of aseasonal polyphenism in Precis coenia (Lepidoptera Nymphal-idae)rdquo Journal of Insect Physiology vol 41 no 11 pp 987ndash9921995

[36] J GlassbergA Swift Guide to the Butterflies of Mexico amp CentralAmerica Sunstreak Morristown NJ USA 2007

[37] P J DeVries The Butterflies of Costa Rica and their NaturalHistory Princeton University Press Princeton NJ USA 1987

[38] K Maeki and C L Remington ldquoStudies of the chromosomesof North American Rhopalocera 4 Nymphalidae CharaxidaeLibytheinaerdquo Journal of the Lepidopteristsrsquo Society vol 14 pp179ndash201 1960

[39] J E Hafernik Phenetics and Ecology of Hybridization in BuckeyeButterflies (Lepidoptera Nymphalidae) vol 96 of University ofCalifornia Publications in Entomology University of CaliforniaPress 1982

[40] S M Paulsen ldquoQuantitative genetics of the wing color patternin the buckeye butterfly (Precis coenia and Precis evarete)evidence against the constancy of Grdquo Evolution vol 50 no 4pp 1585ndash1597 1996

[41] S M Paulsen ldquoQuantitative genetics of butterfly wing colorpatternsrdquoDevelopmental Genetics vol 15 no 1 pp 79ndash91 1994

[42] E Mayr Systematics and the Origin of Species Dover Publica-tions New York NY USA 1942

[43] K de Queiroz and M J Donoghue ldquoPhylogenetic systematicsand species revisitedrdquo Cladistics vol 6 pp 83ndash90 1990

[44] G G Simpson Principles of Animal Taxonomy ColumbiaUniversity Press New York NY USA 1961

[45] A R Templeton ldquoThe meaning of species and speciation agenetic perspectiverdquo in Speciation and Its Consequences DOtte and and J A Endler Eds pp 3ndash27 Sinaur AssociatesSunderland Mass USA 1989

[46] T Dobzhansky Genetics of the Evolutionary Process ColumbiaUniversity Press New York NY USA 1970

[47] C Brevignon ldquoNotes sur les Biblidinae les Apaturinae et lesNymphalinae de Guyane Fran120591aise (Lepidoptera Nymphali-dae)rdquo Labillionea vol 108 no 1 pp 3ndash12 2008

Psyche 19

[48] C Brevignon ldquoNouvelles observations sur le genre Junoniaen Guyane Francaise (Lepidoptera Nymphalidae) PremierePartierdquo Labillionea vol 109 no 1 pp 3ndash7 2009

[49] C Brevignon ldquoDescription de deux nouvelle sous-especesGuadeloupeennes du genere Junonia Hubner 1819 (Lepidop-tera Nymphalidae Nymphalinae)rdquo Labillionea vol CIV no 1pp 72ndash80 1819

[50] N Wahlberg A V Z Brower and S Nylin ldquoPhylogenetic re-lationships and historical biogeography of tribes and generain the subfamily Nymphalinae (Lepidoptera Nymphalidae)rdquoBiological Journal of the Linnean Society vol 86 no 2 pp 227ndash251 2005

[51] U Kodandaramaiah and N Wahlberg ldquoOut-of-Africa originand dispersal-mediated diversification of the butterfly genusJunonia (Nymphalidae Nymphalinae)rdquo Journal of EvolutionaryBiology vol 20 no 6 pp 2181ndash2191 2007

[52] A S Corbet ldquoPapers on Malaysian Rhopalocera V Theconspecificity of the American Precis lavinia (Cramer) with theOriental Precis orithya (Cramer)rdquo Entomologist vol 81 pp 54ndash56 1948

[53] H de Lesse ldquoNote sur les genres PrecisHb et JunoniaHb (LepNymphalidae)rdquo Bulletin de la Societe Entomologique de FranceParis vol 57 pp 74ndash77 1952

[54] E Pfeiler S Johnson and T A Marrow ldquoDNA barcodesand insights into the relationships and systematics of buckeyebutterflies (Nymphalidae Nymphalinae Junonia) from theAmericasrdquo Journal of the Lepidopteristsrsquo Society vol 66 no 4pp 185ndash198 2012

[55] E Pfeiler S Johnson and T A Markow ldquoInsights into popula-tion origins of neotropical Junonia (Lepidoptera NymphalidaeNymphalinae) based onmitochondrial DNArdquo Psyche vol 2012Article ID 423756 6 pages 2012

[56] D H Janzen M Hajibabaei J M Burns W Hallwachs ERemigio and P D N Hebert ldquoWedding biodiversity inventoryof a large and complex Lepidoptera faunawithDNAbarcodingrdquoPhilosophical Transactions of the Royal Society B BiologicalSciences vol 360 no 1462 pp 1835ndash1845 2005

[57] P D N Hebert J R Dewaard and J-F Landry ldquoDNA barcodesfor 11000 of the animal Kingdomrdquo Biology Letters vol 6 no 3pp 359ndash362 2010

[58] K Chan D R Glover C M Ramage and D K HarrisonldquoLowgenetic diversity in the ground parrot (Pezoporuswallicus)revealed by randomly amplified DNA fingerprintingrdquo AnnalesZoologici Fennici vol 45 no 3 pp 211ndash216 2008

[59] J Waldron C P Peace I R Searle et al ldquoRandomly amplifiedDNA fingerprinting A culmination of DNA marker technolo-gies based on arbitrarily-primed PCR amplificationrdquo Journalof Biomedicine and Biotechnology vol 2002 no 3 pp 141ndash1502002

[60] D I Schlipalius J Waldron B J Carroll P J Collins andP R Ebert ldquoA DNA fingerprinting procedure for ultra high-throughput genetic analysis of insectsrdquo InsectMolecular Biologyvol 10 no 6 pp 579ndash585 2001

[61] R Drew S V Siar S Dillon C Ramage C OrsquoBrien and AG C Sajise ldquoIntergeneric hybridisation between Carica papayaand wild Vasconcellea species and identification of a PRSV-Presistance generdquo Acta Horticulturae vol 738 pp 165ndash169 2007

[62] R Mattoni and N Vannucci Garden Butterflies of Buenos AiresLepidoptera Research Foundation Beverley Hills Calif USA2008

[63] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochrome

c oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[64] A Monteiro and N E Pierce ldquoPhylogeny of Bicyclus (Lepi-doptera Nymphalidae) inferred from COI COII and EF-1120572gene sequencesrdquoMolecular Phylogenetics and Evolution vol 18no 2 pp 264ndash281 2001

[65] I Meusnier G A C Singer J-F Landry et al ldquoA universalDNA mini-barcode for biodiversity analysisrdquo BioMed CentralGenomics vol 9 article 214 2008

[66] A V Z Brower and R DeSalle ldquoPatterns of mitochondrialversus nuclear DNA sequence divergence among nymphalidbutterflies the utility of wingless as a source of characters forphylogenetic inferencerdquo Insect Molecular Biology vol 7 no 1pp 73ndash82 1998

[67] Sequencher Version 45 Gene Codes Corporation Ann ArborMich USA 2005

[68] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[69] D L Swofford PAUPlowast Phylogenetic Analysis Using Parsimony(lowastand Other Methods) Sinauer Associates Sunderland MassUSA 1998

[70] R Uma Shaanker K Aravind K Chandrashekara et alJunonia orithya isolate SEC03BP05 cytochrome oxidase sub-unit I Genbank Accession EU7924781 httpwwwncbinlmnihgovnuccore197359534

[71] M Zhang T-W Cao and Y Zhong ldquoMolecular phylogeneticanalysis of the main lineages of Nymphalinae (NymphalidaeLepidoptera) based on the partial mitochondrial COI generdquoAgricultural Sciences in China vol 7 no 6 pp 731ndash739 2008

[72] D H Janzen and M Hajibabaei Junonia evarete cytochromeoxidase subunit I isolates from Area de Conservacion Gua-nacaste Costa Rica Genbank Accessions GU1572880-1573002009 httpwwwncbinlmnihgovnuccoreGU1572801

[73] J M Marcus D D Bell A N Bryant et al ldquoThe Upper GreenRiver barcode of life projectrdquo Journal of the Kentucky Academyof Science vol 70 no 1 pp 75ndash83 2009

[74] X Qin W Ye L Lu et al Junonia almana almana isolateMYJD-001 cytochrome oxidase subunit I Genbank Acces-sion HM446466 2010 httpwwwncbinlmnihgovnuccore301051531

[75] S Schneider D Roessli and L Excoffier Arlequin Ver 2000A Software for Population Genetic Data Analysis Genetics andBiometry Laboratory University of Geneva Geneva Switzer-land 2000

[76] A G F Teacher and D J Griffiths ldquoHapStar automatedhaplotype network layout and visualizationrdquoMolecular EcologyResources vol 11 no 1 pp 151ndash153 2011

[77] M Stephens and P Scheet ldquoAccounting for decay of linkagedisequilibrium in haplotype inference and missing-data impu-tationrdquoThe American Journal of Human Genetics vol 76 no 3pp 449ndash462 2005

[78] F Rousset ldquoGENEPOPrsquo007 a complete re-implementation ofthe GENEPOP software for Windows and Linuxrdquo MolecularEcology Resources vol 8 no 1 pp 103ndash106 2008

[79] M Raymond and F Rousset ldquoGENEPOP (version 12) popula-tion genetics software for exact tests and ecumenismrdquo Journalof Heredity vol 86 pp 248ndash249 1995

20 Psyche

[80] M J Hubisz D Falush M Stephens and J K PritchardldquoInferring weak population structure with the assistance ofsample group informationrdquoMolecular Ecology Resources vol 9no 5 pp 1322ndash1332 2009

[81] J Felsenstein PHYLIP (Phylogeny Inference Package) Version35c Department of Genetics University of Washington Seat-tle Wash USA 1993

[82] P Rice I Longden and A Bleasby ldquoEMBOSS the EuropeanMolecular BiologyOpen Software SuiterdquoTrends inGenetics vol16 no 6 pp 276ndash277 2000

[83] J Felsenstein ldquoEvolutionary trees from gene frequencies andquantitative characters finding maximum likelihood esti-matesrdquo Evolution vol 35 pp 1229ndash1242 1981

[84] H Zhang S Gao M J Lercher S Hu and W-H Chen ldquoEvol-View an online tool for visualizing annotating and managingphylogenetic treesrdquo Nucleic Acids Research vol 40 no 1 ppW569ndashW572 2012

[85] J M S VianaM S F Valente F Fonseca e Silva G BMundimand G P Paes ldquoEfficacy of population structure analysis withbreeding populations and inbred linesrdquo Genetica vol 141 no7ndash9 pp 389ndash399 2013

[86] S A Trewick ldquoMitochondrial DNA sequences supportallozyme evidence for cryptic radiation of New Zealand Peri-patoides (Onychophora)rdquo Molecular Ecology vol 9 no 3 pp269ndash281 2000

[87] M A Smith N E Woodley D H Janzen W Hallwachsand P D N Hebert ldquoDNA barcodes reveal cryptic host-specificity within the presumed polyphagous members of agenus of parasitoid flies (Diptera Tachinidae)rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 103 no 10 pp 3657ndash3662 2006

[88] N D Halbert and J N Derr ldquoA comprehensive evaluationof cattle introgression into US federal bison herdsrdquo Journal ofHeredity vol 98 no 1 pp 1ndash12 2007

[89] NWahlberg EWeingartner A DWarren and S Nylin ldquoTim-ing major conflict between mitochondrial and nuclear genesin species relationships of Polygonia butterflies (NymphalidaeNymphalini)rdquoBMCEvolutionary Biology vol 9 no 1 article 922009

[90] R J Pereira and D B Wake ldquoGenetic leakage after adaptiveand nonadaptive divergence in the Ensatina eschscholtzii ringspeciesrdquo Evolution vol 63 no 9 pp 2288ndash2301 2009

[91] D E Irwin S Bensch J H Irwin and T D Price ldquoSpeciationby distance in a ring speciesrdquo Science vol 307 no 5708 pp 414ndash416 2005

[92] D E Irwin J H Irwin and T D Price ldquoRing species as bridgesbetween microevolution and speciationrdquo Genetica vol 112-113pp 223ndash243 2001

[93] A Martin R Papa N J Nadeau et al ldquoDiversification of com-plex butterfly wing patterns by repeated regulatory evolution ofa Wnt ligandrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 31 pp 12632ndash126372012

[94] M R Kronforst L G Young L M Blume and L E GilbertldquoMultilocus analyses of admixture and introgression amonghybridizing Heliconius butterfliesrdquo Evolution vol 60 no 6 pp1254ndash1268 2006

[95] SW Baxter N J Nadeau L SMaroja et al ldquoGenomic hotspotsfor adaptation the population genetics of Mullerian mimicryin theHeliconius melpomene claderdquo PLoS Genetics vol 6 no 2Article ID e1000794 2010

[96] TMAnderson BMVonHoldt S I Candille et al ldquoMolecularand evolutionary history of melanism in Norths American graywolvesrdquo Science vol 323 no 5919 pp 1339ndash1343 2009

[97] J GlassbergM CMinno and J V Calhoun Butterflies throughBinoculars A Field Finding and Gardening Guide to Butterfliesin Florida Oxford University Press New York NY USA 2000

[98] J Glassberg Butterflies Through Binoculars The East OxfordUniversity Press Oxford UK 1999

[99] C Brevignon ldquoLes papillon diurnes de la Guadelouperdquo Lambil-lionea vol 103 no 2 pp 1ndash29 2003

[100] C B Williams The Migration of Butterflies Oliver and BoydEdinburgh UK 1930

[101] T J Walker ldquoMigration and re-migration of butterflies throughNorth Peninsular Florida quantification with malaise trapsrdquoJournal of the Lepidopteristsrsquo Society vol 32 no 3 pp 178ndash1901978


[103] U Kodandaramaiah T J Simonsen S Bromilow NWahlbergand F Sperling ldquoDeceptive single-locus taxonomy and phylo-geography Wolbachia-associated divergence in mitochondrialDNA is not reflected in morphology and nuclear markers in abutterfly speciesrdquo Ecology and Evolution vol 3 no 16 pp 5167ndash5176 2013

[104] J H Werren D Windsor and L Guo ldquoDistribution of Wol-bachia among neotropical arthropodsrdquo Proceedings of the RoyalSociety B Biological Sciences vol 262 no 1364 pp 197ndash2041995

[105] B K Salunke R C Salunkhe D P Dhotre et al ldquoDetermina-tion of Wolbachia diversity in butterflies from Western GhatsIndia by a multigene approachrdquo Applied and EnvironmentalMicrobiology vol 78 no 12 pp 4458ndash4467 2012

[106] R Ueshima and T Asami ldquoSingle-gene speciation by left-rightreversalrdquo Nature vol 425 no 6959 p 679 2003

[107] R S Peigler ldquoDemonstration of reproductive isolating mech-anisms in Callosamia (Saturniidae) by artificial hybridizationrdquoJournal of Research on the Lepidoptera vol 19 no 2 pp 72ndash811980

[108] M C Minno and T C Emmel Butterflies of the Florida KeysMariposa Press Scientific Publishers Gainesville Fla USA1993

[109] S P Carroll H Dingle and S P Klassen ldquoGenetic differ-entiation of fitness-associated traits among rapidly evolvingpopulations of the soapberry bugrdquo Evolution vol 51 no 4 pp1182ndash1188 1997

[110] S P Carroll J E Loye H Dingle M Mathieson T R Famulaand M P Zalucki ldquoAnd the beak shall inheritmdashevolution inresponse to invasionrdquo Ecology Letters vol 8 no 9 pp 944ndash9512005

[111] C S Mcbride and M C Singer ldquoField studies reveal strongpostmating isolation between ecologically divergent butterflypopulationsrdquo PLoS Biology vol 8 no 10 Article ID e10005292010

[112] A A Forbes G R Hood and J L Feder ldquoGeographic andecological overlap of parasitoid wasps associated with theRhagoletis pomonella (Diptera Tephritidae) species complexrdquoAnnals of the Entomological Society of America vol 103 no 6pp 908ndash915 2010

Psyche 21

[113] J O Stireman III J D Nason and S B Heard ldquoHost-associatedgenetic differentiation in phytophagous insects General phe-nomenon or isolated exceptions Evidence from a goldenrod-insect communityrdquo Evolution vol 59 no 12 pp 2573ndash25872005

[114] Y Abe ldquoHost race formation in the gall wasp Andricus mukai-gawaerdquo Entomologia Experimentalis et Applicata vol 58 no 1pp 15ndash20 1991

[115] G L Bush ldquoHost race foramtion and sympatric speciation inRhagoletis fruit flies (diptera tephritidae)rdquo Psyche vol 99 no4 pp 335ndash357 1993

[116] S P Carroll and C Boyd ldquoHost race radiation in the soapberrybug natural history with the historyrdquo Evolution vol 46 no 4pp 1052ndash1069 1992

[117] D Schluter ldquoEvidence for ecological speciation and its alterna-tiverdquo Science vol 323 no 5915 pp 737ndash741 2009

[118] A Ellis and M D Bowers ldquoEffects of hostplant species andartificial diet on growth of buckeye (Junonia coenia) and paintedlady (Vanessa cardui) caterpillars (nymphalidae)rdquo Journal of theLepidopteristsrsquo Society vol 52 no 1 pp 73ndash83 1998

[119] K L Prudic J C Oliver andMD Bowers ldquoSoil nutrient effectson oviposition preference larval performance and chemicaldefense of a specialist insect herbivorerdquo Oecologia vol 143 no4 pp 578ndash587 2005

[120] M D Bowers ldquoIridoid glycosides and host-plant specificity inlarvae of the buckeye butterfly Junonia coenia (Nymphalidae)rdquoJournal of Chemical Ecology vol 10 no 11 pp 1567ndash1577 1984

[121] D Schluter ldquoEcological speciation in postglacial fishesrdquo inEvolution on Islands P R Grant Ed pp 163ndash180 OxfordUniversity Press Oxford UK 1998

[122] I Emelianov M Dres W Baltensweiler and J Mallet ldquoHost-induced assortative mating in host races of the larch budmothrdquoEvolution vol 55 no 10 pp 2002ndash2010 2001

[123] K W Matsubayashi S Kahono and H Katakura ldquoDivergenthost plant preference causes assortative mating between sym-patric host races of the ladybird beetle Henosepilachna diekeirdquoBiological Journal of the Linnean Society vol 110 no 3 pp 606ndash614 2013

[124] J R Cooley C Simon D CMarshall K Slon and C EhrhardtldquoAllochronic speciation secondary contact and reproductivecharacter displacement in periodical cicadas (HemipteraMagi-cicada spp) geneticmorphological and behavioural evidencerdquoMolecular Ecology vol 10 no 3 pp 661ndash671 2001

[125] S Yamamoto and T Sota ldquoIncipient allochronic speciation byclimatic disruption of the reproductive periodrdquo Proceedings ofthe Royal Society B Biological Sciences vol 276 no 1668 pp2711ndash2719 2009

[126] D P Pashley A M Hammond and T N Hardy ldquoRepro-ductive isolating mechanisms in fall armyworm host strains(Lepidoptera Noctuidae)rdquo Annals of the Entomological Societyof America vol 85 no 4 pp 400ndash405 1992

[127] M Unbehend S Hanniger R L Meagher D G Heckel andA T Groot ldquoPheromonal divergence between two strains ofSpodoptera frugiperdardquo Journal of Chemical Ecology vol 39 no3 pp 364ndash376 2013

[128] F Marcillac Y Grosjean and J-F Ferveur ldquoA single mutationalters production anddiscrimination ofDrosophila sex pherom-onesrdquo Proceedings of the Royal Society B Biological Sciences vol272 no 1560 pp 303ndash309 2005

[129] S Geiselhardt T Otte and M Hilker ldquoLooking for a similarpartner host plants shape mating preferences of herbivorous

insects by altering their contact pheromonesrdquo Ecology Lettersvol 15 no 9 pp 971ndash977 2012

[130] A Hoikkala K Y Kaneshiro and R R Hoy ldquoCourtship songsof the picture-winged Drosophila planitibia subgroup speciesrdquoAnimal Behaviour vol 47 no 6 pp 1363ndash1374 1994

[131] K L Shaw C K Ellison K P Oh and C Wiley ldquoPleiotropyldquosexyrdquo traits and speciationrdquo Behavioral Ecology vol 22 no 6pp 1154ndash1155 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology

2 Psyche

concept that defines species as systems of populations suchthat genetic exchange between these systems is limited orprevented by one ormore reproductive isolatingmechanisms[45 46] Identifying and understanding the reproductiveisolating mechanisms operating in Junonia will be of greatimportance in clarifying Junonia taxonomy

Authorities who favoured the two-species hypothesis inJunonia called the larger form J genoveva and the smallerform J evarete In 1985 Turner and Parnell [26] using spec-imens from Jamaica and Florida USA verified the existenceof two Junonia species in both regions However afterconsulting Cramerrsquos hand-tinted plates and comparing themto specimens from Jamaica and Florida Turner and Parnell[26] switched the names so the larger species was now Jevarete and the smaller species was J genoveva Neild [22]using specimens fromVenezuela (geographicallymuch closerto the type locality of Suriname) also confirmed the existenceof two species However Neild [22] unsatisfiedwithCramerrsquos[25] published plates (copies of which differ fromone anotherdue to variation among the watercolourists who tinted themand differences in how the plates aged) consulted Cramerrsquosoriginal watercolours and Junonia specimens from manylocalities in South America Using this reference materialNeild [22] reversed Turner and Parnell [26] so that the largerspecies was again J genoveva and the smaller species was Jevarete Neild [22] also designated new types for J evarete andJ genoveva to facilitate future taxonomic work

Recently L Brevignon and C Brevignon [23 47 48]identified 5 Junonia species (J evarete J genoveva J wahl-bergi J litoralis and J divaricata) from French Guiana Thisrepresents the most diverse assemblage of Junonia in theNewWorld There are two forms of Junonia known from theCaribbean Islands ldquozonalisrdquo and ldquoneildirdquo which were initial-ly recognized as subspecies of mainland J evarete and Jgenoveva respectively [49] and later as two distinct speciesJ zonalis and J neildi [23] In recognizing J neildi and Jzonalis as distinct species L Brevignon and C Brevignon[23] restricted the use of the species epithets J evarete and Jgenoveva to mainland Central and South American forms Ifit were confirmed that the Caribbean forms are actually dis-tinct species with respect to Junonia from the mainland thiswould explain some of the widespread difficulty of assigningappropriate taxonomic names to specimens from FloridaJamaica and elsewhere in the West Indies Finally there aretwo additional Junonia species J coenia fromNorth Americaand J vestina from the Andes mountains of South Americafor a current total of up to 9 species of NewWorld Junonia

The first molecular phylogenetic approaches to under-standing the relationships among the species discussed hereestablished that the New World fauna appears to be mono-phyletic and that the various New World forms are indeedin the genus Junonia [50 51] (some authorities had pre-viously placed these species in the related genus Precis)[52 53] Unfortunately these early studies which incor-porated data from both mitochondrial and nuclear locihad limited taxon sampling including data from only 3New World species [50 51] More recent studies of themolecular phylogeny of New World Junonia [23 54 55]which have better taxon sampling have focused entirely on

the mitochondrial cytochrome oxidase I (COI) locus whichis widely used as a barcoding locus for animal taxa [56 57]Based on mitochondrial haplotype sequences the relation-ships amongmanyNewWorld Junonia species are ambiguousand most species are not reciprocally monophyletic [23 2454] The degree to which recent divergences retained poly-morphisms andor hybridization events contribute to thesepatterns in Junonia is unknown because only mitochondrialmarkers were considered What is apparent is that thereare two very divergent COI haplotype groups (4 sequencedivergence between them) present in New World JunoniaGroup A which predominates in South America and is alsopresent in the Caribbean and Group B which predominatesin North and Central America but which also occurs in theCaribbean and South America [24 54] Sequences belongingto each of these haplotype groups can occur in differentindividuals of the same species at the same locality [24]

The most successful study to date to distinguish betweenNew World Junonia taxa using molecular markers employeda combination of mitochondrial and nuclear markers toexamine populations of Junonia in Buenos Aires ArgentinaBorchers and Marcus [24] used DNA sequences from thenuclear wingless gene and anonymous nuclear loci identifiedby Randomly Amplified Fingerprinting (RAF) (a techniqueused to assess genetic diversity within populations [58ndash60]and gene flow between populations [61]) in addition tosequences from the mitochondrial COI gene They identified3 distinct populations of Junonia from Buenos Aires onepopulation with dark-coloured wings referred to as J evareteflirtea [62] which Borchers and Marcus [24] suggested maycorrespond to J wahlbergi and 2 light-coloured populationsthat correspond to J genoveva hilaris and either a geneticallydisparate population of J genoveva hilaris or an undescribedcryptic Junonia species However the relationship of theseArgentinian forms with J evarete and J genoveva fromSuriname and French Guiana is not known so we refer tothem as J ldquoflirteardquo and J ldquohilarisrdquo

In the current study we extend the genetic tools that wereemployed by Borchers and Marcus [24] to Junonia popula-tions from French Guiana and the French Antilles in orderto study the distinctiveness of the named taxa within andbetween these two localities This will allow an explicit test ofthe 7-species taxonomic hypothesis (2 species in the FrenchAntilles plus 5 species in French Guiana) of L Brevignonand C Brevignon [23] and also detect possible hybridizationevents between named forms By using a common set ofmarkers we will also be able to compare these populations topreviously studied Junonia from Argentina [24]

2 Materials and Methods

21 Specimens and DNA Preparation A total of 104 Junoniaspecimens were collected from the wild as adults rearedfrom wild-collected larvae or reared from eggs laid by wild-collected adults and frozen at minus20∘C (Table 1) DNA was iso-lated from legs removed from each specimen Some samples(42 specimens) were prepared by the Canadian Centre forDNA Barcoding at the University of Guelph as previouslydescribed [23] The remaining samples (62 specimens) were

Psyche 3



in ourlaboratory











4 Psyche






















Psyche 5






3 Results



6 Psyche








LCB185LCB186

LCB184

LCB196LCB197

LCB190

LCB203LCB204




LCB201

LCB187

LCB199LCB198

LCB180









LCB195

LCB192LCB193

LCB208LCB200

LCB194

LCB209








J evareteSonora MX


05 SRNP 3118405 SRNP 58208

05 SRNP 5799905 SRNP 31186


00 SRNP 3055

05 SRNP 5799595 SRNP 6024

J evareteCosta Rica


















CIAD10 B07



J evareteCosta Rica



































J evarete Sonora MX


















Caribbean


hemisphere



51

73

63

91

88

64

53

56

55

9198

71

80

94

96

100

88

86

98

9699

5295

959377

97

74

76

100

56

80

9356

98

98

98

72

73


Psyche 7

Haplotypegroup A

Haplotypegroup B

ARG 4



LCB 027



LCB 160

LCB 162 LCB 163

LCB 188





LCB 207

LCB 191

LCB 204

LCB 159

LCB 320

ARG 18ARG 19

ARG 16

ARG 17

ARG 2

ARG 3ARG 1

ARG 14

ARG 10

ARG 13

ARG 15


LCB 013LCB 025

LCB 173

ARG 12





(a)





Haplotypegroup A

Haplotypegroup B



LCB 027LCB 166

LCB 169

LCB 173

LCB 167

LCB 192

LCB 320

LCB 162

LCB 160


LCB 157

LCB 188

LCB 163






LCB 159

LCB 207

LCB 025LCB 013

ARG 17

ARG 2

ARG 3ARG 1ARG 16

ARG 15

ARG 13

ARG 14

ARG 10


ARG 18ARG 19

ARG 12

ARG 4

ARG 9

(b)


8 Psyche

Haplotypegroup A

Haplotypegroup B





LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 017 LCB 163 LCB 162

LCB 188



LCB 209




LCB 159

LCB 160

LCB 320

LCB 033LCB 041

LCB 031

LCB 007LCB 029

LCB 032

LCB 036

LCB 037LCB 009

LCB 025LCB 013


LCB 172LCB 175

ARG 4

ARG 9ARG 16 ARG 18

ARG 14

ARG 1

ARG 2

ARG 10

ARG 13

ARG 15


ARG 20

ARG 3

ARG 19

ARG 12

ARG 17

(a)





Haplotypegroup A

Haplotypegroup B

LCB 007

LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 031

LCB 041LCB 033

LCB 017

LCB 320LCB 163

LCB 188

LCB 013

ARG 17

LCB 025

LCB 009LCB 037

LCB 029

LCB 032









LCB 159

LCB 160

LCB 162

ARG 4

ARG 9

ARG 12

ARG 16 ARG 18

ARG 2

ARG 5ARG 6ARG 7

ARG 11ARG 21ARG 22

ARG 10

ARG 13ARG 15

ARG 8

ARG 14

ARG 20

ARG 1

ARG 3

ARG 19

(b)


Psyche 9









10 Psyche






LCB 019LCB 027


LCB 026ARG 2LC B014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095



ARG 2

a3

a7

a21 a20

a22

a31

a27

a4

a14

a11

a18a17

a24

a15

a9a19

a13a28

a10

a23

a16

a2

a26

a34

a35

a12

a29

a33

a30

a32

a25

a8

a7

a5

a9

a6

a1



(a)


LCB 019LCB 027


LCB 026

ARG 2LCB 014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095








ARG 2

a21

a7

a3

a20

a22

a31

a27

a4

a1

a6

a5a8

a7 a9

a23

a16

a2

a12

a10

a29

a33

a30

a32

a13

a28

a9

a19

a15

a24

a17a18

a11

a14

a25

a35

a34

a26

(b)


Psyche 11



Medianln[119875(119863)]



4 minus5368 1664119864 minus 05

5 minus13683 1287119864 minus 41

6 minus13559 4426119864 minus 41

7 minus17316 2136119864 minus 57

8 minus13003 1156119864 minus 38

9 minus14933 4797119864 minus 47

10 minus6183 4806119864 minus 09

11 minus5965 4251119864 minus 08

12 minus5559 2465119864 minus 06

13 minus5393 1296119864 minus 05

14 minus4928 000115 minus4782 0006



RP2 (bp) RP4 (bp) RP6 (bp)35 37 3539 42 3841 46 4743 50 5047 56 6149 58 6351 62 7254 65 7356 68 7758 71 8260 79 8564 82 8966 85 9368 87 14772 89 14975 93 19076 95 19379 99 19780 10084 10386 11287 14188 14590 14992 15295 15598 158100 161107 164135 168136 175148 179150 196158 201160 202162 209182 223200 228228 231233 234234 237240 240241 247

249258261277280287289

12 Psyche

Table 5 Continued

RP2 (bp) RP4 (bp) RP6 (bp)291342345363369371389391394440483






Medianln[119875(119863)]



1 minus23598 9598119864 minus 47

2 minus17872 7078119864 minus 22

3 minus14720 3458119864 minus 08

4 minus14308 2129119864 minus 06



10 minus15254 1659119864 minus 10

11 minus14613 1008119864 minus 07

12 minus16062 5136119864 minus 14

13 minus15026 1630119864 minus 09

14 minus15784 8279119864 minus 13

15 minus157205 1562119864 minus 12


2 minus30045 7665119864 minus 42

3 minus23734 1963119864 minus 14


10 minus31386 1156119864 minus 47

11 minus34271 3398119864 minus 60

12 minus25891 8416119864 minus 24


15 minus27082 5658119864 minus 29


Psyche 13









8 Junonia genoveva

4 Junonia genoveva



2 Junonia zonalis

2 Junonia genoveva

1 Junonia genoveva

1 Junonia zonalis

1 Junonia neildi









2 Junonia zonalis

(a)

POP 3A

POP 3BPOP 3C

POP 5POP 6A

POP 6B

POP 6CPOP 2A

POP 1POP 2B

POP 4

00163

000450006 00242

00172004750043

001350052100549

00414

00142001

00474

001

(b)


14 Psyche


4 Discussion






Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Psyche 3



in ourlaboratory











4 Psyche






















Psyche 5






3 Results



6 Psyche








LCB185LCB186

LCB184

LCB196LCB197

LCB190

LCB203LCB204




LCB201

LCB187

LCB199LCB198

LCB180









LCB195

LCB192LCB193

LCB208LCB200

LCB194

LCB209








J evareteSonora MX


05 SRNP 3118405 SRNP 58208

05 SRNP 5799905 SRNP 31186


00 SRNP 3055

05 SRNP 5799595 SRNP 6024

J evareteCosta Rica


















CIAD10 B07



J evareteCosta Rica



































J evarete Sonora MX


















Caribbean


hemisphere



51

73

63

91

88

64

53

56

55

9198

71

80

94

96

100

88

86

98

9699

5295

959377

97

74

76

100

56

80

9356

98

98

98

72

73


Psyche 7

Haplotypegroup A

Haplotypegroup B

ARG 4



LCB 027



LCB 160

LCB 162 LCB 163

LCB 188





LCB 207

LCB 191

LCB 204

LCB 159

LCB 320

ARG 18ARG 19

ARG 16

ARG 17

ARG 2

ARG 3ARG 1

ARG 14

ARG 10

ARG 13

ARG 15


LCB 013LCB 025

LCB 173

ARG 12





(a)





Haplotypegroup A

Haplotypegroup B



LCB 027LCB 166

LCB 169

LCB 173

LCB 167

LCB 192

LCB 320

LCB 162

LCB 160


LCB 157

LCB 188

LCB 163






LCB 159

LCB 207

LCB 025LCB 013

ARG 17

ARG 2

ARG 3ARG 1ARG 16

ARG 15

ARG 13

ARG 14

ARG 10


ARG 18ARG 19

ARG 12

ARG 4

ARG 9

(b)


8 Psyche

Haplotypegroup A

Haplotypegroup B





LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 017 LCB 163 LCB 162

LCB 188



LCB 209




LCB 159

LCB 160

LCB 320

LCB 033LCB 041

LCB 031

LCB 007LCB 029

LCB 032

LCB 036

LCB 037LCB 009

LCB 025LCB 013


LCB 172LCB 175

ARG 4

ARG 9ARG 16 ARG 18

ARG 14

ARG 1

ARG 2

ARG 10

ARG 13

ARG 15


ARG 20

ARG 3

ARG 19

ARG 12

ARG 17

(a)





Haplotypegroup A

Haplotypegroup B

LCB 007

LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 031

LCB 041LCB 033

LCB 017

LCB 320LCB 163

LCB 188

LCB 013

ARG 17

LCB 025

LCB 009LCB 037

LCB 029

LCB 032









LCB 159

LCB 160

LCB 162

ARG 4

ARG 9

ARG 12

ARG 16 ARG 18

ARG 2

ARG 5ARG 6ARG 7

ARG 11ARG 21ARG 22

ARG 10

ARG 13ARG 15

ARG 8

ARG 14

ARG 20

ARG 1

ARG 3

ARG 19

(b)


Psyche 9









10 Psyche






LCB 019LCB 027


LCB 026ARG 2LC B014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095



ARG 2

a3

a7

a21 a20

a22

a31

a27

a4

a14

a11

a18a17

a24

a15

a9a19

a13a28

a10

a23

a16

a2

a26

a34

a35

a12

a29

a33

a30

a32

a25

a8

a7

a5

a9

a6

a1



(a)


LCB 019LCB 027


LCB 026

ARG 2LCB 014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095








ARG 2

a21

a7

a3

a20

a22

a31

a27

a4

a1

a6

a5a8

a7 a9

a23

a16

a2

a12

a10

a29

a33

a30

a32

a13

a28

a9

a19

a15

a24

a17a18

a11

a14

a25

a35

a34

a26

(b)


Psyche 11



Medianln[119875(119863)]



4 minus5368 1664119864 minus 05

5 minus13683 1287119864 minus 41

6 minus13559 4426119864 minus 41

7 minus17316 2136119864 minus 57

8 minus13003 1156119864 minus 38

9 minus14933 4797119864 minus 47

10 minus6183 4806119864 minus 09

11 minus5965 4251119864 minus 08

12 minus5559 2465119864 minus 06

13 minus5393 1296119864 minus 05

14 minus4928 000115 minus4782 0006



RP2 (bp) RP4 (bp) RP6 (bp)35 37 3539 42 3841 46 4743 50 5047 56 6149 58 6351 62 7254 65 7356 68 7758 71 8260 79 8564 82 8966 85 9368 87 14772 89 14975 93 19076 95 19379 99 19780 10084 10386 11287 14188 14590 14992 15295 15598 158100 161107 164135 168136 175148 179150 196158 201160 202162 209182 223200 228228 231233 234234 237240 240241 247

249258261277280287289

12 Psyche

Table 5 Continued

RP2 (bp) RP4 (bp) RP6 (bp)291342345363369371389391394440483






Medianln[119875(119863)]



1 minus23598 9598119864 minus 47

2 minus17872 7078119864 minus 22

3 minus14720 3458119864 minus 08

4 minus14308 2129119864 minus 06



10 minus15254 1659119864 minus 10

11 minus14613 1008119864 minus 07

12 minus16062 5136119864 minus 14

13 minus15026 1630119864 minus 09

14 minus15784 8279119864 minus 13

15 minus157205 1562119864 minus 12


2 minus30045 7665119864 minus 42

3 minus23734 1963119864 minus 14


10 minus31386 1156119864 minus 47

11 minus34271 3398119864 minus 60

12 minus25891 8416119864 minus 24


15 minus27082 5658119864 minus 29


Psyche 13









8 Junonia genoveva

4 Junonia genoveva



2 Junonia zonalis

2 Junonia genoveva

1 Junonia genoveva

1 Junonia zonalis

1 Junonia neildi









2 Junonia zonalis

(a)

POP 3A

POP 3BPOP 3C

POP 5POP 6A

POP 6B

POP 6CPOP 2A

POP 1POP 2B

POP 4

00163

000450006 00242

00172004750043

001350052100549

00414

00142001

00474

001

(b)


14 Psyche


4 Discussion






Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 Psyche






















Psyche 5






3 Results



6 Psyche








LCB185LCB186

LCB184

LCB196LCB197

LCB190

LCB203LCB204




LCB201

LCB187

LCB199LCB198

LCB180









LCB195

LCB192LCB193

LCB208LCB200

LCB194

LCB209








J evareteSonora MX


05 SRNP 3118405 SRNP 58208

05 SRNP 5799905 SRNP 31186


00 SRNP 3055

05 SRNP 5799595 SRNP 6024

J evareteCosta Rica


















CIAD10 B07



J evareteCosta Rica



































J evarete Sonora MX


















Caribbean


hemisphere



51

73

63

91

88

64

53

56

55

9198

71

80

94

96

100

88

86

98

9699

5295

959377

97

74

76

100

56

80

9356

98

98

98

72

73


Psyche 7

Haplotypegroup A

Haplotypegroup B

ARG 4



LCB 027



LCB 160

LCB 162 LCB 163

LCB 188





LCB 207

LCB 191

LCB 204

LCB 159

LCB 320

ARG 18ARG 19

ARG 16

ARG 17

ARG 2

ARG 3ARG 1

ARG 14

ARG 10

ARG 13

ARG 15


LCB 013LCB 025

LCB 173

ARG 12





(a)





Haplotypegroup A

Haplotypegroup B



LCB 027LCB 166

LCB 169

LCB 173

LCB 167

LCB 192

LCB 320

LCB 162

LCB 160


LCB 157

LCB 188

LCB 163






LCB 159

LCB 207

LCB 025LCB 013

ARG 17

ARG 2

ARG 3ARG 1ARG 16

ARG 15

ARG 13

ARG 14

ARG 10


ARG 18ARG 19

ARG 12

ARG 4

ARG 9

(b)


8 Psyche

Haplotypegroup A

Haplotypegroup B





LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 017 LCB 163 LCB 162

LCB 188



LCB 209




LCB 159

LCB 160

LCB 320

LCB 033LCB 041

LCB 031

LCB 007LCB 029

LCB 032

LCB 036

LCB 037LCB 009

LCB 025LCB 013


LCB 172LCB 175

ARG 4

ARG 9ARG 16 ARG 18

ARG 14

ARG 1

ARG 2

ARG 10

ARG 13

ARG 15


ARG 20

ARG 3

ARG 19

ARG 12

ARG 17

(a)





Haplotypegroup A

Haplotypegroup B

LCB 007

LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 031

LCB 041LCB 033

LCB 017

LCB 320LCB 163

LCB 188

LCB 013

ARG 17

LCB 025

LCB 009LCB 037

LCB 029

LCB 032









LCB 159

LCB 160

LCB 162

ARG 4

ARG 9

ARG 12

ARG 16 ARG 18

ARG 2

ARG 5ARG 6ARG 7

ARG 11ARG 21ARG 22

ARG 10

ARG 13ARG 15

ARG 8

ARG 14

ARG 20

ARG 1

ARG 3

ARG 19

(b)


Psyche 9









10 Psyche






LCB 019LCB 027


LCB 026ARG 2LC B014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095



ARG 2

a3

a7

a21 a20

a22

a31

a27

a4

a14

a11

a18a17

a24

a15

a9a19

a13a28

a10

a23

a16

a2

a26

a34

a35

a12

a29

a33

a30

a32

a25

a8

a7

a5

a9

a6

a1



(a)


LCB 019LCB 027


LCB 026

ARG 2LCB 014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095








ARG 2

a21

a7

a3

a20

a22

a31

a27

a4

a1

a6

a5a8

a7 a9

a23

a16

a2

a12

a10

a29

a33

a30

a32

a13

a28

a9

a19

a15

a24

a17a18

a11

a14

a25

a35

a34

a26

(b)


Psyche 11



Medianln[119875(119863)]



4 minus5368 1664119864 minus 05

5 minus13683 1287119864 minus 41

6 minus13559 4426119864 minus 41

7 minus17316 2136119864 minus 57

8 minus13003 1156119864 minus 38

9 minus14933 4797119864 minus 47

10 minus6183 4806119864 minus 09

11 minus5965 4251119864 minus 08

12 minus5559 2465119864 minus 06

13 minus5393 1296119864 minus 05

14 minus4928 000115 minus4782 0006



RP2 (bp) RP4 (bp) RP6 (bp)35 37 3539 42 3841 46 4743 50 5047 56 6149 58 6351 62 7254 65 7356 68 7758 71 8260 79 8564 82 8966 85 9368 87 14772 89 14975 93 19076 95 19379 99 19780 10084 10386 11287 14188 14590 14992 15295 15598 158100 161107 164135 168136 175148 179150 196158 201160 202162 209182 223200 228228 231233 234234 237240 240241 247

249258261277280287289

12 Psyche

Table 5 Continued

RP2 (bp) RP4 (bp) RP6 (bp)291342345363369371389391394440483






Medianln[119875(119863)]



1 minus23598 9598119864 minus 47

2 minus17872 7078119864 minus 22

3 minus14720 3458119864 minus 08

4 minus14308 2129119864 minus 06



10 minus15254 1659119864 minus 10

11 minus14613 1008119864 minus 07

12 minus16062 5136119864 minus 14

13 minus15026 1630119864 minus 09

14 minus15784 8279119864 minus 13

15 minus157205 1562119864 minus 12


2 minus30045 7665119864 minus 42

3 minus23734 1963119864 minus 14


10 minus31386 1156119864 minus 47

11 minus34271 3398119864 minus 60

12 minus25891 8416119864 minus 24


15 minus27082 5658119864 minus 29


Psyche 13









8 Junonia genoveva

4 Junonia genoveva



2 Junonia zonalis

2 Junonia genoveva

1 Junonia genoveva

1 Junonia zonalis

1 Junonia neildi









2 Junonia zonalis

(a)

POP 3A

POP 3BPOP 3C

POP 5POP 6A

POP 6B

POP 6CPOP 2A

POP 1POP 2B

POP 4

00163

000450006 00242

00172004750043

001350052100549

00414

00142001

00474

001

(b)


14 Psyche


4 Discussion






Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Psyche 5






3 Results



6 Psyche








LCB185LCB186

LCB184

LCB196LCB197

LCB190

LCB203LCB204




LCB201

LCB187

LCB199LCB198

LCB180









LCB195

LCB192LCB193

LCB208LCB200

LCB194

LCB209








J evareteSonora MX


05 SRNP 3118405 SRNP 58208

05 SRNP 5799905 SRNP 31186


00 SRNP 3055

05 SRNP 5799595 SRNP 6024

J evareteCosta Rica


















CIAD10 B07



J evareteCosta Rica



































J evarete Sonora MX


















Caribbean


hemisphere



51

73

63

91

88

64

53

56

55

9198

71

80

94

96

100

88

86

98

9699

5295

959377

97

74

76

100

56

80

9356

98

98

98

72

73


Psyche 7

Haplotypegroup A

Haplotypegroup B

ARG 4



LCB 027



LCB 160

LCB 162 LCB 163

LCB 188





LCB 207

LCB 191

LCB 204

LCB 159

LCB 320

ARG 18ARG 19

ARG 16

ARG 17

ARG 2

ARG 3ARG 1

ARG 14

ARG 10

ARG 13

ARG 15


LCB 013LCB 025

LCB 173

ARG 12





(a)





Haplotypegroup A

Haplotypegroup B



LCB 027LCB 166

LCB 169

LCB 173

LCB 167

LCB 192

LCB 320

LCB 162

LCB 160


LCB 157

LCB 188

LCB 163






LCB 159

LCB 207

LCB 025LCB 013

ARG 17

ARG 2

ARG 3ARG 1ARG 16

ARG 15

ARG 13

ARG 14

ARG 10


ARG 18ARG 19

ARG 12

ARG 4

ARG 9

(b)


8 Psyche

Haplotypegroup A

Haplotypegroup B





LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 017 LCB 163 LCB 162

LCB 188



LCB 209




LCB 159

LCB 160

LCB 320

LCB 033LCB 041

LCB 031

LCB 007LCB 029

LCB 032

LCB 036

LCB 037LCB 009

LCB 025LCB 013


LCB 172LCB 175

ARG 4

ARG 9ARG 16 ARG 18

ARG 14

ARG 1

ARG 2

ARG 10

ARG 13

ARG 15


ARG 20

ARG 3

ARG 19

ARG 12

ARG 17

(a)





Haplotypegroup A

Haplotypegroup B

LCB 007

LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 031

LCB 041LCB 033

LCB 017

LCB 320LCB 163

LCB 188

LCB 013

ARG 17

LCB 025

LCB 009LCB 037

LCB 029

LCB 032









LCB 159

LCB 160

LCB 162

ARG 4

ARG 9

ARG 12

ARG 16 ARG 18

ARG 2

ARG 5ARG 6ARG 7

ARG 11ARG 21ARG 22

ARG 10

ARG 13ARG 15

ARG 8

ARG 14

ARG 20

ARG 1

ARG 3

ARG 19

(b)


Psyche 9









10 Psyche






LCB 019LCB 027


LCB 026ARG 2LC B014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095



ARG 2

a3

a7

a21 a20

a22

a31

a27

a4

a14

a11

a18a17

a24

a15

a9a19

a13a28

a10

a23

a16

a2

a26

a34

a35

a12

a29

a33

a30

a32

a25

a8

a7

a5

a9

a6

a1



(a)


LCB 019LCB 027


LCB 026

ARG 2LCB 014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095








ARG 2

a21

a7

a3

a20

a22

a31

a27

a4

a1

a6

a5a8

a7 a9

a23

a16

a2

a12

a10

a29

a33

a30

a32

a13

a28

a9

a19

a15

a24

a17a18

a11

a14

a25

a35

a34

a26

(b)


Psyche 11



Medianln[119875(119863)]



4 minus5368 1664119864 minus 05

5 minus13683 1287119864 minus 41

6 minus13559 4426119864 minus 41

7 minus17316 2136119864 minus 57

8 minus13003 1156119864 minus 38

9 minus14933 4797119864 minus 47

10 minus6183 4806119864 minus 09

11 minus5965 4251119864 minus 08

12 minus5559 2465119864 minus 06

13 minus5393 1296119864 minus 05

14 minus4928 000115 minus4782 0006



RP2 (bp) RP4 (bp) RP6 (bp)35 37 3539 42 3841 46 4743 50 5047 56 6149 58 6351 62 7254 65 7356 68 7758 71 8260 79 8564 82 8966 85 9368 87 14772 89 14975 93 19076 95 19379 99 19780 10084 10386 11287 14188 14590 14992 15295 15598 158100 161107 164135 168136 175148 179150 196158 201160 202162 209182 223200 228228 231233 234234 237240 240241 247

249258261277280287289

12 Psyche

Table 5 Continued

RP2 (bp) RP4 (bp) RP6 (bp)291342345363369371389391394440483






Medianln[119875(119863)]



1 minus23598 9598119864 minus 47

2 minus17872 7078119864 minus 22

3 minus14720 3458119864 minus 08

4 minus14308 2129119864 minus 06



10 minus15254 1659119864 minus 10

11 minus14613 1008119864 minus 07

12 minus16062 5136119864 minus 14

13 minus15026 1630119864 minus 09

14 minus15784 8279119864 minus 13

15 minus157205 1562119864 minus 12


2 minus30045 7665119864 minus 42

3 minus23734 1963119864 minus 14


10 minus31386 1156119864 minus 47

11 minus34271 3398119864 minus 60

12 minus25891 8416119864 minus 24


15 minus27082 5658119864 minus 29


Psyche 13









8 Junonia genoveva

4 Junonia genoveva



2 Junonia zonalis

2 Junonia genoveva

1 Junonia genoveva

1 Junonia zonalis

1 Junonia neildi









2 Junonia zonalis

(a)

POP 3A

POP 3BPOP 3C

POP 5POP 6A

POP 6B

POP 6CPOP 2A

POP 1POP 2B

POP 4

00163

000450006 00242

00172004750043

001350052100549

00414

00142001

00474

001

(b)


14 Psyche


4 Discussion






Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 Psyche








LCB185LCB186

LCB184

LCB196LCB197

LCB190

LCB203LCB204




LCB201

LCB187

LCB199LCB198

LCB180









LCB195

LCB192LCB193

LCB208LCB200

LCB194

LCB209








J evareteSonora MX


05 SRNP 3118405 SRNP 58208

05 SRNP 5799905 SRNP 31186


00 SRNP 3055

05 SRNP 5799595 SRNP 6024

J evareteCosta Rica


















CIAD10 B07



J evareteCosta Rica



































J evarete Sonora MX


















Caribbean


hemisphere



51

73

63

91

88

64

53

56

55

9198

71

80

94

96

100

88

86

98

9699

5295

959377

97

74

76

100

56

80

9356

98

98

98

72

73


Psyche 7

Haplotypegroup A

Haplotypegroup B

ARG 4



LCB 027



LCB 160

LCB 162 LCB 163

LCB 188





LCB 207

LCB 191

LCB 204

LCB 159

LCB 320

ARG 18ARG 19

ARG 16

ARG 17

ARG 2

ARG 3ARG 1

ARG 14

ARG 10

ARG 13

ARG 15


LCB 013LCB 025

LCB 173

ARG 12





(a)





Haplotypegroup A

Haplotypegroup B



LCB 027LCB 166

LCB 169

LCB 173

LCB 167

LCB 192

LCB 320

LCB 162

LCB 160


LCB 157

LCB 188

LCB 163






LCB 159

LCB 207

LCB 025LCB 013

ARG 17

ARG 2

ARG 3ARG 1ARG 16

ARG 15

ARG 13

ARG 14

ARG 10


ARG 18ARG 19

ARG 12

ARG 4

ARG 9

(b)


8 Psyche

Haplotypegroup A

Haplotypegroup B





LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 017 LCB 163 LCB 162

LCB 188



LCB 209




LCB 159

LCB 160

LCB 320

LCB 033LCB 041

LCB 031

LCB 007LCB 029

LCB 032

LCB 036

LCB 037LCB 009

LCB 025LCB 013


LCB 172LCB 175

ARG 4

ARG 9ARG 16 ARG 18

ARG 14

ARG 1

ARG 2

ARG 10

ARG 13

ARG 15


ARG 20

ARG 3

ARG 19

ARG 12

ARG 17

(a)





Haplotypegroup A

Haplotypegroup B

LCB 007

LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 031

LCB 041LCB 033

LCB 017

LCB 320LCB 163

LCB 188

LCB 013

ARG 17

LCB 025

LCB 009LCB 037

LCB 029

LCB 032









LCB 159

LCB 160

LCB 162

ARG 4

ARG 9

ARG 12

ARG 16 ARG 18

ARG 2

ARG 5ARG 6ARG 7

ARG 11ARG 21ARG 22

ARG 10

ARG 13ARG 15

ARG 8

ARG 14

ARG 20

ARG 1

ARG 3

ARG 19

(b)


Psyche 9









10 Psyche






LCB 019LCB 027


LCB 026ARG 2LC B014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095



ARG 2

a3

a7

a21 a20

a22

a31

a27

a4

a14

a11

a18a17

a24

a15

a9a19

a13a28

a10

a23

a16

a2

a26

a34

a35

a12

a29

a33

a30

a32

a25

a8

a7

a5

a9

a6

a1



(a)


LCB 019LCB 027


LCB 026

ARG 2LCB 014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095








ARG 2

a21

a7

a3

a20

a22

a31

a27

a4

a1

a6

a5a8

a7 a9

a23

a16

a2

a12

a10

a29

a33

a30

a32

a13

a28

a9

a19

a15

a24

a17a18

a11

a14

a25

a35

a34

a26

(b)


Psyche 11



Medianln[119875(119863)]



4 minus5368 1664119864 minus 05

5 minus13683 1287119864 minus 41

6 minus13559 4426119864 minus 41

7 minus17316 2136119864 minus 57

8 minus13003 1156119864 minus 38

9 minus14933 4797119864 minus 47

10 minus6183 4806119864 minus 09

11 minus5965 4251119864 minus 08

12 minus5559 2465119864 minus 06

13 minus5393 1296119864 minus 05

14 minus4928 000115 minus4782 0006



RP2 (bp) RP4 (bp) RP6 (bp)35 37 3539 42 3841 46 4743 50 5047 56 6149 58 6351 62 7254 65 7356 68 7758 71 8260 79 8564 82 8966 85 9368 87 14772 89 14975 93 19076 95 19379 99 19780 10084 10386 11287 14188 14590 14992 15295 15598 158100 161107 164135 168136 175148 179150 196158 201160 202162 209182 223200 228228 231233 234234 237240 240241 247

249258261277280287289

12 Psyche

Table 5 Continued

RP2 (bp) RP4 (bp) RP6 (bp)291342345363369371389391394440483






Medianln[119875(119863)]



1 minus23598 9598119864 minus 47

2 minus17872 7078119864 minus 22

3 minus14720 3458119864 minus 08

4 minus14308 2129119864 minus 06



10 minus15254 1659119864 minus 10

11 minus14613 1008119864 minus 07

12 minus16062 5136119864 minus 14

13 minus15026 1630119864 minus 09

14 minus15784 8279119864 minus 13

15 minus157205 1562119864 minus 12


2 minus30045 7665119864 minus 42

3 minus23734 1963119864 minus 14


10 minus31386 1156119864 minus 47

11 minus34271 3398119864 minus 60

12 minus25891 8416119864 minus 24


15 minus27082 5658119864 minus 29


Psyche 13









8 Junonia genoveva

4 Junonia genoveva



2 Junonia zonalis

2 Junonia genoveva

1 Junonia genoveva

1 Junonia zonalis

1 Junonia neildi









2 Junonia zonalis

(a)

POP 3A

POP 3BPOP 3C

POP 5POP 6A

POP 6B

POP 6CPOP 2A

POP 1POP 2B

POP 4

00163

000450006 00242

00172004750043

001350052100549

00414

00142001

00474

001

(b)


14 Psyche


4 Discussion






Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Psyche 7

Haplotypegroup A

Haplotypegroup B

ARG 4



LCB 027



LCB 160

LCB 162 LCB 163

LCB 188





LCB 207

LCB 191

LCB 204

LCB 159

LCB 320

ARG 18ARG 19

ARG 16

ARG 17

ARG 2

ARG 3ARG 1

ARG 14

ARG 10

ARG 13

ARG 15


LCB 013LCB 025

LCB 173

ARG 12





(a)





Haplotypegroup A

Haplotypegroup B



LCB 027LCB 166

LCB 169

LCB 173

LCB 167

LCB 192

LCB 320

LCB 162

LCB 160


LCB 157

LCB 188

LCB 163






LCB 159

LCB 207

LCB 025LCB 013

ARG 17

ARG 2

ARG 3ARG 1ARG 16

ARG 15

ARG 13

ARG 14

ARG 10


ARG 18ARG 19

ARG 12

ARG 4

ARG 9

(b)


8 Psyche

Haplotypegroup A

Haplotypegroup B





LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 017 LCB 163 LCB 162

LCB 188



LCB 209




LCB 159

LCB 160

LCB 320

LCB 033LCB 041

LCB 031

LCB 007LCB 029

LCB 032

LCB 036

LCB 037LCB 009

LCB 025LCB 013


LCB 172LCB 175

ARG 4

ARG 9ARG 16 ARG 18

ARG 14

ARG 1

ARG 2

ARG 10

ARG 13

ARG 15


ARG 20

ARG 3

ARG 19

ARG 12

ARG 17

(a)





Haplotypegroup A

Haplotypegroup B

LCB 007

LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 031

LCB 041LCB 033

LCB 017

LCB 320LCB 163

LCB 188

LCB 013

ARG 17

LCB 025

LCB 009LCB 037

LCB 029

LCB 032









LCB 159

LCB 160

LCB 162

ARG 4

ARG 9

ARG 12

ARG 16 ARG 18

ARG 2

ARG 5ARG 6ARG 7

ARG 11ARG 21ARG 22

ARG 10

ARG 13ARG 15

ARG 8

ARG 14

ARG 20

ARG 1

ARG 3

ARG 19

(b)


Psyche 9









10 Psyche






LCB 019LCB 027


LCB 026ARG 2LC B014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095



ARG 2

a3

a7

a21 a20

a22

a31

a27

a4

a14

a11

a18a17

a24

a15

a9a19

a13a28

a10

a23

a16

a2

a26

a34

a35

a12

a29

a33

a30

a32

a25

a8

a7

a5

a9

a6

a1



(a)


LCB 019LCB 027


LCB 026

ARG 2LCB 014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095








ARG 2

a21

a7

a3

a20

a22

a31

a27

a4

a1

a6

a5a8

a7 a9

a23

a16

a2

a12

a10

a29

a33

a30

a32

a13

a28

a9

a19

a15

a24

a17a18

a11

a14

a25

a35

a34

a26

(b)


Psyche 11



Medianln[119875(119863)]



4 minus5368 1664119864 minus 05

5 minus13683 1287119864 minus 41

6 minus13559 4426119864 minus 41

7 minus17316 2136119864 minus 57

8 minus13003 1156119864 minus 38

9 minus14933 4797119864 minus 47

10 minus6183 4806119864 minus 09

11 minus5965 4251119864 minus 08

12 minus5559 2465119864 minus 06

13 minus5393 1296119864 minus 05

14 minus4928 000115 minus4782 0006



RP2 (bp) RP4 (bp) RP6 (bp)35 37 3539 42 3841 46 4743 50 5047 56 6149 58 6351 62 7254 65 7356 68 7758 71 8260 79 8564 82 8966 85 9368 87 14772 89 14975 93 19076 95 19379 99 19780 10084 10386 11287 14188 14590 14992 15295 15598 158100 161107 164135 168136 175148 179150 196158 201160 202162 209182 223200 228228 231233 234234 237240 240241 247

249258261277280287289

12 Psyche

Table 5 Continued

RP2 (bp) RP4 (bp) RP6 (bp)291342345363369371389391394440483






Medianln[119875(119863)]



1 minus23598 9598119864 minus 47

2 minus17872 7078119864 minus 22

3 minus14720 3458119864 minus 08

4 minus14308 2129119864 minus 06



10 minus15254 1659119864 minus 10

11 minus14613 1008119864 minus 07

12 minus16062 5136119864 minus 14

13 minus15026 1630119864 minus 09

14 minus15784 8279119864 minus 13

15 minus157205 1562119864 minus 12


2 minus30045 7665119864 minus 42

3 minus23734 1963119864 minus 14


10 minus31386 1156119864 minus 47

11 minus34271 3398119864 minus 60

12 minus25891 8416119864 minus 24


15 minus27082 5658119864 minus 29


Psyche 13









8 Junonia genoveva

4 Junonia genoveva



2 Junonia zonalis

2 Junonia genoveva

1 Junonia genoveva

1 Junonia zonalis

1 Junonia neildi









2 Junonia zonalis

(a)

POP 3A

POP 3BPOP 3C

POP 5POP 6A

POP 6B

POP 6CPOP 2A

POP 1POP 2B

POP 4

00163

000450006 00242

00172004750043

001350052100549

00414

00142001

00474

001

(b)


14 Psyche


4 Discussion






Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

8 Psyche

Haplotypegroup A

Haplotypegroup B





LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 017 LCB 163 LCB 162

LCB 188



LCB 209




LCB 159

LCB 160

LCB 320

LCB 033LCB 041

LCB 031

LCB 007LCB 029

LCB 032

LCB 036

LCB 037LCB 009

LCB 025LCB 013


LCB 172LCB 175

ARG 4

ARG 9ARG 16 ARG 18

ARG 14

ARG 1

ARG 2

ARG 10

ARG 13

ARG 15


ARG 20

ARG 3

ARG 19

ARG 12

ARG 17

(a)





Haplotypegroup A

Haplotypegroup B

LCB 007

LCB 038

LCB 173

LCB 027LCB 166

LCB 177

LCB 169

LCB 167LCB 164

LCB 031

LCB 041LCB 033

LCB 017

LCB 320LCB 163

LCB 188

LCB 013

ARG 17

LCB 025

LCB 009LCB 037

LCB 029

LCB 032









LCB 159

LCB 160

LCB 162

ARG 4

ARG 9

ARG 12

ARG 16 ARG 18

ARG 2

ARG 5ARG 6ARG 7

ARG 11ARG 21ARG 22

ARG 10

ARG 13ARG 15

ARG 8

ARG 14

ARG 20

ARG 1

ARG 3

ARG 19

(b)


Psyche 9









10 Psyche






LCB 019LCB 027


LCB 026ARG 2LC B014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095



ARG 2

a3

a7

a21 a20

a22

a31

a27

a4

a14

a11

a18a17

a24

a15

a9a19

a13a28

a10

a23

a16

a2

a26

a34

a35

a12

a29

a33

a30

a32

a25

a8

a7

a5

a9

a6

a1



(a)


LCB 019LCB 027


LCB 026

ARG 2LCB 014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095








ARG 2

a21

a7

a3

a20

a22

a31

a27

a4

a1

a6

a5a8

a7 a9

a23

a16

a2

a12

a10

a29

a33

a30

a32

a13

a28

a9

a19

a15

a24

a17a18

a11

a14

a25

a35

a34

a26

(b)


Psyche 11



Medianln[119875(119863)]



4 minus5368 1664119864 minus 05

5 minus13683 1287119864 minus 41

6 minus13559 4426119864 minus 41

7 minus17316 2136119864 minus 57

8 minus13003 1156119864 minus 38

9 minus14933 4797119864 minus 47

10 minus6183 4806119864 minus 09

11 minus5965 4251119864 minus 08

12 minus5559 2465119864 minus 06

13 minus5393 1296119864 minus 05

14 minus4928 000115 minus4782 0006



RP2 (bp) RP4 (bp) RP6 (bp)35 37 3539 42 3841 46 4743 50 5047 56 6149 58 6351 62 7254 65 7356 68 7758 71 8260 79 8564 82 8966 85 9368 87 14772 89 14975 93 19076 95 19379 99 19780 10084 10386 11287 14188 14590 14992 15295 15598 158100 161107 164135 168136 175148 179150 196158 201160 202162 209182 223200 228228 231233 234234 237240 240241 247

249258261277280287289

12 Psyche

Table 5 Continued

RP2 (bp) RP4 (bp) RP6 (bp)291342345363369371389391394440483






Medianln[119875(119863)]



1 minus23598 9598119864 minus 47

2 minus17872 7078119864 minus 22

3 minus14720 3458119864 minus 08

4 minus14308 2129119864 minus 06



10 minus15254 1659119864 minus 10

11 minus14613 1008119864 minus 07

12 minus16062 5136119864 minus 14

13 minus15026 1630119864 minus 09

14 minus15784 8279119864 minus 13

15 minus157205 1562119864 minus 12


2 minus30045 7665119864 minus 42

3 minus23734 1963119864 minus 14


10 minus31386 1156119864 minus 47

11 minus34271 3398119864 minus 60

12 minus25891 8416119864 minus 24


15 minus27082 5658119864 minus 29


Psyche 13









8 Junonia genoveva

4 Junonia genoveva



2 Junonia zonalis

2 Junonia genoveva

1 Junonia genoveva

1 Junonia zonalis

1 Junonia neildi









2 Junonia zonalis

(a)

POP 3A

POP 3BPOP 3C

POP 5POP 6A

POP 6B

POP 6CPOP 2A

POP 1POP 2B

POP 4

00163

000450006 00242

00172004750043

001350052100549

00414

00142001

00474

001

(b)


14 Psyche


4 Discussion






Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Psyche 9









10 Psyche






LCB 019LCB 027


LCB 026ARG 2LC B014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095



ARG 2

a3

a7

a21 a20

a22

a31

a27

a4

a14

a11

a18a17

a24

a15

a9a19

a13a28

a10

a23

a16

a2

a26

a34

a35

a12

a29

a33

a30

a32

a25

a8

a7

a5

a9

a6

a1



(a)


LCB 019LCB 027


LCB 026

ARG 2LCB 014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095








ARG 2

a21

a7

a3

a20

a22

a31

a27

a4

a1

a6

a5a8

a7 a9

a23

a16

a2

a12

a10

a29

a33

a30

a32

a13

a28

a9

a19

a15

a24

a17a18

a11

a14

a25

a35

a34

a26

(b)


Psyche 11



Medianln[119875(119863)]



4 minus5368 1664119864 minus 05

5 minus13683 1287119864 minus 41

6 minus13559 4426119864 minus 41

7 minus17316 2136119864 minus 57

8 minus13003 1156119864 minus 38

9 minus14933 4797119864 minus 47

10 minus6183 4806119864 minus 09

11 minus5965 4251119864 minus 08

12 minus5559 2465119864 minus 06

13 minus5393 1296119864 minus 05

14 minus4928 000115 minus4782 0006



RP2 (bp) RP4 (bp) RP6 (bp)35 37 3539 42 3841 46 4743 50 5047 56 6149 58 6351 62 7254 65 7356 68 7758 71 8260 79 8564 82 8966 85 9368 87 14772 89 14975 93 19076 95 19379 99 19780 10084 10386 11287 14188 14590 14992 15295 15598 158100 161107 164135 168136 175148 179150 196158 201160 202162 209182 223200 228228 231233 234234 237240 240241 247

249258261277280287289

12 Psyche

Table 5 Continued

RP2 (bp) RP4 (bp) RP6 (bp)291342345363369371389391394440483






Medianln[119875(119863)]



1 minus23598 9598119864 minus 47

2 minus17872 7078119864 minus 22

3 minus14720 3458119864 minus 08

4 minus14308 2129119864 minus 06



10 minus15254 1659119864 minus 10

11 minus14613 1008119864 minus 07

12 minus16062 5136119864 minus 14

13 minus15026 1630119864 minus 09

14 minus15784 8279119864 minus 13

15 minus157205 1562119864 minus 12


2 minus30045 7665119864 minus 42

3 minus23734 1963119864 minus 14


10 minus31386 1156119864 minus 47

11 minus34271 3398119864 minus 60

12 minus25891 8416119864 minus 24


15 minus27082 5658119864 minus 29


Psyche 13









8 Junonia genoveva

4 Junonia genoveva



2 Junonia zonalis

2 Junonia genoveva

1 Junonia genoveva

1 Junonia zonalis

1 Junonia neildi









2 Junonia zonalis

(a)

POP 3A

POP 3BPOP 3C

POP 5POP 6A

POP 6B

POP 6CPOP 2A

POP 1POP 2B

POP 4

00163

000450006 00242

00172004750043

001350052100549

00414

00142001

00474

001

(b)


14 Psyche


4 Discussion






Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

10 Psyche






LCB 019LCB 027


LCB 026ARG 2LC B014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095



ARG 2

a3

a7

a21 a20

a22

a31

a27

a4

a14

a11

a18a17

a24

a15

a9a19

a13a28

a10

a23

a16

a2

a26

a34

a35

a12

a29

a33

a30

a32

a25

a8

a7

a5

a9

a6

a1



(a)


LCB 019LCB 027


LCB 026

ARG 2LCB 014


LCB 018

LCB 018

LCB 193

LCB 020LCB 020

LCB 008ARG 5ARG 16

LCB 008

LCB 193ARG 19



LCB 041LCB 041

ARG 18

ARG 11

LCB 005

LCB 005



LCB 185LCB 190

ARG 7

ARG 17LCB 095

LCB 095








ARG 2

a21

a7

a3

a20

a22

a31

a27

a4

a1

a6

a5a8

a7 a9

a23

a16

a2

a12

a10

a29

a33

a30

a32

a13

a28

a9

a19

a15

a24

a17a18

a11

a14

a25

a35

a34

a26

(b)


Psyche 11



Medianln[119875(119863)]



4 minus5368 1664119864 minus 05

5 minus13683 1287119864 minus 41

6 minus13559 4426119864 minus 41

7 minus17316 2136119864 minus 57

8 minus13003 1156119864 minus 38

9 minus14933 4797119864 minus 47

10 minus6183 4806119864 minus 09

11 minus5965 4251119864 minus 08

12 minus5559 2465119864 minus 06

13 minus5393 1296119864 minus 05

14 minus4928 000115 minus4782 0006



RP2 (bp) RP4 (bp) RP6 (bp)35 37 3539 42 3841 46 4743 50 5047 56 6149 58 6351 62 7254 65 7356 68 7758 71 8260 79 8564 82 8966 85 9368 87 14772 89 14975 93 19076 95 19379 99 19780 10084 10386 11287 14188 14590 14992 15295 15598 158100 161107 164135 168136 175148 179150 196158 201160 202162 209182 223200 228228 231233 234234 237240 240241 247

249258261277280287289

12 Psyche

Table 5 Continued

RP2 (bp) RP4 (bp) RP6 (bp)291342345363369371389391394440483






Medianln[119875(119863)]



1 minus23598 9598119864 minus 47

2 minus17872 7078119864 minus 22

3 minus14720 3458119864 minus 08

4 minus14308 2129119864 minus 06



10 minus15254 1659119864 minus 10

11 minus14613 1008119864 minus 07

12 minus16062 5136119864 minus 14

13 minus15026 1630119864 minus 09

14 minus15784 8279119864 minus 13

15 minus157205 1562119864 minus 12


2 minus30045 7665119864 minus 42

3 minus23734 1963119864 minus 14


10 minus31386 1156119864 minus 47

11 minus34271 3398119864 minus 60

12 minus25891 8416119864 minus 24


15 minus27082 5658119864 minus 29


Psyche 13









8 Junonia genoveva

4 Junonia genoveva



2 Junonia zonalis

2 Junonia genoveva

1 Junonia genoveva

1 Junonia zonalis

1 Junonia neildi









2 Junonia zonalis

(a)

POP 3A

POP 3BPOP 3C

POP 5POP 6A

POP 6B

POP 6CPOP 2A

POP 1POP 2B

POP 4

00163

000450006 00242

00172004750043

001350052100549

00414

00142001

00474

001

(b)


14 Psyche


4 Discussion






Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Psyche 11



Medianln[119875(119863)]



4 minus5368 1664119864 minus 05

5 minus13683 1287119864 minus 41

6 minus13559 4426119864 minus 41

7 minus17316 2136119864 minus 57

8 minus13003 1156119864 minus 38

9 minus14933 4797119864 minus 47

10 minus6183 4806119864 minus 09

11 minus5965 4251119864 minus 08

12 minus5559 2465119864 minus 06

13 minus5393 1296119864 minus 05

14 minus4928 000115 minus4782 0006



RP2 (bp) RP4 (bp) RP6 (bp)35 37 3539 42 3841 46 4743 50 5047 56 6149 58 6351 62 7254 65 7356 68 7758 71 8260 79 8564 82 8966 85 9368 87 14772 89 14975 93 19076 95 19379 99 19780 10084 10386 11287 14188 14590 14992 15295 15598 158100 161107 164135 168136 175148 179150 196158 201160 202162 209182 223200 228228 231233 234234 237240 240241 247

249258261277280287289

12 Psyche

Table 5 Continued

RP2 (bp) RP4 (bp) RP6 (bp)291342345363369371389391394440483






Medianln[119875(119863)]



1 minus23598 9598119864 minus 47

2 minus17872 7078119864 minus 22

3 minus14720 3458119864 minus 08

4 minus14308 2129119864 minus 06



10 minus15254 1659119864 minus 10

11 minus14613 1008119864 minus 07

12 minus16062 5136119864 minus 14

13 minus15026 1630119864 minus 09

14 minus15784 8279119864 minus 13

15 minus157205 1562119864 minus 12


2 minus30045 7665119864 minus 42

3 minus23734 1963119864 minus 14


10 minus31386 1156119864 minus 47

11 minus34271 3398119864 minus 60

12 minus25891 8416119864 minus 24


15 minus27082 5658119864 minus 29


Psyche 13









8 Junonia genoveva

4 Junonia genoveva



2 Junonia zonalis

2 Junonia genoveva

1 Junonia genoveva

1 Junonia zonalis

1 Junonia neildi









2 Junonia zonalis

(a)

POP 3A

POP 3BPOP 3C

POP 5POP 6A

POP 6B

POP 6CPOP 2A

POP 1POP 2B

POP 4

00163

000450006 00242

00172004750043

001350052100549

00414

00142001

00474

001

(b)


14 Psyche


4 Discussion






Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

12 Psyche

Table 5 Continued

RP2 (bp) RP4 (bp) RP6 (bp)291342345363369371389391394440483






Medianln[119875(119863)]



1 minus23598 9598119864 minus 47

2 minus17872 7078119864 minus 22

3 minus14720 3458119864 minus 08

4 minus14308 2129119864 minus 06



10 minus15254 1659119864 minus 10

11 minus14613 1008119864 minus 07

12 minus16062 5136119864 minus 14

13 minus15026 1630119864 minus 09

14 minus15784 8279119864 minus 13

15 minus157205 1562119864 minus 12


2 minus30045 7665119864 minus 42

3 minus23734 1963119864 minus 14


10 minus31386 1156119864 minus 47

11 minus34271 3398119864 minus 60

12 minus25891 8416119864 minus 24


15 minus27082 5658119864 minus 29


Psyche 13









8 Junonia genoveva

4 Junonia genoveva



2 Junonia zonalis

2 Junonia genoveva

1 Junonia genoveva

1 Junonia zonalis

1 Junonia neildi









2 Junonia zonalis

(a)

POP 3A

POP 3BPOP 3C

POP 5POP 6A

POP 6B

POP 6CPOP 2A

POP 1POP 2B

POP 4

00163

000450006 00242

00172004750043

001350052100549

00414

00142001

00474

001

(b)


14 Psyche


4 Discussion






Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Psyche 13









8 Junonia genoveva

4 Junonia genoveva



2 Junonia zonalis

2 Junonia genoveva

1 Junonia genoveva

1 Junonia zonalis

1 Junonia neildi









2 Junonia zonalis

(a)

POP 3A

POP 3BPOP 3C

POP 5POP 6A

POP 6B

POP 6CPOP 2A

POP 1POP 2B

POP 4

00163

000450006 00242

00172004750043

001350052100549

00414

00142001

00474

001

(b)


14 Psyche


4 Discussion






Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

14 Psyche


4 Discussion






Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Psyche 15








16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

16 Psyche








Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Psyche 17



5 Conclusions




Acknowledgments



References












18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

18 Psyche






































Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Psyche 19


































20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

20 Psyche


































Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Psyche 21




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Molecular Population Structure of Junonia ...downloads.hindawi.com/journals/psyche/2014/897596.pdf · evarete .Neild[ ]alsodesignatednewtypesfor J.evarete and J