butterflies? Inferences from multilocus DNA sequence...

Do pollen feeding and pupal-mating have a single origin in Heliconiusbutterflies? Inferences from multilocus DNA sequence data

Margarita Beltrán1,2,3, Chris D. Jiggins3, Andrew . V. Z. Brower4, Eldredge

Bermingham1, and James Mallet2

1Smithsonian Tropical Research Institute, AA 2072, Balboa, Panama.

2The Galton Laboratory, Department of Biology, University College London,

London NW1 2HE, United Kingdom

3Institute of Evolutionary Biology, School of Biology, University of Edinburgh,

Edinburgh EH9 3JT, Tel: (+44)(0)131 650 8624 Fax: (+44)(0)131 650 6564. Email.

[email protected]

4 Department of Zoology, University of Oregon State University. Corvallis, OR 97331USA

Running Title: Phylogenetic analysis of Heliconius

Phylogenetic analysis of Heliconius 1

AbstractPhylogenetic information is useful in understanding the evolutionary history ofadaptive traits. Here we present a well-resolved phylogenetic hypothesis for

Heliconius butterflies. We use this tree to investigate the evolution of two uniquetraits, pollen feeding and pupal-mating behaviour. Phylogenetic relationships among

60 Heliconiina species (86% of the whole entire subtribe) were inferred from partial

DNA sequences of the mitochondrial genes cytochrome oxidase I, cytochrome

oxidase II and 16S rRNA, and fragments of the nuclear genes elongation factor-1α,

apterous, decapentaplegic and wingless (3834bp in total). The results corroborate

previous hypotheses based on sequence data that Heliconius is paraphyletic, with

Laparus doris and Neruda falling within the genus, demonstrating a single origin forpollen feeding but with a loss of the trait in Neruda. However different genes are not

congruent in their placement of Neruda therefore monophyly of the pollen feedingspecies cannot be entirely ruled out. There is also a highly supported monophyletic

‘‘pupal-mating clade’’ suggesting that pupal mating evolved only once in the

Heliconiina.

Keywords: Ef1α-Heliconius- mtDNA-phylogeny-pollen feeding


IntroductionThe widespread availability of molecular sequence information has greatly facilitatedthe inference of phylogenetic relationships between species. These phylogenetic

hypotheses have been used to investigate the history of ecological and morphologicaltraits (Maddison, 1997; Maddison & Maddison, 1997; Miller and Wenzel, 1995;

Mitter & Brooks, 1983; Sillén-Tullberg, 1988; Wanntorp et al., 1990). In particular,

they have facilitated tests of whether unusual characteristics of particular taxa havearisen through convergent evolution or from a single origin (Miller et al., 1997; Mitter

& Brooks, 1983). In addition, complete species level phylogenetic hypotheses arebeing increasingly used to investigate factors associated with species diversification.

A phylogenetic tree provides evidence on the relative rate of lineage splitting among

clades, and can therefore be used to test whether particular traits are associated withhigher or lower rates of species formation (Barraclough, 1995; Barraclough, 1999;

Barraclough, 2001; Mitter et al., 1988; Barraclough, Harvey and Nee, 1995;

Barraclough, Hogan & Vogler, 1999; Barraclough & Nee, 2001). Species-levelphylogenetic hypotheses can therefore be highly informative, especially in taxa that

have been the object of extensive ecological and evolutionary study.

Unusual ecological and behavioural traits in Heliconius

The genus Heliconius (sensu stricto) or passion-vine butterflies together with theclosely-related genera Laparus Billberg, Eueides Hübner, and Neruda Turner, are one

of the best-known groups of Neotropical butterflies, and have been important instudies of ecological processes such as coevolution between insects and plants

(Brown, 1981). These derived members of the subtribe Heliconiina have undergone

rapid speciation and divergence, while also exhibiting impressive mimeticconvergence in wing patterns. One hypothetically derived group of Heliconius

butterflies have two unique traits that may have facilitated rapid adaptive radiation,pollen feeding and pupal-mating behaviour (Gilbert, 1991).

Most adult lepidopterans feed on fluid resources such as nectar, decomposing animalsand fruit, and dung. However, Gilbert (1972) showed that Heliconius butterflies

collect pollen for its nutritive value, rather than as an indirect result of visits for nectaras had previously been assumed. The butterflies collect and accumulate large loads of


pollen and the production of abundant saliva helps keep pollen attached to the

proboscis, which can gently masticate the pollen load for long periods, allowingbutterflies to obtain amino acids (Gilbert, 1972). Amino acids assimilated from pollen

increase egg production and enable a long adult life span of up to six months (Boggset al., 1981; Gilbert, 1972; Mallet et al., 1998). Also, pollen can provide nitrogen and

precursors for synthesis of cyanogenic glycosides that may increase the concentration

of defensive chemicals in adult butterflies (Cardoso, 2001; Nahrstedt & Davis, 1981).

Morphological studies have revealed no unique structures among the species that usepollen in their diets (Krenn et al., 2001; Penz & Krenn, 2000). However, there are a

combination of features that assist collection and processing of pollen. For example,

Laparus and Heliconius have the second segment of the labial palpi cylindrical ratherthan club-shaped as the rest of the Heliconiina. Penz (1999) suggested that narrow

labial palpi helps Heliconius and Laparus to keep pollen attached to their proboscis.

Behavioural modifications are also important: pollen-feeding species manipulateLantana flowers faster and more thoroughly compared to non-pollen-feeding relatives

(Krenn & Penz, 1998).

A second unusual trait found in some Heliconius species is a unique mating behaviour

known as ‘pupal-mating’. Males of certain species search larval food plants forfemale pupae. The males then sit on the pupae a day before emergence, and mating

occurs the next morning, before the female has completely eclosed (Gilbert, 1976;Deinert, 1994; Deinert, Longino & Gilbert, 1994). Various kinds of pupal-mating

occur scattered across several insect orders (Thornhill & Alcock, 1993); in passion-

vine butterflies almost half the Heliconius species (42%) are pupal-maters (Gilbert,1991). It has long been thought that pupal-mating has a single origin within

Heliconius, without subsequent loss. However, the molecular data do not providestrong statistical support for monophyly of the pupal-mating group (Brower 1997;

Beltrán et al., 2002).

Gilbert (1991) suggested that pupal-mating might play an important role in the

radiation of Heliconius as well as in the packing of Heliconius species into localhabitats. Pupal-mating might enhance the possibility of intrageneric mimicry because


in almost all cases, each mimetic species pair consists of a pupal-mating and a non

pupal-mating species. The strikingly different mating tactics of these groups couldallow phenotypically identical species to occupy the same habitats without mate

recognition errors. Second, this mating tactic may influence host-plant specialisation,because pupal-mating species may displace other heliconiines from their hosts by

interference competition (Gilbert, 1991). Males of these species sit on, attempt to

mate with, and disrupt eclosion of other Heliconius species of both mating types. Thisaggressive behaviour may prevent other heliconiine species from evolving preference

for host plants used by pupal-mating species.

Recent phylogenetic analyses (Brower, 1994a; Penz, 1999) have led to disagreement

over the origin of these two unique characteristics due to discordance among currentphylogenetic hypotheses for the relationships between the heliconiine butterflies. The

purpose of this study was to add new gene sequence data and use newer, model-based

methods of analysis in an attempt to resolve these conflicting ideas.

Systematics in of Heliconius butterflies

In the last sixty years seven major studies have addressed the systematics of the

passion-vine butterflies or Heliconiina (Brower, 1994a; Brower & Egan, 1997;

Brown, 1981; Emsley, 1963; Emsley, 1965; Michener, 1942; Penz, 1999) (Fig. 1).Current taxonomy places the ‘passion vine butterflies’ as a subtribe, Heliconiina,

within the tribe Heliconiini. This tribe includes various other Asian genera as well asthe neotropical genera considered here. The Heliconiini are place in the nymphaline

subfamily Heliconiinae, which also includes the Argynnini or fritillaries, and the

Acraeini (Lamas et al., 2004).

Using 16 characters of adult morphology, Michener, (1942) proposed a key to theidentification of seven subgenera of passion-vine butterflies: Heliconius (including

Eueides), Philaethria Billberg, Podotricha Michener, Dryas Hübner, Dryadula

Michener, Dione Hübner and Agraulis Boisduval and Le Conte. Subsequently,Emsley proposed a phylogenetic hypothesis at a higher level for passion-vine

butterflies (1963) and for the genus Heliconius (1965), based on the Michener (1942)generic revision and using 18 different characters, including the genitalia and other


adult morphological traits, such as the distribution of androconical scales on veins

(Emsley, 1963; Emsley, 1965). The result was a dendrogram including the mainspecies groups but was not based on any quantitative phylogenetic analysis (Fig. 1A

Emsley, 1963; Emsley, 1965).

The Michener (1942) and Emsley (1965) revisions included in Heliconius species that

are currently classified in the genera Eueides, Laparus and Neruda. Turner (1976)formally recognized three subgenera, Neruda, Laparus and Eueides as distinct from

Heliconius. Neruda is characterised by a distinct wing shape, particularly the broadtriangular forewings with very extensive friction patches in the male, although the

females have wings of more normal typical shape for Heliconius. Other characters are

lack of scoli on the head of the larva, pupal morphology, and short antennae in theadult. Turner (1976) also considered Laparus sufficiently distinct to be a candidate for

generic rank, in particular due to the pupae, that lack the gold spots and flanges and

well developed antennal spines of other species. Additionally, Laparus has a markedcolour polymorphism as an adult, is the only species with marked morphological

polymorphism as a pupa, and is the only species apart from N. metharme, to produceblue colour not by iridescence but by laying white scales over black (Turner, 1976).

Turner's four subgenera of Heliconius were later raised to generic rank, so that thepassion-vine butterflies were now considered to contain ten recognised genera:

Philaethria, Podotricha, Dryas, Dryadula, Dione, Agraulis, Eueides, Laparus,Neruda and Heliconius (Brown, 1981). Brown (1981) also collated a number of

changes in species level taxonomy, and erected a new phylogenetic hypothesis using

characters derived from pupal morphology (Turner, 1968), adult behaviour (Gilbert,1972), chemistry and cytogenetics (Brown, 1972). Brown considered Heliconius

(sensu lato) to consist of four separate genera: Eueides (12 species), Neruda (3species), Laparus (1 species), Eueides (12 species) and Heliconius (38 species)

following Turner (1976) (Fig. 1B). Brown (1981) used characters to justify

monophyly of his species groupings but neither he nor any of the earliercommentators had performed any formal, statistically based quantitative phylogenetic

analysis. Cethosia, an Old World heliconiine genus, the nymphaline old world genus,was used to root the tree and place Agraulis, Dione, Podotricha, Dryadula, and Dryas


as a group paraphyletic or "basal" to Heliconius (sensu lato), i.e. the rest of

Heliconius Eueides + Neruda + Laparus + Heliconius. As in Cethosia, in the "basal"group the wing venation of the discal cell of the hindwing is open. These ‘open cell’

heliconiines are generally fast-flying to avoid predation and are relatively edible(Brower, 1995). Also, their highly dispersive populations are associated with open

sunny habitats, where they visit unspecialised butterfly-pollinated flowers with short

corollas and large floral displays (e.g. Lantana) (Gilbert, 1991).

The remaining genera Eueides, Neruda, Heliconius and Laparus (i.e. Heliconius

(sensu lato)), were termed the ‘advanced genera’ and are the most diverse in terms of

numbers of species. All of these possess a closed discal cell (Brown, 1981). Their

wing patterns differ from the general nymphaline ground plan by great simplificationand loss of many elements, as well as by the appearance of several novel mimetic

patterns (Nijhout, 1991). The ‘closed-cell’ genera, Eueides, Neruda, Heliconius and

Laparus are relatively unpalatable, aposematic, and slow-flying. Heliconius andLaparus also feed on pollen from specialised butterfly-pollinated flowers such us

Psiguria (Gilbert, 1991). Within Heliconius, Brown used the absence of a signum onthe female bursa copulatrix as a character to define the pupal-mating group

(erato+sara/sapho group in Fig. 1B).

Recent contributions (Brower, 1994a; Brower & Egan, 1997; Penz, 1999) have

proposed new phylogenetic hypotheses for passion-vine butterflies. All these analysesemployed formal analyses using parsimony or weighted parsimony analysis. Brower

(1994a) presented a cladogram based on parsimony, with successive approximations

weighting, for 35 species of Heliconius and the related genera Eueides, Laparus, andNeruda, based on mitochondrial DNA (mtDNA) sequences from cytochrome oxidase

subunits I and II (950 bp of CoI +CoII ). Brower's study focused on the relationshipsbetween these ‘advanced’ genera and the five-heliconiine genera considered basal to

this clade, Dione, Podotricha, Philaethria, Dryadula, and Dryas (Fig. 1C). The data

supported most traditionally recognised species groups and also the monophyly of thefour closed-cell genera with respect to other heliconiine outgroups. However, in

Brower’s phylogeny Heliconius (sensu stricto) was made paraphyletic by the internal


placement of Eueides, Laparus and Neruda. Most surprisingly Eueides was nested

within the Heliconius pupal-mating group.

Three years later Brower & Egan (1997) published an expanded data set that added tothe mtDNA data a short nuclear protein-coding sequence, from the gene wingless (wg,

375 bp), and this led to a revision of the position of Eueides after adding to the

mtDNA data a nuclear protein-coding gene wingless (wg, 375 bp). This study alsoadded 7 new taxa including Agraulis to complete the ten recognised passion-vine

butterfly genera and used Speyeria cybele (Fabr.) (from the heliconiine tribeArgynnini) as an outgroup to assess relationships among basal Heliconiina. Separate

analyses, of the CoI+CoII sequences showed Eueides still nested within the pupal-

mating Heliconius clade and wg provided a poorly resolved picture of therelationships. Neither of these two gene regions alone supported the monophyly of

Heliconius with respect to Eueides. Nonetheless, simultaneous parsimony analysis of

the two regions together supported a topology largely in agreement with traditionalviews of heliconiine relationships based on morphology, in which Eueides is basal to

Heliconius, Neruda and Laparus. However, Heliconius remained paraphyleticbecause Neruda and Laparus still branched internally to the genus (Fig. 1D). These

results suggested that pollen-feeding behaviour evolved in the common ancestor of

Laparus and Heliconius and was subsequently lost in an ancestor of Neruda.

Most recently Penz (1999) proposed a higher-level phylogeny for the passion-vinebutterflies based on 146 morphological characters from early stages and adults. In this

study 24 exemplar species were analysed representing the ten currently accepted

genera of Heliconiina. The phylogeny derived from the combined analysis ofcharacter sets gathered from different life stages supported the monophyly of all

genera but differed in topology from all previously proposed hypotheses (Fig. 1E).The base of the tree and the relationships among Laparus, Eueides and Neruda

differed significantly from the molecular hypothesis (Brower, 1994a; Brower & Egan,

1997). In Penz's hypothesis, Heliconius was monophyletic with respect to Laparus,Eueides, and Neruda, a grouping supported by three pupal morphology characters.

Penz (1999) and Penz & Peggie (2003) suggested that pollen pollen-feedingbehaviour either evolved independently in Laparus and the ancestor of Heliconius, or


it evolved in the common ancestor of the genera Laparus, Neruda, Eueides, and

Heliconius but was subsequently lost by the ancestor of Neruda and Eueides.

Conflict between phylogenies

In summary, the current phylogenetic hypotheses are in conflict with one another, in

particular with regard to the relationships among the genera Heliconius, Eueides,

Neruda and Laparus. Three features might contribute to this conflict: taxon sampling,number of informative characters and methods of phylogenetic inference

reconstruction (Brower et al., 1996). 1) Sampling selected species in each highertaxon can result in erroneous hypotheses of character state homology that lower

accuracy of phylogenetic inference. Simulations have shown that using species as

terminal taxa gives the most accurate trees under almost all conditions, often by alarge margin (Wiens, 1998). Therefore, the broad species sampling is a positive aspect

of the DNA analysis by Brower (1994a) and Brower & Egan (1997), in contrast with

the morphological analysis of Penz (1999) where just one species per genus wassampled. 2) In molecular systematics the inference of phylogenies can benefit from a

combination of data sets that evolve at different rates (Huelsenbeck et al., 2001). TheBrower & Egan (1997) study clarified the position of Eueides by including the slower

evolving nuclear gene wg (Brower & DeSalle, 1998). However, the number of

characters informative for the basal branches of the Heliconiina remains low, due tosaturation at third positions in CoI and CoII (Brower, 1996a) and short wg sequences

(375bp). Resolution of relationships could clearly benefit from addition of morenuclear gene sequences. 3) Finally, there are many different methods for

reconstructing inferring phylogenies from nucleic acid sequence alignments,

principally maximum parsimony (MP), maximum likelihood (ML), and Bayesianmethods (BAY). Previous species-level phylogenetic analyses of the heliconiines

have all used MP, although recent work suggests that model model-based approaches(ML and BAY) commonly outperform MP with difficult phylogenetic data sets

(Huelsenbeck, et al. 2001). It would therefore benefit our understanding of heliconiine

systematics to apply modern model-based methods to the analysis of molecular data.


Implications for the evolution of key traits

These conflicts and uncertainties in the phylogenetic hypotheses for the heliconiinespecies have implications for our understanding of the evolution of some key traits

found in the group, the major problem being that different characters support differenthypotheses of phylogeny. To establish a useful robust phylogenetic hypothesis for the

heliconiines it would be helpful to add more taxa, more molecular data, and to

compare results from different methods of phylogenetic inference. The principal goalof this study was to construct a species level phylogeny using more data from

mitochondrial DNA and exons of nuclear genes, and include more taxa. Thisphylogenetic hypothesis was then used to address the following questions. Is

Heliconius monophyletic? How many times has pollen feeding arisen in the

Heliconius group? What are the relationships within major clades of Heliconius? Isthe pupal-mating group monophyletic?

Material and methods

Sampling methods

We sampled 122 individual butterflies, representing 38 Heliconius, 10 Eueides, and

10 outgroup species (Appendix 1). According to the Lamas (1998) and Lamas et al.

(2004) classification, only 12 species of Heliconiina are missing from this study: 4species of Heliconius (H. astraea Staudinger, H. lalitae Brévignon, H. tristero

Brower and H. Luciana Lichy), 1 Neruda (N. godmani (Staudinger)), 2 Eueides (E.

emsleyi (Staudinger), E. libitina Staudinger), and 5 outgroup heliconiines (Podotricha

judith (Guérin-Méneville), Philaethria constantinoi Salazar, P. ostara (Röber), P.

Pygmalion (Fruhstorfer), and P. wernickei (Röber) (Appendix 1). To evaluaterelationships between basal Heliconiina, we included Castilia perilla (Nymphalidae:

Nymphalinae: Melitaeini: Phyciodina) as an outgroup. Butterflies collected for thisstudy were preserved in liquid nitrogen and are stored in the Smithsonian Tropical

Research Institute in Panama. Wings of voucher specimens are preserved in glassine

envelopes; pictures are available at “http://www.heliconius.org”. From eachindividual, 1/6 of the thorax was used and the genomic DNA was extracted using the

DNeasy Kit (Qiagen) following the manufacturer’s recommended protocols. Samplesfrom different prior collections were obtained as DNA aliquots (Appendix 1).


Molecular regions and sequencing methods

Mitochondrial DNATwo mitochondrial DNA regions were used: first, a region of cytochrome oxidase

(Co), spanning cytochrome oxidase I (CoI), the mitochondrial gene for leucine

transfer RNA gene (tRNA-leu), cytochrome oxidase II (CoII); and second, the region

coding for 16S ribosomal RNA (16S). Both regions have been used to explorephylogenetic relationships in insects (e.g. DeSalle, 1992; Brower 1994a, 1994b;

Caterino & Sperling, 1999; Smith et al., 2002), although we here used 1611bp ofCoI+CoII compared to Brower's 950bp. Two different sampling strategies were

followed, for CoI+CoII at least 2 individuals per species were sequenced and for 16S

just 12 individuals were sequenced in order to check the relationships withinHeliconius (811 H. melpomene rosina, 346 H. numata, 8560 H. burneyi, 8549 H.

hecuba, 846 L. doris, 8569 N. aoede, 440 H. erato hydara, 8037 H. clysonymus, 842

H. eleuchia, 8562 H. demeter, 320 E. vibilia, and 293 D. iulia).

The mitochondrial CoI+CoII region was amplified using primers and protocolsdescribed previously (Beltrán et al. 2002). A Drosophila yakuba sequence (GenBank

accession number X03240) was used as a reference. The clean template obtained was

sequenced in a 10µl cycle sequence reaction mixture containing 1µl BigDye, 0.3x

buffer, 2mM primer, and 2µl of template. The cycle profile was 96 oC for 30 sec.,

then 96oC for 10 sec., 50oC for 15 sec, 60oC for 4 min. for 30 cycles. This product wascleaned by precipitation using 37.5µl of 70% EtOH and 0.5mM MgCl2. The samples

were re-suspended in 4µl of a 5:0.12 deionized formamide: crystal violet solution,

denatured at 85oC for 2 min. and loaded into 5.5% acrylamide gels. Gels were run on

BaseStation (MJ Research) for 3 hours.

The additional mitochondrial region used was the 16S ribosomal RNA gene. This

region was amplified using 16Sar1 5’-CCC GCC TGT TTA TCA AAA ACA T-3’and Ins16Sar 5’-CCC TCC GGT TTG AAC TCA GAT C -3’. Primers were obtained

by modifying Palumbi (1996) to improve amplification in Lepidoptera. The identity

of this region was confirmed by comparison with Eresia burchellii (GenBankaccession AF186861). Double-stranded DNA was synthesised in 10µl reactions


containing 2µl of genomic DNA, 1x buffer, 1mM MgCl2, 0.8mM dNTPs, 0.5mM of

each primer, and 0.05 u/µl of Qiagen Taq polymerase. DNA was amplified using the

following step-cycle profile: 94oC for 5 min., 94oC for 30 sec., 55oC for 30 sec., 72oCfor 1 min. for 34 cycles. These products were sequenced as described for CoI and

CoII.

Nuclear lociFour nuclear loci were used, elongation factor-1α (Ef1α), apterous (ap),

decapentaplegic (dpp) and wingless (wg). Ef1α is a key factor in protein synthesis

playing a central role in protein chain elongation (Bischoff et al., 2002). This gene hasbeen using in many phylogenetic studies and results showed informativeness of

synonymous nucleotide substitutions up to divergences of 60 million years (Cho et

al., 1995; Mitchell et al., 1997; Reed & Sperling, 1999). The genes ap and dpp, areinvolved in wing development in Drosophila and were isolated in Heliconius by the

Owen Mcmillan laboratory in Puerto Rico (Jiggins et al. 2005; Tobler et al., 2005),but there is no report of their phylogenetic utility. The sampling for Ef1α was the

same as CoI and CoII, for ap and dpp was the same as 16S (just 12 individualsrepresenting the major clades in Heliconius). In addition, wg sequences were included

in the analysis although not for the same individuals. Sequences of wg were loadedfrom Brower’s GenBank accessions AY090135, UO08554, AF014126 to AF014135

and AF169869 to AF169921).

The elongation factor 1α (Ef1α) region was initially amplified and sequenced from

genomic DNA using a mix of primers from Papilio (Ef1-5) (Reed & Sperling, 1999)

and bumble bees (F2-rev) (Walldorf & Hovemann, 1990). The primers were situatedat position 15 (Ef1-5) and 955 (F2-rev) of Papilio glaucus (GenBank accession

AF044826). Then, initial Heliconius sequences were aligned and Heliconius specific

primers were designed to amplify the region consistently using genomic DNAextracts. The specific primers designed were Ef1-H-f 5’- GAG AAG GAA GCC CAG

GAA AT-3’ and Ef1-H-r 5’- CCT TGA CRG ACA CGT TCT TT-3’. DNA wasamplified using the step cycle profile described for 16S and sequenced as for the

mitochondrial region.


The other two nuclear genes sequenced were apterous (ap) and decapentaplegic

(dpp). The gene apterous was amplified using primers ap-f35 5’-TGA ATC CTGAAT ACC TGG AGA-3’ and ap-r224 5’-GGA ACC ATA CCT GTA AAA CCC-3’

and decapentaplegic using dpp-f34 5’-AGA GAA CGT GGC GAG ACA CTG-3’ anddpp-r327 5’-GAG GAA AGT TGC GTA GGA ACG-3’ (Jiggins et al. 2005; Tobler et

al., 2005). The identities of the regions were verified by aligning with Precis coenia

GenBank accession numbers L42140 and L42141 respectively. The products from ap

and dpp were sequenced as a described before.

Alignment and phylogenetic analysesChromatograms were edited and base calls checked using SEQUENCHER 4.1 (Gene

Codes Corporation, Inc). The protein-coding mtDNA and nuclear DNA sequenceswere checked for reading-frame errors and unexpected stop codons by translating the

nucleotide sequences to peptides using MacClade 4.0 (Maddison & Maddison, 1997).

Maximum likelihood models of sequence evolution for each gene were estimatedusing ModelTest 3.04 (Posada & Crandall, 1998). Bayesian analysis (BAY) run in

MrBayes (Huelsenbeck & Ronquist, 2001) was used to infer the phylogeny based onthe best-fit model selected by ModelTest. Specific nucleotide substitution model

parameter values estimated for each gene were used in the combined analysis. Four

chains were run simultaneously, each Markov chain was started from a random treeand run for one million generations, sampling a tree every 100 generations. The log

likelihood scores of sample points were plotted against generation time to determinewhen the chain became stationary. All sample points prior to reaching stationarity

(2,000 trees) were discarded as burn-in samples. Data remaining after discarding

burn-in samples were used to generate a majority rule consensus tree, wherepercentage of samples recovering any particular clade represented the posterior

probability of that clade (Huelsenbeck & Ronquist, 2001). Probabilities ≥ 95% wereconsidered indicative of significant support. Branch lengths of consensus tree were

estimated by maximun likelihood. For comparison, maximun parsimony (MP) trees

were obtained using PAUP* version 4.0b8 (Swofford, 2000) in an equal weightedheuristic search with TBR branch swapping. The consensus tree was calculated using

majority rule. Bootstrap (1000 replicates, heuristic search TBR branch swapping) wasused to assess confidence support for each node.


The Incongruence for Length Difference test (ILD; Farris et al., 1994) implementedby PAUP* was used to test incongruence between the different partitions, (e.g.

CoI/CoII vs. Ef1α; mtDNA (CoI, tRNA-leu, CoII, 16S) vs. nuclear (Ef1α, ap, dpp,

wg); CoI vs. ap; Ef1α vs. ap; etc). This test was applied to a matrix including the

twelve individuals sequenced for CoI, CoII, Ef1α, ap, dpp adding wg sequences of

GenBank for these individuals. Additionally, to test specific hypotheses alternative a

priori scenarios were compared using the method of Shimodaira and Hasegawa(Goldman et al., 2000; Shimodaira and Hasegawa, 1999) and implemented using

PAUP* version 4.0b8. For each genus (i.e. Heliconius, Laparus, Neruda, Eueides),two or three topologies were compared in the same test. To generate trees for each

scenario, the topology shown in Figure 3 was modified using MacClade (Maddison &

Maddison, 1997).

Results

Characterisation of the nucleotide data

The final nucleotide data set contained 3834 positions (2119 mitochondrial, 1716nuclear), translating to 1083 amino acids (511 mitochondrial, 572 nuclear). The

individual sequences are available at GenBank (accession numbers in appendix 1) and

the alignment of full data is available at “htpp:/www.heliconius.org”.

Mitochondrial DNAFor mitochondrial DNA (mtDNA) 1611bp were obtained from the CoI+CoII region

including nucleotides and gaps. These represent 822 bp of CoI corresponding to

position 2191 to 3009 of the D. yakuba sequence (X03240), the complete tRNA-leu

gene (78bp) and 711 bp representing the entire CoII coding sequence, matching

positions 3012-3077 and 3083-3766 in D. yakuba respectively. For 16S ribosomalRNA 512bp were amplified corresponding to positions 26 to 541 in Eresia burchellii

(AF186861). Length variation was concentrated in tRNA-leu and in 16S. At the

beginning of tRNA-leu an insertion of 12bp was found in one individual of H. demeter

(STRI-B-8563) while 7bp of the same insertion was shared by H. charithonia, H.

peruvianus, H. ricini and the second individual of H. demeter (STRI-B- 8562).


Another 3bp insertion was observed at position 71 in H. ismenius. In 16S region a

total of 29 gaps were found located between positions 51 to 63, 241 to 280, and 337 to3511. Additionally, codon deletions were found. In CoI the third codon of the

alignment, corresponding to amino acid position #243 in D. yakuba (X03240), wasdeleted in some Eueides species (E. lineata, E. vibilia, E. lybia, E. aliphera, E.

isabella, and E. tales). There was another codon deletion in H. ismenius just before

the CoI stop codon. In CoII three closely adjacent codon deletions were observed atamino acid position #126 in Dryadula phaetusa, #127 in H. sara and at position #129

in Dryas iulia.

Of the 1611 nucleotide sites examined for mtDNA, 718 (44%) were variable (Table

1). Almost all the variation occurred in the protein-coding region (656 were variablein CoI and CoII). As expected the variation per position was higher at third positions

(70%) (Table 1). In total for mtDNA 30% of the sites were informative.

Nuclear DNAThe nuclear genes Ef1α 876bp, ap 195 bp and dpp 270bp were aligned with Papilio

glaucus (GenBank accession AF044826) at positions 50 to 925, Precis coenia

(L42140) at positions 193 to 387 and Precis coenia (LA42141) at positions 145 to

414, respectively. Only dpp showed length variation with respect to the reference

sequence, a codon deletion at position 196 of Precis coenia (LA42141) was observedin H. cydno chioneus, H. numata and H. burneyi.

There were similar levels of variability across nuclear genes and codon positions

(Table 1). Ef1α showed 21% of sites variable and most of the variation was at third

positions (79%), while ap, dpp, and wg showed similar levels of variation overall

(16%, 21% and 25% respectively) (Table 1), and for ap, dpp, and wg at third positions(85%, 77% and 61% respectively). Also, the relative divergence of first, second and

third positions were similar among nuclear and mtDNA (Table 1).

Among the 12 species sampled for dpp, ap, wg, and 16S divergence ranged from 0.1%

between sister groups (H. m. rosina vs. H. numata) to 26% between Heliconius

species and outgroups (H. m. rosina vs. D. iulia). The complete data for CoI+CoII and


Ef1α showed divergences ranging from 0.3% (between sister groups) to 39%

(between Heliconius species and outgroups) and 0.3% to 32% respectively. The

lowest divergence was at Ef1α second position, which had a maximum pairwise

divergence of <3% within Heliconiina, whereas comparing nuclear and mtDNAregions nearly all third positions exhibited divergences >10%. Additionally,

divergences at the first two codon positions were higher in the mitochondrial data

than in Ef1α, indicating greater rates of protein evolution as well as nucleotide

evolution in the mtDNA region.

Models of sequence evolution

There was some variation between models of sequence evolution selected for each

region (Table 2). Evolution at CoI+CoII and Ef1α were best explained by the six-

parameter general time reversible model of nucleotide substitution (GTR) (Yang,

1994). For 16S and the rest of nuclear genes, simpler models fitted the data (Table 2):TrNef with just three substitution types (Tamura and Nei, 1993), F81 with equal base

frequencies (Felsenstein, 1981) and K2P with a single substitution type (Kimura,

1980). It is perhaps unsurprising that smaller data matrices supported simpler modelsof evolution. As is usual for insects the mitochondrial DNA showed a strong AT bias

(Caterino & Sperling, 1999), AT content for CoI+CoII was 82% and 16S 81%compared with nuclear DNA (50%) (Table 2). Also, inferred transitions were almost

ten times more frequent than transversions in CoI+CoII, Ef1α, and wg, while other

nuclear and mitochondrial regions did not have significant transition: transversion rate

bias, probably due to the lower sample sizes (Table 2). The gamma parameter variedbetween 0.06 and 0.80 (Table 2). Only in CoI+CoII and Ef1α was there a significant

non-zero parameter for proportion of invariable sites (I = 0.50 and 0.54 respectively)

(Table 2).

Congruence test

ILD tests between mitochondrial data (CoI + tRNA-leu + CoII + 16S) vs. nuclear data(Ef1α + ap + dpp + wg) provided no evidence for incongruence between among

partitions based on nucleotides (p=0.08), nor between amino acid sequence partitions

(p=0.23). The lack of incongruence between among amino acid partitions results in

part from low resolution in the nuclear partition because only 13 amino acids were


informative. Comparisons within mtDNA did not show any incongruence either (e.g.

CoI vs. CoII p=0.18 and CoI+CoII vs. 16S p=0.40), and neither did mtDNA vs.individual partitions of nuclear genes. Within nuclear genes only one comparison

showed significant incongruence, Ef1α vs. wg (p=0.01) and it was the only significant

test out of 18 comparisons in total. Therefore, there was no strong evidence forsignificant incongruence between data sets and total data were used to calculate a

combined evidence phylogenetic hypothesis.

Phylogenetic analyses

Topologies for individual data sets are shown in Figure 2 (A. mtDNA, B. Ef1α, and

C. ap, dpp and wg) and the combined hypothesis using all genes described before is

shown in Figure 3. Phylogenetic resolution was somewhat weaker at nuclear locicompared with the mtDNA, for example mtDNA and Ef1α showed a monophyletic

clade that included the sister clades cydno-melpomene and the silvaniforms, but in

Ef1α there was no resolution of species relationships within that clade (Fig. 2A and

B). However resolution increased for clades in which species are more distantly

related such as sara/sapho and erato.

The data also produced well- resolved relationships among genera (Fig. 3).Heliconius, Laparus and Neruda formed a well-supported monophyletic clade with

Eueides basal to this group, in agreement with traditional relationships and prior

molecular hypotheses (Brown, 1981; Brower & Egan, 1997). Laparus fell in a well-supported clade with H. hierax, H. wallacei, and H. hecuba. Also, Neruda fell within

Heliconius closely related to cydno/melpomene and the silvaniform group. Variousalternative topologies were compared using SH tests (Fig. 4 Table 3). The results

showed that a tree constrained to have both L. doris and Neruda basal to Heliconius,

as Brown (1981) was a significantly worse fit both for mtDNA and nuclear DNAalone and also for combined evidence (Fig. 4E p<0.001). When Laparus alone was

forced to be basal to Heliconius the resulting tree was a worse fit to the data based oncombined evidence (Fig. 4B p=0.039). In contrast, even combined evidence could not

exclude the possibility that Neruda was basal to Heliconius (mtDNA p= 0.507,

nuclear p=0.365, combined p=0.329) (Fig. 4G Table 3). This might be a result of thedifferent placements suggested by different genes: mtDNA placed Neruda inside the


Heliconius "primitive" group (Fig. 2A), while nuclear data from Ef1α, ap, dpp, and

wg placed Neruda basal to the silvaniforms + cydno/melpomene group (Fig. 2B and

C).

For comparison we carried out a re-analysis of Brower’s (1994a) mtDNA data. A MLtree was constructed based on general-time-reversible time model of nucleotide

substitution (GTR+G+I) (Yang, 1994) suggested as the best fit for these data by

likelihood (Posada & Crandall, 1998). The ML tree showed Eueides basal toHeliconius. To confirm this pattern we ran a BAY analysis (Huelsenbeck &

Ronquist, 2001), and the tree showed strong support for placing Eueides basal to

Heliconius (Fig. 5). Additionally, the SH results showed that a tree similar to thatinferred using MP by Brower, in which Eueides clustered to Heliconius charitionia

(Brower 1994a), was a significantly worse fit for nuclear DNA and for combinedevidence. However, mtDNA could not exclude the possibility that Eueides was part of

the pupal mating group (mtDNA p= 0.266, nuclear p=0.017, combined p=0.005) (Fig.

4D Table 3).

DiscussionWe have presented a novel phylogenetic hypothesis for the Heliconiina that includes

more species and more phylogenetic information than previous studies. Nine new

species were added to those used by Brower & Egan (1997): five Heliconius, H.

nattereri, H. hierax, H. hecalesia, H. peruvianus, and H. hermathena; four Eueides, E.

lampeto, E. pavana, E. lineata, E. heliconioides, and one outgroup species Dione

moneta. Following Lamas et al. (2004) the species included represent 36 of 40

Heliconius species (90%), and 60 of 69 (86%) of the species in the subtribe

Heliconiina. One of the missing species is H. tristero, recently described by Brower(1996b); however, this species is very close to and/or a hybrid of H. melpomene and

H. cydno. The remaining missing species H. astraea, H. lalitae, and H. luciana, aredifficult to obtain as they are restricted to small areas of Brazil, French Guiana, and

Venezuela respectively; they probably belong to the "primitive" Heliconius group

(Fig. 3; see also Brown, 1981). Additionally, three new nuclear regions were studiedfor this subtribe Ef1α (876bp), ap (195 bp) and dpp (270bp), and 659bp were added to


the 950bp CoI+CoII region reported by Brower (1994a, 1996b) and Brower & Egan

(1997) for Heliconius.

The phylogenetic hypothesis from combined evidence (Fig. 3) largely agrees with thatof Brower & Egan (1997). Of 25 nodes at or above the species level 23 are

concordant including the position of the genera Eueides, Neruda and L. doris. The

position of Laparus doris as a member of Heliconius was well supported. Theposition of Neruda within Heliconius was independently supported by nuclear and

mtDNA data (Fig. 2), but cannot be considered unequivocal, as topology tests failedto rule out the hypothesis that Neruda is sister to Heliconius. The most likely

hypothesis therefore is that pollen feeding arose once but was subsequently lost in

Neruda. Nonetheless, we cannot reject a more parsimonious single-origin no-losshypothesis of pollen feeding, arising in a sister taxon to Neruda that went on to

diversify into present day Laparus and Heliconius.

The first published molecular phylogeny of Heliconius, reconstructed using

parsimony, showed a surprising placement of the genus Eueides within Heliconius

(Brower, 1994). Bayesian reanalysis (Fig. 5) of the same data suggests that this was

an artefact of parsimonious interpretation of homoplastic character states, perhaps due

to "long-branch attraction": fast-evolving sites in the mitochondrial CoI gene happento show convergent evolution between the H. erato group and the genus Eueides, but

that these are outweighed in the Bayesian analysis by information from putativelyslower and more informative sites. In a model-based analysis, the likelihood of

homoplasy is taken into account and inferred rapidly evolving sites are down-

weighted, leading to more realistic phylogenetic reconstruction concordant withresults from other, slower-evolving genes. Like Brower & Egan’s (1997) subsequent

parsimony analysis that included a nuclear gene region, the current results give moresupport for the traditional view of Eueides as a sister group to Heliconius (Fig. 5);

although it is also true to say that even with the extra CoI+CoII data used here, we

cannot rule out Eueides as a part of the pupal-mating group using an SH test (Fig. 4Table 3).


Relationships in the melpomene/cydno and silvaniform group

The melpomene/cydno group and the silvaniform complex consist of a rapidlyradiating group of species with little differentiation at nuclear loci. The combined

analysis reveals two monophyletic groups melpomene/cydno and the silvaniforms,both with a posterior probability support of 1.0 (Fig. 3). This result is mostly due to

information from mtDNA (Fig. 2A), because Ef1α gives little informative variation

(Fig. 2B).

In the melpomene/cydno group, races of H. melpomene cluster into two different

clades. H. melpomene races from west of the easternmost Andean chain in Colombia

clustered with the H. cydno clade, while races of H. melpomene from east of theAndes clustered with H. m. melpomene from French Guiana (see also Brower 1996b;

Flanagan et al., 2004). H. cydno appeared appears paraphyletic with respect to H.

heurippa, H. pachinus, and H. timareta (Fig. 2A). Brower (1994, 1996a) and Lamas

(1998) suggested that H. heurippa, H. tristero, H. pachinus, and H. timareta might

represent well-differentiated races of H. cydno rather than distinct species, becausethey are parapatric or allopatric. Clearly, these taxa are close; however, analyses of

genitalia, allozymes, RAPDs, and mating behaviour have suggested that H. heurippa

is a good species (Beltrán, 1999; Salazar et al. 2005).

The composition of the silvaniform complex agrees with Brower (1994a), (Fig. 3), butthe exact topology differed. The H. numata + H. ismenius and H. atthis + H. hecale

species pairs were the only nodes in agreement with Brower & Egan (1997). It hasbeen considered that H. ethilla is a sister to H. atthis based on their ecology, but H.

atthis and H. hecale are sympatric in Ecuador and it seems possible that their apparent

sister species relationship could be a result of recent gene exchange. Additionally, it isclear that H. elevatus and H. besckei are part of this complex rather than in the

melpomene/cydno group as proposed by Brown (1981). Most of the silvaniforms havea typical ‘tiger’ colour pattern and Brower (1994a, 1997) proposed that the ‘postman’

pattern (red forewing patches and yellow hindwing stripes on a black background) of

H. besckei might be the ancestral colour pattern of this clade. This idea is supportedhere as since H. besckei is placed as sister to the silvaniforms.


‘Pupal-mating clade’

The combined results provided strong support for a monophyletic ‘pupal-mating’clade, suggesting that this trait may have played an important role in the phylogenetic

expansion of Heliconius as well as in the packing of Heliconius species into localhabitats. This group includes sara/sapho, erato/himera and H. charithonia groups

(Fig. 3). All the species in this clade were part of the ‘pupal-mating clade’ in Brown

(1981), Brower (1994a) and Brower & Egan (1997). The sara/sapho clade has thelargest sister species genetic distances between sister species in Heliconius,

suggesting that species such as H. eleuchia, H. congener, and H. sapho are relativelyancient. In this clade the topology remained remains largely unchanged as compared

to the previous hypothesis (Brower & Egan, 1997), and the additional newly-elevated

species H. peruvianus was placed as sister to H. charithonia as expected (Jiggins andDavies, 1998).

In the erato clade three additional species were included, H. hermathena, H.

hecalesia, and H. hortense. The placements of H. hermathena and H. hortense are

different to those previously suggested (Brown, 1981). H. hermathena is restricted tocertain non-forest habitats in the Brazilian Amazon. One of its four subspecies H. h.

vereatta, is mimetic of sympatric H. m. melpomene and H. e. hydara and is very

restricted geographically (Brown & Benson, 1977). The other three are non-mimetic,little differentiated and apparently widespread but the populations are patchy and in

low densities. Their wing colour pattern is black, yellow and red; the forewing isblack with red, resembling H. m. melpomene and H. e. hydara, while the hindwing is

black with yellow bars and spots, resembling H. charithonia. Brown and Benson

(1977) suggested, based on adult morphology and pupal-mating behaviour, that H.

hermathena is closely related to H. erato and H. charithonia. However, the pupae

lack the derived characters of long pupal head appendages, suggesting the species isrelatively primitive, near to the melpomene group, in which all the members show

shortened head appendages. The results shown here demonstrate that H. hermathena

is a member of the ‘pupal-mating clade’, but with some discordance between nuclearand mtDNA results. The nuclear data (Ef1α) place H. hermathena basal to the ‘pupal-

mating clade’ (Fig. 2B), while mtDNA place H. hermathena as a sister to H. erato

(Fig. 2A). Perhaps H. hermathena is a basal member of this clade, which would


explaining the unusual pupal morphology shared with H. melpomene, but has

acquired red colour pattern elements and mtDNA haplotypes via recent hybridizationwith H. erato. Occasional presumptive hybrids between the widely separated species

H. charithonia and H. erato, and between H. clysonymus or H. hortense and H.

hecalesia are known (Mallet et al., 2003).

H. hortense and H. clysonymus have the same colour pattern (red band on forewing,yellow in hindwing) and H. hortense is a geographical replacement of H. clysonymus

found in the cloud forests of Mexico and Nicaragua, with H. clysonymus distributed insimilar habitats through Costa Rica and North Andes. For this reason, H. hortense

was expected to be a sister species of H. clysonymus (Brown, 1981). However, in the

combined evidence hypothesis H. hortense appeared basal to H. telesiphe and H.

clysonymus (Fig. 3). This suggests that the extant H. telesiphe patterns are derived,

although this evidence is largely due to mitochondrial data (compare Figs. 2A and

2B). The race, H. telesiphe cretacea, which has white forewing bands, is perhaps theclosest to the ancestral yellow forewing band state of H. hortense and H. clysonymus.

Paraphyletic taxa

Paraphyly was observed at several different levels. Paraphyly of species relative to

their sisters was observed in the melpomene/cydno group, H. melpomene wasparaphyletic with respect to a clade that includes H. cydno and related species. In the

erato group, H. erato was paraphyletic with respect to H. himera and H. hermathena

(Figs. 2A and B see also Brower 1994a, 1994b, 1996b, Brower & Egan, 1997;

Flanagan et al., 2004). Second at the genus level, Heliconius was paraphyletic with

respect to Laparus and Neruda (Fig. 3).

At the species level, this paraphyly is expected due to hybridization and recentspeciation. Many wild hybrids between H. cydno and H. melpomene (Mallet et al.,

2003) and H. erato and H. himera (Jiggins et al., 1996; Mallet et al., 2003) have been

found, and it is known that these species have strong but incomplete reproductiveisolation (McMillan et al., 1997; Naisbit et al., 2002). For this reason, and because

ancestral polymorphisms may persist after speciation, phylogenies of recently evolvedspecies, which may still exchange genes, are inevitably difficult to resolve and likely


to produce paraphyletic taxa, even in cases where the initial split was a simple

bifurcation. Paraphyletic patterns for the closely related species were observed wherea number of races for each species were included in this study. This paraphyly might

be observed in more pairs of sister species if more geographic populations weresequenced, because around 35% of Heliconius species hybridize in the wild (Mallet

et al., 2003; Mallet, 2005).

At the genus level, it is clear that Laparus doris is part of Heliconius, suggesting only

a single origin for pollen feeding in the Heliconius group. Laparus doris wassuggested as a different genus by Turner (1976), in part due to the marked colour

polymorphism as an adult (red, yellow and the unique blue or green ray pattern in

hindwing). It is the only species within Heliconius with morphological polymorphismas a pupa, and the pupa do not have the gold spots and flanges and well developed

antennal spines of other species. Also, it is the only species apart from N. metharme,

to produce blue colour not by iridescence but by laying white scales over black(Turner, 1976). However, these morphological traits that support the generic status of

Laparus (Turner 1976 and see above) may not be good characters for phylogeneticanalysis. Colour patterns are known to evolve rapidly, and pupal characters may be

derived adaptations to gregarious larval ecology. Neruda was also defined as a

subgenus by Turner (1976), due to its short antennae and, wing shape and pupalmorphology. In particular the broad triangular forewings with extensive friction

patches of the male are very distinctive, although the females have wings of morenormal shape for the genus Heliconius. Additionally, the Neruda larva does not have

scoli on the head, as do other Heliconius species. Again, these may be rapidly

evolving characters perhaps due to sexual selection and therefore misleading. Wehave here retained traditional nomenclature, but it seems likely that the genus

Laparus, at least, should be subsumed within Heliconius.

ConclusionsThe Heliconiina have become an important group in the understanding ofevolutionary biology, in topics as diverse as coevolution, mimicry, behavioural

ecology, hybrid zones, and speciation. Overall, there is a good concordance of themolecular hypothesis presented here with previous molecular phylogenies in this


group of butterflies. However, the inclusion of more species and the addition of more

sequence information, has clarified some relationships within the Heliconiina. Thehypothesis shows that Heliconius as currently defined is not monophyletic, because

Laparus doris and possibly Neruda fall within the genus. These results suggest thatpollen-feeding behaviour evolved only once, in the common ancestor of Laparus and

Heliconius. Pollen-feeding may have been lost subsequently by the ancestor of

Neruda. Additionally, the results provided strong support for exclusion of Eueides

from Heliconius and for a monophyletic ‘‘pupal-mating clade’’ including the

erato/sara/sapho groups. This phylogenetic hypothesis can now be used to testhypotheses regarding evolutionary patterns of rapid diversification and character

evolution across the subtribe Heliconiina.

Acknowledgments

We would like to thank Maribel Gonzales, Oris Sanjur and Nimiadina Gomez for helpin the laboratory; Carla Penz, Luis Mendoza Cuenca, Keith Willmott and Alaine

Whinnet for samples. Funding was received from the Overseas Research Schemeawards, the Smithsonian Tropical Research Institute and a Bogue Fellowship from

University College London.


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Figure 1. Summary of major phylogenetic hypotheses for heliconiine butterflies

published in the last sixty years. Note that the genus name is omitted for Heliconius.A. Emsley (1963). B. Brown (1981). C. Brower (1994). D. Brower & Egan (1997). E.

Penz (1999). Black clubs mark pollen feeding behaviour, black hearts mark ‘pupal-mating clade’s. White clubs and hearts represent loss of the trait.

Figure 2. BAY phylogenies for heliconiine species based on separate data partitionsmtDNA (CoI+CoII and 16S) and nuclear data. A. mtDNA. B. Ef1α. C. CoI+CoII,

16S, Ef1α, dpp, ap and wg for 12 species. Branch lengths were estimated using

likelihood. Values above branches show BAY probabilities and below show

parsimony bootstrap support for the equivalent node, after 1000 replicates. Branches

without bootstrap support reflect differences between BAY and MP trees. P= Panama,E= Ecuador, G= French Guiana, C= Colombia, and Pe= Peru. A. mtDNA. B. BAY

phylogeny based on Elongation factor -1α. C. BAY phylogeny based on Co, 16S,

Ef1α, dpp, ap and wg for12 species representing the major clades within Heliconius

and Dryas as a root.

Figure 3. BAY phylogenetic hypothesis for heliconiine species based on combined

mitochondrial (CoI+CoII and 16S) and nuclear data (Ef1α, dpp, ap and wg). Only one

individual per species was used and the wg sequences aligned correspond to differentindividuals. Branch lengths were estimated using likelihood. Values above branches

show BAY probabilities and below show parsimony bootstrap support for the

equivalent node, after 1000 replicates. Branches without support reflect differencesbetween BAY and MP trees. P= Panama, E= Ecuador, G= French Guiana, C=

Colombia, and Pe= Peru. Black clubs mark pollen-feeding behaviour, black heartsmark pupal-mating. White clubs and hearts represent loss of the trait.

Figure 4. Alternate a priori hypotheses used for SH tests of phylogenetic relationshipsbetween Heliconius, Laparus doris, Neruda and Eueides. A. Best fit ML tree (our

hypothesis) B. Laparus basal to Heliconius. C. Neruda basal to Heliconius andEueides. D. Brower 1994a, Eueides included in pupal mating clade. E. Brown 1981,

Heliconius monophyletic with Eueides, Laparus, and Neruda basal. F. Neruda basal


to cydno-melpomene, Silvaniforms and primitives. G. Neruda basal to pupal mating

clade. H. Neruda basal to Heliconius.

Figure 5. Phylogenetic hypothesis for Heliconiine species based on reanalysis ofBrower’s (1994) sequences data (950 bp of CoI+CoII) with using a Bayesian analysis

(BAY). Values above branches show BAY probabilities.


Table 1. Nucleotide variability over genes and codon position. The values were

calculated for the whole data set in CoI+CoII (CoI +tRNA-leu+CoII) and Ef1α and

just for the 12 species for the remaining genes. CI = consistency index. RI = retentionindex.

CoI+CoII All sites Codon pos. 1 Codon pos. 2 Codon pos. 3No. of characters 1611 511 511 511No. of invariant 955 375 454 66No. variable 656 136 57 445No. informatives 587 106 32 417Tree length 3751 460 109 3086CI 0.262 0.361 0.596 0.236RI 0.715 0.797 0.799 0.704

Ef1α All sites Codon pos. 1 Codon pos. 2 Codon pos. 3No. of characters 876 292 292 292No. of invariant 615 259 271 84No. variable 261 33 20 208No. informatives 186 12 5 169Tree length 734 53 34 647CI 0.47 0.66 0.67 0.44RI 0.83 0.83 0.6 0.83

Gene 16S ap dpp wgNo. of characters 512 195 270 375No. of invariant 413 163 211 281No. variable 99 32 59 94No. informatives 38 15 27 34Tree length 155 54 86 157CI 0.748 0.722 0.837 0.669RI 0.426 0.423 0.745 0.212


Table 2. Best supported models of molecular evolution and estimated parameter

values for the different data sets.

Data set CoI + CoII 16S Ef1α ap dpp wgModel GTR+I+G F81+G GTR+I+G K2P+G K2P+G TrNef+GBase frequenciesA 0.374 0.4421 0.28 0.25 0.25 0.25C 0.1081 0.0649 0.244 0.25 0.25 0.25G 0.0647 0.1234 0.2447 0.25 0.25 0.25T 0.4532 0.3697 0.2313 0.25 0.25 0.25Substitution model All equal ratesTr/tv ratio 1.9162 2.1976Tr [A-G] 15.2471 6.2591 6.2038 [C-T] 27.3268 13.4501 12.7904Tv [A-C] 2.9031 1.6729 1 [A-T] 1.7001 3.3225 1 [C-G] 2.8548 1.7038 1 [G-T] 1 1 1Invariable sites 0.5001 0 0.543 0 0 0Gamma parameter 0.5187 0.1236 0.8076 0.0639 0.2419 0.3561


Table 3. Results of SH tests of alternate a priori hypotheses for phylogenetic

relationships between Heliconius, Laparus doris, Neruda and Eueides.CI=consistency index. RI=retention index.

Data set Hypotheses SH P Length CI RImtDNA Figure 4A. 19105.7 (Best) 3641 0.295 0.489

Figure 4B. 19135.6 0.092 3662 0.294 0.485Figure 4C. 19117 0.507 3647 0.295 0.488Figure 4D. 19127.3 0.266 3655 0.294 0.487Figure 4E. 19197.9 0.000* 3696 0.291 0.478Figure 4F. 19108.8 0.843 3642 0.295 0.489Figure 4G. 19126.5 0.234 3654 0.294 0.487Figure 4H. 19117.6 0.476 3645 0.295 0.489

Nuclear Figure 4A. 9238.92 (Best) 1271 0.505 0.669Figure 4B. 9265.13 0.154 1284 0.5 0.663Figure 4C. 9255.47 0.365 1281 0.501 0.664Figure 4D. 9287.03 0.017* 1306 0.492 0.651Figure 4E. 9340.87 0.000* 1332 0.482 0.637Figure 4F. 9244.86 0.748 1273 0.504 0.668Figure 4G. 9295.76 0.007* 1298 0.495 0.655Figure 4H. 9255.44 0.365 1281 0.501 0.664

Combined Figure 4A. 29376.1 (Best) 4912 0.35 0.539Figure 4B. 29434.4 0.039* 4946 0.347 0.534Figure 4C. 29402.9 0.329 4928 0.348 0.537Figure 4D. 29463.6 0.005* 4961 0.346 0.532Figure 4E. 29592.6 0.000* 5028 0.341 0.522Figure 4F. 29384.3 0.777 4915 0.349 0.538Figure 4G. 29458.4 0.008* 4952 0.347 0.533Figure 4H. 29403.5 0.318 4926 0.349 0.537

Appendix 1. Heliconiina included in the study. ID numbers are STRI collection numbers and should be prefixed by “stri-b” for individuals

belonging to different collections i.e +=A. Brower.

Group Species Name Collection Collection Collection GenBank accession numberID No. locatity date CoI + CoII Ef1α 16S ap dpp

melpomene (L.)/ 553 H. cydno chioneus Bates Panama: Canal Zone, Pipeline Road 22/8/98 STRI AF413672 AY747993cydno Doubleday 570 H. cydno chioneus Bates Panama: Canal Zone, Pipeline Road 25/8/98 STRI AF413707 AY748014

811 H. melpomene rosina Boisduval Panama: Canal Zone, Pipeline Road 25/8/98 STRI AF413673 AY747985 AY747875 AY747887 AY747899544 H. melpomene rosina Boisduval Panama: Canal area, Pipeline road 21/8/98 STRI AF413674 AY748015

436 H. melpomene melpomene (L.)French Guiana: Sablance (La Victoire)Route N1 km 19 30/5/98 STRI AF413675 AY747994

437 H. melpomene melpomene (L.)French Guiana: Sablance (La Victoire)Route N1 km 19 30/5/98 STRI AF413676

8074 H. melpomene cythera Hewitson Ecuador O. McMillan AF413677 AY7479958073 H. melpomene cythera Hewitson Ecuador O. McMillan AF4136788158 H. melpomene malleti Lamas Ecuador: Napo, Rio Quijos 23/5/01 STRI AY748026 AY7479128036 H. pachinus Salvin Panama: Chiriqui, Café Duran Finca 9/9/00 STRI AF413679M26£ H. melpomene mocoa Brower Colombia M. linares AY548117 AY747998M82£ H. melpomene mocoa Brower Colombia M. linares AY548129 AY7480168023 H. pachinus Salvin Panama: Chiriqui, Café Duran Finca 8/9/00 STRI AY748058 AY747944

8 H. heurippa HewitsonColombia: Cundinamarca, Virgen deChirajara 28/2/98 STRI AF413680 AY747997

40 H. heurippa HewitsonColombia: Cundinamarca, Virgen deChirajara 28/2/98 STRI AY748059 AY747945

8520 H. timareta Hewitson Ecuador: Tungurahua, El Topo 16/2/02 STRI AY748028 AY7479148521 H. timareta Hewitson Ecuador: Tungurahua, El Topo 16/2/02 STRI AY748060 AY747946C-1* H. besckei Ménétriés Brazil C. Penz AY748029 AY747915

B-3-2+ H. besckei Ménétriés Brazil A. Brower AY748061 AY747947silvaniforms

346 H. numata (Cramer)French Guiana: Cacao - Cayenne,9.9km 21/5/98 STRI AF413681 AY747986 AY747876 AY747888 AY747900

8130 H. numata (Cramer) Ecuador: Napo, Rio Quijos 23/5/01 STRI AY748062 AY747948

503 H. elevatus NölderFrench Guiana: Camp Caiman, KawMountains 21/5/98 STRI AF413682 AY747999


434 H. elevatus NölderFrench Guiana: Camp Caiman, KawMountains 21/5/98 STRI AY748063 AY747949

665 H. hecale (F.) Panama: Canal Zone, Pipeline Road 27/8/98 STRI AF413683 AY748000608 H. hecale (F.) Panama: Gamboa, El Renacer 29/8/98 STRI AY748064 AY747950666 H. ismenius Latreille Panama: Canal Zone, Pipeline Road 27/8/98 STRI AY748030 AY747916656 H. ismenius Latreille Panama: Canal Zone, Pipeline Road 27/8/98 STRI AY748065 AY747951

8127 H. atthis DoubledayEcuador: Pichincha, Pedro VicenteMaldonado 18/5/01 STRI AY748031 AY747917

8162 H. atthis DoubledayEcuador: Pichincha, Pedro VicenteMaldonado 13/5/01 STRI AY748066 AY747952

JM-23-1+ H. pardalinus Bates Peru A. Brower AY748032 AY7479188014 H. ethilla Godart Argentina: Iguacu Falls 18/8/00 STRI AY748033 AY7479198729 H. ethilla Godart Peru: San Martin, Rio Shilcayo 25/8/02 STRI AY748067 AY747953C2 H. nattereri Felder & Felder Brazil: ES, Santa Teresa 12/4/88 STRI AY748034 AY747920

erato (L.)/ 2980 H. erato petiverana Doubleday Panama: Canal Zone, Pipeline Road 23/7/00 STRI AF413684 AY748001himera 2981 H. erato petiverana Doubleday Panama: Canal Zone, Pipeline Road 24/7/00 STRI AF413685 AY748017

440 H. erato hydara HewitsonFrench Guiana: Sablance (La Victoire)Route N1 km 19 30/5/98 STRI AF413686 AY747987 AY747877 AY747889 AY747901

442 H. erato hydara HewitsonFrench Guiana: Sablance (La Victoire)Route N1 km 20 31/5/98 STRI AF413687

2861 H. erato cyrbia Godart Ecuador: Pichincha, Alluriquin 13/5/01 STRI AF413688 AY7480028075 H. erato cyrbia Godart Ecuador O. McMillan AF413689 AY748018

2842 H. himera HewitsonEcuador: San Pedro de la Bendita,Catamayo STRI AF413690 AY748003

8076 H. himera Hewitson Ecuador O. McMillan AF413691 AY7479548037 H. clysonymus Latreille Panama: Chiriqui, Café Duran Finca 9/9/00 STRI AF413692 AY747988 AY747878 AY747890 AY7479028024 H. clysonymus Latreille Panama: Chiriqui, Café Duran Finca 8/9/00 STRI AY748068 AY747955590 H. hecalesia Hewitson Panama: Canal Zone, Pipeline Road 28/898 STRI AF413693 AY748004856 H. hecalesia Hewitson Panama: Santa Rita ridge 12/12/98 STRI AY748069 AY7479562928 H. telesiphe Doubleday Ecuador: Pastaza, Pastaza STRI AY748036 AY7479228525 H. telesiphe Latreille Ecuador: Tungurahua, El Topo 17/2/02 STRI AY748070 AY747957

Group Species Name Collection Collection Collection GenBank accession numberID No. locatity date CoI + CoII Ef1α 16S ap dpp9111 H. hortense Guérin-Méneville Mexico STRI AY748043 AY747929

charithonia 2923 H. charithonia (L.) Ecuador: Zamora-Chinchipe, Zamora STRI AF413694 AY748005E-3971^ H. charithonia (L.) Ecuador J. Mallet AY748071 AY747958E-190^ H. peruvianus Felder & Felder Ecuador J. Mallet AY748037 AY747923E-288^ H. peruvianus Felder & Felder Ecuador J. Mallet AY748072 AY747959C-3* H. hermathena Hewitson C. Penz AY748027 AY747913

sara/sapho 850 H. sara (F.) Panama: Canal Zone, Pipeline Road 2/12/98 STRI AF413695 AY748019

308 H. sara (F.)French Guiana: St Maurice on road toApatou 24/5/98 STRI AY748038 AY747924

1180 H. ricini (L.) French Guiana: Rio Compte bridge 9/2/99 STRI AF413696 AY748006

8197 H. ricini (L.)French Guiana: Sablance (La Victoire)Route N1 km 19 1/6/01 STRI AY748073 AY747960

842 H. eleuchia Hewitson Panama: Canal Zone, Pipeline Road 27/11/98 STRI AF413697 AY747989 AY747879 AY747891 AY747903857 H. eleuchia Hewitson Panama: Santa Rita bridge 12/2/98 STRI AY748074 AY747961536 H. sapho (Drury) Panama: Canal Zone, Pipeline Road 16/8/98 STRI AF413698 AY7480072739 H. sapho (Drury) Panama: Canal Zone, Pipeline Road 30/1/00 STRI AY748075 AY747962

P-32-6* H. Antiochus (L.) Panama A. Brower AY748039 AY747925C-4* H. Hewitson Staudinger C. Penz AY748040 AY747926C-6* H. Hewitson Staudinger C. Penz AY748076 AY747963

C19-1+ H. congener Weymer Colombia: Provincia del Putumayo A. Brower AY748041 AY747927RB119+ H. leucadia Bates Brazil: Rondônia, Cacaulandia A.Brower AY748042 AY747928

8562 H. demeter StaudingerPeru: San Martin, Tarapoto-Yurinaguas, F. Bio. 18/8/02 STRI AY748022 AY747908 AY747871 AY747883 AY747895

8563 H. demeter StaudingerPeru: San Martin, Tarapoto-Yurinaguas, F. Bio. 18/8/02 STRI AY748077 AY747964

“primitive” 8154 H. hierax Hewitson Ecuador: Napo, Rio Quijos 23/5/01 STRI AF413708 AY748008Heliconius 8147 H. hierax Hewitson Ecuador: Napo, Rio Quijos 23/5/01 STRI AY748078 AY747965

286 H. wallacei ReakirtFrench Guiana: La Desiree nearCayenne 22/5/98 STRI AF413699 AY748009

8212 H. wallacei Reakirt French Guiana: Pointe Macouria 28/5/98 STRI AY748079 AY7479668560 H. burneyi (Hübner) Peru: San Martin, Tarapoto-

Yurinaguas, F. Bio.18/8/02 STRI AY748023 AY747909 AY747895 AY747884 AY747896


Yurinaguas, F. Bio.

8561 H. burneyi (Hübner)Peru: San Martin, Tarapoto-Yurinaguas, F. Bio. 18/8/02 STRI AY748080 AY747967

G-31-2+ H. egeria (Cramer) French Guiana A. Brower AY748035 AY7479218549 H. hecuba Hewitson Ecuador 20/2/02 STRI AY748024 AY747910 AY747873 AY747885 AY7478978550 H. hecuba Hewitson Ecuador 20/2/02 STRI AY748081 AY7479688609 H. xanthocles Bates Peru: San Martin, La Antena 18/8/02 STRI AY748044 AY7479308611 H. xanthocles Bates Peru: San Martin, La Antena 18/8/02 STRI AY748082

Laparus 846 L. doris (L.) Panama: Canal Zone, Pipeline Road 2/12/98 STRI AF413700 AY747990 AY747880 AY747892 AY747904847 L. doris (L.) Panama: Canal Zone, Pipeline Road 2/12/98 STRI AY748083 AY747969

8645 L. doris (L.)Peru: San Martin, Tarapoto-Yurinaguas (Km 28) 21/8/02 STRI AY748084 AY747970

Neruda2949 N. metharme (Erichson)

Ecuador: Napo, Yasuni ResearchStation STRI AY748045 AY747931

RB257+ N. metharme (Erichson) Brazil: Rondônia, Cacaulandia A. Brower AY748085 AY747971

8569 N. aoede (Hübner)Peru: San Martin, Tarapoto-Yurinaguas, F. Bio. 18/8/02 STRI AY748025 AY747911 AY747874 AY747886 AY747898

8198 N. aoede (Hübner) French Guiana: Savane du Galion 2/6/01 STRI AY748086 AY747972

8570 N. aoede (Hübner)Peru: San Martin, Tarapoto-Yurinaguas, F. Bio. 18/8/02 STRI AY748087 AY747973

Eueides2991 E. lineata Salvin & Godman

Panama: Chiriqui, Quebrada HornitoTrail, Fortuna 12/8/00 STRI AF413701 AY748010

320 E. vibilia (Godart)French Guiana: Camp Caiman, KawMountains 25/5/98 STRI AF413702 AY747991 AY747881 AY747893 AY747905

8699 E. vibilia (Godart)Peru: San Martin, Tarapoto-Yurinaguas (Km 78) 23/8/02 STRI AY748088

555 E. aliphera (Godart) Panama: Canal Zone, Pipeline Road 22/8/98 STRI AF413703 AY748011875 E. aliphera (Godart) Panama: Colon, Forest edge Gamboa STRI AY748089 AY747974556 E. lybia (F.) Panama: Canal Zone, Pipeline Road 22/8/98 STRI AF413704 AY748012

8595 E. lybia (F.)Peru: San Martin, Pongo deCainarache - Baranquita 17/8/02 STRI AY748090 AY747975

780 E. Isabella (Cramer) Panama: Canal Zone, Pipeline Road STRI AY748046 AY7479328740 E. Isabella (Cramer) Peru: San Martin, Puente Rio

Serranoyacu27/8/02 STRI AY748091 AY747976


Serranoyacu

2933 E. procula Doubleday Ecuador O. McMillan AY748047 AY747933C-7* E. procula Doubleday C. Penz AY748092 AY747977

2948 E. tales (Cramer)Ecuador: Napo, Yasuni ResearchStation STRI AY748048 AY747934

C-8* E. tales (Cramer) Carla Penz AY748093C-9* E. pavana Ménétriés Carla Penz AY748049 AY747935

8750 E. lampeto BatesPeru: San Martin, Puente AguasVerdes 28/8/02 STRI AY748050 AY747936

8947 E. heliconioides Felder & Felder Ecuador STRI AY748051 AY7479378948 E. heliconioides Felder & Felder Ecuador STRI AY748094 AY748020

Dryadula 2940 D. phaetusa (L.) Panama O. McMillan AF413705 AY748013

2921 D. phaetusa (L.)Panama: Darien, Panama city - Yaviza266km 16/6/00 STRI AY748095 AY747978

Dryas293 D. iulia (F.)

French Guiana: La Desiree nearCayenne 22/5/98 STRI AF413706 AY747992 AY747882 AY747894 AY747906

Philaethria8700 P. dido (L.)

Peru: San Martin, Tarapoto-Yurinaguas (Km 78) STRI AY748052 AY747938

690 P. dido (L.) Panama: Gamboa, El Renacer 14/9/98 STRI AY748096 AY747979

8749 P. diatonica (Fruhstorfer)Peru: San Martin, Puente AguasVerdes 28/8/02 STRI AY748097

Podotricha 2929 P. telesiphe (Hewitson) Ecuador: Pastaza, Pastaza STRI AY748053 AY7479398146 P. telesiphe (Hewitson) Ecuador: Napo, Rio Quijos 23/5/01 STRI AY748098 AY747980

Dione 2970 D. juno (Cramer) Panama: Canal Zone, Pipeline Road 20/6/00 STRI AY748054 AY7479408727 D. juno (Cramer) Peru: San Martin, Rio Shilcayo 25/8/02 STRI AY748099 AY747981

C-10* D. glycera Felder & Felder C. Penz AY748055 AY747941Pe57+ D. glycera Felder & Felder Peru A. Brower AY748100 AY747982

8748 D. moneta HübnerPeru: San Martin, Puente AguasVerdes 28/8/02 STRI AY748056 AY747942

Mex2-1+ D. moneta Hübner Mexico A. Brower AY748101 AY747983Agraulis 8554 A. vanillae (L.) Panama: Gamboa, 183 STRI AY748057 AY747943

8725 A. vanillae (L.) Peru: San Martin, Rio Shilcayo 28/8/02 STRI AY748102 AY747984


Outgroup 8632 Castilia perilla Peru: San Martin, La Antena 19/8/02 STRI AY748021 AY747907

OutgroupAgraulisDionePodotrichaDryasDryadulaPhilaethriaEueidesLaparusxanthocleshecubawallacei groupNerudaSilvaniform group

erato groupcharithonia

melpomene-cydno group

sara-sapho group

D. E.OutgroupAgraulisDionePodotricha

DryasDryadula

PhilaethriaLaparusNerudaEueideswallaceimelpomene

eratocharithoniasara

cydno

DryasDryadulaPhilaethriaPodotrichaLaparusxanthocleswallacei groupNerudaSilvaniform group

erato groupcharithoniaEueidesricinisara-sapho group

Dione

melpomene-cydno group

C.

PhilaethriaPodotrichaDioneAgraulisDryadulaDryasEueidesNerudaLaparushecubaxanthocleswallacei groupSilvaniform groupmelpomene-cydno grouperato groupcharithoniasara-sapho group

B.Philaethria

PodotrichaDryadula

Dryadula

DryasEueidesLaparuswallaceinumatamelpomeneeratoricini

A.

Emsley (1963)6 Heliconius, 2 Eueides, and 9 primitiveHeliconiine spp sampledMorphological characters

Brown (1981)38 Heliconius, 3 Neruda , 12 Eueides, and 11primitive Heliconiine spp sampledMorphology and ecological characters

Brower (1994)30 Heliconius, 1 Neruda , 5 Eueides and 5primitive Heliconiine spp sampledCOI-COII

Brower & Egan (1997)33 Heliconius, 2 Neruda, 6 Eueides and7 primitive Heliconiine spp sampledCOI, COII and Wg

Penz (1999)6 Heliconius, 3 Neruda , 3 Eueidesand 11 primitive Heliconiine sppsampledMorphological characters

♣

♣

♣

♣

♣

♥

♥

♥

DioneAgraulis

♣♣

♣♣

♥♥

Figure 1.

8632 Castilia811 H. melpomene rosina P

8074 H. melpomene cythera E544 H. melpomene rosina P8073 H. melpomene cythera E

436 H. melpomene melpomene G437 H. melpomene melpomene G

553 H. cydno chioneus P8023 H. pachinus P8 H. heurippa C40 H. heurippa C570 H. cydno chioneus P

8036 H. pachinus P8520 H. timareta E8521 H. timareta E

8158 H. melpomene malleti E26 H. melpomene mocoa C82 H. melpomene mocoa C

346 H. numata G8130 H. numata E

666 H. ismenius P656 H. ismenius P

C-1 H. besckei BB32 H. besckei B

503 H. elevatus G434 H. elevatus G

JM23 H. pardalinus665 H. hecale P608 H. hecale P

8127 H. atthis E8162 H. atthis E

8014 H. ethilla A8729 H. ethilla Pe

002 H. nattereri 8560 H. burneyi Pe8561 H. burneyi Pe

286 H. wallacei G8212 H. wallacei Pe

G-31-2 H. egeria8569 N. aoede Pe8570 N. aoede Pe8198 N. aoede G

2949 N. metharme ERB257 N. metharme

8549 H. hecuba E8550 H. hecuba E

8154 H. hierax E8147 H. hierax E

8609 H. xanthocles Pe8611 H. xanthocles Pe

846 L. doris P847 L. doris P

8645 L. doris Pe440 H. erato hydara G442 H. erato hydara GC-3 H. hermathena

2842 H. himera E8076 H. himera E

2980 H. erato petiverana P2981 H. erato petiverana P

2861 H. erato cyrbia E8075 H. erato cyrbia E

590 H. hecalesia P856 H. hecalesia P

8037 H. clysonimus P8024 H. clysonimus P2928 H. telesiphe E8525 H. telesiphe E

9111 H. hortense842 H. eleuchia P857 H. eleuchia E

C191 H. congenerC-4 H. hewitsoni PC-6 H. hewsitsoni P

536 H. sapho G2739 H. sapho P

308 H. sara G850 H. sara P

RB119 H. leucadiaP326 H. antiochus P

8562 H. demeter Pe8563 H. demeter Pe

1180 H. ricini P8197 H. ricini G

2923 H. charithonia E3971 H. charithonia E

190 H. peruvianus E288 H. peruvianus E

320 E. vibilia P8699 E. vibilia P

E. pavana8750 E. lampeto Pe780 E. isabella P8740 E. isabella Pe

2991 E. lineata P2933 E. procula E

C-7 E. procula8947 E. heliconioides Pe

8948 E. heliconioides Pe555 E. aliphera P

875 E. aliphera E556 E. lybia P

8595 E. lybia Pe2948 E. tales E

C-8 E. tales E293 Dryas iulia P2940 Dryas phae P

2921 Dryadula phaetusa P2929 Podotricha telesiphe E8146 Podotricha telesiphe E

2970 Dione juno P8727 Dione juno Pe

C-10 D. glycera8748 Dione moneta Pe

Mex2.1 Dione monetaPe57 Dione glycera

8554 Agraulis vanillae P8725 Agraulis vanillae P

8700 Philaethria dido Pe690 Philaethria dido P

8749 Philaethria diatonica Pe

0.01 substitutions/site

1.98

1.100

.8595

1.88

1.95

.9380.62

50

.9795

.9361 1.

99

1.99

1.99

991.

1.83

1.99

1.9976

1.93 1.

981.97

1.88.99

761.99

991.

1.88

801.

1.88

1.68

1.88

1.

.88

1.

.6564

1.99

.6492

1.99

1.98

1.99

.87901.

98

.99

1.99

1.97

1.72

.9953

1.99

1.98

1.99

1.99

1.94

1.86

.92

1.66

.8365

1.99

991.

1.99

981.

1.99

.72.53

1.100

1.100.99

801.

.9190

991.

.93

1.99

1.99

991.

1.98

1.60

.68

1.99.52

51

1.93.92

.8886

.9799

.9797

1.99.55

1.99

991.

1.79

77.59

1..6055

.5970

1.

99

.76

.74

.9999

.9999

.8680

1.

.9486

.5368

1.99

.63

1.99

.9999

.7961

.9999

Figure 2A.

8632 Castilia811 H. melpomene rosina P

82 H. melpomene mocoa C8014 H. ethilla A

436 H. melpomene melpomene G8127 H. atthis E

8162 H. atthis E8158 H. melpomene malleti E503 H. elevatus G434 H. elevatus G

JM23 H. pardalinus665 H. hecale P608 H. hecale P

346 H. numata G553 H. cydno chioneus P

8074 H. melpomene cythera E8036 H. pachinus P

8023 H. pachinus P8 H. heurippa C

8520 H. timareta E26 H. melpomene mocoa C

C-1 H. besckei BB32 H. besckei B

8729 H. ethilla Pe666 H. ismenius P

656 H. ismenius P570 H. cydno chioneus P

544 H. melpomene rosina P40 H. heurippa C

8521 H. timareta E8130 H. numata E

002 H. nattereri 8569 N. aoede Pe

8570 N. aoede Pe8198 N. aoede G

2949 N. metharme ERB257 N. metharme

440 H. erato hydara G2980 H. erato petiverana P

2861 H. erato cyrbia E2981 H. erato petiverana P

8075 H. erato cyrbia E2842 H. himera E

8076 H. himera E8037 H. clysonimus P8024 H. clysonimus P

9111 H. hortense2928 H. telesiphe E

8525 H. telesiphe E842 H. eleuchia P

C191 H. congener857 H. eleuchia E

308 H. sara GC-4 H. hewitsoni P

C-6 H. hewsitsoni P536 H. sapho G2739 H. sapho P

P326 H. antiochus P2923 H. chari.thonia E

RB119 H. leucadia850 H. sara P

8562 H. demeter Pe8563 H. demeter Pe

1180 H. ricini P8197 H. ricini G

190 H. peruvianus E3971 H. charithonia E

288 H. peruvianus EC-3 H. hermathena

590 H. hecalesia P856 H. hecalesia P

8560 H. burneyi Pe8561 H. burneyi Pe

286 H. wallacei G8212 H. wallacei PeG-31-2 H. egeria

8549 H. hecuba E8550 H. hecuba E

846 L. doris P847 L. doris P

8645 L. doris Pe8154 H. hierax E8147 H. hierax E

8609 H. xanthocles Pe320 E. vibilia P2933 E. procula E8750 E. lampeto PeC-9 E. pavana

C-7 E. procula2991 E. lineata P

780 E. isabella P8740 E. isabella Pe

8947 E. heliconioides Pe8948 E. heliconioides Pe555 E. aliphera P

875 E. aliphera E556 E. lybia P

8595 E. lybia Pe2948 E. tales E

293 Dryas iulia P2921 Dryadula phaetusa P

2940 Dryadula phaetusa P2929 Podotricha telesiphe E8146 Podotricha telesiphe E

8700 Philaethria dido Pe690 Philaethria dido P

2970 Dione juno P8727 Dione juno Pe

C-10 Dione glyceraPe57 Dione glycera

8748 Dione moneta PeMex2.1 Dione moneta

8554 Agraulis vanillae P8725 Agraulis vanillae P


.96.82

.93

.80.96

1.

1.99

.9664

.7151

1..6098

1.55

.9992

1.92

.9066

1.100

.76

511.100

1.67

1.65

.9869

.9874

1.100

1.99

.97

.8054

.67

.91

.5451

.6454

1.

1.

1.

96

100

90

.92

62

.7962

.8851

1.53

1.100

1001.

1.881.

88

1.100

1.79

.9879

.6074

1.

1.99

99

.9886

.6894

.5684

1.

1.77

1.100

.80

54

1.94

1.

1.

1.99

.9089

72

.6968

1.100

1.98

70

.81

1.100

.97

.7657

1.93

1.83

.92621.98

1.52

1.93

1.

1.99

Figure 2B.

H. melpomene

H. numata

N. aoede

H. hecuba

L. doris

H. burneyi

H. erato

H. clysonimus

H. eleuchia

H. demeter

E. vibilia

D. iulia 0.01 substitutions/site

1.100

.9253

1.95

.90

1.

1.78

.9894

1.93

1.100

1.

Figure 2C.

CastiliaH. melpomeneH. cydnoH. heurippa H. pachinus

H. timaretaH. numata

H. ismeniusH. besckei

H. elevatusH. pardalinus

H. hecaleH. atthis H. ethilla

H. nattereri N. aoede

N. metharmeH. burneyi

H. wallaceiH. egeria

H. hecubaL. doris

H. hierax H. xanthocles

H. eratoH. hermathena

H. himeraH. hecalesia

H. clysonimusH. telesiphe

H. hortenseH. eleuchia

H. congenerH. sapho

H. hewitsoniH. sara

H. leucadiaH. antiochus

H. demeterH. ricini

H. charithoniaH. peruvianus

E. vibilia E. lampetoE. pavana

E. proculaE. lineata

E. isabellaE. heliconioidesE. alipheraE. lybia

E. tales Dryas iulia

Dryadula phaetusaPodotricha telesiphe

Phylaethria didoPhylaethria diatonica

Dione juno Dione glycera

Dione monetaAgraulis vanillae


.9254

1.92

.96851.70

1.93

.5558

1.84

1.

1.100

1.100

1001.

1.100

801.

.60

1.

1.

1.

100

99

99

.7881 .97

911.99

1.

88

.93

1.100

1.

98

1.93

1.91

1.100 .69

.84691.

67

1.100.98

55.79

1.100

1.80

1.

.56

.9970

1.100

1.95

64

.57611.

991.

.59

1.

.9975 .96

.51

.97.5458

1.100

1001.

1.

.6754

1.

♣

♣♣

♥

36 Heliconius, 2 Neruda, 10 Eueides and 10 primitive Heliconiine spp sampledCO, 16S, EF1a, dpp and ap.

melpo/cydno

Silv

an

iform

sN

erudaP

rimitiv

eera

to g

roup

sara/saph

oE

ueid

es

outg

roups

Figure 3.

HeliconiusNerudaHeliconiusLaparus

EueidesOutgroupsRoot

A.

HeliconiusHeliconius

HeliconiusNerudaHeliconius

Laparus


B.


Heliconius

Neruda

HeliconiusLaparus

Eueides

Outgroups

Root

C.


HeliconiusNerudaHeliconiusLaparus


D.


Heliconius

Neruda

Heliconius

Laparus


E.


Heliconius

Neruda

HeliconiusLaparus


F.


Heliconius

Neruda

HeliconiusLaparus


G.


Heliconius

Neruda

HeliconiusLaparus


H.


Figure 4.

SpeyeriaH. tristeroH. heurippa

H. timaretaH. pachinus

H. cydno PAH. cydno ECH. cydno CR

H. melpomeneH. atthis

H. hecaleH. elevatus

H. pardalinusH. ethilla

H. besckeiH. numata FG

H. numata EBH. ismenius

N. aoedeN. metharme

H. wallacei ECH. wallacei RB

H. burneyiH. egeria

H. hecubaL. doris

H. xanthoclesH. antiochus PA

H. antiochus FGH. sapho PA

H. sapho ECH. hewitsoni

H. eleuchiaH. congener

H. saraH. leucadia

H. riciniH. demeter

H. charithonia FLH. charithonia PAH. clysonymus

H. telesipheH. erato

H. himeraE. tales 2E. tales 1

E. lybia COE. lybia PA

E. alipheraE. procula

E. isabellaE. vibilia

P. telesipheD. phaetusa

D. iulia ECD. iulia CR

P. didoD. juno

D. glyceraAgraulis


.55

.66

.66 .93

.82

.94

1.

.58

.75

.97

.96

.64

1.1.

1.

.99

1.

.98

.98.98

1.

.96.68

.79

.99

.94

1.

.84

.52

1.

1.

1.

1.

1.

.99

1.

1.

.96

1.

.99

.85

1.

1..87

1.1.

1..81

.58

.99

Figure 5.

butterflies? Inferences from multilocus DNA sequence...

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