Systematic Entomology (2013), 38, 440449

DOI: 10.1111/syen.12008

Molecular systematics of the butterfly tribe Preponini

(Nymphalidae: Charaxinae)
1,2

ELENA ORTIZ-ACEVEDO

1,2

and K E I T H R . W I L L M O T T

McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, University of Florida, Gainesville, FL,
U.S.A. and 2 Department of Entomology and Nematology, University of Florida, Gainesville, FL, U.S.A.

Abstract. The nymphalid butterfly tribe Preponini includes some of the Neotropical
regions most spectacular and familiar butterflies, but the taxonomy of the group
nevertheless remains unstable. Several recent studies of Nymphalidae phylogeny have
suggested that both the tribe itself and several genera might not be monophyletic,
but to date taxon sampling has not been sufficiently comprehensive to allow informed
revision of the groups systematics. We therefore conducted the first complete specieslevel phylogenetic study of the tribe to establish a firm higher classification. We
used DNA sequence data from three genes, the two mitochondrial genes cytochrome
oxidase subunits I and II (COI and COII ), and the nuclear gene elongation factor1 (EF-1), to reconstruct the phylogeny of the tribe using maximum likelihood
(ML), maximum parsimony (MP) and Bayesian inference (BI). We included 48
individuals representing the 22 recognised Preponini species, and an additional 25
out-group taxa to explore taxonomic limits at different levels. Firstly, we found that
Anaeomorpha splendida Rothschild never grouped with remaining Preponini, so that
maintaining monophyly of the tribe requires the taxon to be excluded, and we thus
reinstate the tribe Anaeomorphini stat.rev. Secondly, we investigated generic limits,
in particular the relationship of Noreppa Rydon to Archaeoprepona Fruhstorfer, and
that of Agrias Doubleday to Prepona Boisduval. The molecular results coupled with
previous morphological studies suggest that Noreppa syn.n should be synonymised
with Archaeoprepona, and that Agrias syn.n should be synonymised with Prepona.
We found Prepona pheridamas (Cramer) to be sister to all other Prepona, and
markedly divergent from them in both morphology and DNA sequences, suggesting
the possibility that it should be placed in a separate genus. We also found a number
of cases of significant DNA sequence divergence and paraphyly or polyphyly within
putative species that require further taxonomic attention, including Prepona claudina
(Godart) stat.n. and Prepona narcissus (Staudinger) stat.n., Prepona pylene Hewitson
and Prepona deiphile (Godart). Future research should focus on a broader population
sampling of widespread, polymorphic Preponini species to thoroughly revise the
current species-level taxonomy, thus creating a solid foundation for studies in ecology
and conservation.

Correspondence: Elena Ortiz-Acevedo, McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, University of
Florida, SW 34th Street and Hull Road, PO Box 112710, Gainesville, FL 32611, U.S.A. E-mail: e.ortiz.acevedo@gmail.com; eortiz@ufl.edu

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2013 The Royal Entomological Society

Phylogeny of Preponini
Introduction
The nymphalid butterfly subfamily Charaxinae contains
approximately 400 species within 28 genera (Ackery et al.,
1998; Chacon & Montero, 2007), and is distributed throughout
the worlds tropical regions. Placed within the satyrine clade
(Wahlberg et al., 2009), the spectacular wing patterns of many
charaxines have made them highly popular among collectors
(DeVries, 1987). The subfamily includes five tribes according to the most recent molecular phylogenetic studies (Pena
& Wahlberg, 2008; Aduse-Poku et al., 2009; Wahlberg et al.,
2009). The exclusively Neotropical tribe Preponini contains
22 currently recognised species within five genera (Lamas,
2004): Prepona, Archaeoprepona, Noreppa, Anaeomorpha and
Agrias. Like the majority of charaxines, Preponini are large,
robust, fast-flying inhabitants of the forest canopy, and they
are usually seen when attracted to baits. Agrias in particular is renowned for its conspicuous brilliant wing coloration,
which also shows remarkable geographic variation, and the
rarity of specimens makes them of high commercial value
(Neild, 1996).
Although the biology and taxonomy of preponines has been
the focus of several studies (e.g. Muyshondt, 1974; Johnson
& Descimon, 1988, 1989; Llorente-Bousquets et al., 1992;
Salazar, 1999; DeVries & Walla, 2001; Furtado, 2001, 2008;
Berthier, 2005), historically there has been much disagreement
about the higher-level and species-level classification. The
current generic classification of Preponini (Lamas, 2004)
follows that of other authors (e.g. DAbrera, 1987; DeVries,
1987; Neild, 1996), and has remained moderately stable since
Rydons (1971) morphology-based higher classification of
the Charaxinae. Nevertheless, several studies have suggested
that this classification is in need of revision (Furtado, 2008;
Marconato, 2008; Escalante et al., 2010), but to date studies
of Preponini have only been performed as part of higher-level
studies of butterflies, with relatively few preponine species
represented. Based on partial sequences of the nuclear gene
wingless, and with only a single preponine species included,
Brower (2000) assessed the phylogenetic relationships among
the tribes of the family Nymphalidae, and found Preponini
to be sister to all other charaxines. In a later work,
Freitas & Brown (2004) proposed a phylogenetic hypothesis
of the family Nymphalidae based on morphology, again
including only one Preponini individual, Archaeoprepona
chalciope (Hubner). Later, Marconato (2008) presented a
phylogeny of Charaxinae based on morphological characters,
including the tribe Preponini, which was represented by 13
species. This study provided strong evidence of the need for
further taxonomic study of the tribe. Marconato concluded
that maintaining the monophyly of the tribe required that
Anaeomorpha splendida Rothschild be excluded, and recent
molecular studies have suggested that this taxon may be
related to either the tribe Anaeini or the tribe Pallini (Pena &
Wahlberg, 2008; Wahlberg et al., 2009), with its phylogenetic
placement remaining unclear. Similarly, Marconatos results
challenged the current generic classification, suggesting that
both Prepona and Archaeoprepona might be paraphyletic.

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Further progress in preponine taxonomy requires a comprehensive approach, in terms of both taxa and characters,
using independent datasets that have been proven helpful for
reconstructing patterns of relatedness at both higher and lower
taxonomic levels (Miller et al., 1997; Wahlberg et al., 2005).
Morphology has historically provided the basis for butterfly classification (Ackery et al., 1998), but clearly the value
of such characters may be limited if they are under strong
selection or if divergence has occurred recently and rapidly
(Bickford et al., 2007). Preponini are morphologically quite
homogeneous at both species and genus levels (Fruhstorfer,
1916; Neild, 1996; Marconato, 2008), suggesting close relationships among a number of taxa. Indeed, Furtado (2008)
demonstrated hybridisation between taxa placed in the two genera Prepona and Agrias. Given that morphological characters
alone have so far not convincingly resolved taxonomic problems in the Preponini, our goal was to use molecular sequence
data to infer the first species-level phylogeny for the tribe and
thereby to establish a firm higher classification for the group.
The resulting phylogeny should also provide a valuable foundation for investigating ecological and evolutionary questions,
such as the origins and variation of the bright dorsal coloration
in Agrias (Austin, 2009), its putative involvement in mimetic
rings between Agrias, Callicore Hubner and Asterope Hubner
(Descimon, 1977; Jenkins, 1987), and its potential function
in sexual signalling, as presumed in other butterfly groups
(reviewed in Silberglied, 1989).

Material and methods

Taxon sampling
The analysis included DNA sequence data from 25 outgroup taxa accessed through GenBank, in addition to novel
sequence data, representing a total of 73 individuals, of
which 48 individuals represented the tribe Preponini. Outgroup species were chosen based on Wahlberg et al.s (2009)
phylogeny, comprising individuals from the charaxine tribes
Anaeini (n = 9), Charaxini (n = 8), Pallini (n = 3), Prothoini
(n = 2) and the closely related subfamily Satyrinae (n = 3).
The samples come from several Neotropical countries, and
were collected by the authors, provided by collaborators or
obtained from museum collections (details are available in
Tables S1, S2). Fresh tissue was obtained from preponine
butterflies collected in the field using entomological nets and
Van Someren-Rydon traps hung in the forest canopy and baited
with rotten fish or fermenting fruit. Butterfly vouchers were
stored inside glassine envelopes for transport and then spread
in the laboratory. Two legs were removed from each voucher
specimen and placed in 96% ethanol until ready to process.

DNA extraction, amplification and sequencing

Total genomic DNA was extracted from one leg of
each specimen using the Qiagen DNEasy Extraction Kit,

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E. Ortiz-Acevedo and K. R. Willmott

following the manufacturers protocol (Qiagen Inc., Valencia,

CA, U.S.A.). For old tissue or low-yield DNA samples
the protocol was modified following Iudica et al. (2001).
Using standard PCR we amplified two mitochondrial gene
regions, cytochrome oxidase subunits I and II (COI and
COII ), and one nuclear gene region, Elongation Factor-1
(EF-1), which have proven to be of value in phylogenetic
studies of butterflies (Monteiro & Pierce, 2001; Sperling,
2003; Kandul et al., 2004; Silva-Brandao et al., 2005; Warren
et al., 2008; Hundsdoerfer et al., 2009). The primers used for
each region follow Hebert et al. (2004), Folmer et al. (1994)
and Monteiro & Pierce (2001), respectively, and the PCR
thermocycling profiles are described in Hebert et al. (2004)
and Hillis et al. (1996) (Tables S3, S4). The PCR reaction mix
is provided in Appendix S1. Custom DNA sequencing from
both strands of each gene was carried out by the University of
Floridas Interdisciplinary Center for Biotechnology Research
Sanger Sequencing Group. We used geneious 5.3 (Drummond
et al., 2010) to manually edit both strands of each gene and
check peak calls, with the resulting sequences aligned using
clustalw (Larkin et al., 2007), and then checked by eye;
subsequently a consensus sequence was produced for each
sample and gene. The final edited and aligned sequences
comprised 618 bp for COI, 892 bp for COII and 951 bp for
EF-1, for a complete data set of 2461 bp.

Phylogenetic analyses
We examined the degree of saturation of each codon position for the mitochondrial genes by calculating the transition/transversion (Ti/Tv) ratio in mega 5 (Tamura et al., 2011)
using the Tamura & Nei (1993) distance model. We analysed
each gene individually and a concatenated data set for the three
genes under the maximum parsimony (MP) optimality criterion
using paup* 4.0 (Swofford, 2003), executing multiple heuristic searches with characters equally weighted and gaps treated
as missing data, using the treebisectionreconnection (TBR)
branch-swapping algorithm and random stepwise addition of
1000 replicates. A strict consensus tree was estimated when
multiple equally parsimonious trees were obtained, followed by
a 1000-replicate bootstrap analysis (Felsenstein, 1985), using
the same search routine, as a measure of branch support and
reliability of the resulting tree (Hall, 2008). The data were also
analysed using maximum likelihood (ML) in garli (genetic
algorithm for rapid likelihood inference; Zwickl, 2006)
through the garli Web service (Bazinet et al., 2007; Bazinet
& Cummings, 2011). A partitioned analysis was performed on
the concatenated dataset after the sequence evolution model
for each gene was selected using jmodeltest (Posada & Crandall, 1998). The model GTR + I + G was chosen for the three
genes. We estimated support for the resulting phylogenetic
trees by conducting 1000 replicate bootstrap searches using the
same search routine and parameters. Lastly, we performed a
Bayesian inference (BI) analysis using mr bayes 3.1 (Huelsenbeck et al., 2001; Ronquist & Huelsenbeck, 2003). BI analyses
involved a partitioned analysis with four Markov chain Monte

Carlo simulations of 1 106 generations, sampling every 100

steps with a burn-in of 25% of generations. Finally, a consensus tree with node posterior probabilities was constructed. We
used figtree 1.3.1 (Rambaut, 20062009) to edit the resulting
trees.

Results
The analysis of the degree of saturation of each codon position
of COI and COII genes showed that none of the positions
were saturated, as the Ti/Tv ratio was found to be higher than
1 (Table S5). From the total of 2461 bp used in the analysis
we found 799 bp to be informative. MP analysis resulted in
a total of 54 equally parsimonious trees of 4525 steps. The
MP and BI consensus trees (Figures S1, S2) were generally
consistent with the ML tree, but both showed low resolution
for the relationships of Preponini to the remaining charaxine
tribes (Fig. 1). The ML tree constructed from the concatenated
dataset was the most highly resolved, and is shown in Fig. 1.
None of our analyses found Preponini as currently conceived
to be monophyletic, as Anaeomorpha never grouped with the
remaining Preponini. Instead, the ML tree, MP strict consensus
tree and BI consensus tree for the concatenated dataset showed
this taxon to be placed outside the tribe, but the position
within the subfamily remained unclear. The MP analysis
placed Anaeomorpha as sister to a clade containing all other
charaxines, including monophyletic Pallini, and polyphyletic
Anaeini and Charaxini. Prepona pheridamas (Cramer) was
sister to the clade Polygrapha cyanea (Salvin & Godman,
1868) + remaining Preponini, albeit with weak support for
this topology (Figure S1). The BI analysis did recover a
monophyletic Preponini (node posterior probability 1) with the
exclusion of Anaeomorpha, and with the latter placed within
a polytomy containing all the charaxine tribes (Figure S2).
The ML tree also showed a monophyletic Preponini (bootstrap
value 77), with the exclusion of Anaeomorpha, which was
placed as sister to Anaeini, although there was no strong
support for this latter relationship (Fig. 1). In addition, none
of the analyses of each individual gene found Anaeomorpha
splendida clustering with the remaining Preponini, nor as sister
to the tribe Anaeini (Figures S311).
Within the Preponini (excluding Anaeomorpha) we found
strong support in all three analyses for a clade containing
Archaeoprepona and Noreppa. Although some of the deeper
relationships among species in this clade were not strongly
supported by all three analyses, our results clearly showed that
Noreppa chromus (Guerin-Meneville) is sister to Archaeoprepona licomedes (Cramer) (Fig. 1), a relationship highly supported by bootstrap and posterior probability results. There
was moderate support in the three analyses for Archaeoprepona licomedes + Noreppa being sister to a clade containing Archaeoprepona chalciope, Archaeoprepona amphimachus
(Fabricius) and Archaeoprepona meander (Cramer), showing
Noreppa to be placed well within the genus Archaeoprepona as
currently conceived. The independent analyses for each gene

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Phylogeny of Preponini

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Fig. 1. Maximum-likelihood tree for the 48 preponine individuals plus 25 out-group taxa. Out-group taxa are highlighted with black; the remaining
genera follow the colour code. Numbers above or below branches correspond to maximum likelihood and maximum parsimony bootstrap values,
and Bayesian inference posterior probabilities, respectively. A lack of node support for any methodology is denoted with a hyphen (). The line
weight indicates whether the clade was recovered in one, two or three methodologies. Individuals shown (denoted by ) represent each of the tribes
included; in some cases more than one individual per tribe is shown to highlight the diversity of forms.
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E. Ortiz-Acevedo and K. R. Willmott

also supported the placement of Noreppa within Archeoprepona, even though the analyses for COI found that some
Archaeoprepona individuals clustered with Prepona + Agrias
(Figures S311).
The other major clade recovered with moderate to strong
support in the ML and BI analyses contained the species of
Prepona and Agrias (Fig. 1), and this clade also appeared
in several analyses using individual genes (Figures S311).
Sister to all other Prepona and Agrias is Prepona pheridamas, with the monophyly of the clade containing remaining Prepona and Agrias strongly supported in all three
analyses. Within this latter clade Prepona dexamenus Hopffer is sister to a clade containing the remaining species,
and again this topology is strongly supported in all three
analyses of the concatenated dataset. The type species for
the genus Prepona, Prepona laertes (Hubner), was found
to be sister to a clade containing Agrias + Prepona praeneste Hewitson + Prepona deiphile + Prepona werneri Hering
& Hopp + Prepona pylene Hewitson, a strongly supported
relationship recovered in all analyses for the concatenated
dataset. Relationships among species in the Agrias + Prepona
praeneste + Prepona deiphile + Prepona werneri + Prepona
pylene clade were more weakly resolved. Several analyses for independent genes and our BI and ML results
did not recover Agrias as monophyletic, although none
of these alternative topologies were strongly supported. In
our ML analysis Agrias aedon Hewitson clustered with
two Prepona taxa, Prepona praeneste and Prepona deiphile
neoterpe Honrath, and together these taxa formed a weakly
resolved clade with other Prepona deiphile taxa, Prepona
pylene and Prepona werneri, and remaining Agrias, with
this clade appearing in all analyses with moderate support.
Although resolving relationships among taxa below the
species level was not the focus of our study, 20 species of
preponines were represented by multiple individuals. Conspecific individuals clustered together in 15 species, but not in
the remaining five species. Individuals from Archaeoprepona
amphimachus and Archaeoprepona meander formed a mixed
group, with relatively high bootstrap and posterior probability support (although some nodes lack support). Archaeoprepona amphimachus amphimachus (Fabricius) and Archaeoprepona meander meander (Cramer) formed monophyletic clusters, respectively, but the west Andean taxon Archaeoprepona
amphimachus amphiktion (Fruhstorfer) was in conflict, yielding Archaeoprepona amphimachus as paraphyletic. Prepona
pylene appears as a paraphyletic entity, clustering with individuals of Prepona deiphile, which in turn appears as polyphyletic (Prepona deiphile neoterpe clusters with Prepona
praeneste). In the Prepona pylene + Prepona deiphile clade
the terminal nodes are well supported in all analyses, but the
basal relationships, namely the position of Prepona pylene
philetas Fruhstorfer with respect to the remaining Prepona
pylene + Prepona deiphile ibarra Beutelspacher are still unresolved. Similarly, within the genus Agrias, we found that individuals of both Agrias narcissus and Agrias claudina Godart
failed to cluster.

Discussion
In general, the different methods of analysis of the concatenated dataset produced congruent results for well-supported
nodes, as indicated in Fig. 1. We selected the mitochondrial
markers COI and COII, and the nuclear marker EF-1, based
on the fact that they have been successfully used in studies of
lepidopteran systematics, not only at different taxonomic levels, but also in a broad spectrum of groups within the order
(Caterino et al., 2000; Monteiro & Pierce, 2001; Sperling,
2003; Kandul et al., 2004; Silva-Brandao et al., 2005; Warren et al., 2008; Hundsdoerfer et al., 2009). Previous studies
have shown that the third codon positions of the mitochondrial
genes COI and COII can be saturated (i.e. Ti/Tv < 1), and are
thus phylogenetically uninformative (e.g. Silva-Brandao et al.,
2005); however, our analysis showed that none of the Ti/Tv
ratios for the individual codon positions nor the overall Ti/Tv
ratio for both genes was saturated (Table S5), suggesting that
they retain valuable information worth keeping in our analyses. Additional data, especially from nuclear genes, should of
course help strengthen our results and help resolve some parts
of the topology that are still poorly resolved. However, the
principal results that we report in this paper, and upon which
we base taxonomic decisions, are consistent among genes,
among different methods, and with other morphological and
molecular datasets, with the latter involving more genetic data
but fewer taxa. We are thus confident that our data provide
sufficient resolution and support for us to explore phylogenetic relationships within the tribe. Below we address the two
main subjects of this research study, tribal and generic limits,
and also discuss lower-level taxonomic conflicts that became
evident as a result of this study.

Tribal limits
The first goal of our study was to test the monophyly of the
tribe, in particular by examining the placement of Anaeomorpha splendida. Rothschild (1894) suggested that Anaeomorpha
splendida was related to the tribe Anaeini, but Fruhstorfer
(1916) included it within the genus Prepona, albeit as a group
of species separate from other Prepona (including Archaeoprepona). Rydon (1971) also classified Anaeomorpha with the
other four preponine genera (within the subfamily Preponinae),
although he discussed a number of characters that conflicted
with this placement. Several authors working on phylogenetic
studies have questioned the relationship of Anaeomorpha to
other preponines, suggesting instead a relationship to Anaeini
(Wahlberg et al., 2009) or to Pallini (Pena & Wahlberg, 2008).
Marconatos (2008) study left Anaeomorpha as a member of
Preponini, despite the fact that the taxon does not possess
bunches of hair-like androconial scales on the dorsal hindwing, which she considered a synapomorphy for Preponini.
Her results showed Anaeomorpha to be placed in a polytomy
with representatives of Pallini, Anaeini, Preponini and Charaxini, although after successive weighting approximations, it was
placed as sister to Anaeini.

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Phylogeny of Preponini
Our results show that maintaining the monophyly of
the tribe Preponini requires the exclusion of Anaeomorpha
splendida, and are consistent with current morphological data
suggesting that Anaeomorpha is not particularly closely related
to any extant charaxine tribe. Although the ML analysis
provided weak support for a relationship with the tribe
Anaeini, as suggested by Marconato (2008) and Wahlberg
et al. (2009), we believe that the best current solution is
to exclude Anaeomorpha from Preponini and reinstate the
tribe Anaeomorphini stat.rev., given the lack of agreement
among datasets and analyses, and the weak support for
the current topology. Ultimately, of course, additional genes
and perhaps morphological data, especially from the as yet
unknown immature stages, might firmly resolve the position of
Anaeomorpha within the Charaxinae. Nevertheless, the current
lack of clear morphological characters uniting Anaeomorpha
with other charaxine tribes suggests that treating Anaeomorpha
within its own tribe may remain the best taxonomic solution,
even after its relationships with other charaxines have been
resolved.

Generic limits
The second focus of this study was to test the monophyly of
Preponini genera. In particular, two main issues were evident
in Marconatos (2008) study: (i) the placement of Noreppa
chromus with respect to the genus Archaeoprepona, and (ii)
the relationship of Agrias with the genus Prepona. Rydon
(1971) described Noreppa as a new genus on the basis of its
possession of hairs on the eyes and palpi, and dorsal spines
on the mid and hind tibia and tarsi, but without cladistic
analysis it was not clear whether the alternative states for these
characters in Archaeoprepona represented synapomorphies
or symplesiomorphies. Our results suggest that the latter
hypothesis is correct, as we found strong support for a sister
relationship between Noreppa and Archaeoprepona licomedes
(Fig. 1). Marconato (2008) also noted that the morphological
synapomorphies that characterise Archaeoprepona occur in
Noreppa, whereas there is relatively weak morphological
variation among other Archaeoprepona species. It is possible
that the development of apomorphic morphological character
states in Noreppa chromus is related to this being the
only montane species in the clade. Even though there are
other taxonomic modifications that could solve the current
taxonomic conflict, for instance maintaining Noreppa and
describing additional genera for other monophyletic groups, the
genetic evidence coupled with the morphological data suggest
that placing Noreppa within Archaeoprepona is the most
practical solution. We therefore synonymise Noreppa syn.n.
with Archaeoprepona.
Similarly to Archaeoprepona, Prepona was never found to
be monophyletic in the analyses for the concatenated dataset
nor in the individual analyses for each gene. Instead, our
results corroborate Marconatos (2008) finding that Agrias is
nested within Prepona, and indeed the short branch lengths
between Agrias and related Prepona species suggest that

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Agrias has only recently diverged from its closest relatives.

Although there is only weak support for the relationships
of Agrias to the most closely related Prepona species, our
results show that Agrias is firmly contained within Prepona,
with the four most basal nodes of Prepona + Agrias all
strongly supported. Given the paraphyly of Prepona a number
of taxonomic solutions are possible. To maintain Agrias as
currently conceived would require making Prepona monotypic
and the description of two new monotypic genera for Prepona
dexamenus and Prepona pheridamas. The notable divergence
between Prepona pheridamas and all other Prepona + Agrias,
the lack of the morphological synapomorphies of Prepona
in Prepona pheridamas and the unique genitalic morphology
of the latter species (Marconato, 2008) might support the
recognition of a new genus for Prepona pheridamas. Prepona
dexamenus is, however, morphologically typical of Prepona,
and taxonomic resolution is also needed within the Prepona
werneri, Prepona pylene, Prepona deiphile and Prepona
praeneste assemblage. Our current results suggest that another
three genera would be needed to contain these last four
species, but even if additional data showed them to form just
a single clade, sister to Agrias, the resulting genus would
lack any known morphological or ecological synapomorphies.
Similarly, expanding Agrias to include the Prepona werneri,
Prepona pylene, Prepona deiphile and Prepona praeneste
assemblage would result in a morphologically and ecologically
heterogeneous genus. By contrast, recognising an expanded
Prepona to also contain Agrias requires fewer genus-level
taxonomic changes, is more reasonable in view of the current
classification of other charaxine genera, is consistent with
morphological data (see Marconato, 2008), and reflects the
evident close relationship between Agrias and Prepona, as
demonstrated by intergeneric hybrids (Furtado, 2008). In fact,
almost a century ago Fruhstorfer (1916) suggested that Agrias
might in future be degraded to the denomination of a group of
variegated Prepona, and subsequent authors have expressed
similar sentiments (e.g. DeVries, 1987; Lamas, 2004). We
therefore synonymise Agrias syn.n with Prepona.

Species relationships
We found that in some cases individuals of the same taxon
did not cluster together. In part, this finding probably reflects
the known inability of limited DNA sequence data to diagnose
some closely related species (e.g. Elias et al., 2007; Dasmahapatra et al., 2010), perhaps because of introgression, horizontal
gene transfer or the presence of pseudogenes (Funk & Omland,
2003). For example, individuals of Archaeoprepona amphimachus and Archaeoprepona meander formed a mixed group
with significant node support, suggesting that the results are
not caused by errors in the recovered sequences. This mixed
group was not only found in the individual analyses of the
mitochondrial genes, but also in the ML and BI analyses of
the nuclear gene (MP not resolved), suggesting that our mitochondrial markers are not a source of conflict. Escalante et al.
(2010) also found that Mexican individuals of Archaeoprepona

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amphimachus and Archaeoprepona meander failed to cluster,

but given that known morphological characters support neither
merging of the two taxa nor additional splitting, we suggest
that the current taxonomy be maintained.
Within Prepona, however, there are several cases where
DNA sequence divergence or lack of monophyly correspond
with morphologically well-defined taxa. Prepona pylene and
Prepona deiphile exhibited high genetic divergence, appearing as paraphyletic and polyphyletic, respectively. Escalante
et al. (2010) also found high genetic divergence among different subspecies of Prepona deiphile, even within Mexico.
Increasing the geographic breadth and taxonomic representation within Prepona deiphile, by including Ecuadorian Prepona
deiphile neoterpe, demonstrated even more significant divergence. These results suggest that at least some of the often
isolated, rare and morphologically well-defined populations of
Prepona deiphile and Prepona pylene might represent distinct
species.
The former genus Agrias also showed taxonomic conflicts
regarding two species, Prepona narcissus stat.n. and Prepona
claudina stat.n.. Both species are geographically polymorphic,
and clearly increasing the sampling scope would be useful to
explore whether our results reflect allopatric cryptic species,
ancient polymorphism (Avise & Ball, 1990) or possibly
introgression resulting from interspecific hybridisation (Sota
& Vogler, 2001).

Wing pattern evolution

A large part of the reluctance of previous authors, including
ourselves, to merge the morphologically similar Prepona and
Agrias is the strikingly different wing pattern of the latter
group of species in comparison with the remainder of the
tribe. Fruhstorfer (1916) noted a remarkable similarity between
the latter group of species and two biblidine nymphalid
genera, Callicore and Asterope, a similarity later ascribed
to mimicry (Descimon, 1977), and Jenkins (1987) further
documented putative mimetic relationships among the various
taxa in these genera. There is little information about the
palatability of any of these species, and the basis for mimicry
therefore remains unknown, but strong dorsal and ventral
wing pattern convergence, marked geographic variation and
matching geographic distributions of similar wing patterns are
all consistent with mimicry.
Strong selection for mimicry is, therefore, a likely explanation for the rapid evolution of bright wing colour patterns in
the former Agrias species. Why only some species of Prepona
are mimetic remains to be investigated, but our data suggest
the intriguing possibility that red or orange forewing coloration
arose initially without any (known) function in predator signalling, as in Prepona praeneste and Prepona deiphile, and was
then subsequently modified for mimicry. It may also be significant that both Prepona praeneste and Prepona deiphile occur
in montane regions, with few Callicore or Asterope, suggesting that selection for mimicry may have occurred with a shift
into lowland forests inhabited by these two biblidine genera.

Clearly a better understanding of species limits in Prepona

and an expanded phylogenetic character dataset to more firmly
establish species relationships are needed for further investigation of wing pattern evolution and diversification in this
genus.

Conclusion
Our study indicates that a number of taxonomic modifications are required within the tribe Preponini, ranging from
tribal limits to species boundaries. Based on our results we
move Anaeomorpha splendida from Preponini into the reinstated tribe Anaeomorphini. The inclusion of additional markers and broader taxonomic sampling are needed to determine
the relationships of Anaeomorpha within the Charaxinae. At
the generic level, we synonymise Noreppa with Archaeoprepona. The combined results from genetic and morphological data (Marconato, 2008) support this action, as our
phylogeny shows that Noreppa chromus is embedded well
within Archaeoprepona, and the morphological synapomorphies used to define Archaeoprepona also apply to Noreppa.
We also synonymise Agrias with Prepona, after discussing
and rejecting other possible taxonomic solutions. In contrast, future morphological and ecological study might show
that a new genus is warranted for Prepona pheridamas,
which is sister to all other Prepona, in addition to having distinctive wing pattern characters and genitalia. The
likely need for some species-level taxonomic modifications
is also apparent from our data. Individuals of five out of
20 species represented by multiple samples in our study
failed to cluster together, and significant geographic variation
within species such as Prepona pylene and Prepona deiphile
probably represents taxa that should be treated as distinct
species.
Our results thus clarify the higher classification of Preponini,
and will hopefully provide a basis for future work on species
taxonomy in the tribe and ultimately in systematic revisions
of preponine genera. In addition, more comprehensive taxon
and character sampling should enable us to explore the
phylogeography of the tribe to better understand current
patterns of distribution and species richness, investigate the
evolution of wing pattern diversity and reveal factors that
have been important in the diversification of these spectacular
butterflies.

Supporting Information
Additional Supporting Information may be found in the online
version of this article under the DOI reference:
10.1111/syen.12008

Figure S1. Strict consensus tree of 54 most-parsimonious

trees recovered for the concatenated dataset. Numbers
above branches correspond to bootstrap values. Colour
codes follow Fig. 1 in the main text.

2013 The Royal Entomological Society, Systematic Entomology, 38, 440449

Phylogeny of Preponini

Figure S2. Bayesian inference consensus tree recovered

for the concatenated dataset. Node numbers correspond to
posterior probability values. Colour codes follow Fig. 1 in
the main text.
Figure S3. Majority-rule consensus tree of 195 mostparsimonious trees of 1623 steps recovered for COI. This
dataset had 232 parsimony-informative characters and a
consistency index (CI) of 0.310.
Figure S4. Maximum likelihood tree for COI.

447

del Ambiente for providing research permits for Ecuador, and

A. Navas and Ministerio de Ambiente y Desarrollo Sostenible
for processing the Colombian permits. We thank Dr. G. Lamas
and three anonymous reviewers and the journal editor for their
valuable comments and suggestions for the article. Finally,
we acknowledge funding from Sigma Xi (Grants-in-Aid of
Research G20100315153261), the Center for Systematic Entomology, the Council of the Linnean Society and the Systematics Association for the Systematics Research Fund, the
National Geographic Society and the National Science Foundation (DEB#0103746, 0639861).

Figure S5. Bayesian inference consensus tree for COI.

Figures S6. Majority-rule consensus tree of 27 mostparsimonious trees of 1511 steps recovered for COII. This
dataset had 301 parsimony-informative characters and a
consistency index (CI) of 0.925.
Figures S7. Maximum-likelihood tree for COII.
Figure S8. Bayesian inference consensus tree for COII.
Figure S9. Majority-rule consensus tree of 3817 mostparsimonious trees of 1186 steps recovered for EF-1. This
dataset had 266 parsimony-informative characters and a
consistency index (CI) of 0.769.
Figure S10. Maximum-likelihood tree for EF-1.
Figure S11. Bayesian inference consensus tree for EF-1.
Table S1. Preponine taxa included in the phylogenetic
reconstruction.
Table S2. Out-group taxa included in the phylogenetic
reconstruction.
Table S3. PCR primer names and sequences.
Table S4. Modified PCR thermocycling profiles from
Hebert et al. (2003) and Hillis et al. (1996).
Table S5. Transition/transversion (Ti/Tv) ratio for the
mitochondrial genes COI and COII by codon position.
Appendix S1. Supporting methods.

Acknowledgements
We thank Dr T.C. Emmel and the McGuire Center for Lepidoptera and Biodiversity for supporting this study. We
are grateful to J. Hall, J. Miller, D. Mathews-Lott, C. Pozo,
J. Llorente-Bousquets, D. Janzen, R. Robbins, A. Neild, W.
Zoller, C. Brevignon, M.F. Checa, G. Gallice, C.R. Malaver,
J. Radford, R. Aldaz and Universidad de los Andes Museum
ANDES for making available data and/or preponine material for study. We thank Dr P.C. Zalamea, Dr P. Stevenson,
Dr M. Wolff and Universidad de Antioquia for logistic support. We are also grateful to S. Padron for the pictures of
charaxine specimens, L. Xiao for support in the molecular
laboratory, J.P. Gomez for assistance with artwork and everybody involved in the fieldwork. We also thank S. Villamarn,
the Museo Ecuatoriano de Ciencias Naturales and Ministerio

References
Ackery, P.R., De Jong, R. & Vane-Wright, R.I. (1998) The butterflies:
Hedyloidea, Hesperioidea and Papilionoidea. Lepidoptera, Moths
and Butterflies, Vol. 1: Evolution, Systematics and Biogeography,
Handbook of Zoology, Vol. IV, Part 35 (ed. by N.P. Kristensen),
pp. 264300. de Gruyter, New York, New York.
Aduse-Poku, K., Vingerhoedt, E. & Wahlberg, N. (2009) Out-ofAfrica again: a phylogenetic hypothesis of the genus Charaxes
(Lepidoptera: Nymphalidae) based on five gene regions. Molecular
Phylogenetics and Evolution, 53, 463478.
Austin, G.T. (2009) Nymphalidae of Rondonia, Brazil: variation and
phenology of Agrias (Charaxinae). Tropical Lepidoptera Research,
19, 2934.
Avise, J.C. & Ball, R.M. (1990) Principles of genealogical concordance in species concepts and biological taxonomy. Oxford Surveys
in Evolutionary Biology, 7, 4567.
Bazinet, A.L. & Cummings, M.P. (2011) Computing the tree of life:
leveraging the power of desktop and service grids. Proceedings
of the Fifth Workshop on Desktop Grids and Volunteer Computing
Systems (PCGrid 2011). Anchorage, Alaska, pp. 18961902.
Bazinet, A.L., Myers, D.S., Fuetsch, J. & Cummings, M.P. (2007)
Grid services base library: a high-level, procedural application
programming interface for writing globus-based grid services.
Future Generation Computer Systems, 22, 517522.
Berthier, S. (2005) Thermoregulation and spectral selectivity of the
tropical butterfly Prepona meander: a remarkable example of
temperature auto-regulation. Applied Physics A: Materials Science
& Processing, 80, 13971400.
Bickford, D., Lohman, D.J., Sodhi, N.S. et al. (2007) Cryptic species
as a window on diversity and conservation. Trends in Ecology &
Evolution, 22, 148155.
Brower, A.V. (2000) Phylogenetic relationships among the Nymphalidae (Lepidoptera) inferred from partial sequences of the wingless
gene. Proceedings of the Royal Society B: Biological Sciences, 267,
12011211.
Caterino, M.S., Cho, S. & Sperling, F.A.H. (2000) The current state
of insect molecular systematics: a thriving tower of babel. Annual
Review of Entomology, 45, 154.
Chacon, I. & Montero, J.J. (2007) Mariposas de Costa Rica. INBio,
San Jose, California.
DAbrera, B. (1987) Butterflies of the Neotropical Region. Part IV
Nymphalidae (Partim). Hill House, Melbourne, Victoria.
Dasmahapatra, K., Elias, M., Hill, R., Hoffman, J. & Mallet, J. (2010)
Mitochondrial DNA barcoding detects some species that are real,
and some that are not. Molecular Ecology Resources, 10, 264273.
Descimon, H. (1977) Biogeographie, mimetisme et speciation dans le
genre Agrias Doubleday (Lep. Nymphalidae Charaxinae). Publications du Laboratoire de Zoologie de lEcole Normale Superieure, 9,
307344.

2013 The Royal Entomological Society, Systematic Entomology, 38, 440449

448

E. Ortiz-Acevedo and K. R. Willmott

DeVries, P.J. (1987) The Butterflies of Costa Rica and their Natural
History, Volume I: Papilionidae, Pieridae, Nymphalidae. Princeton
University Press, Princeton, New Jersey.
DeVries, P. & Walla, T. (2001) Species diversity and community
structure in neotropical fruit-feeding butterflies. Biological Journal
of the Linnean Society, 74, 115.
Drummond, A.J., Ashton, B., Buxton, S. et al. (2010) Geneious., v 5.4
[WWW document]. URL http://www.geneious.com [accessed on 7
March 2011].
Elias, M., Hill, R.I., Willmott, K.R., Dasmahapatra, K.K., Brower,
A.V.Z., Mallet, J. & Jiggins, C.D. (2007) Limited performance of
DNA barcoding in a diverse community of tropical butterflies.
Proceedings of the Royal Society B: Biological Sciences, 274,
28812889.
Escalante, P., Ibarra-Vazquez, A. & Rosas-Escobar, P. (2010) Tropical
montane nymphalids in Mexico: DNA barcodes reveal greater
diversity. Mitochondrial DNA, 21, 3037.
Felsenstein, J. (1985) Confidence limits on phylogenies: an approach
using the bootstrap. Evolution, 39, 783791.
Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. (1994)
DNA primers for amplification of mitochondrial cytochrome c
oxidase subunit I from diverse metazoan invertebrates. Molecular
Marine Biology and Biotechnology, 3, 294299.
Freitas, A.V.L. & Brown, K.S. (2004) Phylogeny of the Nymphalidae
(Lepidoptera). Systematic Biology, 53, 363383.
Fruhstorfer, H. (1916) Aegeronia, Brassolidae, Prepona, Morpho,
Adelpha, Agrias. The Macrolepidoptera of the World (ed. by
A. Seitz), p. 1139. Alfred Kernen, Stuttgart.
Funk, D.J. & Omland, K.E. (2003) Species-level paraphyly and
polyphyly: frequency, causes, and consequences, with insights from
animal mitochondrial DNA. Annual Review of Ecology, Evolution,
and Systematics, 34, 397423.
Furtado, E. (2001) Prepona pheridamas pheridamas (Cramer) and its
immature stages (Lepidoptera, Nymphalidae, Charaxinae). Revista
Brasileira de Entomologia, 18, 689694.
Furtado, E. (2008) Intergeneric hybridism between Prepona and
Agrias. Tropical Lepidoptera Research, 18, 56.
Hall, B.G. (2008) Phylogenetics Trees Made Easy: A How-to Manual.
Sinauer Associates, Sunderland, Massachusetts.
Hebert, P., Penton, E., Burns, J., Janzen, D. & Hallwachs, W. (2004)
Ten species in one: DNA barcoding reveals cryptic species in the
neotropical skipper butterfly Astraptes fulgerator. Proceedings of the
National Academy of Sciences of the United States of America, 101,
1481214817.
Hillis, D.M., Moritz, C. & Barbara, K. (1996) Molecular Systematics.
Sinauer Associates, Sunderland, Massachusetts.
Huelsenbeck, J.P., Ronquist, F., Nielsen, R. & Bollback, J.P. (2001)
Bayesian inference of phylogeny and its impact on evolutionary
biology. Science, 294, 23102314.
Hundsdoerfer, A.K., Rubinoff, D., Attie, M., Wink, M. & Kitching, I.J. (2009) A revised molecular phylogeny of the globally distributed hawkmoth genus Hyles (Lepidoptera: Sphingidae), based
on mitochondrial and nuclear DNA sequences. Molecular Phylogenetics and Evolution, 52, 852865.
Iudica, C.A., Whitten, W.M. & Williams, N.H. (2001) Small bones
from dried mammal museum specimens as a reliable source of DNA.
Biotechniques, 30, 732734, 736.
Jenkins, D.W. (1987) Neotropical Nymphalidae VI. Revision of
Asterope (=Callithea Auct.). Bulletin of the Allyn Museum, 166.
Johnson, K. & Descimon, H. (1988) Systematic status and distribution
of the little-known Charaxinae Prepona werneri Hering & Hopp.
Journal of the Lepidopterists Society, 42, 269275.
Johnson, K. & Descimon, H. (1989) Proper generic and specific
status of Antillean Prepona butterflies with description of a new

subspecies from Puerto Rico (Nymphalidae: Charaxinae). Caribbean

Journal of Science, 25, 4553.
Kandul, N., Lukhtanov, V., Dantchenko, A., Coleman, J., Sekercioglu, C., Haig, D. & Pierce, N. (2004) Phylogeny of Agrodiaetus Hubner 1822 (Lepidoptera : Lycaenidae) inferred from mtDNA
sequences of COI and COII and nuclear sequences of EF1-alpha:
karyotype diversification and species radiation. Systematic Biology,
53, 278298.
Lamas, G. (2004) Charaxinae. Checklist: Pt. 4A HesperioideaPapilionoidea. Atlas of Neotropical Lepidoptera (ed. by J.B. Heppner), p. 439. Association for Tropical Lepidoptera/Scientific Publishers, Gainesville, Florida.
Larkin, M.A., Blackshields, G., Brown, N.P. et al. (2007) Clustal W
and Clustal X version 2.0. Bioinformatics, 23, 29472948.
Llorente-Bousquets, J., Luis-Martnez, A. & Gonzalez-Cota, L. (1992)
Diferenciacion de Prepona deiphile en Mesoamerica y descripcion
de dos subespecies nuevas (Lepidoptera: Nymphalidae). Tropical
Lepidoptera Research, 3, 109114.
Marconato, G. (2008) Analise cladstica de Charaxinae Guenee
(Lepidoptera, Nymphalidae). PhD Thesis, Universidade de Sao
Paulo, Sao Paulo.
Miller, J., Brower, A. & Desalle, R. (1997) Phylogeny of the neotropical moth tribe Josiini (Notodontidae: Dioptinae): comparing and
combining evidence from DNA sequences and morphology. Biological Journal of the Linnean Society, 60, 297316.
Monteiro, A. & Pierce, N. (2001) Phylogeny of Bicyclus (Lepidoptera:
Nymphalidae) inferred from COI, COII, and EF-1 alpha gene
sequences. Molecular Phylogenetics and Evolution, 18, 264281.
Muyshondt, A. (1974) Notes of the life cycle and natural history
of butterflies of El Salvador. VIII. Archaeoprepona antimache
gulina, Siderone marthesia, Zaretis callidryas and Consul electra
(Nymphalidae). Journal of the Lepidopterists Society, 30, 159168.
Neild, A. (1996) The Butterflies of Venezuela. Part 1. Nymphalidae I
(Limenitidinae, Apaturinae, Charaxinae). A Comprehensive Guide to
the Identification of Adult Nymphalidae, Papilionidae, and Pieridae.
Meridian Publications, London.
Pena, C. & Wahlberg, N. (2008) Prehistorical climate change increased
diversification of a group of butterflies. Biology Letters, 4, 274278.
Posada, D. & Crandall, K.A. (1998) MODELTEST: testing the model
of DNA substitution. Bioinformatics, 14, 817818.
Rambaut, A. (20062009) FigTree, 1.3.1. Institute of Evolutionary Biology, University of Edinburgh [WWW document]. URL
http://tree.bio.ed.ac.uk/ [accessed on 20 April 2011].
Ronquist, F. & Huelsenbeck, J.P. (2003) MrBayes 3: bayesian phylogenetic inference under mixed models. Bioinformatics, 19,
15721574.
Rothschild, L.W. (1894) On a new genus and species of butterfly.
Novitates Zoologicae, 1, 1786.
Rydon, A.H.B. (1971) The systematics of the Charaxidae (Lep.
Nymphaloidea). Entomologists Record and Journal of Variation, 83,
219283, 283287, 310316, 336388.
Salazar, J.A. (1999) Habitat e ilustracion del sintipo de Anaeomorpha
splendida columbiana Niepelt, 1928 (Lep: Nymphalidae: Charaxinae) para Colombia. Boletn Cientfico Museo de Historia Natural
de la Universidad de Caldas, 3, 2932.
Silberglied, R.E. (1989) Visual communication and sexual selection
among butterflies. The Biology of Butterflies (ed. by R.I. VaneWright and P.R. Ackery), pp. 207233. Princeton University Press,
Princeton, New Jersey.
Silva-Brandao, K.L., Lucci Freitas, A.V., Brower, A.V.Z. & Solferini,
V.N. (2005) Phylogenetic relationships of the new world Troidini
swallowtails (Lepidoptera: Papilionidae) based on COI, COII,
and EF-1alpha genes. Molecular Phylogenetics and Evolution, 36,
468483.

2013 The Royal Entomological Society, Systematic Entomology, 38, 440449

Phylogeny of Preponini
Sota, T. & Vogler, A.P. (2001) Incongruence of mitochondrial and
nuclear gene trees in the carabid beetles Ohomopterus. Systematic
Biology, 50, 3959.
Sperling, F.A.H. (2003) Butterfly molecular systematics: from species
definitions to higher-level phylogenies. Butterflies: Ecology and
Evolution Taking Flight (ed. by C.L. Boggs, W.B. Watt and
P.R. Ehrlich), pp. 431458. The University of Chicago Press,
Chicago, Illinois and London.
Swofford, D.L. (2003) PAUP*. Phylogenetic Analysis Using Parsimony (* and Other Methods), 4. Sinauer Associates, Sunderland,
Massachusetts.
Tamura, K. & Nei, M. (1993) Estimation of the number of nucleotide
substitutions in the control region of mitochondrial DNA in humans
and chimpanzees. Molecular Biology and Evolution, 10, 512526.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M.
& Kumar, S. (2011) MEGA5: molecular evolutionary genetics
analysis using maximum likelihood, evolutionary distance, and
maximum parsimony methods. Molecular Biology and Evolution,
28, 27312739.
Wahlberg, N., Braby, M., Brower, A. et al. (2005) Synergistic effects
of combining morphological and molecular data in resolving the

449

phylogeny of butterflies and skippers. Proceedings of the Royal

Society B: Biological Sciences, 272, 15771586.
Wahlberg, N., Leneveu, J., Kodandaramaiah, U., Pena, C., Nylin,
S., Freitas, A.V.L. & Brower, A.V.Z. (2009) Nymphalid butterflies
diversify following near demise at the Cretaceous/Tertiary boundary.
Proceedings of the Royal Society B: Biological Sciences, 276,
42954302.
Warren, A.D., Ogawa, J.R. & Brower, A.V.Z. (2008) Phylogenetic
relationships of subfamilies and circumscription of tribes in the
family Hesperiidae (Lepidoptera: Hesperioidea). Cladistics, 24,
642676.
Zwickl, D.J. (2006) Genetic algorithm approaches for the phylogenetic
analysis of large biological sequence datasets under the maximum
likelihood criterion. PhD Dissertation, The University of Texas at
Austin, Austin, Texas.
Accepted 29 November 2012
First published online 20 February 2013

2013 The Royal Entomological Society, Systematic Entomology, 38, 440449