ral
ssBioMed CentBMC Evolutionary Biology
Open AcceResearch article
Two sisters in the same dress: Heliconius cryptic species
Nathalia Giraldo1, Camilo Salazar1,3, Chris D Jiggins2, 
Eldredge Bermingham3 and Mauricio Linares*1
Address: 1Instituto de Gen?tica, Universidad de los Andes, Carrera 1 No 18a ? 70, P.O. Box 4976, Bogot? D.C, Colombia, 2Department of Zoology, 
University of Cambridge, Downing Street, Cambridge, CB2 3EJ, UK and 3Smithsonian Tropical Research Institute, Apartado 0843-03092, Panam?, 
Rep?blica de Panam?
Email: Nathalia Giraldo - na-giral@uniandes.edu.co; Camilo Salazar - salazarc@si.edu; Chris D Jiggins - c.jiggins@zoo.cam.ac.uk; 
Eldredge Bermingham - bermingham@si.edu; Mauricio Linares* - mlinares@uniandes.edu.co
* Corresponding author    
Abstract
Background: Sister species divergence and reproductive isolation commonly results from
ecological adaptation. In mimetic Heliconius butterflies, shifts in colour pattern contribute to pre-
and post-mating reproductive isolation and are commonly correlated with speciation. Closely
related mimetic species are therefore not expected, as they should lack several important sources
of reproductive isolation.
Results: Here we present phenotypic, behavioral and genetic evidence for the coexistence of two
sympatric 'cryptic' species near Florencia in the eastern Andes of Colombia that share the same
orange rayed colour pattern. These represent H. melpomene malleti and a novel taxon in the H.
cydno group, here designated as novel race of Heliconius timareta, Heliconius timareta florencia. No-
choice mating experiments show that these sympatric forms have strong assortative mating (?96%)
despite great similarity in colour pattern, implying enhanced divergence in pheromonal signals.
Conclusion: We hypothesize that these species might have resulted from recent convergence in
colour pattern, perhaps facilitated by hybrid introgression of wing pattern genes.
Background
Ecological selection is known to play an important role in
speciation [1,2]. Where ecological traits under divergent
selection also affect the species recognition system, speci-
ation can be rapid [3,4]. Young species pairs provide evi-
dence for this, such as stickleback morphs found in glacial
lakes in which body size diverges through adaptation to
different niches and is correlated with mate choice [5,6].
Similarly, in Darwin's finches adaptive variation in beak
size also promotes reproductive isolation through pleio-
is also under ecological selection for signaling to preda-
tors, crypsis and thermoregulation [11].
In Heliconius butterflies shifts in colour pattern have been
shown to play a major role in speciation [12,13]. Closely
related species and sub-species typically differ in colour
pattern [14] and are adapted to local M?llerian mimicry
rings in which distasteful species converge on a common
pattern [15,16]. The pattern differences between related
forms lead to strong assortative mating [12,13]. Frequency
Published: 28 November 2008
BMC Evolutionary Biology 2008, 8:324 doi:10.1186/1471-2148-8-324
Received: 6 August 2008
Accepted: 28 November 2008
This article is available from: http://www.biomedcentral.com/1471-2148/8/324
? 2008 Giraldo et al; licensee BioMed Central Ltd. 
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), 
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 11
(page number not for citation purposes)
tropic effects on song [7,8]. In many butterflies colour pat-
tern similarly plays a role in mate recognition [9,10] and
dependent selection contributes to maintaining the
mimetic patterns and also causes post-mating isolation
BMC Evolutionary Biology 2008, 8:324 http://www.biomedcentral.com/1471-2148/8/324
[17,18]. Thus, between closely related species such as H.
cydno and H. melpomene, rare hybrid individuals are likely
to be strongly selected against, as their pattern will not be
recognized by predators [12,15]. These species overlap
extensively across Central America and the Andes [19,20].
Through their geographical range, H. melpomene mimics
H. erato with a black background and red, yellow and
orange marks, whereas H. cydno mimics species of the H.
sapho group with a black-blue background and white and
yellow marks [12,21]. Although H. melpomene and H.
cydno occasionally hybridize in nature (less than 0.1%),
reproductive isolation is strong, with ecological isolation
and colour pattern associated mate choice playing the
major role [12,22,23].
The phylogeny of Heliconius supports a key role for pattern
change in speciation, with almost all sister species differ-
ing in colour pattern [24]. Exceptions such as H. sara and
H. leucadia are far more genetically divergent than species
in the H. melpomene clade, implying that speciation was
relatively ancient [24]. It was therefore a surprise when a
putative 'cryptic' species was identified in the H. cydno/
melpomene species complex, implying either mimetic con-
vergence between closely related species, or speciation
without pattern change [25]. H. tristero was described as a
new species from the southeastern Andes of Colombia
with a cydno-like mtDNA haplotype, but a black, red and
yellow "postman" pattern similar to sympatric H. mel-
pomene mocoa [25]. This species was initially met with
scepticism, mainly due to the fact that only two individu-
als of H. tristero were collected, combined with the likeli-
hood of mtDNA introgression between H. cydno and H.
melpomene [26]. It was therefore suggested that the H. tris-
tero specimens most likely represent rare hybrids between
H. cydno and H. melpomene.
The discovery of very closely related sympatric mimetic
forms is therefore of considerable interest as it would
imply either speciation without colour pattern shifts, or
alternatively very recent mimetic convergence between
hybridizing species, possibly through adaptive introgres-
sion [23,26]. Here we present compelling evidence of a
cryptic species pair, in which an H. cydno cognate resem-
bles and coexists in sympatry with a well-known race of H.
melpomene (Figure 1).
Methods
Sampling and species assignment
A total of 196 adult butterflies were collected between
2002?2006 in Sucre (01?48'12?N 75?39'19?W) near Flor-
encia, Colombia. For comparison of larval and adult mor-
phology, an additional 118 butterflies representing H.
cydno weymeri (N = 21), H. c. cordula (N = 19), H. c. zelinde
from various sites in Colombia. Some of the individuals
from Florencia were maintained alive in separate insectar-
ies at La Vega, Cundinamarca for mate and host choice
experiments. Wings were removed from the remaining
specimens and bodies preserved in DMSO or Ethanol
(96%) for subsequent phenotypic (N = 131) and molecu-
lar analysis (N = 142) and stored in the permanent collec-
tion of the Instituto de Gen?tica of Universidad de los
Andes in Colombia (the collection is now part of the
recently created Natural History Museum ANDES). DNA
extractions were made from one-third of the thorax of
each individual using the DNeasy tissue Kit (QIAGEN),
following the manufacturers' protocol. Digital images
were obtained by scanning the wings and quantitative
measurements taken using TpsDig image software [27].
One-way Analyses of Variance (ANOVA) on phenotypic
measurements were performed with SPSS 11.0.4 software
[28].
No-choice mating experiments
Once diagnostic characters for the two morphotypes had
been established we carried out mate choice experiments
to investigate reproductive isolation. No-choice mating
trials were performed in 2 m ? 2 m ? 2 m insectaries in La
Vega, Cundinamarca between June 2004 and October
2005. These experiments are a simulation of a natural sit-
uation where males encounter females singly, and esti-
mate the reluctance of both sexes to mate inter-
specifically. A virgin female (one to three days old) of each
morphotype was presented to ten mature males (more
than 10 days old) in a single insectary for two days. Suc-
cessful and failed matings were recorded every 30 minutes
between 6 am to 2 pm. Males were used only once. In
order to detect matings that were unobserved, females
were checked for the presence of a spermatophore in their
reproductive tract. A binomial mating probability Pixj and
95% confidence intervals were obtained for each combi-
nation of i-type female and j-type male using Maximum
Likelihood as previously described [29].
Host plant choice and larval morphology
Females of both Florencia populations (H. m. malleti and
H. cydno cognate) and other populations of the two spe-
cies were kept in individual insectaries with known host
plants, Passiflora edulis, P. maliformis, P. ligularis, P. arborea,
P. quadrangularis, P. oerstedii [30,31], and as controls two
species used by H. erato, P. suberosa, and P. rubra [32]. The
number of eggs laid per plant by each female was recorded
twice a week. Offspring from the same females were used
for analysis of larval morphology. H. melpomene and H.
cydno larvae are distinguishable by cephalic colour [14]
which is pale yellow in H. melpomene and orange in H.
cydno. Pictures of larvae raised from wild females werePage 2 of 11
(page number not for citation purposes)
(N = 12), H. melpomene vulcanus (N = 17), H. m. mel-
pomene (N = 26) and H. m. mocoa (N = 23) were collected
taken under similar light conditions with a colour stand-
ard. Between four and seven larvae were analysed from
BMC Evolutionary Biology 2008, 8:324 http://www.biomedcentral.com/1471-2148/8/324
each of 21 females collected in Florencia and compared to
those from other localities. Pictures were processed using
Scion Image (Scion Corporation, Frederick, MD, USA)
and four RGB indexes calculated, R' = r/(r+g+b), G' = g/
(r+g+b), B' = b/(r+g+b) and LM = R'-G' [33]. Statistical sig-
nificance of differences in indices was tested with one-way
variance analysis using SPSS 11.0.4 software [28].
Sequence Analysis
We sequenced a region of nuclear DNA spanning the 3'
end of the Z-linked Triose phosphate isomerase (Tpi) gene,
from thirteen female specimens of H. m. malleti and seven
H. cydno cognate from Sucre Florencia, Caquet? and nine
using primers and conditions outlined elsewhere [34].
The PCR products were electrophoretically separated on
1.5% low melting point agarose (Invitrogen), and the
bands were cut from the gel and dissolved in gelase (Inv-
itrogen). Clean PCR products were sequenced using the
DNA sequence Kit (Big Dye 3.1, PE Applied Biosystems),
in an ABI Prism 3100 Genetic Analyzer (PE Applied Bio-
systems).
We also sequenced fragments of two mitochondrial genes
(COI-COII) from seventeen H.m. malleti, seven cydno-like
and seven H. timareta individuals. In addition to
sequences obtained here, we also included CO and Tpi
Species under studyFigure 1
Species under study. a. H. m. malleti from Florencia and b. its co-mimetic H. timareta florencia (Holotype). Left side: dorsal 
view, right side: ventral view. Both individuals are lodged at the Natural History Museum ANDES of Universidad de los Andes 
in Bogot?, Colombia (accession numbers: Andes-L-01370 and Andes-L-01369).Page 3 of 11
(page number not for citation purposes)
specimens of H. timareta from Ecuador (additional file 1).
The Polymerase Chain Reaction (PCR) was performed
sequences from GenBank (additional file 1). PAUP*
v4.0b10 [35] was used to search for a maximum parsi-
BMC Evolutionary Biology 2008, 8:324 http://www.biomedcentral.com/1471-2148/8/324
mony tree, using a heuristic search with TBR branch swap-
ping; bootstrap values were calculated with 5000
replicates using the same search conditions.
MrModeltest v2.2 [36] was used to determine the most
appropriate model of nucleotide substitution based on
hierarchical likelihood ratio tests. For the mtDNA COI/
COII data set MrModeltest identified the GTR+I+G model
[37,38], and for Tpi, the GTR+G model [37,38]. Bayesian
phylogenetic analyses were performed with MrBayes v3.1
[39] following the analytical recommendations of the
authors [40]. The base frequency parameters determined
by MrModeltest were used for analysis in MrBayes v3.1,
with remaining parameters estimated using the GTR+I+G
model for COI/COII and GTR+G for Tpi. Four differen-
tially heated Markov chains were initiated from random
trees, run for 106 generations and sampled every 100
cycles. Likelihood values were plotted against number of
generations to determine the points at which stationary
was reached. All trees sampled before these points were
discarded and the remaining tree samples were used to
generate a 50% majority rule consensus tree (n = 12101
for COI/COII and n = 9981 for Tpi). The posterior proba-
bility of each clade is provided by the percentage of trees
identifying the clade [39,40].
Microsatellite Analyses
A total of 13 microsatellite loci were amplified and scored
as described previously [41]. Hardy-Weinberg equilib-
rium and linkage disequilibrium and their significance
were tested for at each locus using Arlequin v2000 [42].
Two Bayesian model-based clustering algorithms imple-
mented in the programs STRUCTURE 2.2 [43] and BAPS
4 [44] were used to test the hypothesis that the Florencia
population consisted of two clusters. We determined the
number of ancestral clusters, K, using an ad hoc statistic
?K based on the rate of change in the log probability of
data for K between 1 and 5 in multiple runs [45]. Each run
consisted of 106 iterations, after a burning period of 104
iterations. To use these programs, Hardy-Weinberg and
linkage equilibrium are assumed, and the software differ-
entiates mixed populations on the basis of allele frequen-
cies at each locus. Finally, population differentiation (FST)
and genetic distances (DA) among the two Florencia types
were calculated with Arlequin v2000 [42].
Results
One of us (ML) originally noticed that specimens col-
lected in Florencia represented two phenotypes, one of
which was larger, darker red in colour and with a broader
forewing yellow band. However, none of these pheno-
typic characters proved to be reliably diagnostic (Figure
1). Only the red line, probably homologous to the previ-
consistent diagnostic character of two morphotypes in the
Florencia population. There was a clearly bimodal distri-
bution in the length of this red line, measured relative to
the distance between the base of the Discal Cell and its
intersection with the Cubital Vein (Cu2) (Figure 2). This
character was therefore used to assign individuals to the
two morphotypes for further analysis, with the longer red
line being diagnostic of H. melpomene malleti and the
shorter line of the putative H. cydno cognate. There was a
significant difference in wing size between the two mor-
photypes, with H. melpomene malleti having a smaller wing
size similar to other populations analysed (additional file
2). When offspring were raised from field collected
females, the colour of the larval head capsule also differed
significantly between the H. m. malleti and the H. cydno
cognate individuals from Florencia (additional file 3).
Species relationships
In mtDNA, both parsimony and Bayesian methods pro-
duced three well-supported clades (Figure 3): 1) an east-
ern melpomene clade, 2) an H. cydno clade including all the
H. cydno and H. timareta sequences and 3) a western mel-
pomene clade (Figure 3). Within the cydno clade, individu-
als sampled near to Florencia fell into two different clades
corresponding to their phenotypic assignment described
above. Individuals with longer red line phenotypes are to
the eastern H. melpomene and those with the shorter red
line form a monophyletic group inside H. cydno. The H.
timareta samples from Ecuador form a distinct mono-
Histogram of phenotypic measurements of the ventral red lineFigure 2
Histogram of phenotypic measurements of the ven-
tral red line. The measurements taken from 131 forewings 
clearly show a bimodal distribution. The short line individuals 
represent the H. cydno cognate and the long line individuals 
H. m. malleti, respectively. In dark gray are the five individuals 
with intermediate population assignment probabilities deter-Page 4 of 11
(page number not for citation purposes)
ously described "red dot" in H. c. weymeri [46,47], on the
anterior edge of the ventral forewing was identified as a
mined from microsatellite data.
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phyletic group also within H. cydno. At Tpi, two clusters
are well resolved with high posterior probability and
bootstrap support (99), corresponding to H. melpomene
and H. cydno (1.1% net divergence). The individuals from
Florencia fell into the H. melpomene and H. cydno clades as
predicted from their morphological assignment (Figure
Microsatellite Data Analyses
Individuals from Florencia were screened for variation at
thirteen microsatellite loci. The H. cydno cognate was less
variable, with a mean observed heterozygosity of 0.39 as
compared to 0.66 in H. m. malleti. All loci were in Hardy-
Weinberg equilibrium within the two morphotypes,
except Hm19, which showed significant heterozygote def-
icit for both forms, most likely due to null alleles which
are known to be present at this locus [48]. All subsequent
analyses are presented with this locus removed. The mic-
rosatellite FST value between the two morphotypes in Flor-
encia was large and significant (FST = 0.24226, P <
0.0001).
Without reference to the morphological assignment, two
distinct algorithms, STRUCTURE 2.2 and BAPS 4, were
used to test the number of distinct populations (K) sam-
pled. The ad-hoc statistic ?K, estimated using both pro-
grams, indicated that the most likely value of K was two
(additional file 4). This conclusion was robust to different
assumptions made by STRUCTURE (presence or absence
of genetic admixture, and correlated or uncorrelated allele
frequencies). These two distinct populations identified by
STRUCTURE represented the two colour pattern types
already identified a priori, with virtually all individuals
correctly assigned to the appropriate cluster with posterior
probabilities = 0.95 (Figure 5).
Five individuals had clearly intermediate assignment
probabilities, suggesting possible hybrid genotypes. In
support of this hypothesis, these individuals also had
some evidence for colour pattern introgression, with
either somewhat intermediate red line phenotypes (Figure
2) or melanic scales in the forewing band. These individ-
uals represented around 3.5% of the total sample (5/142)
with three of the five being consistent with an F1 genotype
(Figure 5). Five additional individuals in the H. m. malleti
cluster showed posterior probabilities less than 0.95 sug-
gesting that these might represent backcross hybrids.
No-choice mate experiments
A total of 112 trials were performed (Table 1). An initial
null model with a single mating probability (a = b = c = d)
was established across all trials (LnL = -88.5821). To test
different hypotheses, the likelihood model was fitted in a
stepwise manner by adding parameters to the initial
model. When mating probabilities were estimated sepa-
rately for intra and inter morphotypes (2p: a = d and b =
c) this led to a significant improvement in fit of the model
(G = 119.73, d.f. = 1 and p < 0.0001). This reflects the dif-
ferent mating frequencies between inter and intra mor-
photype trials (3.5% and 96.5%, respectively; Table 1). A
more complicated four parameter model with asymmetric
mtDNA Phylogenetic treeFigure 3
mtDNA Phylogenetic tree. Phylogenetic relationships of 
H. m. malleti (green circles) and Florencia H. cydno cognate 
(pale blue squares) with other populations of H. melpomene 
(red circles) and H. cydno (dark blue squares) based on CoI 
and CoII sequences. Also included are specimens of H. tima-
reta collected from eastern Ecuador (yellow squares). In 
additional files (additional file 1) Sequence ID's beginning with 
AF and AY indicate GenBank accession numbers. Branch 
lengths and probability values (over branches) were esti-
mated using Bayesian analysis and bootstrap support (under 
branches) derived from a Maximum Parsimony analysis.Page 5 of 11
(page number not for citation purposes)
4). mating probabilities did not significantly improve fit of
the model (4p: a?b?c?d, G = 4.74, d.f. = 2 and p > 0.09).
BMC Evolutionary Biology 2008, 8:324 http://www.biomedcentral.com/1471-2148/8/324
Host plant preference
Nineteen females from Florencia (10 H. m. malleti and 9
H. cydno-type) were assessed for host preference. Females
assigned as H. m. malleti used primarily two plants (471
eggs in total on P. oerstedii, 71%, and P. ligularis, 28%),
while the H. cydno cognate females oviposited on many
Allele network for nuclear gene TpiFigure 4
Allele network for nuclear gene Tpi. Red, green, dark blue, light blue and yellow are H. melpomene races, H. m. malleti 
from Florencia, H. cydno races, H. cydno cognate from Florencia and H. timareta. Respective alleles are identified with the letters 
M, MF, C, CF and T, followed by the individual number and allele letter. Black dots are hypothetical ancestors. Sizes of the cir-
cles reflect allele frequencies in the population. Networks were constructed with statistical parsimony in TCS v 1.21 [61].
Assignment test analysisFigure 5
Assignment test analysis. Genetic differentiation between H. m. malleti and H. cydno cognate using assignment analysis of 
multilocus microsatellite data in Structure 2.2. The relative genome contribution of the two clusters to each individual are Page 6 of 11
(page number not for citation purposes)
shown in dark and light grey.
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species (729 eggs in total on P. edulis 48%, P. ligularis
15%, P. oerstedii 22%, and P. quadrangularis, P. arborea and
P. maliformis 14%).
Discussion
The colour patterns of H. melpomene and H. cydno are traits
under strong ecological selection that also contribute to
speciation [12,23,26,49,50]. Divergence in mimetic pat-
tern contributes to reproductive isolation by assortative
mating, due to the use of colour as a mate recognition sig-
nal and by frequency dependent mimicry selection
against rare colour pattern hybrids [12,23,26,29]. The fact
that such a major role for mimicry in the reproductive iso-
lation of currently hybridizing species has been clearly
demonstrated has led to an expectation that mimicry is
unlikely between closely related species with incomplete
reproductive isolation [15]. The results of the present
study clearly show that this is not always the case. The but-
terflies collected in Florencia represent two mimetic spe-
cies in the H. melpomene and H. cydno clades respectively.
Despite the extreme phenotypic similarity between the
butterflies studied here, the data indicate clear concord-
ance between nuclear, mtDNA and phenotypic markers in
assigning over 90% of the individuals sampled to one or
the other morphotype. These represent a population of
the widespread race H. m. malleti, and a novel entity
related to H. cydno. Nonetheless, there is clear evidence for
ongoing interspecific hybridization with around 3.5% of
individuals sampled representing clearly identifiable
hybrids.
The strong concordance between markers and relative
scarcity of hybrids implies strong reproductive isolation.
The mating experiments described here demonstrate
strong assortative mating, and host choice experiments
further imply some degree of ecological isolation. The
strength of assortative mating in our experiments is actu-
ally greater than that between the phenotypically very
divergent H. melpomene melpomene and H. cydno cordula (?
82%, Mav?rez et al. 2006). It therefore seems likely that
there has been increased divergence in mating signals
apart from colour ? most likely pheromonal ? to allow the
Florencia species to coexist. As colour is used as a cue in
this is compensated for by divergence in other mating sig-
nals.
H. melpomene and H. cydno are known to differ in habitat
preference and host use [30,51]. Our host choice data
imply that this ecological difference is maintained
between the Florencia species, with the H. cydno cognate
more of a host generalist similar to other populations of
H. cydno. It seems likely that this corresponds to a prefer-
ence for forest habitats, as is the case for other H. cydno
populations, leading to ecological isolation. Although
crosses between the Florencia forms have not been carried
out, there is preliminary evidence that the H. cydno cog-
nate morphotype is compatible with other populations of
H. cydno, while the Florencia H. m. malleti shows hybrid
sterility with H. cydno (Giraldo and Linares, unpub). It
seems likely that the Florencia species show similar
female hybrid sterility as compared to other sympatric H.
melpomene and H. cydno populations.
In order to clarify the role of colour pattern in speciation
we need to consider the order of divergence in different
factors. Divergence might have occurred initially in factors
other than colour pattern, such as habitat preference and
pheromonal mating cues. Divergence in mimicry then
occurred subsequent to speciation in most populations of
the H. cydno group, except those in the eastern Andes such
as that studied here. Alternatively, speciation might have
been initially triggered by divergence in colour pattern
with mimetic convergence a derived state acquired subse-
quent to speciation in the Florencia region. The first expla-
nation is more parsimonious with respect to colour
pattern, but may actually be less likely. The derived posi-
tion of the H. cydno cognate form in the mtDNA phylog-
eny suggests that it has evolved from a more H. cydno like
ancestor (Figure 3). Furthermore, the observation that
intraspecific races of H. melpomene show both divergent
colour patterns and strong assortative mating implies that
mimicry and associated mate preferences are the first steps
in divergence in this group [13].
Under the second scenario, an initial divergence in colour
pattern associated with adaptation to different M?llerian
Table 1: Results from the no-choice mating trials
female male m n t Mating probability C.I (Upper) C.I. (Lower) Parameters
H.m. malleti x H.m. malleti 21 1 22 0.9545 0.9594 0.9506 a
H.m. malleti ? H. cydno-type 1 26 27 0.0370 0.0420 0.0246 b
H. cydno-type ? H.m. malleti 1 23 24 0.0417 0.0714 0.0000 c
H. cydno-type ? H. cydno-type 35 4 39 0.8974 0.8869 0.9313 d
Mating probabilities were estimated using likelihood as described in the text, with upper and lower confidence intervals shown. m: number of trials 
with mating occurring; n: number of trials without mating occurring; t: total number of trials.Page 7 of 11
(page number not for citation purposes)
mate finding, we would predict that males are likely to be
attracted to the pattern of the "wrong" species, but that
mimicry rings became associated with further changes in
ecology and hybrid inviability, eventually leading to spe-
BMC Evolutionary Biology 2008, 8:324 http://www.biomedcentral.com/1471-2148/8/324
ciation [12]. Subsequently, strong mimetic selection in
the sympatric Florencia population has led to conver-
gence of H. cydno onto the H. melpomene pattern. This
might have occurred through adaptive introgression of
colour pattern genes between the species [21,23,26,52-
54]. It seems plausible that the lack of suitable mimicry
models in the H. sapho group has led this east Andean
population to secondarily converge on the H. melpomene
pattern.
Our results raise the possibility that other populations of
cryptic species might exist in Heliconius. Notably the cryp-
tic H. cydno cognate, H. tristero which was described based
on two specimens and shares a red and yellow banded
colour pattern with sympatric H. m. mocoa in Putumayo,
Colombia may represent another example of the same
phenomenon [25] (Figure 6). Another potential case
occurs in Peru, where a "postman" H. cydno has been dis-
covered that mimics H. m. amaryllis (Mavar?z et al.
unpub.).
The new H. cydno cognate from Florencia resembles H.
timareta, a known relative of H. cydno from eastern Ecua-
dor. H. timareta is polymorphic with one orange rayed
form that is very similar to the taxon from Florencia (Fig-
ure 6). Furthermore, a recently described subspecies, H. t.
timoratus Lamas (1998) from Per? is a near perfect mimic
of H. m. malleti, although the two have so far not been col-
lected in sympatry (Figure 6). Due to the colour pattern
similarity with H. t. timoratus of Per? and the east Andean
distribution of both forms, here we propose the H. cydno
cognate from Florencia as a new northern race of H. tima-
reta, which forms a mimicry ring with H. m. malleti and H.
e. lativitta in the southern foothills of Colombia. We pro-
pose to name this form after Florencia, the town where it
was discovered. From a geographical point of view (Figure
6) the H. cydno clade in the eastern Andes is represented
by, from north to south, H. cydno cordula, H. heurippa, H.
timareta florencia and H. tristero in Colombia; H. timareta
timareta in Ecuador and H. timareta timoratus and another
new H. cydno-like taxon in Per? (Mavarez et al., in prep.).
H. timareta florencia is anatomically similar to H. timareta
and H. cydno races, with a FW area of 5,26 to 6,64 cm2
(additional file 2). The FW has two principal pattern ele-
ments in a black background, 1. An irregular yellow post-
medial band extending proximodistally from distal end of
discal cell to R2?R3 fork, and laterally from subcostal to
CU1a, and 2. A red "Dennis" element extending from the
basal end of the discal cell to the CU1b-discal cell fork,
which is generally shorter than the H. m. malleti FW ele-
ment (the approximate basal fourth of the FW). The ven-
tral FW is similar with a slight reduction in the yellow
black background colour and an androconial distribution
as in H. timareta. A reddish dennis-ray element [55] is sim-
ilar in form to the dennis-ray in H. m. malleti, with a nar-
row red longitudinal bar D (Dennis) and six red radiate
marks on the discal part of the upper side [55]. The ventral
HW underside is similar with narrower red areas. Males
and females are phenotypically similar. H. t. florencia is
registered in ZooBank with the unique digital identifier
LSID: 49656F41-E817-4FD6-B28D-6AA362CC5268. In
order to comply with the International Code of Zoologi-
cal Nomenclature, paper copies of this electronic article
have been deposited in the following libraries: The Ento-
mology Library of the Natural History Museum, London,
UK; The Balfour Library, Department of Zoology, Univer-
sity of Cambridge, UK; The Genetics Library, Department
of Genetics, University of Cambridge, UK; Biblioteca Uni-
versidad Nacional de Colombia, Hemeroteca Nacional,
Sede Bogot?; Biblioteca Universidad de los Andes, Bogot?,
Colombia.
Type material: The holotype male specimen is deposited in
the permanent collection of the Natural History Museum
ANDES of Universidad de los Andes in Colombia. The
data label reads: Andes-E-11517, Colombia, Caquet?,
Florencia, Quebrada las Doraditas, 01/09/2008. The red
holotype label reads: Holotipo, Andes-E-11517, Helicon-
ius timareta florencia.
Conclusion
Obviously the study of cryptic species has a long history
and, especially with the advent of modern molecular tech-
niques many previously undescribed taxa have been
detected [56]. However, cryptic species often use sensory
modalities that humans do not readily perceive, such as
pheromones [57,58], toxicity resistance [59] and imper-
ceptible song differences [60]. The surprising aspect of this
study is therefore the discovery of cryptic species in well-
studied taxa where speciation is commonly triggered by
bright visual signals. It is increasingly becoming clear that
tropical biodiversity is severely underestimated and that
combining morphological and DNA sequence analysis is
a powerful tool for species discovery. Finally, our mate
and host choice experiments suggest that the sympatric
species in Florencia combine the same ecological differ-
ences known to occur in other parts of the range of H.
cydno and H. melpomene with enhanced pheromonal sig-
nals to compensate for the lack of colour pattern signals.
Our results therefore highlight the fact that speciation is
the combined result of divergence along multiple pheno-
typic axes.
Authors' contributions
NG carried out laboratory work, mating experiments,Page 8 of 11
(page number not for citation purposes)
band area and the distinguishing red line with length 2,65
to 5,92 mm (Figure 1b and Figure 2). The HW also has a
plant choice trials, larvae and adult morphology measures
and description, and behavioural, morphological and
BMC Evolutionary Biology 2008, 8:324 http://www.biomedcentral.com/1471-2148/8/324
Page 9 of 11
(page number not for citation purposes)
East Andean geographical distribution of known H. cydno clade taxaFigure 6
East Andean geographical distribution of known H. cydno clade taxa. On the left from top to bottom: H. t. timareta f. 
contigua, H. t. timareta f. timareta, H. t. timareta f. timareta, H. t. timareta f. peregrina and H. t. timoratus (Lamas 1998). On the right: 
H. heurippa, H. cydno cognate from Rio Pato (undescribed), H. t. florencia, H. tristero. See text for details.
BMC Evolutionary Biology 2008, 8:324 http://www.biomedcentral.com/1471-2148/8/324
genetic analyses. NG and CDJ designed experiments. CS
contributed with mtDNA sequences of Heliconius timareta
from Ecuador and genetic analysis. CDJ helped in data
analysis and, with NG and CS, drafted the manuscript. EB
and ML participated in the design and coordination. ML
conceived the study, did the field observations and
obtained all the specimens from Florencia used in this
study. All authors read and approved the final manuscript.
Additional material
Acknowledgements
We would like to thank Maribel Gonz?lez and Carlos Arias at the Smithso-
nian Tropical Research Institute for laboratory help with microsatellites and 
mtDNA amplification and Fernando Alda for help in microsatellite scoring 
and analysis. We also thank Mathieu Joron and Jesus Mavarez for ongoing 
discussion of cryptic species in the eastern Andes and for the original sug-
gestion that these might represent sub-species of H. timareta. James Mallet, 
Gerardo Lamas and Jean Fran?ois Le Crom for photographs of H. timareta 
from Ecuador and Per? and H. tristero. Ministerio del Ambiente of Ecuador 
for collecting permitions. This work was carried out with grants from the 
Smithsonian Tropical Research Institute (NG), Instituto Colombiano para 
el Desarrollo de la Ciencia y la Tecnolog?a Francisco Jos? de Caldas COL-
CIENCIAS 7155-CO (CS), Biotechnology and Biological Sciences Research 
Council and the Royal Society (CDJ), Banco de la Rep?blica (ML and NG) 
and private donations (ML) from Continautos S.A., Proficol El Carmen S.A., 
Didacol S.A. and F. Arango, Colombia.
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Additional file 1
Individuals used in phylogenetic analyses. Gene accession number and 
locality of alleles and individuals included in the phylogenetic analysis.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2148-8-324-S1.doc]
Additional file 2
Forewing size between H. cydno and H. melpomene. Wild individu-
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