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Zoological Journal of the Linnean Society, 2014, 170, 690–709. With 8 figures
Species boundaries in Philaethria butterflies: an
integrative taxonomic analysis based on genitalia
ultrastructure, wing geometric morphometrics,
DNA sequences, and amplified fragment
length polymorphisms
KIM R. BARÃO1, GISLENE L. GONÇALVES2,3, OLAF H. H. MIELKE4,
MARCUS R. KRONFORST5 and GILSON R. P. MOREIRA6*
1
PPG Biologia Animal, Departamento de Zoologia, Universidade Federal do Rio Grande
do Sul. Avenida Bento Gonçalves, 9500, Bloco IV, Prédio 43435, Porto Alegre, RS 91501-970,
Brazil
2
PPG Genética e Biologia Molecular, Departamento de Genética, Universidade Federal
do Rio Grande do Sul. Avenida Bento Gonçalves, 9500, Porto Alegre, RS 91501-970,
Brazil
3
Instituto de Alta Investigación, Universidad de Tarapacá, Antofagasta 1520, Arica, Chile
4
Departamento de Zoologia, Universidade Federal do Paraná. Caixa Postal 19020, Curitiba, PR
81531-980, Brazil
5
Department of Ecology and Evolution, University of Chicago, Chicago, IL 60637, USA
6
Departamento de Zoologia, Universidade Federal do Rio Grande do Sul. Avenida Bento Gonçalves,
9500, Bloco IV, Prédio 43435, 91501-970, Porto Alegre, RS 91501-970, Brazil
Received 14 October 2013; revised 11 November 2013; accepted for publication 23 November 2013
Neotropical passion-vine butterflies in the tribe Heliconiini (Lepidoptera: Nymphalidae) are a major focus of
research in ecology and evolution because of their diverse, aposematic wing patterns, extensive Müllerian mimicry,
and coevolution with their Passifloraceae host-plants. However, the basic taxonomy of this group, which is essential
to evolutionary ecology research, has been built over the last two centuries using primarily gross morphological
comparisons, with most species identification being based on wing colour pattern variation. For some taxa, such
as the genus Philaethria Billberg, even the most basic information, such as species limits and geographical
distributions, remains uncertain. Furthermore, descriptions of new species, within Philaethria and beyond, have
generally been based on small sample sizes collected from a restricted area of the full geographical distribution.
To address these issues in the genus Philaethria, here we used an integrative taxonomic approach involving both
morphology (genitalia ultrastructure; linear and geometric morphometric analyses of wing shape) and molecular
data (multilocus DNA sequence data and amplified fragment length polymorphisms). Specifically, we tested the
taxonomic validity of two Philaethria species, Philaethria pygmalion and Philaethria wernickei, described in the
literature as having disjunct distributions, corresponding to the Amazon Basin and the Atlantic Rain Forest of
Brazil, respectively. Our analyses revealed that these two Philaethria species cannot be delimited and diagnosed
using metric and nonmetric morphological characters. Furthermore, they occur in sympatry in the Cerrado biome
of central Brazil, and appear to form a latitudinal cline in wing colour variation across their combined distribution.
These results are further supported by limited genetic differentiation and a lack of reciprocal monophyly between
Amazon and Atlantic Rain Forest populations based on DNA sequence data, and unstructured amplified fragment
length polymorphism variation. Our combined results allow us to clarify species-level limits within the genus
Philaethria, whereby we propose that P. pygmalion is conspecific with P. wernickei (new synonym), and reassess
*Corresponding author. E-mail: gilson.moreira@ufrgs.br
690
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 690–709
SPECIES BOUNDARIES IN PHILAETHRIA
691
the spatial range of P. wernickei by providing a refined mapping of its geographical distribution. Beyond clarifying
the taxonomy of Philaethria, our results provide a solid, integrative framework that could be applied to fully
characterize the taxonomy of other species in the Heliconiini and beyond.
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 690–709.
doi: 10.1111/zoj.12118
ADDITIONAL KEYWORDS: genetic variation – heliconian butterflies – latitudinal clines – phylogeography
– species delineation.
INTRODUCTION
Major questions concerning speciation and diversification processes, population management, and conservation depend on well-informed species-level
designations and detailed knowledge of geographical
distributions. Thus, unambiguous taxa delineation is
an essential component of many aspects of ecological
and evolutionary research. Although the discovery
and description of species has long been considered a
major goal of systematic biology, relatively little published work has focused on methodological procedures
for species delimitation (Wiens, 2007). In general,
insect species are still delimited based on morphological characters; however, recent studies have shown
that gross morphological traits alone may fail to
delineate taxa within a given taxonomic group (e.g.
Ross et al., 2010). Particularly in Lepidoptera, closely
related species are often too similar in their morphology to be markedly delineated (Mutanen, 2005;
Mullen, Dopman & Harrison, 2008). In addition,
characters used to describe species are sometimes
continuous variables, and they may even represent
geographical clines; thus, trait values may overlap
between species, making it difficult to distinguish
between intraspecific and interspecific variation. As
an alternative, molecular approaches have recently
been proposed as a means to identify and characterize
species based on DNA sequence data (Tautz et al.,
2002, 2003; Hebert et al., 2003; Chen et al., 2011).
However, it has been argued that variation in DNA
sequence data alone is not enough to assign species
boundaries in some cases, as it may lead to unstable
results (e.g. Meyer & Paulay, 2005; Raxworthy et al.,
2007; Vogler & Monaghan, 2007; Dasmahapatra
et al., 2010; Powell, 2012; Simonsen et al., 2012). Most
recently, new methods for delineating species that
focus on integrative taxonomy have been adopted,
combining morphological, molecular, and other available data (e.g. Dayrat, 2005; Will, Mishler & Wheeler,
2005; Padial & de La Riva, 2010; Padial et al., 2010;
Schwentner, Timms & Richter, 2011; Bornholdt
et al., 2013; Lopez et al., 2013; Tancioni et al., 2013;
Vences et al., 2013). In this study, we applied an
integrative approach to examine the morphologically
similar Neotropical passion-vine butterflies Philaethria wernickei (Röber, 1906) and Philaethria
pygmalion (Fruhstorfer, 1912).
The genus Philaethria Billberg, 1820, has been
placed as one of the most basal lineages within the
tribe Heliconiini (Penz, 1999; Beltrán et al., 2007),
which encompass nine other genera (sensu Lamas,
2004): Agraulis Boisduval & LeConte, [1835], Dione
Hübner, [1819], Podotricha Michener, 1942, Dryadula
Michener, 1942, Dryas Hübner, [1807], Laparus
Billberg, 1820, Neruda J. R. G. Turner, 1976, Eueides
Hübner, 1816, and Heliconius Kluk, 1780. Previous
studies have addressed phylogenetic relationships
amongst these genera, with the greatest emphasis
placed on relationships within Heliconius (e.g.
Michener, 1942; Emsley, 1963, 1965; Brown, 1981;
Brower, 1994; Brower & Egan, 1997; Penz, 1999;
Beltrán et al., 2007). Little attention has been given
to the other genera, making this the first intensive
study using an integrative taxonomic analysis in
Philaethria. Interestingly, patterns of morphological
diversity differ substantially within Heliconiini. For
instance, Heliconius displays a great diversity of wing
colour patterns (see Holzinger & Holzinger, 1994) and
has c. 39 species formally recognized (Lamas, 2004).
In contrast, Philaethria species exhibit remarkable
phenotypic similarity, yet the genus accounts for
ten recognized species (Constantino & Salazar,
2010), often being considered a ‘species complex’
(Suomalainen & Brown, 1984) of difficult interspecific
designations.
In a recent review of Philaethria (Constantino &
Salazar, 2010), three new species and seven new
subspecies were described based on morphological
characters and haploid chromosomal number, and a
diagnostic key based on ventral hind wing coloration
pattern was also proposed. According to this key,
P. wernickei and P. pygmalion are characterized by
having diffuse and continuous submarginal cellular
spots, which separates them from other Philaethria
species. Furthermore, the authors proposed that the
two can be distinguished from each other by the
proportional length between the inner and medial
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 690–709
692
K. R. BARÃO ET AL.
postdiscal bands on the ventral hind wing, and by
morphological aspects of male genitalia. Therefore,
P. wernickei is thought to be characterized by having
an inner postdiscal band as wide as the medial,
whereas it is reduced in P. pygmalion. However, the
conclusions of Constantino & Salazar (2010) were
based on small sample sizes and thus intraspecific
variation in these characters was not examined.
Importantly, Constantino & Salazar (2010) suggested
that P. wernickei and P. pygmalion are allopatric,
with P. wernickei restricted to the Brazilian Atlantic
Rain Forest (higher latitudes) and P. pygmalion to the
Amazon Basin (lower latitudes).
Taxonomic ranks associated with P. wernickei
(Röber, 1906) and P. pygmalion (Fruhstorfer, 1912)
have moved back and forth throughout history.
Philaethria wernickei was originally described as
Metamorpha wernickei based on specimens collected
from southern Brazil (Rio Grande do Sul and Santa
Catarina states), and P. pygmalion as Metamandana
dido pygmalion, based on specimens from Óbidos,
in northern Brazil (Pará state). Seitz (1913) considered P. wernickei a subspecies of Philaethria dido
(Linnaeus). Subsequently, Brown & Mielke (1972)
revalidated P. wernickei, and considered P. pygmalion
as its subspecies (P. wernickei pygmalion). Finally,
using coloration characters of the fifth larval instar in
comparison to those of P. wernickei, Brown & Benson
(1977) ranked P. pygmalion at the specific level.
In a pilot study, we applied the morphological
criteria proposed by Constantino & Salazar (2010) to
diagnose P. pygmalion and P. wernickei in a broad
sample of individuals, including museum specimens
previously identified as either species, from several
parts of Brazil including both lower (Amazon Basin)
and higher (Atlantic Rain Forest) latitudes. In doing
so, we noted that contrary to what was proposed by
Constantino & Salazar (2010), both species were distributed throughout Brazil, not restricted to specific
biomes (Fig. 1A). Furthermore, individuals showed
marked intraspecific variation (Fig. 1C), overlapping
in a continuous distribution for the traits proposed
to distinguish P. pygmalion and P. wernickei. Thus,
we decided to explore further the corresponding taxonomic identities. We hypothesized that they could
represent a single evolutionary lineage with high
intraspecific variation along its broad geographical
distribution.
In this study, we applied an integrative taxonomic
approach utilizing variation in morphological characters (genitalia, wings), geometric morphometrics
(wings), and genetic data [mtDNA and nuclear gene
sequences, amplified fragment length polymorphisms
(AFLPs)] to comprehensively evaluate the taxonomic
status of P. wernickei and P. pygmalion. We compared
variation in morphology and DNA polymorphism to
distributional data from wild-caught and museum
specimens along a latitudinal gradient, using them to
Figure 1. Geographical distributions of Philaethria wernickei and Philaethria pygmalion, and corresponding variation in
male genitalia ultrastructure and ventral hind wing colour. A, shaded areas show distribution ranges proposed by
Constantino & Salazar (2010) for P. wernickei (green) and P. pygmalion (red); green circles and red triangles represent
collection localities of the material analysed in this study. B, variation in valva’s cucullus, external view. C, variation in
the colour pattern of hind wing surface, ventral view.
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 690–709
SPECIES BOUNDARIES IN PHILAETHRIA
test whether P. wernickei and P. pygmalion represent
independent lineages based on at least one of three
criteria: (1) diagnosability, i.e. the appearance of
fixed differences (Cracraft, 1983; Nixon & Wheeler,
1990), (2) monophyly (Donoghue, 1985; de Queiroz &
Donoghue, 1988), and (3) discrete genetic clustering
(Mallet, 1995). In addition, we addressed the spatial
distribution of our observed variation amongst populations in a phylogeographical context. Our results
demonstrate that the use of multiple data sources,
integrated with advanced analytical methods, provides a powerful means to test taxonomic hypotheses.
MATERIAL AND METHODS
MORPHOLOGICAL ANALYSIS
A total of 299 specimens of P. wernickei (N = 211)
and P. pygmalion (N = 88) was surveyed, either wild
caught or obtained from museum collections. In
addition, P. dido (N = 68) and Philaethria diatonica
(Fruhstorfer, 1912) (N = 1) were incorporated as
outgroups in the analysis. The examined materials are
listed in Appendix S1. Sample sites were plotted on a
map (Fig. 1A) constructed using Quantum GIS
WROCLAW v. 1.7 (Quantum GIS Development Team,
2010) with longitude and latitude coordinates compiled from online gazetteers (http://www.fallingrain
.com/world/ and http://splink.cria.org.br/geoloc).
In order to evaluate the diagnosability of the
morphological traits currently used to identify
P. wernickei and P. pygmalion, we focused our attention on male genitalia and wing characters. The
general and ultrastructural morphology of male genitalia were characterized by optical and scanning
electron microscopy (SEM) and also compared to
P. dido, including specimens from a broad distributional range (Appendix S1). The male genitalia
samples were cleaned in a 10% KOH solution, neutralized in acetic acid, and stored in glycerine. Structural details were analysed using a Leica M125
stereomicroscope. Structures selected to be drawn
were previously photographed with a Sony Cybershot DSC-H10 digital camera mounted on the stereomicroscope. Vectorized line drawings were then
created with the software Adobe ILLUSTRATOR CS5,
using digitized images as a guide. Tegumentary
ultrastructure was prepared, photographed, and analysed using a JEOL JSM5800 scanning electron
microscope at the Centro de Microscopia Eletrônica of
Universidade Federal do Rio Grande do Sul, following
the procedure described in Barão & Moreira (2010).
Nomenclature follows Constantino & Salazar (2010)
and Dias, Casagrande & Mielke (2010). Additionally,
for the study of wing pattern variation, digitized
images of dorsal and ventral wing surfaces were used
in linear and geometric morphometric analyses.
693
Photographs were obtained with a digital camera,
Sony Cybershot H20 (5 megapixel resolution, Iso200,
One-Shot, macro function activated). Images were
taken from a standard distance (20 cm) with the aid
of a tripod. Millimetre graph paper served as the
background and scale factor.
Constantino & Salazar (2010) distinguished P.
wernickei from P. pygmalion by the proportional length
between the inner and medial postdiscal bands on the
ventral hind wing. Thus, we measured these traits in
P. wernickei and P. pygmalion, on the ventral side,
using the software ImageJ (Rasband, 2007). Using a
linear morphometric approach, hind wing length was
taken as the distance between the distal end of the
radial sector (A) and the origin of the subcostal (B)
veins (Fig. 2A). The segments ‘D−F’ and ‘E−F’, referring to the inner and medial postdiscal band widths,
were measured on a straight line from ‘B’ and the distal
end of the cubital anterior 1 (C). We tested for the
existence of bilateral asymmetry and sexual dimorphism for these traits and found none (Student’s
t-tests; P > 0.05). Thus, the mean of each specimen was
used for the analysis. A linear regression was performed with the data, classifying them by (1) species;
(2) latitude grades, (3) AB/DE; and (4) DF/EF ratios.
Unless noted otherwise, results are shown in box plots
in order to demonstrate total variation and the corresponding quartiles. Differences between classification
schemes were explored using a linear discriminant
analysis (LDA). A cross-validation procedure was
adopted to estimate the correct classification percentage of LDA, i.e. to evaluate performance of these linear
measures as diagnosable taxonomic characters.
In addition, we digitized landmark data on a total of
165 specimens of P. wernickei (N = 66), P. pygmalion
(N = 31), and P. dido (N = 68) for the geometric
morphometric analysis. For each specimen, twodimensional coordinates of 19 fore wing and 15 hind
wing landmarks were digitized by the same person
(K. R. Barão), using TPSDig, v. 1.4 (Rohlf, 2006).
Corresponding landmark positions and definitions can
be found in Figure 2B, C and Appendix S2, respectively. We tested first for differences in shape amongst
species. In a follow-up analysis, specimens of P.
wernickei and P. pygmalion were classified by latitude
and AB/DE and EF/DF ratios in order to assess how
variation in shape was spatially distributed.
Prior to the statistical analyses, landmark coordinates were superimposed with a generalized leastsquares Procrustes procedure (Dryden & Mardia,
1998), in which wings were treated as independent
variables. Differences in the wing shape inferred from
statistical analyses were visualized through multivariate regression of shape variables on discriminant
axes. As there was no evidence of bilateral asymmetry
or sexual dimorphism (data not shown), the least
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 690–709
694
K. R. BARÃO ET AL.
Figure 2. Location of linear measurements (A) and schematic representation (B, C) of Philaethria wings showing
veins and landmarks adopted in this study. A, hind wing
dorsal and ventral (detail) views, showing measured
vectors. B, fore wing. C, hind wing. See Appendix S2 for
details on morphological definitions of landmarks.
◀
injured wing of each specimen was used. To assess
phenotypic similarity between species and classification schemes of specimens, principal components (PC)
analysis was calculated on the variance−covariance
matrix of generalized least-squares superimposition
residuals. The PCs were used as new shape variables
in order to reduce the dimensionality of the data set
as well as to take independent variables into account
(Baylac & Friess, 2005). Differences in shape amongst
species were evaluated with a multivariate analysis
of variance (MANOVA). In order to test the ability
to discriminate species and specimens classification
schemes, differences in shape amongst groups were
explored by LDA, calculated on PCs (Cordeiro-Estrela
et al., 2006). To compute correct assignment percentages amongst each classification scheme, we used
a leave-one-out cross-validation procedure, which
allows an unbiased estimate of classification percentages (Baylac, Villemant & Simbolotti, 2003).
All statistical analyses were carried out with R v. 2.9
(R Development Core Team, 2009) and the libraries
MASS (Venables & Ripley, 2002), ape (Paradis et al.,
2006), stats (R Development Core Team, 2009), and
ade4 (Dray & Dufour, 2007). Morphometric analyses
were performed with the Rmorph library (Baylac,
2007).
MOLECULAR
EXPERIMENTAL PROCEDURE
DNA was extracted from one-third of the thorax of each
specimen (N = 20 Philaethria, from northern and
southern portions of the distribution in Brazil; N = 1
P. dido; N = 1 P. diatonica) using a DNeasy Blood
and Tissue Kit (Qiagen) and stored in TE 1X buffer
at −80 °C. We amplified partial fragments of the
mitochondrial DNA (mtDNA) cytochrome oxidase
subunit I (COI) gene and three nuclear loci, triosephosphate isomerase (Tpi), wingless (Wg), and tyrosine
hydroxylase (TH) (Table 1). Tpi and TH are both
Z-linked genes whereas Wg is autosomal. Details of
all PCR primers and reaction conditions are provided
in Appendix S3. PCR products were cleaned with
exonuclease I and shrimp alkaline phosphatase, and
sequenced in both 5′ and 3′ directions using the Big
Dye Terminator 3.1 Sequencing Kit (Applied Biosystems). Sequenced products were analysed on an ABI
Prism 3730xl Genetic Analyzer (Applied Biosystems). Chromatograms were edited and aligned using
CodonCode Aligner (CodonCode Corporation, USA)
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 690–709
SPECIES BOUNDARIES IN PHILAETHRIA
695
Table 1. Genetic characterization of populations from northern and southern latitudes of Philaethria wernickei analysed
Neutrality test
Tajima’s
Fu’s
Marker
bp
S
H
Hd
π
Model
D
D
F
FS
COI
Tpi
Wg
TH
1259
496
453
772
15
60
15
8
9
25
7
10
0.82 ± 0.07
0.98 ± 0.01
0.66 ± 0.05
0.78 ± 0.04
0.003 ± 0.0002
0.026 ± 0.0060
0.002 ± 0.0010
0.001 ± 0.0001
GTR + I
HKY + G
K80
GTR
0.56
−1.74
−0.25
−0.74
−0.18
−2.09
−0.66
−1.44
0.03
−2.34
−0.63
−1.43
−0.14
−9.21
−2.18
−4.11
bp, base pairs; S, number of segregating sites; H, number of haplotypes; Hd, haplotype diversity; π, nucleotide diversity;
Model, substitution model; COI, cytochrome oxidase subunit I; Tpi, triose-phosphate isomerase; Wg, wingless; TH, tyrosine
hydroxylase; GTR + I, general time reversible plus invariant sites; HKY + G, Hasegawa-Kishino-Yano plus Gamma
distributed; K80, Kimura 2-parameters.
and nucleotide sequences were visually inspected for
miscalls. Heterozygous sites in nuclear loci were identified when two different nucleotides were present
at the same position in electropherograms of both
strands, with the weakest peak reaching at least 25%
of the strongest signal. When two or more heterozygote
sites were identified in the same marker, the gametic
phase of the variants was determined computationally
by using PHASE 2.1 (Stephens, Smith & Donnelly,
2001; Stephens & Donnelly, 2003). All sequences have
been deposited in GenBank (Table 2).
In order to more fully evaluate genome-wide patterns of genetic differentiation, we also genotyped 23
specimens (12 from northern and 11 from southern
latitudes) with AFLP markers. We used the Applied
Biosystems AFLP Plant Mapping Kit to generate
markers and fragments were separated with an ABI
Prism 3130xl Genetic Analyzer. Four selective primer
combinations were used to generate fragments: EcoRIACT/MseI-CAT, EcoRI-ACT/MseI-CTG, EcoRI-ACA/
MseI-CAT, and EcoRI-ACA/MseI-CTG. We sized and
scored AFLP fragments using ABI GENEMAPPER
software (www.appliedbiosystems.com).
PHYLOGENETIC
AND GENOTYPING ANALYSIS
Sequence data were used to test the monophyly of
P. wernickei and P. pygmalion using P. dido and
P. diatonica as outgroups. Data partition homogeneity tests (Farris et al., 1995), implemented in
PAUP*4.0b10 (Swofford, 2002), were carried out to
determine whether different genes (CoI, Tpi, Wg, TH)
were congruent and could be analysed together. Separated and combined data sets were then analysed
using MRMODELTEST (Posada & Crandall, 2001;
Nylander, 2004) to determine the sequence evolution
model that best fit the data. The best fit model,
chosen based on the Akaike information criterion, was
employed to perform Bayesian phylogenetic reconstruction using MRBAYES 3.1.2 (Huelsenbeck &
Ronquist, 2001; Ronquist & Huelsenbeck, 2003). The
Bayesian analysis was performed with four runs and
106 generations, every 100th of which was sampled.
After confirming that likelihood values had stabilized
prior to the 10 000th generation, the first 10% of
sampled trees were discarded. Of the remaining 9000
trees, a majority rules consensus tree was calculated.
The posterior probability was used as node support.
The consensus tree was visualized and processed
with FIGTREE 1.3 (Rambaut, 2009). The Kimura
two-parameter model (K2P; Kimura, 1980) was used
to calculate the genetic divergence between Amazon
Forest (northern) and Atlantic Rain Forest (southern) populations, with 1000 bootstrap replicates.
Haplotype networks were constructed for each gene
using the median-joining approach implemented in
the software NETWORK 4.6 (Bandelt, Forster &
Röhl, 1999). In order to infer hierarchical population
structure, analyses of molecular variance (AMOVAs)
were performed considering both genetic distances
between haplotypes and their frequencies, using
ARLEQUIN 3.5 (Excoffier & Lischer, 2010).
For AFLP data, STRUCTURE 2.2 (Pritchard,
Stephens & Donnelly, 2000; Falush, Stephens &
Pritchard, 2007) was used to estimate the number of
genetic groups (K) and the distribution of individuals
amongst these groups. An admixture model with
correlated allele frequencies was used to estimate the
number of genetic clusters, ranging from K = 1 to
K = 4. The use of the admixture model allows the
number of genetic clusters to be estimated and the
ability to detect historical population admixture
(Ostrowski et al., 2006; Falush et al., 2007). For these
analyses, we used a burn-in period of 20 000 generations followed by 105 generations of data collection.
Additionally, fixation indices (FST) were calculated,
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 690–709
ID
01°00′20′′, 50°12′28′′
54°42′30′′
54°42′30′′
54°42′30′′
54°42′30′′
46°10′38′′
46°10′38′′
46°10′38′′
46°10′38′′
46°10′38′′
43°40′11′′
43°40′11′′
49°05′09′′
49°05′09′′
49°16′23′′
49°16′23′′
49°22′43′′
48°50′44′′
50°35′01′′
BR: PA, Ilha de Marajó
02°26′35′′,
02°26′35′′,
02°26′35′′,
02°26′35′′,
06°57′50′′,
06°57′50′′,
06°57′50′′,
06°57′50′′,
06°57′50′′,
19°52′48′′,
19°52′48′′,
24°58′28′′,
24°58′28′′,
25°25′40′′,
25°25′40′′,
26°15′01′′,
26°18′16′′,
29°26′53′′,
−
PA, Santarém
PA, Santarém
PA, Santarém
PA, Santarém
MA, Fortaleza dos Nogueiras
MA, Fortaleza dos Nogueiras
MA, Fortaleza dos Nogueiras
MA, Fortaleza dos Nogueiras
MA, Fortaleza dos Nogueiras
MG, Caeté
MG, Caeté
PR, Tunas do Paraná
PR, Tunas do Paraná
PR, Curitiba
PR, Curitiba
SC, São Bento do Sul
SC, Joinville
RS, São Francisco de Paula
Honduras
BR:
BR:
BR:
BR:
BR:
BR:
BR:
BR:
BR:
BR:
BR:
BR:
BR:
BR:
BR:
BR:
BR:
BR:
Locality
JQ245450
JQ245449
JQ245461
JQ245460
JQ245455
JQ245456
JQ245452
JQ245453
JQ245451
JQ245457
JQ245454
JQ245465
JQ245464
JQ245462
JQ245466
JQ245469
JQ245463
JQ245467
JQ245468
JQ245470
COI
JF267330
−
JF267340
JF267339
JF267336
JF267337
JF267333
JF267334
JF267332
JF267338
JF267335
JF267343
JF267342
−
JF267344
JF267347
JF267341
JF267345
JF267346
JF267348
Wg
GenBank accession number
JQ978718
JQ978717
JQ978727
JQ978726
−
JQ978723
JQ978719
JQ978720
JQ978721
JQ978724
JQ978722
JQ978732
JQ978731
JQ978728
JQ978733
JQ978735
JQ978730
JQ978734
JQ978729
JQ978736
TH
JQ978738
JQ978737
JQ978747
JQ978746
−
JQ978743
JQ978740
JQ978741
JQ978739
JQ978744
JQ978742
JQ978751
JQ978750
JQ978748
JQ978752
JQ978755
JQ978749
JQ978753
JQ978754
JQ978756
Tpi
COI, cytochrome oxidase subunit I; TH, tyrosine hydroxylase; Tpi, triose-phosphate isomerase; Wg, wingless; BR, Brazil; MA, Maranhão; MG, Minas Gerais; PA,
Pará; PR, Paraná; RS, Rio Grande do Sul; SC, Santa Catarina.
Ingroup
Philaethria wernickei
LMCI 94-9
LMCI 94-8
LMCI 94-1
LMCI 104-3
LMCI 117-1
LMCI 117-2
LMCI 117-3
LMCI 117-4
LMCI 117-5
LMCI 43–60
LMCI 43-2
LMCI 52-15
LMCI 52-18
LMCI 23-10
LMCI 21-10
LMCI 110-14
LMCI 111-10
LMCI 27-22
Outgroup
Philaethria diatonica
RH09359
Philaethria dido
LMCI 100-33
Taxon
S (latitude),
W (longitude)
Table 2. Specimens of Philaethria used in the molecular analysis, with sample localities and GenBank accession numbers
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SPECIES BOUNDARIES IN PHILAETHRIA
comparing southern and northern populations, using
ARLEQUIN. Mantel tests, also implemented in
ARLEQUIN, were used to test each species for isolation by distance; i.e. to test for a correlation between
pairwise genetic and geographical distances.
RESULTS
MALE
GENITALIA AND HIND WING VARIATION
Using the diagnostic criteria proposed by Constantino
& Salazar (2010), we found that spatial distributions
of P. pygmalion and P. wernickei were not restricted to
the Amazon Basin (lower latitudes) and Atlantic Rain
Forest (higher latitudes), respectively. Rather, the distribution of each was much broader, including several
parts of central Brazil (Cerrado biome), where they
appear to occur in sympatry (Fig. 1A). Both species
also exhibited marked intraspecific variation in their
medial postdiscal bands on the ventral hind wing and
had a continuous overlapping distribution for the
trait values proposed above (Fig. 1B, C); thus, identification of intermediate specimens based upon such
criteria is unstable.
As expected, P. wernickei and P. pygmalion genitalia were distinct from those of P. dido (Appendix S4).
In the latter, the harpe reached the uncus median
part and the round ampulla bore microspines at distal
and ventral regions in external view; it also had a
fultura inferior with truncated tip, without ornamentations. However, we could not find any trend regarding the subtle, unstable variation found between
genitalic structures of P. wernickei and P. pygmalion.
For both species, tegumen presented setae at the
median and distal areas (Fig. 3A–C). At the valvae,
harpe was curved forward (Fig. 3A–C), surpassing
a little the uncus tip; ampulla shape was variable,
ranging from hollow to round (Fig. 1B), the external
and internal views being covered by similar
microtrichia (Fig. 3D–I). The fultura inferior was
wing-shaped with a pointed dorsal tip, with angle
aperture varying in size and bearing similar
interspecific microspines (Fig. 3J, K). The distal
uncus region presented microtrichia with variable
shape.
Additionally, hind wing length did not differ
statistically between P. wernickei and P. pygmalion
specimens (Student’s t-test, P = 0.57; Fig. 4A). The
postdiscal band and wing length ratio was significantly
different between species (Student’s t-tests; P < 0.001;
Fig. 4B) and the inner and medial postdiscal band
ratio was also significantly different between them
(Student’s t-test, P = 0.049; Fig. 4C). However, trait
values for all measurements showed substantial
overlap across the geographical distribution, and this
appeared to be largely clinal variation associated with
latitude (Fig. 4D–F).
697
The PC analysis of fore and hind wing shape
variables showed differences between P. dido and
the other two species (Appendix S5), with essentially
complete overlap between northern and southern
populations of P. wernickei and P. pygmalion (Fig. 5A,
B). When shape residuals for both wings were taken
into account, PC1 was not able to distinguish between
specimens from the Amazon and those from the
Atlantic Rain Forest, either when they were classified
by latitude (Fig. 5C, D), postdiscal length (Fig. 5E, F)
or by inner and medial postdiscal band ratio (Fig. 5G,
H). PC2 was also unable to distinguish species.
The correct classification percentage by LDA was
90%, when considering P. wernickei and P. pygmalion
as one group, in which all P. dido specimens were
correctly identified. The correct classification percentage of specimens between P. wernickei and
P. pygmalion from south and north was 77.32%,
in which 12 and ten specimens, respectively, were
misclassified.
As the geometric morphometrics analyses and SEM
results based on wing traits and genitalia failed
to detect the existence of any fixed morphological
differences between north and south specimens of
P. wernickei and P. pygmalion, we pooled the samples
of these two species and explored the potential for
latitudinal variation. A linear morphometric analysis,
in which specimens were classified according to
latitudinal classes, showed that hind wing length
(Fig. 4E) decreased with the increase of latitude (y = −0.0113x + 3.2093; r2 = 0.1581; P < 0.001;
N = 288), whereas the AB/DE (y = 0.0602x + 4.5688;
R2 = 0.4944; P < 0.0001; N = 288) and the EF/DF
(y = 0.0064x + 0.4377; R2 = 0.1164; P < 0.001; N = 288)
ratios increased with latitude (Fig. 4F, G), thus demonstrating the existence of latitudinal clines. Interestingly, variation in AB and EF/DF overlapped
between all latitude classes (Fig. 4E–G). The correct
classification percentages by LDA based on the latitude classes for such parameters were 37, 44, and
31%, respectively (Appendix S6).
Classification of specimens by latitude (Fig. 5C, D),
wing, and postdiscal length band ratio (AB/DE)
(Fig. 5E, F) or inner and medial postdiscal band ratio
(EF/DF) (Fig. 5G, H) did not recover any particular
group (Fig. 5C–H). In this case, the correct classification percentages by LDA were 56.7 and 65.97% for
latitude, 65.98 and 67.01% for AB/DE, and 51.5 and
42.3% for EF/DF, for fore and hind wings, respectively
(Appendix S7).
PHYLOGENETIC
RELATIONSHIPS AND
GENETIC DIFFERENTIATION
Our mitochondrial sequence alignment consisted
of 1259 bp, of which 108 (8.6%) were variable sites
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K. R. BARÃO ET AL.
Figure 3. Male genitalia of Philaethria wernickei and Philaethria pygmalion. A, P. wernickei, lateral view. B,
P. pygmalion, lateral view. C, schematic representation of generalized genitalia for both, in lateral view. D, F, H, J,
scanning electron micrographs of P. wernickei; E, G, I, K, scanning electron micrographs of P. pygmalion. D, E, ampulla
external view. F, G, ampulla internal view. H, I, ampulla ornamentation in detail. J, K, fultura inferior distal end. Scale
bars = 150, 30, and 100 μm, for D–G, H–I, and J–K, respectively.
(Table 1). Length variation (indels) between aligned
regions of mtDNA sequences was not observed. In
addition to mtDNA, 1721 bp of nuclear sequence data
(Tpi, Wg, and TH) were analysed (Table 1). Tpi presented the most variable sites amongst these loci, of
which 60 (12%) were informative. Seven indels were
observed at this locus. Additionally, higher nucleotide
and haplotype diversity were observed in this
segment (Table 1). Less variability was found in the
Wg and the TH introns, in which 15 (3.3%) and eight
(1%) variable sites were observed, respectively. In
addition, low nucleotide and haplotype diversity were
found in these two markers (Table 1).
Phylogenetic reconstruction also used mtDNA and
nuclear sequence data jointly, as the partition homogeneity test revealed nonsignificant heterogeneity
(P = 1). The best-fit model chosen to describe the
evolution of this combined data set was the general
time reversible + I model (Yang, 1994).
Nuclear gene trees all resulted in a similar topology, placing specimens from northern and southern
regions in the same well-supported clade (Fig. 6). In
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SPECIES BOUNDARIES IN PHILAETHRIA
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Figure 4. Linear variation in hind wing size and medial postdiscal bands for Philaethria wernickei and Philaethria
pygmalion (left column), and in relation to latitude when samples from the two species are combined (right column). A,
D, hind wing length. B, E, hind wing length/postdiscal band ratio (AB/DE). C, F, inner and medial postdiscal band ratio
(EF/DF). See Fig. 2A for details on wing position of corresponding measurements. Numbers above boxes indicate the
number of specimens measured in each class.
comparison, mtDNA and the consensus phylogeny
(Fig. 7) yielded an internal, strongly supported clade
containing all southern specimens. However, in these
trees, northern and southern specimens were not
reciprocally monophyletic. When we evaluated the
genetic differentiation based on K2P (%) distance and
the fixation index (FST) amongst populations from the
Amazon Forest (northern) and Atlantic Rain Forest
(southern), we also observed, overall, less divergence
for nuclear genes, particularly considering FST (Tpi =
0.3%, FST 0.04, P < 0.05; Wg = 0.2%, FST 0.06, P > 0.05;
TH = 0.2%, FST 0.34, P > 0.05) in relation to mtDNA
(1%, FST 0.85, P < 0.05). A hierarchical analysis of
variance (AMOVA) did not show significant genetic
differentiation between northern and southern populations studied based on nuclear markers, but did find
strong differentiation for COI data (variation allocated amongst groups = 85.87%, P < 0.05). AMOVA
based on Tpi, Wg, and TH nuclear markers provided
evidence that most of the variation occurred within
populations (95.7, 93.8, 65.4%, respectively; P > 0.05
for all comparisons).
Intraspecific genealogy analysis showed nine
haplotypes based on mtDNA data; none of them were
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SPECIES BOUNDARIES IN PHILAETHRIA
701
Figure 5. Two first axes of the principal component (PC) analysis on shape residuals for fore (left column) and hind (right
column) wings of Philaethria dido, Philaethria wernickei, and Philaethria pygmalion. A, B, wing shape variation for
P. dido (d, grey), P. wernickei (w, purple), and P. pygmalion (p, green). C–H, wing shape variation in P. wernickei. C, D,
specimens classified by latitude. E, F, specimens classified by postdiscal length. G, H, specimens classified by inner and
medial postdiscal band ratio. Shape variation is indicated next to each axis, where the dashed line represents the shape
at minimum values and the solid line represents the shape at maximum values.
◀
Figure 6. Evolutionary relationships of Philaethria based on DNA sequences from specimens of Philaethria wernickei
(southern population; Atlantic Rain Forest) and individuals previously described as Philaethria pygmalion (northern
population; Amazon Forest), depicted by the green shading (grey in print version). Philaethria diatonica and Philaethria
dido were used to root the tree. Purple (grey) circles represent individuals from the Atlantic Rain Forest and black
triangles indicate samples from the Amazon Basin. A, consensus Bayesian tree based on mitochondrial (cytochrome
oxidase subunit I, Co-I) and nuclear [triose-phosphate isomerase (Tpi), wingless (Wg), and tyrosine hydroxylase (TH)] DNA
sequences. Posterior probabilities are shown above branches. Bootstrap node support based on maximum likelihood
analysis is indicated below branches. Asterisks indicate node support lower than 70%. B, Median-joining network based
on mtDNA and nuclear loci sequence data describing the relationship between haplotypes (purple indicates southern
population, and black, northern population). Nucleotide substitutions are shown on the branches as small transverse bars.
Circle size is proportional to haplotype frequency.
shared between northern and southern populations (Fig. 6B; Table 1). In nuclear loci at least one
haplotype was shared between individuals from both
regions.
Bayesian clustering with STRUCTURE, based on
68 polymorphic AFLP loci, revealed only one genetic cluster, despite the distinct population sources
(Amazon Basin and Atlantic Rain Forest) (Fig. 8).
Comparison of the −ln likelihood values vs. K
revealed that increasing K yielded a better fit to the
data but clustering with K = 2–4 did not suggest any
population subdivision (Fig. 8). FST estimates based
on AFLP data did not show significant genetic differentiation between populations within the range of
< 10°S and > 20°S (0.03, P > 0.05), except for the com-
parison over the greatest distance (between 0°S and
25°S; 0.06, P < 0.01). Specimens from sites between
20°S and 25°S did not show significant genetic structure (FST = 0.001; P > 0.05).
DISCUSSION
THE
INTEGRATIVE APPROACH OF
SPECIES DELINEATION
In this study, we used a combination of morphological,
morphometric, and genetic data to show that there is
no support to recognize specimens of P. pygmalion
from north Brazil and P. wernickei from south Brazil as
two distinct evolutionary lineages. The corresponding
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K. R. BARÃO ET AL.
Figure 7. Multilocus consensus Bayesian tree based on
cytochrome oxidase subunit I (Co-I), triose-phosphate
isomerase (Tpi), wingless (Wg), and tyrosine hydroxylase
(TH) sequences from specimens of Philaethria wernickei
(Atlantic Rain Forest, purple circles) and individuals previously described as Philaethria pygmalion (Amazon
Forest, black triangles) depicted by the green shading (grey
in print version). Philaethria pygmalion and Philaethria
dido were used to root the tree. Posterior probabilities are
shown above branches and bootstrap node support based on
maximum likelihood analysis is indicated below branches.
Asterisks indicate node support lower than 70%.
synonym is herein thus proposed [P. wernickei (Röber,
1906) = P. pygmalion (Fruhstorfer, 1912)]. Such a
synonym was highly supported not only by the absence
of fixed diagnostic traits but also by a monophyletic
clade including individuals from populations from the
Amazon and the Atlantic Rain Forest of Brazil that
shows very little genetic differentiation.
Butterflies in the tribe Heliconiini have been collected intensively across the Neotropics for well over
a century and their alpha taxonomy has been worked
out during this time (Brown, 1981; Penz, 1999;
Moreira & Mielke, 2010). Based on this extensive
history, the taxonomy of this group is thought to be
very well characterized, but is that really the case?
With the exception of Eueides and Heliconius, the rest
of the group has received minimal study and basic
knowledge, such as species limits and geographical
distributions, remains uncertain in some cases.
Furthermore, certain lineages have been elevated to
the species level based on very slight morphological
differences, primarily related to wing colour. This is
the case in the genus Philaethria, in which there
is conspicuous similarity in wing colour pattern
amongst species (Suomalainen & Brown, 1984; Brown
et al., 1992; Constantino & Salazar, 2010). Apart
from the studies on karyotype variation amongst
Philaethria species by Suomalainen & Brown (1984),
which showed a remarkable diversity in the genus
(haploid number varying from 12 to 88), no additional
criteria other than colour pattern variation have been
used to distinguish species (Constantino & Salazar,
2010). The genitalia of Philaethria species are very
similar in shape, and thus are not useful to key out
species (Constantino & Salazar, 2010). In the case of
P. wernickei and P. pygmalion however, chromosome
number is not a useful character to distinguish them
as both putative species have a haploid count of 29
(Suomalainen & Brown, 1984).
Our findings suggest that the taxonomic status
within Philaethria should be reviewed, in particular
regarding the nine subspecies described in Constantino & Salazar (2010). Living organisms generally fall into largely discrete groups, recognizable
by differences in morphology and/or other traits
(Monaghan et al., 2005). However, in some cases these
subdivisions may be overestimated because diagnostic traits are not consistent. Additionally, biologically
distinct species present difficulties, as their delineation can depend on the evaluation of complex and
variable traits. The accuracy of species delineation
also depends on the degree of sampling, as local
variation may affect the conclusions about population
differences (Davis & Nixon, 1992). Thus, we suggest
that to clarify classification within Philaethria, one
should employ an integrative approach combining
morphological, molecular, and other available data, as
we have done here. In addition, as clines may occur in
this case, not only type material should be considered,
but also a representative number of samples for each
species from a broad area of their distributions.
WING
AND GENITALIA MORPHOLOGY AS
DIAGNOSTIC TRAITS
As hypothesized, we were not able to distinguish
between specimens of P. wernickei from the north
(previously considered as P. pygmalion) and south of
the range using morphological traits. The minor differences in male genitalia described by Constantino &
Salazar (2010) were not evident in the broader data
set that we compiled, even under SEM. This is
perhaps surprising, given that genitalia morphology
is considered one of the most important and useful
species-diagnostic characters in insect taxonomy
(see Tuxen, 1970), and thus routinely used in insect
species-level determination.
The Lepidoptera wing is as important in systematics as the male genitalia. Its colour and venation
patterns are widely used to determine species (e.g.
Mielke, Austin & Warren, 2008; Moreira & Mielke,
2010; Mitter et al., 2011). In the case of Philaethria,
the diagnostic character is based upon the coloration
of the ventral hind wing margin (see Constantino &
Salazar, 2010); P. wernickei and P. pygmalion were
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SPECIES BOUNDARIES IN PHILAETHRIA
703
Figure 8. STRUCTURE-based clustering of Philaethria wernickei individuals from low (0–10°S) to high (20–25°S)
latitudes (north and south populations, respectively) based on amplified fragment length polymorphism loci. Each
individual is represented by a vertical line divided into segments of different colour that represent genetic clusters (K)
from 1–4.
thought to be separated by the length of the inner and
medial postdiscal bands on the ventral hind wing
surface. However, our data showed remarkable variation between them in these characters in such a
way that, by forming a cline, one can clearly recognize
specimens from the extremes of the variation, but
not the intermediate forms, because they overlap in
all colour traits analysed here. Similarly, Prieto,
Munguira & Romo (2009) tried to distinguish two
Cupido species (Lepidoptera: Lycaenidae) by the hind
wing linear morphometry, but were not able because
the traits used were highly variable.
A great variety of biological processes may result
in shape differences amongst individuals or species.
Such differences may signal different functional roles
played by the same parts, different responses to the
same selective pressures, and differences in processes
of growth and morphogenesis (Zelditch et al., 2004).
Geometric morphometric analyses are very sensitive methodological tools, which can detect small
intraspecific differences in shape (e.g. Soto, Hasson &
Manfrin, 2008; Soto et al., 2008; Jorge et al., 2011),
including the existence of hybridizing comimetic
subspecies in the derived genus Heliconius (Mérot
et al., 2013) and within the primitive genus Dione
(D. Massardo, Universidade Federal do Rio Grande
do Sul, unpubl. data). Using these tools, we were
able to distinguish P. dido from P. wernickei and
P. pygmalion, but we could not separate P. wernickei
and P. pygmalion. The PC and linear discriminant
analyses did not distinguish between P. wernickei and
P. pygmalion, for either the fore or the hind wing.
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K. R. BARÃO ET AL.
The most recent taxonomic decision to separate
P. wernickei from P. pygmalion was based on fifth
instar larval coloration (Brown & Benson, 1977).
Barão & Moreira (2010) found considerable variation in that trait for P. wernickei, which overlapped
in those structures thought to separate it from
P. pygmalion. Intraspecific variation in Lepidoptera
larval coloration is known for other species (e.g.
Kaminski, Dell’Erba & Moreira, 2008; Brower, 2010).
It can be affected by host plants and abiotic conditions, and thus may not always serve as a useful
guide for taxonomic decisions.
SPECIES
DELINEATION THRESHOLD AND GENE FLOW
Recently, several studies have shown that a specific
‘barcode’ region of the mitochondrial gene COI is
useful to identify predefined species (e.g. Hebert
et al., 2003, 2004; Hogg & Hebert, 2004; Vences et al.,
2005; Smith et al., 2006). It has been suggested that
approximately 1–2% sequence divergence at the
barcode region is consistent with intraspecific diversity, with values above this threshold indicative of
‘phylogroups’ or separate species (Avise & Walker,
1999). In the case of Philaethria, we found a genetic
divergence of approximately 1% in the COI barcode
region between northern (putative P. pygmalion)
and southern (P. wernickei) samples. The distance
between these specimens and other species of
Philaethria (P. dido and P. diatonica) is 5%.
Furthermore, our phylogenetic reconstruction did
not yield reciprocally monophyletic clades for specimens from the Amazon Basin and the Atlantic
Rainforest. This result reinforces our interpretation
that only one evolutionary lineage should be considered hereafter along a broad distributional area:
P. wernickei. The mtDNA data indicated slight
phylogeographical structure, as an internal clade
including specimens from the south was supported.
The north and south populations analysed did not
show shared mitochondrial haplotypes, but nuclear
loci haplotypes were widespread in both regions. The
north−south shift of mitochondrial haplotypes might
indicate female philopatry (e.g. Girman et al., 2001),
which should thus be carefully evaluated by increasing collection sites from both extremes of the
distribution and intermediate ones. Alternatively, it
could represent an isolation by distance effect, as
P. wernickei occupies a broad range, including more
than 4000 km from north to south of the distribution,
and geographical population structure is expected.
As pointed out by Brown & Mielke (1972), northern
and southern populations of P. wernickei intergrade
in the gallery forests of the Cerrado biome of central
Brazil. Philaethria butterflies are well recognized as
strong fliers when compared to Heliconius species,
which in general show roosting behaviour and have
small home ranges. Gallery and deciduous forests
located in this Chacoan subregion putatively function
as past and present bridges between the Amazonian
and Paraná subregions for a variety of organisms,
including small forest mammals (Costa, 2003).
Furthermore, Moreira et al. (2011) demonstrated that
the southernmost forests of Brazil act as a transition
zone, which, in conjunction with the Chaco province,
maintains contact between passion vine populations
from the Amazon and Atlantic region. In particular,
Passiflora suberosa Linnaeus and Passiflora caerulea
Linnaeus, passion vine species known as hosts for
P. wernickei (Brown & Mielke, 1972), are known to
span this range between biogeographical zones. Thus,
the existence of effective gene flow between northern
and southern extant populations of Philaethria
was expected. The AFLP results further corroborate
this hypothesis, as levels of gene flow were slightly
reduced between the extremes of the distribution, but
higher amongst intermediate populations. Moreover,
STRUCTURE-based clustering did not show differences amongst specimens from the Amazon Basin and
Atlantic Rain Forest.
INTRASPECIFIC
VARIATION OF
P. WERNICKEI
We found variation in all data sets that we examined
for P. wernickei, each with a different geographical
pattern. First, there was no geographical structure in
hind wing shape. Second, two wing size gradients
were observed throughout the range. In addition,
distinct patterns of genetic structure were found. The
quantification of P. wernickei hind wing shape variation along its geographical distribution showed that
there is no differentiation amongst individuals of different localities regardless of how we classified specimens. Thus on the one hand, shape variation appears
to be random across the range of P. wernickei. On the
other, wing size was negatively correlated with latitude, whereas length of the postdiscal band on the
dorsal surface, and the inner and medial postdiscal
bands on the ventral surface, were positively correlated with latitude.
Size variation between populations generally
depends on environmental conditions (Alibert et al.,
2001), and body size frequently varies along latitudinal gradients (Brakefield, French & Zwaan, 2003);
for example, body size in many animals is known
to increase with increasing latitude (Endler, 1977;
Partridge & French, 1996; Gilchrist et al., 2004). In
contrast, we found that hind wing size of P. wernickei
showed the opposite pattern, decreasing in size with
increasing latitude. This inversion of the classic size
cline has been observed for some ectotherms (e.g.
Roff, 1980; Nylin & Svard, 1991; Mousseau, 1997;
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SPECIES BOUNDARIES IN PHILAETHRIA
Blanckenhorn & Demont, 2004; Bidau & Martí,
2007). A positive correlation often exists between
developmental time and body size; thus, a potential
explanation for these inverted clines is the interaction
between the length of the growing season and the
time available to the insects for development. In
butterflies in particular, adult size is strongly influenced by host plants used as larval food (Rodrigues &
Moreira, 2002, 2004); thus, longer growing seasons at
lower latitudes could contribute to larger body size in
these areas.
CONCLUSION
This study investigated whether P. wernickei (Röber,
1906) and P. pygmalion (Fruhstorfer, 1912), two traditionally recognized taxa of nymphalid butterflies
(Lamas, 2004), are valid species according to specific
criteria (diagnosability, monophyly, and genetic
clustering). We evaluated their taxonomic status
using an integrative approach that combined morphology, morphometrics, and molecular data. These
taxonomic units were sampled throughout a broad
distributional area, using wild-caught specimens,
in order to evaluate the intraspecific variation
of morphological traits and genetic variability. We
found no consistency in the previously proposed
diagnostic traits (wing colour and male genitalia)
to delimit the passion-vine butterflies P. wernickei
and P. pygmalion. In addition, our phylogenetic
reconstruction did not reveal reciprocal monophyly
between the putative taxa. Finally, we found very low
levels of genetic differentiation between these previously described taxonomic units. Using the species
delineation criteria that we considered in this study,
we found evidence for only one valid lineage and we
propose that P. pygmalion should now be treated as a
synonym of P. wernickei. Our findings demonstrate
the utility of large sample sizes and an integrative
taxonomic approach when investigating species
boundaries. These important lessons extend well
beyond our specific investigation of nymphalid
butterflies and will be broadly useful for future taxonomic and biogeographical studies.
ACKNOWLEDGEMENTS
We are grateful to the following insect collection curators for making available specimens for this study:
Fernando Meyer (MAPA), Gervásio Carvalho (MCTP),
Orlando Silveira (MPEG), Jacques C. Jauffret
(KAGLESI), Marcelo Duarte (MZSP), and Miguel
Monné (MNRJ). Thanks are also due to Darli
Massardo (UFRGS) and Eduardo Carneiro (UFPR) for
helping in part with the wing photography. Ana Kristina Silva (UFRGS), Carlos Mielke, Eduardo Carneiro,
705
Diego Dolibaina and Fernando Dias (UFPR), Gilberto
Albuquerque (UENF), Fernando Campos (UFMG),
and Patricia Lopes (UFPA) assisted with field collection of specimens used in the molecular analyses. We
are also grateful to the staff members of the Centro de
Microscopia Eletrônica of UFRGS for the use of facilities and assisting with scanning electron microscopy
analyses. We also acknowledge Augusto Ferrari and
Luiz A. Campos (UFRGS), and Taran Grant (USP) for
fruitful comments made on an early version of the
manuscript. The financial support for this study came
in part from a CAPES Master Fellowship granted
to K. R. Barão. Molecular work was supported by
NSF grant DEB-1316037 to M. R. Kronforst. G. L.
Gonçalves and G. R. P. Moreira were supported by
CNPq grants (156153/2011-4 and 309676/2011-8,
respectively).
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
Appendix S1. Philaethria specimens used in the morphological, morphometric, and genetic analyses, listed per
locality and institution.
Appendix S2. Morphological definition of fore and hind wing landmarks depicted in Figure 2B, C.
Appendix S3. Description of primers and conditions used in gene amplification.
Appendix S4. Male genitalia of Philaethria dido. A, schematic representation of generalized condition, lateral
view. B, C, scanning electron micrographs of ampulla in internal view and distal end of fultura inferior in
ventral view, respectively.
Appendix S5. Multivariate analysis of variance table of shape variables (non null principal components)
amongst Philaethria species.
Appendix S6. Tables with assignments of Philaethria wernickei specimens by latitude classes on linear
measurements, obtained by using a linear discriminant analysis followed by a leave-one-out, cross-validation
procedure. A, wing length (AB); B, wing length and postdiscal ratio (AB/DE); C, inner and medial postdiscal
ratio (EF/DF).
Appendix S7. Tables with assignments of Philaethria wernickei specimens by latitude classes on shape,
obtained by using a linear discriminant analysis followed by a leave-one-out, cross-validation procedure. A,
latitudinal classes based on shape; B, wing and postdiscal band length ratio classes based on shape; C, inner
and medial postdiscal band ratio classes based on shape.
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 690–709