Neotrop Entomol (2012) 41:472484

DOI 10.1007/s13744-012-0071-7

SYSTEMATICS, MORPHOLOGY AND PHYSIOLOGY

Taxonomy of Mechanitis (F.) (Lepidoptera: Nymphalidae) from the West

Colombian Andes: an Integrative Approach
CE GIRALDO, SI URIBE
Grupo de Investigacin en Sistemtica Molecular, Lab de Biologa y Sistemtica de Insectos, Univ Nacional de Colombia, Sede Medelln,
Medelln, Colombia

Keywords
Cryptic species, DNA barcoding, host plants,
immature stages
Correspondence
CE Giraldo, Grupo de Investigacin en
Sistemtica Molecular, Lab de Biologa y
Sistemtica de Insectos, Univ Nacional de
Colombia, Sede Medelln, Calle 59A No
63-20. Bloque 16, Medelln, Colombia;
cegiral0@gmail.com
Edited by Fernando L Cnsoli ESALQ/USP
Received 16 January 2012 and accepted 3
July 2012
Published online 24 August 2012
* Sociedade Entomolgica do Brasil 2012
Abstract
Species identification in the butterfly genus Mechanitis (F.) (Lepidop-
tera: Nymphalidae) becomes difficult when it is based only on wing color
patterns, a common practice in butterfly taxonomy. Difficulties in Mech-
anitis taxonomy are related to the widespread mimicry and polymor-
phism among species belonging to this genus. Species recognition and
inventories of Mechanitis genus in geographic areas as the Andean
region of Colombia are of particular interest and the use of more than
one character for taxonomic identification is desirable. In this study, we
included morphological, ecological, and mitochondrial DNA data to
identify the occurring species in this region. Species of Mechanitis were
studied from ecological, morphological, and molecular perspectives
considering host plant identification, oviposition behavior, and life
cycles under laboratory conditions. Immature morphology, patterns of
wing color, and genital structures of adults were also studied. The
genetic barcoding region of the cytochrome oxidase I mitochondrial
gene was sequenced and used to verify the limits between species
previously defined by the other characters and to validate its usefulness
for species delimitation in this particular genus. The integrative ap-
proach combining independent datasets successfully allowed species
identification as compared to the approach based on a single dataset.
Three well-differentiated species were found in the studied region,
Mechanitis menapis (Hewitson), Mechanitis polymnia (Linnaeus), and
Mechanitis lysimnia (Fabricius). New valuable characters that could
improve taxonomic identification in this genus are considered.

Introduction color patterns according to descriptions by Fox (1967) and

Mechanitis (Fabricius) belongs to the tribe Ithomiini (Nym-
phalidae: Danainae), which includes Neotropical butterflies
distributed in humid forests from sea level up to 3,000 m asl
and from Mexico to southern Brazil, Paraguay, and across
three Caribbean islands (Willmott & Freitas 2006). This genus
has been recorded throughout the Colombian Andes and it is
of particular abundance in the coffee-growing areas of Anti-
oquia and Caldas departments (Constantino 1997, Garca et
al 2002, Muriel 2006). Taxonomical identification of Mecha-
nitis has been traditionally based on subtle variability in wing
Brown (1977). However, occurring variation within some
species and subspecies makes difficult accurate species iden-
tification using only this criteria (Brown 1977). Useful taxo-
nomic characters in male genitalia have not been found to
differentiate species in this genus (Fox 1967, Brown 1977,
Neild 2008) and as stated by Brown (1977), additional char-
acters and studies are still needed to better understand the
biology and taxonomy of this group.
Studies available on Mechanitis include Young & Moffett
(1979), Vasconcellos-Neto (1980), Portugal & Trigo (2005)
and Torres et al (2009) among others. In these studies,
Mechanitis spp Identification
immature stages of Mechanitis were collected and while
the biology of some species was described, the biological
data generated was not used for species differentiation.
Hill et al (2012) is nowadays the only known study including
immature stages and other ecological features to better
understand the taxonomy of Mechanitis. There are many
host plant records for Mechanitis throughout Central and
South America (Beccaloni et al 2008), but many of these
records did not establish specific associations of each
Mechanitis species with their host plants. Vasconcellos-
Neto (1980) found ecological differentiation between
Mechanitis lysimnia (F.) and Mechanitis polymnia (L.) in
southeast Brazil based on host plant use, but data are
lacking in other parts of the genus range.
There is limited molecular data available for Mechanitis
(Elias et al 2007, Dasmahapatra et al 2009), the use of
some of the molecular markers traditionally used in mo-
lecular systematics, such as the cytochrome oxidase I mi-
tochondrial gene, has failed to recover the species diversity
expected based on morphology, leading to an overestima-
tion of the number of taxa, whereas AFLPs separated the
four recognized species and suggested the existence of a
new one (Dasmahapatra et al 2009).
The DNA barcoding initiative propose the use of a short
section of the mitochondrial DNA (mtDNA) COI gene to
rapidly and inexpensively identify species (Hebert et al
2003) and has been successfully used for taxonomy in
animals, including butterflies (Hebert et al 2004, Janzen
et al 2005, Hajibabaei et al 2006, Burns et al 2008, Silva-
Brando et al 2009). The usefulness of barcode for Mech-
anitis species delimitation was previously evaluated (Elias
et al 2007, Dasmahapatra et al 2009) and shown to pro-
vide a relative low resolution, but these studies did not
included specimens from Colombia, considered one of the
most diverse regions for the genus, which are considered
comparatively in the present study.
The use of barcoding for separating species is still con-
troversial and it needs to be proved for specific organisms
as the resolution of COI sequences for delimiting species
varies across taxonomical groups (Erpenbeck et al 2006,
Roe & Sperling 2007, Silva-Brando et al 2009). Also, there
are limitations of DNA barcoding that may potentially af-
fect results and need to be considered for analysis. They
include nuclear-mitochondrial sequences (Goios et al
2009), mtDNA hybridization (Verardi et al 2006, Nevado
et al 2009), and parasitic endosymbionts as Wolbachia
(Funk et al 2000). Thus, DNA barcoding needs to be care-
fully used and not as the only criteria for species delimita-
tion, but as a component of an integrative approach where
multiple sources of variation are considered (Hebert et al
2004).
Integrative taxonomy, as defined by Dayrat (2005), is
the science that study the diversity of life from multiple
473
and complementary perspectives. Here, we aim to use
different criteria to define the species of Mechanitis occur-
ring in the Cauca River region, on the east slope of the
western Cordillera of the Colombian Andes. This is the first
attempt to study the Mechanitis genus in Colombia from
an integrative perspective.
Material and Methods
Specimens were collected in Colombia from 4501,500 m
asl in the canyon and valley of the Cauca River, mainly on
the eastern slope of the western Cordillera de los Andes,
including localities in Antioquia, Caldas and Valle del Cauca
departments (Fig 1). Some specimens were collected on the
western slopes of the Cordillera Central to provide addi-
tional comparative material. Geographical coordinates and
altitude for collecting sites are provided in Table 1. Adults,
immature stages, and host plants were collected from each
locality including forest fragments and adjacent grassland
areas. Also, humid areas with Ithomiini congregation
(pockets) were sampled. Sampling was carried out be-
tween 2008 and 2009. Specimens were deposited in enve-
lopes and stored at 20C. Two legs of each specimen were
removed and preserved in 100% isopropanol for DNA ex-
traction. Plants were identified using the keys of Whalen et
al (1981) and Bohs (2005) and by comparisons with speci-
mens deposited at local herbariums [Universidad de Anti-
oquia (HUA) and Herbarium MEDEL, Universidad Nacional
de Colombia, Medelln]. Identification at the species level
was confirmed by Michael Nee, an expert on Solanum
(Leptostemonum subgenus). Voucher specimens were de-
posited in the HUA. Immature stages of Mechanitis were
reared under laboratory conditions at the insectarium lo-
cated in the Universidad Nacional de Colombia, Medelln
(1,538 m asl, 27C and 45% humidity). Larvae were fed with
fresh leaves of the host plants cultivated as described by
Giraldo & Uribe (2010). Adult specimens were mounted by
conventional methods using an entomological pin, and also
on the middle of two transparent plastic slides after re-
moving wing scales to improve wing venation and melanic
color pattern observation (Algarin & Alvarez 2002). Adult
morphology was studied using both wing patterns (color
and venation) and male and female genital structures.
Abdomens were cleared in 10% KOH solution for 10 min
and subsequently stored in glycerol until genitalia was
removed and described. The wing melanic color pattern
was studied after removing wing scales and variability or
species particularities were described. Scales were re-
moved using chemical exposure to 70% ethanol for 1 min,
10% NaOH for 15 s, and distilled water for 1 min.
Molecular analysis was carried out on 36 specimens
representing morphospecies from the sampled localities.
474 Giraldo & Uribe

Fig 1 Geographical illustration

of the sampling locations for
Mechanitis in Cauca region,
Colombia.

Specimens with uncertain morphological identification or
unusual wing patterns were also included. DNA was
extracted from one of the middle legs previously removed
from each specimen using grind buffer (Collins et al 1990
modified by Uribe 1999). DNA was treated with RNAase
Fermentas (10 mg/ml, for 1 h at 37C) and DNA quality
was verified by agarose gel electrophoresis. A region of
475 bp of the mtDNA comprising the 5-end of COI known
as the barcode region was PCR-amplified in thermocycler
PTC 100 (MJ Research) programmed at 95C for 5 min for
one cycle, followed by 40 cycles at 94C for 30 s, 50C for
30 s, and 72C for 1.5 min, with a final cycle for extension at
72C for 10 min, using the primer set used LCO1490 (5-
GGTCAACAAATCATAAAGATATTGG-3) and HCO2198 (5-
TAAACTTCAGGGTGACCAAAAAATCA-3) (Folmer et al 1994).
PCR products were sequenced in both directions. Sequence
editing and manual alignment were done using BioEdit
version 7.0.9.0 (Hall 1999). Edited sequences were depos-
ited in GenBank and accession numbers are from JN815139
to JN815174.
Nucleotide diversity and variability among sequences
were calculated in DAMBE version 5.0.23 (Xia & Xie
2001). Sequences obtained were subjected to similarity
searches using the BLASTn tool available in the NCBI to

Table 1 Geographical location of

the samples sites of Mechanitis in Department Municipality Locality N W m asl
Colombia.
Antioquia Hispania La F 54517.44 75555.77 1,022
Antioquia Santaf de Antioquia Cotov 6329.01 754938.94 510
Antioquia Santaf de Antioquia La Gual 63326.53 75501.26 540
Antioquia Valparaso Taboga 54134.69 753824.59 1,000
Caldas Anserma Canoas 51031.58 754053.96 850
V. del Cauca Cali Saladito 341 5.00 763148.00 1,925
V. del Cauca Buga El Vnculo 352 48.00 761710.00 1,030
Mechanitis spp Identification 475

Table 2 List of host plants of

Mechanitis and their occurrence Host plant species Locality
in different localities in Colom-
bia (O 0 absent; X 0 present Anserma Cali Hispania Santaf de Antioquia Valparaso
without immature stages; I 0
present with immature stages). Solanum hirtum X 0 0 I I
Solanum pseudolulo I I I 0 I
Solanum mammosum I 0 0 0 I
Solanum viarum I 0 0 0 0
Solanum jamaicense I 0 I 0 I
Solanum torvum I X I I I

exclude any sequences carrying DNA sequences from sym-
bionts possibly associated with the specimens studies,
such as Wolbachia. Neighbor-joining (NJ.K2P), maximum
parsimony (MP) and maximum likelihood (ML) analysis
were done in MEGA 5.05 (Tamura et al 2011). A Majority
Rule consensus tree of the 325 most parsimonious trees
was obtained for MP. Also, a ML tree was constructed
using the GTR + G + I model. Branch support was assessed
with 1,000 bootstrap replicates. Trees were rooted using
DNA sequences of Forbestra (Fox) (Lepidoptera: Ithomiini)
and Danaus (Kluk) (Lepidoptera: Danaini) deposited in
Genbank with accession numbers gi|157842142| and gb|
EU069039.1| for Forbestra olivencia juntana Haensch, and
gb|GU333889.1| for Danaus plexippus Linnaeus. Additional
sequences from BOLD (The Barcode of Life Data Systems;
http://www.boldsystems.org/views/taxbrowser.php?
taxid03614), Genbank, and from the Nymphalidae System-
atics Group (http://nymphalidae.utu.fi/list.php?genus0
Mechanitis) were included in complementary analysis to
compare and verify groupings and species placement. Ac-
cession numbers for included sequences are: gb |
GU334058.1|Mechanitis polymnia isthmia Bates,
FJ445944FJ445945Mechanitis menapis mantineus
(Hewitson), and NN61-NN94Mechanitis lysimnia
roqueensis Bryk.
(Table 2). Identified species belong to the two major
groups of Solanaceae. One of them includes species from the
Torva and Micracantha clades (Bohs 2005) and the other
includes species from the Lasiocarpa and Acanthophora
clades. Mechanitis polymnia used host plants of Torva and
Micracantha clades and M. menapis used Lasiocarpa and
Acanthophora clades (Fig 2). Neither host plants nor imma-
ture stages were found for M. lysimnia utemaia, which rep-
resented less than 2% of adult specimens collected in all sites
(Table 3). Host plant and immature stages were very useful to
rapidly differentiate M. polymnia and M. menapis at the field
and under laboratory conditions when compared to the use
of traditional morphological characters from adults only.
Egg clutch size
The oviposition patterns were clearly different among spe-
cies (Fig 3). Females of M. polymnia always laid eggs in
clutches on the upper surface of leaves of Solanum jamai-
cense and Solanum torvum (masses found n041), with all
egg clutches larger than 14 eggs. Clutches were found on
mature leaves, frequently below 1 m above the ground,

Results

Butterflies and host plants

A total of 268 individuals were collected and identified

representing the three species: M. polymnia, Mechanitis
menapis (Hewitson) and M. lysimnia, belonging to the
subspecies Mechanitis polymnia caucaensis (Haensch),
Mechanitis menapis occasiva (Fox) and Mechanitis lysimnia
utemaia (Reakirt). A total of 141 (52.6%) individuals were
collected as adults with sex proportion represented as 1:1.
One-hundred and twenty-seven specimens (47.4%) were
collected as immature stages, including eggs and larvae, Fig 2 Mechanitis and its relationship with Solanum clades in Cauca
and six Solanum species were found as host plants Region.
476 Giraldo & Uribe

Table 3 Immature stage clutches per species of Mechanitis found on Immature morphology
different host plant species. Solanum clade in parenthesis.

Host plant (Clade) Mechanitis Egg morphology did not allow species differentiation by naked
eye or stereomicroscopy. However, the larvae of M. polymnia
Polymnia Menapis and M. menapis were clearly alike until the third instar. The
most visible variation in larvae was the color of the head
Solanum torvum (Torva) 5 0
capsule (Fig 4), which was brown in M. polymnia and black
Solanum Jamaicense (Micracantha) 35 0
in M. menapis. The last two instars had a pale yellow head
Solanum hirtum (Lasiocarpa) 1 68
capsule in M. polymnia and gray in M. menapis, but this
Solanum pseudolulo (Lasiocarpa) 0 8
variation was less conspicuous. After detailed morphological
Solanum mammosum (Acanthophora) 0 5
observation, it was concluded that the abdominal tubercles
Solanum viarum (Acanthophora) 0 4
could also be used for species identification until the third
Total 41 85
instar, being shorter and paler in M. polymnia and larger and
yellow in M. menapis (Fig 4). The length of the larvae also
differed among species from the first to the fourth instar
with more than one mass, only rarely (n03) being found on (Table 4). However, differences were not consistent and both
a single plant. On the other hand, females of M. menapis species eventually reached the approximated same size (Fig 5).
laid eggs individually or in small clutches with an average of
three eggs per clutch (never more than 11 eggs per clutch) Adult morphology
on the upper or lower surface of leaves of their host plants
(masses found n0104). In the case of Solanum hirtum and Wing color patterns showed substantial intraspecific varia-
Solanum pseudolulo, eggs were found on leaves of young tion, even when specimens were obtained from the same
plants, mostly below 0.5 m above the ground, while eggs cohort (egg clutch). Both sexes in M. menapis and M.
of Solanum viarum and Solanum mammosum were usually polymnia, and all females of M. lysimnia showed variation
located below 1 m above the ground, often with more than in wing color patterns. Variation in wing color pattern was
one egg or egg clutches per plant or even per leaf. observed in both forewing and hindwing (Fig 6). Other
previously defined taxonomic characters showed more sta-
bility, including those used by Neild (2008), such as the
Life cycle black protrusion (comma mark) between forewing veins
Cu1-Cu2. In M. polymnia this protrusion is rounded, where-
There were not significant differences on the duration of as in M. menapis it has a hammer head shape. The
the life cycle between the species sampled (P00.139), mounted wings on transparent plastic slides enabled us
although development of M. polymnia was in average to identify additional characters, not used previously, that
1.6 days longer than that of M. menapis (Table 4). Howev- are apparently helpful for species differentiation. One of
er, significant differences in the development of eggs and these characters is situated on the dorsal face of hindwing
of the first and third instars were found (Table 4). Estima- (HW) basal region close to the humeral vein (Fig 7). In M.
tion of the egg duration based on oviposition events was menapis, both sexes have a black spot in this region, which
possible only for M. polymnia and M. menapis because is absent in both sexes of M. polymnia and in females of M.
ovipositing females were observed in the field. lysimnia utemaia (no males were available for study). This

Fig 3 Egg clutches for

Mechanitis polymnia (a) on
Solanum jamaicense and
Mechanitis menapis (b) on
Solanum hirtum.
Mechanitis spp Identification 477

Table 4 Life cycle duration (days) (xsd), immature measurements (mm) (x sd) and comparison between species.

Stage M. polymnia M. menapis Students t test

Length (mm) Duration (d) Length (mm) Duration (d) P value length P value duration

Egg 5.80.33 5.080.50 <0.01

Instar1 3.60.37 3.80.80 5.00.40 2.60.50 <0.01 <0.01
Instar 2 5.90.68 3.90.71 7.00.51 3.60.70 <0.01 1
Instar 3 10.20.62 3.00.67 11.30.61 4.10.80 <0.01 <0.01
Instar 4 18.40.87 3.70.83 16.90.47 3.10.70 <0.01 0.137
Instar 5 24.40.83 2.70.79 24.90.79 2.61.00 0.343 0.813
Prepupa 1.20.43 0.90.10 0.096
Pupa 18.00.60 8.41.02 18.20.75 8.90.90 0.348 0.128
TOTAL 32.61.80 30.91.50 0.139

character appears as valuable and it needs to be further
evaluated for species not available in this study. Another
potentially useful character was found when wing scales
were removed for observation of the melanic pattern. Al-
though females of the three species exhibited a melanic
pattern behind the black scales in every black region of their
wing, there were observable differences between males of
M. polymnia and M. menapis. Males of M. polymnia lack a
melanic pattern beneath the medial hindwing band that is
present in males of M. menapis (Fig 8). Black scales were
particularly hard to remove in the hindwing of M. polymnia,
which has androconial scales (Willmott & Freitas 2006). In
this sense, it is considered that melanic patterns should be
studied for the male of M. lysimnia utemaia and the other
species belonging to the genus in order to verify the useful-
ness for species delimitation in Mechanitis.
There were no differences in venation between species,
except for the discal cell where the anterior edge was
approximately 1 mm longer than the posterior edge in M.
polymnia males (1.5 0.29 mm, n 010), whereas these
edges were approximately equal in length in M. menapis
females (0.80.13 mm, n010).
Male genitalia showed no differences between M.
menapis and M. polymnia, in agreement with reports by
Fox (1967), Neild (2008) and Brown (1977). Neither female
genitalia exhibited variation. Inclusion of two available
specimens of M. lysimnia showed similar results.
Molecular analysis
The mtDNA NJ tree (Fig 9a) showed three major mtDNA hap-
logroups: M. polymnia, M. menapis and Mechanitis lysimnia
(threshold of 1.5% sequence divergence). Distances are consis-
tently lower within groups (average 0.3%) than between groups
(average 3 %), a condition known as the barcoding gap (Meyer
& Paulay 2005), and there was no evidence of intraspecific
subdivision or geographical patterns. The raw average pairwise
mtDNA distance between haplogroups was larger among M.
lysimnia with M. polymnia (3.4%) and M. menapis (3.5%) than
between M. polymnia and M. menapis (2.2%). Distance values
were similar to those calculated by Dasmahapatra et al (2009).
Analysis including sequences of different Mechanitis subspecies
from outside of Colombia failed to recover monophyly for M.
lysimnia and M. menapis (Fig 9b). Similar results were found
when MP and ML analysis were done (Fig 10).
An integrative analysis using morphology, ecology, and
molecular data yielded three main groups identified as M.
menapis occasiva, M. polymnia caucaensis and Mechanitis
lysimnia utemaia (Fig 11).

Fig 4 Useful characters (color

of the head and abdominal
tubercles) for identification of
Mechanitis polymnia (a) and
Mechanitis menapis (b) larvae.
478
Fig 5 Differences in size of
cephalic capsules in larvae of
Mechanitis polymnia (a) and
Mechanitis menapis (b).
Discussion
Some host plants found and reported in this study are
apparently new records for the list of host plants of Neo-
tropical diurnal butterflies. Solanum hirtum was previously
registered for Venezuela as a host plant of M. polymnia
and M. lysimnia (Rathcke & Poole 1975, Guagliumi 1967,
Drummond & Brown 1987), and is first registered in here as
a host plant for M. menapis in America (Giraldo & Uribe
2010). Also, S. pseudolulo is a new record of host plant for
Mechanitis in America and it was found hosting M. mena-
pis larvae (see Beccaloni et al 2008).
Differences in the number of clutches on the different
host plants may suggest ecological differentiation between
the sympatric species in the studied area (Courtney 1984,
Vasconcellos-Neto & Monteiro 1993). A similar ecological
differentiation was mentioned by Vasconcellos-Neto (1980)
for M. polymnia and M. lysimnia in southeastern Brazil.
Figure 12 shows a comparison between distribution of food
Fig 6 Variable wing color
pattern characters in
Mechanitis menapis. Letters
indicate the character state
showed on wings. Arrow points
to character location on wings.
Giraldo & Uribe
plant resources from southeastern Brazil (Vasconcellos-
Neto 1980) and that of the Cauca region (Colombia). It
seems likely that this kind of association is similar for
sympatric species of Mechanitis in their ranges. More con-
clusive analysis to establish specific ecological associations
of Mechanitis with their host plants are complicated be-
cause population studies in most geographic regions are
lacking. One other complicating factor that should be
added is the difficulties found for accurate species and host
plant identification.
The clutch size of sympatric and related species of butter-
flies were first studied decades ago (Stamp 1980). Several
factors may explain these differences. Firstly, different selec-
tive pressures including time span for egg maturation upon
adult emergence, life expectancy of adults, and environmen-
tal conditions affecting mating and dispersion and host plant
viability, can all influence the oviposition behavior of differ-
ent species. In the case of Mechanitis, nothing is known on
the period of time required for egg maturation and physio-
logical studies would be required to establish any
Mechanitis spp Identification 479

Fig 7 Useful character for

identification of Mechanitis
polymnia (a) and Mechanitis
menapis (b) in adults color
wing pattern.

differences. Likewise, the life expectancy of adults requires a
population study to determine the average life span of adult
females in wild. Environmental conditions are also expected
to affect the mating and host selection behavior of M.
menapis and M. polymnia, as host searching requires energy
investment. Thus, oviposition of large groups of eggs, as
observed in M. polymnia, should be favored when the plants
are not abundant or are not uniformly distributed, but occur
in small isolated patches.
Searching for different plants implies in an increase in
energy investment and in a higher probability of death
without achieving the full lifetime fecundity. As a conse-
quence, a female may prefer to lay larger clutches and to
restrict their oviposition to one or a few plants before
searching for new potential host plants. On the contrary,
if host plants are much more available and/or uniformly
distributed, females may adopt a strategy to lay fewer
eggs, individually or in smaller groups, on different host
plants to avoid the risk of losing her reproductive invest-
ment in a single egg clutch (Stearns 2000). In Mechanitis,
this theory could explain why females of two sympatric
species showed different oviposition strategies. Host plants
of M. polymnia (S. jamaicense and S. torvum) grow in
groups and are directly exposed to sunlight, and are not
as frequent as host plants of M. menapis (mainly S. hirtum).
Solanum jamaicence and S. torvum fruits are much
smaller (<1 cm) and less juicy than fruits of Solanum sec-
tions Acanthophora and Lasiocarpa and the number of
seeds per fruit is significantly lower. This could result
in differences in plant dispersal ability and, therefore, a
different pattern of distribution in the field, which
needs to be proved. Larval thermoregulation could also
explain clutch size variation, which could be related to
differences in mean altitude, but no correlation was
found between altitude and clutch size in Mechanitis
(CR00.0233; R2 00.054%).

Fig 8 Melanic wing patterns of

Mechanitis. Mechanitis
menapis occasiva, a male, b
female; Mechanitis polymnia
caucaensis, c male, d female;
Mechanitis lysimnia utemaia, e
female. Arrows point to
similarities among females of
the three species. Ovals point
differences between males of
Mechanitis menapis and
Mechanitis polymnia.
480
Fig 9 mtDNA neighbor-joining tree of Mechanitis from Cauca region (a) and mtDNA neighbor-joining tree of Mechanitis from Cauca region and
subspecies out of Colombia (b). Bootstrap values next to nodes.
The variation in embryonic development time was not
considered a very reliable data as the duration was ob-
served from the time egg clutches were collected in the
field with an unknown oviposition date. Even though there
were no differences in the duration of the total life cycle or
in the final size of the immature stages, larvae of M.
polymnia were shorter than M. menapis until the fourth
instar, probably because there is a trade-off between num-
ber of viable eggs laid and size at eclosion. Although size
could be directly related to the clutch sizes found, it is not
possible to establish how many viable eggs were produced
by each female of both species without dissection. Further
studies of life history attributes will be necessary to com-
pletely understand these aspects. Finally, host plants and
immature stages of M. lysimnia utemaia remain unknown.
Its low frequency in collections throughout this region and
Giraldo & Uribe
a lack of biological studies of this species suggest it should
be a priority for future research in this region.
Wing color patterns showed an enormous variation. For
example, assuming that the variable wing pattern characters,
as the black hammer mark and discocellular spots (see Fig 6),
are independently expressed, the number of possible differ-
ent morphotypes for M. menapis can be calculated using the
General Multiplication Rule [P(A and B)0P(A)P(B|A)]. Thus,
we calculated 384 possible varieties (32242220
384). This number is a remarkably high hypothetical number
of forms for a single species in one region. Some of these
variable characters were used traditionally to identify sub-
species, including the black discocellular spots (band) of the
forewing (FW), as well as the shape of the black band in the
discal and postmedial regions of FW and the anal margin of
HW (Fox 1967, Brown 1977). Therefore, color pattern must be
Mechanitis spp Identification 481

Fig 10 mtDNA maximum parsimony (a) and maximum likelihood (b) trees of Mechanitis from Cauca region. Bootstrap values next to nodes.

Fig 11 Adult butterflies of studied region. a. Mechanitis menapis occasiva. b. Mechanitis polymnia caucaensis. c. Mechanitis lysimnia utemaia.
482
Fig 12 Comparison between uses of food plants in southeast Brazil (a) and Cauca region, Colombia (b).
used carefully to avoid erroneous identification, and its var-
iation must be reviewed throughout the distribution range of
the species. Melanic patterns in the wing membrane, on the
other hand, are apparently useful for species identification
and should be studied throughout the genus.
The study of the genitalia of insects has been widely used in
taxonomy because it is considered a source of diverse char-
acters to reveal differences between cryptic species (Eberhard
1985) and genitalia characters of males have been extensively
used for insect taxonomy (Crdoba 2000). One of three
explanations for the evolution of species-specific genitalia
morphology is the Lock-and-Key Hypothesis, which views
genitalia as a mechanism of reproductive isolation between
species (Eberhard 1985, Arnqvist & Thornhill 1998), preventing
the production of hybrids of reduced fitness (Sota & Kubota
1998). However, the existence of hybrids between species of
Mechanitis (Vasconcellos-Neto & Brown 1982) shows the lack
of prezygotic isolation between the species and it is perhaps
partialy a result of the lack of genitalia differentiation in males.
Another possible explanation is that recent speciation has not
allowed the accumulation of morphological changes in
Giraldo & Uribe
genitalia over time. This situation was noted by Fox (1967)
when he wrote: is it possible that in X numbers of centuries
from now, Mechanitis species are readily distinguishable:
today (1967) there is great difficulty and we could only infer
the inherent genetic differences, which are ultimately the real
criterion of species. The same author also expressed that
Mechanitis is the group that exhibits the greatest evolutionary
exuberance and ecological adaptability of the subtribe Mech-
anitina. His findings support the hypothesis that the group is in
a recent process of diversification and speciation, which per-
haps does not reflect the reproductive isolation in genital
structures. However modern available electron microscopy
techniques could allow a much more detailed morphological
study of the genital structures that could reveal variation that
remains undetected.
The topologies based on mtDNA sequence data from the
barcode region did not show any geographical association.
Genetic distances obtained are lower than those found in
most of other Lepidoptera, but they are in concordance with
those found in other studies of Mechanitis (Dasmahapatra et
al 2009) and with those sequences published in BOLD.
Mechanitis spp Identification
Conversely, Dasmahapatra et al (2009) suggested that the
barcode region could imply in the existence of some un-
real species in Mechanitis, especially within haplogroup of
M. polymnia and M. mazaeus with distances above 2.2%.
However, even though similar genetic distances separated
the clusters found in the present study, morphological and
ecological evidence support the hypothesis that these clus-
ters represent three different species in the region studied.
Topologies constructed using sequences from different sub-
species, outside of Colombia, showed no monophyly for M.
lysimnia and M. menapis. Mechanitis lysimnia roqueensis,
from the Amazon, and M. lysimnia utemaia were divided
into different groups and the genetic distance between them
is higher than the genetic distance between the well-
differentiated and sympatric M. polymnia and M. menapis.
Similarly, M. menapis occasiva and M. menapis mantineus
were divided into different groups, but it is surprising that
distances between M. menapis mantineus and other groups
is noticeably lower than it is among other groups. These
results could suggest that there are problems with the cur-
rent subspecific classification and that some subspecies
should probably be treated as species. There are more than
50 taxa (subspecies) in the genus Mechanitis in Lamas
(2004). The subspecies concept is broadly used in Ithomiini
to designate geographic races (Fox 1955). Some hybrid zones
have been found for Mechanitis subspecies (Neild 2008) but
many hybrid zones are still missing and those geographical
variations are recognized just using color wing pattern. Thus,
many subspecies could be subjective concepts and not have
biological significance.
NJ has been broadly used for studies using bar-
code region with some of them including Mechanitis
(Dasmahapatra et al 2009, Hill et al 2012) and in our case
NJ haplogroups are well supported for the morphological
and ecological data. Similar results were found when used
more robust analysis of phylogeny as MP and ML.
The use of barcode data alone in understanding species
limits in Mechanitis in our study region would have led to
erroneous conclusions about species diversity based on the
low divergence between species. However, the additional
use of morphology and ecological attributes allowed us to
validate the utility of the marker. Indeed, the barcode
region allowed us to identify two specimens that were
otherwise difficult to identify from morphology alone (sam-
ples 9691 and 9829 as M. polymnia).
Two hypotheses could explain the low differentiation of
the molecular marker used: a very low mutational rate in
the group or a very recent process of speciation that has
not been reflect in accumulation of nucleotide changes
among the sequences.
Our study shows how combination of different tools
allows for a better approach to understand the systematic
of a group. Fox (1955) suggested that no characters other
483
than wing pattern could be used to identify Mechanitis
species, but here we demonstrate new characters and
ecological data that can help to understand the evolution-
ary patterns and the ecological diversity of this complex
group of butterflies.
Acknowledgments We thank Keith Willmott for his support, train-
ing and hospitality. We also thank Marianne Elias who encouraged us
to continue studying Mechanitis in Colombia, to Michael Nee for help
with host plant identity confirmation, Jean Francois Lecrom for pro-
viding some specimens and for his appreciations about Mechanitis
taxonomy, Natalia Ruiz for help with lab work and to the Herbariums
MEDEL (Universidad Nacional de Colombia sede Medelln) and HUA
(Universidad de Antioquia). Finally, we thank the Sciences School and
Entomology postgraduate program at the Universidad Nacional de
Colombia sede Medelln and to The Lepidoptera Research Founda-
tion, INC. for its grants for students (Feb 16, 2009). This work was
supported by the Direction for Investigation Universidad Nacional de
Colombia, Medellin, DIME grant 20101007738 and for the Fundacin
BBVA (MARIPOSA Proyect: BIOCON08_021)
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