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Uncovering species boundaries in the Neotropical ant complex Ectatomma ruidum (Ectatomminae) under the presence of nuclear mitochondrial paralogues

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Nuclear mitochondrial (mt) paralogues (numts) are non-functional fragments of mtDNA integrated into the nuclear genome that can overestimate the number of species in analyses based on mtDNA sequences. As numts have relatively slow mutation rates, they can pass undetected by conventional procedures such as inspecting for internal stop codons, indels or apparent polymorphism in chromatograms. Species boundaries based on mtDNA markers therefore require a thorough assessment of numts, especially in insects, where this phenomenon appears to be relatively frequent. Ectatomma ruidum is a widely distributed Neotropical ant species that is distributed from northern Mexico to northern Brazil. Previous behavioural and molecular evidence suggests that this species actually represents a composite taxon. Here we assessed the species boundaries in E. ruidum based on two mt (COI, cyt b) and one nuclear (H3) marker, as well as on external morphology. Ancient and recent mt paralogues were detected in several specimens, although pre-PCR dilution of DNA template helped to recover most of the mt orthologues. Based on the congruence found between our species delineation obtained from the mt genealogies and the discriminated morphospecies, we propose that E. ruidum is actually composed of at least three species. Two of these species have a wide geographical distribution in the Neotropics, whereas the remaining one was restricted to localities situated near the Pacific coast in south-east Mexico. We also found extensive intra- and interspecific variation in the barcoding locus. Moreover, the nuclear evidence suggests the existence of hybrids between two of these species in Oaxaca, south-east Mexico. This study agrees with previous studies of other closely related animal taxa, which have revealed a complex evolutionary history and overlooked species diversity in the latter region.
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Uncovering species boundaries in the Neotropical ant
complex Ectatomma ruidum (Ectatomminae) under the
presence of nuclear mitochondrial paralogues
REINA GABRIELA AGUILAR-VELASCO
1
, CHANTAL POTEAUX
2
, RUBI MEZA-L
AZARO
1
,
JEAN-PAUL LACHAUD
3,4
, DMITRY DUBOVIKOFF
5
and ALEJANDRO
ZALD
IVAR-RIVER
ON
1,
*
1
Colecci
on Nacional de Insectos, Instituto de Biolog
ıa, Universidad Nacional Aut
onoma de M
exico,
3er. circuito exterior s/n, Cd. Universitaria, Copilco Coyoac
an A. P. 70233, C. P. 04510, Ciudad de
M
exico, M
exico
2
Laboratoire d’
Ethologie Exp
erimentale et Compar
ee, EA 4443, Sorbonne Paris Cit
e, 99 avenue J.-B.
Cl
ement, 93430 Villetaneuse, France
3
Centre de Recherches sur la Cognition Animale, CNRS-UMR 5169, Universit
e de Toulouse UPS, B ^
at.
IVR3, 118 route de Narbonne, 31062 Toulouse Cedex 09, France
4
Depto. de Conservaci
on de la Biodiversidad, El Colegio de la Frontera Sur, Avenida Centenario Km.
5.5, AP 424, 77074 Chetumal, Quintana Roo, M
exico
5
Department of Applied Ecology, Faculty of Biology, Saint Petersburg State University, 16th line of
Vasilievsky Island, 29, St. Petersburg 199178, Russia
Received 20 September 2015; revised 25 January 2016; accepted for publication 27 January 2016
Nuclear mitochondrial (mt) paralogues (numts) are non-functional fragments of mtDNA integrated into the
nuclear genome that can overestimate the number of species in analyses based on mtDNA sequences. As numts
have relatively slow mutation rates, they can pass undetected by conventional procedures such as inspecting for
internal stop codons, indels or apparent polymorphism in chromatograms. Species boundaries based on mtDNA
markers therefore require a thorough assessment of numts, especially in insects, where this phenomenon appears
to be relatively frequent. Ectatomma ruidum is a widely distributed Neotropical ant species that is distributed
from northern Mexico to northern Brazil. Previous behavioural and molecular evidence suggests that this species
actually represents a composite taxon. Here we assessed the species boundaries in E. ruidum based on two mt
(COI, cyt b) and one nuclear (H3) marker, as well as on external morphology. Ancient and recent mt paralogues
were detected in several specimens, although pre-PCR dilution of DNA template helped to recover most of the mt
orthologues. Based on the congruence found between our species delineation obtained from the mt genealogies and
the discriminated morphospecies, we propose that E. ruidum is actually composed of at least three species. Two of
these species have a wide geographical distribution in the Neotropics, whereas the remaining one was restricted to
localities situated near the Pacific coast in south-east Mexico. We also found extensive intra- and interspecific
variation in the barcoding locus. Moreover, the nuclear evidence suggests the existence of hybrids between two of
these species in Oaxaca, south-east Mexico. This study agrees with previous studies of other closely related animal
taxa, which have revealed a complex evolutionary history and overlooked species diversity in the latter region.
©2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016
doi: 10.1111/zoj.12407
ADDITIONAL KEYWORDS: DNA sequences – Formicidae – hybridization – pseudogenes – species
delineation.
*Corresponding author. E-mail: azaldivar@ib.unam.mx
1©2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016
Zoological Journal of the Linnean Society, 2016. With 4 figures
INTRODUCTION
Currently, one of the most challenging tasks in sys-
tematics is the development of objective, practical
approaches to delimit boundaries between species
(De Queiroz, 2007). Species descriptions, and there-
fore their delimitation, are traditionally carried out
using only diagnostic morphological features, based
on the premise that intra- and interspecific differ-
ences can be somewhat distinguishable. This prac-
tice, however, is now widely recognized to have
serious limitations in recently diverged species with
conserved morphology (e.g. Ross et al., 2010; Milit~
ao
et al., 2014). The use of taxonomic approaches that
combine information obtained from different sources
of evidence (i.e. integrative taxonomy; Padial et al.,
2010; Schlick-Steiner et al., 2010) therefore repre-
sents a valuable option for the accurate delineation
of species. Assessing different types of data, however,
requires additional precautions about the homolo-
gous nature of the characters that are being
examined.
Mitochondrial (mt) DNA sequences have been by
far the most widely employed molecular markers for
species delineation analyses mainly due to their
matrilineal inheritance and higher coalescence ratio
in comparison with nuclear genes (Avise, 2000;
Palumbi, Cipriano & Hare, 2001). Various phenom-
ena, however, can obscure the accurate delineation
of species using this locus, including incomplete lin-
eage sorting (Funk & Omland, 2003), mt introgres-
sion (Bryson et al., 2014) and nuclear mt
pseudogenes (numts; Song, Moulton & Whiting,
2014).
These numts represent non-functional fragments
of mtDNA integrated into the nuclear genome
(Bensasson et al., 2001). They can be accidentally co-
amplified through conventional amplification using
conserved primers, and their inclusion can overesti-
mate the number of species in analyses based on
mtDNA sequence data (Song et al., 2008). As numts
have relatively slow mutation rates, they can pass
undetected by conventional procedures such as
inspecting for internal stop codons, indels or appar-
ent polymorphism in chromatograms (double peaks)
(Calvignac et al., 2011; Song et al., 2014).
Two main types of numts have been categorized to
occur according to their phylogenetic placement with
respect to the orthologue and their branch length,
paleonumts and neonumts (Song et al., 2014). The
first are ancient paralogues whose duplication took
place before the mtDNA had its current form, and
are characterized by having relatively long branch
lengths and often being grouped in separate clades
that are distantly related to the othologue. The sec-
ond are recent duplication events that have not had
time to accumulate enough mutations. These often
have short branch lengths and are not placed
separately from the mt othologue, making them
extremely difficult to detect in phylogenetic analyses
based on single mt sequence fragments.
Ectatomma Smith (Ectatomminae) is one of the
most common groups of foraging Neotropical ants,
currently comprising 15 recognized species dis-
tributed from northern Mexico to central Argentina
(Fern
andez, 1991; Arias-Penna, 2008; Bolton, 2015;
D. Dubovikoff, unpubl. data). Two species have by
far the widest geographical distribution in the genus,
E. tuberculatum (Olivier) and E. ruidum (Roger),
with the latter ranging from Tamaulipas in northern
Mexico to northern Brazil (Bolton, 2015; D. Dubovik-
off, unpubl. data). Ectatomma ruidum is also one of
the most studied Neotropical ant species in terms of
its behavioural ecology. It is facultatively polygynous
(i.e.with colonies having more than one queen) and
is known to be a generalist predator, scavenger and
collector of extrafloral nectar (Lachaud, 1990; Schatz,
Lachaud & Beugnon, 1995, 1996; Lachaud et al.,
1999).
Despite their abundance and conspicuousness,
the taxonomy of E. ruidum has not been thor-
oughly investigated. This species is morphologically
similar to and appears to be sympatric with E. gib-
bum Kugler & Brown, which has a more restricted
geographical distribution. Moreover, a species from
central Mexico, E. aztecum Emery, was distin-
guished from E. ruidum based on body sculpture
features and the form of the petiolar node (Emery,
1901), although it was subsequently regarded as a
junior synonym of the latter species by Kugler &
Brown (1982).
More recently, a molecular phylogenetic study
carried out for 12 of the 15 recognized species of
Ectatomma found that the members of E. ruidum
were divided into two sister clades that shared the
same wide distribution and were separated by rela-
tively long branch lengths (Nettel-Hernanz et al.,
2015). One of these clades contained specimens
from the only two populations recorded as having
colonies with microgynous queens (i.e. an isometric
reduction of the normal macrogynous queens;
Lachaud et al., 1999), whereas the other one had
specimens from localities without records of microg-
yny. This biological and molecular evidence thus
suggests that E. ruidum actually represents a
taxon composed of more than one evolutionary lin-
eage (i.e. species).
Here we assessed the number of speciation events
that occurred within E. ruidum based on specimens
collected from several localities along its known geo-
graphical distribution. For this, we have followed an
integrative taxonomic approach and examined two
©2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016
2R. G. AGUILAR-VELASCO ET AL.
mt markers and one nuclear marker, as well as
external morphological features. Our preliminary mt
gene genealogies revealed an incorrect phylogenetic
placement for a number of the examined sequences.
We therefore investigated the presence of numts in
the mt data sets using different approaches. Based
on the congruence found between the species delin-
eation analyses carried out with our putative mt
othologues and on the consistent morphological dif-
ferences found, we propose that E. ruidum actually
represents a complex composed of at least three evo-
lutionary units, two of which have a wide geographi-
cal distribution within the Neotropics. The evidence
obtained from the nuclear marker also suggests the
existence of a hybrid zone between two of the
proposed species in the Pacific coast of Oaxaca,
south-east Mexico.
MATERIALS AND METHODS
TAXON SAMPLING
We examined newly generated and previously pub-
lished sequences (Smith, Hallwachs & Janzen, 2014;
BOLD systems, www.boldsystems.org) for a total of
133 specimens originally assigned to E. ruidum and
E. gibbum according to the two available keys to
species of Ectatomma (Kugler & Brown, 1982;
Arias-Penna, 2008). The taxon sampling included
specimens collected from 38 geographically separated
populations in the Neotropics (Fig. 1), with an
emphasis on south-east Mexico, where preliminary
observations carried out by the authors revealed con-
siderable external morphological differences between
the sampled populations.
We also included newly generated and published
sequences (Moreau et al., 2006; Smith et al., 2014) of
33 specimens belonging to E. brunneum Smith,
E. edentatum Roger, E. opaciventre (Roger) and
E. tuberculatum, as well as sequences of two other
ectatommine genera, Typhlomyrmex rogenhoferi
Mayr and Rhytidoponera metallica (Smith). We used
sequences of R. metallica and of E. tuberculatum to
root the COI and cyt btrees, respectively. A list with
the included specimens, their species assignment
after this study, localities and GenBank accession
numbers for the three markers examined is provided
in Table S1.
All specimens were preserved in 95% ethanol and
kept at 20 °C until they were processed for DNA
Figure 1. Map showing the sampled localities for the specimens assigned to Ectatomma ruidum and Ectatomma
gibbum. The taxon names refer to the delimited morphospecies.
©2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016
SPECIES BOUNDARIES IN THE ECTATOMMA RUIDUM COMPLEX 3
sequencing. The examined material is deposited in
the Colecci
on Nacional de Insectos, Instituto de
Biolog
ıa, Universidad Nacional Aut
onoma de M
exico
(IB-UNAM), the National Museum of Natural His-
tory, Paris, France (MNHN), and the Zoological
Institute RAS, St. Petersburg, Russia (ZISP).
LABORATORY PROTOCOLS
DNA sequences of two mt and one nuclear gene frag-
ments were examined. We first sequenced a 632-bp
fragment of the cytochrome oxidase I (COI) mtDNA
gene, which corresponds to most of the barcoding
locus (Hebert et al., 2003). We also amplified a 792-
bp fragment corresponding to part of the cytochrome
b(cyt b) and the tRNA
ser
mtDNA genes, and 260 bp
of the histone H3 protein (H3) encoding nuclear gene
for a subset of 62 and 41 specimens, respectively.
COI and cyt bare the most widely employed mt
markers for analyses of closely related taxa, as well
as for phylogeographical and population genetic stud-
ies in animals, including insects (Simon et al., 1994;
Simmons & Weller, 2001; Lin & Danforth, 2004). We
also generated 32 sequences corresponding to a 748-
bp fragment of the second and third domain regions
of the 28S ribosomal DNA gene (GenBank accession
numbers KU57070637). These sequences, however,
did not show variation among the sequenced
specimens and therefore they were not considered
for the study.
Genomic DNA in most cases was extracted from
one to three legs of each specimen using the E-Z10
Spin Colum Genomic DNA Minipreps kit (Bio Basic,
Amherst, NY, USA) following the manufacturer’s
protocol. Some of the DNA extractions were carried
out by placing the dried samples in 50 lLof5%
(w/v) Chelex (Bio-Rad, Hercules, CA, USA), which
contained 12 lgmL
1
of proteinase K, followed by
digestion for approximately 2 h at 5560 °C and
finally inactivating proteinase K at 96 °C for 15 min.
PCRs were performed in 15 lL of total volume reac-
tions containing 1.25 lLof109PCR buffer, 0.5 lL
of MgCl
2
, 0.2 mMof each dNTP, 0.2 lMof each pri-
mer, 0.125 lL of platinum Taq polymerase (Invitro-
gen, Waltham, MA, USA), 3 lL of DNA template and
9.475 lL of ddH
2
O. Amplifications were carried out
using the following primers: LCO (50-GGTCAA-
CAAATCATAAA
GATATTGG-30) and HCO (50-TAAACTTCAGGGTGA
CCAAAAAATCA-30) (Folmer et al., 1994) for COI;
CB1 (50-TATGTACTACCATGAGGACAAATATC-30)
and tRS (50-TATTTCTTTATTATGTTTTCAAAAC-30)
(Jermiin & Crozier, 1994) for cyt b; and H3F (50-
ATGGCTCGTACCAAGCAGACVGC-30) and H3R (50-
ATATCCTTRGGCATRATRGTGAC-30) (Colgan et al.,
1998) for H3.
PCR cycling conditions were as follows. COI: one
step of 3 min at 95 °C, followed by 30 cycles of 94 °C
for 40 s, 50 °C for 1 min and 72 °C for 40 s, and a
final step at 72 °C for 7 min. Cyt b: one step of
2 min at 94 °C, followed by 35 cycles of 94 °C for
30 s, 50 °C for 1 min and 72 °C for 1 min, and a
final step at 72 °C for 1 min. H3: one step of 1 min
at 94 °C, followed by 35 cycles of 1 min at 94 °C,
1 min at 45.5 °C and 1 min 30 s at 72 °C, and a final
step of 10 min at 72 °C.
Unpurified PCR products were sent for sequencing
at the University of Washington’s High-Throughput
Genomics Unit (http://www.htseq.org). Sequences
were edited with Sequencher version 4.1.4 (Gene
Codes Corp., Ann Arbor, MI, USA) and aligned manu-
ally based on their translated amino acids using Mes-
quite version 2.75 (Maddison & Maddison, 2009). The
fragment belonging to the tRNA
ser
gene was excised
from the alignment containing the cyt bmarker due
to its considerable sequence length variation.
DETECTION OF NUMTS
Nuclear mt paralogues can be detected based on the
presence of codon position substitution bias, distinct
rates of evolution, internal stop codons, indels,
apparent polymorphism in chromatograms (double
peaks) or incongruent phylogenetic placement (Song
et al., 2008, 2014; Calvignac et al., 2011). We there-
fore detected the presence of numts in the two mt
data sets based on the above six criteria and
excluded them from the species delineation analyses.
In particular, several sequences, especially of COI,
had polymorphisms and/or incongruent relationships
after carrying out preliminary phylogenetic analyses.
We therefore performed additional COI amplifica-
tions, following the pre-PCR dilution procedure
described by Calvignac et al. (2011) to recover mt
othologues in samples that had the latter two cases
of paralogy. This procedure has been shown to be the
best one employed to amplify mtDNA sequences
instead of numts, as dilution of the DNA template
considerably decreases the nuclear genome and
therefore reduces the chance of amplifying nuclear
gene fragments in PCRs (Calvignac et al., 2011).
DNA extractions of the selected specimens had
between 3 and 4 ng lL
1
of genomic DNA using
1lL of DNA template with a Nanodrop ND 1000
Spectrophotometer (NanoDrop Technologies, Wil-
mington, DE, USA). Based on this DNA concentra-
tion, we amplified the COI marker for 29 E. ruidum
specimens as described above, employing both 1:10
and 1:30 dilutions of the DNA template.
A partitioned Bayesian analysis was performed
with the parameters described below, including both
the originally generated COI sequences and those
©2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016
4R. G. AGUILAR-VELASCO ET AL.
recovered with the pre-PCR dilution procedure.
Those subsequently generated sequences that had
alternative relationships involving geographically
and morphologically congruent clades were regarded
as orthologues. We followed Calvignac et al.’s (2011)
criterion and refer to all our proposed mtDNA
sequences with the prefix ‘putative’, as we cannot
guarantee their mt origin. For the cyt bdata set we
did not repeat amplifications with pre-PCR dilutions,
although numts were also detected and excised from
subsequent analyses based on incongruent relation-
ships. Sequences considered in this study as nuclear
mt paralogues can be retrieved from GenBank under
accession numbers KU57073862. All the sequences
and locality details are also available in the project
file ‘Species boundaries in the Neotropical ant com-
plex Ectatomma ruidum’ (ECT project), contained in
the projects section of the Barcode of Life Data Sys-
tems (www.boldsystems.org). All the matrices
analysed in this work can be retrieved from Data S1.
NUCLEAR DNA RECOMBINATION
The presence of recombination in the nuclear H3 gene
was assessed using TOPALi version 2.5 (Milne et al.,
2009). This program uses a sliding-window approach
to search for putative recombination breakpoints
along aligned sequences (Milne et al., 2009). We
looked for signal of recombination, which is expressed
by a significant difference in sum-of-squares (DSS)
peak along the sequence alignment (250 bp) using the
DSS with a 75- and 100-bp window and a 10-bp step-
size. The statistical significance of DSS was assessed
using 100 parametric bootstraps. We also visually
examined the aligned sequences to try to identify
positions with inconsistent phylogenetic signal, which
could represent recombination breakpoints.
Sequences with evidence of recombination were
regarded as potential hybrids and were excluded from
the species delineation analyses.
SPECIES DELINEATION BASED ON MTDNA
SEQUENCE DATA
It has been shown that various groups of ants have
complex population genetic processes that promote
the presence of marked geographical genetic structure
(Wild, 2009; Ross et al., 2010). Species delineation
approaches that are not sensitive to intrinsic proper-
ties of mtDNA polymorphism, such as overlap in inter-
and intraspecific divergences and non-monophyly of
species, can thus lead to overestimation of the number
of species within Formicidae. Among these are the
DNA barcoding approach (Hebert et al., 2003) and the
General mixed Yule-coalescent model (Pons et al.,
2006), which have been shown to be highly susceptible
to the presence of high levels of mt genetic structure
(Wild, 2009; Talavera, Dinc
a & Vila, 2013).
Species delineation based on the mt markers was
therefore assessed using Wiens & Penkrot’s (2002)
approach based on DNA haplotype phylogenies. In
this approach, the discordance between haplotype
clades and the geographical provenance of their indi-
viduals is assumed to represent evidence of gene flow
among the examined populations (Wiens & Penkrot,
2002). Therefore, the number of potential species can
be assessed by the presence of lineages that are con-
cordant with geography.
Bayesian phylogenetic analyses were carried out
with MrBayes version 3.2.6 (Ronquist et al., 2012)
for each mt marker and for a concatenated COI +cyt
bmatrix that included only specimens with
sequences generated for both genes. Two different
matrices were employed for the separate analyses of
each mt marker. The first one included all their gen-
erated sequences, whereas the second one excised all
sequences regarded as numts, as well as specimens
with evidence of recombination based on the nuclear
marker. Each analysis consisted of two simultaneous
runs of 20 million generations each, using four
chains and saving trees every 1000 generations.
Three partitions were considered for each marker
according to their codon positions. The most appro-
priate models of evolution for each partition were
obtained with jModeltest version 2.1.7 (Darriba
et al., 2012) (COI: 1st =GTR +G; 2nd =GTR +G;
3rd =GTR +G; cyt b: 1st =GTR +I; 2nd =GTR +
G; 3rd =GTR +I). Burn-in was determined based on
convergence of the two simultaneous runs measuring
the values of the average standard deviation of split
frequencies. A value <0.01 was considered as conver-
gence of the two simultaneous runs. We discarded
the trees sampled during the first 10 million genera-
tions in all analyses, and the remaining sampled
trees were employed to reconstruct a phylogram,
considering clades as significantly supported when
they had a posterior probability (PP) 0.95.
We also calculated corrected pairwise genetic
distances for COI using the Kimura two-parameter
distance model (Kimura, 1980) with the program
MEGA version 6 (Tamura et al., 2013) to compare
the results obtained between the above approach and
the 2% corrected pairwise distance criterion that is
widely employed for this marker in animals (Hebert
et al., 2003).
MORPHOSPECIES DELIMITATION AND INTEGRATIVE
TAXONOMY
Morphospecies were discriminated based on nine
external morphological characters examined from a
vast number of worker specimens belonging to all
©2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016
SPECIES BOUNDARIES IN THE ECTATOMMA RUIDUM COMPLEX 5
the populations sampled for the molecular analyses.
Most of these features have been employed in previ-
ous taxonomic studies to distinguish species in
Ectatomma (Kugler & Brown, 1982). A list describing
the morphological features examined, as well as their
observed variation among the morphospecies
discriminated, is shown in Table 1.
Digital photographs were taken for representative
specimens of each of the discriminated morphos-
pecies with a Leica IC 3D digital camera mounted on
a Leica MZ16 microscope and using the Leica Appli-
cation Suite imaging system. All the examined speci-
mens are deposited in the Colecci
on Nacional de
Insectos, Instituto de Biolog
ıa, Universidad Nacional
Aut
onoma de M
exico (IB UNAM).
We followed an integrative taxonomic approach
by congruence (sensu Padial et al., 2010), and rec-
ognized as evolutionary species (sensu Wiley, 1978;
De Queiroz, 2007) those taxa delimited by the
DNA sequence-based species delineation analysis
that also had consistent external morphological dif-
ferences.
RESULTS
DETECTION OF NUMTS
The initial COI, cyt band H3 data sets consisted of
148, 66 and 41 sequences, respectively. A total of 36
sequences were subsequently added to the COI data
set from the amplifications obtained with the pre-
PCR dilution procedure, increasing its number to
184. Details of the gene fragments examined are
listed in Table S2.
The Bayesian phylograms derived from the sepa-
rate analyses of the COI and cyt bdata sets with all
the examined sequences are shown in Fig. S1. The
COI phylogram recovered most of the specimens
originally assigned to E. ruidum within a major, sig-
nificantly supported clade with strong morphological
and geographical structure. A total of 19 sequences
were regarded as numts according to their incorrect
phylogenetic placement and/or presence of polymor-
phism. Thirteen of these sequences were considered
as paleonumts as they were grouped outside the
major E. ruidum clade and belonged to specimens
coming from distinct localities in Oaxaca and Costa
Rica. The remaining six sequences (five E. ruidum
and one E. gibbum), on the other hand, were
regarded as neonumts due to the fact that they were
nested within geographically and morphologically
congruent clusters but were polymorphic. The pre-
PCR dilution procedure generated four alternative
sequences that were regarded as putative othologues
for specimens whose original sequences had incon-
gruent relationships or polymorphism in their
chromatograms, and also supported the orthologue
nature of 21 original sequences.
The cyt bdata set contained ten probable numts.
One of them is a paleonumt represented by a speci-
men from Oaxaca with an incorrect phylogenetic
placement. The remaining nine are neonumts, five of
which belong to specimens from Jalisco that lack the
stop codon flanking the tRNA
ser
gene, and four to
specimens from Chiapas, Oaxaca and Costa Rica.
NUCLEAR DNA RECOMBINATION
The alignment of the 41 H3 sequences had nine phy-
logenetically variable sites (3.5%). Sequences of three
specimens from Pinotepa Nacional in Oaxaca, Mexico
(DNA voucher nos. CNIN199395), have one of the
two exclusive nucleotide substitutions that charact-
erize two of the delimited morphospecies
(E. sp. ‘ruidum 3’ and E. sp. ‘ruidum 4’; see below),
although in the other position they instead have the
state that is present in E. sp. ‘ruidum 1’ and
E. sp. ‘ruidum 2’. The DSS analyses revealed signifi-
cant signal of recombination in the latter three sam-
ples using the 75-bp window and a 10-bp step-size,
although they had a non-significant signal using the
100-bp window. Based on this result, the latter speci-
mens were regarded as potential hybrids and thus
were excluded from the subsequent species
delineation analyses.
DNA SEQUENCE-BASED SPECIES DELINEATION
ANALYSES
The Bayesian phylograms derived from the separate
COI and cyt band the concatenated data sets exclud-
ing all potential numts and hybrids are shown in
Fig. 2, S2, and Fig. 3, respectively. Ectatomma gib-
bum appears significantly supported in the three
topologies as sister to the clade containing all speci-
mens assigned to E. ruidum. Use of Wiens & Penk-
rot’s (2002) approach in the two mt and the
concatenated analyses consistently delimited two sig-
nificantly supported lineages within the main
E. ruidum clade. Both lineages contain specimens
collected in localities occurring from Mexico to north-
ern South America, appearing as sister taxa in the
cyt b(PP =0.95) and the concatenated (PP =0.93)
analyses, but not in the one performed with COI.
The concatenated phylogram also recovered two sig-
nificantly supported clades represented by the two
morphospecies that appear restricted to localities
near the Pacific coast in south-east Mexico
(E. sp. ‘ruidum 3’ and E. sp. ‘ruidum 4’). However,
members of these two morphospecies appeared inter-
mingled as a grade in the COI and the cyt b
phylograms.
©2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016
6R. G. AGUILAR-VELASCO ET AL.
Table 1. List of the nine external morphological features examined, their states and the morphospecies delimited for specimens originally assigned to Ecta-
tomma ruidum; morphospecies assignments for the specimens examined in this study are given in Table S1.
Species assignment
Space between eye
and frontal carina Clypeus
Posterior
outline of
head Pronotal hump Lateral pronotal teeth
E. gibbum With oblique rugae With oblique rugae Strongly
arched
High, rounded, considerably
higher than lateral teeth
Reduced, rectangular
E. sp. ruidum 1’ 8 or fewer longitudinal
striae; space between
striae rugulose
With few irregular,
longitudinal striae
Straight Low, moderately acute, slightly
higher than lateral teeth
Prominent, pointed
E. sp. ‘ruidum 2’ 8 or fewer longitudinal
striae; space between
striae slightly rugulose
With few irregular,
longitudinal striae
Straight Low, strongly acute, distinctly
higher than lateral teeth
Prominent, pointed
E. sp. ‘ruidum 3’ 1016 longitudinal striae
running from base of
antennae to top level of eye
With well-defined
longitudinal striae
Straight Low, rounded, same level as
lateral teeth
Reduced, rectangular
E. sp. ‘ruidum 4’ 1016 longitudinal striae
running from base of
antennae to top level of eye
With well-defined
longitudinal striae
Straight Low, rounded, same level as
lateral teeth
Reduced, rectangular
E. sp. ‘ruidum 2’
9E. sp. ‘ruidum 3’
1016 longitudinal striae
running from base of
antennae to top level of eye;
space between striae
slightly rugulose
With well-defined
longitudinal striae
Straight Low, rounded, slightly higher
than lateral teeth
Reduced, rectangular
Species assignment Propodeal teeth Propodeum Petiolar node
E. gibbum Reduced, blunt With poorly defined transverse striae
surrounded by strong oblique rugae
Low, wide, strongly arched anteriorly, straight posteriorly
E. sp.ruidum 1’ Distinct, sharp With transverse striae, oblique
rugae anteriorly and/or medially
High, narrow, medially constrained, subparallel dorsally
E. sp. ‘ruidum 2’ Distinct/reduced, sharp With transverse striae, oblique rugae
anteriorly and/or medially
Moderately high, narrow, slightly arched anteriorly,
almost straight posteriorly
E. sp. ‘ruidum 3’ Distinct/reduced, sharp With well-defined transverse striae Low, wide, distinctly arched anteriorly, straight posteriorly
E. sp. ‘ruidum 4’ Absent/reduced, sharp With well-defined transverse striae High, moderately wide, straight, slightly arched anteriorly
E. sp. ‘ruidum 2’
9E. sp. ‘ruidum 3’
Reduced, sharp With transverse, irregular striae Low, wide, distinctly arched anteriorly, straight posteriorly
©2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016
SPECIES BOUNDARIES IN THE ECTATOMMA RUIDUM COMPLEX 7
The corrected genetic distances for the COI data
set excluding all probable numts and hybrids among
and within the four taxa delimited by both the mor-
phological evidence (see below) and the concatenated
analysis are shown in Table S3. The four taxa show
considerably high intra- and inter-lineage COI dis-
tances (09.5 and 3.412.2%, respectively), with the
highest levels of intra-lineage distance occurring in
the two taxa restricted to south-east Mexico (up to
5.6 and 9.5% in E. sp. ‘ruidum 3’ and E. sp. ‘ruidum
4’, respectively).
MORPHOLOGICAL DATA
A list with the morphospecies assignments for the
specimens examined in this study is given in
Table S1. Digital photographs showing some of the
morphological features that characterize the delim-
ited morphospecies are shown in Figs 4 and S3. The
nine external morphological features examined
helped to discriminate four morphospecies that are
concordant with the four species delimited by the cyt
band the concatenated species delineation analyses.
The two species with wide distributions recovered
by the above species delineation approach, E. sp.
ruidum 1’ and E. sp. ‘ruidum 2’, can be morphologi-
cally distinguished from each other by the form of the
pronotal hump and the petiolar node (Table 1;
Figs 4B, C; S3 and S4). These two morphospecies also
differ from the other two by five consistent additional
features. On the other hand, the two morphospecies
restricted to south-east Mexico, E. sp. ‘ruidum 3’ and
E. sp. ‘ruidum 4’, differ from each other by two mor-
phological differences: the form of the petiolar node
and presence/absence of pronotal teeth. However, for
the latter character, the state that distinguishes
E. sp. ‘ruidum 4’ (pronotal teeth absent) is not fixed.
Our morphological examination also supports a hybrid
origin for the specimens from Pinotepa Nacional, Oax-
aca, which share features of both E. sp. ‘ruidum 2’
and E. sp. ‘ruidum 3’ (Figs 4F and S3).
DISCUSSION
DETECTION OF NUMTS IN SPECIES DELIMITATION
ANALYSES
Here we have shown that the use of universal pri-
mers for the two most widely used mt gene markers,
COI and cyt b, could lead in some cases to preferen-
tial amplification of numts in the ant genus Ecta-
tomma. Our results also agree with those of a
previous study that supports the use of the pre-PCR
dilution procedure as a valuable tool to increase
the preferential amplification of mt othologues
(Calvignac et al., 2011).
Species assignment Scultpure of first gastric tergum
E. gibbum With arched, rugulose ‘fingerprint’-like costulae basally, running to apical end laterally; medial area punctate, with fine,
broken costulae
E. sp. ruidum 1’ With arched, rugulose ‘fingerprint’-like costulae running from basal to apical end of tergum
E. sp. ‘ruidum 2’ With arched, rugulose ‘fingerprint’-like costulae running from basal to apical end of tergum
E. sp. ‘ruidum 3’ With arched, well defined ‘fingerprint’-like costulae running from basal to apical end of tergum
E. sp. ‘ruidum 4’ With arched, well defined ‘fingerprint’-like costulae running from basal to apical end of tergum
E. sp. ‘ruidum 2’
9E. sp. ‘ruidum 3’
With arched, slightly rugulose ‘fingerprint’-like costulae running from basal to apical end of tergum
©2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016
8R. G. AGUILAR-VELASCO ET AL.
Most of the nuclear mt paralogues detected in
this study appear to represent paleonumts, as they
were nested in a clade at the base of the main
E. ruidum clade, suggesting that they originated
before speciation events occurred in this species
complex. In contrast, only a few mt paralogues
seem to be neonumts based on their small number
of substitutions. These neonumts were detected by
the presence of internal stop codons and/or
polymorphism in chromatograms. We recognize that
additional neonumts could have passed undetected
by our established criteria. However, as they usu-
ally accumulate few mutations, they are often
nested together with the orthologue (Song et al.,
2014), and thus do not have a strong impact in
species delineation analyses.
0.03 subst./site
E_rui1_ACGAB872-09 Costa Rica
E_rui2_CNIN1822_Costa Rica
E_rui3_CNIN1891_OaxacaMexico
E_rui2_CNIN1814_Nayarit _Mex ico
E_rui3_CNIN1987_30_Guerrero_Mexico
E_rui1_CLeoncillos113Chia pas_M exico
E_gibbum_ASLAM423_11_Honduras
E_rui1_CNIN1748_Chiapas_Mex ico
E_rui1_ACGAB812-09_Costa Rica
E_rui2_CNIN1976_Yucatan_Mexico
E_rui4_CNIN1736_Oaxaca_Mexico
E_rui1_ACGAF453-11_Costa Rica
E_rui1_CLeoncillos115Chia pas_M exico
E_rui_ACGAF378-11_Costa Rica
E_rui2_CNIN1815_Nayarit_Mexico
E_rui1_ACGAF379-11_Costa Rica
E_rui2_Izapa5_Chiapas_Me xic o
E_gibbum_ASLAM1629_11_Nicaragua
E_rui1_CNIN1749_Chiapas_Mex ico
E_rui11_ACGAB870-09_Costa Rica
E_rui1_CLeoncillos114Chia pas_M exico
E_rui2_CNIN1819_Chiapas_ Mexi co
E_rui2_CNIN1746_Jalisco _Mex ico
E_rui1_ACGAE182-10_Costa Rica
E_rui2_ER13ALCO_Ecu ador
E_rui2_Izapa16b_Chiapas_Me xic o
E_rui1_CLeoncillos16Chia pas_M exico
E_rui1_ACGAF452-11_Costa Rica
E_rui4_CNIN1882_Oaxaca_Mexico
E_rui2_CNIN1758_Qunitana Roo_Me xico
E_rui4_CNIN1737_Oaxaca_Mexico
E_rui1_ACGAB827-09_Costa Rica
E_rui2_ER18ALCO_Pan ama
E_rui1_ACGAC854-10_Costa Rica
E_rui2_CNIN1808_Costa Rica
E_rui2_4_2_Costa Rica
E_rui1_20_Colombia
E_gibbum_ASLAM2059_12_Honduras
E_rui2_ER11ALCO_Ecu ador
E_rui2_Chame_2_1Jalisc o_M exi co
E_rui2_CNIN1868_Oaxaca_Mexico
E_rui2_ER16ALCO_Ecu ador
E_rui1_W2LCO_venezuela
E_rui2_PMorellos25 Quintana Roo_Mexico
E_rui1_CNIN2001_Guatemala
E_rui2_CNIN1866_Oaxaca_Mexico
E_rui2_PMorellos26 Quintana Roo_Mexico
E_rui2_CNIN1742_30Jalisc o
E_rui2_CNIN1974_Yucatan_Mexico
E_rui2_CNIN1865_30Oaxaca_Mexico
E_rui_21_Colombia
E_rui2_ER10ALCO_Ecu ador
E_rui2_CNIN1861_Oaxaca_Mex ico
E_rui2_ER17ALCO_Ecu ador
E_rui2_CNIN1975_Yucatan_Mexico
E_rui2_CNIN1996_Costa Rica
E_rui1_CNIN2002_Honduras
E_rui2_CNIN1817_Jalisco _Mex ico
E_rui2_CB225LCO
E_rui1_ACGAC856-10_Costa Rica
E_rui2_CNIN1894_Oaxaca_Mexico
E_rui3_CNIN1890_Oaxaca_Mexico
E_rui1_ACGAJ226-11_Cos ta Rica
E_rui3_CNIN1810_Oaxaca_Mexico
E_rui2_CNIN1741_Jalisco _Mex ico
E_rui3_CNIN1889_30Oaxaca_Mexico
E_rui2_CNIN1744_Jalisco _Mex ico
E_rui3_CNIN1988_Guerrero_Mexico
E_rui2_CNIN1863_Oaxaca_Mexico
E_rui3_CNIN1809_Oaxaca_Mexico
E_rui1_WLCO_Venezuela
E_rui2_4_1_Costa Rica
E_rui2_CNIN1735_Chiapas_ Mexi co
E_rui2_Izapa3b_Chiapas_Me xic o
E_rui2_CNIN1745_30Jalisc o_Me xico
E_gibbum_ASLAM1785_12_Nicaragua
E_rui1_CNIN1754_Chiapas_Mex ico
T. rogenhoferi
E_rui2_CNIN1979_Yucatan_Mexico
E_rui2_ER15ALCO_Ecu ador
E_rui2_1_Costa Rica
E_rui1_ACGAB874_09 Costa Rica
E_rui2_Izapa6b_Chiapas_Me xic o
E_rui4_CNIN1813_Oaxaca_Mexico
E_rui2_CNIN1893_Oaxaca_Mexico
E_rui1_ACGAB847-09_Cost a Rica
E_rui2_CNIN1816_Jalisco _Mex ico
E_rui1_CNIN1750_Chiapas_Mex ico
E_rui2_CNIN1978_Yucatan_Mexico
E_rui2_CNIN1998_30Costa Rica
E_rui2_ER8ALCO_Ecua dor
E_rui2_CNIN1977_Yucatan_Mexico
E_rui2_3_Costa Rica
E_gibbum_CNIN2003_Nicaragu a
E_rui1_24_Colombia
E_rui2_2_Costa Rica
E_rui2_CNIN1490_Yucatan_Mexico
E_rui1_19_Colombia
E_rui11_22_Colombia
E_rui3_CNIN1989_Guerrero_Mexico
E_rui1_CNIN2000_Guatemala
E_gibbum_ASLAM419_11_Honduras
E_rui2_Izapa7b_Chiapas_Me xic o
E_rui2_CNIN1807_30Costa Rica
E_rui4_CNIN1880_Oaxaca_Mexico
R. metallica
E_rui2_CNIN1864_10Oaxaca_Mexico
E_rui2_ER9ALCO_Ecuador
E_rui3_CNIN1851_Guerrero_Mexico
E_rui1_ACGAB871-09_Cost a Rica
E. tuberculatum E. edentatum
E. opaciventre
E. brunneum
COI
putative orthologues
BPP 0.95
E. sp. “ruidum 2”
E. sp. “ruidum 3 +4”
E. sp. “ruidum 1”
Figure 2. Phylogram derived from the Bayesian analysis performed with the COI marker excluding all potential numts
and hybrids. Black circles near branches are Bayesian posterior probabilities 0.95. Colour lines refer to the main lin-
eages recovered. Taxon names refer to the delimited morphospecies.
©2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016
SPECIES BOUNDARIES IN THE ECTATOMMA RUIDUM COMPLEX 9
The presence of numts has been reported to occur
in various insect orders (Leite, 2012). In Hymenop-
tera, they occur in members of Cynipidae (Rokas
et al., 2003), Apidae (Pamilo, Viljakainen & Vihavai-
nen, 2007) and Colletidae (Magnacca & Brown,
2010), and in Formicidae they have been recorded
for species of Dorylus (Kronauer et al., 2007) and in
members of the Attini genera Atta and Acromyrmex
(Martins et al., 2007; Cristiano, Cardoso & Fernan-
des-Salom~
ao, 2014). Here, we propose the existence
of numts within the E. ruidum complex not only
among our generated sequences, but also among
others previously published (Smith et al., 2014). Our
results thus highlight the use of rigorous, objective
approaches that help to detect the presence of numts
in order to avoid overestimation in the number of
independent evolutionary lineages recovered in
species delimitation analyses.
NUCLEAR RECOMBINATION
Our morphological and nuclear DNA recombination
evidence suggests the existence of a hybrid zone
between two of our delineated species, E. sp. ‘ruidum
2’ and E. sp. ‘ruidum 3’, in a locality situated near
the Pacific coast of Oaxaca in south-east Mexico.
Nuclear DNA recombination due to hybridization
could represent a potential problem for DNA
sequences, as traditional phylogenetic methods
assume that a single evolutionary history underlies
the data (Ibrahim, Cooper & Hewitt, 2002; Posada &
Crandall, 2002). In particular, phylogenetic biases
are likely to take place when there is recent recombi-
nation among divergent taxa, and the recombina-
tional breakpoint divides the alignment into two
regions of similar length (Posada & Crandall, 2002).
In this work, the examined nuclear marker did not
provide enough phylogenetic signal for estimating
interspecific relationships within the E. sp. ruidum
complex. Its observed variation, however, suggests the
existence of H3 sequences composed of regions belong-
ing to both E. sp. ‘ruidum 2’ and E. sp. ‘ruidum 3’.
This, to our knowledge, represents the first report
of putative recombination of this protein-coding gene
in insects.
INTEGRATIVE TAXONOMY
Integrative taxonomy has proved to be a powerful
approach for a robust delineation of species,
especially in morphologically conserved groups.
This criterion has been particularly useful in
discriminating overlooked species diversity in ants
(Schlick-Steiner et al., 2006; Seifert, 2009; Ferreira
et al., 2010). The congruence between our species
delineation analysis based both on mtDNA
sequence data and morphological evidence supports
the existence of at least three independent evolu-
tionary lineages (i.e. species) within the E. ruidum
complex. Two of these species are represented by
E_rui_CNIN1749_Chiapas_Mexico
E_rui4_CNIN1880_Oaxaca_Mexico
E_rui3_CNIN1889_Oaxaca_Mexico
E_rui3_CNIN1987_Guerrero_Mexico
E_rui2_CNIN1808_Costa Rica
E_rui2_CNIN1758_QRoo_Mexico
E_rui_CNIN1748_Chiapas_Mexico
E_rui4_CNIN1736_Oaxaca_Mexico
E_rui2_CNIN1816_Jalisco_Mexico
E_rui3_CNIN1809_Oaxaca_Mexico
E_rui2_CNIN1814_Nayarit_Mexico
A_rui3_CNIN1890_Oaxaca_Mexico
E_rui_CNIN1754_Chiapas_Mexico
A_rui3_CNIN1891_Oaxaca_Mexico
E_rui2_CNIN1863_Oaxaca_Mexico
E_rui2_CNIN1815_Nayarit_Mexico
E_rui3_CNIN1989_Guerrero_Mexico
E_rui_CNIN2002_Honduras
E_rui3_CNIN1988_Guerrero_Mexico
E_rui2_CNIN1741_Jalisco_Mexico
E_rui2_CNIN1819_Chiapas_Mexico
E_gibbum CNIN2003_Nicaragua
E_rui2_CNIN1817_Jalisco_Mexico
E_rui_CNIN1750_Chiapas_Mexico
E_rui4_CNIN1813_Oaxaca_Mexico
E_rui3_CNIN1810_Oaxaca_Mexico
E_rui2_CNIN1735_Chiapas_Mexico
E_rui4_CNIN1737_Oaxaca_Mexico
E_rui4_CNIN1882_Oaxaca_Mexico
E_brunneum CNIN2047, 2040
E_rui2_CNIN1807_Costa Rica
COI + Cyt b
putative orthologues
E. sp. “ruidum 2”
E. sp. “ruidum 3”
E. sp. “ruidum 1”
E. sp. “ruidum 4”
E_rui2_CNIN1861_Oaxaca_Mexico
E_tuberculatum CNIN1733, 1743
0.05 subst./site
BPP 0.95
Figure 3. Phylogram derived from the Bayesian concatenated (COI +cyt b) analysis excluding all potential numts and
hybrids. Black circles near branches are Bayesian posterior probabilities 0.95. Coloured lines refer to the main lineages
recovered. Taxon names refer to the delimited morphospecies.
©2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016
10 R. G. AGUILAR-VELASCO ET AL.
morphospecies E. sp. ‘ruidum 1’ and E. sp. ‘ruidum
2’, whereas the third one is provisionally composed
of E. sp. ‘ruidum 3+4’, although further biological,
morphological and molecular studies are needed to
determine whether the latter two morphospecies do
or do not represent valid species.
The delimited species E. sp. ‘ruidum 1’ and
E. sp. ‘ruidum 2’ share a similar, wide geographical
distribution in the Neotropics. Their allospecificity
was significantly supported by the separate and con-
catenated mt analyses, and also was confirmed by
the presence of exclusive external morphological fea-
tures of workers. Roger (1860) described E. ruidum
based on syntypes from Colombia, Brazil and French
Guiana. However, more recently Kugler & Brown
(1982) restricted its type locality to Colombia because
the syntypes from this country ‘would agree with the
present concept of E. ruidum’. According to the scan-
ning electron micrographs included in the latter
study, our proposed diagnostic morphological fea-
tures for the species assigned to E. sp. ‘ruidum 1’
agree with those of E. ruidum. Moreover, E. sp.
ruidum 1’ corresponds morphologically to the origi-
nal description of E. ruidum (Roger, 1860) and to the
photographs of their syntypes provided in the
Antweb website (Bolton, 2015). Ectatomma sp.
AB
CD
EF
Figure 4. Digital photographs showing the head (full face) and pronotal hump of representative specimens belonging to
the four delimited morphospecies originally assigned to Ectatomma ruidum:A,E. gibbum;B,E. sp. ‘ruidum 1’; C,
E. sp. ‘ruidum 2’; D, E. sp. ‘ruidum 3’; E, E. sp. ‘ruidum 4’; F, E. sp. ‘ruidum 29sp. ruidum 3’.
©2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016
SPECIES BOUNDARIES IN THE ECTATOMMA RUIDUM COMPLEX 11
ruidum 2’ thus represents an undescribed species,
which will be described elsewhere.
Based on the specimens examined in this study
and additional material deposited at IB UNAM,
E. sp. ‘ruidum 1’ occurs at least from south-eastern
Mexico on the border between Chiapas and Guate-
mala, to northern and north-eastern South America
(central Colombia, Venezuela and French Guiana) to
the south. On the other hand, our confirmed records
of E. sp. ‘ruidum 2’ indicate that this species extends
from Tamaulipas and Nayarit on the Atlantic and
Pacific coasts of Mexico, respectively, to south-wes-
tern Ecuador. Examination of reported populations
assigned to E. ruidum from northern Brazil (Bolton,
2015) are needed to determine which of the two pro-
posed species extends its southern distribution to
this country.
We decided to maintain the morphospecies
E. sp. ‘ruidum 3’ and E. sp. ‘ruidum 4’ as a single
evolutionary lineage because the haplotype genealo-
gies failed to consistently recover them as recipro-
cally monophyletic. Our records of this proposed
species are restricted to a few localities situated
along the south-eastern Pacific coast of Mexico in
Oaxaca and Guerrero, not far from the state of
Michoacan, the type locality of one of the current
synonyms of E. ruidum,E. aztecum (Emery, 1901).
Our examination of the syntype of E. aztecum
showed that it is quite similar to the specimens
assigned to this third species based on body sculpture
and the form of the petiolar node. Further integrative
biological, molecular and morphological studies are
therefore needed to clarify whether morphospecies
E. sp. ‘ruidum 3’ and E. sp. ‘ruidum 4’ actually rep-
resent separate species, and whether both or one of
them can be assigned to E. aztecum. The presence of
overlooked diversity and potential hybrids in this
group agrees with previous studies of other animal
taxa, which have revealed a complex evolutionary
history and overlooked species diversity along the
Mexican Pacific coast, and in particular near the
Isthmus of Tehuantepec in Oaxaca (e.g. Zarza, Rey-
noso & Emerson, 2008; Pedraza-Lara et al., 2015).
PRESENCE OF MICROGYNY
The two proposed species that share a wide geo-
graphical distribution in the Neotropics appear to
differ from each other by the presence/absence of
microgyny. All populations of E. sp. ‘ruidum 1’
inspected for the presence/absence of microgyny lack
it. These include populations situated in south-east-
ern Mexico, Costa Rica, Venezuela, French Guiana
and eastern Colombia (Breed et al., 1990, 1992,
1999; Gu
enard & McGlynn, 2013; J.-P. Lachaud,
unpubl. data). In contrast, gynecoid workers and
microgynes have been reported for populations
assigned to E. ruidum sp. ‘ruidum 2’ from south-east
Mexico, the Yucatan peninsula and western Colom-
bia (Fern
andez, 1991; Lachaud et al., 1999; Nettel-
Hernanz et al., 2015; F. C. Prada, pers. comm.).
The above two species are known to be sympatric
only in a region situated on the coastal plain near
the border between Chiapas in Mexico and Guate-
mala. Previous studies have revealed that both
species have similar life traits in this region, includ-
ing feigning of death when workers are disturbed
(Cupul-Maga~
na, 2009) and presence of the same par-
asitoid eucharitid wasp species (P
erez-Lachaud et al.,
2006; Lachaud & P
erez-Lachaud, 2015). However,
ongoing research supports that, in addition to the
presence/absence of microgyny, the two species can
also be discriminated from each other by other bio-
logical traits, such as date of sexuals production
(J.-P. Lachaud, unpubl. data), and chemical com-
pounds in hydrocarbon profiles (Gallegos-Aguirre,
1998; C. Poteaux, unpubl. data). Thus, we expect
that the integration of novel biological, morphological
and molecular data, as well as historical museum
material, can help to provide a robust, conclusive
delineation and description of species in this group.
ACKNOWLEDGEMENTS
We thank P. Hanson, J. Longino, F. Fern
andez, D.
Donoso, M. V
azquez-Bola~
nos and J. Ponce-Saavedra
for the loan/donation of specimens; F. C. Prada and
the ‘ants lab’ team in Cali, Colombia, for their help
in the field; C. Mayorga and G. Ortega for helping
with the curation of specimens; A. Jim
enez and L.
M
arquez for their help in the laboratory; and S. Guz-
man for taking the digital photographs. This study
was supported by grants given by the Consejo Nacio-
nal de Ciencia y Tecnolog
ıa (CONACyT: convocatoria
SEP-Ciencia B
asica 2014, proyecto no. 220454; Red
Tem
atica del C
odigo de Barras de la Vida; convocato-
ria fortalecimiento de infraestructura 2014, proyecto
no. 224743) to A.Z.R., and by a grant given by the
Ecos-Nord-CONACyT Program (proyecto no.
M12A01) to A.Z.R. and C.P. This study is part of the
MSc thesis of R.G.A.V., who received an MSc schol-
arship given by CONACyT. R.G.A.V. also thanks the
Posgrado en Ciencias Biol
ogicas, UNAM, for its sup-
port during her studies.
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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:
Table S1. List of specimens, their localities and voucher and GenBank accession numbers for COI, cyt band
H3.
Table S2. Main characteristics for the gene markers examined.
Table S3. Ranges of pairwise COI corrected distances within and among the four main lineages of specimens
assigned to E. ruidum that were recovered both by morphology and by the concatenated (COI +cyt b) data
set.
Data S1. Separate and concatenated matrices examined in this study for the COI, cyt b, 28S and H3 markers.
Figure S1. Bayesian phylograms of the separate analyses derived from the COI and cyt bdata sets including
all the examined sequences. Sequences considered as numts after the phylogenetic analyses are indicated with
a black line. Black circles near branches are Bayesian posterior probabilities 0.95.
Figure S2. Phylogram derived from the Bayesian analysis performed with the cyt bmarker excluding all
potential numts and hybrids. Black circles near branches are Bayesian posterior probabilities 0.95. Coloured
lines refer to the main lineages recovered. Taxon names refer to the delimited morphospecies.
Figure S3. Digital photographs showing side and dorsal views of trunk, petiole and gaster of representative
specimens belonging to the four delimited morphospecies originally assigned to E. ruidum:A,E. gibbum;B,
E. ruidum;C,E. sp. ‘ruidum 2’; D, E. sp. ‘ruidum 3’; E, E. sp. ‘ruidum 4’; F, E. sp. ‘ruidum 29sp. ruidum 3’.
Figure S4. Drawings showing the observed differences in the pronotal hump and petiolar node among the
delimited putative species belonging to the Ectatomma ruidum complex.
©2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2016
SPECIES BOUNDARIES IN THE ECTATOMMA RUIDUM COMPLEX 15
... Currently, E. ruidum is known to occur from northern Mexico in the state of Tamaulipas to central Brazil, and also on some Caribbean islands (Aguilar-Velasco et al., 2016). This species inhabits a wide range of environments from sea level to 1600 m of elevation (Kugler & Brown, 1982;Santamaría et al., 2009). ...
... Ectatomma ruidum is of particular interest in evolutionary studies due to its taxonomic complexity, wide geographic distribution, and presence of heteroplasmy (occurrence of more than one mtDNA type within an individual) in some of its populations. In the first phylogenetic study carried out for this species-complex, Aguilar-Velasco et al. (2016) proposed the existence of four evolutionary lineages plus a presumed hybrid population, based on examination of external morphology and nuclear and mtDNA sequence data. Two of these lineages (E. ...
... We used worker specimens assigned to E. ruidum collected from lo- We examined a 626-bp fragment of the cox1 gene for 250 specimens assigned to E. ruidum and one specimen of E. gibbum, employing the latter as the outgroup. Of these sequences, 107 and 12 were obtained from Aguilar-Velasco et al. (2016) and Meza-Lázaro et al. (2018), respectively, whereas 132 were newly generated. We excluded from the data set all potential nuclear mt paralogous sequences (numts) that were detected based on their presence of internal stop codons or when they had clearly incorrect phylogenetic relationships (Song et al., 2014). ...
Article
Full-text available
Geographic separation that leads to the evolution of reproductive isolation between populations generally is considered the most common form of speciation. However, speciation may also occur in the absence of geographic barriers due to phenotypic and genotypic factors such as chemical cue divergence, mating signal divergence, and mitonuclear conflict. Here, we performed an integrative study based on two genome‐wide techniques (3RAD and ultraconserved elements) coupled with cuticular hydrocarbon (CHC) and mitochondrial (mt) DNA sequence data, to assess the species limits within the Ectatomma ruidum species complex, a widespread and conspicuous group of Neotropical ants for which heteroplasmy (i.e., presence of multiple mtDNA variants in an individual) has been recently discovered in some populations from southeast Mexico. Our analyses indicate the existence of at least five distinct species in this complex: two widely distributed across the Neotropics, and three that are restricted to southeast Mexico and that apparently have high levels of heteroplasmy. We found that species boundaries in the complex did not coincide with geographic barriers. We therefore consider possible roles of alternative drivers that may have promoted the observed patterns of speciation, including mitonuclear incompatibility, CHC differentiation, and colony structure. Our study highlights the importance of simultaneously assessing different sources of evidence to disentangle the species limits of taxa with complicated evolutionary histories. We employed different sources of molecular information in order to assess the species limits of a taxonomically problematic Neotropical ant species complex. We found that species boundaries in this group do not coincide with geographic barriers, and therefore we suggest alternative drivers that may have promoted the observed patterns of speciation, including mitonuclear incompatibility, cuticular hydrocarbon differentiation, and colony structure.
... Currently, E. ruidum is known to occur from northern Mexico in the state of Tamaulipas to central Brazil, and also on some Caribbean islands (Aguilar-Velasco et al., 2016). This species inhabits a wide range of environments from sea level to 1600 m of elevation (Kugler & Brown, 1982;Santamaría et al., 2009). ...
... Ectatomma ruidum is of particular interest in evolutionary studies due to its taxonomic complexity, wide geographic distribution, and presence of heteroplasmy (occurrence of more than one mtDNA type within an individual) in some of its populations. In the first phylogenetic study carried out for this species-complex, Aguilar-Velasco et al. (2016) proposed the existence of four evolutionary lineages plus a presumed hybrid population, based on examination of external morphology and nuclear and mtDNA sequence data. Two of these lineages (E. ...
... We used worker specimens assigned to E. ruidum collected from lo- We examined a 626-bp fragment of the cox1 gene for 250 specimens assigned to E. ruidum and one specimen of E. gibbum, employing the latter as the outgroup. Of these sequences, 107 and 12 were obtained from Aguilar-Velasco et al. (2016) and Meza-Lázaro et al. (2018), respectively, whereas 132 were newly generated. We excluded from the data set all potential nuclear mt paralogous sequences (numts) that were detected based on their presence of internal stop codons or when they had clearly incorrect phylogenetic relationships (Song et al., 2014). ...
Preprint
Full-text available
Reproductive isolation between geographically separated populations is generally considered the most common form of speciation. However, speciation may also occur in the absence of geographic barriers due phenotypic and genotypic factors such as chemical cue divergence, mating signal divergence and mitonuclear conflict. Here we performed an integrative study based on two genome-wide techniques, 3RAD and ultraconserved elements, coupled with cuticular hydrocarbon and mtDNA sequence data, to assess the species limits within the E. ruidum species-complex, a widespread and conspicuous group of Neotropical ants for which heteroplasmy has been recently discovered in some populations from southeast Mexico. Our analyses indicate the existence of at least five distinct species in this complex, two widely distributed along the Neotropics and three that are restricted to southeast Mexico and that apparently have high levels of heteroplasmy. We found that species boundaries in the complex did not coincide with geographic barriers. We therefore consider possible roles of alternative drivers that may have promoted the observed patterns of speciation, including mitonuclear incompatibility, cuticular hydrocarbon differentiation, and colony structure. Our study highlights the importance of simultaneously assessing different sources of evidence to disentangle the species limits of taxa with complicated evolutionary histories.
... Ectatomma ruidum Roger is a mainly Neotropical ant species that exhibits the peculiar intraspecific behaviour named cleptobiosis (food thievery between neighbouring colonies), and that was recently discovered to be a species complex (Aguilar-Velasco et al. 2016). Recent molecular studies have shown that some species within the complex have faced selection in molecular traits (mitochondrial DNA) (Meza-Lázaro et al. 2018). ...
... Cuticular hydrocarbons serve as a chemical signature that conveys information about castes, task specialisation, and reproductive status and allow social insects to discriminate between nestmates and foreigners (Blomquist and Bagnères 2010;Van Zweden and d'Ettorre 2010). In contrast to the molecular and chemical diversification of the E. ruidum species complex, morphology is relatively conserved across the species (Aguilar-Velasco et al. 2016), while knowledge about the acoustic organ morphology and characteristics of the sounds produced is scarce. ...
... Yet, there seems to be no clear association between geographic distance and sound differentiation. According to Aguilar-Velasco et al. (2016), E. ruidum sp. 2 and sp. 3-4 are located at less than 25 km distance from each other, and the current results show that their stridulations did not differ significantly. ...
Article
Social insects communicate by using chemical, visual, tactile, and acoustic signals, including stridulations. Ectatomma ruidum is a mainly Neotropical ant species complex that has faced strong divergence at the genetic level; the species have a highly variable blend of cuticular hydrocarbons and a relatively conserved morphology. Based on evidence for genetic and chemical differentiation, we tested for variation in acoustic traits. We compared the stridulations produced by the species E. ruidum sp. 2, sp. 3–4 and the new putative species E. ruidum sp. 5, as well as the morphology of the stridulatory file. We found that the stridulations produced by E. ruidum sp. 5 were statistically different from those of the other species in a number of traits. The differences in stridulatory traits might rely more on the way the ants produce the sound (rubbed area percentages) than on the morphology of the stridulatory file, for which we did not find variation. Our results highlight the use of acoustic traits as potential taxonomic tools for integrative taxonomic studies and suggest that the acoustic traits of E. ruidum species complex have been subjected to selection.
... Ectatomma ruidum Roger is a mainly Neotropical, widely distributed ant taxon that is likely to include a cryptic species diversity, which only has been recently started to be unveiled. Two recent studies based on mitochondrial (mt) and nuclear DNA sequence data and morphological evidence revealed that this taxon actually represents a species complex (Aguilar-Velasco et al. 2016;Meza-Lázaro et al. 2018), though its species delineation still remains unclear. In the first of these studies, morphological data indicated the existence of four morphospecies, two of which apparently are widespread along the Neotropics (E. ...
... Specifically, we tested for the intraspecific variation of the cuticular hydrocarbon profiles between populations of E. ruidum sp. 3-4 from southeast Mexico, whose boundaries remain controversial (Aguilar-Velasco et al. 2016;Meza-Lázaro et al. 2018). ...
... Ectatomma ruidum is an earth-dwelling ant that occurs in a wide range of habitats in Neotropics, including plantations and damp forests, from sea level up to 1600 m Aguilar-Velasco et al. 2016). Typically, E. ruidum nests have a single entrance and contain between 50 and 200 individuals (Lachaud 1985) and their densities can be very high (up to 11,200 nests per hectare, Schatz and Lachaud 2008). ...
Article
Full-text available
In social insects, chemical communication is the main communication mode among colony members, which use the blends of cuticular hydrocarbons as recognition cues to discriminate between nestmates and non-nestmates and to prevent the exploitation of their nest resources by aliens. The aim of this study was to assess the variation of nestmate recognition cues in the ant Ectatomma ruidum, a species complex with a considerably conserved morphology and one of the few ant species where intraspecific thievery, a form of cleptoparasitism, has been reported. We analyzed the cuticular hydrocarbon profiles of ants collected from a number of geographically separated populations and examined DNA sequence data to assess their species identity. We focused on one species of the complex, E. ruidum sp. 3–4, whose species delineation remains controversial. We documented that several quantitative and qualitative traits of the cuticular hydrocarbon profiles varied significantly between populations, indicating that this species harbors more cuticular chemical phenotypic diversity than expected within a single species. In particular, there was a striking divergence among populations in the proportion of methylalkanes, alkenes, alkadienes and odd-chain components, which likely play a major role in nestmate/non-nestmate discrimination, a process which might have been crucial in these cleptobiotic ants. Further investigations are needed to test the hypothesis that biotic pressures, such as the need to discriminate conspecific intruders and limit thievery, could have played an important role in promoting the evolutionary divergence between populations in this ant species complex.
... Historically, the dominant E. ruidum had been considered as a single, though very variable, species distributed from central Mexico to northern Brazil, from sea level to 2200 m asl 51 . However, molecular analyses have recently shown that the species is a complex of at least three cryptic species with very little morphological variation 30,49,52,53 . Two of these species have a wide distribution in the neotropics; the first one corresponds to the syntype of E. ruidum described by Roger 54 and the second, Ectatomma sp. 2, is considered as a new, still undescribed species. ...
... The third species, Ectatomma sp. 3, also undescribed, is endemic of the southern Mexican Pacific coast of Oaxaca. Based on confirmed records, Ectatomma sp. 2 extends from Tamaulipas and Nayarit on the Atlantic and Pacific coasts of Mexico, to southwestern Ecuador 30 . This species nests in the soil, and colonies show highly variable social structure and behavior 49 . ...
... Molecular analysis of the ant species concerned in the present study was performed within the framework of a previous work focused on delimiting the species boundaries of the E. ruidum complex 30 . Barcoding confirmed that the population sampled in the present study corresponded to Ectatomma sp. 2 (Genbank accession number: KU570627.1) ...
Article
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Ant parasitoidism has been reported in seven of the 26 recognized species of the mite genus Macrodinychus (Machrodynichidae). Macrodynichus sellnicki, previously reported as a parasitoid of the invasive ant Nylanderia fulva in Colombia, is now reported, in the same region, as attacking a native host, Ectatomma sp. 2 (E. ruidum complex). The mite develops within the protective silk cocoon of an Ectatomma pupa and waits for the emergence of the young ant before leaving the cocoon, unmolested. Overall nest prevalence was relatively high (34.6% of the 52 nests containing cocoons) but pupae prevalence was low (4.0%, n = 1401 cocoons). Mite life-history (parasite or parasitoid) was context dependent, shifting according to the intensity of the attack on a same host. Contrary to the strictly parasitoidic association of M. sellnicki with N. fulva, single mite attacks against E. ruidum did not result in host killing and solitary M. sellnicki (78.6% of the cases) behaved as parasites. However, in 21.4% of the attacks (0.9% of all available host pupae) more than one mite was involved and behaved as parasitoids, draining the host of its internal fluids and killing it. This is the first association of a macrodinychid mite with a species of the subfamily Ectatomminae, and the first ant associated mite for which such a context dependent life-style shift is described.
... goninion, E. ruidum, E. tuberculatum) vary widely in color and sculpture, the structure of their populations, their biology, and their reproductive strategies [7]. In fact, such variability hides cryptic species complexes (i.e., organisms that are described as a single nominal species due to their highly similar morphology) [1,6,7], so that several new species will probably be described in the future. Delimiting species can be challenging, as some species exhibit extensive heteroplasmy (i.e., the coexistence of more than one mitogenome within an individual), which is responsible for mtDNA polymorphism. ...
... Based on mtDNA gene analysis, Azuma et al. (2006) proposed that the Asian weaver ant (Oecophylla smaragdina) consists of two major groups with seven geographical subgroups, and inferred its dispersal history over a geological time scale. Indeed, genetic information on widely distributed species can reveal the phylogeographic history of those species and also provide useful taxonomic information for species delimitation (Steiner et al. 2010;Aguliar-Velasco et al. 2016). The 12S ribosomal RNA (12S) region has been widely used to infer the intra-and interspecific relationships of vertebrates (macaques by Evans et al. 1999, Tosi and Coke 2007, Ziegler et al. 2007, Evans et al. 2003b; rabbits by Surridge et al. 1999;squirrels by Mercer and Roth 2003;frogs by Evans et al. 2003a; tigers by Luo et al. 2004; water skinks by Honda et al. 2005; medaka fishes by Takehana et al. 2005) and insects (Pheidole ants by Moreau 2008; paper wasps by Santos et al. 2015; flightless moths by Liu et al. 2015;crickets by Dong et al. 2018; diving beetles by Balke et al. 2018). ...
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The myrmicine species Crematogaster rothneyi is one of the most widely distributed ants in Asia, but it has rarely been collected in the field. Its distribution range covers South and Southeast Asia, extending approximately 5,000 km from India in the west to Sulawesi in the east. Despite this wide distribution range, C. rothneyi has been treated as a single taxonomic species, and no combined morphological or molecular analysis has been conducted to assess whether any intraspecific variation exists. The sequence divergences of C. rothneyi populations, mainly obtained from Southeast Asia, were investigated by analyzing 387 bp and 175 bp sequences of the 12S ribosomal RNA and cytochrome c oxidase subunit (COI) genes, respectively. Phylogenetic analysis indicated that the C. rothneyi populations were separated into three groups: group I from Thailand and Cambodia, group II from Bangladesh and Myanmar, and group III from Krakatau and Sulawesi. Groups II and III were recovered as a single clade, sister to group I. The interspecific divergences were 7.3% to 8.5% for 12S and 14.5% to 23.3% for COI between most C. rothneyi specimens and Cambodian specimens, while divergence for 12S was 3.5% between Thai and Cambodian specimens. The Thai specimens are not morphologically differentiated, and are considered conspecific to C. rothneyi. We describe the Cambodian series as a new species, Crematogaster yaharai sp. nov., based on unique antennal morphology and mesosomal sculpture patterns with molecular evidence. Crematogaster rothneyi civa Forel, 1902 is synonymized under C. rothneyi. Crematogaster rothneyi is widespread and has sister species, suggesting recent diversification within the Indochinese Peninsula. Citation: Hosoishi S, Ogata K. 2019. Cryptic diversity of the widespread Asian ant Crematogaster rothneyi (Hymenoptera: Formicidae) inferred from morphological and genetic evidence. Zool Stud 58:11.
... Previous studies have shown that eusociality in hymenopterans leads to small effective population sizes, since most individuals are sterile workers (Chapman and Bourke 2001;Packer and Owen 2001), which could cause a marked mt genetic structure. A marked genetic structure has been found to occur in some widely distributed Neotropical ant species, such as in members of the S. saevissima (Smith) and Ectatomma ruidum (Roger) species complexes (Ross et al. 2010;Aguilar-Velasco et al. 2016) and in species of Linepithema Mayr (Wild 2009). In our study, we did not observe a considerable genetic structure in N. villosa along its sampled geographic distribution. ...
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