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Extensive mitochondrial heteroplasmy in the neotropical ants of the Ectatomma ruidum complex (Formicidae: Ectatomminae)

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We assembled mitogenomes from 21 ant workers assigned to four morphospecies (E. ruidum spp. 1-4) and putative hybrids of the Ectatomma ruidum complex (E. ruidum spp. 2x3), and to E. tuberculatum using NGS data. Mitogenomes from specimens of E. ruidum spp. 3, 4 and 2 × 3 had a high proportion of polymorphic sites. We investigated whether polymorphisms in mitogenomes are due to nuclear mt paralogues (numts) or due to the presence of more than one mitogenome within an individual (heteroplasmy). We did not find loss of function signals in polymorphic protein-coding genes, and observed strong evidence for purifying selection in two haplotype-phased genes, which indicate the presence of two functional mitochondrial genomes coexisting within individuals instead of numts. Heteroplasmy due to hybrid paternal leakage is not supported by phylogenetic analyses. Our results reveal the presence of a fast-evolving secondary mitochondrial lineage of uncertain origin in the E. ruidum complex.
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Extensive mitochondrial heteroplasmy in the
neotropical ants of the Ectatomma ruidum
complex (Formicidae: Ectatomminae)
Rubi N. Meza-Lázaro, Chantal Poteaux, Natalia J. Bayona-Vásquez, Michael
G. Branstetter & Alejandro Zaldívar-Riverón
To cite this article: Rubi N. Meza-Lázaro, Chantal Poteaux, Natalia J. Bayona-Vásquez, Michael
G. Branstetter & Alejandro Zaldívar-Riverón (2018): Extensive mitochondrial heteroplasmy in the
neotropical ants of the Ectatomma ruidum complex (Formicidae: Ectatomminae), Mitochondrial
DNA Part A, DOI: 10.1080/24701394.2018.1431228
To link to this article: https://doi.org/10.1080/24701394.2018.1431228
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Published online: 31 Jan 2018.
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RESEARCH ARTICLE
Extensive mitochondrial heteroplasmy in the neotropical ants of the
Ectatomma ruidum complex (Formicidae: Ectatomminae)
Rubi N. Meza-L
azaro
a
, Chantal Poteaux
b
, Natalia J. Bayona-V
asquez
c
, Michael G. Branstetter
d
and
Alejandro Zald
ıvar-River
on
a
a
Colecci
on Nacional de Insectos, Instituto de Biolog
ıa, Universidad Nacional Aut
onoma de M
exico, Circuito Exterior s/n, Ciudad Universitaria,
CdMx, M
exico, M
exico;
b
Laboratoire dEthologie Exp
erimentale et Compar
ee E.A. 4443 (LEEC), Universit
e Paris 13, Sorbonne Paris Cit
e,
Villetaneuse, France;
c
Department of Environmental Health Science, University of Georgia, Athens, GA, USA;
d
USDA-ARS Pollinating Insects
Research Unit, Utah State University, Logan, UT, USA
ABSTRACT
We assembled mitogenomes from 21 ant workers assigned to four morphospecies (E. ruidum spp. 1-4)
and putative hybrids of the Ectatomma ruidum complex (E. ruidum spp. 2x3), and to E. tuberculatum
using NGS data. Mitogenomes from specimens of E. ruidum spp. 3, 4 and 2 3 had a high proportion
of polymorphic sites. We investigated whether polymorphisms in mitogenomes are due to nuclear mt
paralogues (numts) or due to the presence of more than one mitogenome within an individual (hetero-
plasmy). We did not find loss of function signals in polymorphic protein-coding genes, and observed
strong evidence for purifying selection in two haplotype-phased genes, which indicate the presence of
two functional mitochondrial genomes coexisting within individuals instead of numts. Heteroplasmy
due to hybrid paternal leakage is not supported by phylogenetic analyses. Our results reveal the pres-
ence of a fast-evolving secondary mitochondrial lineage of uncertain origin in the E. ruidum complex.
ARTICLE HISTORY
Received 1 November 2017
Accepted 18 January 2018
KEYWORDS
Mitochondrial genome;
Formicidae; heteroplasmy;
paternal leakage; numts;
phasing haplotypes;
hymenoptera
Introduction
The tenets of inheritance and evolution of mitochondrial (mt)
DNA in animals are widely accepted and constitute the basis
for its use in phylogenetics and phylogeography. MtDNA is
known to be maternally inherited and nonrecombinant (Birky
1995,2001; Ballard and Rand 2005). As a consequence, all
copies of mtDNA of an individual are expected to be identi-
cal, a condition known as homoplasmy (Ling et al. 2011).
Homoplasmy could be achieved through processes that pre-
vent paternal mtDNA transmission, such as having a low pro-
portion of paternal mtDNA in the gametes, by elimination of
sperm mitochondria within the egg cytoplasm or by reducing
the population of mtDNA molecules during oogenesis and
morphogenesis (Sato and Sato 2013).
Recent advances in DNA sequencing and polymorphism
detection technology have made it possible to test the exist-
ence of heteroplasmy, i.e. the presence of more than one
type of organellar genome within a cell or individual
(Robertson and Williams 2009; Magnacca and Brown 2010).
Heteroplasmy is a common phenomenon in fungi, protists
and plants, and is being increasingly found in animals (Kann
et al. 1998; Barr et al. 2005; Kmiec et al. 2006; Mat
e et al.
2007; Mjelle et al. 2008). In animals, heteroplasmy is known
to occur mainly by paternal leakage of mtDNA, and so far, it
has been reported in mammals, birds, reptiles, fish, mollusks,
nematodes, bivalves and some groups of arthropods
(Skibinski et al. 1994; Chinnery and Turnbull 2000; Magnacca
and Brown 2010). In insects, paternal leakage resulting in het-
eroplasmy has been shown to occur in honeybees (Meusel
and Moritz 1993), flies (Drosophila; Nunes et al. 2013) and
cicadas (Robison et al. 2015).
Detection of heteroplasmy can be challenging because
parental haplotypes are often highly similar, which makes
non-maternal haplotypes difficult to detect, though hybrid
zones can be an exception (Kvist et al. 2003). To date, the
majority of confirmed cases of mtDNA heteroplasmy caused
by paternal leakage are from interspecies or interpopulation
hybridization events, which heighten the genetic dissimilarity
between parental mtDNAs increasing the chance of detection
(Kaneda et al. 1995; Shitara et al. 1998; Sutovsky et al. 2000).
The effects of heteroplasmy and mt recombination on
phylogenetic reconstruction have been poorly explored and
are not well understood. If heteroplasmy is rare and transient
then its effects on evolutionary analyses should be negligible.
It has been shown, however, that in some cases heteroplasmy
can persist in the germ line for several generations and even
remain long enough for recombination (Zsurka et al. 2007).
Therefore, persistent heteroplasmy might have substantial
impact on genealogical analyses based on mtDNA sequence
data, affecting coalescent reconstructions and parameters
that are dependent on such estimates (White et al. 2008). For
instance, it has been suggested that past mt genetic
CONTACT Alejandro Zald
ıvar-River
on azaldivar@ib.unam.mx Colecci
on Nacional de Insectos, Instituto de Biolog
ıa, Universidad Nacional Aut
onoma de
M
exico, Circuito Exterior s/n, Ciudad Universitaria, CdMx, M
exico
Supplemental data for this article can be accessed here.
ß2018 Informa UK Limited, trading as Taylor & Francis Group
MITOCHONDRIAL DNA PART A, 2018
https://doi.org/10.1080/24701394.2018.1431228
exchange and recombination of coexisting mtDNA molecules
can explain reticulations in phylogenetic networks (Anderson
et al. 2001; Arunkumar et al. 2006; Zsurka et al. 2007,2010).
As deep sequencing (i.e. high per base coverage) enables
the efficient detection of even low-levels of DNA hetero-
plasmy, high-throughput DNA sequencers represent a power-
ful tool to identify mitochondrial heteroplasmy and to
quantify its prevalence (Huang 2011). Here, we use Illumina
sequencing to examine mitogenome structure and the occur-
rence of heteroplasmy in a group of Neotropical ants.
With more than 13,000 recognized species (AntCat; avail-
able from https://www.antcat.org. Accessed 2017 Sep 20),
ants represent one of the most successful radiations of terres-
trial Metazoa, monopolizing about 1520% of the terrestrial
animal biomass (Schultz 2000). Despite their importance,
mitogenomes of only a dozen ant species have been
described and annotated thus far with all of them belonging
to the formicoid clade of ants. Ant mitogenomes are known
to vary from 15 to 18.7 Kb (de MeloRodovalho et al. 2014),
with their gene content and order being highly conserved
except for tRNA. Mitogenome size variation in ants has been
associated with the presence/absence of intergenic spacers
(IGSs), which are noncoding DNA regions between genes
(R
edei 2008), and TA tandem repeats in the control region
(de MeloRodovalho et al. 2014; Yang et al. 2016). Several
features of IGSs (e.g. high AT content, presence of hairpin
and cloverleaf secondary structures) suggest that they might
contain an origin of replication (Cornuet et al. 1991). Most
IGSs appear to be unique to each species (Sheffield et al.
2008); however, there are also conserved IGSs present in all
insect groups and these appear to be associated with the
binding site of mtTERM, a transcription attenuation factor
(Taanman 1999; Sheffield et al. 2008; Wei et al. 2010).
The ant genus Ectatomma currently includes 15 described
species and is distributed throughout most of the Neotropics
and the southernmost part of the Nearctic (AntWeb; https://
www.antweb.org. Accessed 2017 May 9). Ectatomma ruidum
(Roger 1860) is, together with E. tuberculatum (Olivier 1792),
one of the most widely distributed species in the genus,
occurring from northern Mexico in Tamaulipas to northern
Brazil (Kugler and Brown 1982; AntWeb; https://www.antweb.
org. Accessed 2017 May 9; unpubl. data). A number of studies
have focused on ecological and behavioural aspects of this
species, since it is regarded as an important natural biological
control agent against cotton, coffee, cocoa and maize pests
in various Neotropical countries (Perfecto and Sediles 1992;
IbarraNu~
nez and Garcia 2001).
Recent phylogenetic studies among populations of E. rui-
dum using mt (cox1,cob) and nuclear (H3, wingless) markers
and morphology revealed that this species actually represents
a complex composed of at least three species
(NettelHernanz et al. 2015; Aguilar-Velasco et al. 2016). Two
of these species have a wide Neotropical distribution,
whereas the third one appears to be restricted to the south-
eastern portion of the Mexican Pacific coast, where it was
found to have considerably high intraspecific mt genetic dis-
tances. Within the latter species, some specimens were pro-
posed to constitute either an additional species or be of
hybrid origin, though this could not be confirmed (Aguilar-
Velasco et al. 2016). The authors also reported polymorphic
sites in the two mt markers and odd phylogenetic positions
for some sequences, which was suggested to occur due to
amplification of nuclear mt paralogues (numts; Song et al.
2014). However, no signals of gene functionality loss, such as
internal stop codons or frame shifts, were detected in the
polymorphic or in the phylogenetically incongruent sequen-
ces, leaving their origin unconfirmed. Re-amplification of
problematic samples with a pre-PCR dilution of DNA template
recovered homozygous chromatograms, which were consid-
ered as putative mt orthologues.
In this study, we investigated whether the origin of
mtDNA polymorphism in the E. ruidum complex is due to
numts or heteroplasmy. To do this, we obtained 24 mitoge-
nomes generated with high-throughput DNA sequencing
(shotgun Illumina sequencing and by-products of ultracon-
served element (UCE) enriched genomic libraries) of speci-
mens assigned to the four species proposed by Aguilar-
Velasco et al. (2016) based on morphology. Mitogenomes
were also characterized with respect to their size, genome
architecture, gene content/arrangement, genetic code and
secondary RNA structure. Our results reveal extensive hetero-
plasmy in the E. ruidum complex and highlight the need for
similar studies to be done across diverse taxa to better
understand the origin and extent of this phenomenon in
metazoans.
Methods
Taxon sampling
We sampled mitogenomes from a total of 21 individual
Ectatomma worker ants, of which 15 were sequenced via
standard shotgun Illumina methods and nine from UCE target
enrichment methods (mitogenome data obtained from off-
target reads). We sequenced three of the ant samples using
both methods to allow us to check for possible sample con-
tamination and library preparation or sequencing errors. We
sampled 15 specimens belonging to the four E. ruidum mor-
phospecies reported in Aguilar-Velasco et al. (2016) (hereafter
referred as E. ruidum spp. 1-4), two specimens representing
possible hybrids between E. ruidum sp. 2 and 3 (2 3), and
four specimens of E. tuberculatum, which we included as the
outgroup. We also generated four cox1 and seven cob
sequences belonging to males of E. ruidum spp. 3, 4 and
23 to explore whether males also had polymorphic sites
(double peaks) and thus also are heteroplasmic (see below).
We generated these sequences with Sanger sequencing and
the same laboratory procedures described in Aguilar-Velasco
et al. (2016). A list of the examined specimens, their locality
details and GenBank accession numbers for the generated
mitogenomes is provided in Supplemental Table S1.
Laboratory procedures
High-molecular weight genomic DNA was extracted using the
Spin Column Genomic DNA Minipreps kit (Bio Basic 5) follow-
ing the manufacturers protocol. Shotgun library preparation
was carried out using 200 ng of DNA that was previously
2R. N. MEZA-L
AZARO ET AL.
sheared by sonication with a Bioruptor Pico instrument
(Diogenode, Liege, Belgium). Sonication was performed using
three to five cycles of alternating 30 s ultrasonic bursts and
30 s pauses in a 4 C water bath. Library preparation was per-
formed using the Kapa Biosystems Hyper Prep Kit (Kapa
Biosystems Inc., Wilmington, MA) following the man-
ufacturers protocol. DNA fragments were attached to custom,
TruSeq-style dual-indexing adapters (Glenn et al. 2016) and
size selected to a range of 200400 bp. Size selected frag-
ments were enriched through PCR and subsequently purified
and normalized. The Illumina NextSeq v2 300 cycle kit was
used for library sequencing to produce paired-end 150
nucleotide reads at the Georgia Genomics Facility, University
of Georgia, Athens, USA.
We extracted additional mitogenomes from samples being
sequenced primarily for UCE data that we will use in a further
phylogenomic study of Ectatomma. Samples sequenced for
UCE loci were prepared following the protocol outlined in
Branstetter et al. (2017). Briefly, for each sample up to 50 ng
of input DNA was sheared to a mean fragment size of
400600 bp using a Qsonica Q800R sonicator (Qsonica 457
LLC, Newton, CT). Sequencing libraries were subsequently
built using the Kapa Hyper Prep kit (Kapa Biosystems Inc.,
Wilmington, MA) and custom, TruSeq-style dual-indexing
adapters (Glenn et al. 2016). Enrichment of UCE loci was per-
formed following a standard enrichment protocol (version 1.5
available from ultraconserved.org) and a custom RNA bait
library developed for ants (Branstetter et al. 2017) and syn-
thesized by MYcroarray (MYcroarray, Ann Arbor, MI). The bait
set included 9898 baits targeting 2524 UCE loci. Following
enrichment, up to 100 samples, including samples not used
in this study, were pooled at equimolar concentrations and
sent to the University of Utah genomics core facility for
sequencing on an Illumina HiSeq 2500 instrument (PE125, v4
chemistry).
Mitogenome assembly and annotation
Raw Illumina data were demultiplexed by the sequencing
centre, and trimmed and filtered using Geneious version
10.0.7 (http://www.geneious.com, Kearse et al. 2012).
Mitogenome assembly was carried out using both de novo
and reference-based assembly approaches with the programs
MITObim version 1.9 (Hahn et al. 2013), CLC Genomics
Workbench version 9.5 (http://www.clcbio.com) and Geneious
version 10.0.7 (http://www.geneious.com, Kearse et al. 2012).
No single strategy allowed for the assembly of complete (cir-
cular and closed) mitogenomes, and thus we used a combin-
ation of approaches. The following five complete ant
mitogenomes were used for reference-based assembly:
Solenopsis richteri (NC_014677.1), Myrmica scabrinodis (NC_
026133.1), Formica selysi (NC_026711.1), Camponotus atrox
(KT159775.1) and Pristomyrmex punctatus (AB556947.1). A
consensus of these mitogenomes was also used as reference.
The longest contigs generated by the de novo and refer-
ence based assemblies were used for assembling longest con-
tigs with the program Geneious v10.0.7. This strategy allowed
for the assembly of contigs that overlapped forming longer
sequences that were then used as templates for subsequent
assemblies. All mitogenomes were independently assembled
because unique gene orders and long tandem TA repeat
regions precluded the use of a single specimens mitoge-
nome as a reference. De novo annotation was accomplished
with the mt genome annotation server MITOS (Bernt et al.
2013) set to use the invertebrate genetic code. The existence
of numts was assessed by examining each mitogenome for
the presence of internal stop codons and frame-shifts. Base
composition was calculated with Geneious version 10.0.7.
Illumina raw data were submitted to the Sequence Read
Archive under the Bioproject accession no. PRJNA431513.
Numts, heteroplasmy and mt recombination tests
From a total of 24 recovered mitogenomes, there were nine
that contained a considerable number of polymorphic sites
across all their genes. These nine were assembled from seven
specimens that were identified as E. ruidum sp. 3, 4 and 2 3
(six mitogenomes obtained with shotgun Illumina sequenc-
ing, three with UCEs, and two with both techniques). Variants
were identified using the Find Variationcommand in
Geneious version 10.0.7 (Kearse et al. 2012) with the follow-
ing parameters: minimum coverage ¼5, minimum variant
frequency¼0.25, maximum variant pvalue ¼610 5
(approximate calculation method).
For the polymorphic mitogenomes, we phased two com-
plete, noncontiguous, protein-coding genes [cytochrome oxi-
dase I (cox1); cytochrome b(cob)] into separate haplotypes.
We then reconstructed gene genealogies for each gene using
all phased haplotypes to evaluate whether or not the
observed polymorphism was the product of heteroplasmy
originating from hybridization and paternal leakage. Phasing
of haplotypes was carried out manually, calling those variants
across different positions to the same haplotype if they were
present in the same read. The two haplotypes from the same
individual were labelled as A and B for genetic distance esti-
mation, recombination and phylogenetic analysis. Both var-
iants were translated to amino acids to search each sequence
for possible frameshift changes and/or internal stop codons,
which would serve as evidence for the presence of numts.
The GenBank accession numbers of the phased haplotypes
are listed in Supplemental Table S1.
We assessed recombination events following two
approaches. First, we carried out phylogenetic network analy-
ses with the phased cox1 and cob haplotypes. This method
provides an explicit representation of incompatibilities within
and between data sets (Huson and Bryant 2006). Unrooted
phylogenetic networks were separately and simultaneously
reconstructed for both mt gene data sets with the program
Splitstree4 version 4.14.4 (Huson and Bryant 2006) using K2P
corrected distances and the NeighborNet algorithm (Huson
1998; Huson and Bryant 2006). A Phi recombination test was
carried out with the latter program.
We also evaluated recombination for the phased haplo-
types with the program TOPALi version 2.5 (Milne et al.
2009). This program searches for a recombination signal,
which is expressed as a significant difference in the sum-of-
squares (DSS) peak along the sequence alignment using the
DSS with a 100-bp window and a 10-bp step-size. The
MITOCHONDRIAL DNA PART A 3
statistical significance of DSS was assessed using 100 para-
metric bootstraps. Codon-based Z-tests of selection were per-
formed to estimate the probabilities of positive, neutral and
purifying selection in MEGA version 7.0 (Kumar et al. 2016)
on phased haplotypes of cox1 and cob.
Genealogical reconstruction
We examined phylogenetic relationships using the following
matrices: (1) a concatenated protein-coding gene matrix for
the complete mitogenomes, (2) separate cox1 and cob matri-
ces including the phased haplotypes, (3) separate cox1 and
cob matrices including the phased haplotypes and all sequen-
ces included in Aguilar-Velasco et al. (2016). Protein-coding
genes were separately aligned with the program MACSE ver-
sion 0.9b1 (Ranwez et al. 2011, available at http://mbb.univ-
montp2.fr/macse). This program aligns protein-coding gene
data sets, taking into account their potential amino acid
translation while preserving their codon structure. For the
concatenated protein-coding gene matrix using the complete
mitogenomes, each consensus sequence that was generated
from the Illumina data was built from the most frequent resi-
dues that were represented in at least 50% of the rows at
each site (alignment column).
The program PartitionFinder version 2 (Lanfear et al. 2017)
was employed to select the best-fit partitioning scheme and
evolutionary models for the concatenated protein-coding
gene matrix based on the Bayesian Information Criterion (BIC)
and the greedy algorithm option. This program was run using
a priori data blocks that corresponded to each gene and
codon position. This yielded a three-partition scheme
(Supplemental Table S2) that we implemented for subse-
quent phylogenetic analyses.
We performed a Bayesian phylogenetic analysis for all
matrices using the program MrBayes version 3.2.6 (Ronquist
et al. 2012). For each MrBayes analysis, two simultaneous runs
of 100 million generations were performed, each with four
chains. Uniform priors were selected for all parameters and
trees were sampled every 10,000 generations. Convergence of
runs was visually confirmed based on the potential scale
reduction factors and the average deviation of split frequen-
cies. Burn-in was determined to occur after 25 million genera-
tions, and the remaining trees were used to reconstruct a
majority rule consensus tree with posterior probability of
clades. We also carried out a Maximum Likelihood (ML) ana-
lysis for the concatenated protein-coding gene matrix using
the program RAxML version 8.2.10 (Stamatakis 2014) and the
GTRGAMMA model of sequence evolution. Node support was
assessed by performing non-parametric bootstrap replicates of
the input data with the number of replicates determined by
the autoMRE function in RAxML.
P-distances for the concatenated coding gene matrix were
calculated within and between morphospecies using MEGA
version 7.0 (Kumar et al. 2016). We also calculated p-distances
separately for the cox1 and cob genes between haplotypes
found within the same individuals (see below). When two
library preparation methods were used for the same sample,
we also obtained p-distances between the most and less fre-
quent haplotypes.
Results
Mitogenome assembly and annotation
A total of 24 mitogenomes were recovered from 21
Ectatomma specimens. The mean sequence coverage was
from 8.5 to 18.5, 26.6 to 70.4 and 38.4 to 153.6X in E. ruidum
spp. 1, 2 and 3, 4 and 2 3, respectively. The main features
(genome size and coverage) of the mitogenomes that were
generated are listed in Table 1. Mitogenomes of E. ruidum sp.
1 and E. tuberculatum could not be closed-circular, because
their control region and tRNAs were not recovered in assem-
bly. The size of the complete mitochondrial genomes varied
from 17,365 to 17,500 bases. Complete genomes contained
13 protein-coding loci, 22 tRNAs and two rRNAs, and had a
considerably high A þT-bias, which is typical of insect
mtDNA. All 13 protein-coding genes of Ectatomma began
with the typical start codon ATN and end with TAA and less
frequently with TAG.
Figure 1 provides a comparison of mitogenome gene
structure between Ectatomma, other ants and an ancestral
arthropod/hymenopteran. All protein-coding genes and
rRNAs for the examined species of Ectatomma are oriented in
the same direction and order as in all other hymenopterans
and the hypothetical ancestral arthropod mitogenome. tRNA
genes of all reconstructed mitogenomes are also oriented in
the same direction; however, they have an arrangement
between rrnS and nad2 (Figure 1(a,b)), which was previously
unknown among insects. The tRNA arrangement for the
amino acids QMI differs from the IQM organization of ances-
tral insects, and MIQ differs from all other ant mitogenomes
assembled to date.
Also, the mitogenomes in the E. ruidum complex have a
tRNA gene arrangement between nad3 and nad5 genes that
was previously unknown among insects (Figure 1(a)): tRNAs of
amino acids SAERNF differs from the ARNSEF arrangement typ-
ical of the ancestral pancrustacean mitogenome. Two overlap-
ping regions in the examined mitogenomes, a three-
nucleotide overlap positioned between atp8 and atp6 and a
2024 nucleotide overlap between trnL1 and trnL
(Supplemental Table S3), and a one-nucleotide overlap
between trnR and trnN, were also detected. All overlapping
genes occupied the same strands. All mitogenomes contained
IGSs that spanned a total 1940 bp (Supplemental Table S3).
IGSs occurred between almost all genes (3134 out of the 37
possibilities), and each ranging from 3 to 134 bp. All predicted
tRNA molecules had the typical cloverleaf structure excluding
trnS1 (data not shown).
All specimens assigned to E. ruidum spp. 3, 4 and 2 3
were found to have from 1.88% to 5.81% polymorphic sites
spread across their mitogenomes (Figure 2,Table 2).
Alternative states of protein-coding genes neither contained
any indels nor nucleotide substitutions that would produce
frameshifts or internal stop codons.
The cox1 and cob genes were phased in all but two
specimens assigned to E. ruidum sp. 2 3 (DNA voucher
nos. CNIN2078 and CNIN2087, from Pinotepa Nacional)
which had overly wide separations between contiguous
variable sites, disallowing assignment of alternative states
to one or the other haplotype. The cox1 translation
4R. N. MEZA-L
AZARO ET AL.
showed that only 2.6 to 13.5% of the nucleotide differen-
ces between the two phased haplotypes of each individual
yielded non-synonymous substitutions and most of these
involved substitutions to amino acids in the same family
(Supplemental Table S4).
No statistically significant evidence for recombination was
found by the phy (phi test: p¼.8706, .8184 and .4936,
respectively) or DSS tests. The Z-tests of selection showed sig-
nificant deviations from neutrality, with d
S
being greater than
d
N
for all comparisons between intraindividual haplotypes,
suggesting that the DNA sequence is under negative or puri-
fying selection (Supplemental Table S5).
Phylogenetic analyses and detection of heteroplasmy
Bayesian and ML phylograms generated from the concaten-
ated protein-coding gene data set have similar topologies,
with all but four of their nodes being strongly supported
(Figure 3(A)). Both analyses strongly support the exclusivity of
each E. ruidum spp. 1 and 2 (PP ¼1.0; BTP¼100). In contrast,
the exclusivity of E. ruidum sp. 3 was not recovered, instead
being paraphyletic with respect to E. ruidum sp. 4 and E. rui-
dum sp. 2 3 with most relationships being strongly sup-
ported. Ectatomma ruidum sp. 2 was recovered as sister to
the clade containing the latter three taxa, though with low
Figure 1. Organization of the E. ruidum and E. tuberculatum mitogenomes compared to the pancrustracean hypothetical ancestral mitogenome and those previously
obtained for other ant species.
Table 1. Main descriptive features of the 24 mitogenomes generated for this study.
Coverage
Sample LP R1 R2 Size (bp) Genes not found mean SD Max
E. ruidum sp. 1 2081 SG 489,440 475,627 16,047
a
18.5 12.5 64
E. ruidum sp. 1 2083 SG 665,337 664,717 15,508
a
trnI, trnM, trnQ, trnT 8.5 7.3 38
E. ruidum sp. 1 2908 SG 325,752 302,808 17,275
a
trnA, trnF, trnH, trnV 14.5 7.6 41
E. ruidum sp. 1 2074 UCEs 1,042,156 968,499 16,715
a
15.2 10.4 46
E. ruidum sp. 2 2066 SG 573,202 568,980 17,490 38.5 9.4 71
E. ruidum sp. 2 2066 UCEs 3,092,030 3,090,281 17,500 70.4 56.8 207
E. ruidum sp. 2 2068 SG 1,842,548 1,639,824 17,498
a
31.4 25.5 101
E. ruidum sp. 2 2068 UCEs 1,985,796 1,984,687 17,872
a
31.3 25.8 100
E. ruidum sp. 2 2076 SG 619,141 608,200 17,518
a
37.8 15.8 82
E. ruidum sp. 2 2077 SG 613,499 608,590 17,488 42.7 12.8 89
E. ruidum sp. 2 2090 SG 537,199 534,336 17,501
a
26.6 10 60
E. ruidum sp. 2 2070 UCEs 2,103,176 2,094,915 17,517
a
43.2 42.2 343
E. ruidum sp. 3 2075 SG 647,323 642,407 17,441 91.2 27.4 155
E. ruidum sp. 3 2075 UCEs 2,362,439 2,354,406 17,458
a
122.2 94 334
E. ruidum sp. 3 2088 SG 1,303,926 1,301,246 17,452 146 92.1 387
E. ruidum sp. 3 2088 UCEs 1,897,210 1,889,558 17,462 91.2 96.1 413
E. ruidum sp. 3 2854 SG 1,008,711 1,005,751 17,443 153.6 61.6 318
E. ruidum sp. 4 2065 SG 797,142 788,517 17,464 98.8 43.5 468
E. ruidum sp. 4 2098 SG 423,283 418,945 17,365 56.5 27.3 278
E. ruidum sp. 23 2087 SG 604,015 598,656 17,444
a
38.4 32.8 151
E. ruidum sp. 23 2078 UCEs 1,785,851 1,779,785 17,625
a
71.3 67.2 269
E. tuberculatum 2085 SG 896,506 892,753 15,101
a
trnI, trnM, trnQ, rrnS 33.1 26.5 120
E. tuberculatum 2916 SG 851,149 849,016 16,611
a
47.9 32 152
E. tuberculatum 2998 UCEs 2,439,158 2,438,134 Fragmented
a
trnC, trnE, trnF, trnI, trnS1, trnW 32.4 43.5 186
E. tuberculatum 3005 UCEs 2,105,371 2,104,140 17,310
a
68.8 62.3 246
Sample, library preparation protocol (LP), number of reads in files R1 and R2 (filtered and trimmed), mt genome size (size), genes that were not found by the
program Geneious, and mitogenome coverage statistics (mean, SD and maximum coverage) are mentioned.
a
Unclosed mitogenomes.
MITOCHONDRIAL DNA PART A 5
support (Pp¼0.99; BTP¼80). In the two phylograms, E. rui-
dum sp. 1 was sister to the remaining members of the com-
plex (Pp¼1.0; BTP¼100).
None of the Bayesian or ML phylograms derived from
phased cox1 or cob data recovered any of the haplotypes
from spp. 3, 4 or 2 3 as nested within the E. ruidum spp. 1
or 2 clades, ruling out paternal linkage by interspecific
hybridization. The cox1 phylograms showed similar topologies
(Figure 3(B)) and supported the exclusivity of E. ruidum spp. 1
and 2 (Pp¼0.99, 1.0; BTP¼73, 98, respectively), with the for-
mer species being sister to the remaining members of the
complex (Pp¼1.0, BTP ¼100). Ectatomma ruidum sp. 2 was
sister to a clade containing exclusively the E. ruidum spp. 3, 4
and 2 3 phased haplotypes (Pp¼0.98; BTP¼60). One of the
two haplotypes of each specimen was characterized by hav-
ing considerably long branches, whereas the alternative hap-
lotypes had short branches and were grouped together with
two E. ruidum sp. 2 3 haplotypes with long branches. The
relationships recovered by cob were mostly congruent with
the ones obtained by cox1, except for the relationships
among the phased haplotypes, where they were divided into
two subclades, each represented by the haplotypes with long
and short branches, respectively (Figure 3(C)).
The integration of our cox1 phased haplotypes with
Aguilar-Velasco et al.s(2016) data set revealed that the two
alternative haplotypes have been independently sequenced
for specimens of E. ruidum spp. 3, 4 and 2 3. Moreover, of
the four cox1 and seven cob chromatograms generated for
Figure 2. From inside out: Map of the E. ruidum sp. 3, 4 and 2 3 mitochondrial genome (tRNAs are designated by single letter amino acid codes and their corre-
sponding anti-codons; arrow heads indicate direction of transcription). The inner track of the outer semicircles represents nucleotide differences (pale bars) between
the two phased haplotypes of the protein-coding genes in a specimen of E. ruidum sp. 3 (DNA voucher no. CNIN2075). The outer tracks of semicircles represent pro-
tein-coding gene translations. Bars represent amino acids. No stop codons were found.
6R. N. MEZA-L
AZARO ET AL.
males of E. ruidum spp. 3, 4 and 2 3, 10 and one were poly-
morphic and monomorphic, respectively. All polymorphic
sequences were represented with IUPAC ambiguity codes
(Supplemental Figure S1 and Supplemental Figure S2).
The phylogenetic networks derived from the cox1 and cob
data sets (Figures 4(A,B), respectively) show a tree-like struc-
ture, which divides the sequences of E. ruidum spp. 1 and 2
into two separate clusters. Also, there is a third cluster com-
posed of the phased haplotypes of specimens assigned to
the E. ruidum spp. 3, 4 and 2 3. Within this cluster, there is
a short-branched group formed by one of the phased haplo-
types of each individual, whereas the remaining phased hap-
lotypes have considerably long terminal branches.
Uncorrected genetic distances based on the concatenated
gene-coding matrix between E. tuberculatum and the mem-
bers of the E. ruidum complex, and among the three con-
firmed species of the latter complex, ranged from 16.47% to
16.72% and 5.13% to 5.48%, respectively (Supplemental Table
S6). Cox1 distances within and between the latter three spe-
cies on the other hand ranged from 1.12% to 4.2% and 5.
30% to 6.29%, respectively, whereas for cob genetic distances
varied from 1.40% to 5.33% and 5.87% to 6.82%, respectively.
Cox1 and cob distances within the groups of phased haplo-
types having short- and long-terminal branches ranged from
0 to 2.6% and 0.09 to 1.55% and from 0 to 8.04% and 4.83 to
9.38%, respectively. Cox1 and cob distances between haplo-
types from same individuals ranged from 3.73% to 6.08% and
5.57% to 7.21%, respectively (Supplemental Table S7).
Discussion
Using high-throughput DNA sequencing, we demonstrate for
the first time the existence of extensive heteroplasmy in ants.
The main features of the newly annotated Ectatomma mito-
genomes and those that have been previously reported for
other ant species are compared and contrasted below. We
also discuss probable causes for the extensive heteroplasmy
within this ant genus.
Mitogenome features in the E. Ruidum complex
Here, we have characterized complete or nearly complete
mitogenomes to assess the origin and extent of intraspecific
mt variation in ants, and we have also reconstructed for the
first time, complete mitogenomes of species belonging to the
ant subfamily Ectatomminae.
Mitogenomes in all organisms, including insects, display
strong base compositional bias (A þTGþC) (Cameron
2014). The Ectatomma mitogenomes exhibit a high A þT bias
(81.483.4%), which falls within the range previously
described for ants (from 69.5% in Leptomyrmex pallens to
83.4% in Formica fusca; Babbucci et al. 2014; Berman et al.
2014; Kim et al. 2015). The size of the complete mitochondrial
genomes in our examined specimens ranges from 17,365 to
17,500 bp. This size variation is mainly due to the presence of
31 to 34 non-coding IGSs, which span from 1650 to 1940 bp.
Large numbers of IGSs also have been reported in the mito-
genomes of Solenopsis geminata,S. invicta,S. Richteri,
Pristomyrmex punctatus,Atta laevigata,A. cephalotes,
Camponotus atrox and Formica selysi (Gotzek et al. 2010;
Hasegawa et al. 2011; de MeloRodovalho et al. 2014; Kim
et al. 2015; Yang et al. 2016).
Protein-coding and rRNA genes in the Ectatomma mitoge-
nomes display the same order and orientation as those pre-
sent in other ants and in the hypothetical ancestral
pancrustacean mitogenome (Str
oher et al. 2017; Zhao et al.
2017). However, we found two rearrangements involving
tRNAs that were previously unknown in ants and other
insects. One of them characterizes the E. ruidum complex
(SAERNF, between ND3 and ND5), whereas the second one
probably characterizes Ectatomma, since it is also present in
E. tuberculatum (QMI, between rrnS and nad2).
All predicted tRNA molecules in Ectatomma had the typical
cloverleaf structure excluding trnS1. A missing D-stem in the
trnS1 gene is a common feature in insects and most metazo-
ans (Sheffield et al. 2008; Cameron 2014; Mao et al. 2014).
Anticodons were also similar to those described for other
ants (Figure 1). The trnN anticodon consists of GTT as in all
Table 2. Number of variable sites, mean distance between contiguous variable sites, number of indels and polymorphic sites pro-
portion between the phased haplotypes across the mitogenome of single individuals of specimens assigned to E. ruidum spp. 1, 2,
3, 4, and 2 3.
Sample Variable sites
Mean distance between
contiguous variable sites Indels Genome size
Polymorphic
sites proportion
E. ruidum sp. 3
CNIN2075
942 17.58 82 17,441 5.40%
E. ruidum sp. 3
CNIN2075 UCE
877 18.839 76 17,458 5.02%
E. ruidum sp. 3
CNIN2088
744 22.4 127 17,452 4.26%
E. ruidum sp. 3
CNIN2088 UCE
554 30.3 30.3 17,462 3.17%
E. ruidum sp. 3
CNIN2854
958 17.319 115 17,443 5.49%
E. ruidum sp.4
CNIN2065
1015 16.31 102 17,464 5.81%
E. ruidum sp.4
CNIN2098
999 16.5 116 17,365 5.75%
E. ruidum sp. 2 3
CNIN2078
332 35.41 35.41 17,625 1.88%
E. ruidum sp. 2 3
CNIN2087
455 33.85 52 17,444 2.61%
MITOCHONDRIAL DNA PART A 7
known ant mitogenomes except for Solenopsis spp. and
Leptomyrmex pallens, which have an ATT anticodon, though
this seems to be a misidentification (Bernt et al. 2013;
Babbucci et al. 2014).
Detection of heteroplasmy
The absence of frameshift mutations or stop codons in the
two phased haplotypes of cox1 and cob for E. ruidum sp. 3, 4
and 2 3, and the strong evidence of purifying selection in
these two alternative haplotypes indicate that both haplo-
types maintain their functionality. This assertion of functional-
ity is reinforced by the translation of cox1 haplotypes, which
reveals that most of the nucleotide substitutions are syn-
onymous, and when non-synonymous, they result in amino
acids from within the same family. This proves that the exten-
sive polymorphism found across the mitogenomes of the
above taxa is due to heteroplasmy, and not by the presence
of numts or mt recombination. Even in very young numts,
one would expect the majority of substitutions to be random
and most often deleterious. We found no evidence of such
mutations in the co-occurring mitogenomes that we
sequenced, even though there was a considerable amount of
genetic divergence between the two recovered haplotypes in
each individual. In addition, at least for the UCE data, its
unlikely that we would sequence numts, given that we did
not enrich for them and they would be in lower numbers as
compared to mtDNA.
In insects, indels and internal stop codons have been used
to prove the existence of numts in orthopterans (Song et al.
2008), dipterans (Aedes aegypti; Black and Bernhardt 2009) and
hymenopterans, including ants (Martins et al. 2007; Cristiano
et al. 2014). As noted above, no stop codons or frameshifts
were detected in the examined specimens of Ectatomma. The
codon-based Z-test also gave strong evidence of purifying
selection operating on the cox1 and cob mt genes.
The heteroplasmy we found in Ectatomma is restricted to
E. ruidum spp. 3, 4 and the putative 2 3 hybrids. In Aguilar-
Velasco et al. (2016), polymorphic cox1 and cob chromato-
grams generated from Sanger sequencing were recovered for
the above taxa. The observed polymorphisms were thought
to be caused by co-amplification of numts, and thus pre-PCR
dilutions were carried out, yielding monomorphic chromato-
grams consisting of sequences with considerably high
E. ruidum sp. 2x3 CNIN2078 UCE B
E. ruidum sp. 2x3 CNIN2087 B
E. ruidum sp. 3 CNIN2088 UCE B
E. ruidum sp. 3 CNIN2088 A
E. ruidum sp. 2x3 CNIN2087 A
E. ruidum sp. 2x3 CNIN2078 UCE A
E. ruidum sp. 3 CNIN2088 UCE A
E. ruidum sp. 3 CNIN2088 B
E. ruidum sp. 4 CNIN2065 A
E. ruidum sp. 4 CNIN2098 A
E. ruidum sp. 3 CNIN2075 A
E. ruidum sp. 3 CNIN2854 A
E. ruidum sp. 3 CNIN2075 UCE A
E. ruidum sp. 4 CNIN2065 B
E. ruidum sp. 4 CNIN2098 B
E. ruidum sp. 3 CNIN2075 B
E. ruidum sp. 3 CNIN2854 B
E. ruidum sp. 3 CNIN2075 UCE B
E. ruidum sp. 1 CNIN2083
E. ruidum sp. 1 CNIN2908
E. ruidum sp. 1 CNIN2074
E. ruidum sp. 1 CNIN2081
E. tuberculatum CNIN2998
E. tuberculatum CNIN2085
E. tuberculatum CNIN2916
E. tuberculatum CNIN3005
E. ruidum sp. 2 CNIN2066 UCE
E. ruidum sp. 2 CNIN2066
E. ruidum sp. 2 CNIN2068 UCE
E. ruidum sp. 2 CNIN2076
E. ruidum sp. 2 CNIN2077
E. ruidum sp. 2 CNIN2090
E. ruidum sp. 2 CNIN2070
1/100
0.86/69
1/100
0.99/73
1/98
0.98/60
0.98/59
0.99/68
1/94
1/84
0.84/56
1/100
1/100
0.72/38
0.64
E. ruidum sp. 3 CNIN2088 A
E. ruidum sp. 3 CNIN2088 B
E. ruidum sp. 4 CNIN2065 A
E. ruidum sp. 4 CNIN2098 A
E. ruidum sp. 3 CNIN2075 A
E. ruidum sp. 3 CNIN2854 A
E. ruidum sp. 4 CNIN2065 B
E. ruidum sp. 4 CNIN2098 B
E. ruidum sp. 3 CNIN2075 B
E. ruidum sp. 3 CNIN2854 B
E. ruidum sp. 1 CNIN2083
E. ruidum sp. 1 CNIN2908
E. ruidum sp. 1 CNIN2074
E. ruidum sp. 1 CNIN2081
E. tuberculatum CNIN2998
E. tuberculatum CNIN2085
E. tuberculatum CNIN2916
E. tuberculatum CNIN3005
E. ruidum sp. 2 CNIN2066 UCE
E. ruidum sp. 2 CNIN2066
E. ruidum sp. 2 CNIN2068 UCE
E. ruidum sp. 2 CNIN2076
E. ruidum sp. 2 CNIN2077
E. ruidum sp. 2 CNIN2090
E. ruidum sp. 2 CNIN2070
1/100
1/100
1/100
1/98
1/96
0.94/85
0.87/80
0.66/38
0.63/55
0.53/52
0.94/68
0.92/72
E. ruidum sp. 2x3 CNIN2078 UCE
E. ruidum sp. 2x3 CNIN2087
E. ruidum sp. 3 CNIN2088 UCE
E. ruidum sp. 3 CNIN2088
E. ruidum sp. 4 CNIN2065
E. ruidum sp. 4 CNIN2098
E. ruidum sp. 3 CNIN2075
E. ruidum sp. 3 CNIN2854
E. ruidum sp. 3 CNIN2075 UCE
E. ruidum sp. 2 CNIN2066 UCE
E. ruidum sp. 2 CNIN2066
E. ruidum sp. 2 CNIN2068 UCE
E. ruidum sp. 2 CNIN2076
E. ruidum sp. 2 CNIN2077
E. ruidum sp. 2 CNIN2090
E. ruidum sp. 2 CNIN2070
E. ruidum sp. 1 CNIN2083
E. ruidum sp. 1 CNIN2908
E. ruidum sp. 1 CNIN2074
E. ruidum sp. 1 CNIN2081
E. tuberculatum CNIN2998
E. tuberculatum CNIN2085
E. tuberculatum CNIN2916
E. tuberculatum CNIN3005
1/100
1/100
1/98
1/100
1/99
1/100
0.98/80
0.99/81
1/100
1/97
0.99/63
1/97
1/91
1/93
1/100
0.88/49
0.93/63
1/100
1/100
(A)
(B) (C)
Figure 3. Bayesian phylograms derived from the (A) protein-coding gene data set and the (B and C) cox1 and cob data sets including the phased haplotypes
(labelled as A and B). Bayesian posterior probabilities (Pp) and Maximum-Likelihood bootstrap values (BTP) values are indicated in the above branches (Pp/BTP).
8R. N. MEZA-L
AZARO ET AL.
intraspecific and intrapopulation genetic distances. Based on
our analyses here, we have confirmed that the latter diver-
gent sequences actually belong to two different haplotypes
present in the same specimen. Aguilar-Velasco et al. (2016)
also reported the existence of cox1 and cob numts in E. rui-
dum spp. 1 and 2. Here, we neither detected numts nor het-
eroplasmy in these two species. A detailed exploration of low
frequency reads generated for the E. ruidum spp. 1 and 2
samples might allow one to uncover the presence of numts
in these species.
Origin of heteroplasmy in the E. ruidum complex
Mitochondrial heteroplasmy appears to be infrequent, even
in organisms where it has been reported, with most examples
coming from experimental hybrids or natural hybrid zones
(Kvist et al. 2003; Fontaine et al. 2007; Paduan and Ribolla
2008). Heteroplasmy by hybridization seems to be caused by
the breakdown of cellular mechanisms that recognize and
remove paternal mtDNA, and is more easily detected by the
heightened genetic dissimilarity between parental mtDNAs
Figure 4. Phylogenetic (Neighbornet) networks estimated from p-distances with the program SplitsTree for (A) cox1 and (B) cob including highly and low divergent
phased haplotypes.
MITOCHONDRIAL DNA PART A 9
(Sutovsky et al. 2000; White et al. 2008). Cases of intraspecific
paternal leakage have been documented for various animal
taxa (Kondo et al. 1990; Gyllensten et al.1991; Gantenbein
et al. 2005; Sherengul et al. 2006; Ujvari et al. 2007). In these
studies, however, paternal leakage is limited and hetero-
plasmy seems to be transient (Preiss et al. 1995; Lightowlers
et al. 1997). The unusual extensive heteroplasmy reported in
our study for the specimens of E. ruidum sp. 3, 4 and 2 3
cannot be explained by occasional paternal leakage. Instead,
we propose that a prevalent source of a second mt lineage
in the progeny of these taxa is needed to explain this
phenomenon.
A similar case of extensive heteroplasmy with considerably
divergent haplotypes found in individuals of marine mussels
in the genus Mytilus, where it was hypothesized to be pro-
duced by a double uniparental mt inheritance (DUI; Hoeh
et al. 1991; Zouros et al. 1992; Zouros 2000). In this mode of
inheritance, females transmit their mtDNA to both male and
female offspring, whereas males only transmit it to male off-
spring (Skibinski et al. 1994; Zouros et al. 1994). The result is
the co-occurrence of two independently evolving mtDNA lin-
eages, one that is transmitted through eggs and another
through sperm (Skibinski et al. 1994). Consequently, females
are basically homoplasmic for the maternal mtDNA but may
contain low amounts of paternal mtDNA and only produce
eggs with maternal mtDNA. In contrast, Mytilus males are het-
eroplasmic both for maternal and paternal mtDNA, but pro-
duce sperm that only contain the latter. Separate maternal
and paternal transmissions allow long-term maintenance of
both mtDNA genomes within mussel populations even in the
presence of genetic drift (Zouros et al. 1992; Zouros 2000).
Our Ectatomma mitogenomes exhibit heteroplasmy with
highly divergent haplotypes, like Mytilus; however, in
Ectatomma, the amount of heteroplasmy is considerably
lower (7% in Ectatomma;>20% in Mytilus). Moreover, the pat-
tern found in Ectatomma differs from DUI in Mytilus, in that
both sexes are heteroplasmic according to the reconstructed
mitogenomes of female workers and the cox1 and cob of
males. Whether or not males are in fact heteroplasmic is still
uncertain. If this is the case, then unfertilized eggs should
also be heteroplasmic because ant males, like most hymenop-
terans, are produced by arrhenotokous parthenogenesis
(males develop from unfertilized eggs; Heimpel and de Boer
2008). Mitochondrial lineage transmission in the heteroplas-
mic populations of Ectatomma therefore requires further
investigation to clarify its origin and maintenance.
Species boundaries in the E. ruidum complex
Reciprocal monophyly and high genetic distances (>5%;
Supplemental Table S6) support the delineation of three of
the E. ruidum species previously proposed by Aguilar-Velasco
et al. (2016). The later study, using long terminal branches
and distinctive morphological features as evidence, also pro-
posed the presence of one additional species (referred to as
E. ruidum sp. 4) as well as hybrids that presumably belong to
E. ruidum sp. 2 3. Here, we confirm the presence of a sec-
ondary mt genome lineage found in E.ruidum 3, 4 and 2 3
from Oaxaca that appears to evolve faster and independent
of other lineages. These results suggest that the taxa involved
represent a single species whose populations share hetero-
plasmy and considerably high genetic distances among alter-
native haplotypes. However, the possibility that populations
assigned to E.ruidum sp. 4 represent a separate evolutionary
lineage originated by rapid speciation promoted by the exist-
ence of a secondary mt genome needs to be assessed based
on additional molecular and morphological information.
Acknowledgements
We thank Javier Ponce and Dmitry Dubovikoff for donating some of the
examined specimens; Cristina Mayorga and Guillermina Ortega for their
assistance at the CNIN IB-UNAM, Laura M
arquez and Gabriela Camacho
for assistance in the laboratory, Susana Guzm
an for taking the digital pic-
ture of the Ectatomma specimen and to Travis C. Glenn from UGA for
library prep and sequencing support. Part of the bioinformatic work was
carried out using the supercomputer resources given by the Direcci
on
General de C
omputo y de Tecnolog
ıas de la Informaci
on (DGTIC-UNAM)
through the project LANCAD-UNAM-DGTIC-339. USDA is an equal oppor-
tunity provider and employer.
Disclosure statement
The authors report no conflicts of interest.
Funding
This study was funded by grants given by the Consejo Nacional de
Ciencia y Tecnolog
ıa (CONACyT, Mexico, Proyecto SEP-Ciencia B
asica no.
220454; Proyecto de cooperaci
on internacional SEP-CONACyT-ANUIES no.
207562), UNAM (PAPIIT-DGAPA convocatoria 2016, proyecto no.
IN207016) to AZR, and the US National Science Foundation (NSF,
DEB#1354739, Project ADMAC). RNML was supported by CONACyT and a
DGAPA-UNAM postdoctoral fellowships to carry out this work.
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12 R. N. MEZA-L
AZARO ET AL.
... The authors also reported considerable variation in the mt locus cox1, and the existence of nuclear mt paralogs (numts; Song et al., 2014) based on the presence of polymorphism in several chromatograms. Meza-Lázaro et al. (2018) subsequently assembled the mitogenomes of workers assigned to the putative species and the hybrid population proposed in the earlier study using NGS data. ...
... 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). ...
... We generated UCE data from libraries following Branstetter et al. (2017), and included previously generated UCE data from four male specimens (Meza-Lázaro et al., 2018). We fragmented up to 50 ng of input DNA to an average fragment distribution of 400-600 bp using a Qsonica Q800R (Qsonica LLC, Newton, CT) or a BioRuptor ® Pico sonicator (Diagenode, Liége, Belgium). ...
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.
... The authors also reported considerable variation in the mt locus cox1, and the existence of nuclear mt paralogs (numts; Song et al., 2014) based on the presence of polymorphism in several chromatograms. Meza-Lázaro et al. (2018) subsequently assembled the mitogenomes of workers assigned to the putative species and the hybrid population proposed in the earlier study using NGS data. ...
... 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). ...
... We generated UCE data from libraries following Branstetter et al. (2017), and included previously generated UCE data from four male specimens (Meza-Lázaro et al., 2018). We fragmented up to 50 ng of input DNA to an average fragment distribution of 400-600 bp using a Qsonica Q800R (Qsonica LLC, Newton, CT) or a BioRuptor ® Pico sonicator (Diagenode, Liége, Belgium). ...
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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.
... In O. glaber and Dolichoderus species, the IGSs showed TA runs with 3 to 22 repeats. The absence of any overlap between the nad4l and nad4 genes was not limited to Dolichoderinae species since it has been observed in species from other ant subfamilies [35,40,41]. In other hymenopteran species, it is also possible to find IGSs in the atp8/atp6 junction. ...
... Table S1: Annotation of the mitogenomes of Tapinoma melanocephalum and Tapinoma sessile; Table S2: Annotation of the mitogenomes of Dolichoderus lamellosus and Dolichoderus pustullatus; Table S3: Annotation of the mitogenome of Leptomyrmex erythrocephalus; Table S4: Gene order in Formicidae regarding the QMI-nad2-WCY cluster of the ancestral insect-pancrustaceus mitogenome. References [15,16,20,[24][25][26][27][28][29]34,35,40,41, are cited in the supplementary materials. ...
Article
Full-text available
The ant Tapinoma ibericum Santschi, 1925 is native to the Iberian Peninsula. This species, as well as other species from the Tapinoma nigerrimum complex, could form supercolonies that make these species potentially invasive and could give rise to pests. Recently a mature colony from this species has been found in the Isle of Wight (United Kingdom). Mitogenomes have been used to study the taxonomy, biogeography and genetics of species, improving the development of strategies against pest invasion. However, the number of available mitogenomes from the subfamily Dolichoderinae is still scarce and only two of these mitogenomes belong to Tapinoma species. Herein, the complete mitogenome of T. ibericum is presented in order to increase the molecular information of the genus. The T. ibericum mitogenome, retrieved by Next-Generation Sequencing data, is 15,715 bp in length. It contains the typical set of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNAs and the A + T-rich control region. Comparisons of the T. ibericum mitogenome with other dolichoderine mitogenomes revealed the existence of four gene rearrangements in relation with the ancestral insect mitogenome. One of these rearrangements, involving the tRNA-Ile, tRNA-Gln and tRNA-Met genes, was found in most of the analyzed ant mitogenomes. Probably this rearrangement was an ancestral or plesiomorphic character in Formicidae. Interestingly, another rearrangement that affects to tRNA-Trp, tRNA-Cys and tRNA-Tyr genes was found only in Tapinoma species. This change could be a synapomorphic character for the genus Tapinoma, and could be used as a phylogenetic marker. Additionally, a phylogenetic analysis was performed using the protein-coding gene sequences from available Dolichoderinae mitogenomes, as well as mitogenomes from representative species from other Formicidae subfamilies. Results support the monophyletic nature of the genus Tapinoma placing it within the same clade as the rest of Dolichoderinae species.
... Whole mitogenome exploration of heteroplasmy is not yet widely performed in insects. Within Hymenoptera, mitochondrial heteroplasmy has been almost ex-clusively reported from bees (with a notable report in ants ( Meza-Lázaro et al., 2018 )). At least 29 bee species from five of the seven bee families have been identified with unusually high rates of mitochondrial heteroplasmy ( Magnacca and Brown, 2010 ;Magnacca and Brown, 2012 ;Françoso et al., 2016 ;Ricardo et al., 2020b ;Songram et al., 2006 ), but the most extensively studied group are the Hylaeus of Hawai'i ( Magnacca and Brown, 2010 ). ...
... Additionally, Ion Torrent shotgun sequencing data obtained from one female, further suggested extensive heteroplasmic sites ( > 200 SNPs) throughout the mitogenome. This is an unparalleled number of mitochondrial variable sites compared with other invertebrate heteroplasmic systems ( Xiong et al., 2013 ;Sriboonlert and Wonnapinij, 2019 ;Meza-Lázaro et al., 2018 ;Moreira et al., 2017 ). High divergences between the heteroplasmic mitogenomes (i.e., 3.8% at COI) might initially suggest that one mitogenome was introduced in the past, perhaps following a hybridization event. ...
Article
Mitochondrial heteroplasmy is the occurrence of more than one type of mitochondrial DNA within a single individual. Although generally reported to occur in a small subset of individuals within a species, there are some instances of widespread heteroplasmy across entire populations. Amphylaeus morosus is an Australian native bee species in the diverse and cosmopolitan bee family Colletidae. This species has an extensive geographical range along the eastern Australian coast, from southern Queensland to western Victoria, covering approximately 2,000 km. Seventy individuals were collected from five localities across this geographical range and sequenced using Sanger sequencing for the mitochondrial cytochrome c oxidase subunit I (COI) gene. These data indicate that every individual had the same consistent heteroplasmic sites but no other nucleotide variation, suggesting two conserved and widespread heteroplasmic mitogenomes. Ion Torrent shotgun sequencing revealed that heteroplasmy occurred across multiple mitochondrial protein-coding genes and is unlikely explained by transposition of mitochondrial genes into the nuclear genome (NUMTs). DNA sequence data also demonstrated a consistent co-infection of Wolbachia across the A. morosus distribution with every individual infected with both bacterial strains. Our data are consistent with the presence of two mitogenomes within all individuals examined in this species and suggest a major divergence from standard patterns of mitochondrial inheritance. Because the host's mitogenome and the Wolbachia genome are genetically linked through maternal inheritance, we propose three possible hypotheses that could explain maintenance of the widespread and conserved co-occurring bacterial and mitochondrial genomes in this species.
... The heteroplasmy in the mitogenome has been found in various insect and mite species, including the bed bug (Cimex lectularius Linnaeus, Hemiptera:Cimicidae) [80], honeybee (Apis mellifera Linnaeus, Hymenoptera:Apidae) [81], a neotropical ant species (Ectatomma ruidum (Roger), Formicidae:Ectatomminae) [82], Anapodisma miramae Dovnar-Zapolskij (Orthoptera:Acrididae) [83], Tetrodontophora bielanensis (Waga) (Poduromorpha:Onychiuridae) [84], and Drosophila melanogaster Meigen (Diptera:Drosophilidae) and is caused by paternal mitochondrial DNA leakage [85,86]. It has been found that the heteroplasmy phenomenon in the mitogenome can be involved in biological functions, including pesticide resistance [87][88][89][90] and xenobiotics detoxification [91]. ...
... The amino acid at the 49th position of COX3 in both forms did not affect their alpha-helix structure (See red parts in Figure 4A,B), indicating that this non-synonymous mutation from heteroplasmy may not affect three-dimensional structure severely. The heteroplasmy in the mitogenome has been found in various insect and mite species, including the bed bug (Cimex lectularius Linnaeus, Hemiptera:Cimicidae) [80], honeybee (Apis mellifera Linnaeus, Hymenoptera:Apidae) [81], a neotropical ant species (Ectatomma ruidum (Roger), Formicidae:Ectatomminae) [82], Anapodisma miramae Dovnar-Zapolskij (Orthoptera:Acrididae) [83], Tetrodontophora bielanensis (Waga) (Poduromorpha:Onychiuridae) [84], and Drosophila melanogaster Meigen (Diptera:Drosophilidae) and is caused by paternal mitochondrial DNA leakage [85,86]. It has been found that the heteroplasmy phenomenon in the mitogenome can be involved in biological functions, including pesticide resistance [87][88][89][90] and xenobiotics detoxification [91]. ...
Article
Full-text available
White-backed planthopper (WBPH), Sogatella furcifera (Horváth), is one of the major sap-sucking rice pests in East Asia. We have determined a new complete mitochondrial genome of WBPH collected in the Korean peninsula using NGS technology. Its length and GC percentages are 16,613 bp and 23.8%, respectively. We observed one polymorphic site, a non-synonymous change, in the COX3 gene with confirmation heteroplasmy phenomenon within individuals of WBPH by PCR amplification and Sanger sequencing, the first report in this species. In addition, this heteroplasmy was not observed in wild WBPH populations, suggesting that it may be uncommon in fields. We analyzed single nucleotide polymorphisms, insertion, and deletions, and simple sequence repeats among the three WBPH mitogenomes from Korea and China and found diverse intraspecific variations, which could be potential candidates for developing markers to distinguish geographical populations. Phylogenetic analysis of 32 mitogenomes of Delphacidae including the three WBPH mitogenomes suggested that Delphacinae seems to be monophyletic and Sogatella species including WBPH are clearly formed as one clade. In the future, it is expected that complete mitogenomes of individuals of geographically dispersed WBPH populations will be used for further population genetic studies to understand the migration pathway of WBPH.
... This pattern also prevailed when we restricted the analyses to common species. This may result from different factors, including selective PCR primer amplification bias 50 , better efficiency of nuclear DNA over mitochondrial DNA to delineate species in ants 69 , amplification bias in AT rich Hymenopteran genomes 70 or extensive heteroplasmy in the mitonchondrial DNA of certain species complex, such as Ectactomma ruidum 71 . The faunal composition of metabarcoding samples was significantly different from that in traditional samples, and this pattern was similar when we restricted the data to common species. ...
Article
Full-text available
The soil fauna of the tropics remains one of the least known components of the biosphere. Long-term monitoring of this fauna is hampered by the lack of taxonomic expertise and funding. These obstacles may potentially be lifted with DNA metabarcoding. To validate this approach, we studied the ants, springtails and termites of 100 paired soil samples from Barro Colorado Island, Panama. The fauna was extracted with Berlese-Tullgren funnels and then either sorted with traditional taxonomy and known, individual DNA barcodes (“traditional samples”) or processed with metabarcoding (“metabarcoding samples”). We detected 49 ant, 37 springtail and 34 termite species with 3.46 million reads of the COI gene, at a mean sequence length of 233 bp. Traditional identification yielded 80, 111 and 15 species of ants, springtails and termites, respectively; 98%, 37% and 100% of these species had a Barcode Index Number (BIN) allowing for direct comparison with metabarcoding. Ants were best surveyed through traditional methods, termites were better detected by metabarcoding, and springtails were equally well detected by both techniques. Species richness was underestimated, and faunal composition was different in metabarcoding samples, mostly because 37% of ant species were not detected. The prevalence of species in metabarcoding samples increased with their abundance in traditional samples, and seasonal shifts in species prevalence and faunal composition were similar between traditional and metabarcoding samples. Probable false positive and negative species records were reasonably low (13–18% of common species). We conclude that metabarcoding of samples extracted with Berlese-Tullgren funnels appear suitable for the long-term monitoring of termites and springtails in tropical rainforests. For ants, metabarcoding schemes should be complemented by additional samples of alates from Malaise or light traps.
... In recent years, the sequence capture of UCEs has become one of the most used methods for obtaining genomic-scale data to investigate evolutionary relationships of several animal taxa, including insects (e.g., [59][60][61]). Regardless of the targeted nature of this technique, raw UCEs datasets can be harvested to recover off-target sequences such as mt DNA, with the possibility of assembled complete mt genomes [62]; thus, the recovery of mt genomes from UCE libraries is currently increasing in phylogenomic studies (e.g., [57,63]). ...
Article
Full-text available
Background: Mitochondrial (mt) nucleotide sequence data has been by far the most common tool employed to investigate evolutionary relationships. While often considered to be more useful for shallow evolutionary scales, mt genomes have been increasingly shown also to contain valuable phylogenetic information about deep relationships. Further, mt genome organization provides another important source of phylogenetic information and gene reorganizations which are known to be relatively frequent within the insect order Hymenoptera. Here we used a dense taxon sampling comprising 148 mt genomes (132 newly generated) collectively representing members of most of the currently recognised subfamilies of the parasitoid wasp family Braconidae, which is one of the largest radiations of hymenopterans. We employed this data to investigate the evolutionary relationships within the family and to assess the phylogenetic informativeness of previously known and newly discovered mt gene rearrangements. Results: Most subfamilial relationships and their composition obtained were similar to those recovered in a previous phylogenomic study, such as the restoration of Trachypetinae and the recognition of Apozyginae and Proteropinae as valid braconid subfamilies. We confirmed and detected phylogenetic signal in previously known as well as novel mt gene rearrangements, including mt rearrangements within the cyclostome subfamilies Doryctinae and Rogadinae. Conclusions: Our results showed that both the mt genome DNA sequence data and gene organization contain valuable phylogenetic signal to elucidate the evolution within Braconidae at different taxonomic levels. This study serves as a basis for further investigation of mt gene rearrangements at different taxonomic scales within the family.
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This new third edition updates a best-selling encyclopedia. It includes about 56% more words than the 1,392-page second edition of 2003. The number of illustrations increased to almost 2,000 and their quality has improved by design and four colors. It includes approximately 1,800 current databases and web servers. This encyclopedia covers the basics and the latest in genomics, proteomics, genetic engineering, small RNAs, transcription factories, chromosome territories, stem cells, genetic networks, epigenetics, prions, hereditary diseases, and patents. Similar integrated information is not available in textbooks or on the Internet.
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Targeted enrichment of conserved genomic regions (e.g., ultraconserved elements or UCEs) has emerged as a promising tool for inferring evolutionary history in many organismal groups. Because the UCE approach is still relatively new, much remains to be learned about how best to identify UCE loci and design baits to enrich them. 2.We test an updated UCE identification and bait design workflow for the insect order Hymenoptera, with a particular focus on ants. The new strategy augments a previous bait design for Hymenoptera by (a) changing the parameters by which conserved genomic regions are identified and retained, and (b) increasing the number of genomes used for locus identification and bait design. We perform in vitro validation of the approach in ants by synthesizing an ant-specific bait set that targets UCE loci and a set of “legacy” phylogenetic markers. Using this bait set, we generate new data for 84 taxa (16/17 ant subfamilies) and extract loci from an additional 17 genome-enabled taxa. We then use these data to examine UCE capture success and phylogenetic performance across ants. We also test the workability of extracting legacy markers from enriched samples and combining the data with published data sets. 3.The updated bait design (hym-v2) contained a total of 2,590-targeted UCE loci for Hymenoptera, significantly increasing the number of loci relative to the original bait set (hym-v1; 1,510 loci). Across 38 genome-enabled Hymenoptera and 84 enriched samples, experiments demonstrated a high and unbiased capture success rate, with the mean locus enrichment rate being 2,214 loci per sample. Phylogenomic analyses of ants produced a robust tree that included strong support for previously uncertain relationships. Complementing the UCE results, we successfully enriched legacy markers, combined the data with published Sanger data sets, and generated a comprehensive ant phylogeny containing 1,060 terminals. 4.Overall, the new UCE bait design strategy resulted in an enhanced bait set for genome-scale phylogenetics in ants and other Hymenoptera. Our in vitro tests demonstrate the utility of the updated design workflow, providing evidence that this approach could be applied to any organismal group with available genomic information. This article is protected by copyright. All rights reserved.
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