Introduction
Speciation in sexual organisms can be defined as the origin of
reproductive isolation between two populations (de Queiroz 2007;
Harrison and Larson 2014; Seehausen et al. 2014). Such
reproductive isolation is thought to occur most often between
geographically separated populations (Boomsma and Nash 2014), and
allopatric speciation is thus considered the most common mode of species
diversification (Coyne and Orr 2004). However, phylogenetic splits
sometimes do not coincide with geographic dispersal barriers (Wollenberg
Valero et al. 2019). In such cases, intrinsic factors may help to
explain patterns of geographic distribution and species divergence
(Seehausen et al. 2014).
Speciation can be driven by intrinsic barriers initiated by genetic
incompatibilities, through genetic drift, as an indirect consequence of
selection, or through genomic conflict (Presgraves 2010; Seehausen et
al. 2014). One proposed mechanism initiated by genomic conflict is the
evolution of incompatibilities in co-functioning mitochondrial (mt) and
nuclear genes (Gershoni et al. 2009; Chou and Leu 2010; Burton
and Barreto 2012; Crespi and Nosil 2013; Hill 2015). In eukaryotes,
mitochondria are almost exclusively inherited through the maternal
gamete (Birky 1995; Sato and Sato 2013; Radzvilavicius et al. 2017).
Co-evolution of nuclear and mt genomes may result in inter-population
hybrids that display cytoplasmic incompatibilities (Grun 1976), which
create hybridization barriers that may contribute to speciation (Hill
2015, 2016, 2019). This hypothesis predicts that the mt genotype of each
species will be functionally distinct, and that introgression of mt
genomes will be prevented by mitonuclear incompatibilities that arise
when heterospecific mt and nuclear genes attempt to co-function to
enable aerobic respiration (Sloan 2015; Hill 2018, 2019).
Any process that promotes
population divergence could also facilitate speciation (Pfennig et al.
2010). At the phenotypic level, communication signals play an important
role in species identification and may contribute to or even drive
reproductive isolation (Bradbury et al. 1998). In insects, cuticular
hydrocarbons (CHCs) play a major role in species recognition, especially
in social insects (Blomquist and Bagnères 2010; Sprenger and Menzel
2020). CHCs expressed on the insect’s cuticle are essential to prevent
desiccation, and have secondarily evolved in communication roles such as
nestmate/non-nestmate discrimination, information about castes, and task
specialization (Blomquist and Bagnères 2010; van Zweden and d’Ettorre
2010). CHCs can also be involved in interspecific discrimination
(Bagnères and Wicker-Thomas 2010; Chung and Carroll 2015; Adams and
Tsutsui 2020), and in ants they have been used as taxonomic tools to
detect morphologically similar but chemically distinct lineages (e.g.,
Schlick-Steiner et al. 2010; Hartke et al. 2019; Lucaset al. 2002).
In the present study, we assessed the species boundaries within theEctatomma ruidum species-complex, a widespread and conspicuous
group of Neotropical ants, through integrative analyses of data from
3RAD, ultraconserved elements (UCEs), a fragment of the cytochrome
oxidase 1 (cox1 ) mtDNA gene, and CHCs. The ant genusEctatomma Smith, 1858 (Ectatomminae) currently includes 15 valid
species with mainly Neotropical distributions (Antweb 2021). Of these,E. ruidum (Roger) is perhaps the most widely distributed. This
species was originally described from localities in Brazil, French
Guiana, and Colombia (Roger 1860), though its type locality was
subsequently restricted to Colombia (Kugler and Brown 1982). 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 and 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) found 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.ruidum spp. 1 and 2) have broad Neotropical distributions,
whereas the others (E. ruidum spp. 3, 4, and 2x3) are
apparently restricted to localities along the Pacific coast in southeast
Mexico. The authors also reported considerable variation in the mt locuscox1 , 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.
The mitogenomes of some of the populations from southeast Mexico
(E. ruidum spp. 3, 4 and 2x3) had a high number of
polymorphic sites, and a detailed examination indicated the presence of
two functional mt genomes within these specimens.
Peña-Carrillo et al. (2021a) analyzed the CHC profiles of specimens
assigned to E. ruidum from populations across its known
geographic distribution, focusing on the heteroplasmic populations from
southeast Mexico. The CHC profiles varied considerably among
populations, supporting the existence of various evolutionary lineages
within the complex. More recently, an acoustic study of the distress
call for members of this complex found that a population in
Huaxpaltepec, Oaxaca, Mexico, differs significantly in this trait from
other examined populations, strongly suggesting that this population
could represent a distinct, undescribed species (Peña-Carrillo et al.
2021b).
Here, we performed a comprehensive integrative systematic study based on
different sources of molecular evidence to assess the species limits
within the E. ruidum complex. Specifically, we coupled DNA
sequence information generated using two reduced genome representation
techniques, 3RAD (Bayona-Vásquez et al. 2019), and ultraconserved
elements (UCEs; Faircloth et al. 2012; Faircloth 2017), with a large
data set consisting of a mtDNA sequence fragment of a commonly barcoded
locus (Hebert et al. 2003) and CHC profiles, with special attention to
heteroplasmic populations from southeast Mexico.