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.