Taxon sampling 
We used worker specimens assigned to E. ruidum collected at localities across the Neotropics from central Mexico to Colombia, but with emphasis on the populations from the state of Oaxaca in southeast Mexico that contain high levels of heteroplasmy (Meza-Lázaro et al. 2018) (Figure 1). Our sample size varied by location, though in all datasets we included representative specimens of the four putative evolutionary lineages proposed in Aguilar-Velasco et al. (2016) study (E. ruidum spp. 1-4 and 2x3).
We examined a 626 bp fragment of the cox1 gene in 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 that were detected based on the presence of internal stop codons or relationships that were evidently incorrect (Song et al. 2014). We also detected and excluded all fast-evolving secondary mt copies for the specimens assigned to E. ruidum spp. 3, 4 and 2x3, employing the phasedcox1 sequences generated in Meza-Lázaro et al. (2018) as a reference. For the 3RAD and UCE data sets, we generated sequences for 35 (34 E. ruidum , one E. tuberculatum as outgroup) and 14 (13 of E. ruidum , one of E. gibbum as outgroup) specimens, respectively.
We also generated CHC profiles of 24 workers from two localities in Mexico (Puerto Morelos, Quintana Roo, 5 nests; Cuatode, Municipality of Santa María Tonameca, Oaxaca, 6 nests) and pooled them with the data set obtained in Peña-Carrilo et al. (2021a). The complete dataset comprised 132 workers and included two to five ants per colony. A list of the specimens used for the different data sets, their taxon assignments, localities information and DNA voucher and GenBank accession numbers is available in the supplementary material (Table S1).