Accurately delimiting species boundaries is essential for understanding biodiversity. Here, we assessed the taxonomy of the leaf-cutting ants in the Acromyrmex octospinosus species complex using an integrative approach incorporating morphological, population genetic, phylogenetic, and biogeographical data. We sampled populations across the species complex’ biogeographic distribution and reconstructed their evolutionary relationships using ultraconserved elements (UCEs) as molecular markers. We evaluated traditional morphological characters used to distinguish putative taxa and performed species delimitation analyses to investigate divergence between evolutionary lineages. Our results support the hypothesis that the A. octospinosus species complex consists of two species: the widely distributed and polymorphic species A. octospinosus and its inquiline social parasite A. insinuator. We consider A. echinatior syn. nov. and A. volcanus syn. nov. as well as the subspecies A. octospinosus cubanus syn. nov., A. octospinosus ekchuah syn. nov., and A. octospinosus inti syn. nov. as junior synonyms of A. octospinosus. We also investigated the biogeographic history of the species complex and the evolutionary origin of the social parasite A. insinuator. We inferred that A. octospinosus originated during the late Miocene ~6.9 Ma ago in the Neotropical rainforest. Acromyrmex insinuator shared a common ancestor with A. octospinosus ~3.4 Ma ago, with a crown-group age of ~0.9 Ma. Our phylogeny supports the hypothesis that the inquiline social parasite speciated via the intra-specific route of social parasite evolution in direct sympatry from its host. Our findings reshape our understanding of the A. octospinosus species complex and provide a foundation for future studies of Acromyrmex leaf-cutting ants.

DNA was extracted non-destructively from adult ants using a DNeasy Blood and Tissue Kit (Quiagen, Inc.) following the manufacturer’s protocol. After DNA extraction, the specimens were re-mounted and preserved as vouchers in our entomological collection.

A total of 50–100 ng of DNA was sheared by sonication (Qsonica) to a target fragment size of 400–600 bp and used as input for genomic DNA library preparation for targeted enrichment of ultraconserved elements (UCEs) (Kapa Hyper Prep Library Kit, Kapa Biosystems). We incorporated "with-bead" (SeaPure) cleaning steps (Fisher et al., 2011). Each library was associated with a unique combination of iTru adapters (Gnirke et al., 2009). We followed the protocol for library preparation as described in Faircloth et al., (2015) and modified by Branstetter et al., (2017a). Libraries were pooled for target enrichment using the hym-v2 set of ant-specific RNA probes (MYcroarray), which target 2,590 UCE loci in the Formicidae. The enrichment protocol followed Faircloth et al., (2015) and modifications described in Branstetter et al., (2017a) and Borowiec et al., (2021).

After the enrichment, we performed quantitative PCR to reliably estimate DNA concentration for the enriched pools. We used a SYBR® FAST qPCR kit (Kapa Biosystems) and a Bio-Rad CFX96 RT-PCR thermal cycler (Bio-Rad Laboratories). Finally, we combined all samples into an equimolar final pool. Final pools for each lane were sequenced at the High Throughput Genomics Core Facility at the University of Utah, Novogene Corporation Inc., and Admera Health Biopharma.