Studying the historical biogeography and life history transitions from eusocial colony life to social parasitism contributes to our understanding of the evolutionary mechanisms generating biodiversity in eusocial insects. The bulldog ants in the genus Myrmecia are a well-suited system for testing competing evolutionary hypotheses about how their species diversity was assembled through time because the genus is endemic to Australia with the single exception of the species Myrmecia apicalis inhabiting the Pacific Island of New Caledonia and because at least one social parasite species exists in the genus. However, the evolutionary mechanisms underlying the disjunct biogeographic distribution of M. apicalis and the life history transition(s) to social parasitism remain unexplored. To study the biogeographic origin of the isolated, oceanic species M. apicalis and to reveal the origin and evolutionary history of social parasitism in the genus, we reconstructed a comprehensive phylogeny of the ant subfamily Myrmeciinae. We utilized Ultra Conserved Elements (UCEs) as molecular markers to generate a comprehensive molecular genetic dataset consisting of 2,287 loci per taxon on average for 66 out of the 93 known Myrmecia species as well as for the sister lineage Nothomyrmecia macrops and selected outgroup taxa. Our time-calibrated phylogeny inferred that: (i) stem myrmeciine ants originated during the Paleocene ~58 Ma ago; (ii) the current disjunct biogeographic distribution of M. apicalis was driven by long-distance dispersal from Australia to New Caledonia during the Miocene ~14 Ma ago; (iii) the single social parasite species, M. inquilina, exploits three host species and evolved directly from one of the known host species, M. nigriceps, most likely via the intraspecific route of social parasite evolution in sympatry; and (iv) 5 of the 9 previously established taxonomic species groups are non-monophyletic. We suggest minor changes to reconcile the molecular phylogenetic results with the taxonomic classification. Our study enhances our understanding of the evolution and biogeography of Australian bulldog ants in the genus Myrmecia, contributes to our knowledge about the evolution of social parasitism in ants, and provides a solid phylogenetic foundation for future inquiries 

Molecular data generation

We used Ultra Conserved Elements (UCEs) as molecular markers. UCEs have been widely used as a reliable source for inferring molecular phylogenies in several taxa including ants (Barrera et al. 2022; Borowiec et al. 2021; Branstetter et al. 2017; Camacho et al. 2022; Faircloth et al. 2015; Hanisch et al. 2022; van Elst et al. 2021; Zhang et al. 2019). UCEs can be extracted from museum vouchers that are old and/or have not been preserved in optimal conditions (Blaimer et al. 2016; Borowiec et al. 2021), which was the case for most of our samples.

Ant DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, GmbH) following the manufacturer’s protocol. We extracted DNA from the three right legs of the ants when the specimens were longer than 15 mm. When the ants were smaller, we extracted DNA non-destructively without removing any appendages. After the DNA extractions, the specimens were re-mounted and preserved as vouchers in their corresponding biological collections.

When DNA was extracted from the legs, the three right legs (including the coxae) were removed and placed in 1.5 ml tubes along with a sterilized stainless-steel bead that facilitated pulverization of the tissue before DNA extraction. Samples were pulverized with a tissue lyser where disruption was carried out at a frequency of 25 Hz for 30 seconds. When DNA was extracted non-destructively from entire specimens, we poked two small holes into the right side of the mesosoma to allow the lysis buffer and enzymes to enter the specimen. Each sample was incubated in a solution of proteinase K and lysis buffer for ~24 hours. For the rest of the extraction, we followed the manufacturer’s protocol. 50–100 ng of DNA were sheared by sonication (Qsonica) to an average fragment size of ~600 bp and used for the library preparation (Kapa Hyper Prep Library Kit, Kapa Biosystems) incorporating "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 by Faircloth et al. (2015) and modified by Branstetter et al. (2017).

Libraries were pooled for target enrichment using the hym-v2 set of ant-specific RNA probes (MYcroarray), which target 2,590 UCE loci in ants (Branstetter et al. 2017). The enrichment protocol followed Faircloth et al. (2015) and modifications described in Branstetter et al. (2017) with the following changes suggested in Prebus (2021): We (1) used iTru blockers instead of the standard blockers, (2) conducted a post-enrichment PCR of the samples, (3) left enriched DNA bound to the streptavidin beads during post-enrichment PCR, (4) cleaned the resulting pools using Speed-Bead magnetic carboxylate beads (Rohland and Reich, 2012), and (5) adjusted the final volume to 22 μl.

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. In total, we sequenced two lanes submitted as two final pools of samples. One of the lanes was sequenced at the High Throughput Genomics Core Facility at the University of Utah and the second lane was sequenced at the Novogene Corporation Inc.