Study site and field methods
Trachymyrmex septentrionalis colonies were found in Florida, USA. Florida is located entirely within the North American Coastal Plain, which is a global biodiversity hotspot owing to its unique geologic history and diverse ecosystems (Griffith, 1994; Noss, 2012; Noss et al., 2015). Samples were collected across two primary regions in Florida that correspond with known biogeographic breaks in the state (e.g., (Seal et al., 2015)); the panhandle (which included two distinct ranger districts within the Apalachicola National Forest and a nearby barrier island) and the peninsula, represented by samples collected in North Central Florida near Gainesville and in Central Florida near Orlando (Figure 1).
In the panhandle, the Apalachicola National Forest (ANF) is divided into two ranger districts that occur on either side of the Ochlockonee River: the Apalachicola Ranger District on the west and the Wakulla Ranger District on the east. The Apalachicola Ranger District (ARD) is comprised mainly of pine flatwood forest with high herbaceous cover, little relief, and a high water table (Abrahamson & Harnett, 1990).Trachymyrmex septentrionalis colonies in pine flatwoods are restricted to ‘habitat islands’ of somewhat higher elevation (relative to the water table) and population sizes likely vary temporally as annual rainfall varies from drought to very wet (Seal & Tschinkel, 2010). On the other hand, the Wakulla Ranger District (WRD) contains significant stands of sandhill forest (i.e., remnants of Pleistocene sand dunes; (Myers, 1990)) that are excessively drained and support many species associated with deserts (Brown, Stone, & Carlisle, 1990).T. septentrionalis colonies are found throughout the sandhills, absent only from frequently inundated areas near ephemeral ponds. Unlike flatwoods populations, those found in sandhills do not likely fluctuate very much in size, at least with respect to rainfall (Seal & Tschinkel, 2006, 2010). Both ranger districts are dominated and characterized by longleaf pine (Pinus palustris ) and sandy soils. Located just south of the ANF, St. George Island is a sandy barrier island approximately 6km from the mainland. Despite its low relief and susceptibility to hurricanes, the island contains several stands of pine forest on elevated regions (Gornish & Miller, 2010; Huffman, Platt, & Grissino-Mayer, 2004) which harbor several species typical of sandhills further inland, such as T. septentrionalis and the seed-harvesting antPogonomyrmex badius . The Florida peninsula is separated from the panhandle by the Suwanee River, a known biogeographic barrier to many species (including ants), owing to different soils and geological history (Brown et al., 1990; Richter et al., 2014; Strehl & Gadau, 2004). Thus, the collections were made across multiple ecological gradients and potential dispersal barriers.
Trachymyrmex septentrionalis colonies were identified in the field by their distinctive and steep crescent-shaped mounds, which are built up by ants piling excavated chamber soil (Seal & Tschinkel, 2006). Individual worker ants were collected directly from colonies in 2013 and 2018. All workers were preserved in 95% ethanol at -20ºC until DNA extraction. Only one individual worker per colony was used for sequencing. All relevant specimen information (e.g., collection date, region/location) can be found in Table S1.