Nutrient preference of Neotropical ant assemblages along a habitat strata gradient and across trophic levels

In different biome, we installed 10 transects as sampling units (6 × 10 = 60 transects in total) separated by at least 1 km on average. Each transect was 740 m long with 75 sampling points, each separated by 10 m. At each sampling point, we provided one of five liquid food resources into one of three habitat strata (subterranean, epigaeic and arboreal). Hence, within each transect, each resource and strata combination (5 × 3 = 15 combinations) were repeated five times in sequence along each transect. Consequently, there were five pseudoreplicates of the 15 resource/habitat strata combinations per transect (5 pseudoreplicates × 5 resource types × 3 habitat strata = 75 sampling points per transect), giving a total of 750 sampling points per biome (75 sampling points × 10 transects = 750) and 4500 sampling points across the study (750 points × 6 biomes = 4500 points in total). We placed the food resources in 50-mL Fisher Scientific polypropylene centrifuge tubes that had a 5 cm cotton ball containing 10 ml of solutions in distilled water for each resource type. The solutions were: 1% sodium (NaCl), 20% sugar (CHO, made with sucrose), 20% amino acids (AA, made with unflavoured whey protein isolate which contains L-glutamine and other branched-chain amino acids such as leucine, isoleucine, and valine), lipids (100% extra virgin olive oil, without water), and distilled water as a control. Analogous resource solutions have successfully been used in previous studies (e.g., Fowler et al., 2014; Peters et al., 2014; Guariento et al., 2021; Lasmar et al., 2021). The placement of the tubes was different for each habitat strata. In the subterranean strata, we placed the tubes in a plastic box (4.5 cm high, 8 cm wide and 15 cm long) with an access hole <em>c</em>. 1.5 cm in diameter on each side. We buried the box <em>c</em>. 20 cm below the soil surface. In the epigaeic strata, we placed the baited tubes horizontally on the ground. For the arboreal strata, we fixed the tubes horizontally <em>c</em>. 2 m above the ground on the tree trunk closest to the sampling point, using a piece of adhesive and a string to keep the tube in place. The placement of the baited tubes began at 7:00 am for each transect except in the Caatinga biome. We delayed the baiting arrangement in Caatinga until 1:00 pm because of morning rainfall that could potentially reduce ant activity. We restricted all of our sampling to take place during only sunny or partially cloudy days and never during rainfall events. We left tubes operating for three hours. After that, we closed the tubes to sample the ants inside. We considered a tube as visited when there was at least one ant individual inside. To understand whether our baits were representatively sampling from the wider ant community we also sampled with pitfall traps. The pitfall trap data act as a proxy of ant abundance for all 60 of the baiting transects across the six biomes. By comparing ant abundance in pitfall traps and the ant foraging activity recorded by the baited tubes, we would know: (i) whether ants did not visit a bait because they did not occur in this habitat or (ii) whether baits did not attract ants due to methodological issues; (e. g. high ant abundance in pitfall traps but low ant foraging in baiting tubes, possibly indicating a problem with our baiting experiment). Thus, we started the pitfall sampling transects after the baiting experiment to avoid possible ant population depletions caused by pitfall traps (Lasmar et al. 2017). Pitfall transects were placed 20 m away in parallel from the corresponding baiting transects, totalling 60 pitfall transects across the entire study. Pitfall transects were 200 m long and had five sampling points separated by 50 m. At each sampling point, we collected ants at three habitat strata using arboreal, epigaeic and subterranean pitfall traps. Arboreal traps were installed at 1.5 m above the ground, tied in the middle of a tree trunk. Epigaeic traps were installed at the ground level and subterranean traps were buried at 20 cm under the ground. Pitfall traps were 8 cm in diameter and 12 cm in depth, and contained a 200 ml solution of water, salt (0.4%) and liquid soap (0.6%). Arboreal and epigaeic traps had a lid to cover and protect them against rain and sunlight. Subterranean traps were closed with lids and had four lateral holes of <em>c</em>. 1.5.cm in diameter on the sides. All pitfall traps remained in the field operating for 48 h.