2.1 | Plant and ant materials
We used the ant species N. flavipes , which is polyphagous and collects plant seeds in the field (Tanaka & Tokuda 2016; Hosoishi et al., 2019). Ten N. flavipes colonies were collected from June to July 2019 from the sites at the Yuminariyama mountain and the Hokkaido University of Education Asahikawa Campus in Hokkaido, Japan. All the colonies collected were queenless, consisting of approximately 60 workers and numerous eggs, pupae, and larvae. The colonies of M. humile and N. flavipes coexist sympatrically at Yuminariyama mountain and we also collected M. humile fruits at Yuminariyama mountain in August 2019 and stored them in a screw tube at 4 ℃.
The ant colonies were kept in a container that consisted of an artificial nest and a foraging arena (Figure 1). The polypropylene box (200 mm length × 136 mm width × 68 mm height) was used as a foraging arena. Fluon was applied to the upper 3 cm inside the feeding arena to prevent ant escape. A vinyl tube (10 mm diam. × 120 mm length) was used as an artificial nest and connected to the side wall of the container at 15 mm height. To prevent the artificial nest from drying out, an absorbent piece of cotton was inserted at the wall-side of the nest and moistened with distilled water. The absorbent cotton was replaced every two weeks.
The feeding arena was divided into four equivalent sections to investigate whether the ants discard seeds in areas with different conditions. The four sections were respectively filled with dry glass beads (1.5-2.5 mm diam.), moistened glass beads, dry cotton wool, and moistened cotton wool.
Glass beads simulated an environment with inorganic substances like sand or small pebbles in the forest floor, which were filled with about 10 g of beads in each section. Cotton wool simulated an environment with organic substances such as fallen and decomposing litter, which were filled with about 0.25 g of cotton in each section. The entrance of the nest was placed at the center of the arena so that the four sections were equally aligned for the ants. The entrance of nest floats was bridged with filter paper at the center of the feeding arena (Figure 1a). In the bioassay, the seeds or the dummy seeds were placed at the center of the arena near where the bridge connected the different sections (Figure 1b).
To moisten two of the sections, 5 mL of distilled water was added every three days. All the containers were kept indoors at 25 ℃. The ants were fed with tuna and honey.