Odor of achlorophyllous plants’ seeds

We performed bioassays using seeds of M. humile, 20 fresh untreated or solvent-treated seeds (odourless seeds) per turn of the bioassay. For the solvent treated seeds, 20 fresh seeds of M. humile were soaked in ethanol (99.5%, Wako Pure Chemical Industries, Ltd.) for 30 min. Four replicate colonies resulted in a total of 80 seeds for each treatment. Untreated and solvent treated seeds were assayed separately to avoid odor transfer, in which the treatment order for each colony was randomized by the function 'sample' 4.0.3 for Mac OSX (R core team, 2018). The colonies were starved (only given water) for 72 h before performing the assays. After the seeds were set at the centre of the arena, the number of seeds carried to the nest by the ants was recorded every 15 min for 90 min. We performed a bioassay using dummy seeds made of a square bit of filter paper (2 mm × 2 mm). Although the size of dummy seeds were larger than the real seeds (Figure 1), the size is the minimum to track the outcome of the dummy seeds. The dummy seeds were numerically numbered with a carbon pencil, enabling to refer the treatment conditions for each assay. The dummy seeds were soaked with the volatile compounds of M. humile seeds (Table 1). These compounds were purchased from Wako Pure Chemical Industries (Osaka, Japan) or Tokyo Kasei (Tokyo, Japan) with >98.0% purity. In this dummy-seed assay, 20 pieces of filter paper were set for each assay. The odor treatment in this assay consisted of eight conditions: each of the six odors identified alone (six conditions), a mixture of equivalent amounts of the six odors (one condition), and the control condition (ethanol alone). Each odor component was diluted to a final concentration of 100 ng/µL using 99.5% ethanol as a solvent. Twenty pieces of filter paper were soaked in 20 µL odor solution (1 µL for each filter paper). Each of the eight odor treatments was replicated six times using six different colonies, and each colony experienced each treatment only once. The colonies were starved (only given water) for 72 h before performing the assays. During each turn of assay (72 h) ant colony were supplied nothing other than the dummy seeds. The interval between assay turns for each colony was about a week in minimum.