Experimental set-up for nestmate recognition assay. For the nestmate recognition assay, focal ants were introduced directly into the incipient colony's nest box. Each colony is housed in a transparent plastic box (10 cm wide × 7 cm high) with a dental plaster floor and a molded nest chamber. The nest chamber is covered with a plate of glass and has only one entrance. In incipient colonies, nearly all the workers are in the nest chamber; very few workers are outside the nest. With clean forceps, we lowered the focal ant (shown in red) into an unoccupied area of the nest box and the observed the reaction of the colony members to the introduced ant for 5 minutes.