2.3 ǀ Colony maintenance
Individual life pairs of a carrying worker and a carried female sexual were kept in 1.5 ml Eppendorf tubes with moist cotton and fed with cookie crumbs. After transfer to the laboratory, we added to each of 43 female sexuals one to three unrelated males from laboratory stock colonies (one male to 16, two males to 13, and three males to 14 female sexuals). Additional 112 female sexuals were left without the chance to mate with additional males. One day later, the males were removed and the female sexuals were individually placed into 94mm x 16mm Petri dishes divided by a separator into two equal halves. One half of the dish was filled with plaster with small tunnels and two or three small chambers serving as the nest, the other served as a foraging arena, into which we added a droplet of honey, dead Drosophila or chopped up cockroaches twice per week. To allow the ants to move from the nest to the foraging arena we made a 5mm wide hole in the plastic separator using a soldering iron. The nesting chambers were covered by red plastic foil. To more closely mimic the architecture of natural nests the Petri Dishes were kept vertically by attaching them to a plaster base. Experimental nests were kept in incubators under near-natural artificial conditions matching those found at the collecting site (i.e., from June to September at summer temperatures of 17°C – 34°C, slowly changing by a weekly temperature decrease of up to 4°C to 5°C – 13°C in December to March). Offspring production of isolated female sexuals was recorded every week for a total of 40 weeks. In addition, we observed offspring production from similarly housed lab-reared female sexuals, which had had only access to brothers. These latter colonies were kept alive only for three months after the hibernation. Note here that we use “female sexuals” even for individuals that have mated and began laying eggs and which would conventionally be referred to as “queens.” However, as many data refer to both mated, fertile and virgin, non-fertile individuals, using both terms would have made the text difficult to read.
In addition to the population genetics software mentioned above, statistical analyses were done with R 4.0.3 (R Core Team 2020) and Statistica 6.0 (StatSoft, Tulsa, OK).