The evolution of cooperation is a fundamental problem in biology, especially for non-relatives, where indirect fitness benefits cannot counter within-group inequalities. Multilevel selection models show how cooperation can evolve if it generates a group-level advantage, even when cooperators are disadvantaged within their group. This allows the possibility of group selection, but few examples have been described in nature. Here we show that group selection can explain the evolution of cooperative nest founding in the harvester ant Pogonomyrmex californicus. Through most of this species’ range, colonies are founded by single queens, but in some populations nests are instead founded by cooperative groups of unrelated queens. In mixed groups of cooperative and single-founding queens, we found that aggressive individuals had a survival advantage within their nest, but foundress groups with such non-cooperators died out more often than those with only cooperative members. An agent-based model shows that the between-group advantage of the cooperative phenotype drives it to fixation, despite its within-group disadvantage, but only when population density is high enough to make between-group competition intense. Field data show higher nest density in a population where cooperative founding is common, consistent with greater density driving the evolution of cooperative foundation through group selection.

Olfactory discrimination tasks can provide useful information about how olfaction may have evolved by demonstrating which types of compounds animals will detect and respond to. Ants discriminate between nestmates and non-nestmates by using olfaction to detect the cuticular hydrocarbons on other ants, and Camponotus floridanus have particularly clear and aggressive responses to non-nestmates. A new method of adding hydrocarbons to ants, the “Snow Globe” method was further optimized and tested on C. floridanus. It involves adding hydrocarbons and a solvent to a vial of water, vortexing it, suspending hydrocarbon droplets throughout the solution, and then dipping a narcotized ant in. It is hoped this method can evenly coat ants in hydrocarbon. Ants were treated with heptacosane (C27), nonacosane (C29), hentriacontane (C31), a mixture of C27/C29/C31, 2-methyltriacontane (2MeC30), S-3-methylhentriacontane (SMeC31), and R-3-methylhentriacontane (RMeC31). These were chosen to see how ants reacted in a nestmate recognition context to methyl-branched hydrocarbons, R and S enantiomers, and to multiple added alkanes. Behavior assays were performed on treated ants, as well as two untreated controls, a foreign ant and a nestmate ant. There were 15 replicates of each condition, using 15 different queenright colonies. The Snow Globe method successfully transfers hydrocarbons, as confirmed by solid phase microextraction (SPME) done on treated ants, and the behavior assay data shows the foreign control, SMeC31, and the mixture of C27/29/31 were all statistically significant in their differences from the native control. The multiple alkane mixture received a significant response while single alkanes did not, which supports the idea that larger variations in hydrocarbon profile are needed for an ant to be perceived as foreign. The response to SMeC31 shows C. floridanus can respond during nestmate recognition to hydrocarbons that are not naturally occurring, and it indicates the nestmate recognition process may simply be responding to any compounds not found in the colony profile and rather than detecting particular foreign compounds.