Within-colony sharing
To evaluate within-colony sharing, we conducted a laboratory experiment
testing polygyne worker discrimination between larvae that are close
siblings and larvae that are not siblings (Fig 2). To do this, we
established six single-lineage experimental colonies by collecting mated
polygyne queens following mating flights. Fire ant queens are typically
singly mated (Ross, 1993; Ross & Fletcher, 1985; but see Fritz, Vander
Meer, & Preston, 2006; Lawson, Vander Meer, & Shoemaker, 2012), so by
maintaining colonies with an individual queen (rather than several
queens), we were able to test worker discernment between brood that are
close siblings (i.e., from the same mother) and brood that are not close
siblings (i.e., from a different mother).
All colonies were kept in standardized laboratory conditions for at
least two years to ensure they were large enough to be divided into
smaller experimental colonies. Colonies were confirmed as the polygyne
social form by screening workers for the presence of theGp-9b allele using the same methods as
described above (see Genetic analysis in the methods section).
Once incipient colonies were large enough, we used food dye to label the
brood of each colony. We dyed brood by giving workers two separate tubes
of 15 mL of water and 15 mL of artificial nectar each containing 0.9 ml
of food coloring (McCormick® Food Colors & Egg Dye, McCormick &
Company, Inc., 18 Loveton Circle, Sparks, MD). We gave three colonies
(colonies A, B, and C) yellow food coloring and three colonies (colonies
D, E, and F) green food coloring (Table 1; Fig 2). Colors were randomly
assigned. During this six-day period of brood dyeing, we did not give
the ants any proteinaceous food so that dye would be highly visible in
the guts of larvae.
Next, we created experimental colonies by combining 0.1g of workers
(~120 workers) with 50 larvae from the same natal colony
as the workers (i.e., close siblings) and 50 larvae from a different
colony (i.e., not siblings; see Table 1 for complete family
combinations). Not all permutations of families were logistically
possible in this experiment, so only larval combinations of different
colors were combined so that all possible two-color combinations were
created (Table 1). In all, there were 18 experimental colonies.
To quantify the feeding of larvae by workers, experimental colonies were
given 7.5 ml artificial nectar containing 0.2 g of non-toxic,
fluorescent dye (DFDRY-C0 UV Dye from Risk Reactor, 2676 S. Grand Ave.,
Santa Ana, CA) for 18 hours. After 18 hours, we recorded the number of
larvae remaining from each family and used a black light to count the
number of larvae from each family fed the fluorescent dye. In order to
ensure accurate results for potentially variable behaviors, data for
experimental colonies were averaged across the three iterations of this
experiment. This allowed us to remove within colony temporal variation
and estimate the general behaviors of each experimental colony instead
of only looking at a single snapshot of their behavior.
All data were analyzed using JMP® 9. Percentage data
were arcsine-square-root transformed. All graphs were produced with
untransformed data. A more detailed description of the methods can be
found in Appendix S4.