found this in Frieseomelitta doederleini; however, we only had individuals of one colony) (Fig. 1f; and Supplementary Fig. 1a). Color differences were most obvious in the head area, the abdomen and the legs. We quantified the differences by measuring the darkness ("melanization"). We photographed each individual in standardized light and magnification conditions and transformed the images to 32-bit grayscale with ImageJ 1.46. We then measured the average gray level in the clypeus. The melanization level mwas calculated as m = 1grgr, where g is the average gray level, and r is the reference gray level measured on the white background43.Quantifying cuticle thickness in Frieseomelitta variaTo test whether the darker Frieseomelitta varia guards had a thicker cuticle, we caught one guard and one forager from nine different colonies (nine guards, nine foragers) and sectioned the cuticle in three different parts of the clypeal area of the head to measure the thickness of the cuticle using transmission electron microscopy (Jeol-Jem-100cx-II Electron Microscope). Thinly cut head slices were fixed in glutaraldehyde 5% and washed three times in sodium cacodylate 0.1 M. Subsequently, the thickness of the cuticle was measured using ImageJ 1.46.Statistical analysisAll tests were done in R 3.044. We used LME models to compare guard and forager head widths in each species. We used a Gaussian error distribution and included colony as a random effect to control for non-independence of data from the same colony45. Since we tested 28 species, we corrected P-values table-wide (Table 1) to avoid false positives (false discovery rate) using the Benjamini and Hochberg correction46. We used an LME and included bee nested within colony as random effects to test if guards in Frieseomelitta varia have thicker cuticles than foragers.To test for allometry within species, we log10-transformed all head width measurements and plotted them against the log10-transformed cube root of the wet weight. This allowed us to determine whether the relationship between the two traits is isometric or allometric. This is because the slope b of the regression log (y) = log (a) + b log (x) equals the power term of the geometric relationship y = axb, with b = 1 indicating allometry and b = 1 indicating isometry4,6. We used reduced major axis regression (model II) to estimate the slopes and to test if they differed significantly from isometry47.When exploring relationships between traits across species, we used GLS models while correcting for phylogenetic dependence and assumed that traits evolve under a Brownian motion model48. Continuous variables were logarithmically transformed before analyses48. The phylogenetic framework for the comparative analysis of our species (Fig. 1a) relied on the phylogenetic results of Rasmussen and Cameron18, complemented with information provided by G.A.R. Melo (pers. communication). The chronogram from Rasmussen and Cameron18 was pruned to only include the taxa relevant for this study. Time-proportional estimates for most branch lengths are available18, except for the relationships among species of Frieseomelitta and Scaptotrigona. We used two trees with different sets of branch lengths for species of these genera (divergence time between species = 2 million years vs.divergence time between species close to 0) to evaluate