In social insects, chemical communication is the main communication mode among colony members, which use the blends of cuticular hydrocarbons as recognition cues to discriminate between nestmates and non-nestmates and to prevent the exploitation of their nest resources by aliens. The aim of this study was to assess the variation of nestmate recognition cues in the ant Ectatomma ruidum, a species complex with a considerably conserved morphology and one of the few ant species where intraspecific thievery, a form of cleptoparasitism, has been reported. We analyzed the cuticular hydrocarbon profiles of ants collected from a number of geographically separated populations, and examined DNA sequence data to assess their species identity. We focused on one species of the complex, E. ruidum sp. 3-4, whose species delineation remains controversial. We documented that several quantitative and qualitative traits of the cuticular hydrocarbon profiles varied significantly between populations, indicating that this species harbors more cuticular chemical phenotypic diversity than expected within a single species. In particular, there was a striking divergence among populations in the proportion of methylalkanes, alkenes, alkadienes and odd-chain components, which likely play a major role in nestmate/non-nestmate discrimination, a process which might have been crucial in these cleptobiotic ants. Further investigations are needed to test the hypothesis that biotic pressures, such as the need to discriminate conspecific intruders and limit thievery, could have played an important role in promoting the evolutionary divergence between populations in this ant species complex.

Methods B and D allowed COI amplification in all insects, while techniques A, C and E were successful in DNA amplification from insects up to 12 years old. The technique F, the fastest, was useful in insects up to 4 months-old. Finally, we adapted permanent slide preparation in Canada balsam for every technique. The results reported allow for combining morphological and molecular methodologies for taxonomic studies.