Maintaining water balance is vital for terrestrial organisms. Insects protect themselves against desiccation via cuticular hydrocarbons (CHCs). CHC layers are complex mixtures of solid and liquid hydrocarbons, with a surprisingly diverse composition across species. This variation may translate to differential phase behaviour, and hence varying waterproofing capacity. This is especially relevant when temperatures change, which requires acclimatory CHC changes to maintain waterproofing. Nevertheless, the physical consequences of CHC variation are still little understood. We studied acclimatory responses and their consequences for CHC composition, phase behaviour, and drought survival in three congeneric ant species. Colony fragments were kept under cool, warm, and fluctuating temperature regimes. Lasius niger and platythorax, both of which are rich in methyl-branched alkanes, showed largely predictable acclimatory changes of the CHC profile. In both species, warm acclimation increased drought resistance. Warm acclimation increased the proportion of solid compounds in L. niger but not in L. platythorax. In both species, the CHC layer formed a liquid matrix of constantly low viscosity, which contained highly viscous and solid parts. This phase heterogeneity may be adaptive, increasing robustness to temperature fluctuations. In L. brunneus, which is rich in unsaturated hydrocarbons, acclimatory CHC changes were less predictable, and warm acclimation did not enhance drought survival. The CHC layer was more homogenous, but matrix viscosity changed with acclimation. We showed that ant species use different physical mechanisms to enhance waterproofing during acclimation. Hence, the ability to acclimate, and thus climatic niche breadth, may strongly depend on species-specific CHC profile.

1. Organismal traits may experience conflicting selection pressures if they fulfil different functions simultaneously. This can require trade-offs between functions or alternatively functional separation between elements of the trait.2. An important multifunctional trait in insects is the cuticular hydrocarbon (CHC) layer. CHCs cover the body of nearly all insects, protect against desiccation and serve as a communication signal. In social insects like ants, they provide cues for nestmate recognition. To maintain their waterproofing function, insects have to adjust CHC composition to current temperatures. These changes might affect information content and interfere with communication, which would be especially detrimental in social insects.3. Here, we studied how acclimation affects nestmate recognition in two sister species of the ant genus Lasius. Colony fragments were exposed to three climate regimes. We analysed behaviour towards same and differently acclimated conspecifics, and determined which CHCs were related to acclimatory changes, colony differences and inter-individual aggression.4. Differential acclimation led to higher aggression and chemical distances among former nestmates. We identified small CHC subsets, which only differed among colonies or among acclimation treatments. Moreover, few compounds sufficed to explain inter-individual aggression, suggesting that ants do not use the entire CHC profile for nestmate recognition and that colony identity is encoded in a redundant way. 5. Across individual CHCs, their contribution to colony differences and to differences among acclimatory treatments was negatively correlated, indicating that there is some degree of functional separation. However, CHC classes could not be clearly assigned to one or another function, indicating that the role of each CHC is idiosyncratic and may differ among species. Acclimatory effects and colony differences were more independent from each other in L. platythorax than in L. niger, indicating that functional separation can differ even among sister species.