Abstract
Cryptic differentiation poses a significant challenge to taxonomy, particularly in morphologically conserved lineages whose divergence is not immediately apparent. In eusocial insects like ants, where species boundaries are often maintained through chemical rather than morphological differentiation, integrative methodologies are essential for resolving taxonomic complexity. Here, we present compelling evidence for the recent divergence of African carpenter ants initially identified as Camponotus maculatus into two distinct sub-populations. This divergence is supported through an integrative approach combining chemical, behavioral, and genetic analyses. Most strikingly, we identify two sharply contrasting chemotypes characterized by categorically different cuticular hydrocarbon (CHC) profiles that unambiguously separate the investigated ant populations. Concordantly, behavioral assays reveal that worker ants consistently exhibit aggression toward individuals with opposing chemotypes, while displaying affiliative behaviors toward shared chemotypes irrespective of colony affiliation. Genetic barcoding further corroborates these findings, indicated by phylogenetic clusters largely corresponding with the two chemotypes. Our results highlight the primary role of chemical differentiation within morphologically indistinguishable populations to resolve cryptic sub-population structures. These findings also provide valuable insights into potential early-stage chemical differentiation mechanisms in eusocial insect populations.