Data and code from: Sharply contrasting chemotypes coincide with aggression and divergence in cryptic African carpenter ant populations

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. # Data and code from: Sharply contrasting chemotypes coincide with aggression and divergence in cryptic African carpenter ant populations Dataset DOI: [10.5061/dryad.rbnzs7hq3](doi.org) ## Description of the data and file structure Chemical data: Separate Master tables for chemotypes (CMT) 1 and 2 as well as the eventual combined master table containing relative peak areas, acquired through Quantitative Analysis MassHunter Workstation Software (Version B.09.00 / Build 9.0.647.0, Agilent Technologies, Santa Clara, California, USA) Behavioral data: Scoring sheets of individual aggression assays based on a modified scale adapted from Krapf et al. (2019), see manuscript for details. Genetic data: Mitochondrial DNA barcoding based on cytochrome c oxidase subunit I (COI), aligned using MEGA-X 10.1.8. (Kumar et al. 2018). Sequence alignment (~658bp) was carried out using ClustalW (version 10.1.8). Statistical and data visualization software: R (Version 4.3.1), R Studio (Version 2023.09.1+494) (R Core Team 2020), R scripts for chemical and behavioral data analysis are given. ### Files and variables ### File: CMT_2_Complete.xlsx **Description:** Master table chemotype 2 ##### Variables: * Sample * \[All further columns] Relative peak area of CHC compound (ng) ### File: CMT_1_Complete.xlsx **Description:** Master table chemotype 1 ##### Variables: * Sample * \[All further columns] Relative peak area of CHC compound (ng) ### File: CMTs MT final.xlsx **Description:** Master table combined ##### Variables: * Sample * \[All further columns] Absolute peak area of CHC compound (ng) ### File: Agg_data_Camp_maj.csv **Description:** Aggression score sheet major workers ##### Variables: * test_ID: Individual pairing number * treatment: Paired chemotypes * aggression_score: -4 to 4 aggression scale ### File: Agg_data_Camp_min.csv **Description:** Aggression score sheet minor workers ##### Variables * test_ID: Individual pairing number * treatment: Paired chemotypes * aggression_score: -4 to 4 aggression scale ### File: acqmeth.txt **Description:**  This constitutes the Method file for the Gas chromatography coupled with mass spectrometry (GC-MS) based chemical analytical approaches for the study ### File: All_in_one_customized.R **Description:** This R script contains all used packages, scripts and automatizations used for statistical data analysis and visualization in our study ### File: Camponotus_Sequences_COI.txt **Description:** Mitochondrial cytochrome c oxidase subunit I (COI) sequences aligned for barcoding