Background While ants are textbook examples of eusocial animals in which altruistic behavior is maintained through kin selection, several ants form genetically diverse colonies that challenge this concept. One example is the Australian green-head ant (Rhytidoponera metallica) whose colonies harbor such extreme genetic variation that they have been speculated to represent an unstable form of eusociality. Yet, R. metallica is among the most successful ants on the Australian subcontinent. This success has been hypothesized to be partly due to the diverse venoms harbored within each colony. However, the genomic basis and evolutionary scenarios that maintain this toxin diversity remain unknown. Results To examine toxin genomic architecture, quantify individual-level genetic variation, and identify both proximate and ultimate mechanisms that have facilitated the toxin diversity in R. metallica, we generate a high-quality draft genome from a single worker. Most ectatotoxin genes are in clusters that contain evidence of multiple, complex gene-family expansions, some of which are likely explained by the presence of transposable elements. We also show that toxin regions of the genome exhibit elevated genetic variation despite being under strong selection and that this variation can translate to phenotypic diversity through toxin alleles with different functional properties. Conclusions Taken together, our results point to classical gene duplication and diversification as the main evolutionary mechanism by which the main toxin family in ant venoms evolves, suggest toxin-gene functional diversification under frequency-dependent selection maintains colony-level venom hypervariability in R. metallica, and provide new insight into the role of multi-level selection in eusocial animals.

Background While ants are textbook examples of eusocial animals in which altruistic behavior is maintained through kin selection, several ants form genetically diverse colonies that challenge this concept. One example is the Australian green-head ant (Rhytidoponera metallica) whose colonies harbor such extreme genetic variation that they have been speculated to represent an unstable form of eusociality. Yet, R. metallica is among the most successful ants on the Australian subcontinent.