Neofunctionalization in Ligand Binding Sites of Ant Olfactory Receptors

Abstract

Chemical communication is fundamental for the operation of insect societies. Their diverse vocabulary of chemical signals requires a correspondingly diverse set of chemosensory receptors. Insect olfactory receptors (ORs) are the largest family of chemosensory receptors. The OR family is characterized by frequent expansions of subfamilies, in which duplicated ORs may adapt to detect new signals through positive selection on their amino acid sequence. Ants are an extreme example with ∼400 ORs per genome-the highest number in insects. Presumably, this reflects an increased complexity of chemical communication. Here, we examined gene duplications and positive selection on ant ORs. We reconstructed the hymenopteran OR gene tree, including five ant species, and inferred positive selection along every branch using the branch-site test, a total of 3326 tests. We find more positive selection in branches following species-specific duplications. We identified amino acid sites targeted by positive selection, and mapped them onto a structural model of insect ORs. Seventeen sites were under positive selection in six or more branches, forming two clusters on the extracellular side of the receptor, on either side of a cleft in the structure. This region was previously implicated in ligand activation, suggesting that the concentration of positively selected sites in this region is related to adaptive evolution of ligand binding sites or allosteric transmission of ligand activation. These results provide insights into the specific OR subfamilies and individual residues that facilitated adaptive evolution of olfactory functions, potentially explaining the elaboration of chemical signaling in ant societies.

Fig. 1.

—OR gene tree including 2,973 genes from seven ants, honeybee, and jewel wasp. The tree was reconstructed using RAxML based on a MAFFT amino acid alignment and divided into 31 clades for subsequent analyses. Formicidae-specific clades are colored red (only ant sequences), Aculeatan-specific clades are colored purple (include bee sequences), and Hymenopteran clades are colored blue (include wasp sequences). Clades colored white were not tested for positive selection.

Fig. 2.

—A representative subfamily from the OR gene tree. (a) Maximum likelihood phylogeny of clade 22. Scale bar represents 0.3 amino acid substitutions per site. Branches inferred to be under positive selection by the branch-site test are colored in red (q value < 0.1). Bootstrap support values are displayed throughout; branch numbers are written above the red branches only. Subtrees of species-specific gene duplications are marked by curly brackets. (b and c) Two sections of the sequence alignment showing positions under positive selection (posterior probability > 0.9) indicated by red arrows for the partitions by branch 9 (b) and branch 121 (c). Species name abbreviations according to table 1.

Fig. 3.

—Mapping of positively selected sites to a 3D structural model of insect ORs. (a) Ribbon diagram of DmOR85b is rainbow colored from the amino terminus (blue) to the carboxy terminus (red), with the seven transmembrane helices numbered. (b) Amino acids under selection in ≥6 branches of the gene tree are shown as spheres and colored orange (between 6 and 8 branches) or red (between 9 and 12 branches). Two clusters of positively selected sites are marked by ellipses. A region in TM3 that affects ligand activation based on the mutagenesis experiments in Drosophila melanogaster is marked by an arrow. (c and d) Top view (from the extracellular side) of (a) and (b), rotated 90^° about the X axis.