Divergence of insulin superfamily ligands, receptors and Igf binding proteins in marine versus freshwater stickleback: Evidence of selection in known and novel genes.

Three-spine stickleback (Gasterosteus aculeatus) is a teleost model for understanding genetic, physiological and morphological changes accompanying freshwater (FW) adaptation. There is growing evidence that the insulin superfamily plays important roles in traits involved in marine and FW adaptation. We performed a candidate gene analysis to look for evidence of selection on 33 insulin superfamily ligand-receptor genes and insulin-like growth factor binding proteins (Igfbp's) in stickleback. Using genotype data from 11 marine and 10 FW populations, we calculated the number of SNPs per site in regulatory and intronic regions, the number of synonymous and nonsynonymous mutations in coding regions, Wright's fixation index (Fst), and performed t-tests to identify SNPs with divergent genotype frequencies between marine/FW versus Atlantic/Pacific populations. Next, we analysed genome-wide transcriptome data from eight tissues to assess differential gene expression. Two Igfbp's (Igfbp2a and Igfbp5a) show evidence of divergent adaptation between life-history types, and a cluster of nonsynonymous mutations in Igfbp5a exhibit high Fst in exons apparently alternatively spliced in gill. We find evidence of selection on the relaxin family ligand-receptor gene pair, Insl3-Rxfp2, known to be involved in male spermatogenesis and bone metabolism, and in the 5' regulatory region of Igf2. We also confirmed the gene and coding sequence of two unannotated relaxin family ligands. These analyses underscore the utility of candidate gene studies and indicate directions for further exploration of the function of insulin superfamily genes in FW adaptation.