Data transformation
Selection was performed based on relative amounts of acetates. However, describing relationships among relative amounts is problematic, because they sum to 100%, thereby mathematically constraining the (co)variation in the pheromone and biasing the analysis. We therefore transformed pheromone measurements to log-contrasts for all down-stream analyses in this study. This approach breaks the interdependency and normalizes the data. Since the divisor used in the contrast of the variable can no longer be part of any downstream analyses, we chose 14:Ald as the divisor, because this component has a small but clearly detectable peak in the chromatogram, while it is irrelevant for male response behavior (Heath et al., 1990). Prior to downstream analyses, samples with a χ2-distributed Mahalanobis distance score (calculated using the ‘mahalanobis’ function in the ‘stats’ package) that exceeded a threshold value corresponding to a Bonferroni-corrected P-value < 0.05 were removed, which resulted in removing 64 out of a total of 2,861 samples. These samples showed abnormal pheromone ratios and were present across all selection and control lines and generations. We are therefore confident that these samples represent outliers, e.g. due to extraction or measurement errors, and are not representative of any relevant biological phenomena.