Integrated Multivariate and Univariate Analysis of Circadian Metabolomic Signatures in Yerba Mate Clones in a Semi‐Hydroponic System

This study presents an integrated multivariate and univariate analysis of circadian metabolomic signatures in Ilex paraguariensis (yerba mate) clones cultivated under semi-hydroponic conditions. Using repeated measures ANOVA-Simultaneous Component Analysis (RM-ASCA+) and hierarchical clustering on principal components (HCPC), we explored clone-specific and photoperiod-dependent metabolic responses across light and dark phases, complemented by high-resolution timepoint sampling (HRS) and targeted screening. Clone EC21 exhibited elevated levels of sugars, amino acids, and organic acids, suggesting a metabolic strategy adapted to saline stress and nocturnal energy demands. Photoperiod effects revealed circadian regulation of central carbon metabolites (e.g., glucose, fructose, maltose) and phenylpropanoid intermediates linked to bioactive compounds such as caffeoyl-quinic acids. Interaction effects highlighted metabolic plasticity, particularly in nitrogen assimilation, with compounds like 2-oxo-glutaric acid, glutamine, and ornithine showing clone-specific temporal patterns. Caffeine, a heritable and physiologically relevant metabolite, displayed distinct circadian profiles. EC24 accumulated caffeine during the day, while EC21 peaked at night. This dynamic distribution, supported by allantoin patterns in caffeine catabolism, suggests divergent nitrogen turnover strategies between clones. These findings underscore the importance of genotype selection and temporal regulation in optimizing yerba mate performance under semi-hydroponic systems. The combined use of RM-ASCA+ and univariate analysis proved to be a powerful approach for profiling metabolomic rhythms, offering valuable insights for breeding programs targeting bioactive compound enhancement, stress resilience, and metabolic efficiency.