Geographical Influence on Metabolite Profiles of Cupressus torulosa: UPLC-QTOF-MS (Positive Mode) and Chemometric Insights

Abstract

C. torulosa, known as the Himalayan or Bhutan cypress, is a significant evergreen conifer that typically reaches heights between 20 and 45 m. This species is primarily found in the Himalayan regions of Bhutan, northern India, Nepal, and Tibet. In this study, we utilized ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) in positive ion mode, along with chemometric analysis, to investigate the metabolomic profiles of C. torulosa needles collected from 14 geographically distinct areas in Uttarakhand and Himachal Pradesh. Various statistical techniques, including ANOVA, Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA), violin plots, scatter plots, box-and-whisker plots, and heatmaps, were employed to illustrate the relative quantitative differences among compounds based on their peak intensities across these regions. Our investigation revealed 34 marker compounds consistently detected across all samples (locations). These compounds were screened using rigorous filtering criteria, incorporating a moderated t-test and multiple testing adjustments using the Benjamini–Hochberg false discovery rate (FDR) approach. Furthermore, we pioneered the identification of the phenylpropanoid and flavonoid biosynthesis pathways in C. torulosa, providing new insights into its metabolic profile. This work establishes a foundational reference for future research into the species metabolome, helping guide studies in areas like genetic diversity, ecological adaptations, and climate resilience in C. torulosa. Mapping these pathways deepens scientific knowledge of C. torulosa's metabolic processes, contributing to a clearer understanding of its unique biochemical makeup.