Does leaf wax isotopic characterisation of gymnosperms and angiosperms capture environmental gradients in Himalayas?

The responses of angiosperms and gymnosperms to environmental variables (e.g., rainfall [MAP], temperature [MAT] and pCO2) remain ambiguous and require systematic investigation. This study monitored the plants' responses to changing environments and measured variations in molecular n-alkane indices, delta 13Cbulk, delta 13Cn-al-kanes, and delta 2Hn-alkanes values in woody gymnosperms (n = 36) and angiosperms (n = 17) from an altitudinal gradient (0.9 to 3.8 km) in the Himalayas The results indicate that delta 13Cbulk values in angiosperms show a moderate correlation with altitude, MAP, and pCO2, while gymnosperms show no response in their delta 13Cbulk. The delta 13Cn-alkanes values in angiosperms remain unaffected by MAP, MAT, and pCO2, whereas gymnosperms exhibit a moderate negative correlation. The fractionations between delta 13Cn-alkanes and delta 13Cbulk values (epsilon alk/leaf) are approximately -4.0 +/- 1.6 %o and -5.6 +/- 1.5 %o for gymnosperms and angiosperms, respectively. In gymnosperms, the fractionation between delta 2Hn-alkane and delta 2Hrain (epsilon alk/rain) is -133.2 +/- 39.8 %o and -122.8 +/- 38.0 %o (C31), while in angiosperms, it is -88.5 +/- 44.6 %o (C29) and -62.4 +/- 22.9 %o (C31). The delta 2Hn-alkane values in gymnosperms and angiosperms are weakly and positively influenced by MAT and MAP, respectively. We found that gymnosperms are enriched in 13C (-1.5-3.0 %o) and depleted in 2H (-56-60 %o) compared to angiosperms; this pattern is consistent throughout the latitudes sampled in this study. The species-specific isotopic response to environmental factors is primarily driven by lower stomatal conductance, smaller leaf-size, and presence of complex tracheids in gymnosperms compared to angiosperms. The statistical K-means algorithm of dual isotope analysis (delta 13Cn-alkanes and delta 2Hn-alkane) provided two distinct clusters with an accuracy of 70 % for angiosperms and gymnosperms, which has implications for studying past vegetation transitions.