Biosynthetic sources of hydrogen isotope variability in acetogenic lipids are driven by phylogeny in eudicot plants

Hydrogen stable isotope analyses of plant derived n‑alkanes have been developed as potential tools for ecological, environmental and palaeoclimatological studies. The hydrogen stable isotope composition (δ²H) of source and leaf water influence n‑alkane δ²H values (δ²H_alkane), but hydrogen isotope fractionation occurs during plant carbon metabolism. Large variation in δ²H_alkane values among species at a single geographic location have been observed, suggesting that metabolic isotope effects are large, but the origin of this biochemical variation remains poorly understood. To explore the variation in δ²H_alkane, and test if phylogenetic relatedness structures variation in δ²H_alkane values across species, we measured δ²H values of n‑alkanes in a total of 218 samples and 183 eudicot plant species grown in a single common garden during the 2019 growing season. Our results show that most variation in δ²H_alkane values could be explained by family and genus identity, and δ²H_alkane values significantly evolved along the phylogeny. To identify the intracellular location where the variation in δ²H_alkane is induced, we measured δ²H_alkane values as well as δ²H values of plastid‑produced precursors, as the acetogenic pathway in higher plants is spatially separated within cells, where fatty acids up to 18 carbon units are produced in the plastid, and additional chain elongation (e.g., for n‑alkane synthesis) occurs in the endoplasmic reticulum. Specifically, during the 2020 growing season, we measured δ²H values of n-alkanes and n‑C_16:0 fatty acids (palmitic acid; δ²H_n‑C16:0) in a total of 61 samples and 58 eudicot plant species grown in a single common garden and determined how the two spatially separated parts of the acetogenic pathway influenced δ²H_alkane values by comparing to δ²H_n‑C16:0 values. δ²H_n‑C16:0 and δ²H_alkane values showed a significant positive correlation, suggesting that δ²H_alkane biosynthetic isotopic variability is largely shaped by processes in the plastid leading up to the synthesis of palmitic acid. Strikingly, δ²H_n‑C16:0 values were almost always higher than δ²H_alkane values, and the offset between these values (as an enrichment factor; Ɛ_{C16:0‑alkane} in ‰) was also related to phylogeny. This illustrates that the extent to which the two spatially separated parts of the acetogenic pathway influence δ²H values of n‑alkanes depends on species identity due to phylogenetic effects. Our results demonstrate that variation in δ²H_alkane values among species is strongly related to phylogenetic effects, and that most of these effects are generated early in lipid biosynthesis. Future studies should address if the observed variability can be explained by metabolic traits.