Stem water cryogenic extraction biases estimation in deuterium isotope composition of plant source water

The hydrogen isotope ratio of water cryogenically extracted from plant stem samples (delta H-2(stem_CVD)) is routinely used to aid isotope applications that span hydrological, ecological, and paleoclimatological research. However, an increasing number of studies have shown that a key assumption of these applications-that delta H-2(stem_ CVD) is equal to the delta H-2 of plant source water (delta H-2(source))-is not necessarily met in plants from various habitats. To examine this assumption, we purposedly designed an experimental system to allow independent measurements of delta H-2(stem_CVD,) delta H-2(source), and delta H-2 of water transported in xylem conduits (delta H-2(xylem)) under controlled conditions. Our measurements performed on nine woody plant species from diverse habitats revealed a consistent and significant depletion in delta H-2(stem_ CVD) compared with both delta H-2(source) and delta H-2(xylem). Meanwhile, no significant discrepancy was observed between delta H-2(source) and delta H-2(xylem) in any of the plants investigated. These results cast significant doubt on the long-standing view that deuterium fractionation occurs during root water uptake and, alternatively, suggest that measurement bias inherent in the cryogenic extraction method is the root cause of delta H-2(stem_ CVD) depletion. We used a rehydration experiment to show that the stem water cryogenic extraction error could originate from a dynamic exchange between organically bound deuterium and liquid water during water extraction. In light of our finding, we suggest caution when partitioning plant water sources and reconstructing past climates using hydrogen isotopes, and carefully propose that the paradigm-shifting phenomenon of ecohydrological separation (two water worlds) is underpinned by an extraction artifact.