 A growing number of field studies report isotopic offsets between stem water and its potential sources that prevent the unambiguous identification of plant water origin using water isotopes. We explored the causes of this isotopic offset by conducting a controlled experiment on the temperate tree species Fagus sylvatica.  We measured δ 2 H and δ 18 O of soil and stem water from potted saplings growing on three soil substrates and subjected to two watering regimes.  Regardless of substrate, soil and stem water δ 2 H were similar only near permanent wilting point. Under moister conditions, stem water δ 2 H was 11±3‰ more negative than soil water δ 2 H, coherent with field studies. Under drier conditions, stem water δ 2 H became progressively more enriched than soil water δ 2 H. Although stem water δ 18 O broadly reflected that of soil water, soil-stem δ 2 H and δ 18 O differences were correlated (r = 0.76) and increased with transpiration rates indicated by proxies.  Soil-stem isotopic offsets are more likely caused by water isotope heterogeneities within the soil pore and stem tissues, which would be masked under drier conditions due to evaporative

Abstract. We investigated plant water sources of an emblematic refugial population of Fagus sylvatica (L.) in the Ciron river gorges in south-western France using stable water isotopes. It is generally assumed that no isotopic fractionation occurs during root water uptake, so that the isotopic composition of xylem water effectively reflects that of source water. However, this assumption has been called into question by recent studies that found that, at least at some dates during the growing season, plant water did not reflect any mixture of the potential water sources. In this context, highly resolved datasets covering a range of environmental conditions could shed light on possible plant–soil fractionation processes responsible for this phenomenon. In this study, the hydrogen (δ2H) and oxygen (δ18O) isotope compositions of all potential tree water sources and xylem water were measured fortnightly over an entire growing season. Using a Bayesian isotope mixing model (MixSIAR), we then quantified the relative contribution of water sources for F. sylvatica and Quercus robur (L.) trees. Based on δ18O data alone, both species used a mix of top and deep soil water over the season, with Q. robur using deeper soil water than F. sylvatica. The contribution of stream water appeared to be marginal despite the proximity of the trees to the stream, as already reported for other riparian forests. Xylem water δ18O could always be interpreted as a mixture of deep and shallow soil waters, but the δ2H of xylem water was often more depleted than the considered water sources. We argue that an isotopic fractionation in the unsaturated zone and/or within the plant tissues could underlie this unexpected relatively depleted δ2H of xylem water, as already observed in halophytic and xerophytic species. By means of a sensitivity analysis, we found that the estimation of plant water sources using mixing models was strongly affected by this δ2H depletion. A better understanding of what causes this isotopic separation between xylem and source water is urgently needed.

Abstract. We investigated plant-water sources of an emblematic refugial population of Fagus sylvatica (L.) in the Ciron river gorges in South-Western France using stable isotopes. The stable isotopes of water are a powerful tracer of water fluxes in the soil-plant-atmosphere continuum. It is generally assumed that no isotopic fractionation occurs during root water uptake, and that xylem water isotopes effectively reflect source water isotopes. However, recent studies showed that under certain conditions the isotopes in plant water do not reflect any of the potential sources considered. Highly resolved datasets covering a range of environmental conditions could shed light on possible plant-soil fractionations processes. In this study, the hydrogen (δ2H) and oxygen (δ18O) isotope compositions of all potential tree water sources and xylem water were measured bi-weekly over an entire growing season. Using Bayesian isotope mixing models (MixSIAR), we then quantified the contribution of the considered sources to xylem water of F. sylvatica and Quercus robur (L.) trees. Based on δ18O data alone, both species used a mix of top and deep soil water over the season, with Q. robur using soil water relatively deeper than F. sylvatica. The contribution of stream water appeared to be marginal despite the proximity of the trees to the stream, as already reported for other riparian forests. Xylem water δ18O could always be interpreted as a mixture of deep and shallow soil waters, but the δ2H of xylem water was often more depleted than any other possible water source. We argue that an isotopic fractionation in the unsaturated zone and/or within the plant tissues could underlie this unexpected δ2H depletion of xylem water, as already observed in halophytic and xerophytic species. By means of a sensitivity analysis, we found that the estimation of plant-water sources using isotope mixing models was largely affected by this isotopic δ2H depletion. A better understanding of what causes this isotopic separation between xylem and source water is urgently needed.