• Stable isotope tracing of plant water use can illuminate plant water sources. • Xylem water isotope values showed strong sorting and niche segregation. • The majority of the observed species relied on 0.0–0.2 m depth soil water. • Tropical forest water uptake depth is largely driven by tree functional traits. Stable isotope tracing of plant water use can illuminate plant water sources. But to date, the number of species tested at any given site has been minimal. Here, we sample 46 tropical hardwood tree species in a 0.32 ha plot with uniform soils. Soil water was characterized at 6 depths at 0.2 m intervals down to 1 m and showed simple and predictable depth patterns of δ 2 H and δ 18 O, and simple and spatially uniform isotope composition at each depth. Nevertheless, tree xylem water δ 2 H and δ 18 O showed remarkable variation covering the full range of soil composition, suggesting strong sorting and niche segregation across the small plot. Wood density, tree size and mean basal area increment together explained approximately 55% of the variance of xylem water isotope composition through principal component analysis. A Bayesian mixing model was applied to the data and showed that sampled trees were either sourcing their water from very shallow or deep soil layers, with very little contribution from the middle portion of the soil profile. The majority of the observed species relied on 0.0–0.2 m depth soil water. This layer contributed approximately 75% of the xylem water which was significantly higher than the contributions from all other depths. The contribution from shallow soil was highest for trees with high wood density, slow-growing trees and small-sized trees. Our work suggests that stable isotope tracers may aid a better understanding of tropical forest water uptake depths and their relation to tree functional traits and potential hydrological niche segregation among co-occurring tropical species.

• Stable isotope tracing of plant water use can illuminate plant water sources. • Xylem water isotope values showed strong sorting and niche segregation. • The majority of the observed species relied on 0.0–0.2 m depth soil water. • Tropical forest water uptake depth is largely driven by tree functional traits. Stable isotope tracing of plant water use can illuminate plant water sources. But to date, the number of species tested at any given site has been minimal. Here, we sample 46 tropical hardwood tree species in a 0.32 ha plot with uniform soils. Soil water was characterized at 6 depths at 0.2 m intervals down to 1 m and showed simple and predictable depth patterns of δ 2 H and δ 18 O, and simple and spatially uniform isotope composition at each depth. Nevertheless, tree xylem water δ 2 H and δ 18 O showed remarkable variation covering the full range of soil composition, suggesting strong sorting and niche segregation across the small plot. Wood density, tree size and mean basal area increment together explained approximately 55% of the variance of xylem water isotope composition through principal component analysis. A Bayesian mixing model was applied to the data and showed that sampled trees were either sourcing their water from very shallow or deep soil layers, with very little contribution from the middle portion of the soil profile. The majority of the observed species relied on 0.0–0.2 m depth soil water. This layer contributed approximately 75% of the xylem water which was significantly higher than the contributions from all other depths. The contribution from shallow soil was highest for trees with high wood density, slow-growing trees and small-sized trees. Our work suggests that stable isotope tracers may aid a better understanding of tropical forest water uptake depths and their relation to tree functional traits and potential hydrological niche segregation among co-occurring tropical species.

Studies of plant water sources generally assume that xylem water integrates the isotopic composition (δ 2 H and δ 18 O) of water sources and does not fractionate during uptake or transport along the transpiration pathway. However, woody xerophytes, halophytes, and trees in mesic environments can show isotopic fractionation from source waters. Isotopic fractionation and variation in isotope composition can affect the interpretation of tree water sources, but most studies to date have been greenhouse experiments. Here we present a field-based forensic analysis of xylem water isotope composition for 12 Eucalyptus tetrodonta and Corymbia nesophila trees . We used a 25-tonne excavator to access materials from the trees' maximum rooting depth of 3 m to their highest canopies at 38 m. Substantial within-tree variation occurred in δ 2 H (−91.1‰ to −35.7‰ E. tetrodonta ; −88.8‰ to −24.5‰ C. nesophila ) and δ 18 O (−12.3‰ to −5.0‰ E. tetrodonta ; −10.9‰ to −0.3‰ C. nesophila ), with different root-to-branch isotope patterns in each species. Soil water δ 2 H and δ 18 O dual isotope slopes (7.26 E. tetrodonta , 6.66 C. nesophila ) were closest to the Local Meteoric Water Line (8.4). The dual isotope slopes of the trees decreased progressively from roots (6.45 E. tetrodonta , 6.07 C. nesophila ), to stems (4.61 E. tetrodonta , 5.97 C. nesophila ) and branches (4.68 E. tetrodonta , 5.67 C. nesophila ), indicative of fractionation along the xylem stream. Roots of both species were more enriched in 2 H and 18 O than soil water at all sampled depths. Bayesian mixing model analysis showed that estimated proportions of water sourced from different depths reflected the contrasting root systems of these species. Our study adds evidence of isotopic fractionation from water uptake and along the transpiration stream in mature trees in monsoonal environments, affecting the interpretation of water sources. We discuss the findings with view of interpreting aboveground xylem water isotopic composition, incorporating knowledge of root systems. • Isotopic fractionation of xylem water may affect plant water source identification. • We analysed xylem δ 2 H and δ 18 O from roots to branches in mature trees in a savanna. • Fractionation increased from below- to aboveground xylem in the dual isotope space • Root structure assessment helped clarify aboveground interpretation of water use. • Future studies should consider xylem water fractionation and include plant traits.

Studies of plant water sources generally assume that xylem water integrates the isotopic composition (δ 2 H and δ 18 O) of water sources and does not fractionate during uptake or transport along the transpiration pathway. However, woody xerophytes, halophytes, and trees in mesic environments can show isotopic fractionation from source waters. Isotopic fractionation and variation in isotope composition can affect the interpretation of tree water sources, but most studies to date have been greenhouse experiments. Here we present a field-based forensic analysis of xylem water isotope composition for 12 Eucalyptus tetrodonta and Corymbia nesophila trees . We used a 25-tonne excavator to access materials from the trees' maximum rooting depth of 3 m to their highest canopies at 38 m. Substantial within-tree variation occurred in δ 2 H (−91.1‰ to −35.7‰ E. tetrodonta ; −88.8‰ to −24.5‰ C. nesophila ) and δ 18 O (−12.3‰ to −5.0‰ E. tetrodonta ; −10.9‰ to −0.3‰ C. nesophila ), with different root-to-branch isotope patterns in each species. Soil water δ 2 H and δ 18 O dual isotope slopes (7.26 E. tetrodonta , 6.66 C. nesophila ) were closest to the Local Meteoric Water Line (8.4). The dual isotope slopes of the trees decreased progressively from roots (6.45 E. tetrodonta , 6.07 C. nesophila ), to stems (4.61 E. tetrodonta , 5.97 C. nesophila ) and branches (4.68 E. tetrodonta , 5.67 C. nesophila ), indicative of fractionation along the xylem stream. Roots of both species were more enriched in 2 H and 18 O than soil water at all sampled depths. Bayesian mixing model analysis showed that estimated proportions of water sourced from different depths reflected the contrasting root systems of these species. Our study adds evidence of isotopic fractionation from water uptake and along the transpiration stream in mature trees in monsoonal environments, affecting the interpretation of water sources. We discuss the findings with view of interpreting aboveground xylem water isotopic composition, incorporating knowledge of root systems. • Isotopic fractionation of xylem water may affect plant water source identification. • We analysed xylem δ 2 H and δ 18 O from roots to branches in mature trees in a savanna. • Fractionation increased from below- to aboveground xylem in the dual isotope space • Root structure assessment helped clarify aboveground interpretation of water use. • Future studies should consider xylem water fractionation and include plant traits.