Collection / processing of soil water isotope samples: Three deep soil cores (2 m length, 5 162 cm diameter) were collected outside of the riparian corridor in watershed N2B. Cores were extracted with a hydraulic-push corer (540MT Geoprobe Systems, Salina, KS). After collection, cores were immediately stored in sealed plastic coring tubes in a laboratory refrigerator at 1-2 °C. Cores were subsampled at 10, 20, and 30 cm, then every 25 cm for the remainder of the core. When the core was cut, root-free subsampled soil was immediately placed into exetainer vials (LabCo Ltd, UK) and stored at 1-2 °C. Soil water was extracted from each soil depth for 55-65 minutes using the cryogenic vacuum distillation method.

Collection / processing of plant xylem water isotope samples: At each site, non-photosynthetic tissue was collected from each species in May, June, July, and August of 2016. For each woody individual, 10-15 cm of stem tissue (from stems ≤1 cm diameter) were collected and immediately placed in an exetainer vial. For grasses, crown tissue was collected and stored in the same way. All samples were immediately put on ice, and then stored at 1-2 °C. Xylem water was extracted using the cryogenic vacuum distillation method.

Isotope analyses: All water samples were analyzed for δ18O and δ2H on a Picarro WS-CRDS isotopic water analyzer. ChemCorrect software was used to identify if spectral interference by organic contaminants occurred during analysis of soil and plant water samples – contaminated samples were removed from further analysis. Isotopic ratios were expressed in per mil (‰) relative to V-SMOW (Vienna Standard Mean Ocean Water). The long-term precision of this instrument using in-house standards was <0.3 ‰ for δ2H and <0.15 ‰ for δ18O.

Methods from: Keen, R.M., Nippert, J.B., Sullivan, P.L., Ratajczak, Z., Ritchey, B., O’Keefe, K. and Dodds, W.K., 2023. Impacts of riparian and non-riparian woody encroachment on tallgrass prairie ecohydrology. Ecosystems, 26(2), pp.290-301.