Regenerating oak trees with different techniques has long-lasting legacy effects on root development, stem growth and plant physiology.

Regeneration methods can have legacy effects on tree physiology and growth via differential root development and affect forest climate sensitivity. However, there are few studies providing a long-term perspective on how forest regeneration techniques affect root development and physiology of established seedlings that have grown into trees. This study investigates leaf carbon (C), oxygen (O) and nitrogen (N) isotope composition (δ13C, δ18O, δ15N) and stem growth in ⁓20-year-old oak (Quercus robur L.) trees regenerated in two stands by three methods: direct acorn sowing in the field, bare-root planting preceded by root pruning and containerized planting with the root plug intact. Leaf isotopic values were measured three times during summer, together with tree-ring δ13C. Ground penetrating radar analyses indicated that acorn sowing resulted in deep root systems compared shallower root systems of bare-root and containerized trees. Higher annual basal area increment was observed in acorn-sown oaks than in bare-root or containerized oaks. Leaf δ13C, δ18O and δ15N values varied among regeneration origins. Trees with deeper root systems consistently showed lower leaf δ18O values, regardless of the season, which suggests higher uptake of water from deep soil horizons and better leaf hydration. In contrast, oaks with shallower root systems exhibited higher leaf δ18O values, which were positively related with δ13C. More negative leaf δ15N values in shallow-rooted oaks pointed to higher water stress. A lack of correlation between N concentration and intrinsic water-use efficiency (iWUE) for containerized trees suggests that variability in stomatal conductance rates governed iWUE in these shallow-rooted oaks. The lack of correlation between leaf and tree-ring δ13C supports that leaf physiological processes do not necessarily explain latewood isotopic composition. We conclude that regeneration techniques have long-lasting, carry-over effects on root development, plant physiology and tree growth. The findings underscore the importance of considering tree regeneration origin and root distribution when assessing the impact of climate change on tree performance and forest vigor.