Species differentiate water uptake depths within a narrow vertical zone in temperate grasslands

Whether and under which conditions coexisting species in temperate grasslands differentiate their water uptake within the shallow soil layer they share is central to understanding hydrological niche differentiation in these ecosystems. In this field study, we quantified water uptake depth of 28 coexisting herbaceous species across 74 temperate grassland plots in three regions of Germany using oxygen stable isotopes and soil water content measured at multiple depths. Species consistently differed in water uptake depth across all regions, consistent with hydrological niche differentiation. This differentiation was largely confined to the upper 30 cm of soil and became stronger as overall soil moisture declined, with plants accessing water at greater depths in drier plots. In contrast, the vertical distribution of soil moisture was unrelated to either uptake depth or species differentiation. Despite pronounced species level variation, broad herbaceous functional classifications, including grasses versus forbs and fibrous versus taprooted species, showed substantial overlap in water uptake depth. Together, our results show that hydrological niche differentiation in temperate grasslands is expressed through fine scale differences among species within a shared shallow soil domain, becomes more pronounced under drier conditions, and is poorly captured by broad herbaceous functional classifications.