Abstract. Increased incidence of drought, as predicted under climate change, has the potential to negatively affect grassland production. Compared to monocultures, vertical belowground niche complementarity between shallow- and deep-rooting species may be an important mechanism resulting in higher yields and higher resistance to drought in grassland mixtures. However, very little is known about the belowground responses in grassland systems and increased insight into these processes may yield important information both to predict the effect of future climate change and better design agricultural systems to cope with this. This study assessed the effect of a 9-week experimental summer drought on the depth of water uptake of two shallow-rooting species (Lolium perenne L. and Trifolium repens L.) and two deep-rooting species (Cichorium intybus L. and Trifolium pratense L.) in grassland monocultures and four-species mixtures by using the natural abundance δ18O isotope method. We tested the following three hypotheses: (1) drought results in a shift of water uptake to deeper soil layers, (2) deep-rooting species take up a higher proportion of water from deeper soil layers relative to shallow-rooting species, and (3) as a result of interspecific interactions in mixtures, the water uptake of shallow-rooting species becomes shallower when grown together with deep-rooting species and vice versa, resulting in reduced niche overlap. The natural abundance δ18O technique provided novel insights into the depth of water uptake of deep- and shallow- rooting grassland species and revealed large shifts in depth of water uptake in response to drought and interspecific interactions. Compared to control conditions, drought reduced the proportional water uptake from 0–10 cm soil depth (PCWU0–10) of L. perenne, T. repens and C. intybus in monocultures by on average 54%. In contrast, the PCWU0–10 of T. pratense in monoculture increased by 44%, and only when grown in mixture did the PCWU0–10 of T. pratense decrease under drought conditions. In line with hypothesis (2), in monoculture, the PCWU0–10 of shallow-rooting species L. perenne and T. repens was 0.53 averaged over the two drought treatments, compared to 0.16 for the deep-rooting C. intybus. Surprisingly, in monoculture, water uptake by T. pratense was shallower than for the shallow-rooting species (PCWU0–10 = 0.68). Interspecific interactions in mixtures resulted in a shift in the depth of water uptake by the different species. As hypothesised, the shallow-rooting species L. perenne and T. repens tended to become shallower, and the deep-rooting T. pratense made a dramatic shift to deeper soil layers (reduction in PCWU0–10 of 58% on average) in mixture compared to monoculture. However, these shifts did not result in a reduction in the proportional similarity of the proportional water uptake from different soil depth intervals (niche overlap) in mixtures compared to monocultures. There was no clear link between interspecific differences in depth of water uptake and the reduction of biomass production under drought compared to control conditions (drought resistance). Cichorium intybus, the species with water uptake from the deepest soil layers was one of the species most affected by drought. Interestingly, T. pratense, which was least affected by drought, also had the greatest plasticity in depth of water uptake. This suggests that there may be an indirect effect of rooting depth on drought resistance, as it determines the potential plasticity in the depth of water uptake.
The effect of drought and interspecific interactions on depth of water uptake in deep- and shallow-rooting grassland species as determined by δ<sup>18</sup>O natural abundance
