Hydrological niche segregation (HNS), specifically the variation in root water uptake depth among coexisting species, is an understudied area of research. This is especially the case in semi-arid ecosystems, such as China’s Loess Plateau (CLP), where seasonal aridity necessitates adaptive water use strategies among plant species. In this study, we conducted a two-year investigation to understand the water sources and intrinsic water use efficiency (WUEi) of four coexisting plant species: Populus simonii (tree), Caragana korshinskii and Salix psammophila (shrubs), and Artemisia ordosica (semi-shrub). We analyzed the isotopic compositions of xylem and soil water (δ2H and δ18O) and leaf δ13C to identify the water sources and WUEi, respectively, of each species. We then used the nicheROVER model to quantify HNS based on the variations in xylem water δ2H and δ18O. Our results show that the four co-existing species occupied distinct positions on a hydrological niche axis, delineated by their respective water sources and disparate WUEi. The tree P. simonii exhibited a preference for deep soil water and demonstrated a high WUEi. Both shrubs, S. psammophila and C. korshinskii, utilized intermediate and deep soil water, respectively, and with comparable WUEi. Conversely, the semi-shrub A. ordosica relied on shallow soil water and displayed a low WUEi. These differences in water sources and WUEi led to HNS between A. ordosica and the other three species in a relatively wet year. However, in a relatively dry year, HNS between A. ordosica and the other three species contracted and WUEi increased as species increased the use of deep soil water. Overall, these results demonstrate that HNS is a dynamic phenomenon that varies on at least an annual basis. It expands and contracts as plants regulate their water uptake and loss in response to changing soil moisture conditions.
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