Moisture conditions are important ecological factors limiting plant growth in karst areas. In karst areas, because bedrock exposure and permeability are significant and soils are dispersed—without spatial continuity—and shallow, the water storage required for plant uptake and growth in rock fissures as well as shallow soils is very limited, and therefore, water conditions are an important factor influencing plant growth. In order to discover the sources of water used by plants in the karst zone ecosystem of southwest China and the differences in plant water use under different lithological conditions, this study selected limestone and dolomite in the karst ecological test site of Maocun, Guilin, Guangxi, for comparison with the clastic rock area. By measuring the δD and δ18O composition of plant stem water and the potential water sources (soil water, groundwater and precipitation) of the dominant species in the study area, and using the IsoSource and soil water excess (SW-excess) models, we analyzed the proportion of water utilization by different vegetation types under different lithological conditions. The results showed that (1) the slope and intercept of the local rainfall line (LMWL) and soil water line (SWL) in the study area were smaller than those of the global rainfall line (GMWL), and also smaller than those of the local atmospheric precipitation line in Guilin (δD = 8.8δ18O + 17.96), indicating that the local rainfall is influenced by evaporation and is formed by nonequilibrium fractionation of isotopes; (2) in general, the plant water sources in the dolomite, limestone, and clastic areas were dominated by rainfall, groundwater, and soil water, respectively; and (3) the fluctuation range of SW-excess in karst areas was significantly greater than that in nonkarst areas, the xylem water of plants in karst areas was more depleted in δD than soil water, and groundwater was more enriched in δD than soil water, indicating that there might be an ecological–hydrological separation phenomenon in karst areas, i.e., the “two water worlds” hypothesis. The results of this study provide scientific data for hydrological regulation in the ecological restoration of karst areas.
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