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The change in land use leads to territorial spatial conflict. Territorial spatial conflicts mainly show that the boundaries of agricultural space, urban space, and ecological space overlap each other and interfere with each other’s functions, which will have a negative impact on regional high-quality development. The Aksu River, the most principal source of the Tarim River, the largest instream river in China, is a key area for maintaining ecological security in Northwestern China. It is highly significant to identify the spatial conflict characteristics of land use in this region to promote the sustainable use of regional land resources, optimization of spatial patterns, and the balance between economic construction and ecological environmental protection in mountainous areas. This paper takes the Aksu River Basin as the research area. Using Arcgis 10.8 software, Yaahp software, and SPSSAU software, it builds a land use spatial conflict measurement model based on a quantitative analysis of land use changes from 1990 to 2020 and explores the spatial and temporal evolution characteristics of land use conflicts in the Aksu River Basin. The results show that: (1) From 1990 to 2020, the spatial conflict pattern of the study area was dominated by ecological spatial conflicts. The spatial conflict area shows dynamic changes, but the overall trend is decreasing. (2) The spatial conflict pattern of territorial space in the Aksu River Basin has basically formed, and the three types of spatial conflicts are closely related to the level of local economic development. (3) Ecological spatial conflicts are mainly distributed in high-altitude mountains, river valleys, and oasis-to-desert transition areas. Urban spatial conflicts are mainly distributed in the middle and lower reaches of the Aksu River plain oasis. Agricultural spatial conflicts are scattered but large in scale, with small differences in the proportion of conflict distribution among the counties.