| Summary: | <i>Research Highlights: </i>Water use efficiency (WUE) varied along a gradient of <i>Juniperus thurifera</i> (L.) forest expansion, being higher in recently colonised areas. <i>Background and Objectives: </i>WUE is a classic physiological process of plants that reflects the compromise between carbon assimilation and water loss and has a profound influence on their performance in water-limited environments. Forest expansion in Mediterranean regions associated with land abandonment can influence the WUE of plants due to the existence of two opposing gradients: one of favourable–unfavourable environmental conditions and another one of increased–decreased intraspecific competition, the former increasing and the latter decreasing towards the expanding front. The main objective of this study was to elucidate how the WUE of <i>Juniperus thurifera</i> varied along the stages of forest expansion and to provide insight on how this variation is influenced by intraspecific competition and abiotic factors. <i>Materials and Methods</i>: Seventeen plots at different distances from the mature forest core were selected at three sites located in the centre of the Iberian Peninsula. For 30 individuals within each plot, we measured biometric characteristics, age, tree vigour, and C/N ratio in leaves, and the leaf carbon isotope signature (δ<sup>13</sup>C (‰)) as a proxy for WUE. Around each individual, we scored the percentage cover of bare soil, stoniness, conspecifics, and other woody species. <i>Results</i>: WUE of <i>J. thurifera</i> individuals varied along the forest expansion gradient, being greater for the individuals at the expanding front than for those at the mature forest. WUE was influenced by the cover of conspecifics, tree age, and C/N ratio in leaves. This pattern reveals that less favourable environmental conditions (i.e., rocky soils and higher radiation due to lower vegetation cover) and younger trees at the expanding front are associated with increased WUE. The increased cover of conspecifics decreases irradiance at the mature forest, involving milder stress conditions than at the expanding front. <i>Conclusions</i>: Lower WUE in mature forests due to more favourable conditions and higher WUE due to abiotic stress at expanding fronts revealed high constraints on water economy of this tree species in these two contrasting situations. Climate change scenarios bringing increased aridity are a serious threat to <i>Juniperus thurifera</i> forests, affecting both mature and juvenile populations although in different ways, which deserve further research to fully unveil.
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