Assessing the impact of land-use history on soil fungal diversity and carbon sequestration across Mediterranean and temperate forests

Forests in south-western Europe are expanding spontaneously as a result of the abandonment of traditional land-uses. Expanding forests can recover aboveground biodiversity, biomass and structure within a few decades. Yet, the impacts of land-use belowground might not recover at the same pace, compromising long-term nutrient cycling, carbon (C) sequestration, resistance and resilience to drought and other extreme climatic events. Such ecosystem functions rely on the activity of soil dwelling organisms and in mid-latitude forests, ectomycorrhizal (ECM) fungi are arguably the most crucial player for maintaining nutrient, carbon and water cycling. Still, the recovery of ECM communities during forest expansion and its link to long term C storage remain underexplored. Here, we assess how land-use history changes the diversity and structure of ECM communities and how these changes have altered the C sink capacity. We selected forests that established after the second half of the XX^th century, following the first massive rural exodus in Spain, and pre-existing forests. Our study encompasses three forest types with contrasting climatic conditions and functional attributes dominated by: Pinus uncinata (pre-alpine, evergreen needleleaved), Fagus sylvatica (temperate, deciduous broadleaved) and Quercus ilex (Mediterranean, evergreen broadleaved). In autumn 2025, we collected soil samples  in these forests to analyse: (1) ECM fungal community composition and structure, using molecular techniques; (2) C storage and the ratio of labile vs. stable soil C; and (3) age of soil bulk C and of respired C, by measuring Δ¹⁴C. Analyses of preliminary data showed that the C:N ratio was higher in mature than in recently established forests, regardless of the dominant species, but the trends for total C and N content varied among forest types: total N content was higher in recently established F. sylvatica forests, and total C was higher in mature Q. ilex forest, whereas in P. uncinate forest, we did not find significant differences for total C or N. We suggest that a combination of differences in land-use history, and functional attributes could underlie these results . In turn, we expect that soil nutrient ratios will underlie functional soil attributes and in future analyses, we expect to find higher relative abundance of ECM types with long hyphae and longer C retention time a s the C:N ratio increases.