Development of ecosystem processes, functions and biodiversity in new forests on post-agricultural land

Afforestation of post-agricultural land offers a promising nature-based solution to address climate change and biodiversity loss. However, understanding the long-term effects of land-use legacies on ecosystem processes and functions in these new forests requires a comprehensive integration of field-based measurements and advanced technologies. This study combines forest inventory data, biodiversity surveys, and soil sampling with remote sensing (LiDAR) and environmental DNA (eDNA) to assess forest structure (FS), biodiversity, and carbon dynamics in forests established on former agricultural land. We conducted a chronosequence study in beech ( Fagus sylvatica ), oak ( Quercus robur ), and spruce ( Picea abies ) stands planted over the past 50 years in the same afforestation area in Denmark. Ground truth data were collected within a circular plot of 15 m radius and included forest inventory, understory vegetation and soil fungal community composition from 2022, while forest floor carbon and mineral soil organic carbon (SOC) stock was from the years 1998, 2011 and 2022. FS was analysed using high-resolution national LiDAR data under leaf-off conditions from 2019, while the PacBio sequencing technology for eDNA analyses was employed to explore belowground fungal diversity and community composition. Preliminary findings suggest that FS and biodiversity are shaped by a combination of tree species types and stand age. Spruce showed rapid vertical development with dense canopy cover, while oak forests supported higher structural heterogeneity and tree species richness due to their multi-layered canopy architecture and light conditions, supporting colonization of other woody species. Beech forests exhibited significant vertical heterogeneity at later stages but tended to develop a more homogeneous structure over time. The development in understory plant and belowground fungal communities reflected land-use legacies, showing a gradual yet slow, recovery over time. In general, open habitat understory species disappeared with canopy closure and forest specialist species slowly increased over time, while generalist species remained abundant in all surveys. Dispersal limitations emerged as a primary constraint shaping the vegetation. Fungal communities transitioned more rapidly, becoming dominated by ectomycorrhizal and basidiomycete species. Forest floor carbon sequestration followed a non-linear temporal trend, stabilizing after about 30 years, suggesting higher SOC stocks under spruce compared to oak. The mineral soil C stocks increased with forest age across the three soil inventories and sequestrated 0.29 ± 0.05 Mg C ha-1 per year, where spruce exhibited the highest rates of soil carbon accumulation and biomass over time. This study emphasizes the value of combining traditional ecological measurements with emerging technologies to better understand the complex interactions driving forest development on post-agricultural landscapes. The findings highlight the potential of afforestation to support biodiversity recovery, carbon sequestration, and ecosystem functioning, while emphasizing the long-term influence of agricultural legacies on forest ecosystems.