Afforestation and subsequent expansion of trees on former grasslands may significantly impact the structure and activity of the soil microbial community, altering soil aggregation and affect its potential to store and cycle organic matter (OM). We investigated OM dynamics in aggregate-size topsoil samples collected along a Mediterranean alpine ecotone consisting of three vegetation types (grassland/shrubland, mixed shrubland-pine, and pine forest) in central Spain. Analytical determinations of soil organic carbon (SOC), total nitrogen (TN), particulate OM (POM), mineral-associated OM (MaOM), and the stable isotopic composition of carbon were conducted in each of the four aggregate-size fractions considered. Additionally, the structure of the microbial community (assessed as PLFA abundance), and the β-glucosidase and β-glucosaminidase activities were determined in bulk soil samples. More than half of the soil mass was contained within small macroaggregates regardless of vegetation type. SOC and TN values increased with decreasing aggregate-size classes across all vegetation types. The stability of microaggregates was negatively affected by the expansion of woody vegetation, which resulted in tree-dominated stands showing comparatively lower SOC and TN values in the smaller aggregate-size classes. On the other hand, these vegetation dynamics promoted soil macro-aggregation. While SOC contents did not show significant differences between land covers, vegetation shifts induced changes in the soil microbial community. Soil δ13C values, the abundance of gram-positive bacteria and β-glucosidase activity were significantly higher in grasslands/shrublands than in forests, while significantly higher fungi/bacteria ratio was observed in forests. Small macroaggregates appear to play a key role in the stabilisation of relatively unprocessed OM across all vegetation types, as suggested by their significantly higher concentrations of POM.
However, this fraction represents the most labile pool of OM, and as such, it is the most exposed to mineralisation. We conclude that the afforestation and potential vegetation shifts experienced in Mediterranean alpine grasslands lead to distinct changes in soil microbial communities, aggregation and soil OM dynamics, which given the strong temperature sensitivity to decomposition commonly reported in cold environments, suggests that soil OM in these high-elevation ecosystems may become highly vulnerable to environmental change.