Soil fungi can help improve ecosystem restoration, yet our understanding of how they reassemble in degraded land is limited. Here, using DNA metabarcoding, we studied the fungal community structure in reforested sites following agricultural abandonment and ungulate overabundance. Two treatments, namely ‘reforestation using different numbers of tree species’ and ‘deer exclusion,’ have been applied for multiple decades in the study sites. We found that local fungal richness (alpha diversity) and total fungal richness (gamma diversity) were 1.9–2.9 and 1.3–1.9 times greater, respectively, in reforested stands than in natural forests. These results were regardless of the number of tree species planted in the reforested stands. Conversely, reforested stands had a spatially homogenized community structure with relatively lower degrees of compositional dissimilarity among sites within each stand (beta diversity). These findings were attributable to lower environmental heterogeneity, stronger dispersal limitation, and a comparatively shorter time since the onset of community assembly in reforested stands. Deer exclosures had no detectable effect on fungal community structure. Overall, the agricultural legacy in fungal community structure appears to have persisted for decades, even under proactive restoration of aboveground vegetation. Direct human intervention belowground may therefore be necessary for the recovery of soil biota once altered.

In May 2013, we collected soil samples at 72 locations across a restoration landscape in northern Japan. The topsoil from 0–5 cm depth were collected using a soil core. Soil samples were put into separate zip-lock bags, transported from the field on ice, frozen within 3 hours of collection, and kept at −20°C until further analysis.

Total DNA was extracted from each of the 72 soil samples (0.25 g sample⁻¹) using the Soil DNA Isolation kit (Norgen Biotek Corp., Canada). A semi-nested polymerase chain reaction (PCR) was then performed to amplify the nuclear internal transcribed spacer 1 (ITS1) region. The pooled amplicons were sequenced with a GS Junior sequencer (454 Life Sciences, USA).

The dataset consists of a fungal community matrix (389 OTU × 72 sites), species’ functional groups, a list of literature we used to determine the functional groups, and consensus sequences.