Summary• Here we examine the effects of increased nitrogen (N) supply on the ectomycorrhizal fungal communities of a temperate oak savanna.• In a 16-yr N-addition experiment in which replicate 1000 m 2 plots received 0, 5.4 or 17 g N m − 2 yr − 1 , ectomycorrhizal sporocarp production was measured in the 14th, 15th and 16th year of fertilization. Ectomycorrhizal fungi (EMF) colonizing roots were examined by morphotyping-PCR-RFLP and sequence analysis in the 14th and 15th year of fertilization.• Total sporocarp richness was reduced by > 50% in both fertilization treatments in all 3 yrs, whereas Russula spp. produced approx. five times more sporocarps with 17 g N m − 2 yr − 1 . Below-ground, treatment-scale species richness and species area curves were lower with 17 g N m − 2 yr − 1 but richness, diversity indices and evenness at smaller spatial scales were not. Dominant fungi colonizing roots included Cenococcum geophilum , common in all treatments, Cortinarius spp., dominant in unfertilized plots, and Russula spp., dominant with 17 g N m − 2 yr − 1 .• Communities of EMF in this temperate deciduous ecosystem responded to N addition similarly to those of coniferous ecosystems in that increased N supply altered EMF diversity and community composition but differently in that dominance of Russula spp. increased.

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Terminal restriction fragment length polymorphism (TRFLP) is an increasingly popular method in molecular ecology. However, several key limitations of this method have not been fully examined especially when used to study fungi. We investigated the impact of spore contamination, intracollection ribosomal DNA internal transcribed spacer (ITS) region variation, and conserved restriction enzyme recognition loci on the results produced by TRFLP to characterize soil fungal communities. We find that (i) the potential for nontarget structures such as spores to contribute DNA to target sample extractions is high; (ii) multiple fragments (i.e. 'extra peaks') per PCR primer-restriction enzyme combination can be detected that are caused by restriction enzyme inefficiency and intracollection ribosomal DNA ITS variation; and (iii) restriction enzyme digestion in conserved vs. variable gene regions leads to different characterizations of community diversity. Based on these results, we suggest that studies employing TRFLP need to include information from known, identified fungi from sites within which studies take place and not to rely only on TRFLP profiles as a short cut to fungal community description.

Summary• How nitrogen (N) deposition impacts ectomycorrhizal (EM) fungal communities has been little studied in deciduous forests or across spatial scales. Here, it was tested whether N addition decreases species richness and shifts species composition across spatial scales in temperate deciduous oak forests.• Combined molecular (terminal restriction fragment length polymorphism (T-RFLP), sequencing) and morphological approaches were used to measure EM fungal operational taxon unit (OTU) richness, community structure and composition at the spatial scale of the root, soil core and forest during a 3-yr N fertilization experiment in Quercus-dominated forests near Chicago, IL, USA.• In N treatments, significantly lower OTU richness at the largest but not smaller spatial scales and a different community structure were detected. The effects of N appeared to be immediate, not cumulative. Ordination indicated the composition of EM fungal communities was determined by forest site and N fertilization.• The EM fungi responded to a N increase that was low compared with other fertilization studies, suggesting that moderate increases in N deposition can affect EM fungal communities at larger spatial scales in temperate deciduous ecosystems. While responses at large spatial scales indicate that environmental factors can drive changes in these communities, untangling the impacts of abiotic from biotic factors remain limited by detection issues.