Vertical niche differentiation of ectomycorrhizal hyphae in soil as shown by T-RFLP analysis.

•   Niche differentiation for different soil substrates has been proposed as a mechanism contributing to ectomycorrhizal fungal diversity. This hypothesis has been largely untestable because of a lack of techniques to study the in situ distribution of ectomycorrhizal hyphae. •   We developed a technique involving soil DNA extraction, PCR and terminal restriction fragment length polymorphism (T-RFLP) analysis for species identification to investigate the vertical distribution of fungal hyphae in four distinct layers of the forest floor (lower litter, F-layer, H-layer, and B-horizon) of a Pinus resinosa plantation. •   Fungal communities differed markedly among the four layers. Cluster analysis suggested six different patterns of resource utilization: litter-layer specialists, litter-layer generalists, F-layer, H-layer, and B-horizon species, and multilayer generalists. Known ectomycorrhizal species were found in all six clusters. •   This spatial partitioning observed among ectomycorrhizal fungi along a single, relatively simple substrate-resource gradient supports the niche differentiation hypothesis as an important mechanism contributing to ectomycorrhizal fungal diversity.