Endemic species of ectomycorrhizal fungi support the exceptional productivity of a temperate rainforest

In the fall of 2020, over a six-week period (October 5-November 15) we visited each plot three times to search for fleshy, terricolous sporocarps. We selected sporocarps that were fresh and free from insect damage and strove to obtain composite samples for each fungal species, typically 3–6 individual sporocarps, from over the entire plot area. Following collection, sporocarps were cleaned of debris and dried in a 60°C convection oven for 24 h (larger sporocarps were sliced in half to facilitate drying). Species identity was confirmed by molecular DNA of the fungal ITS region using a small piece of dried sporocarp, and the remainder of the sample (cap and stipe) was ground to < 2 mm for nutritional analysis. In June 2021, soil cores approximately 12 cm in diameter and 15 cm in depth were removed with a soil knife at 15 random locations in each of the ten plots. The soil core included upper mineral soil and organic horizons. Samples were returned to the laboratory and kept at 4°C during processing. Each core was submerged under water in a large pan and random portions of roots removed for examination under a dissecting microscope. Fresh, vigorous colonies of EMF root tips were detached using tweezers. Composited root tips from three soil cores were combined into one vial, for a total of 5 subsamples per plot (n = 50 for the study). This process was conducted by two personnel for 30 minutes per soil core, and resulted in 7–10 mL of fresh root tips per vial. The samples were then freeze-dried and ground with a ball mill before DNA extraction.