Sampling of root nodules, rhizosphere soils, and riparian soils
In the study sites, we collected root nodules, rhizosphere soils (i.e., inside under host tree crown), and riparian soils (i.e., riparian but outside of alder crown). Root nodules were collected from the roots of 78 adult trees, whose genetic variation was estimated using 1,077 single nucleotide polymorphism (SNP) markers (Kagiya et al., 2018). A nodule cluster was collected from each of seven roots per alder individuals. The sampling was performed in August 2017.
Rhizosphere soils were collected between August and September 2018. Following litter layer removal, we collected three 10-cm3 soil blocks from the inside area under each of the alder crowns using a sterilized shovel. The shovels were sterilized at 121 °C for 20 min before using for the sampling, and disinfected using 70% (v/v) ethanol after every soil sampling event. We changed shovels to new ones when we moved to other sites.
To sample riparian soils, we established 40-m transects along the rivers at the centroid points of the studied alder individuals within each of the study sites. The riparian soils samplings were conducted at every 10 m along the transects (i.e., four points in total per each site). At the points, we collected 10-cm3 soil blocks in the same way of rhizosphere soil samplings. The riparian soils samplings were performed from July to August in 2017. From these samplings, we obtained a total of 12 riparian soil samples. To investigate environmental conditions, at the sampling points, soil pH was measured using a direct soil pH probe kit (Hanna Instruments). After transporting the soils to the laboratory, total inorganic nitrogen, including NH4+ and NO3-, was extracted from 10 g of the sampled riparian soils by 100 mL of 2M KCl, and analyzed with an auto analyzer (AACS-4, BL-TEC Inc., Japan; the detailed method of total inorganic nitrogen analysis was previously described by Fukuzawa et al., 2006).