Preparation of leaf litter leachates
We collected the leaf litter of 6 broadleaf and 5 coniferous species commonly found in the plains and mountainous of northeastern Japan. Although we did not check when they fell, we collected leaf litter on top of the leaf litter layer on the ground so that the litter did not have direct contact with the soil. These fallen leaves were collected at Zao (N38.122, E140.451) on October 3, 2020, at Kawatabi (N38.745, E140.757) on October 15, 2020, and at Aobayama (N38.259, E140.837) on November 19, 2020 in Miyagi Prefecture, Japan (Table 1).
Before use, leaf litter was dried at room temperature (20 °C) and was cut with scissors to mimic the early decomposition processes by detritivores. For each tree species, we weighed the cut leaf litter and placed it in two 1000 mL Nalgene bottles containing 800 ml of distilled water. The weight of leaf litter in each bottle was adjusted to control the final concentration of TDP to approximately 1-4 µM after 7 days of the leaching process based on the release efficiency (Table 1) estimated by Ho et al. (2023). The water in the two replicate bottles was aerated in the dark for 7 days at 20 °C and mixed before being used as leaf litter leachates.
We used Scenedesmus obliquusas the experimental algal species because it has been frequently used in various studies . Prior to the incubation of Scenedesmus , the leaf litter leachate was filtered through 40 µm mesh to remove litter particles. DOC and total dissolved N (TDN) concentrations in the leaf litter leachate were measured using a TOC/TNb analyzer (multi N/C 3100, Analytik Jena GmbH, Jena, Germany). Total dissolved P (TDP) concentrations were measured using the ascorbic acid-molybdenum blue method after persulfate oxidation .