Soil analyses

Forest floor N and P concentrations were determined by Technicon colorimetry (Pulse Instrumentation, Saskatoon, SK) following wet digestion of air-dried material. Percent organic matter of mineral soil samples was estimated by loss-on-ignition (6 h at 400°C). The pH was measured by H⁺ probe on aqueous suspensions (10:1 forest floor; 2:1 mineral soil) of air-dried soil subsamples. Fresh soil subsamples were extracted in 1 N KCl solution, filtered using Whatman No. 5 filter papers, and NH₄⁺ + NO₃^- concentrations (i.e., dissolved inorganic N (DIN)) were determined by Technicon colorimetry. Forest floor KCl extracts were further passed through 0.45 µm low protein-binding syringe filters prior to DIN analysis. Subsamples of these extracts were analyzed for total dissolved nitrogen (TDN), following persulphate oxidation to NO₃^- [24]. Dissolved organic N (DON) was calculated as TDN minus DIN. Soil mineral N fertility (i.e. mineralizable N) in all samples was assessed from DIN concentrations following 30-day fresh soil incubations (20°C), after correcting for initial DIN concentrations [25].

Soil basal respiration (BR) was measured on fresh soil samples (ca. 10 g forest floor; 30 g mineral soil) sealed in 125 mL jars for 12 h. Headspace CO₂ was then determined by Micro-GC (Chrompack, Middelberg, The Netherlands). Ambient CO₂ and temperature were regularly recorded during assays. Ambient CO₂ was subtracted from CO₂ headspace concentrations, with differences adjusted to 22°C using Ideal Gas Laws and assuming Q₁₀ = 2. The following day, the same soil samples were amended with glucose in order to estimate microbial biomass (MB) by substrate-induced respiration, using the protocol described by Bradley and Fyles [26]. Substrate-induced CO₂ production was converted to MB using equations of Anderson and Domsch [27].

Condensed tannins in forest floor samples were measured colorimetrically using the proanthocyanidin assay (butanol-HCl hydrolysis), standardised against purified black spruce and Kalmia tannins for samples respectively collected under black spruce and Kalmia patches [28]. Briefly, samples were freeze-dried, ground in a mortar and pestle, and extracted twice with 70% aqueous acetone, which was then dried down under N₂ for the determination of extractable tannins. The insoluble residue was dried under N₂ for the analysis of residual tannins; butanol-HCl reagent was added directly to the residue. Total condensed tannin concentrations were calculated as the sum of extractable and residual tannins. Total phenolics were determined after rehydrating 0.5 mL aliquots of dried acetone-water extracts with 1.0 mL distilled water, then adding 0.5 mL Folin-Ciocalteu reagent (Sigma) and 2.5 mL of aqueous Na₂CO₃ (20% w/v). Solution absorbance (750 nm) was read on a spectrophotometer standardised against tannic acid (Sigma-Aldrich), as per Waterman and Mole [29].

The activities of β-glucosidase and acid phosphatase, two extracellular soil enzymes involved respectively in C and P cycling, were measured in forest floor extracts using a microplate-based assay described by Joanisse et al. [7]. Changes in fluorescence of 4-methylumbelliferone (MUB) that was cleaved by the enzymes from their respective substrates (4-MUB-β-d-glucoside, 4-MUB-phosphate) were measured at 10 min intervals over 60 min.