Trees affect organic matter decomposition through allocation of recently fixed carbon belowground, but the magnitude and direction of this effect may depend on substrate type and decomposition stage. Here, we followed mass loss, chemical composition, and fungal colonization of leaf and root litters incubated in mountain birch forests over four years, in plots where belowground carbon allocation was severed by tree girdling or in control plots. Initially, girdling stimulated leaf and root litter mass loss by 12 and 22%, respectively, suggesting competitive release of saprotrophic decomposition when tree-mediated competition by ectomycorrhizal fungi was eliminated (Gadgil effect). After four years, girdling instead hampered mass loss of root litter by 30%, suggesting late-stage priming of decomposition in the presence of trees, in parallel with increased growth of shrubs and associated fungi following tree elimination. Hence, different mechanisms driving early- and late-stage litter decomposition should be considered in climate-feedback evaluations of forest expansion.
The study was carried out in Abisko, Swedish Lapland (68°20´60´´ N, 18°49´48´´ E), on a mountain slope 500-600 m above sea level 5 km southeast of Abisko (68°19´08´´to 68°18´31´´N and 18°49´00´´to 18°50´24´´E), in the treeline ecotone, with patchy stands of mountain birch (*Betula pubescens* Ehrh. ssp. *czerepanovii* (Orlova) Hämet Ahti). Mean annual temperature and annual precipitation at the Abisko Scientific Research Station during the study period (2018-2022) were 0.69 °C and 269 mm, respectively. On September 9, 2018, leaf and root litter was collected from the study area. Leaves of mountain birch were collected from the ground beneath several birch individuals and were brought back to the laboratory. To obtain root litter, organic layer material was sieved in the field, and root material was brought back to the laboratory, where fine roots (≤ 2 mm in diameter) were recovered and cleaned in distilled water to remove external organic and inorganic particles. Roots and leaves were dried at 40 °C for 48 hrs.
To measure litter mass loss in the field, we used litter bags (10.0 x 6.4 cm, internal dimensions, and 1.0 x 0.1 mm in mesh size). In each litter bag, we placed either 1.0 g of leaf litter or 0.35 g of root litter (± 0.001 g, for both litter types), and then closed the top with a wire. The leaf litter was 100% mountain birch leaves, whereas the root litter consisted of a mixture of the most common vascular plant species in the mountain birch forest at the study area (Parker *et al*. 2020), i.e., 75% of *Empetrum nigrum* L. ssp. *hermaphroditum* (Hagerup) Böcher, 15% mountain birch, 7% *Vaccinium vitis-idaea* L., and 3% *V. myrtillus* L., with proportions estimated from DNA analyses (see below) of pre-incubation litter. Subsamples of both litter types were dried at 60 °C for 48 hrs, and weighed, to determine the dry mass. Based on this, the dry mass of litter in each of the litter bags was 0.929 g and 0.330 g for leaf and root litter, respectively.
In early June 2017, six paired plots of 20 m diameter were set up in the study area. Each pair consisted of one control (untreated) plot and one plot in which the mountain birches were girdled in mid-June 2017, to reduce the transfer of photosynthate from the canopy to belowground. For more detailed information on the study site and the plots. In September 2018, two root litter bags were deployed in the upper organic soil layer (~5 cm below the soil surface), together with two leaf litter bags on top of the organic soil, in each of these paired plots. Each of the four litter bags were attached with a metal wire (~5 cm long) to a single, plastic stick stuck into the ground, to ease detection, and a metal clamp was placed over each leaf litter bag to keep it in place and to ensure contact with the organic soil surface. Such a set of four litter bags was deployed at four locations (approximately 5 m apart) in each plot, making it a total of 16 litter bags in each plot. Retrievals of litter bags were made over two years, once in early summer and once in early autumn, i.e. June 6 and September 10, 2019, and June 17 and August 20, 2020. At each date, one randomly chosen pair of leaf and root litter bags were retrieved from each plot. At retrieval, the litter bags were brought back to the laboratory, freeze dried, and then stored in a freezer (-20 °C) before being sorted from exogeneous material and weighed. In addition, on August 10, 2022, 47 months after deployment, the remaining litter bags were retrieved from all plots.
Analyses of litter chemistry and fungal abundance and community composition
For organic matter quality evaluation, we performed solid state Nuclear Magnetic Resonance (NMR) and C and N analyses of litters before deployment as well as after incubations up to 23 months. Fungal abundance was estimated in all litters based on quantitative real-time PCR assays of the fungal ITS2 region, and fungal community composition was based on PacBio sequencing of the same marker gene. Our PCR primers also targeted all dominant plant species in our system, and the relative abundance of different plant species in the root mixture before deployment as well as in incubated litters was estimated based on the ITS2 sequencing output.
