Revealing the transfer pathways of cyanobacterial-fixed N into the boreal forest through the feather-moss microbiome

Abstract

Introduction: Biological N₂ fixation in feather-mosses is one of the largest inputs of new nitrogen (N) to boreal forest ecosystems; however, revealing the fate of newly fixed N within the bryosphere (i.e. bryophytes and their associated organisms) remains uncertain.

Methods: Herein, we combined ¹⁵N tracers, high resolution secondary ion mass-spectrometry (NanoSIMS) and a molecular survey of bacterial, fungal and diazotrophic communities, to determine the origin and transfer pathways of newly fixed N₂ within feather-moss (Pleurozium schreberi) and its associated microbiome.

Results: NanoSIMS images reveal that newly fixed N₂, derived from cyanobacteria, is incorporated into moss tissues and associated bacteria, fungi and micro-algae.

Discussion: These images demonstrate that previous assumptions that newly fixed N₂ is sequestered into moss tissue and only released by decomposition are not correct. We provide the first empirical evidence of new pathways for N₂ fixed in feather-mosses to enter the boreal forest ecosystem (i.e. through its microbiome) and discuss the implications for wider ecosystem function.

Keywords: NanoSIMS; Pleurozium schreberi; biological N2 fixation; boreal forest; moss microbiome; moss-cyanobacteria associations; nitrogen cycling.

Figure 1

¹⁵N accumulation (atom% ¹⁵N) of metabolically active cell components of the bryosphere of two northern Sweden boreal forest after ¹⁵N₂ incubation. (A) Upper younger dark green segment of Pleurozium schreberi shoot from Njällatjirelg, an open canopy forest with high moss N₂ fixation, and (B) from Reivo, a variably dense canopy forest with moderately high N₂ fixation in the moss layer, and (C) lower older brown segment of P. schreberi shoot from Reivo. Data from unlabeled replicates from each site used to generate ¹⁵N natural abundance values of each component - shown as black dots. Only data from the active (i.e. ¹⁵N enriched, red dots) cells is presented, > 95% confidence intervals of unlabeled controls and error (2σ, Poisson) smaller than the difference between the atom % of the labeled sample and the mean atom % of unlabeled control samples. Box plots summarize the quartiles of the target components of the bryosphere where boxes and whiskers encompass 25–75% and 5–95% quantiles of the data, respectively, with the median indicated by a dark horizontal line. Samples from the brown segments of P. schreberi from Njällatjirelg were removed from data analysis after NanoSIMS measurements since we could not detect ¹⁵N enrichment despite the presence of cyanobacteria. Branch location along the moss shoot selected for NanoSIMS analysis and raw images of rastered sections used for data analysis can be found in Figure S4 and Figure S6 respectively.

Figure 2

Spatial distribution of ¹⁵N accumulation in younger dark green segments of Pleurozium schreberi shoot and associated microbiome.¹⁵N concentration (¹⁵N/¹⁴N) of representative samples of an unlabeled shoot (A–C) and shoot labeled with ¹⁵N₂ and incubated for one week (Reivo forest site d-f; Njällatjirelg forest site g-i). ¹⁵N enrichment can be distinguished in cyanobacteria cells (CyanoCell) and in the moss cell wall (MossCW) and cytoplasm (MossCyto) in both forest sites (f and red arrow in e, h and i for moss cells). Epiphytic bacterial cells in direct contact with cyanobacteria can be seen enriched in ¹⁵N (D–F). Fungal hyphae attached to the moss leaf close to the cyanobacteria cells can be distinguished (G). Cellular structures are visible in the greyscale ¹²C¹⁴N images (A, D, G), with corresponding ¹⁵N/¹⁴N (B, E, H) images reflecting levels of ¹⁵N enrichment. The HSI colour scale (0.0037–0.01; ¹⁵N/¹⁴N natural abundance = 0.0037) applies to all HSI images. Dots and line scans show numerical levels of ¹⁵N enrichment (¹⁵N/¹⁴N) across the subcellular regions (C, F, I), with data acquired from the line (512 × 512 pixels) (I) indicated on the respective ¹²C¹⁴N image (G). Structural features: cyanobacteria (cy), bacteria (b), fungal hyphae (fh), and moss leaf cell (mlc). Bars, 10 µm (for all images).

Figure 3

Spatial distribution of ¹⁵N accumulation in older brown segments of Pleurozium schreberi shoot and associated microbiome. Enrichment of both moss cells and moss-associated microbiome can be distinguished after one week of continuous exposure to ¹⁵N₂ (a-i). Low ¹⁵N enrichment within the moss cell can be detected in some organelles (red arrow in B, C and E, F) whereas the cell wall and lipid bodies are not enriched (< 0.0037 ¹⁵N/¹⁴N, f). Epiphytic bacterial cells in direct contact with cyanobacteria can be seen enriched in ¹⁵N (A–C); the uptake of ¹⁵N fixed by cyanobacteria cells into green algae (A–C) and a hypha from a Basidiomycete (distinguished by a clamp connection at the hyphal septa, white arrows in G–I) were also detected. In (D–F) an extracellular gradient of ¹⁵N enrichment is visible over the moss cell (indicated by two white arrows). Epiphytic microbes seated on the moss leaf have access to the fixed ¹⁵N and were enriched (E, F), whereas the moss cell walls are not enriched (< 0.0037 ¹⁵N/¹⁴N). Cellular structures are visible in the greyscale ¹²C¹⁴N images (A, D, G), with corresponding ¹⁵N/¹⁴N (B, E, H) images reflecting levels of ¹⁵N enrichment. The HSI color scale (0.0037–0.01; ¹⁵N/¹⁴N natural abundance = 0.0037) applies to all HIS images. Line scans (512 x 512 pixels) show numerical levels of ¹⁵N enrichment (¹⁵N/¹⁴N) across the subcellular regions (C, F, I), with data acquired from the lines indicated on each of the respective ¹²C¹⁴N images. Structural features: cyanobacteria (cy), bacteria (b), fungal hyphae (fh), unicellular green algae (ga), unidentified microbes (um) and moss leaf cell (mlc). Bars, 10 µm (for all images).

Figure 4

Bacterial and fungal community shifts along the Pleurozium schreberi shoot profile revealed by 16S and ITS analysis from two northern Sweden boreal forests. Pie charts represent relative abundance of the major bacterial taxa (brownish pie charts) and fungal trophic mode (blueish pie charts) associated with the light green (new growth tissue), dark green (mature tissue) and brown (senescent tissues) segments of the moss P. schreberi. Samples collected in Njällatjirelg, open canopy forest with high nitrogenase activity, and Reivo, variably dense canopy and moderately nitrogenase activity. Results are based on the number of sequence reads from taxa that could be assigned to genus level or lower (detailed results on Supplementary Table 2 and Supplementary Table 3 ). Bacterial taxa marked with asterisks were present in the nifH data set (Cyanobacteria 99.90 % and Alphaproteobacteria 0.10 % of the total number of nifH sequences).