Mosses contribute an average of 20 % of boreal upland forest net primary
productivity and are frequently observed to degrade slowly compared to
vascular plants. If this is caused primarily by the chemical complexity of
their tissues, moss decomposition could exhibit high temperature sensitivity
(measured as
Boreal forests account for over half of global forest soil carbon (C) stocks, with areal soil C densities 2–3 times higher than temperate or tropical forests (Malhi et al., 1999). Many factors contribute to high C stocks, including low temperatures, high soil moisture in many regions, and the high relative abundance of plants with relatively slow-to-decay litter such as mosses (Coûteaux et al., 2002; Hobbie, 1996; Hobbie et al., 2000; Wetterstedt et al., 2010). However, the boreal region is warming more rapidly than the global mean (IPCC, 2013), which could lead to C losses both from the direct effects of warming and drying (Kane et al., 2005) and from the indirect effects of changing C sources due to vegetation change (Gornall et al., 2007; Kohl et al., 2018; Turetsky et al., 2012).
Mosses contribute an average of about 20 % of the total NPP in upland boreal forests (Turetsky et al., 2010) and can locally exceed vascular plant NPP (Frolking et al., 1996; Gower et al., 2001). Despite this, the unique dynamics of moss biogeochemistry are not typically included in models of boreal forest C cycling, which could result in considerable biases. For example, Bona et al. (2013) used a literature review of the rates of primary production and decomposition of upland mosses to estimate the range of moss C that could be stored in the soils of black spruce-dominated boreal forests, and the result (31 %–49 % of total soil organic carbon – SOC) was comparable to the difference between modeled and observed C stocks for those forests. This demonstrates that accurately representing moss production and decomposition is essential for modeling the C cycle in moss-rich boreal forests. While many studies estimate moss primary production, only three estimates of degradation rates were available for this meta-analysis, and none estimated the temperature sensitivity of moss decomposition (Bona et al., 2013). This is significant, because the limited numbers of studies of upland mosses show slower decomposition than vascular plant litter incubated under similar conditions (Harden et al., 1997; Moore and Basiliko, 2006). Omitting moss-specific dynamics could therefore overestimate decomposition and underestimate C storage in moss-rich soils.
Predicting the temperature sensitivity of moss decomposition is difficult,
because it is not clear if the apparent recalcitrance of moss tissues is due
to chemical complexity, physical and structural characteristics impeding
microbial decomposition, or a combination of the two. Most studies of moss
biochemistry have focused on the peat-forming
Distinct from moss chemical characteristics, the physiochemical matrix of
moss cell walls also could play a role in limiting microbial access to moss
tissues otherwise useful as microbial resources. Indeed, scanning electron
micrographs of a slowly decomposing
Study site characteristics including mean annual temperature (MAT) and mean annual precipitation (MAP).
We observed the decay of upland boreal forest mosses collected from the Newfoundland and Labrador Boreal Ecosystem Latitudinal Transect (NL-BELT) for more than 2.5 years to investigate (1) the temperature sensitivity of moss tissue decomposition and (2) the relationship between moss chemical composition, cell-wall structure, and its decomposition. We combined chemical characterization with scanning electron microscopy (SEM) to determine both chemical and physical changes in the moss tissues during decomposition. In doing so we investigate the relative importance of these factors contributing to the slow turnover of moss tissues in these forests.
Moss samples were collected in July 2011 from two balsam-fir-dominated forest
sites within the Newfoundland and Labrador Boreal Ecosystem Latitudinal
Transect (NL-BELT). One site was located in the Salmon River watershed near
Main Brook on Newfoundland's northern peninsula (hereafter “northern
forest”), and one was located in the Grand Codroy watershed in southwestern Newfoundland
(“southern forest”). The mean annual temperature (MAT) in the northern
forest is 3.2
Incubations were designed to include four destructive-sampling time points,
which occurred at 69, 283, 648, and 959 days from the beginning of the
experiment, starting in October 2011. The green portion of moss tissues from
both regions was incubated in the dark in sealed glass jars at 5
and 18
Following inoculation, jars were sealed and incubated, with half the jars at
5
To determine how decomposition affected organic matter composition, the elemental and stable carbon and nitrogen isotopic composition of the moss samples were analyzed on a Carlo Erba NA 1500 Series elemental analyzer interfaced to a Delta V Plus isotope ratio mass spectrometer via a ConFloIII interface (ThermoFisher Scientific). In total 48 samples were analyzed, plus 6 initial samples were taken as random triplicates from the homogenized prepared moss tissue from each site.
Sub-samples of the initial and final (959 days) moss samples were analyzed
using solid state cross-polarization magic-angle-spinning (CPMAS)
Water-soluble inorganic nitrogen, measured as nitrate plus nitrite and
ammonium, was determined using a Lachat 8500 flow injection analyzer. All
samples were first extracted using Nano-UV water. Briefly, 300 mg of the
ground moss material was shaken with 10 mL of water for 2 min at room
temperature then centrifuged and filtered using a glass fiber filter (GF/F;
nominal pore size of 0.45
Total hydrolyzable amino acids (THAAs) were analyzed following the method
outlined in Philben et al. (2016), using the EZ:Faast kit for amino acid
analysis (Phenomenex, USA). Briefly, a 20 mg subsample of each moss sample
was mixed with 1 mL of 6M HCl acid in a 1 mL ampule, which was sealed,
shaken, and heated at 110
We performed scanning electron microscopy (SEM) on prepared fragments of the
initial and final moss tissues from this experiment using a JEOL JSM 7100F
Field Emission SEM equipment with a Thermo EDS. Three random subsamples
were taken from the homogenized
whole dried (40
The percent mass remaining was calculated using the dry weights of the initial
and final mass at the four time points of the incubation. The mass remaining was
then fit to the exponential decay equation to calculate the rate of decay:
Moss tissue C remaining
normalized to the initial tissue C (black circles). The solid black line
represents the exponential fit used to assess the decay constant (
THAA data were analyzed to determine the percentages of total C or N as amino
acids and their change over incubation time, using the equation from Philben
et al. (2016):
To test for the effects of incubation temperature, site, and their
interaction on all quantified variables, we applied a two-way ANOVA using a
mixed effects model. Additional two-way ANOVA tests were conducted for each
time point to determine if any observed treatment effects changed over the
course of the incubation. A two-way ANOVA was conducted to test the effects
of site and temperature on
Results from two-way ANOVA tests of the effects of collection site
and incubation temperature on mass, C and N remaining, molar C to N ratio
(
n/a – not applicable.
Both C and mass loss measured in this experiment were fit to an exponential
equation as described in the methods (Fig. 1). When incubated at
18
Percentage of initial nitrogen remaining (circles and solid lines)
and
The N remaining did not follow the same trend as mass and C and could not be
fitted to an exponential curve but rather exhibited both increases and
decreases over the course of the incubation (Fig. 2). One treatment (the
southern forest at 18
The CPMAS
Mosses collected from the two sites differed in initial N concentration
(
Molar carbon-to-nitrogen ratio of moss tissues from the cooler
The initial
Nitrogen was further characterized into four compound classes, namely
nitrate
Fraction of the total moss tissue N content as nitrate, ammonium,
total hydrolyzable amino acids (THAAs), and molecularly unidentified N (MUN)
in each sample. Total hydrolyzable amino acids were only reported for one
triplicate of the 18
The CPMAS
SEM imaging revealed few apparent differences between the physical structure of moss tissues before and after incubation (Figs. 7, S1, and S2). Intact moss cell walls were visible in both sets of images, with few signs of structural change. There was no evidence of warping, gouging, or pitting from the microbial degradation of the cell wall following the incubation of mosses from either region.
The low decay rates observed are consistent with previous studies of moss
decomposition (Fyles and McGill, 1987; Hobbie et al., 2000; Hogg, 1993; Hagemann and Moroni, 2015). Decay
rates of moss tissues are typically lower than rates for vascular plant decay
under similar conditions (Fyles and McGill, 1987; Hagemann and Moroni, 2015;
Hobbie, 1996). Mass loss during the 1-year litter bag decomposition of balsam
fir needles averaged 27 % at the northern forest and 35 % at the
southern forest (Kate A. Edwards,
unpublished data). The needles therefore experienced a similar mass loss in 1
year to the mosses after 959 days, indicating more rapid
decomposition. The moss decay rates can also be compared to the incubations of
The northern forest mosses, but not the southern forest mosses, exhibited
higher
Hydroxyproline yields in the moss tissues as a percentage of total
hydrolyzable amino acids. Error bars for the initial and final time points
(0 and 959 days) indicate standard deviation (
We did not observe significant differences between sites' total C loss or
Examples of the CPMAS
Representative scanning electron micrographs of moss tissues
before
The low
There is also no evidence that decay-inducing microorganisms were limited by
N availability. Indeed, varying concentrations of moss tissue N and
Panels
The changing composition of N during the incubations suggests that the rapid
turnover of the N pool could have reduced microbial N limitation. The %N
as THAAs declined from
Despite the rapid turnover of the bulk amino acid pool, changing AA
composition during the incubation indicates that the cell-wall matrix
protected some proteins as well. Hyp is found in glycoproteins in the plant cell wall, but it
lacks major microbial sources (Philben and Benner, 2013). The increase in Hyp relative to other amino
acids in the incubations of the GC mosses therefore indicates the selective
preservation of these cell-wall proteins. The northern forest incubations
exhibit an increase in mol% Hyp, followed by a decline to a value near to
that of the pre-incubation sample. This
regional difference is likely due to the difference in bulk N dynamics; the
decline in
The persistence of the physical structure of the cell wall likely explains
the observed combination of slow decomposition and its low temperature
sensitivity. The SEM images indicate that microbial access to chemically labile C
is inhibited by the biochemistry and the physical matrix of the cell wall of the moss.
The lack of microbial access to otherwise reactive microbial resources
appears to be a bottleneck to decomposition that is not alleviated by
increasing temperature, explaining our observation of low
Although they share a backbone of cellulose microfibrils, there are important
chemical and structural differences between moss and vascular plant cell
walls that could contribute to the persistence of the cell wall of the moss
during the incubation (Roberts et al., 2012). Uronic acids (glucuronic acid
and galacturonic acid) are more abundant in mosses than in vascular plants
(Popper and Fry, 2003). The NMR spectra indicated that O- and di-O-alkyl
functional groups account for
The NMR spectra also indicate a substantial contribution of phenolic and aromatic C (7 % of the total C), despite the lack of lignin. The relative abundance of this fraction increased during decomposition, indicating selective preservation. Tsuneda et al. (2001) identified an amorphous phenolic coating on the outside of the moss cell wall and proposed that a specialized fungal consortium is required to break it down. This is analogous to the accelerated degradation of lignin in wood by white rot fungi (Rice et al., 2006) and is a plausible explanation of the lack of cell-wall degradation observed in the SEM images as well as the accumulation of phenolic and aromatic C. If key fungal species were excluded from the soil slurry used as a microbial inoculum in the incubations, or if incubation conditions were not favorable for their growth, then their absence could lead to the persistence of the cell wall's structural integrity.
Overall, our data suggest that some combination of the inherent molecular resistance to decomposition and the molecular architecture of the cell-wall matrix makes it difficult for microbes or their exoenzymes to access. While these analyses can identify biochemical differences between moss and vascular plant cell walls, we cannot identify which of these differences specifically contribute to their apparent recalcitrance. This presents an intriguing avenue for future research, as the persistence of the physiochemical integrity of the moss cell-wall matrix is likely important for maintaining the globally significant pools of moss-derived C in both peatlands and forested uplands.
The contrasting responses of the bulk C composition and the THAA-based
indices during moss decomposition could complicate the interpretation of
decay-focused data sets in moss-rich boreal forest soils. The decomposition of
vascular plant tissues is typically characterized by the selective loss of
carbohydrates. Indices such as the carbohydrate yield and the O-alkyl-to-alkyl ratio of
Our measurements of the decay rates and
All data are included in the paper tables and the Supplement.
The supplement related to this article is available online at:
SEZ, SAB, RB, and KAE designed the research with further input from MP. SB and MP analyzed data with NMR and SEM contributions from SEZ. MP and SB prepared the paper, with editing from SEZ and further contributions on final drafts from RB and SAB.
The authors declare that they have no conflict of interest.
Comments from two anonymous reviewers helped to clarify and improve the
paper. We thank Jamie Warren for overseeing the maintenance of the moss
incubations and for assisting with sample processing and analysis.
Julia Ferguson assisted with moss sample preparation and mesocosm setup and
monitoring. Alex Morgan also assisted with incubation monitoring. We also
thank Celine Schneider for conducting the