Ecology, 91(8), 2010, pp. 2356–2365
Ó 2010 by the Ecological Society of America
The role of Sphagnum mosses in the methane cycling of a boreal mire
TUULA LARMOLA,1,6 EEVA-STIINA TUITTILA,1 MARJA TIIROLA,2 HANNU NYKÄNEN,2 PERTTI J. MARTIKAINEN,3
KIM YRJÄLÄ,4 TERO TUOMIVIRTA,5 AND HANNU FRITZE5
1
Department of Forest Ecology, FI-00014 University of Helsinki, Finland
Biological and Environmental Science, FI-40014 University of Jyväskylä, Finland
3
Department of Environmental Science, University of Eastern Finland, FI-70211 Kuopio, Finland
4
Department of Biosciences, FI-00014 University of Helsinki, Finland
5
Finnish Forest Research Institute, Vantaa Research Unit, FI-01301 Vantaa, Finland
2
Abstract. Peatlands are a major natural source of atmospheric methane (CH4 ). Emissions
from Sphagnum-dominated mires are lower than those measured from other mire types. This
observation may partly be due to methanotrophic (i.e., methane-consuming) bacteria
associated with Sphagnum. Twenty-three of the 41 Sphagnum species in Finland can be
found in the peatland at Lakkasuo. To better understand the Sphagnum–methanotroph
system, we tested the following hypotheses: (1) all these Sphagnum species support
methanotrophic bacteria; (2) water level is the key environmental determinant for differences
in methanotrophy across habitats; (3) under dry conditions, Sphagnum species will not host
methanotrophic bacteria; and (4) methanotrophs can move from one Sphagnum shoot to
another in an aquatic environment. To address hypotheses 1 and 2, we measured the water
table and CH4 oxidation for all Sphagnum species at Lakkasuo in 1–5 replicates for each
species. Using this systematic approach, we included Sphagnum spp. with narrow and broad
ecological tolerances. To estimate the potential contribution of CH4 to moss carbon, we
measured the uptake of d13C supplied as CH4 or as carbon dioxide dissolved in water. To test
hypotheses 2–4, we transplanted inactive moss patches to active sites and measured their
methanotroph communities before and after transplantation. All 23 Sphagnum species showed
methanotrophic activity, confirming hypothesis 1. We found that water level was the key
environmental factor regulating methanotrophy in Sphagnum (hypothesis 2). Mosses that
previously exhibited no CH4 oxidation became active when transplanted to an environment in
which the microbes in the control mosses were actively oxidizing CH4 (hypothesis 4). Newly
active transplants possessed a Methylocystis signature also found in the control Sphagnum spp.
Inactive transplants also supported a Methylocystis signature in common with active
transplants and control mosses, which rejects hypothesis 3. Our results imply a loose symbiosis
between Sphagnum spp. and methanotrophic bacteria that accounts for potentially 10–30% of
Sphagnum carbon.
Key words: Lakkasuo mire, southern Finland; methane; methane oxidation; methanotrophs; peat;
Sphagnum.
INTRODUCTION
Northern mires play a dual role in atmospheric
carbon cycling. Mire vegetation fixes atmospheric
carbon dioxide (CO2) into organic carbon via photosynthesis. Unequal rates of production and decomposition have, over thousands of years, resulted in one third
of the global soil carbon being stored in northern mires
(Gorham 1991). Although capable of significant carbon
fixation, decomposition of organic matter under anaerobic conditions means that mires are a major net source
of methane (CH4 ), an important greenhouse gas.
Up to 100% of the CH4 formed in mire ecosystems
does not reach the atmosphere but is oxidized by
methanotrophic bacteria to CO2 in the uppermost
Manuscript received 23 July 2009; revised 15 October 2009;
accepted 9 November 2009. Corresponding Editor: J. B. Yavitt.
6 E-mail: tuula.larmola@helsinki.fi
aerobic layer of mire (Whalen 2005). Thus, the net flux
of CH4 to the atmosphere is determined by the activities
of microbes producing and consuming CH4. The aerobic
layer is roughly defined by the water table position
(Bubier and Moore 1994), and with water tables at or
near the surface, the Sphagnum moss layer represents
most of the aerobic environment where CH4 oxidation
can occur (Basiliko et al. 2004).
Submerged Sphagnum mosses were recently shown to
be able to consume CH4 through association with partly
endophytic methanotrophs in the water-filled, hyaline
cells of leaves and stems (Raghoebarsing et al. 2005).
Raghoebarsing et al. (2005) found CH4 to be a
significant (10–15%) carbon source for Sphagnum
cuspidatum Hoffm. in peat bogs. A close association
between methanotrophs and Sphagnum would facilitate
recycling of oxygen derived from photosynthesis and
CH4 derived from decaying plants. Raghoebarsing et al.
(2005) suggested that such an association could explain
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CH4 CYCLING IN SPHAGNUM
both the efficient recycling of CH4 and high organiccarbon burial in mire ecosystems. Over half of the peat
in the world was once Sphagnum (Clymo and Hayward
1982). The efficient recycling of peat decomposition
products such as CH4 may mechanistically explain the
paradox of mires as ecosystems with apparent low
primary productivity combined with high carbon burial
(Raghoebarsing et al. 2005). Furthermore, CH4 emissions from Sphagnum-dominated bogs are lower than
those from Carex-dominated fens (Nykänen et al. 1998).
One reason for this could be the methanotrophic activity
of Sphagnum mosses.
The importance of Sphagnum as a habitat for
methanotrophic bacteria and the uptake of CH4 on an
ecosystem scale are poorly understood. Basiliko et al.
(2004) surveyed CH4 oxidation in five Sphagnum species
and found that rates varied from similar to up to 30
times higher than peat on a dry-mass basis. Basiliko et
al. (2004) further suggested that differences in coexisting
species are small compared to differences across
habitats. The extent of CH4 oxidation in mosses and
its contribution to net CH4 flux is likely determined by
spatial and temporal variation in environmental conditions, such as water-table depth and mire type.
Our aim was to study the ecological importance of
CH4 oxidation in Sphagnum on an ecosystem scale.
Based on the work of Basiliko et al. (2004) we addressed
the following hypotheses: (1) all Sphagnum species
support methanotrophic bacteria; (2) water level is the
key environmental determinant of methanotrophic
activity in Sphagnum across habitats. Based on the
finding that CH4 oxidation rate does not always differ
from zero (Basiliko et al. 2004), we expected that (3)
under dry conditions Sphagnum species will not host
CH4-oxidizing bacteria. Because hyaline cells of Sphagnum contain permeable pores (Hayward and Clymo
1982), we hypothesised that (4) methanotrophs can
move from one Sphagnum shoot to another via a
continuous bridge of water.
To test hypotheses 1 and 2 we sampled 23 different
Sphagnum species in the Lakkasuo mire with a 100 3 100
m grid. With this approach we detected Sphagnum spp.
with narrow and broad ecological tolerances. Common
garden transplantations were employed to test hypotheses 2–4. Transplantations allowed us to separate the
effect of host moss species from abiotic environmental
factors on CH4 oxidation and to study the methanotrophic community in Sphagnum species before and after
transplantation.
METHODS
Study site and sampling design
The study was carried out at the Lakkasuo mire
(61848 0 N, 24819 0 E; 150 m a.s.l.), a boreal raised bog
complex in southern Finland. It contains a large variety
of mire site types of different nutrient status, and 23 of
the 41 Sphagnum species known to occur in Finland can
be found there (Laine et al. 2004).
2357
We based the sampling design for Sphagnum mosses
on a recent vegetation inventory (Laine et al. 2004) in
which plants were mapped with a systematic sampling of
100-m grid intervals. Projection cover of all plants and
lichens was recorded on 1-m2 plots and the water level of
the mire had been monitored for three growing seasons
from water wells next to each plot. In June 2006 we
sampled for the common species from five locations
along a hydrological gradient from the wettest to the
driest plot and including the plot with highest amount of
each moss. For the Sphagnum mosses that were found
on 5 plots, we sampled from every location. The
sampling thus covered 40 plots (60% of the inventory
plots).
The summer of 2006 was extremely dry in the study
area. The total precipitation for July was 37 mm, 60%
lower than the 30-year average at the nearby meteorological station (1971–2000, Drebs et al. 2002). Additionally, during 1–15 August precipitation was only 1.2 mm
(Finnish Meteorological Institute, unpublished data).
During the early sampling season (June), the water level
was close to the median over three growing seasons.
After that, the water level drew down until the end
August.
The dry period late in the summer allowed us to
examine the effects of long-term (three-growing-season
median) and short-term (day of sampling) water level on
methanotrophic activity. To monitor oxidation potential
over the season, 15 patches that showed activity in June
were re-sampled in mid-August and early October.
These patches included eight Sphagnum species belonging to the two most common sections (i.e., taxonomical
subgroups within the genus Sphagnum) Acutifolia and
Cuspidata at the Lakkasuo mire. The patches covered
the gradient from species growing on dry hummocks to
species growing on bog hollows and fen flarks, i.e., wet
depressions in the ombrotrophic or minerotrophic part
of the mire, respectively. Nomenclature for Sphagnum
follows Koponen et al. (1977).
Measurement of oxidation potential
To determine the potential of different Sphagnum
species to oxidize CH4 in different habitats, we collected
patches of each moss species for incubation and
measured the prevailing water level. Concurrently, we
took separate volumetric samples (diameter, 5 cm) of
moss from the uppermost 10 cm to determine density
and water content in the field.
We transported moss samples to the laboratory in a
lightproof and insulated box where their CH4 oxidation
potential was measured via a flask incubation technique
using 30 g of moss washed with deionized water
(Basiliko et al. 2004, Raghoebarsing et al. 2005) and
dried overnight at 48C. During incubation, the water
content was 1536% 6 594% (mean 6 SD) of moss dry
mass. After sealing a 600-mL flask with a septum, the
CH4 concentration was adjusted to 10 000 ppm and CH4
oxidation was monitored at 0.5 h, 24 h, and 48 h for a
2358
TUULA LARMOLA ET AL.
two-day incubation in the dark at 158C, which is the
median peat surface temperature during the growing
season at the Lakkasuo mire (Riutta 2008). Two control
sets of flasks were also incubated: (1) flasks without
moss and with initial CH4 concentration of 10 000 ppm
and (2) flasks with moss but without added CH4 (initial
CH4 concentration was 2 ppm). Oxidation rate was
calculated from the decrease in concentration over time.
Methane concentration was determined using two gas
chromatographs (Hewlett-Packard [HP] 5890A and HP
6890; Hewlett-Packard, Palo Alto, California, USA).
Both gas chromatographs were equipped with flame
ionization detectors and had helium as carrier gas.
HP5890A had a 1-mL loop, 1.8 m 3 3 mm packed
column Poropak Q, and HP 6890 had a 0.5-mL loop, 1.8
m 3 3 mm packed column Hayesep Q (see Jaatinen et al.
[2005] and Riutta [2008] for details).
During the incubations, CH4 concentration was
nonlimiting for low affinity methanotrophs (never under
6000 ppm; Sundh et al. 1995). The linear decrease in
CH4 concentration in the flasks further implied that
neither oxygen nor methane supply was limiting the
methane oxidation process. After the two-day incubation the headspace/moss volume of each flask was
measured and the incubated mosses were dried at 608C
to calculate potential oxidation rate per moss dry mass.
Volumetric samples of moss were initially weighed for
fresh mass after removing debris and other mosses than
Sphagnum, then dried at 608C for 48 h and weighed.
Moss water content and density (g/m2) in the uppermost
10 cm were determined based on fresh and dry mass.
The latter was also used to convert the incubation results
(lmol/ g dry moss biomass) to a potential rate per
surface area of the mire (mol/m2).
Concentration of CH4 in pore water
To establish the possible link between CH4 oxidation
and CH4 concentration, the CH4 concentration in the
pore water of the sites was determined in October (n ¼ 2
or 3 samples). Water samples of 30 mL were drawn in
syringes with a metal pipe from ground water wells. The
CH4 concentration in water was determined using a
headspace equilibration technique (McAuliffe 1971).
Nitrogen (30 mL) was added and equilibrated with
water by shaking vigorously for 3 min. The concentration of CH4 was analyzed in the N2 head space.
Dissolved CH4 concentration was calculated from the
headspace concentrations according to Henry’s law
using the values after Lide and Fredrikse (1995).
Transplantation
To separate the effect of different host species from
abiotic factors on CH4 oxidation and to test whether
methanotrophs can move from one Sphagnum shoot to
another with the water, we conducted a transplantation
experiment. On 31 August 2006 we transplanted three
Sphagnum species in three replicates from sites that
showed no methanotrophic activity to high-activity sites,
Ecology, Vol. 91, No. 8
namely wet depressions. A fen species (Sphagnum
girgensohnii ) was transplanted to two fen flarks, and
two bog species (S. rubellum and S. balticum) to two bog
hollows. To control for effects of the transplantation, we
included two sets of control samples in the experiment:
one of which was transplanted within each high-activity
site (n ¼ 3 samples of the native moss species of the site)
and another of which was not transplanted and thus left
intact (n ¼ 3 samples). Native mosses for the highactivity fen flarks were Sphagnum flexuosum and
Sphagnum squarrosum whereas those for the bog sites
were Sphagnum balticum and Sphagnum cuspidatum. All
the moss patches were sampled at the end of the
experiment (1 October 2006).
Stable carbon isotope ratio analyses
of moss, CH4, and CO2
To estimate the potential contribution of CH4 to
carbon uptake for submerged and partly submerged
Sphagnum spp. in different habitats, stable carbon
isotope ratio analyses were done for the moss (d13CC), for dissolved CH4 (d13C-CH4 ) and CO2 (d13C-CO2)
in mire water. The d13C-C was determined from a set of
15 Sphagnum species showing varying oxidation activities using FlashEA 1112 elemental analyzer coupled to
a Thermo Finnigan DELTAPlus Advantage continuousflow isotope mass spectrometer (Thermo Finnigan,
Bremen, Germany). For the analysis, moss samples
were dried at 608C, ground to a fine powder and
portioned (1.5-mg subsamples) into tin cups for the
analysis. Since the mean 6 SD for triplicate subsamples
was ,0.04%, only duplicates were measured for each
sample. From the same sites, mire water was collected by
syringe from the 0–10 cm moss layer, and d13C-CH4 and
d13C-CO2 were analyzed with isotopic ratio mass
spectrometer (Thermo Finnigan DELTAPlusXP) having
a pre-concentration unit (PreCon). The method was
similar to that described in Kankaala et al. (2007) except
for the CO2 analyses, in which a water trap replaced the
CO2 scrubber and the temperature of the catalyst was
decreased to 1008C. All the isotopic values are expressed
relative to Vienna-PeeDee Belemnite.
Using d13C in CH4 and CO2 as end members and d13C
in moss we calculated the potential contribution of CH4derived carbon in moss (a) with the following equation
(Raghoebarsing et al. 2005):
d13 CSphagnum ¼ ad13 Crespired CH4 þ ð1 aÞd13 CCO2 Ep
where a fractionation factor (Ep) of 7% was used for
moss carbon uptake (4–10%; Keeley and Sandquist
1992).
Analysis of the methanotrophic community
To study the methanotrophic (methane-consuming)
community in Sphagnum spp., plants were carefully
washed with distilled water before microbial DNA
isolation. DNA was isolated from Sphagnum spp.
(;0.5 g wet mass) using the FastDNA kit for soil (MP
August 2010
CH4 CYCLING IN SPHAGNUM
biomedicals, Solon, Ohio, USA) according to Yeates
and Gillings (1998; modified as in Tuomivirta et al.
[2009]). A region of subunit a of particulate methane
monooxygenase ( pmoA) was polymerase chain reaction
(PCR) targeted with the primer pairs A189f/A682r
(Holmes et al. 1995) and A189f/A621r with a GC-clamp
attached to the reverse primer (see Tuomivirta et al.
[2009] for a comparative study of the two pmoA primer
pairs, the PCR program, and the GC tail).
Fingerprinting of the methanotrophic community
diversity was performed by denaturing gradient gel electrophoresis (DGGE) as described in Jaatinen et al.
(2005). Single DGGE bands of interest were excised from
the gel, eluted into distilled water, and re-amplified in
PCR as above using 15 cycles following the touchdown.
Products were rerun in DGGE, re-excised, and reamplified 2 to 3 times to reach a clean product for
sequencing. Purified DGGE amplicons were amplified
using M13fA189f (5 0 -CACGACGTTGTAAAACGAC
GGNGACTGGGACTTCTGG-3 0 ) and M13rA621r
(5 0 -GGATAACAATTTCACACAGGCGCTCGACCAT
GCGGAT-3 0 ) primers. These M13-sequencing-primertailed products were direct sequenced according to the
manufactures instructions (SequiThermEXCEL II DNA
Sequencing Kit-LC; Epicentre Technologies, Madison,
Wisconsin, USA) and M13 forward and reverse reactions were performed separately. The respective sequencing reactions were combined during the stop solution
addition step and run on a Long Reader 4200DNA
Sequencer (LI-COR, Lincoln, Nebraska, USA). Nucleic
acid and putative amino acid sequences were screened
using BLAST (Altschul et al. 1997) analysis of the
National Center for Biotechnology Information (NCBI).
Statistical analyses
In order to identify the factors behind variation in
CH4 oxidation potential, the relationship between the
rate of oxidation and environmental factors was
determined using regression modeling. The relationship
between long-term or actual water level was described
with a Gaussian response. The Julian day (day 1 ¼ 1
January) was used to describe the phase of the growing
season. Residuals of the models were analyzed for
possible correlation with CH4 concentration, moss
density, and water content. The possible differences
among species in the transplant experiment were
explored with analysis of variance. The contribution of
CH4 in moss carbon was analyzed for possible
correlation with oxidation activity. All the statistical
analyses were made using SPSS 15.0 (SPSS 2006).
RESULTS
The role of Sphagnum species and abiotic environment
All 23 Sphagnum species supported methanotrophs
(Fig. 1). Nine Sphagnum species growing in the wettest
environments showed the ability in every location
sampled but the rest of the species in only 30–90% of
sampling locations. The rates of potential oxidation
2359
FIG. 1. Frequencies of the Sphagnum moss samples showing
methanotrophy in June 2006 at the Lakkasuo mire, Finland.
(Methanotrophy is the biological consumption of methane as a
source for carbon and energy.) N ¼ 1–5 locations; the number of
locations, n, is indicated in parentheses (below) for species with
,5 locations. The 23 Sphagnum species are: ter, S. teres; ang,
S. angustifolium; gir, S. girgensohnii; wul, S. wulfianum; cus,
S. cuspidatum; bal, S. balticum; fus, S. fuscum; fal, S. fallax;
rip, S. riparium; rus, S. russowii; pap, S. papillosum;
mag, S. magellanicum; rub, S. rubellum; cap, S. capillifolium;
cen, S. centrale; squ, S. squarrosum; fle, S. flexuosum (n ¼ 1);
maj, S. majus (n ¼ 2); subf, S. subfulvum (n ¼ 2); subn, S. subnites
(n ¼ 2); subs, S. subsecundum (n ¼ 1); ten, S. tenellum (n ¼ 1); and
war, S. warnstorfii (n ¼ 2).
ranged from 0 to 62 lmolg1day1, based on dry mass
(Fig. 2).
Among factors tested, water level was the key
environmental factor regulating methanotrophy in
Sphagnum. Spatial variation in the long-term water level
of the microhabitat was related to variation in the
occurrence of methanotrophy. In 80% of the plots, every
Sphagnum species supported methanotrophic activity, at
least in early summer. The remaining 20% of plots with
some or none of the Sphagnum species supporting active
methanotrophs were either located in spruce-dominated
mire margins or high bog hummocks (Appendix: Fig.
A1).
Seasonal variation was related to changes in water
level. After a six-week period with very low precipitation, only 74% of 15 patches of moss that had been
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TUULA LARMOLA ET AL.
FIG. 2. Response of potential CH4 oxidation rates to
median water level of the microhabitat over three growing
seasons at Lakkasuo mire. Positive values indicate that the
water table is above the moss surface. Measured data (solid
circles) and the Gaussian response model are shown. The two df
values are regression and residual.
active early in the season showed a CH4 oxidation
potential, and the rates of potential CH4 oxidation in
these active samples had declined to 10% of the
corresponding rates earlier in the season. Later, when
the water levels had risen, rates had recovered to 90% of
those measured earlier. All samples were wetted in the
laboratory, so their water content was similar during
incubations.
In the complete survey data set, variability of the
long-term water level of the microhabitat and variability
of actual water level explained 10% and 20%, respectively, of the variability in the potential rates of
methanotrophic activity. The response to both waterlevel variables took a bell-shaped form (Fig. 2;
Appendix: Table A1). Early in the season, long-term
median water level of the microhabitat and actual water
level of the sampling day equally explained 13% of the
variation in potential activity. For mosses sampled in
mid-August or early October, water level of microhabitat explained 31% of the variation and the actual water
level 79% of the variation. Variation unrelated to water
level was associated with CH4 supply; residuals of the
late-season model positively correlated with the concentration of CH4 in the pore water (r ¼ 0.278, P ¼ 0.038, n
¼ 56 observations).
We attempted to refine the models to include
variation within microhabitat (grid points), but treating
field water content of the mosses or the density of the
moss patch as independent variables did not significantly improve explanatory power. The residuals of the
seasonal model, including long-term median water level
of the microhabitat and Julian day (since January 1st) as
independent variables, were, however, positively correlated with water content of mosses in the field (r ¼ 0.179,
P ¼ 0.021, n ¼ 165 observations) and the density of the
moss patch (r ¼ 0.224, P ¼ 0.002, n ¼ 182 observations).
Ecology, Vol. 91, No. 8
No additional variation could be attributed to moss
species. Our sampling design for the 23 Sphagnum
species resulted in 26 microsites with .2 coexisting
Sphagnum species. To compare variation in CH4
oxidation rate within one species in a microsite and
among several species in a microsite we calculated
coefficients of variation in respective CH4 oxidation
rates. The coefficients of variation were on average 95%
6 37% (mean 6 SD) within one species in a microsite
and 93% 6 34% among all species within a microsite.
When we averaged the CH4 oxidation in different
species to microsite-specific rates, long-term median
water level and the actual water level explained 27% and
23%, respectively, of the spatial variation in the early
season peak activity (Appendix: Table A1).
Transplants
Regulating factors were further studied with the
transplantation experiment. When individuals showing
no CH4 oxidation activity were transplanted to an
environment where Sphagnum spp. had shown CH4
oxidation activity, all transplants had become CH4
oxidizers after one month. The methanotrophic activity
of the transplants did not significantly differ from the
native surrounding species (Fig. 3). Furthermore, there
were no significant differences among oxidation rates in
the bog species S. rubellum and S. balticum when
transplanted in a common hollow. Nor did transplantation within the high-activity site significantly affect the
oxidation rate. The oxidation rates did not differ
between the bog and the fen transplants (F ¼ 1.902, df
¼ 3, 25, P ¼ 0.155).
Molecular analysis of the methanotrophic community
using the primer pair A189f/A621r targeting pmoA
produced clear amplicons while A189f/A682r primers
produced only weak and diffuse ones in polymerase chain
reactions (PCR; not shown). The clear amplicons of the
methanotrophic community of one bog-transplantation
experiment were investigated using PCR-DGGE (denaturing gradient gel electrophoresis) fingerprinting and
direct sequencing of DGGE bands. Inactive S. rubellum
and S. balticum species were transplanted to hollows
containing active S. cuspidatum as controls. PCR
indicated that all Sphagnum species had methanotrophic
DNA. DGGE revealed that inactive and active transplants and controls hosted a similar methanotroph
sequence (Genbank GQ121280). In addition, active
transplants hosted a methanotroph sequence (GQ121283)
that they did not have before transplantation and that
existed in the active S. cuspidatum serving as controls
(Fig. 4). Sequencing identified these bands as putative
members of the genus Methylocystis with respective
identities of 100% (FJ930091; Tuomivirta et al. 2009)
and 99% (DQ379514; Dumont et al. 2006) to the closest
gene-bank sequence. DGGE bands occurring in the
region of the first three ladder bands could not be
successfully sequenced and DGGE bands above the first
ladder band are heteroduplexes.
August 2010
CH4 CYCLING IN SPHAGNUM
2361
FIG. 3. Potential CH4 oxidation rates one month after transplantation in the active sites at the Lakkasuo mire. Replicates (n ¼
3) of three Sphagnum species were transplanted: fen species S. girgensohnii to two fen flarks and two bog species, S. rubellum and S.
balticum, to two bog hollows. Native species both transplanted within the site and intact (not transplanted) served as controls. The
oxidation rates of the transplants did not significantly differ from those of the controls. Error bars indicate þSD.
Methane-derived carbon in Sphagnum
In Sphagnum, 10–30% of carbon potentially originates
from CO2 derived from CH4 oxidation. This calculation
was based on d13C in pore water CH4, CO2, and in
selected Sphagnum mosses, assuming a fractionation
factor of 7% in moss photosynthesis. When the range of
4–10% for fractionation (Keeley and Sandquist 1992)
was taken into account, variation in contribution of
CH4-derived carbon in Sphagnum ranged from 5% to
38%. In fen and bog water, d13C-CH4 was 49% to
68% and 71% to 80%, respectively. In water d13CCO2 was on average 10% (2–16%), the range in fen
and bog sites overlapping. In Sphagnum d13C-C ranged
from 23.1% to 30.8%. The share of potentially CH4derived carbon in Sphagnum had no correlation with the
concurrent potential rate of oxidation (r ¼ 0.05, P ¼
0.867, n ¼ 15 observations).
DISCUSSION
The role of Sphagnum species
In accordance with our first hypothesis, we found that
all of the 23 studied species could support methano-
trophs. The frequency and magnitude of potential
methanotrophic activity was controlled by environmental conditions of the site, primarily by water level, rather
than by Sphagnum species per se. Our results, including
the same species, are in contrast to the previous finding
of Basiliko et al. (2004) that in the same habitat and with
the same water content, CH4 consumption potential in
the two morphologically different Sphagnum species
could differ significantly. Basiliko et al. (2004) suggested
that interspecific differences may have resulted from
differences in gas–water transfer of CH4 in moss or from
plant physiology constraining the CH4 oxidizing mass.
Instead, we found that functional moss characteristics
related to water-holding capacity, i.e., the water content
and the growth density, correlated weakly but positively
with the variation in CH4 oxidation potential unrelated
to water level. A large part of the variation in water
content may be due to differences in extracellular waterholding capacity (Rice et al. 2008). For example, some
species in the Sphagnum section Acutifolia form dense
hummocks with an efficient capillary network (Hayward
and Clymo 1982) and thus have a greater capacity to
transport water than loosely growing species. Greater
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TUULA LARMOLA ET AL.
Ecology, Vol. 91, No. 8
FIG. 4. Denaturing gradient gel electrophoresis analysis of pmoA amplicons of different Sphagnum samples. The lanes from left
to right are as follows: Ladder (L); empty lane; inactive Spaghnum rubellum (); active transplanted S. rubellum, n ¼ 3; active
transplanted S. cuspidatum control, n ¼ 3; ladder (L); active S. cuspidatum control, n ¼ 3; active transplanted S. balticum, n ¼ 3;
inactive S. balticum (); empty lane; ladder (L). Sequenced DGGE bands are identified by arrows and GenBank accession numbers.
Electrophoresis direction was from top to bottom.
water-holding capacity maintains more stable water
conditions and could thus support a denser methanotrophic population. The study by Rice et al. (2008)
further suggested that a smaller part of the variation in
water content among Sphagnum spp. was related to the
volume of hyaline cells: species with large hyaline cells
have high water content (Rice et al. 2008). Large hyaline
cells could also provide a disproportionally larger
habitat for associated microbes.
The role of environment
Our results agree with the previous finding that CH4
oxidation potential was at its highest when the water
table was at the moss layer (Basiliko et al. 2004). Water
table roughly defines the active zone where both CH4
and oxygen were present (Bubier and Moore 1994) and
thus constrained the substrate supply. The two types of
environment, high hummocks and mire margins (where
methanotrophy in Sphagnum mosses was less frequent
or the oxidation rates were not detectable) typically had
low concentration of CH4 at the moss layer (Appendix:
Fig. A1). In high bog hummocks, water level and the
aerobic zone for potential CH4 oxidation is deeper in the
peat rather than at the moss layer (Jaatinen et al. 2005).
In spruce-dominated mire margins, water is flowing and
CH4 is presumably rapidly oxidized. In various peats,
potential CH4 oxidation is controlled by the same
factors influencing CH4 production, implying that CH4
oxidation is primarily controlled by CH4 availability
(Basiliko et al. 2007). In our study, the bell-shaped
response of oxidation to water level was similar to that
found for net CH4 flux (e.g., Juutinen et al. 2003), which
might imply regulation by substrate availability. Moreover, the finding that CH4 oxidation-potential measurements relate to water level in a pattern one might expect
corroborates that, in spite of occurring in vitro and with
substrate (O2 and CH4 ) supply, these potential measurements can be used to infer relative rates of in situ
processes or at least methanotroph biomass.
At Lakkasuo mire the ranges for rates of oxidation in
ombrotrophic bog and minerotrophic fen sites overlapped. In contrast, Basiliko et al. (2004) observed the
smallest CH4 oxidation potentials in bog hummocks and
hollows, and the potentials increased across fen hummocks and flarks. One reason for similar oxidation rates
in bogs and fens could be that a high nitrogen
requirement of methanotrophic bacteria could have
been compensated for by their fixation of atmospheric
nitrogen (Bodelier and Laanbroek 2004), and hence the
low concentration of inorganic nitrogen in the bog sites
August 2010
CH4 CYCLING IN SPHAGNUM
2363
did not necessarily lead to nitrogen limitation and
reduced CH4 oxidation. Recently, Opelt et al. (2007)
detected that Sphagnum mosses are home to a high
diversity of nitrogen-fixing bacteria.
unpublished results). These findings imply that bacteria
more likely change activity with fluctuating water level
rather than that bacteria remain as tightly associated
symbionts of certain moss species.
Methanotrophs in transplants
Sphagnum-hosted CH4 oxidation in mire carbon cycling
Raghoebarsing et al. (2005) reported that CH4
oxidizing S. cuspidatum hosted a 16S rDNA sequence
showing 93% identity to methanotrophs Methylocella
palustris and Methylocapsa acidiphila. The low identity
suggests that their methanotroph remained unidentified.
In this study S. cuspidatum hosted Methylocystis
sequences. The PCR was performed to detect the a
subunit gene of the particulate membrane-bound
enzyme methane monooxygenase (pMMO) responsible
for the first step in CH4 oxidation. The pMMO is
present in all known aerobic methanotrophs (Hanson
and Hanson 1996), with the exception of Methylocella
species (Dedysh et al. 2000, Dunfield et al. 2003), which
have only the soluble cytoplasmic form (sMMO).
Therefore possible Methylocella sequences would have
not been detected in this study.
The occurrence of the same Methylocystis sequence in
active transplanted and control Sphagnum species that
was not observed in the inactive Sphagnum spp. before
transplantation supports hypothesis 4, which states that
methanotrophic bacteria can move in water from plant
to plant. It has to be mentioned that the DGGE band
(sequence GQ121283) could have been present in the
inactive Sphagnum before transplantation but was below
the PCR-DGGE detection limit. On the other hand,
Sphagnum spp. that were inactive before transplantation
housed another distinct methanotroph (sequence
GQ121280) also belonging to the genus Methylocystis.
Thus, it cannot be ruled out that inactive methanotrophs
that existed prior to transplantation became active in the
new environment.
The hypothesis of microbial dispersal from plant to
plant is in line with several independent findings. First,
wetting alone did not induce oxidation activity in
incubated mosses. Second, when the critical water-level
threshold for maintaining an active methanotrophic
population was presumably achieved, a large part of the
variation in potential oxidation remained unexplained
(Fig. 2). In other words, the occurrence and the level of
methane oxidation appeared to be more unpredictable
than what is to be expected if there is a tight link
between Sphagnum mosses and methanotrophic microbes. For example, oxidation rates among replicates of
S. cuspidatum from the same hollow varied considerably
(Fig. 3). Third, no correlation existed between oxidation
activity and the share of CH4-derived carbon. Fourth, in
a separate trial we watered inactive Sphagnum spp. with
0.45-lm filtered and nonfiltered mire water collected
from active methane oxidation sites. Only Sphagnum
spp. sprinkeled with non-filtered water became active
(A. Putkinen, T. Larmola, H. Siljanen, E.-S. Tuittila, T.
Tuomivirta, A. Saari, P. J. Martikainen, and H. Fritze,
Our estimate of a 10–30% contribution of CH4derived carbon in Sphagnum agrees with previous
studies on refixation of respired carbon into living
Sphagnum. In 14C labeling experiments, it has been
found that 5–8% of the carbon fixed in photosynthesis
by Sphagnum is derived from respired carbon (Rydin
and Clymo 1989, Turetsky and Wieder 1999). Using a
30-year record of atmospheric 14C activity and peaks of
14
C concentrations measured in peat, Tolonen et al.
(1993) estimated that 20% of the carbon originating
from decaying peat would be refixed in living vegetation.
Combining the measurements of natural 13C abundance
in moss and a 13CH4 labeling experiment, Raghoebarsing et al. (2005) showed that CO2 derived from CH4
oxidation could contribute 10–15% of carbon in S.
cuspidatum in peat bogs.
Contribution of the CH4-derived carbon in Sphagnum
supports the idea that an association with the inhabiting
bacteria benefits Sphagnum photosynthesis (Raghoebarsing et al. 2005). The rationale behind this is that the
water layer can limit substantially the diffusion of CO2
in moss (Williams and Flanagan 1996), and thus the
CO2 production within the plant cell from CH4
oxidation could overcome this. The assimilation of
atmospheric CO2 has been observed to decline in
Sphagnum moss when water contents increased above
an optimum. The reduction in fixing atmospheric CO2
through photosynthesis was reversed only by exposing
the moss to very high atmospheric-CO2 concentration,
thus removing the CO2 limitation (Silvola 1990).
Interestingly, a diffusional limitation to moss photosynthesis caused by excess water has also been demonstrated in declining discrimination of 13C-CO2 as water
content increased above optimum (Rice and Giles 1996).
Consequently, moisture can also contribute to variation
in the moss 13C signal (Rice 2000).
The isotopic signature of moss tissue results from the
shifting balance between two distinct processes: diffusional limitation and on overall metabolic slowdown of
photosynthesis due to desiccation. Metabolic slowdown
of photosynthesis due to desiccation also yields organic
carbon enriched with 13C (Williams and Flanagan
1996). These interactions could partly explain why no
correlation was found between the calculated share of
CH4-derived carbon in the moss and the measured
potential oxidation rate. Carbon isotopic ratios and
concentrations of CH4 and CO2 in the water surrounding the mosses agreed with the current understanding of
the effect of oxidation of CH4 (e.g., Chanton et al.
1997). In water, CH4 was more enriched by 13C when
the concentration of CH4 in the pore water of the moss
layer was low, and more depleted CH4 was associated
2364
TUULA LARMOLA ET AL.
with high concentration. Interestingly, Rice (2000)
demonstrated that when growing in a greenhouse, all
Sphagnum had a d13C close to ambient air, whereas at
the field site the mosses were more depleted with 13C.
This could agree with the idea that CH4 is a greater
potential carbon source in the field than in the
greenhouse.
Our most rapid oxidation rates for Sphagnum
exceeded the potentials previously reported from bog
and fen habitats (0–28 lmolg1d1, dry mass; Basiliko
et al. 2004, Raghoebarsing et al. 2005), but they were
only 30% of those observed for a beaver pond (197
lmolg1d1; Basiliko et al. 2004). The potential to
oxidize CH4 in the uppermost 10 cm of the moss layer
was 20–530 lmolm2h1, 0.4–5 fold of the net CH4
flux measured in respective ombrotrophic to mesotrophic sites at Lakkasuo (20–800 lmolm2h1; Nykänen
et al. 1998, Riutta 2008). The actual rates of CH4
oxidation at the Lakkasuo mire are likely to be lower in
part due to possible substrate limitation. Nevertheless,
CH4 oxidation is potentially a major regulator of CH4
emission from Lakkasuo. Similarly, from CH4 concentration profiles in Sphagnum hollows of a hemiboreal
raised bog, Frenzel and Karofeld (2000) deduced that
CH4 emission is 99% controlled by oxidation in the moss
layer.
In conclusion, all 23 Sphagnum species showed
methanotrophic activity, and thus hypothesis 1 was
supported. With respect to hypothesis 2, water level was
the key environmental determinant of methanotrophy in
Sphagnum. According to hypothesis 4 of microbial
dispersal from plant to plant, the active transplants
hosted a Methylocystis sequence that they did not have
before transplantation but that was also found in the
control Sphagnum. As the inactive transplants also had a
Methylocystis sequence in common with the active
transplants and the control mosses, hypothesis 3 was
rejected. These results imply a loose symbiosis between
Sphagnum spp. and methanotrophic bacteria resulting in
10–30% of Sphagnum carbon potentially originating
from CO2 derived from CH4 oxidation.
ACKNOWLEDGMENTS
Sari Juutinen helped with the water sampling, and Sirpa
Tiikkainen with the sequencing. We greatly appreciate Nathan
Basiliko’s and another reviewer’s comments. We acknowledge
funding from Academy of Finland for H. Fritze (Project
109816), T. Larmola (Project 121535), and E.-S. Tuittila
(Project 118493) and from Helsinki University Environmental
Research Centre for consortium ‘‘REBECCA—Responses of
northern ecosystem carbon exchange to changing environment,
in different spatio-temporal scales’’ for E.-S. Tuittila.
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APPENDIX
Potential methane oxidation rates detailed by Sphagnum species, habitat, water level, and methane concentration (Ecological
Archives E091-168-A1).