ORIGINAL RESEARCH ARTICLE
published: 23 Janua y 2012
doi: 10.3389/ micb.2012.00015
Wa e dispe sal o me hano ophic bac e ia main ains
unc ional me hane oxida ion in Sphagnum mosses
Anuliina Pu kinen1*,Tuula La mola2†,Te oTuomi i a1, Hen i M. P. Siljanen3, Le en e Bod ossy 4,5,
Ee a-S iinaTui ila2,6 and Hannu F i ze1
1Finnish Fo es Resea ch Ins i u e, Sou he n Finland Regional Uni , Van aa, Finland
2Depa men o Fo es Sciences, Uni e si y o Helsinki, Helsinki, Finland
3Depa men o En i onmen al Science, Uni e si y o Eas e n Finland, Kuopio, Finland
4Depa men o Bio esou ces/Mic obiology, Aus ian Ins i u e o Technology, Seibe sdo , Aus ia
5CSIRO Ma ine and A mosphe ic Resea ch, Hoba , TAS, Aus alia
6School o Fo es Sciences, Uni e si y o Eas e n Finland, Joensuu, Finland
Edi ed by:
S e lana N. Dedysh, Russian
Academy o Sciences, Russia
Re iewed by:
S e lana N. Dedysh, Russian
Academy o Sciences, Russia
Yin Chen, Uni e si y o Wa wick, UK
*Co espondence:
Anuliina Pu kinen, Finnish Fo es
Resea ch Ins i u e, Sou he n Finland
Regional Uni , P.O. Box 18
(Jokiniemenkuja 1), 01301 Van aa,
Finland.
e-mail: anuliina.pu kinen@me la.fi
†P esen add ess:
Tuula La mola, Moun Holyoke
College, Sou h Hadley, MA 01075,
USA.
I is known ha Sphagnum associa ed me hano ophy (SAM) changes in ela ion o he
pea land wa e able (WT) le el. A e d ough , ising WT is able o eac i a e SAM. We
aimed o e eal whe he his eac i a ion is due o ac i a ion o indigenous me hane (CH4)
oxidizing bac e ia (MOB) al eady p esen in he mosses o o MOB p esen in wa e . This
was es ed h ough wo app oaches: in a ansplan a ion expe imen , Sphagna lacking
SAM ac i i y we e ansplan ed in o fla k wa e nex o Sphagna oxidizing CH4. Al eady
a e 3 days, mos o he ansplan s showed CH4oxida ion ac i i y. Mic oa ay showed
ha he MOB communi y composi ions o he ansplan s and he o iginal ac i e mosses
had become mo e simila wi hin 28 days hus indica ing MOB mo emen h ough wa e
be ween mosses. Me hylocys is- ela ed ype II MOB domina ed he communi y. In a
ollowing expe imen , SAM inac i e mosses we e ba hed o e nigh in non-s e ile and
s e ile-fil e ed SAM ac i e si e fla k wa e . Only mosses ba hed wi h non-s e ile fla k wa e
became SAM ac i e, which was also shown by he pmoA copy numbe inc ease o o e
60 imes. Thus, i was e iden ha MOB p esen in he wa e can colonize Sphagnum
mosses.This coloniza ion could ac as a esilience mechanism o pea land CH4dynamics
by allowing he e-eme gence o CH4oxida ion ac i i y in Sphagnum.
Keywo ds: plan –mic obe in e ac ion, pea land, pmoA, mic oa ay, qPCR, ecosys em esilience
INTRODUCTION
Pea lands s o e o e one hi d o global e es ial ca bon
(Go ham, 1991). Al hough hese ecosys ems a e ca bon dioxide
(CO2) sinks hey a e also a majo sou ce o me hane (CH4),
o med as he final p oduc o anae obic deg ada ion o o ganic
ma e . Mos ca bon in hese sys ems is de i ed om Sphag-
num mosses (Clymo and Haywa d, 1982), he dominan plan in
bog- ype no he n pea lands. Mosses seques e a mosphe ic CO2
di ec ly h ough pho osyn hesis. Me hano ophic bac e ia (MOB)
li ing inside he moss hyaline cells and on lea su aces (Raghoe-
ba sing e al., 2005;Kip e al., 2010) also play an impo an ole
in ca bon binding. These bac e ia p o ide CO2 o he plan ia
CH4oxida ion, a mechanism ha is especially impo an in sub-
me ged condi ions whe e CO2di usion is slow (Kip e al., 2010).
This phenomenon is o local and global impo ance as i has been
de ec ed in all 23 Sphagnum species o a pea land a ea (La mola
e al. (2010)) and in geog aphically dis an pea lands (Kip e al.,
2010) and may be pa ly esponsible o he lowe CH4emissions
o Sphagnum bogs in ela ion o o he pea land ypes (Nykänen
e al., 1998).
Abou 10–15 (Raghoeba sing e al., 2005) o 10–30% (La mola
e al., 2010)o Sphagnum biomass ca bon is om CH4oxida-
ion by MOB. Thus, i seems clea ha mosses benefi om hei
pa ne s and he ela ionship has been discussed o be symbi-
o ic (Raghoeba sing e al., 2005). S ill, he e is e idence ha he
bac e ia in ol ed a e only loosely connec ed o Sphagnum (Basi-
liko e al., 2004;La mola e al., 2010). The s udy by La mola
e al. (2010) showed ha pea land wa e able (WT) le el is he
main ac o influencingMOB ac i i yinmosses.Sphagnum associ-
a ed me hano ophy (SAM) became de-/ eac i a ed upon na u al
WT fluc ua ion. Howe e , La mola e al. (2010) did no p o ide
e idence whe he eac i a ed CH4oxida ion was caused by eac-
i a ion o he o iginal MOB communi y, in asion o new MOB
om he su ounding wa e o by bo h mechanisms. The abili y o
MOB o colonize Sphagnum om su ounding wa e would make
ecosys em CH4dynamics less ulne able o ex ended pe iods o
d ough han a igh symbiosis be ween MOB and Sphagnum o
elaying on he eac i a ion o o iginal communi y. To es he
impo ance o coloniza ion we examined he ques ion mo e ho -
oughly. Fi s , we conduc ed a simila ansplan a ion ial as in
La mola e al. (2010) whe e inac i e mosses we e plan ed nex o
ac i e ones. Coloniza ion p ocess was ollowed by measu emen o
CH4oxida ion po en ials and communi y analysis by a mic oa ay
ha p ofiles di e si y wi hin he pmoA gene coding o pa icu-
la e me hane mono-oxygenase (pMMO), a key enzyme in CH4
oxida ion (Bod ossy e al., 2003). By using his me hod co e ing
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Pu kinen e al. Sphagnum and me hano ophy
a wide ange o MOB di e si y han La mola e al. (2010) we
aimed o e eal mo e de ailed changes in communi y composi-
ions. We hypo hesized ha coloniza ion o MOB h ough he
wa e phase is a subs an ial eason o me hano ophic eac i a-
ion. Since we p esume ha all mosses a e colonized h ough he
same pa hway his should be eflec ed in MOB o he neighbo ing
mosses influencing he mic obial communi y o he ansplan ed
moss. Second, we es ed he hypo hesis in he labo a o y by ea -
ing inac i e Sphagnum mosses wi h wa e om a we dep ession
(fla k) ha bo ing me hano ophic ac i e Sphagnum mosses. As a
con ol, pa allel samples we e ea ed wi h he same wa e a e
MOB emo al h ough fil a ion. CH4oxida ion po en ials we e
measu ed and MOB communi ies analyzed by pmoA-based quan-
i a i e PCR (qPCR) and dena u ing g adien gel elec opho esis
(DGGE) analysis ollowed by sequencing.
EXPERIMENTAL PROCEDURES
TRANSPLANTATION EXPERIMENT
Sphagnum ansplan a ion
The expe imen was conduc ed a he Lakkasuo mi e (61˚48N,
24˚19E; 150 m. a.s.l), a bo eal aised bog complex in sou he n
Finland. On 7 July 2008, pa ches (8 cm in diame e ) o inac i e
Sphagnum ubellum om si e O we e ansplan ed o six di e en
fla k si es (A–F) showing high Sphagnum associa ed me han-
o ophic (SAM; CH4oxida ion) ac i i y (Figu e 1). To con ol he
e ec o ansplan a ion, S. ubellum was eplan ed in he o iginal
si e and he na i e Sphagnum species (Table A1 in Appendix) ga h-
e ed om each o six di e en fla k si es we e also e u ned o hei
o iginal places. Thus, all samples we e o ansplan ed Sphagnum.
Moss samples we e ga he ed a he beginning o he expe imen
(0 day), a e 3 days, and 28 days. A e ga he ing, mosses we e
insed wi h deionized wa e and d ied o e nigh a +4˚C. Only
FIGURE 1 | Diag am o he ansplan a ion expe imen . CH4oxida ion
po en ials a he ime o sampling a e included (±; alues o e
0.005 μmol g d y mass−1h−1we e conside ed posi i e). Dominan
Sphagnum species on ansplan a ion si es we e S. papillosum (si es A–C),
S. cuspida um (si es D and E), and S. majus (si e F).The o iginal si e O and
he ansplan ed immig an mosses consis ed o S. ubellum.
uppe 10 cm o he moss plan s we e included in he ollowing
analysis. Ecological a iables o he ansplan a ion si es a e lis ed
in Table A1 in Appendix.
Me hane oxida ion po en ial
Me hane oxida ion po en ials we e measu ed as desc ibed in La -
mola e al. (2010). B iefly, 30 g o moss was incuba ed in a 600 mL
flask wi h an ini ial CH4concen a ion o 10000 ppm in he da k
a +15˚C and he oxida ion was moni o ed a e 24 and 48 h by
gas ch oma og aphy. Resul s a e p esen ed in mic omole CH4pe
g am d y weigh pe hou (μmolgdw
−1h−1).
Analysis o me hano ophic communi y composi ion by
pmoA-mic oa ay
Communi y composi ion o MOB in Sphagnum samples was
in es iga ed using a mic oa ay (Bod ossy e al., 2003) designed
o de ec di e si y wi hin he pmoA gene. DNA was isola ed as in
Siljanen e al. (2011). A agmen o he pmoA gene was amplified
using a semi-nes ed PCR app oach wi h p ime pai s A189 /T7–
A682 and A189 /T7–mb661 as in Siljanen e al. (2011) wi h he
excep ion ha a e he fi s PCR-s ep, p oduc s no de ec able on
he gel we e dilu ed 1:10 be o e being used as empla es in he sec-
ond PCR. Concen a ion o PCR p oduc s was quan ified using a
Qubi fluo ome e (In i ogen, Ca lsbad, CA, USA). In i o an-
sc ip ion and hyb idiza ion was pe o med as in S alis-Pa ese
e al. (2004) and he applied p obe se was simila o ha applied
by Abell e al. (2009). One o h ee pa allel o iginal inac i e moss
samples om he 0 day ime poin could be success ully analyzed
(see La mola e al. (2010) ha SAM inac i e Sphagnum mosses
hos MOB DNA). P obing pmoA di e si y canno de ec Me hy-
locella o he ecen ly disco e ed Me hylo e ula (Vo obe e al.,
2011) me hano ophs as he pMMO enzyme is no p esen in
hese bac e ia. I should be no ed ha ano he newly disco e ed
me hano oph g oup, Ve ucomic obia, is also no de ec able by
he p obes we used.
S a is ical analysis o mic oa ay da a
The quan i a i e na u e o he mic oa ay da a was con e ed o
a bina y ma ix (p esence =1, absence =0) o e eal he commu-
ni y changes caused by di e en g oups o MOB colonizing mosses
a e ansplan a ion, and also o p e en alse in e p e a ion o igi-
na ing om non-quan i a i e nes ed PCR app oach. The da a we e
hen analyzed using p incipal componen analysis (PCA) ca ied
ou wi h CANOCO Ve sion 4.52 ( e B aak and Smilaue , 2002).
Si es A–F (Figu e 1) we e analyzed sepa a ely (n=1) and as pa al-
lel samples (n=6). Uni e sal MOB p obes (posi i e con ols) and
p obes no hyb idizing o any o he samples we e excluded om
he analyses ( h eshold o posi i e samples ≥3 a e no maliza ion
o he da a o he scale o 0–100).
BATHING EXPERIMENT
Fla k wa e ba hing
Sphagnum mosses we e ga he ed om Sallie’s Fen in NH, USA
(43˚12.5N, 71˚03.5W 110 m. a.s.l.). T iplica e (n=3) esh sam-
ples o app oxima ely 30 mL ( olume based on he olume o wa e
eplaced by he mosses) o inac i e S. magellanicum we e subjec ed
o he ollowing ea men s: (I) no ea men ; (II) o e nigh (11 h)
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Pu kinen e al. Sphagnum and me hano ophy
incuba ion in SAM ac i e S. majus 200 mL fla k; (III) o e nigh
incuba ion in 200 mL 0.45 μm fil e ed SAM ac i e S. majus fla k
wa e ; (IV) o e nigh incuba ion in 200 mL SAM ac i e S. majus
fla k wa e ollowed by insing wi h deionized wa e . In he final
ea men , S. majus ga he ed om he ac i e (fla k) si e was
included as a posi i e con ol (n=3) in he analyses desc ibed
below. SAM ac i e fla k wa e was collec ed di ec ly om a we
dep ession nex o S. majus ege a ion and did no con ain any
mac oscopic plan ma e ial. Each o e nigh incuba ion was con-
duc ed in he da k a +20˚C. Following ea men , all mosses we e
d ied o e nigh a +4˚C. Only uppe 10 cm o he moss plan s
we e included in he ollowing analysis.
Me hane oxida ion po en ial and s a is ical analysis
Me hane oxida ion po en ials we e measu ed as abo e in he
ansplan a ion expe imen bu on a Shimadzu 14A gas ch oma o-
g aph equipped wi h a flame ioniza ion de ec o (Shimadzu Co p.,
Kyo o, Japan). The esul s a e p esen ed in mic omole CH4pe
g am d y weigh pe hou . The di e ence be ween sample ea -
men s was es ed using K uskal–Wallis non-pa ame ic analysis
o a iance ollowed by Nemenyi es o pai wise compa isons
(p<0.05; Za , 1999).
Analysis o communi y composi ion by DGGE and sequencing
Di e si y o MOB in Sphagnum samples om he ba hing expe -
imen was explo ed by pmoA-based PCR–DGGE analysis and
sequencing as p e iously desc ibed (Tuomi i a e al., 2009;La -
mola e al.,2010) using he p ime pai A189 /GC-621 designed o
a ge me hano ophs abundan in bo eal pea lands (Tuomi i a
e al., 2009). DNA was isola ed as abo e in he ansplan a ion
expe imen . De e mined pmoA gene sequences we e submi ed o
Genbank unde accession numbe s HQ651182 and HQ651183.
Quan ifica ion o pmoA genes and s a is ical analysis
Quan i a i e PCR was ca ied ou as p e iously desc ibed
(Tuomi i a e al., 2009) using he same p ime pai as in he
DGGE analysis (A189 /GC-621 ). Resul s a e exp essed in pmoA
copy numbe pe g am d y weigh . To es he di e ence be ween
sample ea men s, alues we e ln ans o med o no malize he
da a ollowed by ANOVA and Tukey’s HSD es (p<0.05).
RESULTS AND DISCUSSION
In he ansplan a ion expe imen SAM inac i e Sphagnum ubel-
lum mosses we e plan ed on six di e en SAM ac i e si es. Mos
o he o iginally inac i e mosses showed de ec able CH4oxida ion
po en ials (>0.005 μmol CH4g−1h−1) a e 3 days and 28 days
(Figu e 1). Compa ing ansplan a ion si es indi idually indica ed
ha ini ially di e en MOB communi ies became mo e simila
wi h ime (Figu e A1 in Appendix). A e aging o e he en i e
da a se , he pmoA-mic oa ay showed ha he MOB commu-
ni y o he o iginally inac i e mosses s a ed o esemble ha o
he na i e mosses in he ac i e si e a e 3 days (Figu e 2). A e
28 days, MOB communi ies om he majo i y o inac i e Sphag-
num mosses ansplan ed o ac i e si es (immig an s in Figu e 1)
we e mo e simila o hose o na i e ac i e mosses han o hose
in he inac i e si e a 0 day. Thus, his field expe imen indica ed
ha MOB could be ans e ed be ween mosses h ough he wa e
FIGURE2|P incipal componen analysis (PCA) o bina y mic oa ay
da a. Sample means om di e en sampling imes in days (d) a e showed
[o iginal 0 days =o iginal inac i e moss (n=1), o iginal 28 days =o iginal
moss ansplan ed back o he o iginal si e (n=3), immig an =o iginal
moss ansplan ed o he ac i e fla k si es (n=6), na i e =na i e mosses o
he ac i e fla k si es (n=6)].The fi s and second PCA axes explain 25 and
11% o he composi ional a ia ion, espec i ely.
phase. In addi ion, he o iginal inac i e Sphagnum (si e O) became
ac i e a e ansplan a ion in i s o iginal si e and had a di e -
en MOB communi y han be o e ansplan a ion, demons a ing
possible new MOB mo emen h ough he wa e phase (Figu es 2
and 3and Figu e A1 in Appendix). Howe e , al hough SAM ac i -
i y was induced in mos o he samples oge he wi h in asion o
new MOB his was no always he case and SAM ac i i y could also
be induced wi hou majo changes in communi y composi ion
(Si e E, Figu e A1 in Appendix). Thus he e a e me hano ophs
ha mo e h ough wa e bu we canno s a e ha o all commu-
ni y membe s. Some membe s o he me hano oph communi y
seem o be pe manen ly associa ed wi h he mosses ega dless o
whe he condi ions a o CH4oxida ion o no . In ou ans-
plan a ion expe imen his ac o hinde ed us om seeing he
in ading membe s o he communi y and pa ly explains why la ge
changes in communi y composi ion we e no always seen when
CH4oxida ion was eac i a ed. As Me hylocys is- ela ed ype II
MOB we e p esen in p ac ically all samples in he ansplan a ion
expe imen (Figu e 3 and Figu e A2 in Appendix), as shown by
he p obes Mcy413, Mcy522, Mcy270, Mcy459, Msi232, Pea 264,
II509, and II630, hei mo emen be ween mosses could no be
clea ly de ec ed. S ill, p obes a ge ing Me hylocys is s ain M
(McyM309), Me hylocys is subg oup B (McyB304), and he genus
Me hylosinus (Ms 214, Ms 314,MsS475,Msi294,NMsiT.271) ga e
mo e a iable p esence pa e ns among he ype II me han-
o ophs, sugges ing wa e -media ed mo emen . Compa ed o α-
p o eobac e ial ype II MOB, he o he main me hano ophic
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Pu kinen e al. Sphagnum and me hano ophy
FIGURE 3 | Bina y map o mic oa ay esul s. Each si e (A–F and O) is
showed sepa a ely oge he wi h he o iginal 0 day sample da a o
compa ison. Mic oa ay analysis was success ul o only one o h ee
pa allel inac i e o iginal_0day samples. Samples ha we e inac i e in CH4
oxida ion a he ime o sampling a e ma ked wi h a black do (•). Only
p obes wi h a posi i e hyb idiza ion signal (≥3 a e no maliza ion o he
da a o he scale o 0–100) on any o he samples a e shown (uni e sal
MOB p obes we e no included). Bina y esul s we e con e ed om he
semiquan i a i e da a p esen ed as a hea map in he Figu e A2 in
Appendix.
lineage, he γ-p o eobac e ial ype I, was p esen mo e a ely and
wi h mo e a iabili y. P io o ansplan a ion, no ype I me han-
o ophs we e de ec ed in inac i e mosses. A e 28 day, hey we e
p esen in ou o six “immig an ” samples ansplan ed o ac i e
si es. Type Ia subg oup was ound only in mosses ansplan ed in
he ac i e fla ks and he na i e mosses o hese si es, sugges ing
ha his g oup mo ed om he na i e mosses o he ansplan ed
“immig an s.”Type I MOB could no be clea ly linked o he eme -
gence o CH4oxida ion ac i i y, al hough hey we e p esen in
mos o he ac i e samples and absen om mos o he inac-
i e ones. Consequen ly, based on he ansplan a ion expe imen
alone, we canno exclusi ely s a e ha in asion by nea by MOB is
an impe a i e ou e in he eac i a ion o SAM ac i i y.
To examine he hypo hesis “ he coloniza ion o MOB h ough
he wa e phase is a subs an ial eason o me hano ophic
eac i a ion” u he , a mo e simplified expe imen was con-
duc ed in labo a o y condi ions. In his ba hing expe imen ,
SAM inac i e S. magellanicum mosses exposed o unfil e ed,
SAM ac i e fla k wa e began CH4oxida ion wi hin 11 h, as
indica ed by CH4oxida ion po en ial measu emen s and com-
muni y analyses including pmoA-based qPCR, DGGE finge -
p in ing and sequencing. Maximum CH4oxida ion po en ials
and pmoA copy numbe s we e measu ed o mosses ea ed
wi h unfil e ed wa e (a e ages o insed mosses 0.33 μmol
CH4g−1h−1, 1.9 ×107pmoA copies g dw−1, and o un insed
0.29 μmol CH4g−1h−1, 2.6 ×107pmoA copies g dw−1) and pos-
i i e con ol S. majus mosses (a e age 0.63 μmol CH4g−1h−1,
20 ×107pmoA copies g dw−1) om he ac i e fla k si e (Figu e 4).
Respec i e alues o mosses ea ed wi h fil e ed wa e (no
CH4oxida ion de ec ed, 0.03 ×107pmoA copies g dw−1) and he
nega i e con ol (<0.005 μmol CH4g−1h−1, 0.06 ×107pmoA
copies g dw−1) we e clea ly lowe . The pmoA copy numbe
be ween fil e ed and non-fil e ed fla k wa e ea ed S. magellan-
icum inc eased in a e age by a ac o o 63. Also, DGGE e ealed
he ans e o wo Me hylocys is- ela ed me hano ophs h ough
unfil e ed wa e (Figu e 5). Fil e ed wa e did no induce CH4oxi-
da ion ac i i y. This expe imen clea ly demons a ed he wa e -
media ed dispe sal o MOB, bu i also showed ha compa ed o
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Pu kinen e al. Sphagnum and me hano ophy
in asion by new MOB, eac i a ion o he o iginal MOB was no
a majo mechanism in he eac i a ion o CH4oxida ion p ocess
in he s udied mosses. In he DGGE gel he wo Me hylocys is
bands a e ain ly p esen al eady in he unba hed nega i e con-
ol. Ba hing o he mosses wi h s e ile-fil e ed wa e caused hese
bands o ade away as shown also by he qPCR. On he con a y,
ea men wi h unfil e ed wa e caused eme gence o high num-
be s o MOB and also high SAM ac i i y. Thus he eac i a ion o
he CH4oxida ion ac i i y mus ha e been b ough up by MOB
in ading he moss h ough he wa e phase o he g ow h in MOB
numbe s should ha e been seen also in he moss ba hed wi h
fil e ed wa e . Mo eo e , known Me hylocys is s ains ha e dou-
bling imes o se e al hou s when g owing on CH4in labo a o y
FIGURE4|CH
4oxida ion po en ials (A) and pmoA copy numbe s (B) o
di e en ly ea ed moss samples (S. mag. =Sphagnum magellanicum,
S. maj. =Sphagnum majus). S anda d e o ba s a e shown (n=3). Same
le e supe sc ip s deno e non-significan di e ences among ea men s in
he po en ial oxida ion a es. No e he loga i hmic scale on he y-axis (B).
condi ions(Wisee al.,1999;Dedyshe al.,2007;BaaniandLiesack,
2008) sugges ing ha i would be highly unlikely o hese MOB
o inc ease hei numbe s o e 60 imes highe in 11 h, as seen in
he ba hing expe imen . The e o e we accep he posed hypo hesis
wi h a mino modifica ion: MOB coloniza ion h ough he wa e
phase occu s and i ob iously suppo s he eac i a ion o CH4
oxida ion in Sphagnum mosses, bu ou expe imen s canno ule
ou he possibili y o eac i a ion o o iginal communi y membe s.
This could be in es iga ed in a p olonged ba hing expe imen in
combina ion wi h diu nal ligh hy hm, bu was beyond he scope
o his in es iga ion.
Based on ou esul s ha MOB colonize he mosses om wa e ,
he ela ionship be ween MOB and Sphagnum seems o be a loose,
mu ually beneficial associa ion a he han a igh symbiosis. This
esul is in line wi h a ecen finding by B agina e al. (in p ess)
who showed by py osequencing ha some bac e ia a e passed
om he Sphagnum spo ophy e o he game ophy e bu no known
me hano ophs we e among hem. Rep esen a i es o he genus
Me hylocys is, howe e , we e de ec ed in he game ophy e. S ill,
e en hough me hano ophs may no be obliga ely dependen
on he mosses, hey mos likely p e e he plan cells o e li e
in he wa e phase. This is suppo ed by ou esul s om he
ba hing expe imen . Despi e ha ing sligh ly lowe ed he amoun
o pmoA de ec ed, mosses insed wi h s e ile wa e had almos he
same po en ial CH4oxida ion ac i i y as un insed ones, indica -
ing ha loosely a ached me hano ophs play only a mino ole
in he p ocess. In addi ion, he apid (<11 h) inc ease in pmoA
copy numbe sugges s ha me hano ophs p esen in he wa e
phase quickly colonize Sphagnum. Compa ed o ee-li ing bac-
e ia, hose associa ed wi h plan s may gain an ad an age om
a s able CH4g adien and supply o oxygen om pho osyn he-
sis, bu i has ye o be demons a ed. In he ba hing expe imen
me hano ophs mo ed o he mosses e en in he da k when oxygen
was no o med in pho osyn hesis, indica ing ha a he e y leas
oxygen is no he only ad an age bac e ia gain om he mosses.
In ano he s udy no CH4oxida ion ac i i y was de ec ed in pea
wa e su ounding Sphagnum mosses (Kip e al., 2010), also indi-
ca ing ha , al hough p esen , MOB a e no ac i ely oxidizing CH4
in he wa e phase. Since he only MOB, Me hylocys is, de ec ed in
he ba hing expe imen , is non-mo ile (Dedysh, 2009), i emains
open how hese MOB cells end up on he moss su ace and inside
he hyaline cells. On he o he hand ano he pea land inhabi ing
ype II MOB genus, Me hylosinus (Dedysh e al., 2003;Chen e al.,
FIGURE 5 | Dena u ing g adien gel elec opho esis gel showing
di e en ly ea ed samples.Two Me hylocys is bands a e ma ked by xand y
(S. mag. =Sphagnum magellanicum,S. maj. =Sphagnum majus). Closes
da abase ma ch o sequence x(HQ651182) was me hano ophic sequence
om an acidic en (FR726177) wi h 95% simila i y. Sequence y(HQ651183)
was iden ical o a Me hylocys is sequence p e iously ound on Finnish
pea lands (GQ468279, GQ279344, FJ930091) and Sphagnum mosses
(GQ121280).
www. on ie sin.o g Janua y 2012 | Volume 3 | A icle 15 | 5
Pu kinen e al. Sphagnum and me hano ophy
2008), does con ain mo ile species (Bowman e al., 1993) and has
been isola ed om Sphagnum mosses (Kip e al., 2011a).
Simila o p e ious s udies (Tuomi i a e al., 2009;La mola
e al., 2010;Y jälä e al., 2011) o Finnish pea lands, DGGE o
ou ba hing expe imen samples om Sallie’s Fen, loca ed in NH,
USA, de ec ed only Me hylocys is-like MOB. These we e also he
dominan me hano ophs in ou ansplan a ion expe imen , un
on he Finnish Lakkasuo aised bog complex, when he pmoA-
mic oa ay was used. Dominance o ype II MOB in ou samples
is in line wi h p e ious s udies. Especially he high p e alence o
Me hylocys is in Sphagnum samples is no su p ising as i is com-
monly ound in no he n pea lands (McDonald e al.,1996;Mo is
e al., 2002;Jaa inen e al., 2005;Dedysh e al., 2006;Chen e al.,
2008). Al hough Kip e al. (2010) ound, in con as o ou esul s,
a high di e si y o ype I me hano ophs, Me hylocys is was s ill
he dominan species in hei globally ga he ed Sphagnum sam-
ples (Kip e al., 2010) and also e y abundan in mosses om a
Du ch pea bog (Kip e al., 2011b).
We ha e demons a ed ha wa e se es as an essen ial ou e o
me hano oph dispe sal and is hus an impe a i e pa o Sphag-
num–me hano oph associa ion. This is likely o ac as a backup
mechanism o pea land CH4dynamics. D ainage o pea lands can
al e he me hano oph communi y composi ion (Jaa inen e al.,
2005;Y jälä e al., 2011) and educe Sphagnum co e age (Y jälä
e al., 2011), consequen ly comp omising his mu ualis ic associ-
a ion. A case s udy (Y jälä e al., 2011) ound ha he pa icula
Me hylocys is sp., which was ound now also in he mosses o Sal-
lie’s Fen (no heas e n USA) o ou ba hing expe imen and in he
mosses o Lakkasuo (La mola e al. (2010)), was los when he WT
d opped by 14 cm, which is simila o he p edic ed d awdown
o no he n pea lands in he global wa ming scena io by 3˚C
(Roule e al., 1992). Res o a ion o d ained pea lands aims o
eac i a e ecosys em unc ion and es a me hanogenesis (Tui -
ila e al., 2000). Any pea land es o a ion p og am should also
aim o e-es ablish he condi ions o he mu ualis ic associa ion
be ween me hano ophs and Sphagnum. Ou s udy indica es ha
his could be done ia ansplan a ions o Sphagnum om dono
si es wi h undis u bed CH4dynamics. In na u al en i onmen s
Sphagnum associa ed me hano ophic communi ies may educe
he me hane flux by as much as 80% (Kip e al., 2010). I is no
ye known whe he his phenomenon can each ha scale also in
comp omised ecosys ems.
CONCLUSION
He e we showed, by wo complemen ing expe imen s, ha in a-
sion o new MOB h ough wa e occu s and ha i can be an
impo an mechanism in he eac i a ion o CH4oxida ion in
Sphagnum mosses. Based on his esul , he ela ionship be ween
Sphagnum and me hano ophs is a loose,mu ually beneficial asso-
cia ion, al hough some me hano ophs may ha e an e en igh e
connec ion o he mosses.
ACKNOWLEDGMENTS
We hank T. Ronkainen and L. Maana ilja o field and labo a o y
assis ance, R. Va ne , J. Bubie , A. Saa i, and P. J. Ma ikainen o
acili ies, P. C ill, S. Whi low, J. Digg, and N. Blake o access o
Sallie’s Fen, M. Ha dman o checking he language, and S. Elo-
maa o he g aphics. This wo k was mainly unded by Maj and
To Nessling Founda ion and he Academy o Finland (P ojec
121535). Addi ional unding was ecei ed om Maa- ja esi ek-
niikan uki Founda ion, Kone Founda ion, and he Academy o
Finland (P ojec s 118493 and 218101).
REFERENCES
Abell, G. C. J., S alis-Pa ese, N., Ses-
si sch, A., and Bod ossy, L. (2009).
G azing a ec s me hano oph ac i -
i y and di e si y in an alpine
meadow soil. En i on. Mic obiol.
Rep. 1, 457–465.
Baani, M., and Liesack, W. (2008). Two
isozymes o pa icula e me hane
monooxygenase wi h di e en
me hane oxida ion kine ics a e
ound in Me hylocys is sp. s ain
SC2. P oc. Na l. Acad. Sci. U.S.A.
105, 10203–10208.
Basiliko, N., Knowles, R., and Moo e,
T. R. (2004). Roles o moss species
and habi a in me hane consump-
ion po en ial in a no he n pea -
land. We lands 24, 178–185.
Bod ossy, L., S alis-Pa ese, N., Mu -
ell, J. C., Radajewski, S., Weilha -
e , A., and Sessi sch, A. (2003).
De elopmen and alida ion o a
diagnos ic mic obial mic oa ay o
me hano ophs. En i on. Mic obiol.
5, 566–582.
Bowman, J. P., Sly, L. I., Nichols, P. D.,
and Haywa d, A. C. (1993). Re ised
axonomy o he me hano ophs:
desc ip ion o Me hylobac e gen.
no ., emenda ion o Me hylococ-
cus, alida ion o Me hylosinus and
Me hylocys is species, and a p oposal
ha he amily Me hylococcaceae
includes only he g oup I me han-
o ophs. In . J. Sys . Bac e iol. 43,
735–753.
B agina, A., Be g, C., Ca dinale, M.,
Shche bako , A., Chebo a , V.,
and Be g, G. (in p ess). Sphag-
num mosses ha bou highly
specific bac e ial di e si y du -
ing hei whole li ecycle. ISME J.
doi:10.1038/ismej.2011.151
Chen, Y., Dumon , M. G., McNa-
ma a, N. P., Chambe lain, P. M.,
Bod ossy, L., S alis-Pa ese, N., and
Mu ell, J. C. (2008). Di e si y o
he ac i e me hano ophic commu-
ni y in acidic pea lands as assessed
by mRNA and SIP-PLFA analyses.
En i on. Mic obiol. 10, 446–459.
Clymo, R. S., and Haywa d, P. M.
(1982). “The ecology o Sphag-
num,” in B yophy e Ecology, ed. A.
J. E. Smi h (London: Chapman and
Hall), 229–289.
Dedysh, S. N. (2009). Explo ing
me hano oph di e si y in acidic
no he n we lands: molecula and
cul i a ion-based s udies. Mic obiol-
ogy 78, 655–669.
Dedysh, S. N, Belo a, S. E, Bodelie , P.
L. E., Smi no a, K. V., Khmelenina,
V. N., Chid haisong, A., T o senko,
Y. A., Liesack, W., and Dunfield, P. F.
(2007). Me hylocys is heye i sp. no .,
a no el ype II me hano ophic bac-
e ium possessing ‘signa u e’ a y
acids o ype I me hano ophs. In .
J. Sys . E ol. Mic obiol. 57, 472–479.
Dedysh, S. N., Dunfield, P. F., De -
akshani, M., S ubne , S., Heye , J.,
and Liesack, W. (2003). Di e en-
ial de ec ion o ype II me han-
o ophic bac e ia in acidic pea lands
using newly de eloped 16S RNA-
a ge ed fluo escen oligonucleo ide
p obes. FEMS Mic obiol. Ecol. 43,
299–308.
Dedysh, S. N., Pank a o , T. A., Belo a,
S. E., Kuliche skaya, I. S., and
Liesack, W. (2006). Phylogene ic
analysis and in si u iden ifica ion
o bac e ia communi y composi-
ion in an acidic Sphagnum pea
bog. Appl. En i on. Mic obiol. 72,
2110–2117.
Go ham,E. (1991). No he n pea lands:
ole in he ca bon cycle and p obable
esponses o clima ic wa ming. Ecol.
Appl. 1, 182–195.
Jaa inen, K., Tui ila, E. S., Laine,
J., Y jälä, K., and F i ze, H.
(2005). Me hane-oxidizing bac e-
ia in a Finnish aised mi e
complex: e ec s o si e e ili y
and d ainage. Mic ob. Ecol. 50,
429–439.
Kip, N., Ouyang, W., an Winden,
J., Raghoeba sing, A., an Ni ik,
L., Pol, A., Pan, Y., Bod ossy, L.,
an Donselaa , E. G., Reicha ,
G.-J., Je en, M. S. M., Sin-
ninghe Dams é, J. S., and Op den
Camp, H. J. M. (2011a). De ec-
ion, isola ion and cha ac e iza ion
o acidophilic me hano ophs om
Sphagnum mosses. Appl. En i on.
Mic obiol. 77, 5643–5654.
Kip, N., Du ilh, B. E., Pan, Y., Bod ossy,
L., Ne eling, K., Kwin , M. P., Je -
en, M. S. M., and Op den Camp,
H. J. M. (2011b). Ul a-deep py ose-
quencing o pmoA amplicons con-
fi ms he p e alence o Me hy-
lomonas and Me hylocys is in Sphag-
num mosses om a Du ch pea
bog. En i on. Mic obiol. Rep. 3,
667–673.
F on ie s in Mic obiology | Te es ial Mic obiology Janua y 2012 | Volume 3 | A icle 15 | 6
Pu kinen e al. Sphagnum and me hano ophy
Kip, N., an Winden, J., Pan, Y.,
Bod ossy, L., Reicha , G.-J., Smol-
de s, A. J. P., Je en, M. S. M., Sin-
ninghe Dams é, J. S., and Op den
Camp,H. J. M. (2010). Global p e a-
lence o me hane oxida ion by sym-
bio ic bac e ia in pea -moss ecosys-
ems. Na . Geosci. 3, 617–621.
La mola, T., Tui ila, E.-S., Tii ola, M.,
Nykänen, H., Ma ikainen, P. J.,
Y jälä, K., Tuomi i a, T., and F i ze,
H. (2010). The ole o Sphagnum
mosses in he me hane cycling o a
bo eal mi e. Ecology 91, 2356–2365.
McDonald, I. R., Hall, G. H., Pickup,
R. W., and Mu ell, C. J. (1996).
Me hane oxida ion po en ial and
p elimina y analysis o me han-
o ophs in blanke bog pea using
molecula ecology echniques.
FEMS Mic obiol. Ecol. 21, 197–211.
Mo is, S. A., Radajewski, S.,Willison, T.
W., and Mu ell, J. C. (2002). Iden-
ifica ion o he unc ionally ac i e
me hano oph popula ion in a pea
soil mic ocosm by s able-iso ope
p obing. Appl. En i on. Mic obiol.
68, 1446–1453.
Nykänen, H., Alm, J., Sil ola, J., Tolo-
nen, K., and Ma ikainen, P. (1998).
Me hane fluxes on bo eal pea lands
o di e en e ili y and he e ec o
long- e m expe imen al lowe ing o
he wa e able on flux a es. Global
Biogeochem. Cycles 12, 53–69.
Raghoeba sing, A. A., Smolde s, A.
J. P., Schmid, M. C., Rijps a, W.
I. C., Wol e s-A s, M., De ksen,
J., Je en, M. S. M., Schou en, S.,
Sinninghe Dams é, J. S., Lame s,
L. P. M., Roelo s, J. G. M., Op
den Camp, H. J. M., and S ous,
M. (2005). Me hano ophic sym-
bion s p o ide ca bon o pho o-
syn hesis in pea bogs. Na u e 436,
1153–1156.
Roule , N., Moo e, T., Bubie , J., and
Lafleu , P. (1992). No he n ens:
me hane flux and clima ic change.
Tellus B Chem. Phys. Me eo ol. 44,
100–105.
Siljanen, H. M. P., Saa i, A., K ause, S.,
Lensu, A., Abell, G. C., Bod ossy, L.,
Bodelie , P. L. E., and Ma ikainen,
P. J. (2011). Hyd ology is eflec ed
in he unc ioning and communi y
composi ion o me hano ophs in
he li o al we land o a bo eal lake.
FEMS Mic obiol. Ecol. 75, 430–445.
S alis-Pa ese, N., Sessi sch, A., Weil-
ha e , A., Reichenaue , T., Riesing,
J., Cson os, J., Mu ell, J. C.,
and Bod ossy, L. (2004). Op i-
miza ion o diagnos ic mic oa -
ay o applica ion in analysing
landfill me hano oph communi ies
unde di e en plan co e s. En i-
on. Mic obiol. 6, 347–363.
e B aak, C. J. F., and Smilaue , P.
(2002). CANOCO Re e ence Man-
ual and CanoD aw o Windows
Use ’s Guide: So wa e o Canonical
Communi y O dina ion. Ve sion 4.5.
I haca, NY: Mic ocompu e Powe .
Tui ila, E.-S., Komulainen, V.-M.,
Vasande , H., Nykänen, H., Ma -
ikainen, P. J., and Laine, J. (2000).
Me hane dynamics o a es o ed cu -
away pea land. Glob. Chang. Biol. 6,
569–581.
Tuomi i a, T. T., Y jälä, K., and F i ze,
H. (2009). Quan i a i e PCR o
pmoA using a no el e e se p ime
co ela es wi h po en ial me hane
oxida ion in finnish en. Res. Mic o-
biol. 160, 751–756.
Vo obe , A. V., Baani, M., Do on-
ina, N. V., B ady, A. L., Lie-
sack, W., Dunfield, P. F., and
Dedysh, S. N. (2011). Me hylo e -
ula s ella a gen. no ., sp. no .,
an acidophilic, obliga ely me han-
o ophic bac e ium possessing only
a soluble me hane monooxygenase.
In . J. Sys . E ol. Mic obiol. 61,
2456–2463.
Wise, M. G., McA hu , J. V., and
Shimke s, L. J. (1999). Me han-
o oph di e si y in landfill soil:
isola ion o no el ype I and ype II
me hano ophs whose p esence was
sugges ed by cul u e-independen
16S ibosomal DNA analysis.
Appl. En i on. Mic obiol. 65,
4887–4897.
Y jälä, K., Tuomi i a, T., Juo onen,
H., Pu kinen, A., Lappi, K., Tui ila,
E.-S., Pen ilä, T., Minkkinen, K.,
Laine, J., Pel oniemi, K., and F i ze,
H. (2011). CH4p oduc ion and oxi-
da ion p ocesses in a bo eal en
ecosys em a e long- e m wa e
able d awdown. Glob. Chang. Biol.
17, 1311–1320.
Za , J. H. (1999). Bios a is ical Analy-
sis. Uppe Saddle Ri e , NJ: P en ice
Hall.
Conflic o In e es S a emen : The
au ho s decla e ha he esea ch was
conduc ed in he absence o any com-
me cial o financial ela ionships ha
could be cons ued as a po en ial con-
flic o in e es .
Recei ed: 20 Decembe 2011; accep ed:
09 Janua y 2012; published online: 23
Janua y 2012.
Ci a ion: Pu kinen A, La mola T,
Tuomi i a T, Siljanen HMP, Bod ossy
L, Tui ila E-S and F i ze H (2012)
Wa e dispe sal o me hano ophic bac e-
ia main ains unc ional me hane oxida-
ion in Sphagnum mosses. F on . Mic o-
bio. 3:15. doi: 10.3389/ micb.2012.00015
This a icle was submi ed o F on ie s
in Te es ial Mic obiology, a special y o
F on ie s in Mic obiology.
Copy igh © 2012 Pu kinen, La mola,
Tuomi i a, Siljanen, Bod ossy, Tui -
ila and F i ze. This is an open-access
a icle dis ibu ed unde he e ms o
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Comme cial License, which pe mi s non-
comme cial use, dis ibu ion, and ep o-
duc ion in o he o ums, p o ided he
o iginal au ho s and sou ce a e c edi ed.
www. on ie sin.o g Janua y 2012 | Volume 3 | A icle 15 | 7
Pu kinen e al. Sphagnum and me hano ophy
APPENDIX
Table A1 | Ecological a iables o Lakkasuo si es in he beginning (0day) and he end (28 days) o he ansplan a ion expe imen .
Si e Sphagnum species WTa(cm) Pea empe a u e (˚C) CH4concen a ion in he po e wa e (ppm)
S a End S a End S a End
AS.papillosum −5−3 13.1 15.7 390 51.5
BS. papillosum −4−8 13.8 15.7 3960 915
CS. papillosum −5−6 13 15.1 5710 689
DS. cuspida um −6−5 12.1 14.8 397 201
ES. cuspida um −7−7 13 15.7 205 4.22
FS. majus −5−5 13.3 15.4 3460 274
0S. ubellum −12 −22 15.0 15.3 16800 8740
aWT =wa e able le el as measu ed om he pea su ace.
Si es A–F =ac i e si es whe e inac i e o iginal Sphagna we e ansplan ed om si e O.
F on ie s in Mic obiology | Te es ial Mic obiology Janua y 2012 | Volume 3 | A icle 15 | 8
Pu kinen e al. Sphagnum and me hano ophy
FIGURE A1 |The p incipal componen analysis (PCA) esul s o
he bina y mic oa ay da a di ided in o ansplan a ion si es
(A–F) (o iginal 0 day =o iginal inac i e moss, o iginal
28 days =o iginal moss ansplan ed back o he o iginal si e,
immig an =o iginal moss ansplan ed o he ac i e la k si e,
na i e =na i e moss o he ac i e la k si e).The fi s and second
PCA axes explain 25 and 11% o he composi ional a ia ion,
espec i ely.
www. on ie sin.o g Janua y 2012 | Volume 3 | A icle 15 | 9
