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New insigh o he ole o mic obes in he me hane exchange in ees : e idence om
me agenomic sequencing
© 2021 The Au ho s New Phy ologis © 2021 New Phy ologis Founda ion
Published e sion
Pu kinen, Anuliina; Siljanen, Hen i M.P.; Laihonen, An i; Paasisalo, Inga; Po kka,
Kaija; Tii ola, Ma ja; Haika ainen, Iikka; Tenho i a, Salla; Pihla ie, Ma i
Pu kinen, A., Siljanen, H. M., Laihonen, A., Paasisalo, I., Po kka, K., Tii ola, M., Haika ainen, I.,
Tenho i a, S., & Pihla ie, M. (2021). New insigh o he ole o mic obes in he me hane
exchange in ees : e idence om me agenomic sequencing. New Phy ologis , 231(2), 524-536.
h ps://doi.o g/10.1111/nph.17365
2021
Viewpoin s
New insigh o he ole o
mic obes in he me hane
exchange in ees: e idence om
me agenomic sequencing
Summa y
Me hane (CH
4
) exchange in ee s ems and canopies and he
p ocesses in ol ed a e among he leas unde s ood componen s o
he global CH
4
cycle. Recen s udies ha e ocused on quan i ying
ee s ems as sou ces o CH
4
and unde s anding abio ic CH
4
emissions in plan canopies, wi h he ole o mic obial in si u CH
4
o ma ion ecei ing less a en ion. Mo eo e , despi e ini ial epo s
e ealing CH
4
consump ion, s udies ha e no adequa ely e alua ed
he po en ial o mic obial CH
4
oxida ion wi hin ees. In his pape ,
we discuss he cu en le el o unde s anding on hese p ocesses.
Fu he , we demons a e he po en ial o no el me agenomic ools
in e ealing he in ol emen o mic obes in he CH
4
exchange o
plan s, and pa icula ly in bo eal ees. We de ec ed CH
4
-p oducing
me hanogens and no el monooxygenases, po en ially in ol ed in
CH
4
consump ion, in coni e ous plan s. In addi ion, ou ield lux
measu emen s om No way sp uce (Picea abies) canopies demon-
s a e bo h ne CH
4
emissions and up ake, gi ing u he e idence
ha bo h p oduc ion and consump ion a e ele an o he ne CH
4
exchange. Ou indings, oge he wi h he eme ging di e si y o
no el CH
4
-p oducing mic obial g oups, s ongly sugges mic obial
analyses should be in eg a ed in he s udies aiming o e eal he
p ocesses and d i e s behind plan CH
4
exchange.
In oduc ion
The i s e idence on ae obic me hane (CH
4
) emissions by
e es ial ege a ion was p o ided by Kepple e al. (2006),
es ima ing ha plan s –including woody and g ass species –a e a
la ge sou ce o CH
4
. Since hen, nume ous s udies (e.g. Kepple
e al., 2008; Wang e al., 2008; B €uggemann e al., 2009; B uhn
e al., 2009, 2014; Ma el & Qade i, 2017, 2019) ha e con i med
ae obic CH
4
emissions om e es ial plan s. Du ing he pas
decade, ee s ems om opical o bo eal o es s and ees g owing
unde a ying hyd ological condi ions ha e been ound o emi
CH
4
h ough mul iple mechanisms behind he emissions
(Ca michael e al., 2014; Ba ba e al., 2019). Al hough he CH
4
emissions om ee s ems and om ae obic p oduc ion in plan
canopies a e widely ecognized, nei he o hese sou ces a e ye
included in he global CH
4
budge (Saunois e al., 2020).
O e all, discussion on ae obic plan CH
4
p oduc ion has mainly
concen a ed on plan physiology, which was ecen ly e iewed by
L. Li e al. (2020), whe eas a mo e gene al iew o he cu en
unde s anding o ee-de i ed CH
4
luxes, magni udes, p ocesses,
and d i e s has been p esen ed by Ca michael e al. (2014), Co ey
& Megonigal (2019) and Ba ba e al. (2019). Po en ial mic obial
CH
4
p oduc ion wi hin he abo eg ound ee habi a emains less
s udied in compa ison wi h o he mechanisms. So a , he p esence
o he mos -well known CH
4
p oduce s – he me hanogenic
a chaea –has been epo ed only om b oadlea ee s ems: i s ,
based on basic cul i a ion me hods (Zeikus & Wa d, 1974; Zeikus
& Henning, 1975), and ecen ly based on molecula biology (Yip
e al., 2019; H-L. Li e al., 2020). T ee-canopy-de i ed CH
4
emissions a e conside ed o be o med mos ly by abio ic/plan
physiological p ocesses (B uhn e al., 2014; Lenha e al., 2015a),
whe eas he po en ial ole o mic obial CH
4
p oduc ion has been
o e looked –a leas pa lydue o heassump ion ha heanae obic
me hanogens would no h i e wi hin he oxygen-p oducing
canopy habi a . Recen ly disco e ed, ae obic CH
4
-p oducing
mic obial g oups –such as ungi (Lenha e al., 2012) and
cyanobac e ia (Bizice al., 2020) –ha e no ye been ho oughly
conside ed as sou ces o CH
4
in li ing ee s ems o canopies.
A mosphe ic hyd oxyl (OH) adicals a e ecognized as he main
sink o a mosphe ic CH
4
, whe eas he la ges biological sink is
mic obiological CH
4
oxida ion ha occu s mos ly in soils
(Ki schke e al., 2013). CH
4
consump ion in plan s has been
obse ed bo h in he ield and in labo a o y s udies (Ki schbaum &
Walc o , 2008; Sundq is e al., 2012; Zhang e al., 2014;
Halmeenm€aki e al., 2017; S e
zpniewska e al., 2018). Resea ch has
mainly concen a ed on CH
4
- ich en i onmen s, such as pea lands,
whe e he impo ance o Sphagnum moss-associa ed me han-
o ophic bac e ia is well ecognized (e.g. La mola e al., 2010).
Al hough bo eal ee shoo s ha e been shown capable o in si u CH
4
consump ion (Sundq is e al., 2012), no much esea ch has been
done o e eal hemechanisms behind his p ocess. Iden i ica ion o
wi hin- ee me hano ophs (Do onina e al., 2004; Van Aken e al.,
2004; Iguchi e al., 2012) poin s o mic obial CH
4
oxida ion, bu a
possibili y o a nonmic obial sink canno be uled ou ei he .
In his iewpoin , we discuss he magni ude and cu en p ocess-
le el unde s anding o CH
4
exchange o ees, wi h an emphasis on
he ole o mic obes in he so a conside ed nonmic obial CH
4
p oduc ion in plan issues. We p opose ha he lack o mic obial
obse a ions is no caused by he absence o hese popula ions, bu
a leas pa ly due o unde eloped me hods wi h poo de ec ion
limi s. To demons a e he po en ial o no el molecula biology
ools, we p o ide wo ypes o me agenomics da a om coni e ous
ee issues: (1) unc ional genes de ec ed h ough an ex ensi e
524 New Phy ologis (2021) 231: 524–536 Ó2021 The Au ho s
New Phy ologis Ó2021 New Phy ologis Founda ion
www.newphy ologis .com
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use,
dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Fo um
sc eening o public me agenome en ies (Na ional Cen e o
Bio echnology In o ma ion (NCBI) Sequence Read A chi e
(SRA)) published so a ; and (2) unc ional gene de ec ion h ough
a no el p obe- a ge ed me agenome sequencing me hod. In
addi ion, we p esen CH
4
lux da a om No way sp uce (Picea
abies) shoo s indica ing he occu ence o bo h CH
4
p oduc ion
and consump ion in he ee canopy.
Th ough ou indings om he ee canopies, we highligh he
need o ex ending he discussions o ae obic CH
4
p oduc ion om
abio ic and plan physiological p ocesses o plan –mic obe in e -
ac ions. Fu he mo e, we discuss how mode n ools could ad ance
ou knowledge on he CH
4
cycling mic obes om de ec ion o
p esence o he unde s anding o ac i e p ocesses, and o he
cha ac e iza ion o no el mic obial g oups in ol ed in he ee
CH
4
exchange.
Cu en unde s anding o he me hane p oduc ion in
ees
T ees as sou ces o me hane
T ee s em lux measu emen s indica e ha ees ac oss ege a ion
zones and g owing habi a s a e mos ly sou ces o CH
4
(Ca michael e al., 2014; Ba ba e al., 2019). The s em CH
4
emissions a e p oposed o o m ei he h ough in e nal CH
4
o ma ion wi hin ee issues (e.g. Wang e al., 2016) o anspo
o soil-de i ed, mic obially o med CH
4
emi ed h ough ee
s ems (e.g. Rusch & Rennenbe g, 1998), o a combina ion o hese
wo as e iewed ecen ly by Ba ba e al. (2019). The numbe o
ield-scale s udies on canopy CH
4
exchange emain low despi e he
nume ous labo a o y s udies epo ing ae obic, p esumably no-
mic obial CH
4
p oduc ion in plan lea es i s p esen ed by
Kepple e al. (2006). Exis ing ield e idence on canopy CH
4
exchange indica es bo h emissions and up ake o CH
4
and calls o
u he s udies (Sundq is e al., 2012; Machaco a e al., 2016;
Pangala e al., 2017; his s udy).
Ae obic me hane p oduc ion h ough nonmic obial
mechanisms
Inc easing e idence sugges s ha ae obic CH
4
o ma ion in plan
lea es may be an in eg al pa o cellula esponses o changing
edox condi ions in all euka yo es, and ha his common
biochemical CH
4
sou ce may exis in all euka yo es –plan s,
animals, ungi, and algae (Kepple e al., 2009; Liu e al., 2015).
Se e al s udies also sugges ha ae obic nonmic obial CH
4
o ma ion occu s h ough eac i e oxygen species (ROS) gene a-
ion and a subsequen elease o CH
4
om p ecu so compounds,
such as pec ic me hyl g oups, me hionine, o o he subs a es (e.g.
Kepple e al., 2008; McLeod e al., 2008; Vigano e al., 2008;
B uhn e al., 2009; Lenha e al., 2015a).
Al hough plan ROS a e p oduced du ing ae obic espi a ion
and pho osyn hesis as a no mal by-p oduc o ae obic plan
me abolism, ROS p oduc ion can be induced by di e en
en i onmen al s esso s (Huang e al., 2019). Simila ly, en i on-
men al s esso s like ul a iole (UV) adia ion and ele a ed
empe a u e (e.g. McLeod e al., 2008; Vigano e al., 2008; B uhn
e al., 2009, 2014; Qade i & Reid, 2009), physical inju y o he
plan – o example, lea damage caused by cu ing and hypoxia
(Wang e al., 2009) –wa e s ess (Qade i & Reid, 2009), and low
ligh le els (Ma el & Qade i, 2017) ha e been obse ed o
s imula e ae obicnonmic obial CH
4
p oduc ion inplan s. S ill, he
biochemical pa hways behind s ess-induced CH
4
o ma ion and
i s po en ial occu ence in na u al condi ions emain unknown.
Mic obes as po en ial me hane p oduce s in ees
Di e en pa s o he ees (lea es, s em, ba k, oo s) se e as
unique habi a s o a a ie y o mic obial communi ies ha can
li e ei he as epiphy es on he plan su ace, o as endophy es inside
he plan issues –used he e o include also pa hogens, as
a ionalized by G i in & Ca son (2018). Toge he , di e en
mic obes, including bac e ia, a chaea, and ungi, o m he ee
mic obiome (Te honen e al., 2019). S udies on ee mic obiomes
ha e la gely concen a ed on ungi and less on bac e ia o
a chaea (G i in & Ca son, 2018; Ha ison & G i in, 2020)
and ee-s em-associa ed p oka yo es especially a e s ill poo ly
cha ac e ized (Bald ian, 2017). Geog aphically, endophy e s ud-
ies ha e ocused on he opical and empe a e egions, lea ing
bo eal and alpine ecosys ems poo ly examined (Ha ison &
G i in, 2020).
Many o he ee-associa ed mic obes a e impo an o he hos
plan s ia p omo ing plan g ow h and inc easing esis ance o
s ess and pa hogens, whe eas some o hem can nega i ely a ec
plan g ow h (F ank, 2018; Te honen e al., 2019; Chaudh y e al.,
2020). Owing o a ying condi ions caused by bo h hos me abolic
p ocesses and abio ic s ess ac o s, such as d ough and UV
adia ion, ee oliage mic obiomes especially a e highly dynamic
sys ems (Chaudh y e al., 2020). In addi ion, coloniza ion pa e ns
a ec he mic obiome composi ion: whe eas some endophy es can
s ay wi h hei hos he whole plan li e cycle ( e ical ansmission),
mos a e es ima ed o o igina e om he en i onmen h ough
ho izon al ansmission ei he om he soil o h ough he ai
(F ank e al., 2017).
On i s hough , ees and o he plan issues seem o be mos ly
ae obic en i onmen s and, as such, unsui ed habi a s o anae obic
o ganisms, like me hanogenic a chaea (Ki schke e al., 2013). As
ecen ly e iewed by Co ey & Megonical (2019), howe e , anoxia
can p e ail inside bo h heal hy and in ec ed ee s ems, leading o
CH
4
p oduc ion (Fig. 1b). In bo h si ua ions, anoxia could be
c ea ed h ough oxygen-consuming me abolic p ocesses o he ee-
associa ed endophy ic mic obes, such as ungi, and also h ough
ac i e s em and oo espi a ion (Teskey e al., 2008), helping o
main ain a o able condi ions o me hanogenesis. In pa icula ,
ungal-media ed decay o hea wood (i.e. hea o disease) has been
sugges ed as an impo an d i e o CH
4
emissions om li ing ees
(Co ey e al., 2012). Mo eo e , anae obic me hanogens ha e also
been de ec ed in he oo s o No way sp uce, Sco s pine (Pinus
syl es is), sil e bi ch (Be ula pendula), and black alde (Alnus
glu inosa) (Bombe g & Timonen, 2009; Bombe g e al., 2011;
Fig. 1c). As in he s ems, oo -associa ed a chaea a e p edic ed o
bene i om he O
2
consump ion o o he mic obes, and also
©2021 The Au ho s
New Phy ologis ©2021 New Phy ologis Founda ion
New Phy ologis (2021) 231: 524–536
www.newphy ologis .com
New
Phy ologis Viewpoin s Fo um 525
di ec ly om he ca bon (C) compounds exuded om he oo s
(Bombe g e al., 2011).
Local anae obic mic oen i onmen s could exis also in he
needles and lea es o ees, whe e he ac i i y o endophy es, and
hus O
2
consump ion, is enhanced by he esh, pho osyn hesis-
de i ed C compounds (Fig. 1a). This ype o in e ac ion has been
epo ed a leas om g amineous plan s (Minamisawa e al.,
2004), whe e anae obic ni ogen (N)- ixing clos idia a e sup-
po ed by o he , nondiazo ophic endophy es. Al hough di ec
canopy-de i ed e idence is s ill lacking, anae obic N ixa ion occu s
also in coni e ous needles (Moyes e al., 2016), demons a ing he
po en ial o o he anae obic p ocesses as well.
Al hough oxygen e ec i ely inhibi s a chaeal CH
4
p oduc ion
(Fe ze e al., 1993; Yuan e al., 2009), a leas some me hanogens
can s ill ole a e oxic condi ions –as p e iously obse ed, o
example, in upland soils (Pe e s & Con ad, 1996; Angel e al.,
2012). Lyu & Lu (2018) e alua ed he mechanisms behind his
ole ance in hei ecen me a-analysis o bo h genomic and
en i onmen al da a. They ound s ong e idence o wo dis inc
me hanogen clus e s, wi h one o hem ha bo ing expanded
ca ego y o oxygen ole ance ea u es, including ways o comba
ROS-de i ed oxida i e s ess. This spli in o clus e s was la gely
in line wi h he classi ica ion in o phylogene ic me hanogen
o de s and hei e olu iona y his o y in ela ion o a mosphe ic
O
2
le els. Mo eo e , global analysis o me hanogens de ec ed in
oxic habi a s ga e u he e idence ha hese pa icula
me hanogens ha e he po en ial o su i e in he p esence o
oxygen (Lyu & Lu, 2018) –and hus possibly e en wi hin he
canopy habi a .
In addi ion o me hanogenic a chaea, ee-de i ed CH
4
could
be p oduced by o he mic obial g oups, be e sui ed o a li e in
ae obic condi ions. Fi s , sap ophy ic ungi p oduce CH
4
a leas
in nonli ing wood ma e ial (Lenha e al., 2012) . The ungal
CH
4
was shown o de i e om me hionine (Lenha e al.,
2012), a p ecu so compound linked wi h plan s ess-induced
ae obic CH
4
p oduc ion (Lenha e al., 2015a). On he o he
hand, Lenha e al. (2012) also sugges ed ha he ungal CH
4
p oduc ion can be connec ed o chlo ome hane (CH
3
Cl)
(a) FOLIAGE
(b) TRUNK
(c) ROOTS
CH4-p oducing sap o ophic ungi
CH4-p oducing cyanobac e ia
Me hanogenic a chaea
Me hano ophic bac e ia
S ong di ec e idence
CH4PRODUCTION
CH4OXIDATION Po en ial loca ions
Indi ec /weak e idence
Sapwood Hea wood Hea wood in ec ed
by pa hogenic ungi 10 cm
1 mm
1 cm
1 mm
Fig. 1 Loca ions o di e en me hane (CH
4
)-p oducing and he so a known CH
4
-consuming mic obes, he me hano ophic bac e ia, p esen wi hin di e en
ee compa men s: (a) oliage,(b) unk, (c) oo s, as de e mined based on he e e ences and new esul sp esen ed in his iewpoin . The loca ions a e ma ked
wi h do s colo ed based on he le el o scien i ic e idence: s ong e idence indica es mo e han one s udy and/o de ec ed in se e al ee species; indi ec /weak
e idence indica es only one s udy o one ee species, o de ec ed ac i i y (e.g. lux measu emen s); po en ial loca ions indica es po en ially sui able condi ions
o he gi en mic obial g oup.Ni ogenase- ela ed CH
4
p oduc ion wasno included in he igu e owing o he s ill low unde s anding o i spo en ial occu ence
in he ee habi a . Red a ows, CH
4
p oduc ion; blue a ows, CH
4
consump ion.
New Phy ologis (2021) 231: 524–536
www.newphy ologis .com
©2021 The Au ho s
New Phy ologis ©2021 New Phy ologis Founda ion
Viewpoin s
Fo um
New
Phy ologis
526
o ma ion and he ype o subs a es a ailable, which migh limi
his p ocess o he wood-decay ungi. As some o he wood-
decaying ungi can also in ec li ing ees (Asiegbu e al., 2005),
and since needles ha bo complex ungal mic obiomes (Pi il€a
&W€ali, 2009), hei ole in he ee s em and canopy CH
4
exchange may be signi ican , bu his emains o be esol ed
(Fig. 1b).
Ano he ecen ly disco e ed CH
4
-p oducing g oup a e
cyanobac e ia, which we e linked o his p ocess bo h unde oxic
and anoxic condi ions (Bizice al., 2020). CH
4
p oduc ion was
sugges ed o occu h ough gene al cell me abolism, such as
pho oau o ophic C ixa ion, and mechanisms ha a e dependen
on pho osyn he ic p oduc s du ing ligh , and on s o age
compounds du ing da k. Since pho osyn hesis-pe o ming
chlo oplas s in plan s a e known o ha e e ol ed om cyanobac-
e ia h ough endosymbiosis (Ra en & Allen, 2003), hese
bac e ia could be connec ed o CH
4
p oduc ion o land plan s as
well (Fig. 1a,b). This ela ion u he unde lines he decadal
discussion on mechanis ic unde s anding o ae obic CH
4
o ma-
ion in plan s (e.g. Kepple e al., 2009; Liu e al., 2015). So a ,
he mos di ec link be ween cyanobac e ia and ee- ela ed CH
4
emissions a e he cyanobac e ia-con aining c yp ogamic co e s,
such as lichens, which can g ow on ee s ems and ha e shown
small CH
4
emissions in labo a o y incuba ions (Lenha e al.,
2015b; Fig. 1b). Finally, CH
4
is also p oduced du ing he p ocess
o N ixa ion when i in ol es he i on ni ogenase, and o a lesse
ex en he anadium ni ogenase (Zheng e al., 2018). These
enzymes a e ound in a ious species ep esen ing bo h a chaea
and bac e ia (McRose e al., 2017). This inding is in e es ing
owing o ecen indica ions ha endophy ic diazo ophs a e
essen ial o coni e ous ees g owing on nu ien -poo soil
(Moyes e al., 2016; Pu i e al., 2020) and also conside ing
c yp ogamic co e s, whe e cyanobac e ia u ilize hese enzymes
(Bellenge e al., 2020). Fu he mo e, ano he ni ogenase- ype
enzyme sys em, ound in a ious mic obial g oups, was ecen ly
epo ed o p oduce CH
4
om dime hyl sul ide (No h e al.,
2020), p oduced, o example, by a ious bac e ia in e es ial
en i onmen s (Ca ion e al., 2015, 2017). Al hough he link
be ween all o hese nona chaeal g oups and CH
4
p oduc ion in
he li ing ees is unce ain, he a o emen ioned indings sugges
ha CH
4
o ma ion in e es ial ecosys ems is a a mo e
widesp ead ai han p e iously hough and also wa an s hei
e alua ion in he ee CH
4
s udies.
Me hanogenic mic obes in canopies o coni e ous ees
Compa ed wi h po en ial ae obic CH
4
p oduc ion in plan s, he
ole o me hanogenic a chaea is o en le unde e mined in
cu en CH
4
-exchange s udies ocusing on he pho osyn hesizing
plan pa s. This si ua ion s ems a leas pa ly om p ac ical
easons, as we ha e lacked me hods wi h adequa e esolu ion o
iden i y mic obial popula ions behind locally ela i ely small, bu
globally signi ican emissions, and me hods ha allow linking
p e iously un ela ed o ganisms wi h pa icula unc ions, like
CH
4
-p oduc ion. PCR-based me hods, such as amplicon
sequencing, a e a s anda d ool in mic obial ecology. Ye ,
co e age limi a ions o en make hem un i o he analysis o
a e, poo ly cha ac e ized endophy es. Acco dingly, only wo
PCR-based s udies (and only wo molecula analyses in gene al)
ha e been published on ee-dwelling me hanogens, and only
om ee s ems (Yip e al., 2019; H-L. Li e al., 2020). The ecen
de elopmen s in high- h oughpu sequencing echniques ha e led
o he ise o me agenomic me hods, which can po en ially
e olu ionize he analysis o a ious mic obiomes, such as ee
endophy es. Compa ed wi h PCR, me agenomic app oaches
en ail a a wide pe spec i e: whole mic obiomes wi hin plan
issues can be sequenced, and wi h he igh analy ical ools e en
genes om no el axa can be e ealed.
We e alua ed wo di e en me agenomic sequencing
app oaches in e ealing po en ial CH
4
-p oducing mic obes in
ees, wi h a ocus on bo eal ee canopies. Fi s , we conduc ed an
ex ensi e me a-analysis o me hanogenic unc ional genes in
al eady published da a-en ies in he SRA (h ps://www.ncbi.nlm.
nih.go /s a) ela ed o pine and sp uce issues (a de ailed
desc ip ion is gi en in Suppo ing In o ma ion Me hods S1),
simila ly as p e iously o he me hanogens in he SRA-da a om
pea lands (B €aue e al., 2020). In b ie , gene agmen s o CH
4
-
p oducing a chaea (mc A, coding o he me hyl-coenzyme M
educ ase) we e sea ched wi h HMMER (hidden Ma ko model
sea ch o gene s uc u es) om he published SRA da abase
(NCBI), and phylogene ics o hem we e analyzed agains
ob ained cul u ed and candida e di isions o unc ional genes.
Second, we u ilized a no el ‘p obe- a ge ed cap u e’ me hod
(Aal o e al., 2020; Siljanen e al., 2021) o analyze genes ela ed
o CH
4
cycling om No way sp uce needles collec ed om
eas e n Finland (Kuopio). The same me hod was used ecen ly
o he de ec ion o N-cycling mic obes in plan biomass (Aal o
e al., 2020). He e, cap u e eac ion was ca ied ou wi h 12 190
unique p obes, which we e designed based on he cu en ly
known mc A gene di e si y in he public da abases (Siljanen e al.,
2021). Be o e he analysis, sp uce b anches we e incuba ed in
ae obic condi ions in a medium o sodium ace a e con aining
dilu ed ni a e mine al sal s o 14 d wi h 100 ppm CH
4
in he
headspace o enhance he de ec ion o bo h me hanogens and
me hano ophs ( esul s o he me hano ophs a e epo ed la e
in his pape ).
In e es ingly, bo h o ou app oaches e ealed known
me hanogen species wi hin he sp uce canopies (Figs 1a, 2, S1;
Table 1). An SRA da abase sea ch e ealed signs o me hanogenic
mc A genes in bo h pine and sp uce ees (Fig. 2; Tables S1, S2).
Howe e , al hough SRA sequences ga e indica ions o a wide
di e si y (o de s Me hanosa cinales, Me hanomic obiales, and
Me hanobac e iales o sp uce-de i ed en ies and Me hanomi-
c obiales o pine-de i ed en ies), he numbe o quali y-checked
sequences was small (Table 1).
Ou cap u ed me agenome analysis included No way sp uce
needles only om one loca ion (Kuopio, Finland) and, as a smalle
sample se , was expec ed o exp ess lowe mc A di e si y han he
global da abase sea ch. S ill, by p o iding much highe leng h
sequences (a e age 250 bp s 100 bp o he SRAs) speci ically
en iched by mc A- a ge ing p obes, his me hod was able o gi e
mo e eliable e idence on he p esence o me hanogenic a chaea
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New
Phy ologis Viewpoin s Fo um 527

wi hin he coni e habi a (Fig. 2; Table 1). The as majo i y o he
sequences belonged o o de s Me hanomic obiales and
Me hanosa cinales wi hin he class Me hanomic obia. Wi hin
he o me o de , mos sequences we e ela ed o he genus
Me hano egula (1799 ou o 4066 sequences om sp uce 1
ma ched wi h Candida us Me hano egula boonei wi h a likeli-
hood-weigh a io LWR >0.95; scale o LWR: 0–1). Wi hin
Me hanosa cinales, sp uce mc A sequences g ouped wi h
Me hano h ix ( o me ly Me hanosae a) species (Me hano h ix
soehngenii linked wi h LWR >0.95 wi h 303/4066 sequences om
sp uce 1 and 101/1865 om sp uce 2). All o hese well-desc ibed
gene a/species a e common inhabi an s in, o example, he
wa e logged laye s o pea lands, whe e hey pe o m anae obic
me hanogenesis as he las s ep o o ganic ma e deg ada ion
(B €aue e al., 2020). The la ges g oups ound, Me hano egula and
Me hano h ix, p oduce CH
4
by educing ca bon dioxide (CO
2
)
wi h hyd ogen (H
2
), o by spli ing o ace a e o CH
4
and CO
2
,
espec i ely. Acco dingly, he de ec ion o he Me hano h ix
L77117 1 771823-773477 Me hanocaldococcus jannaschii DSM 2661
CP000477 1 590544-588887 Me hano h ix he mophila PT
JX141395 1 1-464 Me hanobac e ium o micicum KOR-1 m A
CP000300 1 2545676-2543963 Me hanococcoides bu onii DSM 6242
KM041257 1 2-488 Me hano ollis limina ans A mc A
CP009515 1 197568-195858 Me hanosa cina lacus is Z-7289
AB479391 1 2-721 Me hano egula o micicum SMSP mc A
CP003117 1 500591-502252 Me hano h ixh a undinacea 6Ac
AF313803 1 2-471 Me hano h ix soehngenii VeAc9 mc A
KM041254 1 2-504 Me hano he mobac e ma bu gensis mc A
CP002737 1 1238720-1240374 Me hano o is igneus Kol 5
CP002069 1 861025-859314 Me hanohalobium e es iga um Z-7303
AF414041 1 1-437 Me hano ollis limina ans DSM 4140 mc A
EU715818 1 744-2 Me hanolobus zinde i SD1 mc A
KT387805 1 1141805 Uncul u ed Ba hya chaeo a cloneCX10 BA1 24 9
CP003167 1 861950-863646 Me hano egula o micicum SMSP
CP017921 1 61885-63601 Me hanohalophilus halophilus Z-7982
DQ229161 1 1-519 Me hanogenium boonei mc A
AB703644 1 1-1170 Me hano ollis e hanolicus mc A NBRC 104120 mc A
JQ511369 1 1-425 Me hanocalculus alkaliphilus AMF2 mc A
CP000559 1 1595689-1593992 Me hanoco pusculum lab eanum Z
KT387806 1 1661817 Uncul u ed Ba hya chaeo a cloneCX10 BA2 13 151
CP002057 1 1273204-1274859 Me hanococcus ol ae A3
CP000780 1 552905-551251 Ca. Me hano egula boonei 6A8
CP009517 1 3100739-3099032 Me hanosa cina ba ke i 3
AB496719 1 1-746 Me hanolinea mesophila mc A
CP010070 1 210620-212263 Ca. Me hanoplasma e mi um MpT1
CP003167 1 2552112-2553766 Me hano egula o micicum SMSP
CP002009 1 260010-258356 Me hanocaldococcus in e nus ME
HE964772 2 1548527-1546837 Me hanoculleus bou gensis MS2T
CP001696 1 393468-392637 Me hanocaldococcus e ens AG86
AY386125 1 2-1123 Me hanobac e ium aa husense mc A
AB288270 1 2-742 Me hanoculleus chikugoensis mc A
U22244 1 2-489 Me hanolubus inda ius mc A
CP002117 1 2417258-2415575 Me hanoplanus pe olea ius DSM 11571
HQ896500 1 455-1 Me hanomassiliicoccus luminyensis B10 mc A
CP004049 1 363749-365391 Ca. Me hanome hylophilus al us Mx1201
CP002278 1 713931-715572 Me hano he mus e idus DSM 2088
AF414037 1 1-416 Me hano h ix soehngenii DSM 3671 mc A
AB703641 1 1-1169 Me hanolinea a da NBRC 102358 mc A
AP011532 1 507632-509298 Me hanocella paludicola SANAE DNA
EF026570 1 3-668 Me he micoccus shengliensis ZC-1 mc A
CP002278 1 756135-754476 Me hano he mus e idus DSM 2088
CP002737 1 455360-457008 Me hano o is igneus Kol 5
AB542746 1 1-1144 Me hanobac e ium alcaliphilum NBRC 105226 mc A
AE009439 1 614620-616275 Me hanopy us kandle i AV19
LT608329 1 1413251-1411597 Me hano he mobac e wol eii SIV6
CP001710 1 1394951-1393301 Me hano he mobac e ma bu gensis s Ma bu g
CP002565 1 676986-675313 Me hano h ix soehngenii GP-6
KM259864 1 1-442 Me hanosalsum na onophilum AME9 Mc A
CP001710 1 1425701-1424056 Me hano he mobac e ma bu gensis Ma bu g
AB542760 1 2-1139 Me hanobac e ium palus e NBRC 105230 m A
CP002551 1 338949-340594 Me hanobac e ium lacus AL-21
CP009516 1 105661-107367 Me hanosa cina ho onobensis HB-1
CP003362 1 1659823-1661533 Me hanome hylo o ans hollandica DSM 15978
CP009509 1 92002-93709 Me hanosa cina mazei WWM610
EU919432 1 1-467 Me hanob e ibac e woesei GS mc A
U22245 1 2-489 Me hanolobus ulcani mc A
AB842184 1 3-1141 Me hanobac e ium alcaliphilum NBRC 109449 m A
AB703638 1 1-1147 Me hano he mobac e wol eii NBRC 100332 mc A
CP005934 1 1549873-1548225 Ca. Me hanomassiliicoccus in es inalis Issoi e-Mx1
AF414044 1 1-438 Me hanomic obium mobile DSM 1539 mc A
AB679169 1 2-1148 Me hano h ix pelagica mc A
AM114193 2 1035015-1033351 Me hanocella a o yzae MRE50
CP002551 1 2316117-2317758 Me hanobac e ium lacus AL-21
CP014265 1 1666298-1664653 Me hanob e ibac e olleyae YLM1
T ee scale: 1
Me hanosa cinales
Me hanomic obiales
Me hanocellales
Ba hya chaeo a
The moplasma a
Me hanopy ales
Me hanobac e iales, m A
Me hanococcales, mc A
Me hanococcales, m A,
Me hanobac e iales, mc A
Fig. 2 Phylogene ic placemen s o he me hanogenic mc A gene (coding o he alpha subuni o he me hyl-coenzyme M educ ase (MCR))sequences
e ie ed h ough heSequenceReadA chi e(SRA) da abasesea chandcap u edme agenomicsequencing om wopooledsp uce needle samples(sp uces1
and 2) among he known me hanogen and Ba hya chaeo al mc Aand m A (coding o he isozyme o he MCR) sequences in e ed using he iTOL- ee
(Le unic & Bo k,2019) wi h RAXML (S ama akis, 2014). In he SRA-da abase sea ch, 4822 pine and 1215sp uce SRA iles in o al we e sc eened wi h HMMER o
mc A genes (de ails in he Suppo ing In o ma ion Me hods S1). Only he bes phylogene ic placemen is shown o each que y sequence, and only sequences
wi h likelihood-weigh a ios >0.2 a e included.Size o he placemen icons e lec s he ela i e numbe o sequences o a gi en placemen wi hin each sample.
As an excep ion, he smalles size icon is used o posi ions wi h one o i e sequence placemen s. The o al numbe o sequences o each sample ypeis lis ed in
Table 1. O iginal e e ence ee wi h 189 sequences oge he wi h boo s ap alues is in Fig. S1. Ca., Candida us.
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Viewpoin s
Fo um
New
Phy ologis
528
sequences may ha e been enhanced by he sp uce incuba ion
ea men wi h ace a e in he g ow h medium. Howe e , ou esul s
s ill e lec he axa p esen wi hin un ea ed needles. In soils, bo h
ace oclas ic and hyd ogeno ophic me hanogenic pa hways a e
sus ained by he ac i i y o o he mic obio a, such as syn ophic
mic obes (B €aue e al., 2020). This is likely he case also in he
needle habi a and needs o be in es iga ed h ough a wide analysis
o he whole mic obiome and ela ed mic obe–mic obe in e ac-
ions. I should be no ed ha plan me abolic p ocesses could also
se e as a sou ce o subs a es o he mic obial me hanogenesis. Fo
example, ace a e is con inuously ecycled wi hin he plan cells
(Zhang e al., 2017) and could, hus, be a ailable o he ace oclas ic
me hanogens such as he Me hano h ix species.
Conside ing he la gely oxygenic condi ions wi hin sp uce
needles, de ec ion o Me hanomic obiales and Me hanosa cinales
is i ing: hey belong o he speci ic clus e o me hanogens
sugges ed o con ain enhanced O
2
ole ance mechanisms (Lyu &
Lu, 2018). Fo hem o be ac ually ac i e in CH
4
p oduc ion, a
leas empo ally anoxic mic ohabi a s a e needed –po en ially
in ol ing he oxygen-consuming ac i i y o o he endophy es.
Me hane consump ion wi hin ee s ems and
canopies: an un ecognized me hane sink?
E idence o me hane consump ion by ees om lux
measu emen s
Despi e nume ous CH
4
lux s udies on ee s ems, consump ion o
CH
4
in s ems has been a ely epo ed (Ba ba e al., 2019; Welch
e al., 2019; Moldaschl e al., 2021). As he ne CH
4
exchange is he
sum o bo h p oduc ion and consump ion p ocesses, i emains
unclea whe he consump ion exis s bu is mos ly o e come by a
highe CH
4
p oduc ion a e. The ew exis ing s udies on ee
canopy CH
4
exchange show ha ee canopies can ac as bo h
sou ces and sinks o CH
4
(Sundq is e al., 2012; Machaco a e al.,
2016; Halmeenm€aki e al., 2017; Pangala e al., 2017; his s udy).
Based on ield measu emen s, canopy CH
4
consump ion has, o
ou knowledge, been epo ed only by Sundq is e al. (2012). All
he ee species hey measu ed –coni e ous ees: No way sp uce
and Sco s pine; and b oadlea ees: bi ch (Be ula pubescens) and
owan (So bus aucupa ia)–we e obse ed o mos ly consume CH
4
wi h an a e age CH
4
consump ion a e o 11.2 µgh
1
m
2
lea
a ea (LA) a lowe b anches o he ees du ing au umn pe iod.
Sundq is e al. (2012) es ima ed ha , wi h he up ake a e hey
measu ed, he ee canopy CH
4
sink could be o simila s eng h o
he soil sink.
He e, we p esen esul s om wo No way sp uce ield
campaigns, which u he demons a e he unc ioning o shoo s
as bo h sinks and sou ces o a mosphe ic CH
4
(Fig. 3; de ails in
Me hods S1). The a e age CH
4
exchange a e was
0.3 ng g
1
DW h
1
o ma u e sp uce ees (Skoga yd, Swe-
den), and 1.4 ng g
1
DW h
1
o 2 o 3-y -old ee saplings
(Helsinki, Finland). The scale and a ia ion o he luxes was
clea ly highe in he young samplings, possibly e lec ing hei
g ow h phase and dynamic condi ions du ing he sp ing pe iod.
Howe e , ou luxes om bo h ma u e sp uce shoo s and om
he saplings we e ma kedly smalle han hose measu ed by
Sundq is e al. (2012). As in e p e ed om Sundq is e al.
(2012, Fig. 1), hei CH
4
luxes om ma u e sp uce shoo s
anged om c.40 o 32 µgh
1
m
2
LA, which scales o a ange
o c.200 o 160 ng g
1
DW h
1
(Hage & S e ba, 1985). This
high a iabili y be ween he s udies unde lines he need o mo e
lux measu emen s om ee canopies, and he need o conside
bo h CH
4
p oduc ion and consump ion when e alua ing he ole
o ees in he o es CH
4
balance. Mos impo an ly, o unco e
he d i e s o hese p ocesses, po en ial in ol emen o mic obes
should be s udied using simul aneous collec ion o ee issue
samples.
Table 1 Numbe o quali y-con olled (likelihood-weigh a io LWR >0.20) sequences acqui ed/ e ie edin his s udy oge he wi h hei LWR alues om he
phylogene ic placemen analysis (de ails in Suppo ing In o ma ion Me hods S1).
Sample and analysis
ype
Ma ke
gene Hos ee species (plan pa used in he sequencing) No. o sequences
LWR
ange
LWR mean 
SD
Sp uce 1, cap u e
sequencing
mc A Picea abies (needles) 4066 0.26–1.00 0.83 0.21
mmoX 3782 0.20–1.00 0.67 0.20
Psm_mmoX 1 0.55 —
Sp uce 2, cap u e
sequencing
mc A Picea abies (needles) 1856 0.20–1.00 0.56 0.22
mmoX 270 0.21–0.99 0.69 0.24
Psm_mmoX nd nd nd
SRA da abase, pine mc A Pseudo suga menziesii (megagame ophy e) 2 0.22–0.29 0.26 0.05
mmoX Pinus aeda (needles), Pseudozuga menziesii (needles) 4 0.24–0.43 0.35 0.08
Psm_mmoX Pinus syl es is (needles), Pinus cana iensis (cambial cells), Pinus
con o a ( oliage), Pseudozuga menziesii (megagame ophy e)
4 0.22–0.97 0.56 0.35
SRA da abase, sp uce mc A Picea abies (megagame ophy e) 6 0.21–0.36 0.28 0.06
mmoX Picea abies (megagame ophy e) 2 0.27–0.62 0.45 0.24
Psm_mmoX Picea abies (megagame ophy e) 50 0.02–1 0.57 0.28
In he Sequence Read A chi e (SRA)-da abase sea ch, 4822 pine and 1215 sp uce SRA iles in o al we e sc eened wi h HMMER o mc A,mmoX, and pmoA
genes. A e age leng hs o cap u e and SRA sequences we e 250 bp and 100 bp, espec i ely. No adi ional pmoA gene agmen s we e de ec ed.
nd, no de ec ed.
Psm_mmoX,Pseudomonas sp.- ela ed no el mmoX.
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New
Phy ologis Viewpoin s Fo um 529
E idence o me hane consump ion by ees om de ec ion o
ee-associa ed me hano ophs
Me hano ophs a y in hei p e e ence o he concen a ion o
CH
4
and can hus be di ided in o high (a mosphe ic CH
4
) and
low-a ini y oxidize s –al hough some o hem oxidize CH
4
bo h
in high and low concen a ions (Chowdhu y & Dick, 2013; Ho
e al., 2019). High-a ini y me hano ophs a e esponsible o he
CH
4
sink o he upland soils, whe eas low-a ini y popula ions
h i e, o example, in wa e logged soils wi h high in si u CH
4
p oduc ion (Knie e al., 2003; Chowdhu y & Dick, 2013). T ees
could po en ially p o ide mic ohabi a s o bo h ypes o
me hano ophy. In he canopies, CH
4
concen a ions a e likely
close o a mosphe ic concen a ions, and hus hey migh ha bo
high-a ini y- ype oxidize s as bo h epi- and endophy es. Ou
esul s, howe e , poin o he p esence o me hanogenic ac i i y in
he needles, which migh suppo low-a ini y oxidize s. In line
wi h his, Iguchi e al. (2012) epo ed isola ion o common
me hano ophs, Me hylomonas sp. and Me hylocys is sp., om
No way sp uce and Pinus pa i lo a needles (Fig. 1a). Al hough
hei CH
4
oxida ion capaci y was no es ed, he isola es we e
ob ained using a high CH
4
concen a ion (20%). Likewise,
Do onina e al. (2004) we e able o isola e a Me hylocys is- ela ed
s ain om he needles o Picea pungens. All he isola es
men ioned also g ew wi h me hanol (CH
3
OH), and hus
ep esen ed he so-called acul a i e me hano ophs, po en ially
suppo ed by CH
3
OH o ma ion in he plan physiological
p ocesses (Do okho e al., 2018). Mo e ecen ly, simila
acul a i e me hano ophs we e de ec ed h ough 16S ibosomal
RNA ( RNA) gene sequencing om he needles o Pinus adia a
(Rua e al., 2016) and No way sp uce (Haas e al., 2018). T ee
s ems ha e been shown o occasionally hold e y high CH
4
concen a ions (Co ey e al., 2012) and migh , hus, se e as a
habi a o low-a ini y oxidize s, simila o, o example,
Sphagnum mosses in pea lands (e.g. La mola e al., 2010;
Pu kinen e al., 2012). This is suppo ed by he de ec ion o
bo h 16S RNA genes ela ed o common alpha and gammap o-
eobac e ial me hano ophs and me hanogens (as discussed ea lie
in he pape ) in he s ems o Populus del oides (Yip e al., 2019;
Fig. 1b). In addi ion o li ing s ems, me hano ophs ha e been
ound in allen logs in ec ed by ungi (M€akip€a€ae al., 2018) –
ano he ee habi a whe e in si u CH
4
p oduc ion likely akes
place (Co ey e al., 2012), and, in unquan i iable amoun s, in
Sco s pine oo s (Halmeenm€aki e al., 2017, Fig. 1c).
Based on hese indings, and he CH
4
consump ion de ec ed in
he ield measu emen s, we sugges ha me hano ophs a e
p esen in he coni e habi a and ha hei in-dep h cha ac e -
iza ion is possible wi h he imp o ed me agenomics ools now
a ailable. Fo his pu pose, we used he same wo me agenomic
app oaches as wi h he me hanogens (de ails in Me hods S1;
B €aue e al., 2020). Fi s , he SRA da abase was sea ched o
me hano ophic unc ional genes pmoA and mmoX, coding o he
pa icula e and soluble o ms o me hane monooxygenase
(MMO), espec i ely. Second, he same genes we e a ge ed wi h
he cap u e en ichmen app oach (Aal o e al., 2020) o de ec
me hano ophs in sp uce shoo s, which we e i s incuba ed o
enhance he de ec ion o CH
4
-cycling mic obes (as desc ibed in
he sec ion Me hanogenic mic obes in canopies o coni e ous
ees; Dun ield e al., 2003; Dedysh e al., 2005). Cap u e eac ion
included 640 unique p obes o mmoX and 19 900 p obes o
pmoA (Siljanen e al., 2021).
Bo h analyses e ealed simila pa e ns: monooxygenase (MO)
genes we e ound, bu excep o wo pine-de i ed SRA-
sequences, simila o alphap o eobac e ial mmoX genes, hey
we e no ela ed o pmoA o mmoX genes o known me han-
o ophs (Figs 4, S2; Tables 1, S1, S2). Almos all o he SRA
sequences wi h p ope likelihood weigh alues (Table 1) we e
om he same p ojec a ge ing he genome o he hos ee,
P. abies, wi h sequenced DNA de i ing om he sp uce megaga-
me ophy e (Nys ed e al., 2013) –likely e lec ing he lack o
mic obiome a ge ing analyses in gene al. Excep o one
ac inobac e ial p opane MO (PMO) ma ch, hese SRA sequences
we e ela ed o no el Pseudomonas sp.- ela ed MO genes, which,
in addi ion o bu ane, ha e been linked wi h CH
4
oxida ion
(Cooley e al., 2009). By con as , P. abies-de i ed sequences,
cap u ed wi h mmoX p obes, all g ouped ei he wi h ac inobac-
e ial o p o eobac e ial PMOs. As wi h he mc A analysis, he
quali y o he sho SRA-da abase- e ie ed sequences was lowe
han he ones p oduced in he cap u e sequencing (Table 1). No
simila i ies o ‘ adi ional’ pmoA genes we e ound wi h he
cap u e app oach.
Taken oge he , ou analysis e ealed only mino indica ions o
cu en ly known, ‘ adi ional’ me hano ophs in he analyzed
coni e s. Howe e , he no el MOs, de ec ed bo h in he SRA and
in he cap u ed me agenomics da a, migh ha e he po en ial o
consume CH
4
in he ee canopies. In gene al, unde s anding o
he alkane/alkene monooxygenases is a om comple e and hei
unc ioning wi hin he ees has no been examined. Recen
analysis indica es ha PMO and MMO enzymes sha e a common
ances o bu ha e e ol ed in di e en di ec ions. Consequen ly,
only MMO and bu ane MO (BMO) seem o be capable o
b eaking he C–H bond o CH
4
(Osbo ne & Ha i os, 2019).
PMOs and BMOs can p ima ily b eak he molecule a he
seconda y C, which is es ima ed o equi e a maximum clea age
ene gy o 400 kJ mol
1
. B eaking o he C–Hbondo CH
4,
wi h
an es ima ed clea age ene gy equi emen o 431 kJ mol
1
, would
a leas be a lo less ene ge ically e icien by he PMOs han by
MMOs. Ye , we canno ule ou he possibili y o PMOs o
BMOs oxidizing CH
4
as a co-subs a e o unspeci ically, as
p e iously shown wi h BMO om Pseudomonas bu ano o ans
(Cooley e al., 2009).
E iden ly, we need a deepe unde s anding o he ee-associa ed
CH
4
consump ion mechanisms and mic obial communi ies
in ol ed. Excluding he wo alphap o eobac e ial mmoX SRA-
agmen s, ou me agenomic app oaches could no de ec me han-
o ophs ela ed o p e ious needle isola es (Do onina e al., 2004;
Iguchi e al., 2012). This likely e lec s he well-known challenge o
cul i a e single s ains om complex en i onmen al communi ies:
he a o emen ioned acul a i e me hano oph isola es likely ep-
esen s ains adap ed o highe CH
4
concen a ions (low-a ini y
oxidize s). They ha e p o en easie o g ow in he labo a o y
condi ions han high-a ini y me hano ophs – he i s s ain able
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o g ow in a mosphe ic CH
4
concen a ion was isola ed by T ei
e al. (2019). Sequence eads gained in ou s udy could ep esen so
a uncul i a ed CH
4
oxidize s adap ed o low/ ace le el concen-
a ions o CH
4
(induced by ou incuba ion wi h CH
4
a
100 ppm). I should be no ed ha , owing o he p esence o
ace a e in he g ow h media, ou incuba ion migh ha e a o ed
acul a i e me hano ophs adap ed o he use o his al e na i e C
sou ce.
Fu u e di ec ions o mo ing beyond desc ip i e
s udies
Cu en ly, we s ill need mo e esea ch e en on he p esence o CH
4
-
p oducing and consuming mic obes in he abo eg ound ee
habi a . As e iewed in his pape , he ew exis ing s udies on his
opic ha e been la gely based on ei he cul i a ion, which is biased
owa ds dis inc species h i ing in he labo a o y, o on he
sequencing o uni e sal 16S RNA genes, which lacks in o ma ion
on speci ic unc ions and he sensi i i y o he a e species. Mode n
me agenomic ools ha e he po en ial o mo e de ailed cha ac e -
iza ion o ee mic obiomes, gi ing insigh s o bo h axonomy and
unc ion. S ill, based on he p ojec desc ip ions behind he
e ie ed SRA-da abase en ies, me agenomic sequencing is s ill
mos ly a ge ing he hos ee genomes mo e han he associa ed
mic obiomes. In addi ion, owing o he la ge genome size o he
hos ee compa ed wi h he epi- and endophy es, mic obiome
sequencing h ough he egula sho gun app oach is hinde ed by a
low signal- o-noise a io (Schneide e al., 2021). In ha sense,
a ge ed cap u e me agenomics shows g ea e po en ial o unco e
e en a e mic obial genes among he plan -cell DNA, as we showed
he e o he sp uce shoo s.
Though he me agenomic ools can gene a e a as amoun o
genomic da a, linking unknown DNA agmen s o gi en unc ions
and species is limi ed by he low amoun o anno a ed e e ence
sequences/genomes in he da abases (Kaul e al., 2016; Schneide
e al., 2021). To sol e his, adi ional cul i a ion app oaches a e
s ill needed o complemen he sequencing me hods. Success ul
isola ion o he ele an mic obial s ains would allow e alua ion o
he ole o pu a i e enzymes in he CH
4
cycle, such as Pseudomonas-
ela ed MOs. Wi h pu e cul u es, ull bac e ial/a chaeal genomes
can be acqui ed, allowing he analysis o no only CH
4
me abolism
bu also o he ai s ela ed o, o example, su i al in he plan
habi a and in e ac ions wi h he hos (F ank, 2018). Genomes o
uncul i a ed o ganisms can be de i ed also h ough single-cell
me hods (Rinke e al., 2014), and by building hem om
me agenomic da a (i.e. me agenome assembled genomes; Pa ks
e al., 2017). As an al e na i e, no el genes/enzymes can be
connec ed o pa icula unc ions wi h he help o me agenomics-
based unc ional sc eening app oaches (Nga a & Zhang, 2018) and
by he use o iso ope applica ions, such as nanoscale seconda y ion
mass spec ome y and s able iso ope labeling, o hei combina-
ions (Musa e al., 2016).
08/06/18 10/06/18 30/07/18 31/07/18 01/08/18 04/08/18
−15
−10
−5
0
5
10
15
ng CH4h−1 gDW
−1
Jun Jul / Aug
(a)
(b)
01/04/20 08/04/20 15/04/20 22/04/20 29/04/20 06/05/20 13/05/20 20/05/20 27/05/20 03/06/20 10/06/20
−60
−40
−20
0
20
Da e
ng CH4h−1 gDW
−1
Fig. 3 Sp uce shoo me hane(CH
4
) luxes (median, qua ilesand in e qua ile anges) measu eda (a) Skoga yd Resea ch Ca chmen sp uce o es , Sweden, in
June–Augus 2018, and (b) Helsinki ya d saplings, Finland, in Ap il–June 2020. Shoo luxes we e measu ed using manually ope a ed anspa en shoo
chambe s, as in Machaco a e al. (2016), connec ed o an online CH
4
/CO
2
g eenhouse gas analyze (UGGA; ABB - Los Ga os Resea ch, San Jose, CA, USA). In
o al, he e we e34 sepa a e shoo lux measu emen swi h ma u e ees in Skoga yd and89 sepa a e shoo lux measu emen swi h sp uce saplings inHelsinki.
De ails o he measu emen se up and da a p ocessing a e gi en in he Suppo ing In o ma ion Me hods S1. No e he di e en y-axis scales in he wo g aphs.
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Phy ologis Viewpoin s Fo um 531