Rapid Nitrogen Fixation by Canopy Microbiome in Tropical Forest

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This article isThis article by protected copyright. All rights reserved. 10.1002/ecy.doi: d to lead copyediting, paginationbeen throughthe andproofreadingtypesetting, process,may which Accepted This article acceptedhas been for publication andundergone fullpeer review buthasnot of and leaves branches that make forest areprone canopy. upa Forest canopies to especially little about the factors that control the microbial nitrogen nitrogenBiological fixation of iscriticalforthenitrogen cycle forests tropical Abstract: RUNNINGHEAD: 55108,USA. Email:Paul, MN Phone: author: Corresponding Stanton,Daniel Lab,1479Gort 140Gortner 5 Department ofBiology, University, ColoradoState CO,USA. FortCollins, 4 Smithsonian Tropical Research Institute, Ancon, Panama Leeds, U.K. 3 SchoolofGeographyInternat andPriestley Article Paul,MN,Saint USA. 2 Department ofEcology, Evolution andBehavior,Universityof Minnesota USA. 1 Department Biology, ofEcology andEvolutionary Princeton University, Princeton NJ, Article type :Articles DR. DANIEL STANTON (Orcid ID :0000 Rapid nitrogen bycanopymicrobiome fixation forest tropical determined in byboth

ifferences between the of thisversion citeth and Version Record. Please

D. E. Stanton

2795 Tropical canopy nitrogen fixation Tropical

1,2 , S.A.Batterman phosphorus phosphorus and molybdenum - 0002 [email protected] - 6713 1,3,4 ional Centrefor Climate,of University Leeds, -

9328) , J.VonFischer

fixers thatcolonize the microbiome

and ner Laboratory,ner Saint L. O. Hedin - Twin Cities,

, yet weknow is article asis article 1

This article isThis article by protected copyright. All rights reserved. from pools(although indirect nutrient links soil throughfrom nutrient leaching may leaves Accepted canopyForest environments arepronetonutrientlimitation theybecause (1) are disconnected 2004). ha/ha) and structurallycomplex biological environments worldwide (Lowman and Rinker ~2billion (Pan cover haglobally largest surface that areas by canbecolonized canopyforest environment (phyllosphere).Tropical forest ofthe canopiesrepresentone microbiomethe area ofleaf vast (treethatmake the surfaces andepiphyte) in andbark upa about factorsthatcontroltheknow little microbialnitrogen the fi Introduction limitation, biogeochemistry, tropical canopy,forest phyllosphere Article nitrogen fixation,Keywords: biological cyanobacteria, phosphorus of nutrient significant N fluxesthe at ecosystem asbursts level,especially following atmospheric inputs rates.fixation Ac canopy cyanobacteria,but asecondary role also molybdenumof indetermining per ofphosphorusOur findingsdemonstrateaprimary role inconstrainingoverall N unavailable. We the present r here N controlling biological toleaching.subject Earlierofphosphorus have studies arole molybdenum suggested and in limitationnutrient becauseare (1) they disconnecte

Life onaleaf inthe nitrogenBiological fixation (BNF) for isnitrogen critical tropical cycling, butwe - rich dust.

onservative onservative evaluation suggests canopy that cancontributeto fixation

- fixation fixation butexperimentalrates, confirmation hasbeen hitherto

canopy canbestrikingly different from forest understory. the et al.et esults ofamanipulation of canopynutrient availability

2013) and 2013) and one possess of mostmultilayered the (5

free - living fixers inthe land biosphere: they d from pools,(2) nutrient and often soil

xers that freelywithin xers live limitation, molybdenum - fixation by - cell cell - .

15 This article isThis article by protected copyright. All rights reserved. 1998,Freiberg 1999). arepoikilohydric;(bacteria Bentleyand Fritz Carpenter 1980; forenergy photosynthesis; Carpenter 1992; Freiberg Reed 1998; phyllosphere distributed lowlandlikely factors forests, thatgovernfixation by free 2001), and where free cyanolichens lowland tohot forests where macrolichenscan be range forest”physiognomic of“tropical nitrogen forforest of One the consequence cycle. of source istheclimaticvariation and environmentaland whichlocal needed fixation factorsare totrigger rates large (>5kgha influential insignificant (<0.1kgha al. contributing tothe tropical forest nitrogen cycle (Freiberg 1998; Cusack fromdecoupled nutrient cycles soil (Hedin andnutrients atmospheric sources. Thus, canopy biogeochemical cycles arepartially Nutrientretention. inputs therefore are limite majoritythe of canopy area the surface littlephysical has structure for nutrient water or developasepiphytes structuressuch phytotelmata forexternal storage water 2016) (Zotz and mitigateconstraints these al exist, . 1996, Van Stan et al.. 1996,VanStanet 2015 butsee Runyan etal.2013

Accepted Article 2009; Sullivan

see Benner etal.2007), (2) to and oftensubject leaching byprecipitation Less clear isLess clear whyfixation rates appeartorangeso between widelywithinand forests, Microbial canopy nitrogen notably fixation, bycyanobacteria, h nutrients nutrients (Benner et al.et - 1

yr living bacteria primary are the canopyN

2014); however,2014); reported arehighly variable, values from ranging - - 1 1 ; Forman 1975). yr - 1

; e.g.Carpenter (Nadkarni et al.et

2007;

– et al

from montane withabundantfoliose forests cool

et al. et Reed d to leachates fromd toleachates access plants with the tosoil . 2004; Cardelús 1992; Reed

2009). et al. et

2013) aswellsourcelight (a of

). Canopy soils can develop ). Canopy candevelop soils locally

et al.et

- et al et Sheridan Sheridan and 1987; Portecop

- nearly absent(Lange 2008) to biogeochemically2008) to fixers. fixers. widelyIn the et al. et . 2 - 009) and some vascular living bacteria include

2008) water and as been positedas as been et al. et

2009; Hedin

(e.g. Knops (e.g. enough tobe et al. et , but

et et et et This article isThis article by protected copyright. All rights reserved. and ag Accepted approach that canovercome causedvariation natural and/orleafchemistry, bybark texture microbiomes canopies forests that inhabit the oflowlandtropical foresttropical canopies, influence forestnitrogencycle. and, byextension, the molybdenumand/or may canopynitrogen availability govern withinandacross fixation intropicfixation ofphosphorusmolybdenum andsupply may episodic up trigger intheforests AmazonCentral and basin America. newpossibility Thisraisesthe that dust like of (and phosphorus long microbiome. findings However,recent illustrate episodicthat inputs dust as leachates key sources of nutrients Article nutrient proposetissue concentrationsto thatco moist lowland communities,Reed phosphorus (Benneraddition forest, fixerstropical where aredominated bycyanolichens,fixation response in increased to (Barron and tofuelingcentral fixationthe reaction andoftentheconstraining has led to particularinterestin two elements ecosystem nutrientsforest offer isconsidered, a constraint.more universal observation This the latterthe co a - range in transportfromAfrican the continent sources e (Fritz Light an Light Addressing t Addressing These studieshave generally emphasizedcombination the and ofleaf/bark rainfall et al.

- 2009; Wurzburger 2009; Wurzburger Sheridan andPortecop 1987, Reed d water vary betweenavailability microsites, canopy entire andwhenthe al forestal Morebroadly, canopies. observationsimply these that phosphorus - factor nitrogenase inthe he role ofnutrientshe role in influencing ly molybdenum)ly areas widespread ofnutrient over et al.et et al.et

et al 2007). aCosta In Ricanforest, more characteristic of such as phosphorus ormolybdenumsuch as canopy tothe

2012; Reed . (2013) used. (2013) e nzy –

phosphorus and molybdenum andphosphorus - me limitation byP on andMomayoccur leaves. et al. et et al. et

that catalyzes the fixation the reaction catalyzes that correlations between BNF andleaf rates the cyanobacteria

2013). Because canopy

2013). In

–

can inject significantcan inject quantities -

regulation ofnitrogen nutrient nutrient in tropical forests, demands experimental an a Hawaiian montane - rich epiphytic – - poor tropical

delivered delivered via –

the formerthe

This article isThis article by protected copyright. All rights reserved. Acceptedtotal, 0.15g/m2 resin The site withis anOxisol high 3200mm) by dominated access to the canopyof0.92hahumid (Mean evergreen forest Annual Rainfall ofPanamaRepublic 9.28 (N ShermanSanas Fort Canopy Crane)in Park Lorenzo National on Caribbean the coast ofthe Site. andMaterial methods of fixation. fixation overall constraining the rate and molybdenum perthe controlling We show further this that nutrient isinteraction mechanistically complex, withphosphorus phospho isthe to study first experimentally demonstrate that fixation is toco subject Article approaches toestimatescaling ecosystem molybdenum onther experiments andevaluatedtheindividualinteractive effects ofnitrogen, and phosphorus, throughoutcolonization substrates the (15 canopy microbiomemoist ofa lowland f the ofnutrients evaluate in determiningroles growthand ofthe the activity N intoinsights community responses to nutrients. newcolonize possible surfaces,itis nutrientto use microorganisms to rapid turnoverthe adapted ofleaves are in tropical thus trees, and rapidly

Fertilization experiments at conducted SanLorenzo were the (also CanopyCrane know We results report here from an

rus rus and molybdenum canopy initially forest (as inthe proposed by Reedetal.2013).

ate of atmospheric - extractable; Cusack et al. 2018).

Brosimum utile 0172º, 140m0172º, W The provides 79.975393º, elevation). crane N (1g/m2 andlowphosphorus availability (230 g/m2 total) orest canopy. Using a 50m crane, we installed microbial canopy.Usinga50morest weinstalled crane, in

15 (Moraceae) and - - situ N fixationbymicroorganisms.We two use then wide impacts of our findings ofourfindings wide impacts more broadly. Our

manipulative allows experiment, which usto

- - 40m in two separate 6 height) infused standardized substrates to

Manilkara bidentataManilkara

- bacte - limitation by 2

- (Sapotaceae) fixing rial cell rate - month ∼

gain gain This article isThis article by protected copyright. All rights reserved. minimal.These substrates above, installedas were from 2011 to February removal for experiment1,historical were effects oflocations likely fromreuse satsubstrates the above surfacebranch allowing for nutrient runoff following substrate experimentreused from locations minimize experiment 1in to order site effect (15subcanopy used. Thesame five canopylocations inExperiment1were reused, withanadditional four TocontrolMo). forcounter 0.1M KPO pr Experiment 2:Molybdenum andphosphorusadditions: were never located directly belownutrientadditions. assigned to ofthefoureach branches tree, with per the constraint that the control treatments andJuly 2010, harvested ofeach inFebruary2011.Onesubstrate tr weresubstrates attached upperto the sideofmedium branches(7 sized 30 cm werechosen circumference thewithin midcanopy(30 species, prior to installation. Five ofcanopy height individuals mostabundant the trees locally KPO oneofthefor 12hoursin followingtreatments: purifiedwater (control),orPO vermiculite to formlongcylindersof4cm diameter.was 40cm soaked Eachsubstrate then by prepared methodsadapting the of(Benner Experiment 1:Nitrogen andPhosphorusadditions.

Acceptedepared asinExperiment 1,withthe followingtreatments: purifiedwater (control),PO Article 4

(+P), NH (+P), Brosimum utile 4

(+P), 0.0025M(+P), NaMoO - 25 m) chosentorepresent wetter,shaded branches canopy environments. We 4 +

as 0.8M CO(NH as 0.8M

(Moraceae), were chosen,four andwithin each, branche - ion effects, fouradditional substrates 0.1M with NaPO 2 ) 4 2

(+Mo) or(+Mo) 0.1MKPO (+N) or0.1MKPO(+N) et al.

2007).

Epiphyte growthsubstrates were 4

4 and 0.8MCO(NH Nylon were filled stockings with

and 0.0025MNaMoO Epiphyte growth substrates were growthwere Epiphyte substrates - 40 m ground). the The above eatment wasrandomly - 10 cm in diameter)

September 2011. 2 ) 2 4 s. Because the the s. Because

− (+P +(+P N)

s ofapprox. as 0.1M as 0.1M 4 (+P + (+P 4

were were 4 −

This article isThis article by protected copyright. All rights reserved. SoilInstitute Laboratory usingacoupled inPanama Flash City, Panama, HT Elemental Acceptedpre usingcarefully scraped from surface afineblade the and material (~1 oven individualdrying paper for 72hoursin envelopes. Once biofilmsdried, the were incubation. PARTemperatures the within remained lamps. bottles constant to33ºC) (30 during the than rather determine background 99 % wassuspendedand wetted. frominside This strip asealed100ml andml ahook bottle 25 vermiculite. substrate a50cmFor each fromretrieved open thecanopy, slit andgentlyrinsed purified in toremove water the measured nitr Article assays: fixation Nitrogen occurs.fixation aswell as algae, to countfrequencycells the ofoccurrenceheterocyst where nitrogen examined acompound under microscope toc using grid a light table. threeAt least subsamples taken were from growthsubstrate and each microscope.dissecting Coverofcyanobacteria on andalgaewas assessed a 15cm cm x20 vermiculite offlecks. Individual mossethalli liverworts, vermiculitethe wasremoved andthe lightlynylon surfaces rinsed in watertap to remove processingSample - weighed aluminium Samples tins. a were

15N After thestrips incubation removed were from th 2

15 gas N ogen fixation on rates the from substrates experiment 2.Growthsubstrates were 2

was injected intowas injected tocreate each20% bottle a N labelled

gas. All weregas. All bottles for underincubated constant 12hours illumination with : Substrates were after~6monthsharvested ofgrowth.the In laboratory, 15 N levels, duplicate stripsN levels, alsobut were prepared, in

In addition evaluation ofandheterocyst tothe cover counts, we 2

rectangle was cut from the area of from growt wascut thearea densest rectangle nalysed Smithsonian the in Tropical Research onfirm presence ofcyanobacteria the and/or s and ferns werecountedusings andferns a e bottles andat50ºCe bottles dried ina

headspace. Toheadspace. - 3 mg) was sealed 3 mg)sealed in was jected with air h

of This article isThis article by protected copyright. All rights reserved. Acceptedp <0.0002,Fig1A;Mass ratesincreased whether expressed on ora an area mass (Area basis, Fixation rates inthe controland+Mo treatments low,whereas +P were the and+P+Mo by ordersincreasing ofmagnitude several with theaddi Results: of normality andheteroscedasticity. arcsine scores andin Criterion all cases the simple Covermodels werepreferred. linear datawere position as random were compared Akaike the variables models to using linear Information rate).fixation Linear mixe predictorsinteracting were appliedtoeachofthevariablesresponse (cover,heterocyst count, Data analysis Article indistinguish ofthesubstratewerevalues (~ low contaminating thebiological material withfibers from nylonsubstrate;however, the theδ mean ofu value where δ ofProportion Nfixed =(δ end andAnalyzer Thermo Delta VAdvantage IRMS. Scientific - membermixing model withnodiscrimination onfixation: - To calculate the proportionofTo calculate the sampleNfixedtheincubations,w during Biological nitrogenBiological fixation rateswere greatly enhanced bynutrient additions, 15 square roo

N air able fromable under an

was calculated fora20%was calculated : All data: All were analyzed models R.Linear using Mo with (or N)andPas nlabeled samples ( t transformed data andfixation werelog - normalized: F 15 d effects models(packagenlme) including tree andcanopy N sample - estimate rates. fixation of

- - – 1.4 ‰,n=3).Greatcare towas taken avoid 1 make ‰,n= 3), which wouldcontamination nylon

δ 15 15 N 3,10 N control 2

= headspace (53,570 δ ‰)and

8.07, p=0.005). )/(δ 15 N air

tion (Fig.of alone phosphorus 1). –

- 15 transformed meet to assumptions

N control

) -

normalized: F 15 N control e assume a2

is from the 3,10 =18.77, 15 N This article isThis article by protected copyright. All rights reserved. Fürnkranz Acceptedsimilar tothe nitrogen coveredIn contrast, +P treatments were inathickmat offilamentous cyanobacteria (Fig 3) recoverable from with substrates measure even the which to fixation rates 2mg(< in5 filamentous Incontrol andmolybdenum cyanobacteria. there was treatments material little se vascular recoveredorganisms were from substrates =and Mo(p 0.67). frequencyheterocyst withMoaddition (p ofabundance was heterocysts. Incontrast, there a non Phosphorus significantlyaddition (p< and 0.0003) (~50%) increasedrelative greatly the significantly infrequencytreatments between (F Heterocysts were present ason control asnutrient well Article ofabundance heterocysts potentialeither fixationactivity ormicrobial abundance(cha nitrogenbiological fixation) aper or expressed as greaterratesonaper treatments additionofmolybdenum the increased efficiency the substantially of fixation, forfixation inwas atendency presence higher the ofelevatedmolybdenum. in+P However, rate fixation higherwas notstatistically in +Mothe +Pvs.t effectinteractive and nitrogenfixers. Thepermolybdenum onbacterial ofphosphorus Moreover, our show fixationMoreover, results thatthe overall response emerged an from Microbial Microbial community varied also cover greatly treatments.between range of Awide We onfixation decomposed addition into further impact effects ofnutrient on the et al. edlings (

2008). colonization was Fixer approximated bya

Peperomia - fixing communities ontropical forest provides a a provides p

sp.) sp.) - heterocyst – -

mass (Fig. basis nitrogen 2). the majoritythe of were surfaces colonized dominated by roxy

= 0.15), nosignificant and betweeninteractions P – for

bacteria, bryophytes, fernprotonemata andfew

(non

maximum fixation potential) rates. (or 3,36 - photosynthetic cells

= 13.22, p <0.0001; Fig. 1B). - - significant trend towards decreased enriched substrates, butvaried

racterized asracterized cover). Therelative leav reatment 1),but (Fig. there es (Freiberg 1998,1999;

ssessing of arealextent the specialized in 0 cm - area 2 ). This article isThis article by protected copyright. All rights reserved. Acceptedcontrasts with common forests the of characterization tropical generally as “nitroge ecologicallybeing important epiphytic throughout the canopy environment. Thisobservation ofregulation het accompanied consistent bythe productionofheterocysts and down a trend towards nitrogen suggesting a substrates, molybdenum. Cyanobacteria were the predominant organism ourexperimental colonizing foresttropical canopies by phosphorusavailability,also but byanadditional interaction with Discussion: and molybdenum. the ofcountereffect and choice wasweak, frequencyheterocyst of the +Ptreatment the but magnitude (F=13.93,p0.0013), of the ArticleSimilarly, the presence of ofNaasacounter Kinstead (Fhowever, effectconsidered =5.438,p0.03); the much thanthat ofKwas ofP. smaller but signi phosphorus 0.807) (p = had anyeffectoncyanobacterialcover 4B). wasa (Fig. There weak al. et Contrary to our exp toin contrast additionof the which hadanegative nitrogen, impact 0.0243,Fig.4A). (p= approximately doubled the extentcolonization ofcyanobacterial area surface within6monthscolonized in Experiment25.26, p<0.0001; 2:F cover,whichrespondedcyanobacterial strongly totreatments nutrient (Experiment 1:F

2012), neither molybdenum northeinteraction= 0.811) (p ofmolybdenum and Our results show Our results strong evidence - ficant effect positive acounter ofK as fixing organisms tropical inthese dominancecanopies. This fixers, bynitrogen

erocyst in nitrogencover response to addition, poin all

ectations from leaf litter st nearby high potential forcoloniza 3,36

= 9.45,p< 0.0001),withupto90% of available the for of regulation microbial fixers within nitrogen +P treatments of 4).Theaddition phosphorus (Fig. - ion did not bias theion didnotbias effects strong ofphosphorus - ion on cover when only+Ptreatments were tion u

- dies (Barron of available surfaces byfree ion significantly increa

(p < 0.001,(p < Fig.4Aand4B), et al. t to

2009; Wurzburger

nitrogen limitationnitrogen sed the - n - living - rich”; rich”; 3,31

= This article isThis article by protected copyright. All rights reserved. Accepted phosphorus. increasedthis efficiency growth necessarilytranslate into does not the of in absence nutrientmolybdenum responsesuggests that this influences fixationefficiency,nitrogen but growth that isnotresolved alleviation through of nitrogen limitationbutalsoamore general constraint onfixer biomass The structure. phosphorus response suggests that phosphorus effects solely donot arise nitrogenase phosphorusenzyme, is whereas Molybdenum haslittle biological importance outside ofits asaco role mechanisms ofphosphorus andmolybdenum onfixersandfixationrates. aslimitingnutrients or per efficiency ofnitrogen Articlefou trend towards decreasing investmentheterocysts incyanobacteria 1b). (Fig. Interestingly, we molybdenum alonehad little effect 4) ontotal cyanobacteriacover(Fig andinfactshowed a (Fig. frequecover 4) and The impact ofP microbial wasprimarily throughgrowth, addition oftotal increased both different responsesandappeared to influence different fixation through quite mechanisms. whole(Hedinas a nitrogencontinued canopyenvironmentthe appears tobe from decoupled conditions,favoring nutrient soil nd a highly significantnd ahighly impact ofmolybdenum waslimited that enhancing the to - mass basis 2). nitrogen (Fig. Although both phosphorusandmolAlthough both We differences that these propose reflect the

et al. et - fixing despite highstatus activity the ofsoils ecosystem nitrogen and the - fixation in phosphorus

2009). ncy of costly heterocysts (Fig. 1b). ncy ofcostlyheterocysts (Fig. Inconstrast, the addition of

by increased nitrogen availability In alone. contrast, the

ybdenum constrained fixation, theyelicitedybdenum quite constrained

essential tometabolicactivity andcellular - rich conditions, expressed onrich conditions,expressed aper very different biochemical roles and

- factor in factor the - heterocyst This article isThis article by protected copyright. All rights reserved. Accepted compared andsoil litter in same to the (Reedal. forests et 2013). inwhich molybdenuminputs relative constrained isstrongly when to phosphorus, even stoichiometry tissue,wecanestimate ofleaf communities that epiphyte experience nutri ecologically Byassuming significantrates. the P:Mo leachate that P:Mo mirrorsthe molybdenumknown aboutwhether belostcan also from inthe tissue at plant canopy that leachingfromrecognized phosphorus, butlesscan deliver tissuesofhostplants the is afeedback andindicating nutrientstatus betweensoil epiphyte inthatsystem. iswell It toexpe responded betweencoupling nutrientsand soil canopy montane BNF; tropical cyanolichen fixation rates phosphorus and molybdenum. from planttissuesmaysome Leaching host of degree provide interactively nitrogen govern fixation. Article alterunlikely identification to our of phosphorus and molybdenumthat factors as community development. Theseare for important directions future investigation, buttheyare development, whichmay ormay differ not fromleaves and bark with microbialthe experiment community. Yet,our waslimitedmonths to 6 ofcommunity rates.fixation our Asfor leaves, new substrates were colonized isdifficultit for toseparatethis effect us ofsimple from up that fixationmaynitrogen beencaused have bydifferences ininitialcommunity composition, but Bernecke artificial substrate inother tropical forests (Sanders andLücking2002, consistentcyanobacteria, withreports(Freiberg from 1999) use and the of phyllospheres Th Our results raisethef Our results r - e microbialcommunities inall treatments dominated were byfilamentous Lücking ofthe 2005).Part effect nutrient and oncyanobacteriacolonization rimental manipulations oftree phosphorus content undamental question of where the undamental ofwherethe canopy microbiome question obtains

- de novo regulation of

(Benner etal.2007),

more advanced

by rapid dispersalof by rapid growth and growth and - ent ent This article isThis article by protected copyright. All rights reserved. Accepted of~1inputs forfertilized rates a even few weeks(5 S1) that any withaevent of quantity inputsufficient dust stimulate to fixation tofully area surface by covered microbes andtall tropical We indense forests. estimated (Appendix 2.4by roughly g ha biomasson the intercepteddust bythe availablecanopy wouldbe for assimilationbycyanobacteria. Based inputfromdust phosphorus possibility usingcalculations: Fi two forest nitrogenthe cycle across large ofneotropical forests.areas We this explored molybdenum offers amechanism bywhichepisodesdeposition ofAfrican dust could impact Article molybdenum additions. incanopyfixation,similarsudden bursts those to inourphosphorus and observed Westrichexperiment (e.g. phosphorus and molybdenumfollowing stoichiometric ratios similartotho and/or South to Central America, cancausemany ofquantities rock Atlanticalgae in the tropical ocean(Bristow et al. 2010).Theseevents, inwhichlarge (Okin molybdenum - 1 , which, based on , which,based our fertilizationexperiment,could increase canopy nitrogen fixation et al.et We next explored whether asecond We explored next mechanism The up

- 2004, Yu et al. 2015) 2004, Yuetal. 5 kgha - rich atm rich - - - regulation of fixation nitrogen that 15 gNha t o - - derived by istransported from dust air phosphorus phosphorus ratio dustwe estimate that deposition 700could support - 1

y ospheric particles tothecanopyduringospheric African - 1

1 day assuming microbial moderate ofavailable cover(~30% area). et al. et - rich regions of Africa (7 - 1 (1 2016, Yu –

- could increasedfixation, trigger ashas suggestedfor been 5 kgNha rst, weusedrst, Yu - 10% ofthe issufficien year) et al. et - 1 yr 2015). events cancause likely Such dust - fold increases inatmospheric inputs of - we observed inresponsephosphorus to and 1). Second, theextensi weconsidered – et al et 39 gPha –

the deliveryofthe and phosphorus .’s (2015) 7 - 1

yr - 1 t to producet to nitrogen ) and ) and assumed most that - - - rich areas ofthe rich areas Sahara derived “dustevents” yr average measureyr average of se used in in se used our

- 3900

ve This article isThis article by protected copyright. All rights reserved. Accepted bysupported aPrinceton University President's Award. draft first DS wrotethe of manuscript, all the authors and contributed to DSwasrevisions. Acknowledgments: regional cycle) and transport)(i.e., phosphorus dust and molybdenum cycles. associatedfixation with butalsomediatesplants), coupling the (i.e., oflocal nutrient forest microbiome only not partially is decoupledfrom conditions symbiot (unlike soil componentcrucial oftropicalforest biogeochemist hig surfaceextensive ofcolonizable area canopiesin tropical forest means transiently even that phosphorus fertilization, in withenhanced rates the presenceofmolybdenum. added The amicrobialhouses commu dynamicand highly forest of tropical component ecosystems. Thecanopy environment Articleevents. thecanopy fixationin n origin (Yu phosphorus/molybdenum deposition causedepisodic inputs bythe ofdust ofwestern African apotentialfindings pointto tel symbiotic over fixation decadal time (Batterman scales al.2013;Sullivan et (Barron aresignificantvalues compared other measures to ofnitr h nitrogenrates fixation micro bycanopy

While our calculationsWhile our arepreliminary, arriveatsimilar Moreover, both values. these

DS and LH designed DSthe study,DSsetupexperiment andLHdesigned et al. et al et

2009), atmospheric deposition2009), atmospheric (Cleveland Our findings offer new evidence ofthe microbiomeOur findingsoffer canopy animportant newevidence as . 2015). studies Future shouldseektofurther resolve the importance of

itrogen budget of tropical forests, and its responsiveness to dustitrogen budgetof andresponsivenessto tropical forests, its nity that that is able nity to rapidly econnection betweeneconnection cycle tropicalnitrogen and the - organismsmay an represent but overlooked ry

. This biogeochemical . Thisbiogeochemical oftherole canopy e stab SAB acknowledges funding fromSAB acknowledges a et al. ogen fixation by layer inputs litter

lish

1999) and potentially1999) and even and all authors collected data. authors and all and fix nitrogen in responseand fixnitrogenin to et al. et

ic nitrogenic

2014). Our This article isThis article by protected copyright. All rights reserved. Accepted byEpiphylls in MicrobialFixation Rainforest. Ecology6:109 a Tropical B.L.Bentley, andE.J. Pseudocyphellaria crocata theFertilization Increases Activity AbundanceandNitrogenase oftheCyanolichen Benner, J.W., C symbiotic dinitrogenfixationforestNature secondarysuccession.502:224 in tropical Batterman, S.A.,Hedin Ransijn, L.O., M., vanBreugel, J., and J.S.Hall. of 2013. Keyrole Geoscience 2:42 2009. Molybdenum ofnitrogen fixa asymbiotic limitation Article A.R.,Wurzburger,Barron, N.,Bellenger,J.P., Wright, Kraepiel, A.M.L.andL.OHedin. S.J., reviewers for helpful their comments. Hedinlabmembersthe of forhelpfuldi and Turner D. Agudo assistance for with isotopeWurzburger N. sampleand analyses, Wagner, withK. canopyaccess andE.Sanchezforassistance andexperimental set Wright,like to thank J. O.N. Salazar, Aceve and NE/N012542/1) British the (award(NE/M019497/1; Council 275556724). AcademicLeeds University Natural Environment the Fellowship, Council Research Carbon Mitigation young Initiative investig onroy, S.,Lunch,C.K.,Toyoda, 2007. N.andP.M.Vitousek. Phosphorus – 45.

Carpenter. 1980. Effects andRehydrationon Nitrogen ofDesiccation

in Hawaiian Montane Biotropicain Hawaiian Forests. 39: 400

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This article isThis article by protected copyright. All rights reserved. treatments. Phosphorus significantlyaddition increasedboth densi heterocyst Accepted cyanobacterialsubaerial communities fromamended Mo with substrates x Paddition 1.ResponsesFigure oflog Data are available fromRepository: DryadDigital the Amazon the Rainforest. ResearchLettersGeophysical 42:1984 from Cloud ofAfricanrole in dust the multiyear Amazon Afirst rainforest: assessment ondata based Yu, 8:988 Ecohydrology DYNAMICS. maritimetwo TILLANDSIA oakforest settings: WATER USNEOIDES AND LEACHATE Article usneoidesTillandsia T.Bittar,Van Stan,J.T.,A.Stubbins, J.S. ONE 7:e33710. NitrogenPhosphorus toConstrainAsymbiotic FixationInteract inTropical PLoS Forests. Wurzburger, N., Bellenger,J.P.,Kraepiel,A.M.L. 2012.Molybdenum andL.O.Hedin. Proceedings oftheNational Academyof Scien 2016. Saharandustnutrientspromote W.M., J.R.,Westrich, Ebling,A.M.,Landing,J.L., Joyner, Kemp, Griffin, K.M., D.W., H., Chin, M.,Yuan,T.,Bian, H.,Remer,H., Chin, L.A., Prospero, J.M., - Aerosol Lidar andInfraredAerosol PathfinderSatelliteObservations: D

(L.) L. (Spanish moss) storageandleachatecharacteristics L.(Spanish (L.) water from - transformed fixation rates (A)and heterocyst density (B) of – D 1004. ATA ATA Vibrio

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