Functional Complementarity of Ancient Plant-Fungal Mutualisms

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This is a repository copy of Functional complementarity of ancient plant-fungal mutualisms: contrasting nitrogen, phosphorus and carbon exchanges between Mucoromycotina and Glomeromycotina fungal symbionts of liverworts.

White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/144418/

Version: Accepted Version

Article: Field, K.J., Bidartondo, M.I., Rimington, W.R. et al. (6 more authors) (2019) Functional complementarity of ancient plant-fungal mutualisms: contrasting nitrogen, phosphorus and carbon exchanges between Mucoromycotina and Glomeromycotina fungal symbionts of liverworts. New Phytologist. ISSN 0028-646X https://doi.org/10.1111/nph.15819

This is the peer reviewed version of the following article: Field, K. J., Bidartondo, M. I., Rimington, W. R., Hoysted, G. A., Beerling, D. J., Cameron, D. D., Duckett, J. G., Leake, J. R. and Pressel, S. (2019), Functional complementarity of ancient plant‐ fungal mutualisms: contrasting nitrogen, phosphorus and carbon exchanges between Mucoromycotina and Glomeromycotina fungal symbionts of liverworts. New Phytol., which has been published in final form at https://doi.org/10.1111/nph.15819. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.

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[email protected] https://eprints.whiterose.ac.uk/ London, 2AZ,UK SW7 S10 2TN,UK; UK Author ORCIDiD: Road,SW7 5BD, London, UK Leeds, LS2 9JT, UK;Leeds,LS2 9JT, 1 Grace Hoysted - - Hoysted Grace This article isThis article by protected copyright. All rights reserved. 10.1111/nph.15819 doi: thisarticle differences as to lead between the of version cite this and Record.Please Version copyediting, pagination the been through andproofreadingtypesetting, may process, which This article acceptedhas been forpublication andundergone full peer review buthasnot - Rimington William - Bidartondo Martin - Field Katie Regular - Manuscript type Article : JAYNEDR FIELD ID (Orcid KATIE :0000-0002-5196-2360) David Beerling - David Beerling Duncan Cameron - - Cameron Duncan Jeffrey Duckett - - Duckett Jeffrey Silvia Pressel - - Pressel Silvia - Leake Jonathan Katie Field J. Centre for Plant Sciences, Faculty Biological Universityfor PlantCentre of Leeds, of Sciences, Sciences, David Beerling J. Functional complementarity of ancient plant-fungal mutualisms:contrastingFunctionalcomplementarity plant-fungal ancient of Accepted Articlenitrogen ; ; 4 DepartmentUniversitySheffield, AnimalPlantof of and Sciences, Sheffield, , 0000 phosphorus and carbon exchanges between exchanges carbon and phosphorus 1 0000 *, Martin Bidartondo I. *, 5

Department of LifeDepartmentof Natural Sciences, HistoryMuseum,Cromwell 0000 0000 0000 Glomeromycotina fungalGlomeromycotinasymbionts of liverworts - 0000 0002 0000 0000 4 0000 -0001- , Duncan D. Cameron Duncan D. ,

- - - 2 0002 0001 0003 - - 0001 Departmentof LifeSciences, Imperial London, College 5196 - - ; - 0002 0003 0002 3 9652 Jodrell Laboratory, Royal Botanic Gardens, Kew,3DS,Gardens, TW9 JodrellRoyal Laboratory, Botanic - - - 3469 7101 1869 - - 8364 2360 - - - 5439 3172 0813 - 6338 - - - 2549 6673 4314 -

7616 - - - 6544 3036 9950

Silvia Pressel

2,3

William R. Rimington William 4 , Jeffrey G. Duckett Jeffrey ,

5 * * Mucoromycotina Mucoromycotina 2,3 5 , Jonathan , R.Leake ,5 , GraceHoysted A. and 4 1 ,

Accepted Article    Liverworts, which are amongst the earliest-diverg the amongst are which Liverworts, We tested this hypothesis by hypothesis this tested We Only Mucoromycotina fungal partners provid partners fungal Mucoromycotina Only which which ecosystem pioneers ecosystem forming Glomeromycotina Glomeromycotina forming diver complementarily improve improve complementarily algal fungi was traced using using traced was fungi 15 by atmosphere a modern under partnerships fungi. to symbiotic allocation photosynthate and greatest use pool ofnutrient complementarity showed 33 N- P uptake, uptake, P early land plants. Access to soil nutrients via fungal partners was investigated with investigated was partners via fungal nutrients to soil Access plants. land early labelled algal necromass gent 15 co N, irrespective of atmospheric CO atmospheric of irrespective N, -evolved with early land plants land early with -evolved plants, harbour harbour plants, but Glomeromycotina were were Glomeromycotina , often form nutritional mutualisms with arbuscular mycorrhiza- with arbuscular mutualisms nutritional form often 14 CO both plant and 2

. gr and

fungal groups, suggesting suggesting groups, fungal fine root endophyte Mucoromycotina Mucoromycotina root endophyte fine access to different soil nutrients soil different to access owing liverworts in single and single in liverworts owing 33 P orthophosphate . often Some liverworts, in common with manylater- with common in Some liverworts, 2 concentration and ed more effective under under liverworts with substantial access to to access substantial with liverworts ent

plant lineages and important 1500 ppm . Photosynthate allocation to to allocation Photosynthate . the Both symbionts increased increased symbionts Both . se Dual partnerships partnerships Dual dual

. fungi [ CO

fungal 2 fungi may ] , as experienced experienced , , both of

t al., et (Bidartondo lycophytes and hornworts thalloids), and (Haplomitriopsida liverworts i.e. symbionts, fungal supporting phylogeny plant land the of branches extant divergent between soil fungi and most land plant phyla (Wang & Qiu, & (Wang phyla plant land most and fungi soil between are mycorrhizas) lack as that plants in to associations mycorrhizal-like (and referred associations Mycorrhizal also henceforth roots, Introduction Glomeromycotina-like fungal associations in some of the first vascular plants plants vascular first the the of some in associations fungal Glomeromycotina-like of fungi symbiotic understood, poorly and Mucoromycotina. studied, less much the fungal ancient two of and Glomeromycotina, the within falling members fungi (AM) mycorrhizal arbuscular lineages, with symbioses form to shown been recently T 2008). Read, & (Smith health and nutrition plant in roles key with exchange,nitrogen, orthophosphate, fine rootendophyte, carbon-for-nutrient Symbiosis, arbuscular mycorrhiza, Key words: This article isThis article by protected copyright. All rights reserved. early Devonian (Strullu-Derrien (Strullu-Derrien Devonian early engagement with both fungal partners might have been a common strategy during during strategy common a been have Acceptedmight partners fungal both with engagement Article 

01 Desirò 2011; These findings, together with fossil evidence of Mucoromycotina-like and and Mucoromycotina-like of evidence fossil with together findings, These the evolution of land plants. plants. land of evolution the Glomeromycotina and Mucoromycotina fungal groups that form that symbiose groups fungal and Mucoromycotina Glomeromycotina of biology the functional into new insights providing tested, symbioses plants We show there are important functional differences between between differences functional important are there show . This may explain may. This explain

t al., et 03 Rimington 2013; the persistence of both fungal lineages in symbioses across across symbioses in lineages fungal both of persistence et al., et CO

2014) 2 , liverworts , have led to the novel hypothesis that that hypothesis novel the to led have , t al., et 05 Field 2015; near 200 -ubiquitous mutualisms mutualisms -ubiquitous the 6; Wang 6; t al., et

plant -fungal -fungal 2015a) et al., et he s with with earliest- ,

of 2010 have the the ) ) Derrien both (Strullu- past ancient the with from persisted has symbioses Glomeromycotina and Mucoromycotina form to plants of ability the that suggest discoveries these day 2017a Glomeromycotina fungi (Field fungi Glomeromycotina such as species in the genus genus the in species as such plants. flowering for established recently 2016; Rimington Rimington 2016; 2016; Pressel 2016; (Rimington lycophyte one only in far so reported been have symbioses Haplomitrium This article isThis article by protected copyright. All rights reserved. (Field evolution plant land early lycophytes, hornworts and (thalloid) liverworts (e.g. the complex thalloid thalloid complex so- in simultaneously the (e.g. liverworts mirabilis Neohodgsonia (thalloid) and hornworts lycophytes, nohts FE, rgnly lsiid as classified originally root fine (FRE), widespread globally endophytes the that finding recent the with consistent is This evidence reveals the same Mucoromycotina fungal symbionts are shared by by shared are symbionts early-diverg liverworts, fungal Mucoromycotina same the reveals evidence 2017a Planticonsortium tenue tenue Planticonsortium (Orchard (Orchard em o las oocr ih lmrmctn i te ae ot (Orchard host same the in Glomeromycotina with co-occur always to seem Accepted Article assumed hitherto as Glomeromycotina , being now common in both natural and agricultural ecosystems (Orchard (Orchard ecosystems agricultural and natural both in common now being 21b. 2017b). ; ; 2017b; Hoysted Hoysted 2017b; ; et al., et et al., and 2014; Field 2014; et al., et 2017a; 2017b)2017a; t al., et The Treubia

called ‘dual’ symbiosis (Desirò symbiosis ‘dual’ called 2016 paet essec o smiss ih oh ugl groups fungal both with symbiosis of persistence apparent ent 05 21; Pressel 2016; 2015; ) engage in partnerships with both fungi, sometimes sometimes fungi, both with partnerships in engage ) aclr lns n agoprs (Hoysted angiosperms and plants vascular . The majority of early-divergent mycorrhizal plant clades, clades, plant mycorrhizal early-divergent of majority The . et al., et ) and in the earliest-divergent Haplomitriopsida liverworts liverworts Haplomitriopsida earliest-divergent the in and ) (Walker (Walker t al., et et al., et . In angiosperms Mucoromycotina-FRE, when present, present, when Mucoromycotina-FRE, angiosperms In

2015a; Marchantia

t al., et 2015a; Rimington 2015a; 2019 t al., et 2015b; Field & Pressel, 2018) to the present present the to 2018) Pressel, & Field 2015b; , but instead fall within the Mucoromycotina Mucoromycotina the within fall instead but ), while exclusive Mucoromycotina-FRE Mucoromycotina-FRE exclusive while ), 05) Furthermore 2015a).

Only a few complex thalloid liverworts, liverworts, thalloid complex few a Only appear to associate exclusively with with exclusively associate to appear 2018) t al., et Glomus tenue tenue Glomus ) , 2016), paralleling the situation situation the paralleling 2016), et al., et r nt ebr o the of members not are et al., et 2013; Field 2013; 2018). ,

mr recently (more Taken together, together, Taken new et al., et t al., et et al., et molecular molecular 2015a; 2015a;

2015; 2019 t al., et et al., et ) .

experimental simulations of the largethe atmospheric CO in experimentalchanges simulationsof inresponse to types ofsymbiotic revealed were partnership these three theySignificant between functionalnutrients haveacquired from soil. differences mineral withthe like fungi photosynthateinexchangemutualisms receiving plant for 33 to their hosts undera[CO their hosts theto modern receivedat Liverworts associating only with Glomeromycotinafungi onlywith gain Liverworts associating (Field a greatersymbiosiscarbon cost at in but fungi dual occurrence ofGlomeromycotina a[CO (1,500 ppm firstevolved landunder which Paleozoic plants In contrast,MucoromycotinaIn fungal symbionts Lenton experienced during the long co-evolutionary history of plantsandof mycorrhizal fungi history experienced duringthelong co-evolutionary groups (Fieldgroups These Pgains liverwort undermodern a[CO Mucoromycotina (Field to test this hypothesis. test this hypothesis. to and evidence limited is there date to However, groups. Glomeromycotina fungal these of other or one only both with with associations over associations advantages functional provides likely form fungi Mucoromycotina to ability the suggests today lineages plant diverse in simultaneously them of occurrence widespread the This article isThis article by protected copyright. All rights reserved. 500 the through

Acceptedper unitphotosynthate of viafungal their partners tracer orthophosphate P Article et al., et Studies on liverwort-GlomeromycotinaStudies (Humphreys on et al., et CO

2016)

2018 et al., et 2 concentrations simulating the atmosphere of the early-mid concentrations early-mid simulating the atmosphere of the my . . ) 2016) haveconfirm -long evolutionary history of land plants (Morris plants land of history evolutionary -long than a[CO modern-day under et al., et 2015a; anddualwith both fungal 2015b) symbioses 2 ] ed comparedthePaleozoic to simulated a[CO thatare mycorrhiza-all three partnerships 2 ] were] further co- by the enhanced , maintained orincreased 2 ] of 440 ppm (Fieldof ] 440ppm et al.,et ed considerably more considerably more 2010), liverwort- 2010), 2 2 ]; Berner2006 ]; concentrations concentrations et al., et et al., et 33 P transfer P transfer 2018) and and 2018) 2016). C ; 2 . ]

dual symbioses between FRE and Glomeromycotina fungi (Rimington (Rimington FRE fungi Glomeromycotina between and dual symbioses al., Thirkell &Fitter, 2010;2015; Bücking(Hodge 2015;clearHodge &Kafle, &Storer, groups of fungi, either singly or in dual symbiosis, to utilisein singlyto soilfungi, dualgroups of or nutrients withdual symbiosis, either differencesbe liverwortsto expect in abilitythere associatedwith the of the two P Mucoromycotina do not amplify arbuscularcharacterize DNA mycorrhizal from symbionts usedMucoromycotinaprimers as mostdue undetected to havebeen to may fungi to achieveto as it hassometimes2015), (Lindhal &Tunlid, beenpossiblefree-living saprotrophs organic fornutrients from and matter assimilate useinmetabolism asimilarwayto in fungi are functionally distinct with respect to their provisioning of host plants withto theirrespect plants provisioning distinct are functionally host fungi of Mucoromycotinaurgency oftheneedresolve whether to the andGlomeromycotina Orchard Orchard This article isThis article by protected copyright. All rights reserved. (Smith is well established Read, 2008) (Smith& saprotrophic by organic extracellularnutrients assimilate of matter digestion unableconsideredto organicand plantsymbiosis thus for carbon withaliving obligateentirelycontrast,Glomeromycotinastrict on In reliant fungiare biotrophs, access organic N than the Glomeromycotina. Glomeromycotina. the access organic Nthan

Acceptedand Article

2016 N Mucoromycotina symbionts may be facultative saprotrophs, i.e. able access Mucoromycotina i.e. to be facultativesaprotrophs, may symbionts Whilst the importance of Glomeromycotina AM symbioses inGlomeromycotinaWhilstthe importance of plantP nutrition AM , ) et al., . both of which are major macronutrients that are often plant growth limiting.are which plantmajor of often both growth macronutrients are that in vitro vitro in This ambiguity has increased with the recentevidenceThiswidespread hasincreasedwith the of ambiguity ; it ispossible ablethe Mucoromycotina bebetter that may therefore to

2017a DNA isolationthese fungi and (Field of axenic culturing ) (Bidartondo . Nutritionalpreviously Glomeromycotina effects ascribed to et al., et 2015), their role in host plant N nutrition is muchinN nutritionis rolehost plantlesstheir 2015), et al et ., 2011). This uncertainty., 2011). increasesmajor If this were might the case, thenwe et al., et et al., et 2015b). 2015b). 2015, of both et green alga This article isThis article by protected copyright. All rights reserved. ( single naturally support associations to ofand organicNcompared only type to fungus. with one through inorganic providingincreasingsymbioses access bothto complementarity P paleacea provided as provided as the fungal phosphorus with wasinvestigated via Access soil partners to nutrients mycorrhizosphere soilcontainingnative on non-sterile microbial communities. Mucoromycotina partnerships, andGlomeromycotina)

Lenton 2006;(Berner, cyclemodels carbon by geochemical indicated as Devonianperiods previously relationtoPassimilation (Field in a[CO is changesinfluenced bythe inallocationfungal externalmycelium of to symbionts, andphotosynthate liverwort soil, of functioningin uptakefrom terms fungal nutrient the 1500 ppm a[CO the 1500ppm Acceptedhyphae by fungal couldbeaccessed that compartments addedrhizoid-excluding soil to aquatic terrestrial habitats(Edwards to Article withlandis likely tohave plantswhen transitioned co-existed from theyfirst from 2 ] thatthroughwe shown ] haveoccurred 2006), have Palaeozoic (Berner, as We set out to test this hypothesis by growing wild-collected liverworts, that liverworts, outwild-collected We set by growing totestthis hypothesis We conducted these experiments under both a modern 440 ppm a[CO a modernunder 440 both We conductedtheseexperiments ppm et al., et gr -Glomeromycotina) or dual fungal ( or -Glomeromycotina)dual fungal owing from their plantowingpartners from Chlorella 33

2018) P orthophosphate . This. mycorrhizalthe allowedto test us that secondhypothesis 2 (Trebouxiophyceae). ] experienced by early land plants from the Ordovician/early the experiencedfrom byearlylandplants Haplomitrium and 15

N in labelled necromass of the unicellular oftheNinlabelled necromass gibbsiae et al., et .

We selected

et al., et Neohodgsoniamirabilis 2015).BothN and were Psources -Mucoromycotina; 2012;2015b). in Chlorella experimental microcosms microcosms experimental as a substrate that as asubstratethat Marchantia

–

], and ], This article isThis article by protected copyright. All rights reserved. Glomeromycotina(Field symbionts mirabilis Mucoromycotina2011)(Bidartondoal., and et fungal symbionts Haplomitrium gibbsiae materialPlant conditions and growth and Materials Methods of New Zealand in December Newof December Zealand in microorganisms andsaprotrophs damage and prevent substrate. Native surrounding liverwort inplace soil rhizoids left to was retentionthe aid of to water silica sandandpot volumeIrish 5% moss-peat (120 mm diameter (120 mm °C humidity, 15 12h, °Cday, 12 night. Humphreys Humphreys (thatexclusivelyGlomeromycotina plants with fungal symbionts Bertol. associate whichthe NaturalHistoryLondon. Museum,deposited in are of 50 of bryophytes conditionsfor usingsettings: following chamber the halflight-saturating growth chambers environment (Convironcontrolled BDR16,Conviron, Canada) in together with the base, were covered with nyloncovered withwere mesh withtogether the base, 20 mmxwith that two length, cores (85 windows 50mm 15mmdiameter) mm ingrowth of liverwort rhizoids while allowing penetration by fungal hyphae (Fig. 1 fungal(Fig. by hyphae liverwortingrowthrhizoids of allowing penetration while

Accepted Articlethallisymbioticgemmae propagatedmaintained Mexico in Veracruz, from and  Shortly after collection, the liverworts wereliverworts plantedmicrocosmsthe collection, after Shortly into directly Based on the methods of FieldBased of onthemethods mol m Perss. (known with associateboth Mucoromycotina to Perss. and et al., et -2 to s actincluding asa inoculum, fungi,associated natural mycorrhizal - 1

irradiance (Nobel, 1999; Fletcherirradiance 1999; (Nobel, , 2010 100containingdepth pots) mm pot volumeacid-washed 95% (Steph.) R.M. Schust. (known to exclusively associatewith R.M. Schust.exclusively (Steph.) (known to ) were originally collected from cool temperate cloud forests were from originallycloudcool collected forests temperate 2013 .

(for location details please see SI), vouchersfor (for location SI), details see please et al., et et al. et 2016) SouthwereIsland the collected from (2012), w et al.,et e of constructed cylindrical constructed plastic 10 µm poreprevent sizeto 10 µm

2006 Marchantia paleacea ), 70%relative), Neohodgsonia Neohodgsonia , a) , .

below-ground gassampling during insertedmesh-coveredwas andfilled windowed third core wool withglass toallow This article isThis article by protected copyright. All rights reserved. (Quirk intothe core fungal growth of hyphae encourageto core volume) mineral grains(1% basaltvolume), finely tertiary ground Both core cores werefilledacid-washed silica amixture sand(89% of with cement. placedfast-setting verticallyand Portex, centre securedtothebasalmesh UK) inthe (100 mm length, capillarytube internal fine-bore perforated, mm 1.02 diameter; microcosms,eachthese the twoof of Three coreswereinsertedinto having a (“ tested, 20 to be maintained under ambientatmospheric 20tobemaintained tested, under CO inoculum containing fungi, hyphal-associated containinghyphal-associated inoculum fungi, commencement of isotopeof commencement labelling Upon contamination.isotope to immediately prior labelling avoid label-cross to all species. microorganismsto plant andhyphal-associated saprotrophic the inthereby drip eachcabinet,providing poolof acommon share acommon tray effects.microcosms the Acrosswere to avoid three plantspecies, arranged cabinet weeksto andthechamberevery two effects, andcontentsswappedover settings wereavoidthe pots. Microcosms rotated withinto regularly positional cabinets acclimation cabinetsallow to and fungalto mycelialto networks plant within develop and anyseedlingsregularlymosses, remove 12weeks to weeded maintainedfor or ( from around from rhizoids ( ppm) andppm) 20underanelevatedCO Haplomitrium Accepted static Article A total of 40 werepreparedA totalof microcosms eachthe liverwort for species of After the acclimation period, microcosms were moved to individual drip-trays drip-trays moved the toindividual After were microcosms acclimation period, core”),ensuring hyphal connections between liverworts, ) of wild plants (10% core volume) to actto wild asanaturalof microbial ) core volume) plants(10% Marchantia

and 14 , 2 atmosphere of 1,500 ppm. atmosphere1,500 Microcosms of were ppm. one core inone was eachmicrocosm core static left C labelling. C labelling. Neohodgsonia et al., et and saprotrophic microorganisms. microorganisms. saprotrophic

2012) ) and underground axes and underground ) and nativesoil gathered 2 concentrations (440 fungi andcore The contents (Fig. contents 1b, c) andliverwort core the between the fungalsevering connectionsthe thereby hyphal In half of the microcosms for each CO microcosms halfof the In and additions1) stable coresinmicrocosms(Fig. Radio- isotopetracer to theand contentsof core. the forming between continuous hyphalprevented from liverwort host the connections cultures of cultures This article isThis article by protected copyright. All rights reserved. contents remained intact CCAP UK)andculturedin (Argyll, inner core was filled with a inner wasfilled with core The sand/basalt/soilfilled the betweenand with outer substrate. cores inner the withthe spacecores soil-filled one the of corewasinserted intointernal plastic plastic inner core remainedcore plastic inner solid-walled1b) usingasmall Fig. The funnel. intocore (seethe innerintroduced then drywere Thisgenerated sandparticles,three hours. which algae-covered 4 gof °C) (80 drying before acid-washedg sand inanoven and200 mixed wellwith for liquidrinsedlabelled re-suspended the werefiltered, withwater, medium, cultures 0.05 g algaeg 0.05 supplied as Na supplied as the periodfor thesame time the the symbiotic to fungifungi, accessible as hadaccess to and of therefore withthe rest was the necromass onlyinalgalcontact core contents, Accepted Article 33 P tracer (see Fig.(see P tracer 1a). Chlorella vulgaris and 15 NO 2800 . 3 3 This acts as a control as acontrol This wherein were acts symbionts fungal (  

98 g . A second core in eachwa in pot A second core 15 atom % 15 insitu N per pot). To prepare the Nper pot). mixture,necromass-sand N-labelled algal necromass-sand mixture(1.25% algal algae; N-labelled var. var. viridis until immediately before before untilimmediately 3N 15 N; SigmaAldrich, UK). -BBM +V medium (see SI),where+V medium-BBM NaNO 2 treatment ( treatment Chodat (CCAP 211/12) were obtained from obtained(CCAP from were Chodat 211/12) n = 10 ineach CO s rotated (“ rotated s 33

P labelling After growth in After growth the r otatedcore”), 2 condition), an condition), , ensuring the ensuring 3 was This article isThis article by protected copyright. All rights reserved. DNA fungalof Molecular associates identification Neohodgsonia liverworts maintained their previously identified fungal previously associates(Bidartondo identified liverwortsmaintained their primers NS1, NS3 and NS5 (White NS1,(White NS3 primers NS5 and reamplification The sequencedusingkit. from fungal was JumpStart the DNA samecolonies andusing the on four the Sigma primers performed persample wasand fungal DNA TA reamplification of TOPOusing cloning the kit(Invitrogen) and1990)(Smit EF3 colonised by Glomeromycotina fungi (Humphreys (Humphreys Glomeromycotinacolonised by fungi 2011; Field laboratory-grown laboratory-grown Similarly, we samples took of establishtheir nativefungal partners. to collection et al., et Biosystems). Sequences contigsusing7 (Kearse wereassembled v. Geneious into of collections andcollections samplingof Wild periods. using the Sigma JumpStart kitand universal JumpStart (White the fungal NS1using the Sigma primers kit The(QBioGene). GeneClean fungalthe II extractwas DNA the then amplified in section liverwort & thallus of (Gardes Bruns, 1993) extractionchloroform using Accepted(Rimington anduniversal using molecular primers symbionts wasperformed fungal cloning fungalSequencing the of labellingimmediately following ourisotope experiments. wereused the species alsotaken inourexperiments liverwort across eachof three Article individualsamples offive plants Additional started. representative the experiments analyses were conducted on five samples (thalli wereconductedon of analyses 2012) and identified to the level of fungal subphylum using BLAST searches of the level2012) to searches andidentified using fungal subphylum BLAST of et al et et al., et Haplomitrium , underground axes of axes underground , .,

2016;Humphreys Marchantia 2018 et al., et ). Firstly, a Firstly, ). full DNA was genomic extraction performed on a ) and and 1999) were prepared for preparedfor were plants that have previously been confirmed to be to thatconfirmed havepreviouslybeen plants Neohodgsonia . PCR products from. PCR allwere products cloned samples et al et et al., et Haplomitrium .,

1990) 2010) throughoutthe experimental 2010) DNA plants (please refer to SIto (pleasefor details plants refer and BigDye v. 3.1 (Appliedand BigDyev. et al.,et ) to confirm that the confirm three to ) analyses of oneday within 2010), immediately2010), before Marchantia and et al., et al., et and

MF621059) and MF621059) et al et Neohodgsonia described previously (Duckett previouslydescribed(Duckett scanning processedfor Wild-collected plants microscopywere as electron (SEM) confirmand to the plants Microscopy cytology of mycorrhizal status FR690120).(accession number This article isThis article by protected copyright. All rights reserved. in Rimingtonbe foundin GenBank(Altschul uploaded GenBank while from to sequences Glomeromycotina Haplomitrium gibbsiae were identical to those previouslyby Humphreys foundintheseplants those were to identical through an ethanol series, critical-point dried usingdriedcritical-point CO through anethanol series, wereplant fixed species) dehydrated each glutaraldehyde, in (10 samples 3% of orthophosphate solution( orthophosphate After 12 weeks of growth, we introduced 100 µl, carrier-free carrier-free weµl, introducedgrowth, 12weeksof 100 After exchangeandQuantification fungi betweenliverworts carbon-for-nutrient of Kv. 10 electron microscopeat Quanta scanning andviewedunderaFEI palladium-gold 390nm coated with the same time as introducing timethe same as the (Fig. 1b, Beaconsfield,At inc). core eachmicrocosm oneintoUK)Perkin soil Elmer, labelled algal necromass in pots with this treatment was removed towasinallow removed the potswith necromass labelled thistreatment algal

Accepted ArticleHaplomitrium, ., 2015, 2016). New2015,2016). sequences for –

Neohodgsonia mirabilis Glomeromycotina in in line with previous molecular and microscopy investigations (Field withpreviousandinvestigations line microscopy molecular et al., et and of thalli et al. et 1997). Further detail fungal1997).Further can method the sequencing of 33 P (2018). This(2018). methodfound Mucoromycotina colonisation specific activity 148 GBq mmol specific 148 activity GBq et al.,

33 P tracer, the plasticcore separatingPtracer, inner the Marchantia paleacea

2006) Marchantia, Haplomitrium gibbsiae

(MF . Briefly,underground. axes of 621060 and MF621061) have621060 andMF621061) been and both fungal lineagesandfungal in both 2 as a transfusion fluid, sputter as atransfusionsputter fluid,

and 33 -1 Neohodgsonia mirabilis , total 223 ng total , P-labelled H P-labelled (accession number number (accession Marchantia et al. et 2

PO paleacea 33

(2010) P 4 ;

15 N-

(specific activity 2.04 (specific activity GBq mmol This article isThis article by protected copyright. All rights reserved. eachcombinationa[CO treatment ambient(440 ppm at (Fig. 1c). For eachliverwortspecies fungal were5replicatesthere action for pots effectscontrol forthe ofmicrobialserve asto cycling a andsaprotrophic nutrient cores soil inliverwort connectionsand the between host symbiotic fungal the hyphal toseverthe werethen rotated labelled andhalfof thenon-labelled cores cores Half of the isotope- the of soil-filled part core. fungi growintothis symbiotic to three-litre gas-tight chamber and added 2 ml 10% lactic acid to 15 gas-tight 10%lacticacidto chamber andadded ml three-litre 2 Twenty- uptake. Ptherefore included for pool effects werenot calculations dilution inthe S1 (Fig. whereto wasnotincludedcores algal included necromass compared differences Pbetweenno significant intotal content were coreswherealgae (1,500 ppm a[CO (1,500 ppm regular below-ground gas samples to be taken. We then placedbe to We then each taken. gas samples regular below-ground septum(SubaSeal, to allow Sigma) cores weresealed arubber wool-filled with Glass caps. plastic werein sealedwith all cores lanolinanhydrous microcosms and allsandthe andsoilto mycorrhizal of patches fungal filled the tops colonization, a colorimetric method adapted from Leake, ( Leake, adapteda colorimetric method from Total poolspotential determined phosphorus using pooldilution and was effects. for digestedconcentrated4) were using determine total acidto sulphuric phosphorus Sub-samples of cores 18potswherealgal of Sub-samples from necromass wasincluded (Treatme access to Accepted Article fungal to access nt 33 s 1 and 3) and 18 pots where algae were not includedand1 and3) 2 wheres werenot 18pots algae (Treatments P andnoalgae; one 33 2 ]): P and noalgae. days after introducing the 1 . Fungal to access 3. No fungal access to -1 ) in a cuvette withinincuvette thechambe ) a 33 P

1988 and 33 P and exposing the algal necromass algal necromass P andexposing the 15 ) (see SI for details). There werefor details). (see SI ) N-labelled algae; 33 P or 2 ]) andsimulated Paleozoic 15 N-labelled algae; r. This resulted in l ofNa 2. pot Fungal 33 into a P tracer 14 CO 4 ), . No. 3 Fungus and Plant tissue analyses harvest Chesterfield, 3100TR,UK).Isotech, (PackardTri-Carb scintillation using liquid UK)Beaconsfield,and radioactivity 10 mlUltimaGold(PerkinElmer, the determined of theKOHwas to then transferred An aliquotperiod6 hours. (1 ml) of subsequent were homogenised inaYellowline A10Analytical Grinder (IKA, Germany) Plant tissuesand weighed. Allfreeze-dried tissues andsoils plant wereseparated, introduced into vials within the labelling chambers to trap anyremaininginto thelabelling to vialsintroduced within chambers KOHstopped2M samples was 2 ml increasing, flux gas inbelow-ground detected Chesterfield, 3100TR,UK)(Packard Tri-Carb Isotech, counting Beaconsfield,Elmer, UK) wasmeasured viascintillation before radioactivity spectrometry planttissues in ratio mass was (ng) abundance isotope determined using (Perkin Elmer, Beaconsfield, UK) andBeaconsfield, then mix (Perkin Elmer, UK) vials scintillation Carbosorb intowere containing10ml gas-evacuated injected 17 hours. ca. Gas samples for 1hourandthen corethereafter hours after everytwo available reference gases. To calculate gases. available reference reference standard was regularly andthedetector commercially calibrated to UK). Ltd., AirwasusedastheSercon (PDZ IRMS 2020, using flow acontinuous into6 xultra-clean tincapsulesweighed Ltd., UK)and 4mm out (Sercon, analysed ground respirationgroundof published equations were used (see SI) (Cameron (see used published SI) equations were This article isThis article by protected copyright. All rights reserved. 1.1 MBq Accepted Article - to 14 -plant CO . 2 Between 2 and 5 mg of homogenised, freeze-dried plantBetweentissue washomogenised, of andmg freeze-dried 2 5 being released within the chambers (Fig. 1d) thechambers releasedbeing within 15 N and and 14 CO 33 2 P , 1 ml glasswassoil 1 ml viathe of wool-filled sampled air , transfer transfer 15 N content of samples, previouslycontent ofsamples, N ed with 10 ml Permafluor 10 (Perkin with et al.,et . At the pointthe At whichthe at

2006) . To below-monitor .

14 . CO The 2

ov 15 14 er N C a subtracting the subtracting the were obtained by The equivalentanalysisaccounted algaepresent was for. when 1). contentsand core between(Treatment remained intact liverworts plant tissues from thevalues3)from where (Treatment hyphalconnections fungal to theisotopewhere bycore access microcosms fungal wasrestricted rotation pots, ensuringpots, only This controls diffusion(Treatmentnutrient cycling 2). for microbial isotopesand in of remained intact contents hyphal andcore connectionsbetween liverworts (Treatment 4) weresubtracted from the (Treatment 4) microcosmswhere fungal the free access to isotopecore rotation restricted by was (Cameron SI) equationspreviously (see published using the liverworts wasdetermined to 3100TR, Isotech, Chesterfield, UK). Total 3100TR, Chesterfield,UK). Isotech, radioactivitymeasurethe to Tri-Carb countingUK) vialiquid (Packard scintillation Beaconsfield,Emulsify wasSafe10 mlof then added(Perkin Elmer, to this solution tissue where algal necromass was not a waswhere algaltissue not necromass digests were diluted up to 10 ml digests 10ml were to total diluted usingdistilled up volume H minutes before coolingminutes before withH andclearing 365 charred inml conc. H 1 charred This article isThis article by protected copyright. All rights reserved. Accepted Article on 33 °C15 for Cambridgeblock(Grant Instruments, UK) BT5D-26L, heater in a P To determine the To determine uptake viathe fungaluptake partners et al., et 33

2006) P

values calculated from plant tissues in algae-containing planttissues in from algae-containing values calculated 33 P gained by the plantP gainedby the connectionsis viafungal intact hyphal . 2 To determinesymbiotic fungal-acquired total SO 33 P content of liverwortsand the algalof theeffectP contentof additions 4 for two hours. two then heatedto Charredsamples were hours. for , between 10-30 mg of liverwort biomassliverwort of wasbetween mg 10-30 33 dded P 2 33 O valuestissues where inplant fungal P transferred from fungi within cores P transferred from the the 2 2 to givedigestion to complete 33 P values in 2 O. plant tissues in algae- plant A 2 ml aliquot of aliquot of A 2ml 33 P in plant . Clear Theof the activity described previouslydescribed scintillation counting of countingscintillation through sampleoxidation (Packard307Sample Isotech,UK) Oxidiser, and liquid wassubstrateandthe mesh determined remaininghyphae core into substrate this in This article isThis article by protected copyright. All rights reserved. Plant- within the3- were assumptions ANOVA.Where there for meet the significantinteractionto terms werelog10 required, data Where transformed for fungal the variables Callocation. (GLM), where core rotation,a[CO (GLM), three-wayGeneral LinearAnalysis conductedwithANOVA, Model variance. was analysisAll homogeneityto checkedfor normality prior datawere varianceand of of Statistics contentsand core between remained intact. liverworts where thesubtractedcores hyphal fungal values were rotation from in connections by core connections hyphae toplants severedfrom symbiotic fungal where were cores the valuescalculated from symbiotic ineachpot, withindue to soil fungi cores published(Cameron equations previously duringwas thelabelthe liverworts that fixed labellingperiod by using plant and CO (Minitab, (Minitab, these interactions.the nature v.17 using Minitab of All werecalculated statistics Acceptedplant Article 33 15 to P uptake; and presence of algae, a[CO algae, and of presence P uptake; N uptake; presence algae,N uptake; a[CO of -fungus carbon transfer intomesh transfer cores carbon -fungus 2 content of thetheand proportionof labellingcontent of supplied the chamber PA, wayANOVA’s, additional 2

US A ). 14 C fixed by the liverworts and transferred via the external fungal externalviaCfixedthe fungal liverwortsand bythe transferred

. Total function carbonwasthen asa calculated the of volume 14 CO 2 trapped in 10 ml Carbosorb and 10 ml Permafluor as Permafluor and10ml trappedml Carbosorb in10 et al., 2 ], and symbiont werethe variables identity for

2008) -way ANOVAs were conducted to understand to ANOVAs conducted were -way 2 ], .

and identity symbiont werevariables for To determine total plant-fixedtotal To determine carbon 2 ], and fungal identitywere symbiont 14 CO 2

This article isThis article by protected copyright. All rights reserved. analyses Microscopy the confirmed 2) (Fig. methods and cytological fungal identityusingConfirmation of partner molecular Results publicand repositorywillthe theDOI article. the endof data at beincluded manuscript beaccepted, Should allwill this appropriate data inthe bearchived Data accessibility patterns oftheundergroundpatterns axes of commencement of the experiments. of the commencement theat biomassin eachmicrocosminitial wasfeasible not measure to oftheMost plantbiomassthe experimentswas andit developed commenced, before BiomassS2)(Fig i). 2h,and arbuscules coils (Fig. and(ca.1.5 finer in diameter) had 2e, f).Dual fungalhyphae, vesicles colonisations arbuscules in (Fig. and comprisingplant-Glomeromycotina associations, intracellular relativelycoarse c). 2b,Fungal lumps of (Fig. with terminal colonisation (Carafa withobservations previous significant effects ofaccessbiomass( algalon final necromass significanteffects to studied 0.283; three-way0.283; ANOVA;TableS1) ( Neohodgsonia

Accepted Article features of both fungal symbionts, particularly in the presence of coarse (>3 both particularly of coarse fungalfeatures presenceof symbionts, inthe ; Fig. S2,Table Fig. S1 ) had significantly greater biomassthan greater had the Glomeromycotina- significantly

). The Mucoromycotina with liverwort associatedonly  m informing hyphae intracellular diameter) et al et DNA

, H. gibbsiae Unsurprisingly therefore, we didUnsurprisinglydetect not therefore, for any of theliverwort-fungalfor any partnerships ., 2003), including., 2003), coils intracellularfungal -based results and showed results and colonisation -based by Mucoromycotina fungiconsistentby Mucoromycotina M. paleacea F 1, 48 was typicalwas of

= 1.18, = 1.18, N. mirabilis and P =  m 0.027 concentration of concentration of Glomeromycotina fungi, this effect of symbionts beingeffect Glomeromycotinaofthis( significant fungi, symbionts those to associatedcompared onlywith withassociatedMucoromycotina fungi Mucoromycotina fungi (i.e. (i.e. Mucoromycotina fungi way interaction effects of core rotation,interaction ofcore waya[CO effects type (Table 1: however there were no significant effects of however werenosignificant there a[CO 440 ppm a[CO 440 ppm more than 2.4 times those of the Glomeromycotina-only colonised colonised than the Glomeromycotina-only thoseof 2.4 times more This article isThis article by protected copyright. All rights reserved. ( liverworts dual symbiont P< three-way ANOVA; Table S1) but there was ANOVA; of nooverallTablethree-way effect but S1) for the elevated the ambientcompared to for Neohodgsonia concentrations in liverwort thalli grown within thalliconcentrations cores(Table liverwort static 1: total total f Significant 3)(Fig.via plants symbiotic in Nitrogenuptake fungi plantspecies ( F the alga

Accepted 1, Article 48 0.001, three-way ANOVA). The three-way 0.001, ANOVA). uptakeof

15 = ; N

23.10 three-way ANOVA; Table 1). three-way ANOVA;Table1). e in the liverworts inthe within static cores (Fig. 1 and3(Fig. Table cores within static ungus , P< F F 2 also showed a significant interaction effect with lower final biomass finalbiomass also effectwithshowedinteraction asignificantlower ] (Fig. 3a), rising(Fig. 3a),> to] 1 1, 48 0.001, three-way0.001, ANOVA) ,48 15 uptake and transfer to plantsdetermined to uptake andfromalgal transfer by necromass N =4.79, = 2.83, in the liverwort thalli (Fig. 3c,d) in liverwort the thalli(Fig. , F was limited to plants whose fungal partners had whosewasplants accessto fungal limitedto partners 2 P Neohodgsonia P , 48 =0.018).Singleand with dual partnerships = 0.099, three-way ANOVA; Table S1). = 0.099,three-way Table ANOVA; S1).

= 8.20 90 , CO P -fold times -fold 1,500 ppm a[CO more at =0.01,three-way Table ANOVA; S1 2 and and , . treatments treatments ( This resultedin higher significantly 2 15 ] or symbiontThetype (Table] 1). Haplomitrium N differed bysymbiont significantly ; effect of static versuseffectstatic of rotated cores: 2 ], andtwo or], three- nosignificant , was F 2 35 , 48 - ) had mean had )

90 = 4.41, = a -fold higher in plants higherplants -fold in [ CO F F M. 2, 48 1 ,48 2 P ] across ] all the paleacea

= = = 15 49.26 0.018 4.37 N contents 2 ], ). , , , P at 15

= N

dilution effects in wheredilution treatments the effects would appear to be negligible,be to wouldappear radioactive) P contentor radioactive) algal additions on algal additions in ordirection a[CO magnitude onthe depending liverwortsdiffered algaeshowedassociatedMucoromycotina-only to that responses of algaleffects 4c,d), and additions (Fig. whereas the Glomeromycotina-only The x (Table 2, interaction algae symbiont identity(Table 2, symbiont identity, a[COof significantmain orTableno other buttherewere way interactiveeffects ANOVA; 2), Glomeromycotina-only CO and between symbionts ThisTable inasignificant interaction resulted (Fig. 4b; S2). a[CO Total grown at 440 ppm a[COgrown at440ppm association with Mucoromycotina with association This article isThis article by protected copyright. All rights reserved. uptake Phosphorus Accepted Articlehigh inthe Glomeromycotina-only 2 significant interactionssignificantthe arose from treatment on total treatment ontotal 2 33 ] Algal additions to cores also did not have a significant effect on total (non- haveAlgalsignificantalsocoreson total additions a did to effect not The P uptake by the liverworts grown at1500 bythe liverwortsP uptake ppm a[CO interaction(Table 2, 33 P concentrations in liverwort biomass affected wasliverwort by in significantly P concentrations 33 P concentrations (Table 2). P concentrations (Table 2). by plants via symbiotic fungi (Fig. 4) 4) plants via symbiotic (Fig. fungi 33 M. paleacea 2 P uptake by the liverworts( P uptakebythe ] there was no significant difference betweenthere wassignificant symbionts] the no P 2 ] F concentrations of cores (Fig. S1). Phosphorusconcentrations pool S1). (Fig. of cores

and(Table algae availability of 2). F 2,48 F 2, 48 2, 48 and

= = 5.24, = 5.24, , = 4.60, = 4.60, 8.99 M. paleacea (Fig. (Fig. therefore unlikely to affect plant affect unlikely therefore to . In addition, , 15 P 4a P N labelled algal necromass wasincluded P = 0.001), being= 0.001), highestthe in - note the lognote the scale), forthe liverworts- but = 0.009) dual =but 0.015) -symbiotic liverwort showing no -symbiotic than in the liverworts inor dual single in liverworts than the

2 there wasa significantsymbiont ], butof was ], there effect no overall , F as xwell asasymbiont a[CO 2, 48 no

= 2 5.39 other significanteffects other ] wasnearly ] twice as , P = 0.008, three- 0.008, = 33 P tracer P tracer uptake. x 2 . ] This article isThis article by protected copyright. All rights reserved. wasliverwortphotosynthatethat detected by Thethe proportion of 5)(Fig. externalsymbioticcarbon allocationmycelium fungi Liverwort to of 2), and was <2% of the total C fixed<2% oftheand C was total 2), (Fig. 5a,b- type ofsymbiosiswas by (Tableaffected significantly the unrotated the meshcores three-way ANOVA), but there wasa[CO but there ANOVA), three-way nosignificant effect of Glomeromycotina only or Mucoromycotina only (Table 2; (Table Glomeromycotinaor Mucoromycotina only only proportiongreater photosynthatethan mycelia of associating liverworts of with in Fig. 5b) interactions between the symbionts or other variablesinteractions betweenthesymbionts liverworts (Tableliverworts 2; receiving thatmore type of single-fungal than 5times partnerships onaverage the withdual with symbiosisthe liverworts by the c,d), (Fig. 5 affected significantly was a significant effect of a[CO was effect asignificant of 48 a[CO compared to440 ppm 1,500at across symbiosesalgal treatments ppm allocation fungi and the to total photosynthate allocated into the mesh-walled cores (Tableof effect 2: algae photosynthate mesh-walled cores total allocatedintothe between the symbiont types and the effects of algae, or between algae and a[CO algae and between symbionttypes andthe algae, the of between effects or 6.87, treatments, andnothree-way interactions

Accepted= Article 10.57, P The total = 0.012, three-way ANOVA). The addition of algae significantly increased Thethree-way= 0.012, addition algae significantly of ANOVA). . P External mycelium of dual partnerships in meshcoresreceived the dualExternalmyceliumof partnerships = 0.002, three-way ANOVA), and there werenosignificantinteractions ANOVA),= 0.002, three-way andthere 14 C traced into static cores intostatic C traced viafungal partners was highly F 2, 48 2,

= 49.84, = 49.84, 2 ] (Compareof TableFig.] CO effect 5cand 2: d; 2 ], with an average 4-fold increase in carbon increase average4-fold withan in ], P < 0.001; three-way ANOVA)< 0.001; .

note the dataareonalogscalethat .

F 2,

2 = ], algae,or . 4.96 Inthere addition, 14 C tracing into , P = 0. 2 0

F 11 a 1, 48 , 2 F ] ] = 1, biomass not performed biomassnot and fungal quantification of plantspecies in numberof Although tested the limited partners. inorganic P to host plants inhost soilto non-sterile inorganic plants P fungiwithboth groupsof assimilated and transferred significantand assimilated transferred amounts of Glomeromycotina whenpartnershipswith bothin occurringsinglyand partners, dual in soiltheir liverwort hosts non-sterile to the Mucoromycotina fungi compared to only Glomeromycotinamight bedue tothese the Mucoromycotina only fungi compared to N uptake via of higherrates The fungi. than Glomeromycotina organic Nresources moresuccessfulcompeting Mucoromycotinamicrobes for in withsoil are fungi microorganisms (Thirkell Mucoromycotina fungi enhance transfer of enhance Mucoromycotinatransfer fungi according typeof fungal the are distinct present.responses to partner tested andliverwortbetweenfungalhosts andthat the these their symbiont(s) availablethe nutrient inare the form whichaffects dynamics nutrients exchange thatsuggestvariation experiments carbondioxide inatmospheric concentration and 15 This article isThis article by protected copyright. All rights reserved. Glom fungal Mucoromycotina, symbioses involving differencesthere results important between in are Our functionality plant- showthat Discussion detection small of amounts of flow and diffusion of flow anddiffusion meshgrowingwere the cores into rotation severedby are likely beduetomass to

Acceptedto the the plantin transfer N Article

15 N-containing molecules resulting from mineralization molecules bysoil resultingN-containing from , o et al., et in ur experiments demonstratedur experiments that Mucoromycotina fungal the effectiveness of N transfer from organicN of from the effectiveness transfer and matter 15 Marchantia 2016). Our data support the hypothesis that theOur datasupport hypothesis 2016). N in liverworts where the hyphae of symbiotic hyphae N the fungi of where in liverworts . In contrast, we contrast, In negligible detected fungal -Glomeromycotina symbiosis. N . The datafrom from algal necromass to liverwortfromto necromass algal 15 er N omycotina,and dual symbioses supplied algal necromass as our microcosm The in andcompetitive/ mutualistic complexresult of between soilorganisms symbioticfungiand the interactions other the overallConsequently, inmycorrhizas Nuptakebyplants functioning of isthe divergent land plant groups in non-sterile soil. This is particularly pertinent to pioneer This to isparticularly pertinent soil. innon-sterile groups divergent land plant early- extant membersof in ecologythese two symbiosis fungal groups withof algalconstituents of necromass lipidsN suchasproteins, andnucleicacids, Mucoromycotinafacultative are main fungi saprotrophs depolymerise the and able to It remains the whether in unclear interactingwith soilmicroorganisms. partnerships overallthe mycorrhizal required basis resolvethemechanistic functioningof the to of studies mycorrhizalthe role of now interpreting fungi inare uptake, nutrient further Givencomplexitydepolymerisation, this mineralizationfor andimmobilization. structural carbohydrates such as cellulose cell walls. carbohydratesstructural such ascellulose cell walls. supported by organic C supplied by AM fungal mycelium (Zhang AM fungal mycelium by organicsupported by Csupplied growth promoting and P-solubilizing bacteria bacteria P-solubilizing and growth promoting asnutrients, plant- mineralization of and be involved may in mobilization increasing This article isThis article by protected copyright. All rights reserved. Accepted Articleexample, For 1991). can beutilized (Vitousek&Howarth, potentially mineralization beforeit microbial and plant depolymerisation originating andthat from microbial require litter of theNinsoilas mostin isnaturally organicforms present microorganisms, mycorrhizal fungi inuptake nutrient their interactions arisesfrom soil withother role of depolymerisationIt iswell-established ormineralization that the and/ N. of that acceleratesa mycorrhizosphere-microbialbetter supporting community (b) competingfornecromass, N mineralizedor (c) better saprotrophs, bythe withsoil competingfungi organic (a) better saprotrophs the for Nfrom algal Nonetheless, Nonetheless, AM fungi develop“mycorrhizosphere” microbial specific our findings provide new insights intoand the new functionalfindings provide insights biology ant agonistic processes of activities inthe Pseudomonas

and Burkholderia et al., et populations that populations that 2014, 2016). and can be

competition between plants and soil microbes (Hodge between andsoilcompetition plants microbes al., ofpart soilsas cryptogamiccolonisingpoorly-developed groundcovers(Edwards assumed to not dissimilarexperiencedthat to have byearlylandplants been a situation providelikelyalgaeto are cyanobacteria important and Nrich microsites, habitats microbial decomposers microbialdecomposers priming of myceliumintosoilmycorrhizalhelp C effluxfungal may the Increased from 2013 et al., et (Govindarajulu complex and releasedecompose organic inorganic sources form Nin mayalsoAM fungi rely onmembersof thesaprotrophic to microbial community arbuscules has been discovered (Guether (Guether has been discovered arbuscules containing cells activated in thatis mycorrhiza-specific andpreferentiallytransporter 15 This article isThis article by protected copyright. All rights reserved. givenlike plants liverworts, t availability (de Graaff availability al., elevated a[CO observed here of plant photosynthate (Hodge, 1996; Johnson(Hodge,1996; plantphotosynthate Hodge and Fitter (2010) showed that whilst AM fungal hyphae proliferated on fungalwhilstshowedAM on hyphae(2010) that proliferated Fitter Hodge and of no ornegligible transport reported

Accepted theirangiosperm to plants, host organicpatches andaplantammoniumN-labelled Article 15 2015). Where organic N is available in such systems, there is likely strong there is such systems, Where organic2015). Nisavailable in 2009; Hodge & Fitter, 2010) have thatfungi 2010) 2009; AM transferHodge & Fitter, demonstrated N ) 2005;Leigh . to theliverwortto and host plant- corresponding In the the In of Glomeromycotina association

(O llivier 2 ar ] and higher C fixation by plants, AMandfixationby plants, ] higherfungi C sinks become major of et al., et e consistent with previous studies that have found especially undere consistentwith previous havefound studies that et al., et et al., et

2011) –

2011) a common soil-biotic response to increased organic matter a common response matter increased soil-biotic to organic 2010;Drigo he in which soil crusts containing fast-turning over green containing over crusts in fast-turning soil which for hyphal capture. While(Leigh for hyphal capture. severalstudies low N status of disturbed, primary disturbed, primary successionalof N status low 15 N from fungus to host (Hodge N from &Fitter, fungus to host 2010) et al., et et al., et al., et Marchantia

2010; 2009a;others have 2009b), 2002; Herman to Verbruggen -fungus C allocation patterns Callocation-fungus patterns et al., et , t he

2000 limited fungal transfer et al., et ; Kuzyakov & Xu,Kuzyakov ; etal.,

2013) 2012) 15 N from . Thus,. . and

et et . organic matter complexes organic matter (Lindahl recalcitrant metabolicthe N lockedof Cbut mobilization upinnon-hydrolysable, being withtheorganicthe acquisitionof oxidationof main not benefit matter symbiont contributes to soil N immobilization, limitingto contributes symbiont soil Nimmobilization, decomposition (Bué decomposition organic saprotrophs ingaining Ccompoundsfree-living from behave like matter putativelyfacultative saprotrophicthe general that fungithat ectomycorrhizal idea In this it issource ofmetabolic to be elucidated.regard, note Cisyet interestingto use thethepatch Mucoromycotina Whether organicnutrient fungal symbionts its as C orremains onthe of entirely maindependent host liverwort as asource groups. fungi,mayGlomeromycotinasometimeshave unknowingly AM involvedboth in thatattributed effects nutritional pastexclusively havebeen effects the of to plant haveGlomeromycotina test may colonized andboth sothat roots, FRE that in It should alsobenotedthat nutrition. host than contributinghost rather liverwort N to Lindahl and Tunlid (2015) TunlidLindahl and (2015) remain dependent on their host plants as their principal source of metabolicprincipal of host plantsastheirremain dependent ontheir C, source (F with access to to withaccess the dynamics in This article isThis article by protected copyright. All rights reserved. from acquired N Glomeromycotina rather than transferring it to hostplant transferring than the it rather previous studies using non-sterile soil or root piecesfungalsoilroot from as inocula soilor using non-sterile studies previous

Accepted Articleig . . 3c ). Thus, it is plausible that under elevated a[CO under). Thus,is plausible elevated it that an Marchantia a organic elevatedat a[CO patch AM 15 e N-labelled organic patch the fungus retained mostof the fungus the N-labelled patch organic , given , the increasethe major in Callocationto fungus plant et al., et pr 2005; Courty2005; -Glomeromycotina symbiosis as for angiosperm- for symbiosisas -Glomeromycotina oposed that ectomycorrhizaloposed that co-metabolic fungi perform et al., et 2007).Thus, ectomycorrhizal most fungi et al., et . Our results pointOurresults similar toward N

2007) 2 ] (Fig. 15 has beenchallenged. recently N transfer to the liverwort tothe N transfer 2 ] the ] Glomeromycotina 5c ) for little) for no to 15 N return N return 15 N source. Thiseffective is very source. symbiosis in largely independent of whetherof isadded fresh algallargely necromass independent soil to paleacea supplying P from soil to plants. soil P from supplyingplants. to fungithese highlyinand evolved role of their obligately nature with biotrophic the However, theof amount However, host plantsby eachof thesingle-fungus host partnerships i.e. benefits to the combine afforded the nutritional Glomeromycotinafungal partners compared to Glomeromycotina fungi. Glomeromycotina compared to Pinitsmore ownbiomassand ontothehostplant passing asmaller proportion not at 1500ppma[CO not at but 440at ppm necromass algal was containing in reduced patches fungal partner This article isThis article by protected copyright. All rights reserved. Accepted Articleespecially C, host inexchange a[CO modern relatively plant-fixed for little under access to investigated(Table symbioses plant-fungal 3): amongsymbioses functionaldifferencesFundamental partnership. similardynamics C intheliverwort-Mucoromycotina patch an organicMucoromycotinasymbionts withaccessto (F nutrient 2015) (Lindahl decomposersrather thanbehaving &Tunlid, facultative as saprotrophs . Our results, showing Our results, increasedC allocation. or host stable to 3) Dual symbioses involving simultaneous Mucoro 3) Dualsimultaneoussymbiosesinvolving 2) In Glomeromycotina-only associations, the fung the Glomeromycotina-only2) In associations, 1) Associations involving Mucoromycotina-only partners provide substantial Associations 1) partners Mucoromycotina-only involving indicateresults amongthe three of Overall, our types functional differences do not appear to facilitate significant plantN acquisitionsignificant from appear not to facilitate do N supplied as algal necromass, andPreturns increase to the liverwort as algalsupplied necromass, 2 ], 33 possibly as a result of less effective P uptake or retainingpossiblyless asaresultof effective Puptakeor P gained by a plant host per unit of Pgained byaplanthost

33 P transfer toP transfer hostplantis the and this my al 15 symbionts of symbionts N transfer from availableN transfer from cotina and C invested inthe – ig an consistent consistent . 5), point to . M. organic N 2 ].

patchy, whether spatially or temporallywhetherpatchy, spatially or symbioses (Fieldsymbioses engage landcladesdual plant known in liverworts and early-divergent to other through to contemporary agro-ecosystems (Orchard (Orchard agro-ecosystemsthrough to contemporary simultaneoussymbiosis dual Mucoromycotina andGlomeromycotinawhether singly fungal partners, in or toremain colonisations beestablished. within dual eachandfungal nutrient gains partner from carbon host Theto, allocation relative considerably higher carbon ‘cost’ to the host thanconsiderablyhigher hostthe single carbon ‘cost’ to are available.nutrient sources aHowever, at these come nutritional benefits habitats where N islimitinghabitats where providecompetitive inmay a with N advantage liverwortorganically-derived partners Glomeromycotina Mucoromycotinaof fungi overthe inabilityfungi supplying tested specificallyhavemicrobiota (which investigated). not we plants from the early stages of terrestrial ecosystems (Strullu-Derrien plants earlystagesfromterrestrial ecosystems(Strullu-Derrien the of bothwith of apparentpresence and fungal lineagespartnerships the persistence in withrespect topartners host of provisioning plantswithN and help explain Pmay to This article isThis article by protected copyright. All rights reserved. constant the more by the with Mucoromycotinasymbiosistogether sources organic nutrient Accepted our provided by localized as resource-patches (such photosyntheticfungal capacity, type, symbiont includingand abiotic biotic factors, by acomplex a[CO of interplay thesethat theoutcomefrom results indicate optionsmultiple isinfluenced symbiotic Article Our findings support ourOur the engage hypothesis that ability to withboth 33 P returns fromthe Glomeromycotina P returns whether not or organic symbiosis, 15 et al., et N algalwell as necromass), interactionssoil as withother 2015a; Desirò and , offers additionaloffers intermsplasticity of nutrient uptake to /or where the organicof is distribution nutrients /or . et al., et The apparent complementarity of the The fungal apparentcomplementarity 2013; Rimington2013; et al., -fungus symbioses (Table 3) (Table symbioses -fungus The

2017a et al., et apparentsuperior ; 2017b). 2017b). ; 2 ] and] henceplant 2015 et al., et ) . Our 2014) .

Thedeclare authors version nocompeting this of manuscript. interests. the constructive commentsfor provided andeditoronanearlier the referees by fungi in modern ecosystems (Field fungi modern ecosystems in 2019 symbiosis earlyvascular andangiosperms (Hoysted withliverworts, plants biogeochemistry an biogeochemistry among plantsanddiverse nutritional fungi for of consequences mutualisms symbionts (Bidartondo symbionts Mucoromycotina are vascular the members of plants, widespread ecologicallyand across Pressel,201 roles of Glomeromycotina fungi in planthost Nnutrition inroles ofGlomeromycotina fungi and fernsgroups ofland lycophytes, suchas plants fungalin symbionts later-divergent Orchard Heather Walker for technicalHeatherfor assistance sample Walker with and Conservation Dr DepartmentNew of bryophyte Zealand for collecting permits and a Philip Leverhulme Prize(PLP-2017- andPhilip Leverhulme a Researcha BBSRC by Fellowship Translational KJF issupported (BB/M026825) Acknowledgements from the NERCthe from (NE/N009665/1 KJF,SP, JGD,studentship. MIB, DDC,and JRLgratefully DJB acknowledge funding This article isThis article by protected copyright. All rights reserved. Accepted Article angiosperms. Such investigations together with a Suchinvestigations appreciationwithbetter together angiosperms. the of ). La This finding greatly expands the potential significance of Mucoromycotinaof This finding potential the greatlyexpands significance et al., test evidence indicates that fine root endophyte fungi(FRE) endophytethat evidence test fine root indicates 8)

. 2017 Thus,it is now critical functioning the toexplore Mucoromycotina of and d a) ecosystems in the past, present and future. presentin past, andfuture. ecosystems the closely related to liverwort and Mucoromycotina lycophyte liverwort closely to related . et al., et Furthermore, the sameFurthermore, Mucoromycotinathe into fungi canenter 2011;Rimington , NE/N009665/1 et al., et 2015a;Hoysted 079 ). WRRa NERC issupportedby ). DTP et al., et

and NE/I024089/1). We and NE/I024089/1). thank the

2015 15 N analyses , are key to understandingthe are key ; 2016;Field; et al., et 2018; Fieldand . We are grateful , which are et al., et et al., et 2015b;

1137 dawnsymbiosisand of betweenplants fungi. ReadBidartondoMI, DJ,Trappe JM, Merckx LigroneDuckett V, JG. 2011. R, The atmospheric O Berner,2006. R.A.,GEOCARBSULF: combinedPhanerozoic a model for current knowledge and research gaps. gaps. research and knowledge current of plants: uptake nitrogen in fungi mycorrhizal arbuscular of Role 2015. H, A. Kafle Bücking plants under different phosphorousplants different andnitrogenunder concentration. AzcónAmbrosanoCharest R, acquisition C. E, 2003.Nutrient lettuce inmycorrhizal arbuscular fungus. mycorrhizal This article isThis article by protected copyright. All rights reserved. SP SPanalyses. conductedconducted molecular cytological andJGD the the analyses. MIB and conducted isotopeandWRR the tracer work GAH conductedPanalyses. MIB, research.KJF,JRL, JGD the and DDC,designed KJF DJB, SPconceived and contributionsAuthor of nitrogen from two nitrogen from of Ames RN,1983.Reid C. HyphalPorter LK,Cambardella CPP, uptakeandtransport Accepted programs. Articlesearch protein database of generation 1997.new andPSI-BLAST:Gapped a BLAST AA,AltschulMillerJH, Zhang TL,Schaffer Madden Lipman Zhang SF, Z, W, DJ. References manuscript. , KJF, MIB and JRL led the writing; all authors providedMIBKJF,all led andJRL comments writing; onthe the authors – 1145. 1145. Nucleic Research Acids 2 and CO 15 N-labelledsources of 2 Geochimica Cosmochimica Acta et New Phytologist 25 Agronomy : 3389- : Glomusmosseae 3402

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: This article isThis article by protected copyright. All rights reserved. sylvatica the of ectomycorrhizalpotential inabeech( metabolic community activity monitoring diversityand BueeGarbaye J.2005.Year-round Vairelles of D, M, from rootsinto microbialelevatedfrom inresponseto communities associated AS,van BodelierWhiteley 2010.Shifting Veen PLE, GA. JA,Kowalchuk carbonflow DrigoPijl B, AS,DuytsH, Kielak GamperHA,HoutekamerMJ,Boschker AM, HTS, 280: chequeredsymbioses in a history. hornworts: DesiròDuckett JG,Pressel Bidartondo, A, MI. 2013.Fungal S,VillarrealJC, measuring the carbon cost of mycorrhizasmeasuring thecarboncost greenorchid inthe I, Read2008.GivingLeake Johnson Cameron DJ, JR. andreceiving: DD, rates. rates. decomposition compositionmediate thesoilmicrobialresidue andplant community MA,de Graaff Classen AT,Castro 2010. HF,Schadt CW. Labile carboninputs soil terrestrial orchid green-leaved inthe andnitrogentransfers evidenceplant-fungus from carbon 2006. Mutualistic LeakeJR,ReadDJ. Cameron DD, orchids: mycorrhiza in aseptate fungi. Haplomitrium harbourunique primitive liverwort endophytic a of with type association before and during bud break. before andduring bud New Phytologist Carafa A, Carafa Duckett JG Acceptedorganicsecretion of degrading matter by enzymes PE,CourtyBredaGarbaye 2007. N, J. Relationand betweenoaktree phenology the Article 20130207. New Phytologist ) forest subjected to forest two thinning regimes. ) New Phytologist

Goodyera repens 180 : 176 : ,

Ligrone2003.Subterranean R. gametophyticaxes inthe 188 – : 1055: 184. 184. SoilBiochem Biology and

160 – . 1064 : New Phytologist 185

- 197.

Proceedingsthe B of Royal Society: Lactarius quietus Mycorrhiza

171 istry istry : 405 : 39 – Goodyera repens

: 1655 15 416. 416. : 235 ectomycorrhizas – 1663. 1663. – 245. 245. Fagus . association with the mucilage-secreting, primitive mucilage-secreting, withthe antipodeanliverwort association A, LigroneR.2006.AhighlyCarafa glomeromycotean differentiated Duckett, 58 States of AmericaStates of PalaeozoicCO symbiosisand withGlomeromycota simulated fungi Mucoromycotina under a liverwortsinof dual LeakeDuckett 2016.Functional JG, JR,PresselS. analysis FieldBidartondo AllisonRimington WR, MI, KJ, KE,Beerling Cameron DD, DJ, options for the conquest ofoptions theconquest for land. JG,Field 2015a. S,Duckett RimingtonWR, BidartondoPressel Symbiotic MI. KJ, This article isThis article by protected copyright. All rights reserved. 154 simulated changes to its response Palaeozoic CO inatmospheric Mucoromycotina and and fungi ancient lineages (Haplomitriopsida plant liverworts) mutualismbetweenevidenceof Pressel 2015b.First Leake Duckett S. JG, JR, FieldBidartondo AllisonRimington WR, MI, KJ, KE,Beerling Cameron DD, DJ, PalaeozoicCO a simulated to plants arbuscular vascular mycorrhizalof Contrasting andnon-vascular responses LeakeJR,Tille BeerlingField2012.Bidartondo DD, MI, Cameron S, DJ. KJ, atmospheric CO (Treubiaceae): cluesmycorrhizas.(Treubiaceae): to origins of the ecosystems have bioengineered the planet in mid-Palaeozoic times? have planet mid-Palaeozoic ecosystems bioengineeredthe in early photosynthesizing Edwards ChernsL,Raven land-based E, JA.2015Could Accepted Article797 : 803 : : 743 : – 813. 813. – 837. 837. – 756. 756. 2 2 decline. . Proceedingsthe NationalSciencesof of the United Academy of 107 : 10938: The 2 decline.

ISME Journal – 10942. 10942. Trends in Ecology and EvolutionTrends in and Ecology Nature Communications

10:

1514 American Journal Botany of – 1526. 1526.

3 : 835. :

2 30 . New Phytologist : 477 : Palaeontology Treubia – 486

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HodgeImpactof1996. elevated A. CO for plantgrowth for Lammers PJ,ShachLammers PfefferGovindarajulu DoudsPE, M, JinJ, DD, Allen HR,Abubaker Bücking JW, H, Ecology application - the rusts. to mycorrhizaeBasidiomycetes identification and of Gardes M symbiosis. New Phytologist decomposition. arbuscular fungusandsoil mycorrhizal mediating a microbial community litter DJ,Firestone Herman NuccioMK, Hodge 2012.Interactions E, between A. an This article isThis article by protected copyright. All rights reserved. Field KJ A mycorrhizal-specific ammonium transporter ammonium romA mycorrhizal-specific M, NeuhaüserGuether B, Balestrini LudewigBonfante U, R,DynowskiM, P.2009a. membrane biogenesis during arbuscular mycorrhizal symbiosis mycorrhizal membrane in duringbiogenesis arbuscular transport, cell ofregulatorynetworks, reprogramming Genome-wide wall and M, HeJ,Udvardi BalestriniGuether M, R, Hannah 2009b. MK, Bonfante P. and temperature. and temperature. based model of thallose liverwort isotope fractionationresponse toCO in liverwort ofthallose isotope based model Phytologist functional anancientpartnership betweeninto insights plantsand fungi. released by arbuscular mycorrhizalreleased arbuscular fungi. by Accepted ArticleFletcherBJ, BrentnallQuick Beerling SJ, WP,

, 2 Pressel S.2018. Pressel : 113- : , Bruns TD. (1993).ITS withenhanced for specificity primers Nature 220: . Biology. Soils andFertility of 118

FEMSEcol Microbiology 182 996 GeochimicaCosmochimica et Acta

435

ar : 200 : - 1011 -Hill Y. 2005.Nitrogen in -Hill Y. transfer the arbuscularmycorrhizal : 819 : –

. . Tansley Review - Tansley Review 212. 212. – 823.

2 on mycorrhizal associationson mycorrhizal and implications ogy ogy Plant PhysiologyPlant

Unity structural indiversity: and 23 80 DJ , 388 , : 236 : . 2006.BRYOCARB:. aprocess- Lotus japonicus –

– 70 398. 398. 247. : 5676:

150 – : 73 : 5691. 5691. Lotus japonicus acquires nitro – 83. 83. New 2 , O Molecular 2 , light gen . sequencedata. software platformand for extendable desktop theorganization and of analysis Cooper A, Markowitz S, Duran C, Thierer CooperS, DuranC, A, Markowitz Buxton SturrockStones-Havas S, CheungWilsonM, S, Kearse MoirR, A, S, M, Johnson D, Leake JR, Ostle N,InesonsituRead2002.In LeakeJR,Ostle DJ. P, Johnson CO D, arbuscular mycelia mycorrhizal to the soil. alabellingupland grassland of rapid pathwayofcarbon flux demonstrates from plants through to ecosystems. to ecosystems. through plants from individual implications nitrogen: and mycorrhiza Arbuscular K.2015. Storer A, Hodge National Academy of Science of the United States of Americathe UnitedStatesof Science of National Academy of organicfungicycling. from N material hasimplications for AH. Hodge 2010.Substantial byarbuscular Fitter nitrogen mycorrhizal acquisition A, This article isThis article by protected copyright. All rights reserved. CommunicationsNature ancientMutualistic symbiosis land mycorrhiza-like inthemost group of plants. Leake Bidartondo JR,Beerling PJ,ReesM, MI, 2010. CP,Franks DJ. Humphreys nitrogen? for competing at plants than moreeffective microorganisms Are D, A.2000. Fitter Robinson A, Hodge Plant Biology Field 2018.Ryan Bidartondo Amycorrhizal M, MI. KJ, revolution. Hoysted GA Acceptedplants. Mucoromycotinaendophyte fine root form nutritional fungi withvascular mutualisms G, RimingtonGebauerField KJ.2019. Schornack WR, S,Pressel Hoysted JacobAS,KowalDuckettGiesemannBidartondo GA, J, JG, P, MI, Article bioRxiv , Kowal J, Jacob A, Rimington WR, Duckett JG,Kowal JacobOrchard Pressel J, A,RimingtonWR, Duckett S, 44 , https://doi.org/10.1101/531103 https://doi.org/10.1101/531103 Bioinformatics : 1 - 6.

Trends in Plant Science Plant in Science Trends

1 : 7. : Plant and Soil and Plant

28 : 1647- : 1649.

386 T. New Phytol

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107 : 327 : Current OpinionsCurrent in : 13754: – 2 334. 334. -C 13 – pulse 13759. 13759. Amsterdam, the Netherlands:Amsterdam, Academic Press. evolution. evolution. YangSchneider Z, Donoghue H, PC.2018.Theearly land timescale of plant PresselWellman JL, S, Morris CH, Puttick MN,Clark EdwardsJW, KenrickP, D, Nobel PS.1999. boreal forest. boreal forest. in a litter decomposition2007. of uptake Spatial separation nitrogen andmycorrhizal Lindahl BD, Tunlid A. 2015. TunlidLindahlEctomycorrhizal BD, A. fungi Lindahl Ihrmark BD, Boberg Trumbore K, J, StenlidSE, P, Högberg J FEMS Microbiology Ecology inarbuscular competition fungusin soil mycorrhizal organicmatter bacteria. with AH,HodgeLeigh Fitter 2011.Growth effectiveness J, A. andan of symbiotic biogeochemicalEarth cycling overPhanerozoic time. Science Reviews An of improved model Daines2018. COPSE MillsLenton BJW. TM, SJ, reloaded: decomposers, yetnotdecomposers, saprotrophs. This article isThis article by protected copyright. All rights reserved. reference. ecological and mechanisms nitrogen: for microorganisms and roots between Competition X. Xu 2013. Y, Kuzyakov Accepted ArticlePhytologist host organicamounts ofnitrogentotheirsubstantialplant from material. AH.2009.ArbuscularFitter Leigh Hodge A, mycorrhizal J, fungitransfer can UniversitySheffield, DepartmentPlantof of Sciences). Hull:the With to Special RoleMycorrhizas Reference of 1988.Leake, JR. Proceedings oftheNational AcademyProceedings Sciences of

181 New Phytologist : 199 : Physicochemicalphysiology andenvironmental plant Causes and Effects ofSoilCallunaAcidification vulgaris Causes andEffects by (L.) – 207.

173 : 428 : (3) New Phytologist New Phytologist – : 438. 438. 611 -620.

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(10):.E2274-E2283. (10):.E2274-E2283. , , 2nd, edn. Finlay RD. 178 New : 1 - 28

vascular plants: Reopening vascular plants: book? aclosed JG,Duckett Bidartondo 2015.WR, PresselS, Rimington Fungaldiversityinearly MI. Systematics andEvolution knowledge perspectives.fungalfuture Current and associations: JG. 2016. Duckett WR, Field PteridophyteRimington PresselBidartondoMI, S, KJ, 619 weathering. weathering. silicatemycorrhizal2012. fungi intensifies Evolution mineral trees and of LeakeJR. Beerling ME, J, Romero-Gonzalez Quirk G, Kakonyi DJ,BanwartSA, Powell JR,WalkerC,BassSimoninMonk MH D, J, A,Ryan DickieS,Gleesons Standish DB,RP,JefferyOrchard, HiltonBending IA, RJ, S, GD, This article isThis article by protected copyright. All rights reserved. Ecology NitrogenSchloter insoilM. 2011. turnover andglobal change. ToeweHaiOllivier MeyerA, J, B,KastlEM, A, Bannert Su S, MX, Kleineidam K, New Phytologist producing fungi recently suggested to belongto theMucoromycotina to suggested producing recently fungi Fineendophytesunder root literature onarbuscule- a review scrutiny: the of RentonD, S,Standish2017b.Dickie M,WalkerC,Moot Orchard RyanMH. RJ, IA, Ancient plants with ancient fungi: liverworts associate with early-diverging arbuscular with arbuscular ancient Ancient fungi: liverworts early-diverging associateplants with JG, FieldBidartondo (2018).Read Duckett KJ, MI DJ, WR, PresselS, Rimington pp. 31-32Hoboken,NJ, USA:F Martin). (ed. John &SonsInc. Wiley 2016. Reappraising mycorrhizas. In origin the of BidartondoDuckett JG, C, Field MI. KJ, Strullu-Derrien WR, PresselS, Rimington Accepted Articleendophytes ( - 638

7 8: 3 8: Biology letters Glomus tenue – 16

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. 205 2017a. Fine FEMS MicrobiologyFEMS : 1394: Journal of .

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: it occur? Howarth 1991.Vitousek sea; Nitrogenlimitation onland PM, the RW. how andin can MC. 2013. ArbuscularMC. fungi mycorrhizal VeresoglouVerbruggen Rillig E, Caruso Hammer AndersonIC, J, T, EC,Kohler SD, for plantnutrition andgrowth. for fertilizationarbuscular withorganic mycorrhizas: bringsconsiderable matter benefits ThirkellCameron T, Hodge 2016. Resolving DD, ‘nitthe A. This article isThis article by protected copyright. All rights reserved. 20181600 mycorrhizal fungi. Environmental Microbiology gradient gelelectrophoresis. temperature encoding rRNA and 18S diversity in wheatamplified genes rhizosphere the bysequencingcloned PCR- of AnalysisK. 1999. fungal Wernars van Elsas of JD, LeeflangB, GlandorfSmit E, P, for soil carbonfor storage? Strullu acquisition: from cells to ecosystems. cells from acquisition: toecosystems. Smith SE,AndersonSmith FA. IC, 2015.Mycorrhizal andphosphorus associations Press. 2008. ReadDJ. Smith SE, Accepted plant ancestral year old)closely resemble those inextantland novelintolower insights plants: Fungal in associations Article Derrien C, Kenrick P, PresselDuckettDerrienKenrick JG, RioultStrullu C, S, 2014. P, JP, DG. Biogeochemistry .

– fungus symbioses. symbioses. fungus Proceedings the of RoyalSciences SocietyB: Biological Horneophyton ligneriHorneophyton New Phytologist

13 Mycorrhizal Symbiosis 65 : 87 Plant Cell Environment : 2614-: – 115. New Phytologist 2621. Annual Plant Reviews

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short-term liability but long-term benefits liabilitylong-term but short-term 197

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phosphate-solubilizing bacterium.phosphate-solubilizing betweenanarbuscularenable fungusandcooperation exchangemay mycorrhizal a Y, Liu Zhang2016.Xu Carbon L, Zhang M, Hodge andphosphorus F, Feng A, G, eds. (Orlando: pp.315- Academic Press), MethodsSninskyM.A. Innis, andApplications, Gelfand, J.J. White, D.H. andT.J. phylogenetics.In fungal PCR RNA genesfor a Guide ribosomal Protocols: to White TJ,BrunsT,LeeS Mycorrhiza 2006.PhylogeneticWang B,Qui distribution Y-L. and mycorrhizas. evolution of mycorrhizas in colonizationmycorrhizas land the of byplants. ofland in commonancestor suggestsmycorrhizala keyrole of plants genes the Qiu Y-L. 2010. LiuY,AnéJ-M, PresencethreeWang B,YeunLH,XueJ-Y, of ZhangFan L, J (Mucoromycotina), andnewcombination promote phytatemineralizationpromote soil. in mycorrhizal arbuscular fungusandsolubilizingphosphatebetween an a bacterium This article isThis article by protected copyright. All rights reserved. Accepted Article (basionymGlomus Rhizophagus tenuis). tenue WalkerC, Gollotte A,Redecker A D (2018). newgenus,

16 : 299 : , Ding2014.ZhangHyphosphere X,He interactionsFeng F, G, – 363.

, Taylor JW. 1990. Amplification and direct sequencingAmplification of anddirect JW. 1990. Taylor New Phytologist Soil Biology &Biochemistry (P. tenue), (P. 322.

Mycorrhiza New Phytologist

210 for the fine root endophyte root for thefine : 1022: Planticonsortium

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– – 183. 183. 525. 525. , plantbiomass assumptions. toANOVA conform transformed to log10 are Data treatments. (S) static core within S2. Table a[CO and TableS1. Notes S2 fungus- S1Notes- fungalwithout access algal to necromassa. This article isThis article by protected copyright. All rights reserved. Accepted Articleatmospheric [CO Figure S2. included algae). (+ algalalgae) necromasswas andwhere wasabsent(- necromass where algal Figure S1. Supporting Information to 2 -plant ] on liverwort biomass at end of experiment. experiment. at endof biomass liverwort ] on Two-way ANOVA (GLM) showing effects of fungal symbiont and access to algae to algae access and fungal symbiont of effects showing (GLM) ANOVA Two-way – Three Total mean biomass of liverworts inambient plantsgrown underliverworts Total biomassof mean Mean total andphosphorussubstrate in content core concentration

Supplementary results (total phosphorusSupplementaryresults(total content ofmesh-walled cores,

Supplementary materialsSupplementary total of (analysis biomass, and P, methods ).

15 -way ANOVA (GLM) showing effects of fungal symbiont, inclusion of algae, of algae, inclusion fungal symbiont, of effects showing ANOVA (GLM) -way 2 N and N and ] (440 andelevated ppm) [CO atmospheric 33 P transfer andplant- to -fungus Ctransfer). 2 ] (1,500with(and ppm) 15 (b) experimental haveaccess whereliverworts microcosm to column core; the within greenLocationalgalthe running indicatedby of of length necromass soil shading This article isThis article by protected copyright. All rights reserved. containing 1. Diagramsofexperimental microcosms Fig. Figure Legends mirabilis symbioses. AM astypicalof large inf), vesicles(arrowed patch and fungi. Mucoromycotina colonise symbionts with cytology consisting of coarse consistingof 3 hyphaesymbionts withcytology (> Marchantia paleacea experimentalwhere liverworts microcosm do not haveaccessto (c) andconnectionsbetween coreliverwort core host contents(static treatment); treatment) prior to isotope introduction (red arrow), and (d isotope arrow), treatment) prior to (red introduction plantachievedcontents andhost by rotating core(blue rotated the core arrow; liverwort endophytesh, c,e,f, fungal (b, i). wild whole (a,respective andscanning plants d,g) micrographs electron of hyphae (0.5-1.5 µm hyphae(0.5-1.5 Fig. 2 Fig. to fungicompounds cores. static within set-up showingset-up of fixation

Accepted Article algal necromass N-labelled . Liverwortsand symbionts fungal studied in the presentinvestigation; in dual within Glomeromycotina symbiosis andMucoromycotina fungi Haplomitrium gibbsiae 33 15 P-orthophosphate via severed fungalsevered P-orthophosphate connections betweencore via hyphal N-labelled algal necromass (right)and(left) coresalgal additionalgal without necromass N-labelled ) harbouring inthallusharbouring onlyGlomeromycotina its fungal form coils with swellings (arrowed in b and enlarged in c). (d-f (arrowed form coils withswellings inand inc). b enlarged 14 CO and 2 (a) associates exclusively (a) withMucoromycotina-FRE ofby liverworts labelled andtransfer carbon Haplomitrium 33 P-orthophosphate via intact fungal hyphal fungal intact hyphal via P-orthophosphate (a -c) The-c) earliest-divergentHaplomitriopsida underground axes where their fine their axeswhere underground showing (a) contents of cores contentsshowing cores (a) of (g ) µm 14 -i) -i) C labelling experimental ), arbuscules (e) and), arbuscules (e) Neohodgsonia 15 N-labellednutrient ) . plants (c, d) at simulated d)at plants(c, Palaeozoic a[CO nutrient patch at (a, c) 1,500at [CO nutrient patch ppm with(black(whitewithinbars) andwithout presence oforganic soil cores bars) carbon transferredtofungald) partners allocationb) andtotal liverwort-fixed (c, (a, simulated atmospheric [CO simulated andmodernambient Palaeozoic a[CO simulated Palaeozoic a[CO indicate ± S.E., This article isThis article by protected copyright. All rights reserved. (Mucoromycotina,i). (Glomeromycotina, structures h) and finehyphae witharbuscule-like its thallusshowing hyphaetypical in structures trunk fungi: arbuscules ofboth on fungal-acquired Fig. 5 Fig. fungal-acquired ppm (b, d). d). ppm (b, bars) or was( not bars) simulated atmospheric [CO simulated andmodernambient Palaeozoic Fig. 3 Fig. Fig. 4. Fig. simulated Palaeozoic and modern ambient atmospheric [CO atmospheric simulated andmodernambient Palaeozoic was present, “ waspresent, Accepted Error barsnot present. indicate ±S.E., Article 2 . . ] of(b, d)where ] 440ppm Plant- Fungus Fungus “+ to

- algae” algae”representing fungal - - -fungusbetween liverworts carbontransfer and at fungi n to to 33 15 = 5 (see Table statistics) 1for “Rotated” P (a, b) and concentrationandfungal-acquiredof b) P (a, N (a, b) and concentration of fungal-acquiredconcentrationand b) of N (a, -plantnitrogen liverworts transfer between andat fungi -plantphosphorus between transfer liverwortsat andfungi

values fungal-acquired represent Scale bars: (e) 200 µm; (b) 100 µm; (f, h, i) 50 µm; (c) 20 µm. (c) 20 µm. i) (f,h, 50 µm; (b)100 µm; Scale 200µm; (e) bars: ; white bars) accessible to fungal symbionts. Errorbars white accessible; bars) fungal to symbionts. 2 ] ofc)] and 1,500ppm(a, modern ambienta[CO 15 N labelled algal necromass was ( N labelledalgal necromass 2 ] and 440 ppm] (b,d) [CO n = 5 (see Table statistics) 2for -acquired -acquired 2 ] of 1,500 ppm (a, c)] of1,500 andmodern ppm ambient .

33 P where algal necromass was necromass where algal P 33 P where algal necromass 33 15 2 2 2 2 ]. Notedifference in P in plants (c, d) at d) inplants (c, P N ]. ]. ]. ]. “Static”; black “Static”; detected indetected Mean totalMean Percentage Mean totalMean .

2 ] of 440of ] Plant Plant Plant [ Plant (See Fig. 3c,d) (See Fig. (See Fig. 3a, (See Fig. rotation on rotation Table1. Tables statistics) This article isThis article by protected copyright. All rights reserved. and between liverworts Data tomeetwere ANOVA. log10transformed assumptions of Hyphal connections scale of liverwort to be severed. Experimentsliverwortcarried to be a[CO severed. out under440ppm treatments treatment causeswhereas core connections between rotated hyphal

ppm a[CO 15 15 N (ng) N (ng) N] (ng g N] (ng

Accepted Article Variable b)

-1 ) Summary of three-way ANOVA (GLM) results the of testing Summary core effectsthree-way ANOVA of (GLM) y . -axis in figures (c) and (d). Error -axis bars figures indicate (c) and(d). in ±S.E., 2

]. Significantresults ( ]. 15 N contentand[ Symbiosis (S) (S) Symbiosis 4.37 4.79 F d.f. 2,48

0.027 0.018 15 P P

N-labelled algal necromass are in core preserved N-labelled algal static necromass

15 0.52 0.30 N] of liverworts (Fig. 3) d.f. 1,48 F CO P<

2 (C) (C) 0.05) are indicated in0.05) areindicated n.s n.s P P . . .

49.26 23.10 Core rotation Core rotation (R) F d.f. 1, 48 d.f.

<0.001 <0.001 <0.001 P P

11.34 2.45 . d.f. 2,48 F

S x C S xC

bold n.s n.s P P . . .

2. 1.33 n.s. n d.f. 2,48 F 44 S x R S xR = 5(see Table 2for

= n.s n.s P P . . .

P 2 ] and] 1,500 >0.05. 0.9 1.31 d.f. 1,48 F F C xR 3 3

n.s n.s

P P . . .

2.04 0 S x C x R R S xC .75 d.f. 2,48 F

n.s. n.s. n.s. P P

within fungi(ng) cores Plantof allocation % Callocationto cores Plant [ Plant

33 33 P (ng) P (ng) P] (ngP] g Variable Variable Table3 supplied to host plant suppliedplant host to This article isThis article by protected copyright. All rights reserved. suppliedplant host to 33 15 plant host to external mycelium costsof Relative carbon Functions ( Data tomeetwere ANOVA. log10transformed assumptions of Significantresults a[CO 440 ppm inplantconducted experimentscarbon under for 5) (Fig. (Fig. 4) phosphorus inclusion fungal onfungal-acquired symbiont(s) and exchange of 2. Table symbiosis fungiinMucoromycotina andGlomeromycotina with liverworts. ppm a[CO P<

Accepted P orthophosphate N Article

0.05) areindicatedin from algal necromass from -1 ) ) C . Summary of three-way ANOVA (GLM) results the of testing Summary algae effectsthree-way ANOVA of (GLM) to Summaryof keyand single findings benefitsof ofthecosts anddual 2 ].

49.84 Symbiosis (S) (S) Symbiosis 4.96 0.96 8.99 F d.f. 2,48

<0.001 <0.001 0.011 0.00 n.s. n.s. P P

1 1

bold 6.87 0.08 4.42 1.50 F at ambientat CO algal necromass Reducedby Low High Low fungi Mucoromycotina elevated CO at but not d.f. 1,48 CO

, n.s. , 2 (C) (C) 0.012 -High -High

n.s. n.s. n.s. n.s. P P

= 10.57 P 0.04 2.23 0.50 F >0.05. Algae (A Algae d.f. 1,48 2

0.002 n.s. n.s. n.s. n.s. P P )

algal necromass algal necromass Unaffected by High Low Low fungi Glomeromycotina

0.59 2.87 5.24 5.39 F

d.f. 2,48 Mycorrhiza type Mycorrhiza type

S x C S xC 0.009 0.008 n.s. n.s. n.s. P P

1.49 1.39 1.97 0.54 d.f. 2,48 F S x A S x

necromass necromass Unaffected byalgal Moderate-High High High Glomeromycotina MucoromycotinaBoth and n.s. n.s. n.s. n.s. n.s. P P

0.15 0 0. 1.84 2 .98 F F d.f. 1,48 10 10 ] and] 1,500 C x

A n.s. n.s. n.s. n.s. n.s. P P

1.77 1.53 4.60 1.13 F S x C x A S xC d.f. 2,48

0.015 n.s. n.s. n.s. n.s. P P