Spatial Separation of Litter Decomposition and ...

Spatial Separation of Litter Decomposition and Mycorrhizal Nitrogen Uptake in a Boreal Forest Author(s): Björn D. Lindahl, Katarina Ihrmark, Johanna Boberg, Susan E. Trumbore, Peter Högberg, Jan Stenlid and Roger D. Finlay Source: New Phytologist, Vol. 173, No. 3 (2007), pp. 611-620 Published by: Wiley on behalf of the New Phytologist Trust Stable URL: http://www.jstor.org/stable/4131193 . Accessed: 14/06/2014 16:27 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp

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New

Re

e

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Phytologist

Spatial separation nitrogen uptake

of in

litter a

boreal

and mycorrhizal decomposition forest

Bjorn D. Lindahl1, Katarina Ihrmark', Johanna Boberg', Susan E. Trumbore2, Peter H6gberg3, Jan Stenlid' and Roger D. Finlay1 of Earth Box7026,SE-75007 Uppsala, of ForestMycology Swedish of Agricultural Sweden; Sciences, & Pathology, University 2Department 'Department ofAgricultural of California of ForestEcology, Swedish atIrvine,Irvine,CA92697-3100,USA;3Department Sciences, University SystemScience,University SE-901 83 Umef, Sweden

Summary Authorforcorrespondence: Bj6rnLindahl Tel:+46 18 672725 Fax:+46 18 673599 Email:[email protected] Received:30 August2006 Accepted:22 September2006

* Our understandingof how saprotrophicand mycorrhizalfungi interactto recirculatecarbonand nutrientsfrom plantlitterand soil organicmatteris limitedby poor understandingof theirspatiotemporaldynamics. * In orderto investigatehow differentfunctionalgroups of fungi contributeto carbon and nitrogen cycling at differentstages of decomposition,we studied changes in fungal communitycompositionalong verticalprofilesthrougha Pinus sylvestrisforestsoil.We combinedmolecularidentificationmethodswith 14Cdating of the organic matter,analysesof carbon:nitrogen(C:N) ratiosand 15Nnatural abundancemeasurements. * Saprotrophic confinedto relativelyrecently(< 4 yr)shed litter fungiwere primarily the forest on the surface of floor,whereorganiccarbonwas mineralized components while nitrogenwas retained.Mycorrhizal fungi dominatedin the underlying,more litter and where humus, they apparentlymobilizedN and made it decomposed availableto theirhost plants. * Our observationsshow that the degradingand nutrient-mobilizing components for of the fungalcommunityarespatiallyseparated.Thishas importantimplications forest studies of boreal ecosystems. biogeochemical Keywords: carbon, community, fungi, nutrient cycling, podzol, soil, terminal restrictionfragmentlengthpolymorphism(T-RFLP). New Phytologist (2007) 173: 611-620

? TheAuthors(2006). Journalcompilation? New Phytologist(2006) doi: 10.1111/j.1469-8137.2006.01936.x

Introduction Borealforestscover c. 11% of the land surfacesof the earth and rivaltropicalrainforestsas the largestterrestrialbiome. by cold climate,a long periodof snow They arecharacterized cover,slow decompositionof organicmatterand poor plant nitrogen(N) supply(Persson,1980). In borealforests,fungi play pivotal roles in the circulation of carbon (C) and nutrientsthroughthe ecosystem.Saprotrophicfungi arethe principaldecomposersof wood and litter (Rayner& Boddy, 1988) and obtain energyby degradingdead organicmatter. Mycorrhizalfungi, in contrast, obtain energy mainly as

photoassimilatesprovidedby symbioticallyassociatedplants and in return provide their plant hosts with soil-derived nutrients (Smith & Read, 1997). More than half of the biological activity in boreal forest soils is driven by the to roots, supply of recentlyphotosynthesizedcarbohydrates mycorrhizalfungi and other root-associatedmicroorganisms andmycorrhizal (H6gbergetaL,2001). Althoughsaprotrophic fungi are likely to play fundamentallydifferentroles in the ecosystem, many ecological theories and methods treat as a single,ubiquitously them and othersoil microorganisms occurring, functional group. Little is known about the spatiotemporaldistributionof these functional groups of 611

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fungi and how they interact during decomposition of plant litter. We used DNA-based identification methods to study the vertical distribution and temporal succession of fungi within a boreal Pinus sylvestrisforest soil. Typical boreal forest podzol soils are characterized by accumulation of organic matter on top of the mineral soil, and the absence of mixing soil animals leads to a profound vertical stratification, with the age of the organic matter increasing with depth (Trumbore & Harden, 1997). The temporal succession of fungi within the organic matter may therefore be studied by analysing the vertical distribution of fungal taxa within the soil. Previous studies of vertical niche differentiation among fungi have been limited to easily culturable saprotrophic microfungi (Sbderstrdm & Baith, 1978) or ectomycorrhizal fungi (Dickie et al., 2002; Landeweert et al., 2003; Rosling et al, 2003; Tedersoo et al., 2003; Genney et al., 2006). The present study encompasses the total fungal community, focusing on the upper organic soil horizons. Frequent switches between saprotrophism and symbiosis have occurred during evolution, and both strategies may be represented within groups of relatively closely related fungal taxa (Hibbet et al., 2000). It is therefore necessary to use molecular techniques to analyse communities down to the level of genera and species in order to draw any conclusions about their functional potential. Here, polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region of the ribosomal genes was combined with the community fingerprinting technique terminal restriction fragment length polymorphism (T-RFLP) (Dickie etal., 2002). In order to identify taxa in the T-RFLP profiles, PCR products from representative samples were cloned, enabling sequencing of the ITS region of the ribosomal RNA (rRNA) genes for individual taxawithin the mixed samples (Landeweert et at, 2003; O'Brien et al, 2005) and subsequent identification by database comparisons. To investigate how different fungi influence C and nutrient cycling at different soil depths, we compared the fungal

community composition in the samples with vertical patterns in soil C:N ratio and 15N abundance (Nadelhoffer & Fry, 1988). Analysis of 14Cderived from nuclear bomb tests (Gaudinski et al., 2001) enabled us to determine the age of the organic matter and convert the vertical gradient to a true temporal gradient.

Materialsand Methods Samplingand DNAextraction Soil samples were collected from a Pinus sylvestrisL. forest with an understory of ericaceous dwarf shrubs (Vaccinium vitis-ideae L. and Calluna vulgaris (L.) Hull) and mosses (Pleurozium schreberi(Bridel) Mitten and Dicranum majus Turner).The study was conducted in Jidrals IhV (60o49' N, 16030' E, altitude 185 m), which is a well-documented (Persson, 1980) field site in central Sweden. In total, 27 soil cores (28 mm diameter) were collected and subdivided into eight verticalhorizons each, according to Table 1. Soil samples were freeze-driedand ground in a ball mill. DNA was extracted from 50 to 100 mg of dry matter (500 mg of mineralsoil fractions) in a CTAB buffer (3% cetyltrimetylammoniumbromid, 2 mM EDTA, 150 mM Tris-HCI and 2.6 M NaC1, pH 8) at 65'C for 1 h and precipitated from the supernatant with 2-propanol. The extracts were further purified through the Wizard Plus Minipreps DNA Purification System (Promega, Madison, WI, USA), the Geneclean Kit (Qbiogene, Irvine,CA, USA) or the QIAprep Spin Miniprep Kit (Qiagen, Venlo, the Netherlands), all of which are commercially available DNA cleaning kits.

PCR,cloning,sequencingand sequenceanalysis DNA from all the different horizons was used for PCR, cloning and sequencing (Landeweert et al., 2003; O'Brien et al, 2005). In total, PCR products from 38 samples were

Table1 Subdivisionof soil coresintoverticalhorizons Thickness (mm)

Layer

Description

Needles Litter1 Litter2 (needles) Litter2 (mosses) Fragmentedlitter Humus1 Humus2 E-horizon B-horizon

Recentlyabscisedneedles Pineneedlesamonggreen partsof mosses intactneedlesamongdead mosses Structurally Dead but structurally intactpartsof mosses The upperhalfof the organichorizon The uppertwo-thirdsof the lowerhalfof the organichorizon Lowerthirdof the lowerhalfof the organichorizon The paleeluvialhorizonof the mineralsoil The rust-redilluvialhorizonof the mineralsoil

A14C (%o) 86 (84-90) 95 (93-96) 103 (93-130)

15 (7-25) 10 (5-15) 5 (3-10)

155 (125-181) 211 (184-247) 174 (144-230)

Estimated age (yr) 0 1 3 10 16 > 45

Thethicknessof the organichorizonsis givenas the meanvaluewith the rangein parentheses.The 14Cabundanceis givenas the meanA14C of threesamplesperhorizon(fivefor litter2 (needles))withthe rangein parentheses.Theestimatedage is basedon the meanA14Cvalues, and needleabscissionage (3 yr) is subtractedfor allfractions.

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cloned, and up to 25 clones were sequenced from each sample, resulting in 248 sequenced clones. PCR was carried out using the primers ITS1-F (Gardes & Bruns, 1993) and ITS4 (White et al., 1990) with 550C annealing temperature. PCR products were cloned with the TOPO TA Cloning Kit with the pCR?2.1-TOPO vector and One Shot TOP10 chemically competent Escherichiacoli (Invitrogen, Carlsbad, CA, USA). Small amounts of bacteria were used directly for PCR, which was carried out us above, but with primers M 13 Forward (GTAAAACGACGGCCAG) and M13 Reverse (CAGGAAACAGCTATGAC). The PCR products were purified with the QIAquick PCR purification kit (Qiagen) and sequenced with a CEQ 8000 Genetic Analysis System with the CEQ DTCS Quick Start Kit (Beckman Coulter, Fullerton, CA, USA). Sequences from cloned fragments were compared with database sequences (NCBI) using the BLASTNalgorithm (Altschul et al., 1997), and database sequences with high homology to soil-derived sequences were selected as references. Sample sequences and database referenceswere aligned (DNAStar Inc., using the CLUSTALWalgorithm of MEGALIGN Madison, WI, USA) with the 'gap penalty' parameterreduced to 6 for pair-wise alignment and 10 for multiple alignment. Aligned sequences were compared for similarity by neighbour 4.0b10 (Swofford, 2002). joining using PAUP*

T-RFLP In order to enable processing of a large set of samples, the cloning approach was combined with T-RFLP analysis (Dickie et al., 2002). All samples (27 cores x 8 horizons = 216 samples) were used for T-RFLP analysis. In addition, to enable matching of T-RFLP patterns with sequences, all clones that were sequenced were also subjected to T-RFLP analysis. PCR was carried out as above, but the primers were labelled with WellRED fluorescent dyes, ITS 1-F with D3-PA and ITS4 with D4-PA (Proligo, Boulder, CO, USA). The PCR amplicons were digested with restriction enzymes according to the manufacturers' instructions. The library clones were digestedwith AluI (AmershamBiosciences,Freiburg,Germany), BsuRI(Fermentas,Burlington, Canada), CfoI(Promega),Hinfl (Promega) and TaqI (Fermentas), separately. PCR products from soil samples were digested with TaqIand Cfol, separately, and, when further resolution was needed, with one or more of the other enzymes included in the library. The T-RFLP patterns were analysed with a Beckman Coulter CEQ 8000 Genetic Analysis System, using the CEQ DNA Size Standard Kit-600. Sample T-RFLP patterns were compared with the reference database constituted by T-RFLP patterns from the clones using the program TRAMP(Dickie et al., 2002). To account for base calling during analysis on the Beckman Coulter CEQ 8000 Genetic Analysis System and for withintaxa variation of the ITS region, the threshold level for was set to +2 bp. fragment size in TRAMP

Elementand isotope analyses Threesamplesfromeachof the uppersix organicsoil horizons (five samples of 'litter 2 (needles)')were analysedfor 14C content at the Centerfor AcceleratorMass Spectrometryat LawrenceLivermoreNational Laboratory(Gaudinskiet al., 2001). Radiocarbon data are expressed as A14C, defined as the

differencein permillebetweenthe 14C:12C ratioin the sample and thatof a universalstandard(oxalicacidI, decay-corrected to 1950). The age of the organicmatterwas estimatedby relating the

14C

content of the samples to published records

of atmospheric '4C concentrations from the last 50 yr (Levin& Kromer,2004). Averageresidencetime on theforest floor for the sampled organic matter was estimated by subtractingthe age of recentlyabscised needles from the averageage of the samples.All sampleswere analysedfor N and C content and 615Nusinga C and N elementalanalyser coupledon-line to an isotoperatiomassspectrometer(Model 20-20 IRMS;EuropaScientificLtd,Crewe,UK) accordingto the proceduresoutlined in Ohlsson & Wallmark(1999). Resultsare expressedin 815N deviationsfrom the standard - 1) 1000, where atmospheric N2: g15N = X (Rsample/Rstandard R denotesthe ratio15N: 14N.

Statisticalanalyses The fungal community compositionwas relatedto spatial locationof the samplesandchangesin elementalcomposition by canonicalcorrespondence analysis(CCA)(terBraak,1986) version 4.5 for Windows (Microcomputer using CANOCO Power, Ithaca, NY, USA). Two separateCCA tests were conducted,one with soil horizonsincludedin the analysisas dummyvariables,and one with changesin C:N ratioand 15N natural abundanceas explainingvariables.In the second analysis,only litter 1 (LI), litter2 (needles)(L2), fragmented litter (F) and humus 1 (H1) sampleswere included.Changes in C:N ratioand15Ncontentthroughoutspecifichorizonswere estimatedby comparingdatafrom the horizonsimmediately above and below a subsample.For L1 samples,valuesfrom recentlyabscisedneedleswere subtractedfrom L2 (needles) values.ForL2 (needles)samples,L1valuesweresubtracted from F values. For F samples,L2 values (averageof needles and mosses)weresubtractedfromH1 values,and forHi samples, valuesfromF samplesweresubtractedfromH2 values.Monte Carlo analysiswith 1000 permutationswas used to test for statisticalsignificanceof the environmentalparametersin explaining the variationin community composition. Two taxa,whichwerehighlydominating Thelephoralean mat-forming in two soil cores,wereexcludedfromthe multivariateanalyses, as the samplesfrom these coresdrasticallydeviatedfrom the othersin communitycomposition.Differencesbetweensoil horizonsin C:N ratiosand 15Nnaturalabundanceweretested for statisticalsignifianceusing analysisof variance(ANOVA) and Fisher'sprotectedleastsignificantdifferencetest (PLSD).

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Results PCR productsandT-RFLPcommunityprofileswereobtained from 204 of the 216 collected samples. Among the 248 sequencedclones, 100 differentgenotypeswere identified, which all yielded unique combinations of restriction fragments,and 91 of thesewereencounteredin the sampleTRFLPprofiles.The nine taxathatwerenot retrievedprobably had T-RFLP patterns with amplitudes below the 5% detectionthreshold. The samplesseparatedinto two distinctgroupsaccording to the compositionof the fungalcommunity,with samplesof intact needles containing different fungi from those in samplesof fragmentedlitter,humus and mineralsoil. Taxa recordedfrom intactneedleswereonly detectedsporadically in the more degradedmaterialand vice versa.Dead partsof mossescontainedan intermediatefungalcommunity(Fig. 1). Withinthe deeperhorizons,changesin the fungalcommunity with soil depth could be observed,with some speciesfound predominantlyin the fragmentedlitterandhumusandothers being most common in the mineralsoil. Canonicalcorrespondenceanalysisattributed10%of the totaleigenvalue(the variationin species composition between samples)of the datasetto the verticalpositionof the samples.A Monte Carlo permutation test found the relationshipbetween vertical positionandcommunitycompositionof samplesto be highly significant(P= 0.001). The 'early'fungalcommunity,definedasthe taxaoccurring with a higherfrequencyin littersamplescomparedwith older organicmatterandmineralsoil,wasdominatedby unidentified ascomyceteswithin the Helotiales and Dothideomycetes. Known saprotrophicfungi, such as Mycenaand Marasmius spp., as well as plant parasiticLophodermium spp., were also found in the surfacelitter. In the 'late'fungal community, defined as taxa occurringwith a higher frequencyin fragmented litter and humus than in litter, ectomycorrhizal fromthe generaCortinarius and basidiomycetes, predominantly Piloderma,constituteda significantfraction.Ascomycetous ericae(D.J.Read)W.Y.Zhuang Capronia spp.andRhizoscyphus & Korf,whichboth formmycorrhizalassociationswith ericaceousplants(Allenet al, 2003), werealsocommon.The 'late' fungalcommunitycontainedtaxafromwithin the Helotiales, buttheseweredifferenttaxafromthehelotialeanfungidetected in surfacelitter. Zygomycetes(Mortierellaand Umbelopsis spp.) as well as a distinctgroup of unidentifiedascomycetes (groupG) alsocontributedto the 'late'community(Fig. 1). The average14Cabundanceof the samplesincreasedwith depth and peakedin the upperhumus layer,but was lower again in the bottom part of the humus, where the organic matterwasassumedto be derivedfromplantmatterproduced before the rapid increase in atmospheric 14C content associ-

ated with thermonuclearweaponstestingduringthe 1960s. The averageage of the C in the upperhumuswas estimated at 16 yr, whereas the fragmentedlitter, older litter and


youngerlitter were estimatedto have residedon the forest floorfor on average10, 3 and 1 yr,respectively(Fig.2). Throughoutthe upperlitter layers,C:N ratiosdecreased threefoldfrom recentlyshed litter down to the fragmented litter.Fromthe fragmentedlitterdown to the oldesthumus, C:N ratiosincreasedsignificantlyby22%,followedbya minor but significantdeclinein the mineralsoil. In contrast,the 15N abundancewasconstantin the litterlayers,but increasedwith soil depthfrom the fragmentedlitterand downward(Fig.2). In a canonical correspondenceanalysis,where the fungal communitiesin litter, fragmentedlitter and upper humus sampleswere relatedto changesin organicmatterC:N ratio and15Ncontentacrossthehorizons,7.3%of thetotaleigenvalue of the datasetwas attributedto thesetwo variables.A Monte Carlo permutationtest found the relationshipbetweenthe community composition of samples and changes in C:N ratioor 15Nabundanceto be highly significant(P= 0.001). Accordingto the multivariateanalysis,fungaltaxaknownto be mycorrhizalwereassociatedwith positivechangesin C:N ratio and largeincreasesin 15N naturalabundance,whereas known saprotrophicfungi were associatedwith negative changes in C:N ratio and small changes in 15N natural abundance(Figs2, 3).

Discussion We found a clear shift in fungal community composition betweenthe surfacelitter(the L horizon)and the underlying F horizon,in which the litterhad lost its structuralintegrity (Figs1, 2). Accordingto the 14C enrichmentof the organic matter,the oldestmaterialin the L horizonhad beenresiding on the forestfloor for c. 3 yr, whereasthe averageage of the organicmatterin the F layerwas 10 yr.As the C in the uppermost partof theF layeris youngerthanthe averageageforthe entirehorizon,the shiftin fungalcommunitycompositionis likelyto takeplaceafterc. 3-5 yr of decomposition. species,which werefrequentlyrecordedin Lophodermium the L1 samples,are well-knownneedle endophytes,which havealreadycolonizedthe needleswhen they enterthe forest floor, but seem to be replacedby other fungi at a relatively earlystageof decomposition(Fig. 1). The only saprotrophic basidiomycetethatwasrecordedwith a high frequencywasa Mycenaspecies.The most commonly detected taxa in the 'early'communitywereascomyceteswithinthe Helotialesand Dothideomyceteswith uncertain taxonomy and ecology. However,astheyrarelyextendedbelowthe surfacelayerof the forestfloor,which is devoidof roots,it seemslikelythatthey aresaprotrophs. A large proportionof the 'late' community consists of ectomycorrhizalspecieswithin the genera Cortinariusand Piloderma. Whereasborealtreesgenerallyformectomycorrhizal associations,ericaceousdwarf-shrubs,such as Callunaand Vaccinium spp., formericoidmycorrhizalassociations(Smith & Read,1997). In thisstudy,Rhizoscyphus ericae,whichis the




New Phytologist Sequence

I

ReseBr

Taxon

test2.2b.2 Venturiaceae test2.1b.11 Helotiales(Phialophora sp.) Blz 2.1 HelotialesgroupA A2z 3.6 HelotialesgroupA B2z 2.5 O Mycenasp. A3x2.1 Helotiales test2.1b.12 Helotiales Aix 3.13 Dothideomycete groupB test2.lb.9 Lophodermium pinastri O]Cantharellales Aly 2.11 test2.2b.11 El Marasmius androsaceus Cantharellales A3y2.14 A3x2.3 ElO Lophodermium sp. A2z 3.10 Helotiales testl.8a.27 Helotiales test2.2b.3 Rhytismataceae groupC C3x 1.9 Cantharellales A2z 5.3 Basidiomycete Blz 2.3 Basidiomycete Mollisiasp. A3y3.11 A2x2.8 Helotiales A2z 3.11 Caproniasp. Clz 3.5 Cantharellales HelotialesgroupD A3y3.5 test2.2b.15 Ascomycete A2z 3.5 Caproniasp. A3x2.2 HelotialesgroupA Venturia A3y3.14 sp. Unknown B2z 2.4 Dothideomycete groupB A2z 4.7 Rhytismataceae groupC A2x2.7 Cladosporium sp. 3.4 Venturiaceae A3y test2.3b.16 M Sebacinasp. HelotialesgroupA A3y3.3 A2x2.6 Basidiomycete test2.1b.10 HelotialesgroupD Clz 3.1 Caproniasp. Aix 3.16 Cortinarius sp. Clz 3.6 O Galerinaatkinsoniana B3z4.13 Basidiomycete testl.8a.2 Helotiales testi.8a.25 Tremellales Blz 4.9 Cortinarius sp. Cortinarius armeniacus C2y4.9 A2z 5.8 HelotialesgroupE Unknown Biz 4.3 testl.8a.6 Caproniasp. Sebacinasp. test2.3b.13 A2z 5.4 Unknown A2z 3.3 Helotiales testl.8a.8 Morteriella sp. test2.3b.15 Dermocybesp. testi.8a.5 Basidiomycete Helotiales Aly 2.14 test2.1b.15 Basidiomycete 8.3 C2y Caproniasp. test2.3b.6 Caproniasp. Cly 8.5 AscomycetegroupG Cortinarius A3y3.7 sp. Clz 5.10 Ascomycete Aix 3.11 Atheliacceae A2x5.1 Thelephorales Cly 8.4 Umbelopsisisabellina A3x7.3 Umbelopsissp. test2.3b.10 Basidiomycete A2z 5.13 Umbelopsissp. C3z4.1 Mollisiaminutella A2z 4.9 A Rhizoscyphusericae testl.5b.1 Morteriella sp. aurantiacum Aly 5.3 Hydnellum C3z 4.2 Dermocybesemisanguinea B3z4.4 Cortinarius sp. Helotiales Aly 5.4 A3z 7.4 fortinii Phialocephala testl.5b.4 Unknown U Cortinarius Blz 4.2 armillatus testl.8a.1 HelotialesgroupF U Cortinarius A3z 6.7 sp. C2y8.6 Umbelopsissp. test2.4b.10 A2z 3.1 test1.8a.4 C2y 8.2 A2z 5.11 A3z 7.2 Clz 5.6 A3z 6.8 Clz 5.7 test2.4b.9 A2z 4.6

U

A U

Rozites caperatus Capronia sp. Helotiales group F Ascomycete group G Ascomycete group G Piloderma sp. Helotiales group E Ascomycete group G Unknown (Ascomycete group G) Piloderma reticulatum Ascomycete group G

Litter1

Litter2 (needles)

0.69 0.38 0.35 0.12 0.23 0.27 0.31 0.04 0.46 0.15 0.15 0.12 0.19 0.19 0.19 0.08 0.08 0.04

0.85 0.41 0.30 0.48 0.33 0.22 0.22 0.44 0.04 0.15 0.11 0.07

0.04 0.04 0.04 0.08 0.08 0.08 0.12 0.04 0.04

0.04 0.07 0.04 0.07 0.19 0.07 0.04 0.04 0.04 0.04 0.07

Litter2 Fragmented Humus1 litter (mosses) 0.70 0.48 0.41 0.59 0.26 0.22 0.04 0.30

0.11 0.07 0.11 0.11 0.11

0.05

0.04 0.04 0.11

0.04 0.04 0.04 0.04

0.04

0.26 0.11 0.04 0.07 0.37

E-horizon B-horizon

0.04 0.07

0.07

0.04 0.26 0.30 0.15 0.07 0.07 0.19 0.04

Humus2

0.04

0.05 0.05

0.04 0.04 0.11

0.04 0.07 0.11 0.04

0.04

0.04

0.04

0.07

0.04

0.04 0.04 0.11 0.04 0.04 0.04

0.08

0.11

0.04 0.04 0.04 0.04 0.04 0.04

0.04

0.04 0.11 0.04 0.11 0.07 0.04 0.15 0.07 0.07 0.04 0.04

0.07 0.04

0.04 0.04

0.04

0.07 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04

0.04 0.07

0.04

0.04 0.04 0.04

0.04

0.08 0.08 0.04

0.04 0.04

0.22 0.19 0.19 0.11

0.19 0.04 0.07 0.04 0.04 0.07

0.04 0.08 0.05

0.04 0.07 0.04

0.04 0.08

0.15 0.07

0.26 0.04 0.04

0.04 0.04

0.22 0.11

0.04 0.12

0.04 0.04 0.11

0.15 0.07 0.37 0.15 0.04

0.04 0.08 0.04

0.07 0.04

0.04 0.04

0.04 0.07

0.11 0.04 0.07 0.04 0.11 0.07 0.19 0.19 0.04 0.07 0.15 0.41 0.04 0.15

0.04

0.15

0.26 0.04

0.04 0 44 0.04

0.11 0.15 0.04 0.07 0.30 0.22

0.30 0.07 0.33 0.59 0.74

0.05 0.05

0.04 0.07 0.04 0.04 0.07 0.04 0.07 0.04 0.07 0.07 0.07 0.22 0.30 0.22 0.11 0.07 0.11 0.04 0.15 0.15 0.26 0.04 0.04 0.11 0.19 0.30 0.56 0.56 0.85

0.04 0.04 0.04 0.04 0.12 0.04 0.04 0.12 0.08 0.04 0.08 0.04

0.04 0.04 0.04 0.04 0.08 0.08 0.08 0.04 0.15 0.19 0.15 0.08

0.12 0.04 0.08

0.12 0.04

0.04 0.08 0.20

0.12 0.08 0.08 0.08 0.19 0.15 0.38 0.27 0.31 0.46 0.81

0.12 0.08 0.20 0.16 0.16 0.16 0.12 0.16 0.04 0.12 0.48

0.05 0.32 0.16 0.42 0.37 0.26 0.42 0.16 0.05 0.21

of fungaltaxa in soil samplesfroma Pinussylvestrisforest.Columnsrepresentdifferentverticalhorizonsranging Fig.1 Verticaldistribution from'litter1', whichis needlesat the surface,down to the 'B horizon'of the mineralsoil.Cellfiguresareobservationfrequenciesandthe cells are coded as follows:lightgrey< 0.1; mid-grey0.1-0.3; darkgrey> 0.3. Open squares,knownsaprotrophic taxa;closedsquares,known taxa. taxa;triangles,knownericoidmycorrhizal ectomycorrhizal




616

..

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Estimated age

0

20 40

60 80 100% 0

0

Needlesat abscission

aa

I1

Litter1

b

3Litter2 10 16 > 45

C:Nratio

composition Fungalcommunity

(yr)

30 60

o, .a

cda

needles mosses

e

Humus 1 Humus 2

de

E horizon

a a

g

cde

litter Fragmented

abundance 15Nnatural

90 120 150 -5-4-3-2-1 0 1 2 3 4 5 6

bMiib c

c

d

e

e

Mineral soil B horizonf

m

'early'fungi (I knownsaprotrophic fungi) 'late'fungi(gl knownmycorrhizal fungi) (C:N)ratioand 15Nnaturalabundancethroughoutthe uppersoil profilein a Fig.2 Fungalcommunitycomposition,carbon:nitrogen Scandinavian Pinussylvestrisforest.Differentlettersin the diagramsindicatestatistically significantdifferencesbetweenhorizonsin C:Nratios and 15Nabundance,and the standarderrorof the meanwas < 0.3%ofor 15Nnaturalabundanceand < 3 for C:Nratio(n = 19-27, for recently abscisedneedlesn = 3). Theage of the organicmatteris estimatedfromthe averageA14Cof threesamplesfromeach horizon(fivesamplesof the litter2 (needles)fraction)and needleabscissionage (3 yr)is subtracted.Communitycompositiondataareexpressedas the frequencyof totalobservations.'Early'fungiaredefinedas those occurringwitha higherfrequencyin littersamplescomparedwitholderorganicmatterand mineralsoil. 'Late'fungiarethose occurringwith a higherfrequencyin olderorganicmatter.

LO

C:N increase

Cortinariussp

Cortinanrius

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most thoroughly studied ericoid mycorrhizal fungus, was recorded less often than Capronia taxa, which have only recently been confirmed as ericoid mycorrhizal symbionts (Allen etal., 2003). In contrast to the helotialean taxa found in the surface litter, helotialean taxa in the 'late' community generally show a high degree of sequence homology to taxa recorded from coniferous or ericoid roots, suggesting that the


Fig.3 Canonicalcorrespondenceanalysis depictingthe relationbetweenfungal communitycompositionand changesin (C:N)ratioand 15Nnatural carbon:nitrogen abundancein a borealforestsoil.Thearrows representincreasingC:Nratioor 15N abundancewith depthand age of the organicmatter.Open squares,known taxa;closedsquares,known saprotrophic taxa;triangles,known ectomycorrhizal ericoidmycorrhizal taxa;grey squares,taxa with uncertainecology.

'late' helotealean taxa are mycorrhizal or in other ways associated with roots (Fig. 4). The zygomycetous Umbelopsisspecies found in the 'late' community show a high degree of sequence homology to fungi isolated from surface-sterilized, healthy roots of Pinus ponderosa,suggesting that these fungi are root endophytes (Hoff et al., 2004). The unknown ascomycetes (group G) that were detected in the deeper samples belong to




New Phytologist

I Rese~~ss

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New Phytologist

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a recentlydiscoveredlineage(Schadtet al., 2003) thathitherto has been recordedonly as environmentalDNA and seemsto be phylogeneticallyseparatefrom otherknown ascomycetes. DNA from this group has frequentlybeen obtained from ectomycorrhizalconiferousroots (e.g. Rosling et al., 2003), or root suggestingthat these fungi are also ectomycorrhizal 4). endophytes(Fig. Basedon mycelial13Cabundanceand drasticallyreduced mycelialproductionin plots with severedroot connections, Wallanderet al. (2001) claimedthatmost of the myceliumat This claim the humus/mineralsoil interfacewas mycorrhizal. is now supportedby our observationthatthe vastmajorityof DNA belowtheuppermostmossandlitterlayermaybe assigned to knownmycorrhizal fungior otherwiseroot-associated fungi. In particular,the absence of DNA from nonhelotialean such as Trichoderma ascomycetes,including'soilsaprotrophs' or Penicilliumspp., in the moredecomposedsamplesis noteworthy.The scatteredrecordsof 'early'fungi in humus and mineralsoil samples(Fig. 1) maybe an artefactcausedby litter fragmentsbeingpusheddownwardsby the samplingtool. The findingthat dead partsof mosses,in additionto the 'early'fungal community of the surroundingneedles, also containmycorrhizalfungi is in accordancewith earlierobservationsthatmyceliumof Cortinarius spp. often extendsfrom roots in deepersoil horizonsto colonize dead moss tissues (Genney et al, 2006). Mycorrhizalfungi have also been shownto efficientlycolonizedeadmossesin soil microcosms (Carleton& Read, 1991). Samplescolonizedbythe'early'and'late'fungalcommunities differedsignificantlywith respectto theirC andN dynamics, as shown by the canonicalcorrespondenceanalysis(Fig.3). Throughoutthe upperlitterlayers,wherethe 'early'and presumablysaprotrophicfungal communityis active,the C:N ratio decreaseswith time. DecreasingC:N ratioswith progressing decomposition are commonly observed (Berg & McClaugherty,2003) and indicaterespiratoryremovalof C in combinationwith retentionof N within the litterand its microbialcommunity(Lindahlet al, 2002). In contrast,the 'late'fungalcommunityof mycorrhizalandotherpresumably root-dependentfungi, which was found in the fragmented withincreasing litterandhumus,wasassociated C:N ratioswith of N driven removal matter selective indicating organic age, C (Figs2, 3). Thispresumptionis corroborated by root-derived by the simultaneouslargeincreasein the stableisotope 15N, whichmustbe drivenby fractionationagainstthe heavierisotope duringtransferof N from the soil throughmycorrhizal fungi to their host plants (Hbgberget al., 1996; H6gberg et al., 1999; Hobbie & Colpaert,2003). Duringthis transfer, ectomycorrhizalfungi become c. 10%omore enrichedthan theirhostplants,whichis approximately the differencebetween the litterandthe deepersoil layersobservedhere(Fig.3). The significantenrichmentin 15Nnaturalabundancein horizons dominatedby ectomycorrhizal fungithus indicatesthatplant N is mobilizedvia root-associatedmycorrhizalfungi.


In a classicpaper,Gadgil& Gadgil(1971) suggestedthat ectomycorrhizaland saprotrophicfungi competewith each other for N, and such competitiveinteractionscould act to maintainthe partitioningof the fungalcommunityinto two verticallyseparatedand functionallydistinct subcommunities, as observedin the presentstudy.In soil microcosmswe have previouslyobservedantagonisticinteractionsbetween saprotrophicfungi and ectomycorrhizal fungi (Lindahlet al., 1999), in which the outcomewas stronglyinfluencedby the amount of substrateavailableto the saprotrophs(Lindahl et al., 2001). Saprotrophicfungi are more efficient than mycorrhizal fungiin colonizingandutilizingfresh,energy-rich litter (Colpaert& vanTichelen, 1996) and may thus be able to outcompetemycorrhizalfungi from the upperpartof the forest floor. However, as the C:N ratio decreasesand the substratebecomes depleted in availableenergy,the saprotrophsshould become less competitive,which is consistent with the observedreplacementof saprotrophsby mycorrhizal fungi thatdo not dependon litter-derivedenergy. Ligninand humus arebuilt up from polyphenolicstrucand turesthatrequireoxidativeenzymesfortheirdegradation, it hastraditionallybeen assumedthat saprotrophic'whiterot fungi' are the only organismswith the capacityto degrade theserecalcitrantcompoundsfully (Rayner& Boddy,1988). In this study,saprotrophicfungi appearedto be confinedto the surfacelitter,and humusdegradationand nutrientmobilizationfrom deeperorganicmatterwould thereforehave to be ascribedto other functionalgroups of microorganisms suchasbacteriaor mycorrhizal fungi.Somebacteriahavebeen shown to utilize productsof lignin degradation(Merkens et al., 2005) but reportsof lignin and humusdegradationby bacteriaarescarce(Berg& McClaugherty,2003). However, andericoidmycorrhizal the capacityof ectomycorrhizal fungi to produceenzymesinvolvedin degradationof organicmatter and to mobilizeorganicformsof N is well documentedfrom laboratoryexperiments(Abuzinadahet al, 1986; Read & Perez-Moreno,2003; Lindahl etal., 2005). Melillo et al. (1989) emphasizedthe importanceof well-degradedorganic matterand humus in supplyingplant N and suggestedthat labileC enteringthe soil via rootsand associatedmycorrhizal fungi may play an importantrole in drivingmobilizationof thisN. The dominanceof mycorrhizalfungiin well-degraded litterand humus observedin the presentstudysupportsthe hypothesisthat mycorrhizalfungi play a significantrole in mobilizing N from well-decomposed organic matter in borealforestsoils. In less degradedlitterthe lack of mycorrhizal fungi implies that the major part of needle litter decompositionis carriedout by saprotrophson the surface of the forestfloor. Borealforestshavebeenidentifiedas a majorglobalC sink (Myneniet al., 2001), and interactionsbetweenthe cyclesof C andN havebeenof paramountinterestin discussionsabout predictionsof effectsof increasingatmosphericCO2 concentrationandN depositionon borealandtemperateforests(e.g.




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Townsend et al., 1996; Nadelhoffer et al., 1999). Our observations highlight the need for further studies of C and N cycling in temperate forest ecosystems to acknowledge the spatial separation of major functional components of the microbial community.

Acknowledgements Financial support from the Swedish University of Agricultural

Sciencesis gratefullyacknowledged.Thanksare also due to Andy Taylorand David Read for good advice and critical readingof the manuscript.

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