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Soil Biology & Biochemistry 43 (2011) 1350e1355

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Soil Biology & Biochemistry

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Species richness of ectomycorrhizal hyphal necromass increases soil CO2 effluxunder laboratory conditions

Anna Wilkinson*, Ian J. Alexander 1, David Johnson 1

Institute of Biological and Environmental Sciences, Cruickshank Building, University of Aberdeen, Aberdeen AB24 3UU, UK

a r t i c l e i n f o

Article history:Received 8 October 2010Received in revised form2 March 2011Accepted 7 March 2011Available online 22 March 2011

Keywords:Ectomycorrhizal myceliumDecompositionSoil respirationBiodiversityCarbon cyclingForest soils

* Corresponding author. Tel.: þ44 (0) 1224 273857E-mail addresses: a.wilkinson@abdn.ac.uk (A. Wil

uk (I.J. Alexander), d.johnson@abdn.ac.uk (D. Johnson1 Tel.: þ44 (0) 1224 273857; fax: þ44 (0) 1224 272

0038-0717/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.soilbio.2011.03.009

a b s t r a c t

The ectomycorrhizal mycelium is a large component of boreal and temperate forest soil microbialbiomass and the resulting necromass is likely to be an important source of nutrients for saprotrophicmicroorganisms. Here we test the effects of species richness of ectomycorrhizal mycelial biomass onshort-term CO2 efflux by amending forest soil with necromass from 8 fungal species added separatelyand in mixtures of 2, 4 and 8 species. All additions of necromass rapidly increased soil CO2 effluxcompared to unamended controls but CO2 efflux increased significantly with species richness. Efflux ofCO2 did not correlate with the carbon (C) or nitrogen (N) contents or the C:N ratio of the added nec-romass. The study demonstrates that species diversity of dead ectomycorrhizal fungal hyphae can haveimportant consequences for soil CO2 efflux, and suggests decomposition of hyphae is regulated byspecific constituents of the nutrient pools in the necromass rather than the total quantities added.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Ectomycorrhizal (EM) fungi are a keystone group of microor-ganisms in forest ecosystems where they undertake importantfunctional roles, notably in regulating biogeochemical cycles andimproving plant nutrition (Smith and Read, 2008). The length of EMmycelium can be up to 8000m per metre of root (Leake et al., 2004)and evidence from girdled Pinus sylvestris forest plots in northernSweden suggest that EM mycelium may constitute a third ofmicrobial biomass in coniferous forests (Högberg and Högberg,2002). It has been estimated that around 20e25% of photosyn-thate carbon (C) transferred belowground is allocated to EM fungi(Smith and Read, 2008) and estimates of respiration from externalmycelium of EM fungi range from 3 to 25% of total soil CO2 flux(Heinemeyer et al., 2007;Moyano et al., 2008). Högberg et al. (2001)found that soil respiration rapidly decreased by up to 56% after treeswere girdled due to the termination of photosynthate flow to EMroots. EM-derived C also enters soil microbial pools through therelease of exudates and hyphal turnover (Finlay and Söderström,1992; Godbold et al., 2006). Godbold et al. (2006) found thatexternal EM mycelium was a dominant pathway through which

; fax: þ44 (0) 1224 272703.kinson), i.alexander@abdn.ac.).703.

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C entered the soil organic matter pool (62%), and quantitativelymore important than C fluxes from leaf litter and fine root turnover.It has been suggested that large amounts of mycorrhizal littercould contribute to lower heterotrophic respiration rates in EM-dominated ecosystems due to their apparent recalcitrant forms(Langley and Hungate, 2003). In contrast, other authors claim thatEM fungal hyphae turned over in days rather thanweeks (Friese andAllen, 1991; Godbold et al., 2006), unlike EM root tips that areestimated to turnover between 139 days and 5 years (Majdi et al.,2001; Rygiewicz et al., 1997; Treseder, 2004).

In forests EM mycelium also represents a significant source ofnitrogen (N) to microorganisms involved in decomposition (Conantet al., 2000; Wallander et al., 2004). Bååth and Söderström (1979)estimated that the N stored in EM mycelium makes up 20% of thetotal soil N in the A horizon. A large proportion of fungal N is in theform of chitin, which contains 6.5% N, and makes up 60% of fungalcell walls (Swift et al., 1979). Evidence suggests that some of thisN is recalcitrant to decomposition and therefore doesn’t controldecomposition rates (Langley and Hungate, 2003), whilst otherwork has shown that some fungi are able to utilise pure chitin andfungal mycelium necromass as their sole sources of nutrients(Kerley and Read, 1997; Leake and Read, 1990). Furthermore, it hasbeen suggested that mycelium might be a rich source of otherpolymeric N sources, for example, the insoluble protein foundin cell walls as glycoprotein in which the chitin microfibrils areembedded (Wessels, 1992). Despite the clear nutritional value and

A. Wilkinson et al. / Soil Biology & Biochemistry 43 (2011) 1350e1355 1351

biomass of EM external mycelium, there is little empirical evidenceto show how it affects microbial activity once it enters soil.

Late-successional boreal P. sylvestris forest plots have been found tosupport tens of species per100m2 (Jonssonet al.,1999). Analysis of thediversity of EM fungal mycelium using fine-scale spatially-explicitsampling in a stand of P. sylvestris clearly shows the potential for thefungi to interact within small (8 cm3) patches of soil (Genney et al.,2006). Whilst the effects of EM diversity on the productivity of thehost plant have received limited attention (Baxter and Dighton, 2001;Jonsson et al., 2001), even less is known about the effects of EMdiversity on C cycling in soil once the fungal hyphae die. Experimentsusing mixtures of plant litter suggest that decomposition of lowerquality litter is accelerated when diversity is greatest because there isagreaterprobabilityofhighquality litterbeingpresent (Seastedt,1984;Wardle et al., 1997). However, decomposition of diverse litter is pre-dicted to be less compared to the most labile litters in monoculture.Koide and Malcolm (2009) found that there was substantial variationin the decomposition rate of a variety of species of EM fungi isolatedfrom a Pinus resinosa plantation. They also found that decompositionrate was positively correlated with the fungal tissue N concentration.However, whether similar patterns of litter decomposition found indiverse plant litter mixtures are seen for fungal litter is unknown.

In this study we test how manipulation of the species richness ofEM fungal necromass addedas the sole inputof C andNaffects soil CO2efflux. We used an experimental design according to Jonsson et al.(2001) in which all of the fungi were represented in monoculture aswell as in combinations of 2, 4 and 8 species. We predicted that EMfungal species in monoculture would lead to differences in soil CO2efflux associated with variations in concentrations of C and N.Therefore, CO2 efflux should be greatest in treatments that receivedhigher quality fungal necromass (i.e. fungi with low C:N ratios). Wealso predicted that microbial activity would increase with the speciesrichness of fungal necromass due to the increased likelihood of a ‘highquality substrate’ species being present in themixed communities (i.e.sampling effects). Whilst there may be possible complementarityeffects, we predict that the most diverse mixtures would not decom-pose faster than the most labile substrate added in monoculture.

2. Materials and methods

2.1. Microcosms

Soil CO2 efflux was quantified using the MicroResp� microplatewhole soil respiration system (Campbell et al., 2003). This systemconsists of two microtiter plates held face to face and sealed witha silicone rubber gasket, with interconnecting holes between the

Table 1Treatment combinations of the 8 ectomycorrhizal fungal species and the total added C a

Treatmentidentity

Speciesrichness

Species combinations

A 1 Cenococcum geophilumB 1 Amanita muscariaC 1 Lactarius rufusD 1 Hebeloma crustuliniformeE 1 Laccaria bicolorF 1 Cortinarius glaucopusG 1 Paxillus involutusH 1 Suillus bovinusFH 2 C. glaucopus þ S. bovinusAD 2 C. geophilum þ H. crustuliniformeCE 2 L. rufus þ L. bicolorBG 2 A. muscaria þ P. involutusADFH 4 C. geophilum þ H. crustuliniforme þ C. glaucopus þ S.BCEG 4 A. muscaria þ L. rufus þ L. bicolor þ P. involutusALL 8 All species

a Measured from a subsample of the added necromass.

corresponding wells of the plates. The rubber gaskets remained inplace for the duration of the experiment because they substantiallyminimise loss of moisture as water vapour (Campbell et al., 2003).The weight of the plates was also checked throughout the experi-ment to confirm this. One of the plates (the ‘detector’ plate) containsa bicarbonate solution that changes colour due to a pH changeinduced by absorption of CO2. The second platewas a deepwell platewith a capacity of 1.2 ml inwhich the soil and fungi were incubated.Each well was filled with 1 g fresh weight (fwt) of sieved (<2 mm)soil obtained fromCulbin forest National Nature Reserve (57�380800N,3�4200700W) in Morayshire, Scotland. The soil was removed froma 120-year-old stand of P. sylvestris, with understorey vegetationdominated by mosses (Rhytidiadelphus triquetrus (Hedw.) Warnst.,Pleurozium schreberi (Brid.) Mitt. andHylocomium splendens (Hedw.)Schimp.) and occasional vascular plants (Goodyera repens (L.) R. Br.,Calluna vulgaris (L.) Hull and Betula pendula Roth. seedlings). The soilwas collected from the upper 15 cm of aeolian sand deposits that liedirectly below the shallow surface organic horizons (Gauld, 1981)andwhich is known to contain a diverse communityof EMmyceliumbut few mycorrhizal root tips (Genney et al., 2006).

The wells of the plate each received a total of 100 mg of fungalnecromass, and the species present depended on the individualtreatment (Table 1; there was also an unamended control). Thelength of incubation and the amount of fungi used in the treatmentswere determined in a preliminary study. The amounts used here areabout twice those estimated to be contained in a sandy forest soil byWallander et al. (2001). A gradient of species richness was createdusing 8 different species of EM fungi (Table 1). Fifteen uniquetreatments were created of which 8 were single species mono-cultures (treatments AeH), 4weremixtures of 2 species (treatmentsFH-BG), 2 were mixtures of 4 species (treatments ADFH and BCEG),and 1 comprised all species (treatment ALL). The 2 and 4 speciesmixtures were drawn at random without replacement. Each treat-ment was replicated 6 times. Individual isolates of the fungi weregrown on modified Melin Norkrans (MMN; Marx, 1969) growthmedia and then killed following the technique used by Kerley andRead (1997). Fungi were immersed in 70% (v/v) ethanol:distilleddeionized water (DDH2O) for 16 h at 2e3 �C, followed by 10 washesof DDH2O to remove ethanol before being lightly macerated witha glass tissue macerator to homogenise samples, from which themixtures were created. The resulting necromass provided the soleadded sources of N and C to the soil.

The MicroResp� plates were incubated at 25 �C for 4 weeks.During the first week, the detection plate was placed above theMicroResp plate for 24 h and for the remaining weeks it was addedfor 6 h once a week. The absorbance of the indicator solution was

nd N and C:N quotienta of necromass used in each treatment.

Total added N (mg) total added C (mg) C:N quotient

0.02 1.51 74.00.24 3.73 15.20.13 2.72 20.10.06 2.47 40.00.11 2.64 24.10.21 3.31 15.60.24 4.32 17.70.16 2.97 18.70.19 3.14 16.90.04 1.99 48.50.07 2.68 39.60.24 4.02 16.5

bovinus 0.11 2.56 22.60.18 3.35 18.30.15 2.96 20.0

Treatment identity

A B C D E F G H FH AD CE

BG

ADFH

BCEG AL

L

CO

2 effl

ux (u

g C

O2-C

g-1

soi

l d-1)

0

5

10

15

20

25

30

Fig. 1. Soil CO2 efflux (mg CO2-C g�1 soil d�1) in response to treatment identity of thefungal necromass (�SEM). Treatments AeH are monocultures of 8 EM species, treat-ments FH-BG are mixtures of 2 species, treatments ADFH and BCEG are mixtures of4 species and treatment ALL contains all 8 species. Treatment identity had a significanteffect on CO2 efflux (L-ratio ¼ 105.8, d.f. ¼ 16, P < 0.001).

week 1 week 2 week 3 week 4

CO

2 effl

ux (µ

g C

O2-

C g

-1 s

oil d

-1)

0

5

10

15

20

25

Unamended controlSR = 1SR = 2SR = 4SR = 8

Fig. 2. Soil CO2 efflux (mg CO2-C g�1 soil d�1) in response to the species richness (SR) ofEM fungal mycelial necromass (solid symbols) during a 4-week incubation (�SEM). EMspecies richness had a significant effect on soil CO2 efflux (L-ratio ¼ 177.1, d.f. ¼ 20,P < 0.001) and there was an overall effect of time (L-ratio ¼ 81.6, d.f. ¼ 20, P < 0.001).

A. Wilkinson et al. / Soil Biology & Biochemistry 43 (2011) 1350e13551352

determined colorimetrically using a Vmax microplate reader(Molecular Devices, USA) and a 570 nm filter. Absorbance valueswere normalised (Ai) by multiplying by the average well colourdevelopment (AWCD) of the plates using the equation:

Ai ¼ ðAt=At0Þ � AWCDðAt0ÞWhere At0 ¼ absorbance at 570 nm (A570) at time zero; At ¼ A570at the end of the study. The A570 data were converted to % CO2using calibration constants:

%CO2 ¼ Aþ B=ð1þ D� AiÞWhere A ¼ �0.2265, B ¼ �1.606, D ¼ �6.771.

The % CO2 was then used to calculate CO2 efflux (mg CO2-C/g/d)using gas constant and constants for headspace volume in the well(ml), fresh weight of soil per well (g), incubation time (d), temper-ature and soil sample % dry weight.

Six replicates of each EM fungal species were dried in an oven at70 �C for 24 h in order to calculate the moisture content of the tissue.Both fresh material and dry material of each fungal species, plus thefresh forest soil were analysed for percentage N and C by flashcombustion in an oxygen-enriched furnace (1700e1800 �C) followedby reduction and separation of the gaseous components by gas chro-matography using a thermal conductivity detector (TCD). This wascarriedoutusinganNCSanalyser (Fisons Instruments,NA1500Series).

2.2. Statistical analysis

CO2 efflux across all time points was analysed using a general-ized least squares (GLS) statistical mixed modelling approach(Bulling et al., 2008). This method is unaffected by the unequalvariance arising from the experimental design because it usesvariance-covariate functions, and so the structure of the data can bemaintainedwithout theneed for transformation.We considered theexperimental additions of fungal biomass as 15 independenttreatments (treatment identity; TID, Table 1) and as 4 levels ofspecies richness (SR); these were analysed in separate models dueto collinearity between the variables. We also accounted for effectsof time and its interaction with both TID and SR (models 1 and 3,supporting information). The fixed structure of the model wasestablished by applying backward selection using the likelihoodratio test obtained by Maximum Likelihood (ML). The numericaloutput of the minimal adequate model was obtained using REMLestimation (West et al., 2007). These analyses were all performedusing the ‘nlme’ package (ver. 3.1) in the ‘R’ statistical andprogramming environment (Pinheiro et al., 2006). The statisticaltests used cannot be applied directly to mean values with standarderrors but instead relate to model predictions; these are presentedin Supplementary material (Table S1). As similar patterns in CO2

efflux between species richness and treatments were observedthroughout the time course (Fig. 2) we performed more in-depthanalyses of the effects of species richness on CO2 effluxusing theGLSmixed modelling approach described above applied to the timepoint when maximal CO2 efflux occurred, i.e. week 2. Finally,a backward stepwise linear regression was undertaken to testwhether the amount of C andNand the C:N ratio of the added fungalnecromass could explain the variation in CO2 efflux in week 2.

3. Results

3.1. Effects of fungal necromass treatment identity (TID)on soil CO2 efflux

Despite being grown under identical nutrient conditions, thetotal C and N contents and the resulting C:N ratios of added fungal

necromass varied with each treatment (Table 1). In particular,Cenococcum geophilum and Hebeloma crustuliniforme both had lowN contents and high C:N ratios (74:1 and 40:1 respectively). Wewould therefore expect this to be reflected in the CO2 effluxobtained from each treatment. However, regression analysisrevealed there was no significant effect of the C (P ¼ 0.589;R2 ¼ 0.023; T¼ 0.55) and N (P¼ 0.308; R2 ¼ 0.08; T¼ 1.06) content,and the C:N ratio (P ¼ 0.392; R2 ¼ 0.057; T ¼ �0.89) of the addedfungal mycelium on CO2 efflux.

Soil CO2 efflux was calculated from measurements made overa 6 h incubation every week for 4 weeks. Throughout the studyperiod both time (L-ratio ¼ 358.54, P < 0.001) and treatmentidentity (L-ratio ¼ 355.64, P < 0.001), were found to have strongeffects on CO2 efflux, with a weaker but still significant interactioneffect between time and treatment identity (L-ratio ¼ 182.88,P < 0.001). A more in-depth analysis of the effects of treatmentidentity and species richness on CO2 efflux was carried out on datafromweek 2. With the exception of treatment B (Amanita muscaria)

A. Wilkinson et al. / Soil Biology & Biochemistry 43 (2011) 1350e1355 1353

and treatment H (Suillus bovinus), this time point was whenmaximal CO2 efflux occurred (Fig. 2). In week 2, strong effects ofindividual treatments were observed to drive CO2 efflux(L-ratio ¼ 105.8, P < 0.001, Fig. 1). Of fungal necromass in mono-culture, certain species induced little CO2 efflux (Fig. 1; treatmentsC, E, G and H) compared to others (treatments A, B, D and F). Inparticular, soil containing Cortinarius glaucopus (treatment F)produced the greatest amount of CO2. Not surprisingly thereafterthe two-species fungal combination containing C glaucopus(treatment FH) stimulated greater CO2 efflux than the other threetwo-species treatments. These differences were significant in thecases of treatments AD (t ¼ 3.58, P ¼ 0.0006) and CE (t ¼ 3.42,P ¼ 0.001), although not significant in the case of treatment BG(t ¼ 0.95, P ¼ 0.346). However, at the 4-species richness level thetreatment that did not contain C. glaucopus (treatment BCEG)actually led to slightly greater CO2 efflux than treatment ADFH,although this was not significant (t ¼ 0.94, P ¼ 0.352).

3.2. Effects of species richness of fungal necromass on soil CO2 efflux

Over the course of the 4-week timeperiod species richness of themixtures had the largest effect on CO2 efflux (Fig. 1; L-ratio ¼ 177.1,P < 0.001) followed by time (L-ratio ¼ 81.64, P < 0.001), with thetwo factors interacting significantly (L-ratio ¼ 25.12, P ¼ 0.003). Inthe second week, when maximum CO2 efflux occurred, the speciesrichness of the fungal necromass had a significant impact on CO2efflux (L-ratio¼ P< 0.001, Fig. S1). Therewas a small increase in CO2

efflux between soil containing the monoculture treatments (treat-ments AeH, Table 1) and the 2 species treatments (treatments FH-BG) although this was found not to be significant (t ¼ 0.95,P ¼ 0.341). However, there was a significant increase in the soil CO2effluxwhen fungal necromass richness increased from1 to 4 speciesand 8 species (t ¼ 2.90, P ¼ 0.005 and t ¼ 4.60, P < 0.001 respec-tively) and also from 2 to 8 species (t ¼ 3.30, P ¼ 0.001). When theadded fungal necromass consisted of all 8 species, soil CO2 effluxreached over 18 mg CO2-C g�1 soil d�1, whichwas 7 mgmore than themean CO2 efflux of the monocultures.

4. Discussion

4.1. Effects of species richness of fungal necromass on soil CO2 efflux

Whilst previous studies have demonstrated that increasing thespecies richness of EM fungi can have important effects on plantnutrition and growth during their life (Baxter and Dighton, 2001;Jonsson et al., 2001), our study provides clear evidence that thediversity of EM fungi also has significant effects on C fluxes whenthe mycelium enters soil as necromass.

Although this study is unique from a mycological perspective,previous investigations have tested how diversity of plant litteraffects its decompositionwith often contradictory results (Bardgettand Shine, 1999; Hector et al., 2000; Knops et al., 2001; Rustad,1994; Wardle et al., 1997). Why increased diversity of EM necro-mass would induce CO2 efflux compared to monocultures isunclear. Results from plant litter experiments have led to thesuggestion that the presence of high quality litter could enhancethe decomposition of other litters, while poor quality litter mayhave negative effects (Seastedt, 1984). Similarly, Wardle et al.(1997) found that when litter of plants with high N were mixed,synergistic responses were observed. In our experiment, we wereunable to separate out the individual effects of species on CO2 effluxin the mixed communities and it was therefore not possible todistinguish any effects of biodiversity into sampling or comple-mentarity effects (that may arise because of positive or negativeinteractions between microbes decomposing the different species

of fungal necromass). However, CO2 efflux from the mixed EMfungal treatments was on average less than the highest singleperforming component species in monoculture, with no propensityto increase with species richness, so the data suggest that samplingeffects are driving increased microbial activity. This supports ourhypothesis that increased richness would lead to greater CO2 effluxdue to the increased likelihood of a more nutritionally rich fungalspecies being present in the mixed necromass. It has been sug-gested that sampling effect may be a legitimate mechanism drivingdiversity effects (Tilman,1997) although others suggest this ignoreshow communities are structured (Wardle, 1999).

4.2. Effects of fungal necromass treatment identity on soil CO2 efflux

The strong effects of EM fungal species richness driving CO2efflux were underpinned by the effects of the individual treat-ments, with microbial activity varying greatly in response todifferent species and mixtures of EM fungal necromass as initiallyhypothesised. This supports recent work by Koide and Malcolm(2009) who also found that there was substantial variationamong different EM fungal strains in decomposition rate(measured as % biomass loss of dried fungi buried in litter bags inforest soil). It is noteworthy that the effects of the necromass of thedifferent species on CO2 efflux did not correspond to growth ratesseen in a previous experiment that used the same isolates. C. glau-copus (treatment F), which induced the greatest respiratoryresponse, was previously among the least productive, whereasPaxillus involutus (treatment G) was previously a highly productivespecies but induced the weakest respiratory response by soilmicrobes when added as necromass, despite containing thegreatest amounts of C and N. This suggests an uncoupling of thetraits that promote dominance when the fungi are physiologicalactive compared to those of hyphal necromass that drive sapro-trophic microbial activity.

Different species of EM fungi not only vary in their mycelialmorphology (Agerer, 1995, 1991) but also in physiological attri-butes, such as nutrient uptake (Simard et al., 2002; Thomson et al.,1994), nutrient transfer capacities (Colpaert et al., 1996) andN tolerance (Lilleskov et al., 2002). From the plant literature, it hasbeen suggested that early decomposition rate is controlled by tissueN concentration or C:N ratio (e.g. Berg, 2000; Cotrufo et al., 1995;Taylor et al., 1989), yet lignin concentrations become increasinglymore important at regulating decomposition rates in the laterstages (Berg, 1984). Likewise in fungi, Koide and Malcolm (2009)found that variation in decomposition correlated significantlywith the N concentration of the fungal tissue. We hypothesised thatCO2 efflux would be greater in treatments with higher qualityfungal necromass (i.e. lower C:N ratio). In contrast to our expec-tations there was no relationship between CO2 efflux and theamount of fungal C and N added to the soils or the C:N ratio of thenecromass, despite the large degree of variability in C andN contentamong species. However we made no attempt to measureconcentrations of recalcitrant N forms in the fungal necromass,such as chitin, and it is possible that factors determining soil CO2

efflux are more complex than the initial C and N concentrations ofthe fungal necromass.

We also focused on only the initial respiratory responses andestimate that the quantity of CO2 produced represents about 10% ofadded C in the necromass. It may well be the case that total C andN have more predictive power over longer timescales because theinitial respiratory responses are being driven by the content ofmore labile (e.g. sugars) C sources in the necromass. The C contentof the freshly killed fungi added to the soil ranged between 1 and4 mg g�1 soil, and this induced CO2 production rates in the range6 (treatment G and H, P. involutus and S. bovinus) to 25 (treatment

A. Wilkinson et al. / Soil Biology & Biochemistry 43 (2011) 1350e13551354

F - C. glaucopus) mg CO2-C g�1 soil d�1. High diversity treatmentswere in the range 13e19 mg CO2-C g�1 soil d�1. These values areapproximately a third of those seen upon addition of similaramounts of glucose calculated to relieve saprotrophic microbes ofany C limitation (Anderson and Domsch, 1978).

It has been suggested that EM fungi are a relatively recalcitrantsource of C to soil microorganisms due to their thick walls, septabetween cells, (Langley and Hungate, 2003) and high concentra-tions of complex pigments and chitin (Swift et al., 1979). However,chitin concentrations of EM root tips have been found to varybetween species (Ekblad et al.,1998;Wallander et al., 1997) and fineroot tips associated with P. involutus have higher chitin concen-trations than other species such as Piloderma croceum and Suillusvariegatus (Ekblad et al., 1998). Melanins are also resistant to decay(Butler and Day, 1998) and may enable fungal hyphae and root tipsto persist in soil for several years after death (Bloomfield andAlexander, 1967). These pigmented substances, found in highconcentrations in fungal tissue of some EM species may explainwhy the hyphae from dark-coloured species such as P. involutus andS. bovinus induced less CO2 efflux. However, this was clearly not theonly explanation in our experiment because some light colouredfungal hyphae such as that produced by Lynx rufus and Laccariabicolor also resulted in little CO2 efflux and the pigmented fungusC. geophilum led to large CO2 effluxes, suggesting that the drivingfactors are more complex and interchangeable. Other factorsaffecting microbial activity may include the presence of antibiotic(as seen in P. involutus,H. crustuliniforme, L. bicolor and S. variegatus;Olsson et al., 1996) and antifungal compounds (e.g. P. involutus,Duchesne et al., 1989; Rasanayagam and Jeffries,1992), variations inthe concentration of Ca (Langley and Hungate, 2003) and thehydrophobicity of the fungal tissue (Lützow et al., 2006). Theseobservations suggest the need to obtain data on a range of keyfungal ‘traits’ before reliable predictions can be made on the effectsof particular species in regulating CO2 efflux.

Our study suggests that soil CO2 efflux will increase with fungalbiodiversity, although how this will be affected by other abiotic andedaphic factors in the field is an unknown yet important area ofresearch. Due to the small amounts of soil used in this study it is likelythat certain trophic levels of the soil food web, such as mites or col-lembola, were excluded from the treatments, or did not survive untilthe end of the study. Production of CO2 in the field may differ to ourresults because some species may be preferentially decomposed byfungivorous invertebrates (Hiol et al., 1994; Schneider et al., 2005).

The influence of individual species on CO2 efflux suggests thatother components of EM fungal diversity like evenness may beimportant. Many EM species such as P. involutus form densely packedrhizomorphs (Read, 1992), and these thickened structures havea decreased surface area:volume ratio which could make them moreresistant to degradation (Langley and Hungate, 2003). In addition,despite thehigh levels of species richness in EMcommunities (Jonssonet al.,1999), they tend tobedominatedby fewspecies,withmany ‘rare’species present in low abundance (Erland and Taylor, 2002). If EMfungal species that induce microbial activity are present in lowabundance in the field thismay have little effect on net soil CO2 efflux.Likewise, obtaining reliabledecayconstants for EMhyphaewill help todetermine whether mycorrhizal fungi (and their diversity) warrantinclusion as a specific pool in models of terrestrial C dynamics. Wetherefore need to test the effects of fungal diversity further by varyingthe evenness of the community and assessing the effects of the nec-romass of dominant EM fungal species on CO2 efflux.

5. Conclusions

Our study demonstrates that the species richness of fungalnecromass can increase soil CO2 efflux in laboratory conditions.

This suggests a hitherto unforeseen mechanism by which EMfungal diversity has potential to affect ecosystem functioning. Thisfinding also raises questions about whether particular saprotrophicmicrobial groups can selectively use C and N from the necromass ofdifferent EM species. The application of stable isotope probingtechniques (Radajewski et al., 2000) may enable us to determinethe fate of nutrients from dead mycelium to soil microorganisms.We now need to determine whether the patterns seen in thelaboratory in response to species richness are also seen in nature.

Acknowledgements

We thank Clare Cameron and Prof. Colin Campbell at theMacaulay Land Use Research Institute for help with the MicroRespsoil respiration system, Dr Andy Taylor for providing some of thefungal isolates, Forest Research for access to Culbin Forest NNR,and the Natural Environment Research Council for a doctoralstudentship to AW.

Appendix. Supplementary material

Supplementary data related to this article can be found online atdoi:10.1016/j.soilbio.2011.03.009.

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