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Soil Biology & Biochemistry 48 (2012) 60e68

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Soil Biology & Biochemistry

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Controls of isotopic patterns in saprotrophic and ectomycorrhizal fungi

Erik A. Hobbie a,*, Fernando S. Sánchez b, Paul T. Rygiewicz b

aComplex Systems Research Center, University of New Hampshire, Durham, NH 03824, USAbUS Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Western Ecology Division, 200 SW 35th Street, Corvallis, OR 97333, USA

a r t i c l e i n f o

Article history:Received 9 October 2011Received in revised form17 January 2012Accepted 18 January 2012Available online 3 February 2012

Keywords:Organic nitrogen useSporocarpsProteinChitinNitrogen isotopesCarbon isotopesConiferous forests

* Corresponding author.E-mail address: erik.hobbie@unh.edu (E.A. Hobbie

0038-0717/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.soilbio.2012.01.014

a b s t r a c t

Isotopes of nitrogen (d15N) and carbon (d13C) in ectomycorrhizal and saprotrophic fungi contain importantinformation about ecological functioning, but the complexity of physiological and ecosystem processescontributing to fungal carbon and nitrogen dynamics has limited our ability to explain differences acrosstaxa. Here, we measured d15N and d13C in needles, litter, soil, wood, fungal caps, and fungal stipes atnumerous forested sites in Oregon, USA to determine how functional attributes and biochemical processesmay influence isotopic values. Ectomycorrhizal fungi were classified by hydrophobicity of ectomycorrhizaeand by patterns of hyphal exploration; saprotrophic fungi were classified into wood decay and litter decayfungi. For d15N, caps of hydrophobic taxa averaged 8.6&, hydrophilic taxa 3.2&, and saprotrophic taxa0.5&, whereas needles averaged 3& and soil at 5e12 cm averaged 2&. Caps were higher in d15N, d13C, %N,and %C than stipes by 1.7&, 0.6&, 1.75%, and 2.61%, respectively, presumably because of greater proteincontent in caps than stipes. Isotopic enrichment of caps relative to stipes was greater in hydrophobic taxa(3.1& for 15N and 0.8& for 13C) than in hydrophilic taxa (1.1& for 15N and 0.5& for 13C). In multipleregressions, 45% of variance in d15Ncapestipe and 30% of variance in d13Ccapestipe was accounted for byvarious elemental, isotopic, and categorical variables.We estimated that fungal proteinwas enriched in 15Nrelative to fungal chitin by 15& in hydrophobic taxa and by 7& in hydrophilic taxa. Fungal protein wasenriched in 13C by 4.2 � 0.5& relative to carbohydrates. Isotopic signatures of sources and isotopic frac-tionation during metabolic processing influence both isotopic patterns of sporocarps and the isotopicpartitioning between caps and stipes; functional groups differed in processing of both nitrogen isotopesand carbon isotopes.

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1. Introduction

Comparing isotopic patterns in fungal sporocarps to those ofecosystem components has provided insight into the roles of bothectomycorrhizal and saprotrophic fungi in ecosystem carbon andnitrogen cycling (Hobbie et al., 1999; Högberg et al., 1999a; Kohzuet al., 1999; Taylor and Fransson, 2006). Here, we use an extensivedata set of isotopic ratios in soil, wood, foliage, and sporocarps ofmany taxa of ectomycorrhizal and saprotrophic fungi to assess theprobable mechanisms controlling fungal d15N and d13C. We willassess how internal processes in sporocarps affect d15N and d13C bycomparing isotopic patterns in caps and stipes to elemental patternsand life history strategies.

Several factors control d15N values in fungi. The d15N of sourcenitrogen should influence fungal d15N, with bulk d15N increasingwith depth in soil profiles (Hobbie and Ouimette, 2009). In

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addition, the d15N of organic nitrogen is generally higher thaninorganic nitrogen (Koba et al., 2003; Takebayashi et al., 2010), butvaries with the relative fluxes of ammonification, nitrification, anddenitrification (Houlton et al., 2007). For ectomycorrhizal fungi, theisotopic signature of the original nitrogen source can be modifiedby fractionation against 15N during transfer of nitrogen compoundsto host plants (Hobbie et al., 2000). Relative to available nitrogensources, ectomycorrhizal fungi will then be enriched in 15N andtheir ectomycorrhizal hosts will be depleted in 15N. However, evenin saprotrophic fungi, sporocarps are enriched at least 3& in 15Nrelative to bulk soil nitrogen, despite not transferring 15N-depletednitrogen to plants (Hobbie et al., 2005). Internal 15N fractionationmay contribute to this enrichment if fungi partition nitrogen into15N-depleted chitin and 15N-enriched protein (Taylor et al., 1997)and the protein is preferentially mobilized for sporocarp formation.

The growth patterns of ectomycorrhizal fungi also appear toinfluence d15N of sporocarps. Agerer (2001, 2006) classified ecto-mycorrhizal fungi based onwhether mycorrhizaewere hydrophobicor hydrophilic and the extent and form of hyphal development

Fig. 1. Theoretical relationship between nitrogen isotope ratios in protein and chitin foropen versus closed systems as the proportion of systemnitrogen as chitin increases from0 to 1 (expresses as the transfer ratio, Tr). This assumes that fractionation against 15N is10& during synthesis of chitin from protein. a. Open system, d15Nproteinechitin¼Dchitin. b.Closed system, d15Nprotein-accumulated chitin ¼ Dchitin/Tr$ln [1/(1 e Tr)].

E.A. Hobbie et al. / Soil Biology & Biochemistry 48 (2012) 60e68 61

(termed exploration type). This morphological classification corre-sponds to functionality, with the hydrophobic exploration types(medium-distance fringe, medium-distancemat, and long-distance)putatively possessing better proteolytic capabilities than hydrophilicexploration types (contact, short-distance, and medium-distancesmooth) (Lilleskov et al., 2011). Nitrogen isotope patterns inectomycorrhizal sporocarps correspond with these explorationtypes,with hydrophobic exploration types generally 4e7& higher ind15N than hydrophilic exploration types (Hobbie and Agerer, 2010).

Differences in isotopic ratios between caps and stipes of sporo-carps could potentially provide additional insight. These compo-nents differ in their chemical constituents, nitrogen concentration,and d15N. In four ectomycorrhizal species, Taylor et al. (1997)attributed an average 15N enrichment of caps relative to stipes of2.2 � 0.2& to a concurrently measured 15N enrichment of proteinrelative to chitin of 9.9� 0.5& and a greater proportion of protein incaps than in stipes. Because only three species (all ectomycorrhizal)have been tested to date, these 15N enrichments of caps relativeto stipes have not been compared across different functional clas-sifications, such as between saprotrophic and ectomycorrhizalfungi, or among ectomycorrhizal fungi of different exploration type.13C enrichment patterns in caps and stipes have yet to bemeasured,but 13C enrichment in protein relative to chitin in other organismssuch as locusts (Webb et al., 1998), suggests that caps should alsobe enriched in 13C relative to stipes. Relative to stipes, caps appearhigher in protein and lipids and lower in fiber (Alam et al., 2008).

The mechanism of N-acetyl glucosamine synthesis (Carlile et al.,2001) suggest that fractionation against 15N during aminotrans-ferase of nitrogen from glutamine to N-acetyl glucosamine accountsfor the consistent depletion of 15N in chitin relative to protein,muscle, or bulk tissue in diverse heterotrophic organisms (Mackoet al., 1986; Webb et al., 1998; Hobbie and Colpaert, 2003). No CeNbond is involved in chitin synthesis from N-acetyl glucosamine, sothe 15N depletion of chitin must arise during N-acetyl glucosamineformation. Similarly, 13C enrichment of amino acids relative to chitinand other carbohydrates during metabolic processing drives the13C depletion of these carbohydrates relative to protein and musclein heterotrophic organisms (Schimmelmann and DeNiro, 1986;Webb et al., 1998; Schimmelmann, 2011).

According to the above arguments, %N and d15N of caps willdiffer from stipes if the relative proportion of chitin and protein inthese two fungal components also differ, even if the 15N differencebetween protein and chitin is held constant. However, the d15N ofthe two compound classes (protein and chitin) can also vary (Tayloret al., 1997). For a given relative difference in %N between caps andstipes, an increased difference in d15N indicates increased isotopicseparation between chitin and protein, if the relative increase inprotein is similar.

How isotopes partition between two components (such as chitinand protein) depends on whether the system is treated as openor closed. For an open system, isotopic differences between thetwo components will remain constant (Fig. 1a), whereas isotopicdifferences for the closed system will vary depending on the rela-tive allocation between the two components (Fig. 1b). Closed-system mathematics have been used to explain d15N patterns insporocarps (Hobbie et al., 2005), in which increased sequestrationof 15N-depleted chitin in fungal hyphae increases the 15N enrich-ment of sporocarps relative to available nitrogen sources (Trudellet al., 2004; Hobbie and Agerer, 2010). We suggest that the exten-sive hyphal development in hydrophobic systems will lead toincreased chitin sequestration and consequently increased 15N and13C enrichment of fungal protein relative to hydrophilic systems,and can be treated as a closed system. This isotopic partitioningwill accordingly increase the isotopic enrichment of caps relative tostipes.

Mathematical formulations for isotopic patterns in open andclosed systems are given in Fry (2006). For an open system inwhichsome nitrogen from protein can be transformed into chitin witha fractionation against 15N during chitin synthesis of Dchitin, thefollowing equations apply, in which Tr is the proportion of reactanttransformed into product:

d15Nchitin ¼ d15Nsource N � ð1� TrÞ$Dchitin (1)

d15Nprotein ¼ d15Nchitin þ Dchitin (2)

Thus, the 15N enrichment of protein relative to chitin isa constant, equivalent to Dchitin.

For a closed system, the 15N enrichment of protein relative tochitin is not a constant, although the 15N depletion during chitinsynthesis remains the same.

d15Nprotein ¼ d15Nsource N � Dchitin$lnð1� TrÞ (3)

d15Naccumulated chitin ¼ d15Nsource N

þ Dchitin$ð1=Tr � 1Þ$½lnð1� TrÞ� (4)

d15Nprotein�accumulated chitin ¼ Dchitin=Tr$ln½1=ð1� TrÞ� (5)

E.A. Hobbie et al. / Soil Biology & Biochemistry 48 (2012) 60e6862

In this study, we collected sporocarps together with otherecosystem components at 30 sites inOregon, USA. Sites ranged fromhumid coastal forests to the drier interior forests east of the CascadeMountains (Bowling et al., 2002). We compared the d15N and d13Cof ectomycorrhizal and saprotrophic sporocarps with foliage, litter,wood, and soil. We assessed differences in %N, d15N, %C, and d13Cbetween caps and stipes for insight into mechanisms creatingisotopic patterns in different fungal taxa.

Based on the above arguments, we propose the followinghypotheses:

(1) 15N enrichment in caps versus stipes corresponds to the rela-tive increase in %N between caps and stipes. This arises becauseincreased %N is caused by increased protein, and protein is highin d15N.

(2) Because protein is also high in d13C, caps will be higher in d13Cthan stipes.

(3) Isotopic patterns during fungal metabolism can be treatedassuming a closed system during sporocarp formation, andassuming greater chitin sequestration in hydrophobic taxa thanin hydrophilic taxa. Accordingly, 15N and 13C enrichment incaps versus stipes will be higher in hydrophobic taxa than inhydrophilic taxa.

2. Methods

2.1. Sample collection

At 30 different locations across Oregon during 1998 and 1999,sporocarps of 193 taxa of ectomycorrhizal fungi, litter decay fungi,andwood decay fungi were collected, totalling 337 specimens. For 21of the collections, exact location was not recorded. Most collectingsites had forests dominated by Pseudotsuga menziesii (Mirb.) Franco(Douglas-fir), Tsuga heterophylla (Raf.) Sarg. (western hemlock), Piceasitchensis (Bong.) Carr. (Sitka spruce), or Pinus ponderosa C. Lawson(Ponderosa pine). Collections were identified, cleaned of litter andhumus, and freeze-dried. One specimen of each collection wasdivided into cap and stem and ground using a mortar and pestle.Ectomycorrhizal species were classified by exploration type andhydrophobicity according to Agerer (2001, 2006) and Hobbie andAgerer (2010). Species of unknown exploration type were assignedto the dominant exploration type in the genus. The genera for thethree hydrophilic exploration types were (1) contact: Chroogomphus,Hygrophorus, Lactarius; (2) short-distance: Inocybe, Rozites; (3)medium-distance smooth: Albatrellus, Amanita, Cantharellus, Gom-phidius, Laccaria, Russula. The genera for the three hydrophobicexploration types were (4) medium-distance fringe: Cortinarius,Hebeloma,Hydnum, Tricholoma; (5)medium-distancemat: Gomphus,Ramaria, Sarcodon; and (6) long-distance: Boletus, Leccinum, Paxillus,Suillus. Because of uncertain species identification, all Entolomasamples were excluded from analysis. The pick-a-back explorationtype classification for Chroogomphus and Gomphidius was not used,since assigning fungi to this type requires examination of root tips.

At 21 sites (17 of which were also sampled for fungi), soilsamples (five per site, approximately 200 g fresh weight) werecollected at two depths, 0e5 cm and 5e12 cm. Five samples of greenmature needleswere also collected at each site from the bottom5mof canopy. Most forests sampled were dominated by a single treespecies, but collections were from a range of species in mixedforests. Five samples of surface litter (mixed foliar and twig litter)were also taken from each site (100 g dry weight). We collected fivesamples per site (100 g dry weight) of downed, decayed wood.Samples were dried at 60 �C for 48 h, and then ground usinga rotating sample mill.

2.2. Sample analysis

Samples were analyzed for d15N, d13C, %N, and %C on a FinniganDelta-Plus isotope ratio mass spectrometer linked to a CarloErba NC2500 elemental analyzer (Finnigan MAT GmbH, Bremen,Germany) located at the U.S. EPA, Corvallis, Oregon, USA. Theinternal standards for isotopic and concentration measurementswere acetanilide and spinach (NIST 1570a).We report stable isotopeabundances as d15N (or d13C) ¼ (Rsample/Rstandard � 1)$1000, whereR¼ 15N/14N or 13C/12C of either the sample or the reference standard(atmospheric N2 for nitrogen, PeeDee belemnite for carbon).The precision of isotopicmeasurements based on duplicate sampleswas �0.2& for 15N and 13C. When comparing between samples,samples with more of the heavy isotope are referred to as heavier,or enriched; samples with more of the light isotope are lighter,or depleted. If isotopic fractionation (D) is calculated between twopools (such as between protein and chitin), it is calculated asD ¼ (dsubstrate � dproduct)/(1 þ dsubstrate/1000&). Nitrogen concen-trations in wood were too low for adequate %N and d15N analyses.To compare the relative difference in nitrogen between caps andstipes across taxa, the concentration difference was calculated as(1 � %Nstipe/%Ncap).

Most data analyses used the statistical package Statview (AbacusConcepts, Berkeley, California). Elemental and isotopic differencesbetween caps and stipes were also calculated. Differences in isotopicvalues and elemental concentrations were evaluated using one-wayANOVAs or t tests if only two groups were compared, and variabilitywithin groups was compared using an F-test. Means among classi-fied groups were compared using a post hoc TukeyeKramer testat the 0.05 significance level. Relationships between variableswere compared using correlations. Stepwise, forward linearmultipleregressions only used data from known locations (the 21 sporocarpsof unknown location were excluded), and explained variabilityin d15Ncapestipe and d13Ccapestipe using the SigmaPlot program (Systat,San Jose, California). Both regression models used the followingcontinuous variables: %Ncapestipe, 1 � %Ncap/%Nstipe, and %Ccapestipe.The model to explain d15Ncapestipe included d13Ccapestipe as an inde-pendent variable and the model to explain d13Ccapestipe includedd15Ncapestipe as an independent variable. To determine whichindependent variables to retain in the multiple regression, we usedthe SigmaPlot defaults of setting the “F-to-remove” at a value of4.0, and the “F-to-Enter” at 3.9. Three categorical variables werealso included in themultiple regressions, corresponding to themainclassifications of the fungi, such as having hydrophilic or hydro-phobic ectomycorrhizae, or being a litter decay fungus, with a valueof 1 assigned to the appropriate categorical variable and othercategorical variables assigned a value of 0. Wood decay fungi wereassigned a value of 0 for all three categorical variables.

2.3. Theoretical relationship between nitrogen isotopecomposition in caps and stipes

We sought to relate cap and stipe isotopic composition to theisotopic composition of protein and chitin and stipe and cap %N.Nucleic acids appear to be less than 10% (bymass) of protein contentin gills of Coprinus lagopus (Iten and Matile, 1970; they recordedmaximum concentrations of 10%, 2%, and 0.8% for protein, N-acetylglucosamine, and RNA in cell-free extracts). Because gills are the siteof fungal reproduction, nucleic acid content should be higher in gillsthan in other tissues. For this analysis, we assume that nitrogenin fungi is composed of two main pools, chitin and protein,and assume that stipe nitrogen consists of a fungal chitin pool(a, stipechitin) and a fungal protein pool (b, stipeprotein). Cap nitrogenretains these two pools but adds additional protein (c, capprotein).We also assume that d15N values of protein and chitin do not change

Fig. 2. d13C and d15N values of different ecosystem pools (needles, litter, wood, 0e5 cmsoil, and 5e12 cm soil) and of caps and stipes of different fungal groups. Fungi areseparated into wood decay fungi (wood d), litter decay fungi (litt d), fungi withhydrophobic ectomycorrhizae (ECM-ho), and fungi with hydrophilic ectomycorrhizae(ECM-hi). Names of genera are indicated on the Figure. Wood d15N was not measured;it is indicated by a box spanning its probable range between the d15N of needles andthe d15N of caps of wood decay fungi.

E.A. Hobbie et al. / Soil Biology & Biochemistry 48 (2012) 60e68 63

between caps and stipes, with protein 10& enriched in 15N relativeto chitin. Under these assumptions, stipe and cap d15N can beexpressed as:

d15Nstipe ¼ ½b=ða þ bÞ�$ 10%þ d15Nchitin (6)

d15Ncap ¼ ½ðb þ cÞ=ða þ b þ cÞ�$ 10%þ d15Nchitin (7)

The difference between these two measurements, d15Ncap �d15Nstipe, can be expressed as:

d15Ncap � d15Nstipe ¼ a=ða þ bÞ$c=ða þ b þ cÞ$10% (8)

%Nstipe equals 100%$(a þ b)/total biomass and %Ncap equals100%$(aþ bþ c)/total biomass. Accordingly, the quantity, c/(aþ bþ c),is equivalent to 1 � %Nstipe/%Ncap.

d15Ncap � d15Nstipe ¼ a=ða þ bÞ$�1�%Nstipe=%Ncap�$10% (9)

This can also be expressed as:

d15Ncap � d15Nstipe ¼ stipechitin=�stipechitin þ stipeprotein

�

$�1� %Nstipe=%Ncap

�$10% ð9aÞ

In this analysis, the isotopic difference between caps and stipescan be reduced to three factors: 1 � %Nstipe/%Ncap, the proportion ofstipe nitrogen that is chitin, and the 15N enrichment of proteinrelative to chitin.

3. Results

3.1. Plant litter and soil

Plant litter, wood, and soil were collected at 21 sites. Concen-trations of nitrogen and carbon decreased from litter (0.99 � 0.24 %N, 44.39� 4.52 %C) to 0e5 cm soil (0.69� 0.44%,17.00� 11.78 %C) to5e12 cm soil (0.29 � 0.27 %N, 7.20 � 7.86 %C). d13C values increasedfrom needles to litter to soil to wood, with overall averagesof �28.8 � 1.7& for needles, �27.1 � 0.9& for litter, �26.3 � 0.9&for 0e5 cm soil,�25.5� 0.8& for 5e12 cm soil, and�24.8� 1.1& forwood (all values�SD). d15N values increased fromneedles to litter tosoil, with overall averages of 3.3�1.3& for needles,�2.3� 0.9& forlitter, 0.6 � 1.2& for 0e5 cm soil, and 2.6 � 1.2& for 5e12 cm soil.Elemental and isotopic data for the 21 sites are given in Appendix 1.

3.2. Fungi

A total of 337 collections of fungi were made at 27 sites, ofwhich 227 were ectomycorrhizal, 63 were litter decay, and 47 werewood decay fungi. Ectomycorrhizal fungi were classified by hydro-phobicity of ectomycorrhizae and by exploration type (Hobbie andAgerer, 2010), with 157 specimens classified as having hydrophilicectomycorrhizae and 70 as having hydrophobic ectomycorrhizae. Themost common exploration type was medium-distance smooth, ofwhich 98were collected, followed in abundancebymedium-distancefringe (43), contact (33), short-distance (26), long-distance (23), andmedium-distancemat (4). Complete details on locations sampled andspecies collected are given in Appendix 2.

Nitrogen and carbon isotopic signatures and concentrationsvaried across functional class, exploration type, and fungal tissue.Caps averaged 4.49 � 1.57% (SD) in %N and 41.16 � 2.75 (SD) in %C,whereas stipes averaged 2.83 � 1.19% (SD) in %N and 38.57 � 2.36(SD) in %C. In caps of sporocarps, d15N increased from wood decay(�0.4 � 0.4&) to litter decay (1.3 � 0.5&) to ectomycorrhizal fungi(4.9� 0.3&) and d13C decreased fromwooddecay (�22.4� 0.3&) to

litter decay (�22.8� 0.3&) to ectomycorrhizal fungi (�24.7� 0.1&)(Fig. 2). In ectomycorrhizal fungi, caps of hydrophobic taxa averaged8.5& for d15N and �24.6 � 0.1& for d13C, whereas caps of hydro-philic taxa averaged 3.2 � 0.2& for d15N and�24.8 � 0.1& for d13C.Caps averaged higher than stipes by 1.7& in d15N, 0.6& in d13C,1.75%in %N, and 2.61% in %C. The isotopic enrichment of caps relativeto stipes (designated d15Ncapestipe and d13Ccapestipe) was higher inectomycorrhizal fungi than in litter decay or wood decay fungi, andin ectomycorrhizal fungi, was significantly greater in hydrophobicexploration types (3.1& in d15N and 0.8& in d13C, p< 0.001 for both)than in hydrophilic exploration types (1.1& in d15N and0.5& in d13C)(Fig. 2). d15Ncapestipe varied within the different exploration types,ranging from 1.0& for medium-distance smooth to 3.6& formedium-distance fringe. d13Ccapestipe also varied by explorationtype, ranging from 0.3& for contact exploration types to 0.9& formedium-distance fringe exploration types (Table 1).

The relative differences in nitrogen between caps and stipes wascalculated as (1 � %Nstipe/%Ncap) and varied by exploration type inectomycorrhizal fungi. It ranged from0.29 for short-distance to 0.45for medium-fringe exploration types, with litter decay fungi lower(0.36) thanwood decay fungi (0.42). This quantity was significantlyhigher in fungi with hydrophobic ectomycorrhizae (0.436 � 0.020)than in fungi with hydrophilic ectomycorrhizae (0.351 � 0.012)(t-test, df ¼ 139, p < 0.001) (Table 1).

d15Ncapestipe and d13Ccapestipe were positively correlated inectomycorrhizal fungi (r2¼ 0.16, n¼ 210, p< 0.001) andwooddecayfungi (r2 ¼ 0.38, n ¼ 39, p < 0.001) but were uncorrelated inlitter decay fungi (r2 ¼ 0.01, n ¼ 59, p ¼ 0.451). The relationshipbetween d15Ncapestipe and d13Ccapestipe in different genera is shownby nutritional source, hydrophobicity, and exploration type in Fig. 3.

We used multiple regressions to explore the potential controlsand covariates of the isotopic difference between caps and stipesfor both nitrogen and carbon (d15Ncapestipe and d13Ccapestipe). Themultiple regressions explaining the maximum amount of varianceare shown in Table 2. Only the statistically significant variables havebeen retained in the table. For d15Ncapestipe, the significant factors(in descending order of importance) were the relative proportion ofnitrogen in caps versus stipes, %Ncapestipe, d13Ccapestipe, the hydro-phobicity of ectomycorrhizae, %Ccapestipe, and the hydrophilicity of

Table 1d15N and d13C in caps, the enrichment in caps versus stipes for 15N, 13C, %N, and %C, and the relative enrichment of caps versus stipes for nitrogen concentrations. Because ofmissing variables during measurement or, in some fungi, the absence of stipes, the number of measurements varies for different variables. Number of samples (n) is given inparentheses for each parameter in the table, number of samples for each fungal type is given in the second column. Ectomycorrhizal fungi are classified by exploration type,saprotrophic fungi by substrate (litter or wood). Significance values are given in italics for ANOVAs within exploration types for ectomycorrhizal fungi or between the twosaprotrophic decay types (unpaired t-test); significance values are bolded if p < 0.05. Significant differences among exploration types or between the two saprotrophic typesare indicated by different superscripted letters (a, b, and c), with no letter indicating no significant differences.

Exploration or Decay Type n d15Ncap (&) d13Ccap (&) d15Ncapesti (&) d13Ccapesti (&) %Ncapesti (%) %Ccapesti (%) 1 � %Nsti/capn (333) (336) (309) (335) (312) (334) (312)

Ectomycorrhizal <0.001 0.002 <0.001 <0.001 <0.001 0.036 <0.001contact 33 2.8 � 0.6a �25.0 � 0.3a 1.4 � 0.2b 0.3 � 0.1a 1.8 � 0.1 2.9 � 0.2 0.42 � 0.02bc

medium-smooth 98 3.5 � 0.3a �24.8 � 0.1 1.0 � 0.1a 0.5 � 0.1a 1.4 � 0.1a 3.2 � 0.2b 0.35 � 0.01ab

short 26 2.4 � 0.4a �24.0 � 0.3b 1.5 � 0.2ba 0.6 � 0.1 1.4 � 0.1a 2.9 � 0.4 0.29 � 0.02a

medium-fringe 43 8.5 � 0.5b �24.8 � 0.2 3.6 � 0.3c 0.9 � 0.1b 1.7 � 0.1 1.9 � 0.8 0.45 � 0.02c

medium-mat 4 13.5 � 1.3c �24.3 � 0.3b 3.6 � 0.1cb 0.9 � 0.2 1.7 � 0.2 3.0 � 0.9 0.43 � 0.05long 23 7.8 � 0.6b �24.3 � 0.3b 2.3 � 0.3b 0.7 � 0.2 2.2 � 0.2b 1.6 � 0.3a 0.40 � 0.03bc

Saprotrophic 0.022 0.334 0.022 0.968 0.178 0.284 0.045litter decay 63 1.3 � 0.5b �22.8 � 0.3 1.4 � 0.2 0.7 � 0.1 2.1 � 0.1 2.5 � 0.4 0.37 � 0.01a

wood decay 45 �0.4 � 0.3a �22.4 � 0.3 1.9 � 0.2 0.7 � 0.1 1.8 � 0.2 1.6 � 0.4 0.42 � 0.03b

E.A. Hobbie et al. / Soil Biology & Biochemistry 48 (2012) 60e6864

ectomycorrhizae. For d13Ccapestipe, the significant factors were %Ncap-stipe, d15Ncapestipe, and %Ccapestipe. Adjusted r2 versus the inde-pendent variables is 0.452 for d15Ncapestipe and 0.306 for d13Ccapestipe(n ¼ 286, p < 0.001 for both). Intercepts for both multiple regres-sions were not significantly different from zero.

4. Discussion

4.1. Ecosystem patterns in d13C

Within a site, increases among ecosystem pools in d13C goingfrom needles to litter to deeper soil horizons presumably reflect thetransport of 13C-enriched carbohydrates within plants from leavesto roots and wood (Hobbie and Werner, 2004) and the routing ofroot-derived carbon into deeper soil horizons via mycorrhizal fungiand root decay. In addition, the retention of 13C-enriched carbonderived from microbes and microbial protein will increase d13Cvalues of soil as material is increasingly processed by microbes

Fig. 3. Isotopic differences in carbon and nitrogen between caps and stipes, plotted bygenus for n > 1. Mean values for the species � location combinations within a genusare plotted, standard errors are plotted in grey in the positive direction only for clarity.Black symbols represent taxa with hydrophobic mycorrhizae, for medium-distancefringe taxa, squares (-); medium-distance mat, triangles (:); and long-distance,circles (C). Clear symbols represent taxa with hydrophilic mycorrhizae, for medium-distance smooth taxa, squares (,); short-distance, triangles (D); and contact, circles(B). Litter decay taxa are indicated with a gray x, wood decay fungi with a grey cross.Representative genera are named on the graph. Complete data are given in Appendix 2.

(Boström et al., 2007; Sollins et al., 2009). Similar d13C values forectomycorrhizal fungi and wood may reflect the common carbonsource (tree-transported sugars) and similar levels of fractionationduring metabolism by trees during wood formation and by fungiduring sporocarp formation, despite the rather different chemicalconstituents of these two pools (cellulose and lignin for trees,carbohydrates and protein for fungi). d13C values for needles areprobably somewhat low relative to other ecosystem pools becausewe were unable to sample overstory foliage, which is generally13C-enriched by 1& or more relative to mid-story and understoryfoliage (Gottlicher et al., 2006).

The higher d13C values of litter and wood decay fungi thanectomycorrhizal fungi presumably reflect the incorporation ofcarbon into saprotrophic biomass from 13C-enriched wood celluloserather than from 13C-depletedwood lignin and lipids (Gleixner et al.,1993; Hobbie, 2005). Tree-transported sugars assimilated by ecto-mycorrhizal fungi are probably 13C-depleted relative to those used tomakewood cellulose because a substantial portion of the sugars usedforwood are diverted tomake 13C-depleted secondary compounds inwood, resulting in 13C enrichment of the remaining sugars used tomakewood cellulose (Hobbie andWerner, 2004). These explanationsare shown schematically in Fig. 4, with the small difference in 13Cenrichment between hydrophobic and hydrophilic taxa explained inSection 4.5. The similar d13C values for litter decay and wood decayfungi reflect the common carbon source, cellulose, for both thesefungal types. Although division of saprotrophic fungi into only twotypes simplifies the continuum of decay types and nutrient sourcesin these fungi, this approach is nonetheless useful as a classificationtool.

4.2. Ecosystem patterns in d15N

Progressive 15N enrichment from needles to litter to deeper soilhorizons (Fig. 2) reflects repeated transfer of 15N-depleted nitrogenfrom ectomycorrhizal fungi to trees and 15N enrichment duringmicrobial processes (Hobbie and Ouimette, 2009). The resulting15N-depleted litter then accumulates at the soil surface and15N-enriched fungal residues accumulate in deeper soil horizons(Högberg et al., 1996; Wallander et al., 2009).

The higher d15N of saprotrophic fungi growing on humus andlitter compared to wood decay fungi has been reported previously(Gebauer and Taylor, 1999; Kohzu et al., 1999; Taylor et al., 2003;Trudell et al., 2004). This pattern could reflect differences inthe nitrogen sources used by these fungal types or in the relativeproportions of 15N-enriched protein and 15N-depleted chitin in

Table 2Stepwise multiple regressions to explore controls over d15Ncapestipe and d13Ccapestipe (n ¼ 286). The model explained 0.452 of the variance for d15Ncapestipe and 0.306 of thevariance for d13Ccapestipe (adjusted r2). Dr2 is the increase in the % variance explained by adding the specified independent variable in the stepwise regression. Only statisticallysignificant variables have been retained in the table.

Independent variable d15Ncapestipe Coefficient d13Ccapestipe Coefficient

�SE Dr2 p �SE Dr2 p

Intercept 0.300 � 0.242 e 0.215a 0.073 � 0.069 e 0.289a

%Ncapestipe �0.781 � 0.126 4.9% <0.001 0.253 � 0.033 17.5% <0.001%Ccapestipe 0.123 � 0.034 1.7% <0.001 �0.034 � 0.012 1.9% 0.0061-%Nstipe/%Ncap 5.171 � 0.706 11.0% <0.001 e e e

d13Ccapestipe 0.853 � 0.149 4.6% <0.001 e e e

d15Ncapestipe e e e 0.121 � 0.017 11.9% <0.001Hydrophobic 1.084 � 0.207 22.0% <0.001 e e e

Hydrophilic �0.571 � 0.175 2.2% <0.001 e e e

a p-values for intercepts are taken from non-stepwise multiple regression analyses.

E.A. Hobbie et al. / Soil Biology & Biochemistry 48 (2012) 60e68 65

different taxa. For example, in this study litter decay fungiwere 1.3& and 2.1% higher in 15N and %N than wood decay fungi,and Taylor and Fransson (2006) reported quite similar enrichmentpatterns (1.8& and 2.5%). Thus, we cannot rule out that sourcedifferences in d15N between the two saprotrophic types maybe small, and sporocarp d15N primarily reflects relative proteincontent, which will correlate with %N. However, %N and d15Nof litter decay fungi were not highly correlated and the highervariability of litter fungi than wood decay fungi for these twoparameters (F-test: %N, variance ratio ¼ 2.31, p ¼ 0.004; d15N,variance ratio ¼ 2.02, p ¼ 0.015) suggests that the d15N of sourcesfor litter decay vary widely relative to those for wood decay fungi.

The 15N enrichment of ectomycorrhizal fungi relative to sapro-trophic fungi of about 5& is a well-established pattern across manystudies (Mayor et al., 2009), but the underlying controls remainunclear. Here, saprotrophic fungi averaged 0.5 � 0.3&, hydrophilicectomycorrhizal fungi averaged 3.2 � 0.2&, and hydrophobicectomycorrhizal fungi averaged 8.6� 0.4& (caps for all). The similar15N enrichments for caps versus stipes for saprotrophic fungi andfor hydrophilic ectomycorrhizal fungi suggests similar internalprocesses in these two fungal groups that govern the partitioning ofnitrogen isotopes in these fungi. Based on the 15N enrichments fromthe litter layer to deeper soil horizons that were measured here,the 15N enrichment of hydrophilic ectomycorrhizal taxa relative tosaprotrophic taxa of w3& could arise solely from differences insource nitrogen. Observations in boreal forest by Lindahl et al. (2007)that saprotrophic fungi predominated in 15N-depleted surface litterwhereas ectomycorrhizal fungi predominated at lower depthswhere d15Nwas higher also support interpreting 15N enrichments inhydrophilic ectomycorrhizal fungi relative to saprotrophic fungias partially reflecting source differences. However, the transfer of15N-depleted N by ectomycorrhizal fungi to host plants has been

Fig. 4. Movement and isotopic fractionation of carbon isotopes in different compo-nents of plants, ectomycorrhizal fungi, and saprotrophic fungi. SAP ¼ saprotrophic,ECM ¼ ectomycorrhizal. CHO ¼ chitin and other carbohydrates. Protein (ho) indicatesfungal protein from hydrophobic exploration types; protein (hi) indicates fungalprotein from hydrophilic exploration types. Modified from Hobbie (2005).

shown in culture studies using isotopic mass balance to decreaseplant d15N and increase fungal d15N (Hobbie and Colpaert, 2003) andmust also contribute to the 15N enrichment of ectomycorrhizal fungirelative to saprotrophic fungi.

15N enrichment of sporocarps, rhizomorphs, and mycelia ofthe hydrophobic ectomycorrhizal fungus Suillus relative to suppliedN (Högberg et al., 1999b; Kohzu et al., 2000; Hobbie and Colpaert,2003) and 15N enrichment of gills relative to stipes (Zeller et al.,2007) indicate that internal processes can create 15N differences infungi even when the external source N is uniform. Accordingly, 15Npartitioning among different compound classes must contribute tothe high d15N of many hydrophobic taxa, with one mechanism thesequestration of relatively 15N-depleted nitrogen in chitin and wall-bound protein in extraradical hyphae. Greater nitrogen sequestra-tion in hydrophobic taxa than in hydrophilic taxa could lead toprogressive 15N enrichment in the pool of internal nitrogen availablefor sporocarp formation. As depicted in Fig. 1b, 15N and 14N parti-tioning in a closed system could produce such enrichment patterns.

4.3. Explaining isotopic patterns in caps and stipes

Differing compositions in caps and stipes of protein and carbo-hydrates should influence elemental and isotopic values in thesefungal tissues. Taylor et al. (1997) attributed the higher %N and d15Nin caps than in stipes to greater 15N-enriched protein and less15N-depleted chitin in caps than in stipes. In that study, caps were2.4 � 0.3% higher in %N and about 2& enriched in 15N relative tostipes, and proteinwas 9.9 � 0.5& (n ¼ 6) higher in 15N than chitin,with a resulting calculated Dchitin of 9.8 � 0.5&. Here, we showthat the 15N enrichments follow regular patterns and correlate withexploration type and the hydrophobicity of ectomycorrhizae. Theseare presumably important functional attributes that correlate withenzymatic activity, sensitivity toNdeposition, and explorationdepth(Hobbie and Agerer, 2010; Lilleskov et al., 2011; Peay et al., 2011).

The few studies comparing the compositions of caps and stipeshave focusedon edible, saprotrophic species that are readily cultured.In Volvariella volvacea, protein content of capswas about twice that ofstipes at the elongation stage, but differed less in later stages (Changand Chan,1973). Chitin content of capswas similar to that of stipes inAgaricus bisporus (chitincap/stipe¼ 0.91) but significantly lower in twoother species, Pleurotus ostreatus (chitincap/stipe ¼ 1.35) and Lentinulaedodes (chitincap/stipe¼ 1.20) (Vetter, 2007). In three Pleurotus species,proteinwas significantly higher (w60%) in caps than in stipes in threePleurotus species but not in Calocybe indica, whereas carbohydratecontent did not differ significantly between caps and stipes in anyof these four species (Alam et al., 2008). In Lentinus subnudus,Psathyrella atroumbonata, and Termitomyces striatus, protein contentdoubled from stipes to caps whereas non-protein nitrogen contentstayed roughly constant (cap concentration 90% that in stipes,

E.A. Hobbie et al. / Soil Biology & Biochemistry 48 (2012) 60e6866

calculated from Alofe et al., 1996). Based on these studies, weconclude that assuming a constant chitin content between caps andstipes is a reasonable simplification, with large increase in proteincontent from stipes to caps driving changes in %N and d15N.

The 13C increase of caps relative to stipes presumably reflectshigher 13C values of protein than in carbohydrates (Webb et al.,1998;A. Ouimette, pers. comm.). The 13C enrichment of protein relative tocarbohydrates may primarily arise from the loss of 13C-depleted CO2during respiration in the tricarboxylic acid (TCA) cycle (O’Leary andYapp, 1978; Grissom and Cleland, 1988; Tcherkez et al., 2003),leaving a pool of 13C-enriched, TCA-derived carbon skeletons toconstruct many amino acids. The C:N ratio of protein is lower thanthe C:N of sporocarps (w3 versus w9; calculated from %C and %Ndata), therefore the difference in 13C between caps and stipes causedby differences in protein content is small relative to the 15N differ-ence. In addition, protein is only several per mille higher in 13C thancarbohydrates (Winkler et al.,1978;Webb et al., 1998) but up to 12&higher in 15N than chitin (Taylor et al., 1997; Webb et al., 1998).

The higher calculated isotopic enrichment for both 15N and 13C inhydrophobic exploration types than in either hydrophilic explora-tion types or in saprotrophic fungi implies that patterns of internalsequestration and remobilization greatly influence 15N patterns and13C values in sporocarps. Studies that specifically measure isotopicpatterns in fungal protein, carbohydrates (including chitin), andnucleic acids would help to further resolve these issues.

4.4. Explaining d15Ncapestipe through multiple regressions

For two of the variables used in the multiple regression, 1 � %Nstipe/%Ncap and %Ncap-stipe, multicollinearity is a potential problem.However, given the relatively modest level of multicollinearity (r2ofthe two variables is 0.50) and the very high sample size (n ¼ 286),it appears that the chance of this inducing substantial errors is verysmall (Mason and Perreault, 1991). We have accordingly retainedboth of these variables in themultiple regression, and interpret themseparately.

The multiple regression analysis presented in Table 2 confirmsthe importance of the relative nitrogen concentration in caps versusstipes (1 � %Nstipe/%Ncap) in explaining d15Ncapestipe, as this valueshould correlatewith the relative proportion of 15N-enriched proteinand 15N-depleted chitin in these two fungal components. If shifts in %N between stipes and caps were only caused by adding protein tocaps, with chitin content held constant, then the coefficient for thisvariable should equal the 15N enrichment of protein relative to chitinof 9.9 � 0.5& (Taylor et al., 1997), multiplied by the fraction ofstipe nitrogen that is chitin (Equation (9)). Since 5.171&/9.9&¼ 0.52,about half of the stipe nitrogen is chitin. Little data exist forcomparison. Non-protein nitrogenwas 33% of total nitrogen inwholemushrooms (Fujihara et al.,1995). Non-protein nitrogen in stipeswas51%, 17%, and 14% of total nitrogen for three saprotrophic species(calculated fromAlofe et al.,1996), whereas 39% of stipe nitrogenwaschitin in ectomycorrhizal fungi (calculations in Appendix 3 usingdata from Taylor et al., 1997).

We expected that %Ncapestipe would positively correlate withd15Ncapestipe, given the higher %N in protein than in chitin, whereasthe actual coefficient in the regression was negative (�0.78&/%N).One plausible explanation is that stipes with low %N should averagea greater proportion of nitrogen as chitin than stipeswith high %N. Ifincreases in the %N of caps relative to stipes are primarily attributedto added protein, then for a doubling of protein from stipes to caps,the relative shift in the fraction of nitrogen attributed to protein(and consequently, the shift in d15N)will be less formushroomswithhigher stipe %N. For example, with chitin at 6.89%N, protein at 16.7%nitrogen (calculated from the inverse of nitrogen:protein in Fujiharaet al., 1995), and chitin at 8% of biomass, doubling protein from 7% to

14% from stipes to caps increases %N by 1.17% and increases d15N by1.28&, whereas doubling protein from 14% to 28% increases %Nby 2.33% andonly increases d15N by 0.85&. For the values given here,the shift in d15Ncapestipe for increases in %Ncapestipewould be�0.63&per %N.

The positive correlation of d13Ccapestipe with d15Ncapestipe reflectsthat the increased protein in caps relative to stipes is high in both15N and 13C. Given an assumed 15N enrichment of 9.9& (Taylor et al.,1997) and a calculated 13C enrichment of 4.2& (see first paragraphof next section), the expected coefficient is 2.36, if shifts in proteinequally affect d15N and d13C. From the actual coefficient of 0.89, wecalculate that the fraction of new carbon that is protein is 0.89/2.36,or 37.7%, of the fraction of new nitrogen that is protein. Given a C:Nof protein of 2.86 (calculated from amino acid composition of fourtaxa in Mattila et al., 2002; calculations in Appendix 3), the esti-mated cap C:N is then 2.86/0.377, or 7.59, which is about 17% lowerthan the value of 9.17 calculated here from average %C (41.46%) and%N (4.49%).

Having hydrophobic ectomycorrhizae increased d15Ncapestipe by1.1& and accounted for about 15% of the explained variance, whereashaving hydrophilic ectomycorrhizae decreased d15Ncapestipe by 0.6&and accounted for 7% of the explained variance. The other categoricalvariables (litter decay fungi or wood decay fungi) were not signifi-cantly correlated with d15Ncapestipe, suggesting that these fungiprocessnitrogen similarly between caps and stipes. The coefficient forthese variables can be explained as reflecting variable 15N enrichmentof protein relative to chitin among the four functional groups.If protein as a percentage of total nitrogen is 21%higher in caps than instipes (calculated from Taylor et al., 1997), then the additional 15Nenrichment in hydrophobic taxa of 1.084& in our regression reflectsan additional enrichment of protein relative to chitin of 5.1&. Simi-larly, the coefficient in hydrophilic taxa of �0.571& reflects anadditional enrichment of protein relative to chitin of �2.7&. If thebaseline fractionation between protein and chitin is 9.9&, then thefractionation of chitin versus protein in hydrophobic taxa will be15.0&, in hydrophilic taxawill be 7.2&, and in saprotrophic fungiwillbe 9.9&. The only comparable data are in Taylor et al. (1997), inwhichprotein differed from chitin by 8.45& (Amanita, hydrophilic),10.85&,and 10.3& (both Suillus species, hydrophobic). These differences infractionation attributed to hydrophilic and hydrophobic ectomycor-rhizal taxa suggest that patterns of nitrogen storage prior to sporo-carp formation and nitrogen transfer to host plants can influenceisotopic partitioning between caps and stipes, and presumablyinfluence the underlying isotopic partitioning between fungal proteinand chitin.

4.5. Explaining d13Ccapestipe through multiple regressions

Correlations of d13Ccapestipewith %Ncapestipe could be attributed tothe higher d13C and %N in protein than in chitin and other carbo-hydrates. If %N increases in caps because of added protein (assumedto be 16.67% nitrogen) and chitin content remains constant betweencaps and stipes, then the coefficient of %Ncapestipe, 0.253&/%N,can be used to estimate the 13C enrichment of protein relative tonon-chitin components of 0.253 � 0.033& �16.67, or 4.2 � 0.5&.Carbohydrates dominate fungal composition, averaging 62% in fourspecies (Mattila et al., 2002), and losses fromstipes to caps to balanceprotein gains must necessarily be largely of carbohydrates (Alofeet al., 1996). Fungal carbohydrates and protein have yet to becompared isotopically; Webb et al. (1998) reported that locustprotein was 1.6& enriched in 13C relative to chitin and 3.4&enriched in 13C relative to trehalose.

The positive correlation of d13Ccapestipe with d15Ncapestipe reflectsthat the increased protein in caps relative to stipes is high in both15N and 13C. A coefficient of 0.42 is expected for the shift of

E.A. Hobbie et al. / Soil Biology & Biochemistry 48 (2012) 60e68 67

d13Ccapestipe with d15Ncapestipe if protein C/N and cap C/N are similar,since the increased protein in caps will be 4.2& higher in 13Cand 9.9& higher in 15N than carbohydrates. The actual coefficient of0.121 � 0.017 therefore suggests that cap C:N is 2.86 � 0.42/0.121,or 9.93, which is 8% higher than the actual value of 9.17, and 31%higher than the estimated C:N (7.59) from the coefficient ofd13Ccapestipe when regressed against d15Ncapestipe.

It is possible to combine information in the coefficient ofd13Ccapestipe against d15Ncapestipe and the coefficient of d15Ncapestipeagainst d13Ccapestipe. We have expressed the coefficient ofd13Ccapestipe as:

0:853 ¼ d15Nprotein�chitin=d13Cprotein�chitin � C=Nprotein=C=Ncap

(10)

For the coefficient of d15Ncapestipe against d13Ccapestipe, we have:

0:121 ¼ d13Cprotein�chitin=d15Nptotein�chitin � C=Nprotein=C=Ncap

(11)

Solving for C/Ncap gives:

C=Ncap ¼ C=Nprotein=ð0:853 x 0:121Þ0:5 (12)

For a C/Nprotein of 2.86, the estimated C/Ncap is then 8.90, which iswithin 3% of the value of 9.17 calculated from average %N and %C ofcaps. This close agreement suggests that our assumptions about thecauses of shifts in d15N, d13C, and %N are internally consistent.

The negative correlation between d13Ccapestipe and %Ccapestipe ispresumably caused by variations in lipid content, as lipids are bothhigher in %C and lower in 13C relative to carbohydrates and protein(Poorter et al., 1997; Hobbie andWerner, 2004). Caps appear higherin lipid content than stipes in mushrooms (Alam et al., 2008), withthree species in Alofe et al. (1996) averaging 3% lipids in stipesand 5% in caps. Information is lacking on the 13C depletion of fungallipids relative to carbohydrates or protein in sporocarps, althoughphospholipid fatty acids were 2.4& and 7.4& depleted in 13Crelative to bulk mycelia in two cultured taxa (calculated from Ruesset al., 2005).

Although no categorical variables correlated with d13Ccapestipe,shifts in d13Ccapestipe caused by the categorical variables are effec-tively incorporated into the multiple regression via the coefficient ofd15Nproteinechitin. That is, any shift in d15Nproteinechitin caused by beinghydrophobic or hydrophilic will then change d13Ccapestipe accordingto the given coefficient (0.121). If an additional factor was alteringd15N thatwas not altering d13C, then the estimated C/Ncapwould havenot agreed so closely with the measured value. We conclude thatprocesses differing in magnitude between hydrophobic and hydro-philic taxa that alter nitrogen isotopes also alter carbon isotopes.

4.6. Conclusions

Patterns of nitrogen mobilization and isotopic fractionationbetween protein and other nitrogen-containing molecules appearto be themain driver of the 15N enrichment of caps relative to stipes.Although the fractionation is framed as reflecting two main pools infungi, protein and chitin, other nitrogen-containing molecules couldalso contribute, such as nucleic acids (Gottlieb and Van Etten, 1964;Iten and Matile, 1970). For example, Fujihara et al. (1995) estimatedthat the relative contributions of different nitrogen forms to sporo-carps in 13 species varied from 67% (protein) to 2% (NH3), withintermediate amounts for chitin (9%), nucleic acids (6%), andunknown nitrogen (16%). Chikaraishi et al. (2007) suggested that 15Ndepletion during aminotransferase reactions (such as movement ofan amido group from glutamine to glucose to form glucosamine)

drive many d15N patterns during amino acid metabolism. It ispossible that such aminotransferase reactions control the 15Ndepletion of both fungal chitin and ectomycorrhizal plants relative tofungal protein.

If nitrogenwere delivered as a single isotopically uniform sourceto sporocarps and then differentiated into protein or chitin, wewould expect that 15N differences between caps and stipes wouldbe similar across exploration types when corrected for differencesin %N. Instead, our results suggest two non-exclusive possibilities:(1) the two main nitrogen pools in sporocarps, chitin and protein,can have different internal sources and are delivered separatelyto the developing primordia, and (2) source nitrogen is processeddifferently in hydrophobic ectomycorrhizal fungi relative to otherfungal types into protein and chitin.

The mechanistic and conceptual explanations offered here onthe causes of isotopic differences in sporocarps are unlikely to bethe full story. However, we hope that these ideas will stimulatefurther critical research and analyses of the underlyingmechanismscausing these patterns, and therefore provide additional insightsinto the belowground functioning in carbon and nitrogen dynamicsof ectomycorrhizal and saprotrophic fungi.

One promising avenue is indicated by the existing literature onfungal metabolism during sporocarp formation (Moore,1998). Suchstudies have revealed extensive differences in enzymatic processesbetween caps and stipes; for example, activities of NADP-glutamatedehydrogenase, glutamine synthetase, ornithine acetyltransferase,and ornithine carbamyltransferase are higher in the cap than thestipe of Coprinus cinereus fruitbodies while urease activity is muchhigher in stipes (Moore, 1998). The reactions catalyzed by theseenzymes will influence the movement of amino acids, ammonia,and other compounds that ultimately control nitrogen and carbonisotope distributions. Studies under controlled conditions in whichisotopic patterns of stipes, caps, protein, and chitin could becompared against differences in composition and metabolic path-ways would also be informative.

Acknowledgements

We thank John Hobbie, Luke Nave, Andy Ouimette, Don Phillips,Stephen Trudell, and two anonymous reviewers for comments onprior versions. The US Environmental Protection Agency partiallyfunded this research, which has been reviewed and approved forpublication as an EPA document. Mention of trade names orcommercial products does not constitute endorsement or recom-mendation for use. This work was also supported by two grantsfrom the U.S. NSF Division of Environmental Biology, by a researchfellowship to F.S. by the Scientist Committee of NATO, and bya Bullard Fellowship to E.H. from Harvard University.

Appendix. Supplementary material

Supplementary material associated with this article can befound, in the online version, at doi:10.1016/j.soilbio.2012.01.014.

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