13c and 15n in microarthropods reveal little response of douglas-fir ecosystems to climate change

13C and 15N in microarthropods reveal little response ofDouglas-fir ecosystems to climate change

E R I K A . H O B B I E *, PA U L T . R Y G I E W I C Z w , M A R K G . J O H N S O N w and

A N D R E W R . M O L D E N K E z*National Research Council, US Environmental Protection Agency, Corvallis, OR 97333, USA, wNational Health

and Environmental Effects Research Lab, US Environmental Protection Agency, Corvallis, OR 97333, USA,

zDepartment of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97333, USA

Abstract

Understanding ecosystem carbon (C) and nitrogen (N) cycling under global change

requires experiments maintaining natural interactions among soil structure, soil com-

munities, nutrient availability, and plant growth. In model Douglas-fir ecosystems

maintained for five growing seasons, elevated temperature and carbon dioxide (CO2)

increased photosynthesis and increased C storage belowground but not aboveground.

We hypothesized that interactions between N cycling and C fluxes through two main

groups of microbes, mycorrhizal fungi (symbiotic with plants) and saprotrophic fungi

(free-living), mediated ecosystem C storage. To quantify proportions of mycorrhizal and

saprotrophic fungi, we measured stable isotopes in fungivorous microarthropods that

efficiently censused the fungal community. Fungivorous microarthropods consumed on

average 35% mycorrhizal fungi and 65% saprotrophic fungi. Elevated temperature

decreased C flux through mycorrhizal fungi by 7%, whereas elevated CO2 increased it

by 4%. The dietary proportion of mycorrhizal fungi correlated across treatments with

total plant biomass (n 5 4, r2 5 0.96, P 5 0.021), but not with root biomass. This suggests

that belowground allocation increased with increasing plant biomass, but that mycor-

rhizal fungi were stronger sinks for recent photosynthate than roots. Low N content of

needles (0.8–1.1%) and A horizon soil (0.11%) coupled with high C : N ratios of A horizon

soil (25–26) and litter (36–48) indicated severe N limitation. Elevated temperature

treatments increased the saprotrophic decomposition of litter and lowered litter C : N

ratios. Because of low N availability of this litter, its decomposition presumably

increased N immobilization belowground, thereby restricting soil N availability for

both mycorrhizal fungi and plant growth. Although increased photosynthesis with

elevated CO2 increased allocation of C to ectomycorrhizal fungi, it did not benefit plant

N status. Most N for plants and soil storage was derived from litter decomposition.

N sequestration by mycorrhizal fungi and limited N release during litter decomposition

by saprotrophic fungi restricted N supply to plants, thereby constraining plant growth

response to the different treatments.

Keywords: ecosystem response, food webs, global change, soil carbon, stable isotopes

Received 28 September 2006; revised version received 6 February 2007 and accepted 23 February 2007

Introduction

Sequestration of a significant amount of atmospheric

carbon dioxide (CO2) by vegetation and soils is an im-

portant assumption in the Kyoto Protocol for carbon (C)

accounting and for mitigating anthropogenically caused

increases in atmospheric CO2 (Kimble et al., 2002).

In particular, because properly managed reforestation

may increase both aboveground and belowground C

storage, many governments are focusing on reforestation

as a mechanism to meet their Kyoto protocol obligations.

Studies suggest that C sequestration by vegetation is

limited primarily by nutrient supply (Hungate et al.,

2003), whereas C storage in soils is influenced by

Correspondence: Present address: Erik Hobbie, Morse Hall, CSRC-

EOS, University of New Hampshire, Durham, NH 03824, USA,

fax 11 603 862 0188, e-mail: [email protected]

Global Change Biology (2007) 13, 1386–1397, doi: 10.1111/j.1365-2486.2007.01379.x

r 2007 The Authors1386 Journal compilation r 2007 Blackwell Publishing Ltd

above- and belowground inputs (Schlesinger & Lichter,

2001), soil texture (Giardina & Ryan, 2000), and the

functioning of soil organisms (Hu et al., 1999). Soil organ-

isms influence decomposition, mineralization, and

nutrient availability (Copley, 2000), and thereby indirectly

influence aboveground productivity. An improved under-

standing of soil food web functioning may therefore

increase our ability to manage C stocks stored in

vegetation and soils and increase our ability to predict

belowground responses to climate change (Hunt & Wall,

2002).

We undertook an experiment lasting five growing

seasons at the US Environmental Protection Agency’s

mesocosm facility in Corvallis, Oregon to study the

effects of elevated atmospheric temperature and CO2

on C and nitrogen (N) cycling in Douglas-fir seedlings

and the associated soil ecosystem (Tingey et al., 1996). In

this study, we determined how elevated CO2 and

temperature affected belowground soil food webs and

how these influences altered primary production under

conditions of low N availability. By using enclosed

chambers, a continuous 13C label could be applied in

both ambient and elevated CO2 treatments (Hobbie

et al., 2004a), allowing soil food web functioning to be

probed with a high degree of resolution.

In Douglas-fir forests, fungi dominate the microbial

community (Ingham & Thies, 1997) and serve as the

initial energy source for the soil food web. Fungi can be

functionally classified into mycorrhizal fungi and sa-

protrophic fungi, with mycorrhizal fungi symbiotic

with plants and supplying host plants with most essen-

tial nutrients in return for plant sugars. In contrast,

saprotrophic fungi are free-living microbes that rely on

the decay of dead organic matter for nutrition. Because

of broad taxonomic overlap between these two func-

tional groupings (Hibbett et al., 2000), the two types

cannot generally be quantified separately in the soil.

However, in CO2 fertilization studies that use 13C-

depleted CO2 to maintain or increase CO2 levels, recent

photosynthate can be traced belowground (Schlesinger

& Lichter, 2001), and can be used to separate food

sources of heterotrophic organisms into recent photo-

synthate (13C-depleted) used by mycorrhizal fungi and

organic material (13C-enriched) used by saprotrophic

fungi that existed before CO2 fertilization. In addition,

the ectomycorrhizal fungi symbiotic with Douglas-fir

are enriched in 15N relative to both plants and sapro-

trophic fungi (Hobbie et al., 1999; Kohzu et al., 1999;

Hobbie & Colpaert, 2003). These differences can serve

as tracers of fungal composition.

In this study, we used isotopic analyses of fungivor-

ous microarthropods collected during the final growing

season to census the fungal community. Such isotopic

analyses have been used in field studies to examine

trophic niches of microarthropods (Schneider et al.,

2004; Chahartaghi et al., 2005). Because of the presumed

high functional redundancy of the microarthropod

community (Liiri et al., 2002), we assumed that mycor-

rhizal and saprotrophic fungi were consumed by the

microarthropod community in proportion to their

abundance and that fungivorous microarthropods did

not discriminate between the two types of hyphae. The13C and 15N content of our two fungal types differed in

our system, so we used 13C and 15N content (expressed

as d13C and d15N values) of microarthropods to parti-

tion their diets into sources from mycorrhizal vs. sapro-

trophic hyphae.

To reveal patterns of C and N flow through the

belowground ecosystem, this information was then

combined with d13C values, d15N values, and C seques-

tration data for the vegetation, the litter layer and the A

horizon. Because C allocation to ectomycorrhizal fungi

decreases at high N availability relative to low N avail-

ability (Hobbie & Colpaert, 2003) and elevated tempera-

ture increases decomposer activity and N cycling

(McGuire et al., 1996; Melillo et al., 2002), we hypothe-

sized that C flux to ectomycorrhizal fungi relative to

saprotrophic fungi should decrease with elevated tem-

perature. In contrast, because elevated CO2 should

decrease N availability, allocation to ectomycorrhizal

fungi should increase in elevated CO2 treatments rela-

tive to ambient CO2 (Alberton et al., 2005).

Methods

Twelve sun-lit, controlled-environment mesocosms

(1 m� 2 m footprint with a 1 m deep soil compartment

and a 1.3–1.5 m tall enclosed canopy space) contained

soil collected from an old-growth Douglas-fir (Pseudot-

suga menziesii) forest. The soil was reconstructed by

horizon and then planted with 14 two-year-old Dou-

glas-fir seedlings. The treatments were a 2� 2 factorial

of temperature treatments (ambient, and ambient plus

4 1C) and CO2 treatments (ambient, and ambient plus

200 ppm). Treatments started in late summer 1993 and

the mesocosms were continuously monitored over five

growing seasons until July 1997. At that time, a com-

plete destructive harvest of plants, litter layers, and soil

horizons was done on the instrumented half of each

mesocosm (see Tingey et al., 1996 for a detailed descrip-

tion of the mesocosm instrumentation). C and N ele-

mental and isotopic composition in needles, roots, litter

layers, and soil horizons were measured (Hobbie et al.,

2001, 2002a, b, 2004a) and changes in C stocks during

the experiment calculated. Chamber CO2 levels were

maintained at near target values by adding 13C-de-

pleted CO2 (d13C 5�35%) and through the venting of

chamber air with dump valves. The native litter layer

B E L O W G R O U N D R E S P O N S E S O F D O U G L A S - F I R S Y S T E M S 1387

r 2007 The AuthorsJournal compilation r 2007 Blackwell Publishing Ltd, Global Change Biology, 13, 1386–1397

from the Douglas-fir forest was also included after

sieving to 2.5 cm to remove coarse debris. As the seed-

lings generated only a small amount of litter during the

exposure, additional litter was added to the chambers

after 2 years when the volume of the litter layer had

decreased by 50%.

In the spring and summer of the final (fifth) growing

season, soil microarthropods were extracted from soil

and litter using Tullgren funnels, taxonomically identi-

fied, and classified as to their food sources based upon

the morphology of their mouth parts and natural his-

tory (Dindal, 1990; Coleman & Crossley, 1996). Taxa

classified as fungivores and root feeders were analyzed

for d13C and d15N signatures. The root feeders were all

weevils in the family Curculionidae, whereas fungi-

vores were primarily oribatid mites of the genera Odon-

todamaeus, Caenobelba, Eremaeus, Kartoeremaeus, and

Propelops, in addition to springtails of the genus Isotoma.

Single individuals of weevils were isotopically ana-

lyzed, whereas from 10 to 50 individual mites or spring-

tails of a single taxon were combined for one isotopic

analysis. Soil, litter and microarthopods were analyzed

for d13C and d15N signatures on a Finnigan Delta-Plus

linked to a Carlo Erba NC2500 elemental analyzer

(Finnigan MAT GmbH, Bremen, Germany) located at

the US Environmental Protection Agency in Corvallis,

Oregon. Laboratory standards for isotopic measure-

ments were pine needles (NIST 1575) and acetanilide.

The average difference of duplicate samples was 0.1%for d13C and 0.2% for d15N.

After calculating the d13C of microarthropods only

consuming mycorrhizal fungi (d13CM) or saprotrophic

fungi (d13CS), the contribution of mycorrhizal fungi to

the diet of fungivorous microarthropods was calculated

from the mean d13C of fungivorous microarthropods

using the following equation, fM 5 (d13CF�d13CM)/

(d13CS�d13CM), where fM equals the proportion of fun-

givorous diet derived from mycorrhizal fungi and d13CF

equals the mean d13C of harvested fungivores.

C sequestration data for the litter layer, A horizon,

and vegetation were combined with C : N ratio data to

estimate N fluxes to and from these pools. Isotopic

signatures of ecosystem pools provided additional con-

straints on and insights into these fluxes.

Results

Estimating diets of fungivorous microarthropods

We needed to estimate the isotopic endpoints for micro-

arthropods consuming only saprotrophic or only my-

corrhizal fungi to calculate the proportion of

mycorrhizal vs. saprotrophic fungi in the diets of

fungivorous microarthropods. As saprotrophic and

mycorrhizal fungi differed isotopically in our system,

a dietary isotope mixing line of fungivorous microar-

thropods was constructed. As the isotopic compositions

of mycorrhizal fungi and roots also differed, a similar

dietary mixing line was constructed for root-feeding

weevils consuming both mycorrhizal and plant

material. The intersection of these two mixing lines then

represents the calculated isotopic signature of an organ-

ism consuming only mycorrhizal fungi. This value was

calculated to be d15N 5 6.9 � 1.6%, d13C 5�34.3 � 0.5%(Fig. 1, Point A, shown for elevated CO2 and tempera-

ture treatments).

The 13C content of mycorrhizal fungi differed be-

tween ambient and elevated CO2 treatments because

mesocosm CO2 was more depleted in 13C under ele-

vated CO2 than under ambient CO2 due to greater

inputs of 13C-depleted CO2. Therefore, a separate

mixing line of fungivorous microarthropods could be

constructed for the ambient CO2 treatments. The

intersection of the dietary mixing lines of fungi-

vorous microarthropods at ambient vs. elevated CO2,

therefore, represented the theoretical isotopic signature

of an organism consuming only saprotrophic fungi.

This value was calculated to be d15N 5 0.2 � 0.9%,

d13C 5�23.1 � 1.4% (Fig. 1, Point B).

The calculated value for a fungivore feeding only on

mycorrhizal fungi under elevated CO2 (�34%) was 1%enriched relative to root d13C (�35%); root feeders were

similarly enriched (1.2%) in 13C relative to roots. Ac-

cordingly, to estimate treatment effects on fungivorous

diets, we adopted the direct approach of calculating

d13C signatures for pure mycorrhizal fungivores from

the d13C signature of roots for each chamber, plus a

constant fractionation factor of 1.0%, as shown in Fig. 1.

The d13C of fungivores only consuming saprotrophic

fungi was assumed to be the same as above, �23.1%(Fig. 1) as very little new litter was produced in the

mesocosms and the saprotrophic fungi primarily fed

on old litter whose d13C value was not affected by

tank CO2.

We calculated the relative contribution of mycorrhizal

hyphae to the diet by treatment from the average d13C

value of fungivorous microarthropods and from our

estimates of isotopic endmembers for fungivorous mi-

croarthropods feeding solely on saprotrophic or mycor-

rhizal fungi (Table 1 and Fig. 1, as discussed above).

This apportioning served as a relative measure of

hyphal abundances of mycorrhizal vs. saprotrophic

fungi. The proportion of fungivorous diet derived from

mycorrhizal fungi varied among fungivorous species

and treatment but averaged 35% for the experiment.

Our results show nonsignificant trends due to treat-

ments in the proportion of fungal hyphae that was

mycorrhizal: an increase of 4% with elevated CO2 and

1388 E . A . H O B B I E et al.


a decrease of 7% with elevated temperature. Both

changes agreed with our hypothesized patterns.

Several previously measured parameters correlated

with the dietary estimates. The proportion of mycor-

rhizal fungi in the diet correlated with total plant

biomass (Fig. 2a, r2 5 0.96, n 5 4, P 5 0.021), with the

ratio of needle mass to fine root mass (Fig. 2b, r2 5 0.98,

P 5 0.011), with needle mass (r2 5 0.88, P 5 0.082, data

not shown), and with the fraction of C derived from

recent photosynthate in the A horizon (r2 5 0.84,

P 5 0.120, data not shown).

C and N cycling under elevated CO2 and temperature

This study was part of a comprehensive investigation

into the effects in Douglas-fir ecosystems of elevated

CO2 and elevated temperature on microbial commu-

nities, plant responses, C cycling, and N cycling

(Rygiewicz et al., 2000; Hobbie et al., 2001; Lin et al.,

2001; Olszyk et al., 2003; Tingey et al., 2006). It, therefore,

draws on extensive measurements of many different

parameters, allowing a more complete picture of how

the ecosystem functioned than normally possible.

In other research from this investigation, measurements

of 13C and 18O content were used to partition soil-

respired CO2 among litter respiration (60–65% of total),

rhizosphere respiration (16–32% of total), and soil or-

ganic matter (SOM) oxidation (7–18% of total) (Lin et al.,

2001). Soil CO2 efflux (SCE) increased about 20% with

elevated CO2, 26% with elevated temperature, and 54%

with elevated CO2 and temperature relative to controls

during Years 2 and 3 of the study (Lin et al., 2001;

Year 3 values are shown in Table 2). However, by

Years 4 and 5, SCE was slightly lower (o10%) in

elevated CO2 treatments than in ambient CO2 treat-

ments, with no response in temperature treatments

(Tingey et al., 2006).

Fig. 1 Isotopic patterns in root-feeding weevils (circles) and fungal-feeding soil arthropods (upside-down triangles) under ambient

(open symbols) and elevated carbon dioxide (CO2) (closed symbols) treatments reflect the proportion of mycorrhizal and saprotrophic

fungi in their diet. By constructing intersecting regression lines, we calculated that point ‘A’ represents the isotopic signature of a

hypothetical fungivore feeding solely on mycorrhizal hyphal tissue, whereas point ‘B’ represents the isotopic signature of a hypothetical

fungivore feeding solely on saprotrophic fungal tissue. Also shown are d13C and d15N values for foliage (squares), fine roots (diamonds),

and litter (triangles) from ambient CO2 and elevated CO2 treatments, � standard error (SE), with ambient CO2 treatments given by open

symbols and elevated CO2 treatments given by closed symbols. Data on fungal feeders at elevated CO2 were taken from elevated CO2

and temperature treatments, all other values were not separated by temperature treatments.

Table 1 Proportion of mycorrhizal fungi in fungivorous diets

under elevated CO2 and temperature

Treatment

Proportion (%) of

fungivorous diet from

mycorrhizal fungi (%)

Control 36.3 � 5.3

Elevated temperature (ET) 29.5 � 3.5

Elevated CO2 (EC) 40.6 � 4.7

EC and ET 33.1 � 3.3

Values are given � standard error.

CO2, carbon dioxide.



Soil and plant C storage integrate many processes,

including photosynthesis and plant respiration, soil

sequestration and release (via autotrophic and hetero-

trophic respiration; e.g., litter decomposition and SOM

oxidation), and C allocation and partitioning patterns.

Elevated CO2 (P 5 0.027) and elevated temperature

(P 5 0.056) affected the amount of total C held in the

litter layer and A horizon soil (Table 3). Both treatments

reduced the overall loss of C from these horizons

compared with the control, primarily by increasing

the amount of C held in the A mineral horizon. The

litter layer lost about 1500 g C m�2. Increased soil C

storage with elevated temperature was balanced by a

slight decrease in plant biomass, so that temperature

did not significantly affect overall system C. Neither

CO2 nor temperature treatments significantly affected

plant biomass or N in the mesocosms (Olszyk et al.,

2003; Tingey et al., 2003; Table 4).

Soil and litter N content and C : N ratios control how

quickly these pools become net sources of N for uptake

(Berg & Staaf, 1981; Knops et al., 2002). The C : N ratio of

the lower litter layer ranged from 36 to 41 and was

lower in elevated temperature treatments (P 5 0.040)

and higher in elevated CO2 treatments (P 5 0.037) than

under ambient conditions (Table 4). In contrast, the

C : N ratio of the upper litter layer ranged from 42 to

48, the C : N ratio of the A horizon ranged from 25 to 26,

and the N content of the A horizon ranged from 0.105%

to 0.120%, with none of these three parameters differing

significantly by treatment (data not shown). Litter C : N

values in Douglas-fir sites of >35 correlate with no

significant N mineralization (Prescott et al., 2000). Simi-

larly, a study of 22 Douglas-fir sites in Oregon indicated

that soil nitrate is low relative to soil ammonium when

soil C : N is 25–28 or when soil %N is o0.25% (Perakis

et al., 2006). The C : N and %N values in litter and A

horizon soil in our study accordingly indicate that N is

highly limiting, a result confirmed by the very low

foliar %N of o1% (Table 4).

C and N concentrations in different soil pools (Table

5) can be used to infer patterns in C and N movement.

The lower litter layer had a lower %N than the upper

litter layer (0.80% vs. 0.92%, paired t-test, n 5 12,

P 5 0.004), suggesting that 13% of lower litter N had

700

650

600

550

500

450

400

2.0

1.8

1.6

1.4

1.2

1.0

0.8

25 30Proportion of diet (%) from mycorrhizal fungi

35 40 45

25 30Proportion of diet (%) from mycorrhizal fungi

35 40 45

Pla

nt b

iom

ass

(g m

−2)

Nee

dle

: fin

e ro

ot r

atio

ElevatedCO

Elevated CO

Elevatedtemperature

Elevatedtemperature

Elevated CO and temperature

Elevated COand temperature

Control

Control

(a)

(b)

Fig. 2 Proportion of dietary carbon for fungivorous microar-

thropods derived from mycorrhizal fungi, as given in Table 1,

correlates with both total plant biomass and with the foliage : fine

root ratio (data from Table 3, values are shown � SE). (a) Total

plant carbon vs. percent of diet from mycorrhizal fungi (r2 5 0.96,

P 5 0.021). (b) Needle : fine root ratio vs. percent of diet from

mycorrhizal fungi (r2 5 0.98, P 5 0.011).

Table 2 Overall and treatment-specific partitioning of soil CO2 effluxes during Year 3 among litter respiration, rhizosphere

respiration, and SOM oxidation

Treatment

Total soil

CO2 (%)

Litter

respiration (%)

Rhizosphere

respiration (%)

SOM

oxidation (%)

Overall – 60–65 16–32 7–18

Elevated temperature (ET) 1 26 1 36 1 28 1 14

Elevated CO2 (EC) 1 20 1 4 1 61 �44

EC and ET 1 54 1 51 1 115 �30

Changes in soil CO2 effluxes for elevated CO2 and temperature treatments are relative to control plot values. Values are from

Lin et al. (2001).

CO2, carbon dioxide; SOM, soil organic matter.

1390 E . A . H O B B I E et al.


Fig. 3 (a) %N and %C of the lower litter layer are plotted for each mesocosm, giving a regression of %C 5 43.2�%N�3.5% (r2 5 0.89,

n 5 12, Po0.001). Treatments are: filled symbols, elevated CO2; clear symbols, ambient CO2; circles, ambient temperature; triangles,

elevated temperature. (b) %N and %C of the A horizon are plotted for each mesocosm, giving a regression of %C 5 36.5�%N�1.3%

(r2 5 0.88, n 5 12, Po0.001). Treatments are indicated as in Fig. 3a.

Table 3 Carbon storage or loss in response to elevated CO2 and temperature

Treatment

Carbon storage (1) or loss (�) (g C m�2)

Litter A horizon Total

Control �1390 � 200 �195 � 375 �1590 � 175

Elevated temperature (ET) �1500 � 192 1 77 � 317 �1420 � 255

Elevated CO2 (EC) �1660 � 7 1 345 � 145 �1310 � 152

EC and ET �1360 � 149 1 793 � 197 �570 � 79

Negative values represent decreases in carbon (C) storage from the initial to the final harvest.

Values are given � standard error.



been exported from the lower litter layer into the A

horizon. C concentrations correlated strongly with %N

in the lower litter layer (r2 5 0.90, n 5 12, Po0.0001,

Fig. 3a), with a slope of 45 � 3, meaning that the

average C : N ratio of organic matter lost from the litter

layer was 45. This contrasts with an average C : N ratio

of 39. C and N concentrations in the A horizon were

also highly correlated (r2 5 0.88, n 5 12, Po0.0001, Fig.

3b), with a slope of 36 � 4. This suggests an average

C : N ratio of added organic matter of 36, vs. a measured

C : N of 25.

Isotope patterns in different soil pools can provide

additional insight into transfers of C and N among

pools. 15N content increased slightly (0.4%) from the

upper litter layer to the lower litter layer, increased

dramatically (5.6%) from the lower litter to the A

horizon, and then increased moderately in the B and

C horizons (1.5–2%) (Table 5). 15N content did not vary

by treatment for any soil pool. The d15N and d13C of the

A horizon were correlated within each CO2 treatment

(Fig. 4), reflecting the linked inputs to the A horizon of13C-depleted recent photosynthate and 15N-depleted

litter in these N-limited systems.

Discussion

Determining proportions of ectomycorrhizal andsaprotrophic fungi in diets

Both C and N isotopes have been used qualitatively to

determine ectomycorrhizal vs. saprotrophic life history

strategies in studies of fungal fruiting bodies (Hogberg

et al., 1999; Kohzu et al., 1999). Measurements of 13C in

hyphae collected from in-growth cores have been used

to separate mycorrhizal from saprotrophic fungi and

suggest that all fungal hyphae in the cores could be

attributed to mycorrhizal fungi (Wallander et al., 2001).

Extrapolations were then made to estimate plant alloca-

tion to ectomycorrhizal fungi at the ecosystem scale.

However, such scaling up is difficult because the effects

on mycorrhizal allocation of using nutrient-poor sand

to fill in-growth cores are unknown. In addition, ecto-

mycorrhizal fungi differ from saprotrophic fungi in

d13C by only about 3% (Hobbie et al., 1999; Hogberg

et al., 1999; Kohzu et al., 1999), making it difficult to use

such measurements quantitatively. Here, a relatively

large difference in d13C between ectomycorrhizal and

saprotrophic fungi of 6–13% resulted from using CO2

derived from fossil fuels to maintain either ambient or

elevated concentrations in semiclosed chambers. By

using fungivorous microarthropods as our sampling

devices, we could estimate the relative abundance of

saprotrophic vs. ectomycorrhizal fungi. This approach

could potentially be used in other sites that have used

Fig. 4 d15N and d13C of the A horizon are plotted for each

mesocosm, giving a regression for ambient CO2 treatments of

d15N 5 1.944� d13C 1 52.9% (r2 5 0.86, n 5 12, P 5 0.008) and

for elevated CO2 treatments of d15N 5 0.725� d13C 1 22.8%(r2 5 0.64, n 5 12, P 5 0.056). Treatments are indicated as in Fig. 3.

Table 4 C : N of the lower litter layer and other ecosystem properties at the final harvest

Treatment

C : N of

lower litter

Plant biomass

(g m�2)

Plant nitrogen

(g N m�2)

Needle biomass

(g m�2)

Needle: fine

root mass

New C in

A horizon (%)

First-year

foliar N (%)

Control 38.1 � 1.5 3860 � 850 25.6 � 6.2 910 � 308 1.40 � 0.53 14.0 � 2.6 0.93 � 0.09

Elevated

temperature (ET)

36.3 � 0.6 3370 � 500 23.6 � 4.0 721 � 98 0.95 � 0.04 10.7 � 4.7 1.07 � 0.12

Elevated CO2 41.2 � 0.8 4350 � 410 22.9 � 2.8 1092 � 189 1.60 � 0.27 15.0 � 2.3 0.80 � 0.09

EC and ET 38.0 � 0.9 3510 � 440 23.2 � 3.0 707 � 105 1.20 � 0.38 13.7 � 2.2 0.90 � 0.10

The C : N of the upper litter layer is reported in the text. Values are given � standard error.

Values of plant biomass, needle biomass, and needle : fine root biomass are calculated from Olszyk et al. (2003), plant nitrogen is

calculated from Tingey et al. (2003), % new carbon in the A horizon is from Hobbie et al. (2004a), and foliar %N for first-year needles

is from Hobbie et al. (2001). The litter was split into an upper and lower layer, with the two layers analyzed separately Hobbie et al.

(2004a).

1392 E . A . H O B B I E et al.


13C-depleted CO2, such as open-top chambers or Free-

Air CO2 Enrichment (FACE) sites, although in these

cases labeling control plots (i.e. ambient CO2 levels)

with 13C-depleted CO2 is problematic.

Changing the 13C enrichment from roots to fungivor-

ous microarthropods affected the calculated patterns of

fungal consumption only slightly. Average proportion

of mycorrhizal hyphae in the diet across all treatments

was 34.8% at a 13C enrichment from roots to fungivor-

ous microarthropods of 1.0%; at an enrichment of 0.5%the calculated proportion decreased by 1.8% to 33.0%

and at an enrichment of 1.5% the calculated proportion

increased by 2.0–36.8%, with little change among treat-

ments in relative consumption of mycorrhizal hyphae.

Our estimated 13C enrichment of 1% from roots to

fungivorous microarthropods agrees with culture stu-

dies that examined 13C fractionation from food sources

to fungi and from fungi to fungivorous microarthro-

pods. For example, Ruess et al. (2005) measured an

average 13C enrichment in the collembolan Protaphorura

firmata relative to its diet of the fungi Chaetomium

globosum and Cladosporium cladosporioides of

0.9 � 0.7%. Given that the 13C enrichment of fungi

relative to supplied simple C sources was about 0%across three separate studies (0.3 � 0.5% for 12 strains

of saprotrophic soil fungi supplied with sucrose,

Hobbie et al., 2003; 0.0 � 0.2% for three fungal strains

supplied with 90% sucrose/10% malt extract, Henn &

Chapela, 2000; and �0.3 � 0.7% for 10 strains of ecto-

mycorrhizal fungi supplied with glucose, Hobbie et al.,

2004b), a total 13C enrichment of 1% from roots to

fungivorous microarthropods appears reasonable.

We assumed that fungivorous microarthropods on

average did not discriminate between ectomycorrhizal

and saprotrophic fungi. Although some feeding trials

have demonstrated that Collembola (Scheu & Simmer-

ling, 2004) or oribatid mites (Schneider & Maraun, 2005)

may discriminate against particular fungi when given a

choice, it is unlikely that fungivorous microarthropods

discriminate against ectomycorrhizal or saprotrophic

fungi as a group. Overall discrimination should be

minimal because the large taxonomic overlap between

saprotrophic and ectomycorrhizal fungi (Hibbett et al.,

2000) reduces large-scale morphological or biochemical

differences by which fungivorous microarthropods

could discriminate as a group between these two fungal

life history strategies. In addition, a slight preference for

one of the life history strategies would not unduly bias

the conclusions of our study as long as that preference

does not change greatly across the treatments.

Fig. 5 Schematic of fluxes of carbon (C) and nitrogen (N) among litter, the A horizon, saprotrophic fungi, mycorrhizal fungi, and plants,

as derived from consumption patterns of fungivorous mites and carbon and nitrogen elemental and isotopic patterns of litter and soil

horizons.

Table 5 Elemental and isotopic values for the litter layer and soil horizons, averaged across all treatments (n 5 12, standard errors

also given)

Pool %N d15N (%) %C d13C (%)

Upper litter (Oi) 0.92 � 0.03 �1.9 � 0.1 41.3 � 1.7 �27.6 � 0.1

Lower litter (Oe) 0.80 � 0.03 �1.5 � 0.1 30.7 � 1.6 �27.5 � 0.1

A horizon 0.115 � 0.011 4.1 � 0.1 2.89 � 0.12 �25.5 � 0.1

B1 horizon 0.091 � 0.003 5.6 � 0.1 2.14 � 0.08 �24.7 � 0.1

B2 horizon 0.086 � 0.001 5.8 � 0.1 1.98 � 0.04 �24.5 � 0.1

C horizon 0.073 � 0.001 6.0 � 0.1 1.60 � 0.05 �24.5 � 0.1

For analyses, the B horizon was split into the B1 (upper 30 cm) and B2 (lower 30 cm) horizons.

C, carbon; N, nitrogen.



If the fungivore dietary fraction of mycorrhizal vs.

saprotrophic fungi is a good proxy for the proportions of

these two fungal types in soil, then the hypothesis that

mycorrhizal hyphae would increase in relative abun-

dance in elevated CO2 and decrease in relative

abundance in elevated temperature treatments was

supported in the trends in allocation patterns (Table 1).

Given the complexity of interactions and the many

confounding factors that could affect isotopic patterns,

it is perhaps not surprising that a sample size of three

was insufficient to show a clear treatment effect.

C and N cycling among ecosystem pools

N released during decomposition of either litter or SOM

could both potentially contribute to the added N in the

A horizon. However, the substantially greater CO2

release from litter decomposition than from SOM oxida-

tion (approximately five times greater, 60–65% of total

respiration for litter decomposition vs. 7–18% for SOM

oxidation) indicates that litter should be the main

source of new N to the A horizon. If true, then this

new N should have a d15N signature similar to that of

the lower litter (�1.4%).

Prior results indicating that the sources of new C to the

A horizon consisted of 36% new photosynthate and 64%

litter (Hobbie et al., 2004a) can be used to estimate the C : N

ratio of litter-derived material added to the A horizon. If

we assume that recent photosynthate did not include

added N, then the C : N ratio of litter-derived material

added to the A horizon would be the C : N of new A

horizon material (36, from Fig. 3b) times the proportion of

new C derived from litter (0.64), or 23. Given an average

C : N of organic matter lost from the litter layer of 43 (Fig.

3a), then the efficiency with which C was transferred from

the litter layer to the A horizon was 23/43, or 53%.

We can estimate the d15N and d13C of new organic

matter in the A horizon by using the regression of d15N

vs. d13C in the A horizon under ambient CO2. New

photosynthate in the A horizon was enriched 2% in 13C

relative to roots (Hobbie et al., 2004a). If we assume that

litter-derived C is also 2% enriched in 13C (�25.5%)

relative to the original litter (�27.5%), and the partition-

ing of new C to the A horizon is 36% new photosynthate

and 64% litter, then the estimated d13C of added C is

(�30%� 0.36) 1 (�25.5%� 0.64) 5�27.1% for ambient

CO2 treatments. Based on our regression in Fig. 4 for

ambient CO2, the added N has a d15N signature of 0.2%,

which is slightly higher than the ‘litter-only’ source of

�1.5%, and agrees well with the dominant N source for

the A horizon being the litter layer. This presumably

reflects the addition of 15N-enriched N derived from

either ectomycorrhizal fungi or from deeper soil hori-

zons (d15NB horizon 5 5.7%, Table 5).

Linking fungal processes to ecosystem C and N cycling

Previous measurements of CO2 efflux and ecosystem

sequestration of new C support our interpretations of

activity by saprotrophic and mycorrhizal fungi. The

average proportions of 35% of fungal hyphae derived

from recent photosynthate vs. 65% of fungal hyphae

from old C are very similar to the relative contributions

of recent photosynthate (36%) vs. litter C (64%) to new

A horizon C (Hobbie et al., 2004a). Rhizosphere respira-

tion ranged from 18% to 32% of total soil-respired CO2

in Years 2 and 3 (Lin et al., 2001) and probably increased

as a proportion of total soil-respired CO2 during the last

2 years of the study as the seedlings grew.

The response to temperature of SCE in the different

treatments provides additional insight into the relative

activity of saprotrophic and mycorrhizal fungi. SCE

sensitivity to temperature was 48% higher with ele-

vated CO2 than in ambient CO2 treatments, and 19%

lower in elevated temperature than in ambient tem-

perature treatments (Tingey et al., 2006). Both mycor-

rhizal and saprotrophic fungi will increase their

metabolic activity with increased temperature. Net

photosynthesis also increases with increased tempera-

ture, and here increased 21% under elevated CO2 re-

lative to ambient CO2 (Lewis et al., 2001), presumably

also increasing the supply of labile carbohydrates avail-

able to mycorrhizal fungi. In contrast, the source C for

saprotrophic fungi (primarily litter) was independent of

temperature and CO2 level. Accordingly, we interpret

the SCE temperature response as reflecting elevated

activity of mycorrhizal fungi in elevated CO2 relative

to ambient CO2, and decreased activity of mycorrhizal

fungi relative to saprotrophic fungi with elevated tem-

perature relative to ambient temperature.

Correlations between fungivore diet and various

measured parameters provide insight into how plant

and soil C and N cycles are linked. Here, the strong

correlation between diet and total plant biomass (Fig.

2a) suggests that plant allocation of C to mycorrhizal

fungi was tightly coupled to total plant production. The

correlation between diet and needle biomass (r2 5 0.88,

data not shown) and between diet and the needle : fine

root ratio (Fig. 2b, r2 5 0.98), but not with diet and fine

root biomass (r2 5 0.27, data not shown), implies that

allocation to mycorrhizae is more important than allo-

cation to roots for plant growth under these N-limited

conditions, and supports our understanding that my-

corrhizal fungi and roots compete for the same pool of

plant-derived C. Decreases in root biomass with ecto-

mycorrhizal colonization have been reported and attrib-

uted to the high sink strength of mycorrhizal fungi for

plant carbohydrates (Rygiewicz & Andersen, 1994;

Hobbie & Colpaert, 2003).

1394 E . A . H O B B I E et al.


CO2 efflux patterns and C : N ratios of potential N

sources can provide additional insight into N cycling.

CO2 was released from litter decomposition vs. SOM

oxidation in about a 5 : 1 ratio (Table 2). N mobilization

should accompany these processes; given the lower

C : N of SOM (about 25) than litter (about 40), the

proportions of potentially available N released from

litter decomposition vs. SOM oxidation are about 3 : 1.

Accordingly, the main potential source of N for plant

and mycorrhizal uptake in this study is from litter

decomposition. Many studies indicate that the litter

C : N ratio must drop below 25–35 before net N release

occurs (Bloemhof & Berendse, 1995; Knops et al., 2002);

at higher values available N is generally immobilized

during decomposition. Because of the high C : N ratio of

litter (averaging 45 for the upper litter, 38 for the lower

litter, and 43 for organic matter lost from the lower

litter), litter decomposition here was unlikely to release

inorganic N directly for plant uptake, but it appears that

the extensive surface area and enzymatic capabilities of

mycorrhizal fungi (Lindahl et al., 2002) permit N uptake

at these high C : N ratios. Direct plant uptake is also

unlikely here because fine roots were nearly completely

colonized by mycorrhizal fungi (97–100% colonization,

Hobbie et al., 2001).

Although elevated temperature initially increased

litter decomposition (Lin et al., 2001) and may therefore

have decreased the litter C : N ratio, it did not appear to

influence the ultimate amount of litter decomposed,

which was relatively constant across all treatments

(Table 3). In contrast to effects of elevated temperature,

elevated CO2 may indirectly increase the C : N ratio of

litter by increasing the supply of labile sugars allocated

from Douglas-fir seedlings to mycorrhizal fungi. This

could enhance immobilization of labile N derived from

litter by mycorrhizal fungi, thereby restricting access of

saprotrophic fungi to this N source. Competition be-

tween saprotrophic and mycorrhizal fungi for N ap-

pears to be an important control over N dynamics in

N -limited forest soils (Lindahl et al., 2002).

Overall patterns of C loss from the litter layer and A

horizon indicate that the system had not achieved a

steady state. The relatively high losses of C from the

litter layer arose because the amount of litter present

was similar to that in a mature Douglas-fir forest while

the amount of new litter generated from the young trees

was quite small. Accordingly, decomposition of old

litter was much higher than inputs of new litter. Differ-

ences among treatments in C loss from the litter layer

were small, perhaps because the N available from

deeper horizons to facilitate litter decomposition was

quite limited. In contrast to the lack of treatment effects

in the litter layer, C accumulation in the A horizon

under elevated temperature and elevated CO2 presum-

ably reflects increased allocation belowground during

Years 2 and 3 of the study, when rhizosphere respiration

increased on average by 35% with elevated tempera-

ture, 71% with elevated CO2, and 120% with elevated

CO2 and temperature relative to controls (Lin et al.,

2001; Table 2). During this period, the numbers of

mycorrhizal root tips and mycorrhizal morphotypes

also increased (Rygiewicz et al., 2000).

We propose that C allocation to ectomycorrhizal fungi

and competition between ectomycorrhizal and sapro-

trophic fungi for N controlled ectomycorrhizal re-

sponses to treatments. The increased rhizosphere

respiration with elevated CO2 during Years 2 and 3

(Lin et al., 2001) and a lack of root growth response

(Olszyk et al., 2003) suggest that plants initially allo-

cated more C to mycorrhizal fungi at elevated CO2 than

at ambient CO2 concentrations (Rygiewicz et al., 2000).

The low foliar N concentrations of around 1% (Hobbie

et al., 2001; Table 3) and the high C : N ratio of plant litter

and the A soil horizon strongly suggest that nutrient

availability may have been sufficiently low that the

extra C primarily served to immobilize available N

rather than increase plant N supply. This may account

for the quite modest aboveground plant growth re-

sponse to elevated CO2 (Olszyk et al., 2003) and the

small decrease in total SCE in elevated CO2 treatments

during the last 2 years of the study. Foliar %N of o1.4%

indicates N limitation in Douglas-fir (Hopmans &

Chappell, 1994).

Based on the data and conclusions discussed above,

we propose that the C and N fluxes in the soil–plant

system can be diagrammed as in Fig. 5. Litter is decom-

posed primarily by saprotrophic fungi (Lindahl et al.,

2007). Much of this decomposed litter is ultimately

transferred to the A horizon concurrently with added

C from recent photosynthate with a relatively high C : N

ratio of 36. This decomposition should also make N

potentially available for uptake by mycorrhizal fungi.

Here, most N to supply ectomycorrhizal fungi is de-

rived from the litter layer, with a smaller amount

derived from the oxidation of SOM. A portion of this

N is ultimately transferred to Douglas-fir to support

new growth. With an approximate ratio of litter-

respired CO2 to SOM-respired CO2 of 5 : 1, the decom-

position of about 1500 g C m�2 of litter and 300 g C m�2

of SOM per mesocosm released about 54 g N m�2 that

was potentially available for uptake (based on a C : N of

respired litter of 36 and C : N of respired SOM of 25).

The total N in plant components at the final harvest was

about 24 g N m�2 (Table 3, Tingey et al., 2003). Thus,

about 53% of litter- and soil-derived N was ultimately

transferred to plants, with the remainder presumably

taken up and sequestered primarily by saprotrophic

and mycorrhizal fungi.



Conclusions

Our results suggest that N retention by both mycor-

rhizal and saprotrophic fungi restrict plant growth

under conditions of elevated atmospheric temperature

and CO2. Similar mechanisms may explain the general

pattern of little plant response to elevated CO2

in nutrient-limited conditions (Markkola et al., 1996;

Kasurinen et al., 1999; Kytoviita et al., 1999, 2001; Rouhier

& Read, 1999; Gill et al., 2002). Increased soil C storage in

response to rising CO2 concentrations and temperature is

likely under nutrient-limited conditions, at least in the

short term. Models have predicted that increased nutri-

ent availability under elevated CO2 is crucial to increased

aboveground storage of C in terrestrial ecosystems (Ras-

tetter et al., 1997). Our results confirm these predictions,

and suggest that without exogenous nutrient inputs,

competition between saprotrophic and mycorrhizal

fungi for available nutrients will limit plant growth

responses to anthropogenic climate changes in ectomy-

corrhizal forests of low N availability.

Acknowledgements

The US Environmental Protection Agency has funded this re-search. It has been subjected to review by the National Healthand Environmental Effects Research Laboratory’s Western Ecol-ogy Division and approved for publication. Approval does notsignify that the contents reflect the views of the Agency, nor doesmention of trade names or commercial products constituteendorsement or recommendation for use. This research wasinitiated while the senior author was a National ResearchCouncil associate. Previous versions were improved by com-ments of David Coleman, Bob McKane, and two anonymousreviewers. The assistance of Bill Griffis with isotopic analysesand during microarthropod extraction from soil is gratefullyacknowledged.

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13c and 15n in microarthropods reveal little response of douglas-fir ecosystems to climate change

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