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MINIREVIEW
Natural Abundance of
15
N
in Nitrogen-Limited Forests
and Tundra Can Estimate Nitrogen
Cycling Through Mycorrhizal
Fungi: A Review
E. A. Hobbie
1
* and J. E. Hobbie
2
1
Complex Systems Research Center, University of New Hampshire, Durham, New Hampshire 03824, USA;
2
Marine Biological
Laboratory, Ecosystems Center, Woods Hole, Massachusetts 02536, USA
ABSTRACT
The hyphae of ectomycorrhizal and ericoid
mycorrhizal fungi proliferate in nitrogen (N)-lim-
ited forests and tundra where the availability of
inorganic N is low; under these conditions the most
common fungal species are those capable of protein
degradation that can supply their host plants with
organic N. Although it is widely understood that
these symbiotic fungi supply N to their host plants,
the transfer is difficult to quantify in the field. A
novel approach uses the natural
15
N:
14
N ratios
(expressed as d
15
N values) in plants, soils, and
mycorrhizal fungi to estimate the fraction of N in
symbiotic trees and shrubs that enters through
mycorrhizal fungi. This calculation is possible be-
cause mycorrhizal fungi discriminate against
15
N
when they create compounds for transfer to plants;
host plants are depleted in
15
N, whereas mycor-
rhizal fungi are enriched in
15
N. The amount of
carbon (C) supplied to these fungi can be stoi-
chiometrically calculated from the fraction of plant
N derived from the symbiosis, the N demand of the
plants, the fungal C:N ratio, and the fraction of N
retained in the fungi. Up to a third of C allocated
belowground, or 20% of net primary production, is
used to support ectomycorrhizal fungi. As anthro-
pogenic N inputs increase, the C allocation to fungi
decreases and plant d
15
N increases. Careful analy-
ses of d
15
N patterns in systems dominated by
ectomycorrhizal and ericoid mycorrhizal symbioses
may reveal the ecosystem-scale effects of altera-
tions in the plant–mycorrhizal symbioses caused by
shifts in climate and N deposition.
Key words: nitrogen isotopes; nitrogen dynam-
ics; carbon allocation; nutrient supply; ectomycor-
rhizal; ericoid mycorrhizal; fungal symbiont.
INTRODUCTION
Most plants are obligate symbionts with mycorrhi-
zal fungi. Of the three main classes of mycorrhizal
fungi, ectomycorrhizal fungi and ericoid mycorrhi-
zal fungi often possess the enzymes to access re-
calcitrant forms of nitrogen (N); in contrast, the
Received 6 December 2007; accepted 28 April 2008; published online 30
May 2008
Electronic supplementary material: The online version of this article
(doi:10.1007/s10021-008-9159-7) contains supplementary material,
which is available to authorized users.
*Corresponding author; e-mail: erik.hobbie@unh.edu
Ecosystems (2008) 11: 815–830
DOI: 10.1007/s10021-008-9159-7
815
arbuscular mycorrhizal fungi associated with most
herbaceous plants and tropical trees lack such
enzymatic capabilities (Chalot and Brun 1998).
Tundra soils of the arctic and forest soils of tem-
perate and boreal regions typically contain high
amounts of organic N and low concentrations of
inorganic N (Aerts 2002); growth of trees and
shrubs is strongly N-limited (Nadelhoffer and others
1992). One way that plants can relieve N limitation
is to take up amino acids from the soil. This pathway
is well documented in many experiments using
15
N-
labeled substrates. However, Jones and others
(2005a,b) questioned its importance to the N
budget of plants because experiments usually do
not reflect the very low concentrations of free
amino acids in the soil; such cases greatly favor
microbial uptake over plant uptake. Read and Per-
ez-Moreno (2003) point out that the large surface
area for absorption and the enzymatic capabilities of
ectomycorrhizal or ericoid mycorrhizal fungi could
extend the sources of N important to their plant
hosts beyond inorganic forms to include amino
acids, amino sugars, protein, or chitin. In return for
N and other nutrients, plants probably allocate up to
20% of net primary production to their mycorrhizal
symbionts (Hobbie 2006). Despite the key role of
mycorrhizal fungi as carbon (C) sinks and N sour-
ces, ecosystem ecologists and modelers have largely
ignored C allocation to mycorrhizal fungi and up-
take of organic N in their conceptual picture of how
terrestrial systems operate.
Knowledge of N dynamics in this symbiosis
comes primarily from studies of the ability of cul-
tured ectomycorrhizal and ericoid fungi to grow on
various sources of N or to transfer N to tree seed-
lings. In the field, the high spatial heterogeneity of
the soil environment and the cryptic nature of
interactions among roots, mycorrhizal fungi, and
the soil have slowed progress toward understand-
ing the functioning of this symbiosis (Taylor and
Fransson 2007). One promising approach to probe
the function of the symbiosis relies on fungal dis-
crimination against
15
N during synthesis of transfer
compounds (Hobbie and others 2000,2005; Hobbie
and Colpaert 2003). This process causes N trans-
ferred to plants to be depleted in
15
N relative to the
source N, whereas N remaining in mycorrhizal
fungi is enriched in
15
N. Based on the depletion of
15
N in host plants in nature, we believe that the
transfer process is similar in ectomycorrhizal and
ericoid mycorrhizal symbioses. Of the main types of
mycorrhizal fungi, only ectomycorrhizal fungi
produce macroscopic fruiting bodies that can be
readily sampled for the relative amount of
15
N and
14
N(
15
N:
14
N ratios, expressed as d
15
N values); d
15
N
data for these fruiting bodies has accordingly pro-
vided additional insight into ectomycorrhizal
functioning that is unavailable for ericoid mycor-
rhizal fungi and arbuscular mycorrhizal fungi.
By analyzing d
15
N values in soil, plant foliage, and
ectomycorrhizal fruiting bodies, the amount of N
entering plants can be quantified (Hobbie and others
2000; Hobbie and Hobbie 2006). These
15
N natural
abundance analyses are free of artifacts from adding
15
N-labeled N or from disturbance during sampling.
In this review, we present recent evidence from
laboratory studies of the interactions of mycorrhizal
fungi and their plant hosts in N cycling. We then
review field and laboratory evidence that d
15
N
values can be used to investigate the key role that
mycorrhizal fungi play in supplying N to their plant
hosts in N-limited ecosystems. Such investigations
require some knowledge about d
15
N values of po-
tential N sources in the soil, such as ammonium,
nitrate, amino acids, and other organic N forms. We
therefore address some of the difficulties in making
such measurements and in determining the actual
sources taken up by plants and their associated
mycorrhizal fungi. Finally, we use d
15
N values to
quantitatively link N dynamics and C dynamics in
the plant–mycorrhizal symbiosis.
MYCORRHIZAL SYMBIOSES AND NCYCLING:
EVIDENCE FROM GENETIC,ENZYMATIC,
STRUCTURAL,FUNCTIONAL,AND
15
N
LABELING STUDIES
Enzymatic Activity and C Allocation
to Mycorrhizal Fungi
In N-limited ecosystems, mycorrhizal and sapro-
trophic fungi are the main sources of enzymes to
degrade organic compounds, although bacteria also
undoubtedly contribute. Read and Perez-Moreno
(2003) argued for the importance of ectomycor-
rhizal and ericoid mycorrhizal fungi in decompo-
sition from the following: (1) much of the microbial
biomass in most terrestrial ecosystems consists of
hyphae of mycorrhizal fungi; (2) mycorrhizal fungi
in culture can degrade a wide variety of organic N
compounds; and (3) genes encoding for a variety of
hydrolytic enzymes are present in cultured fungi
and in the fungal DNA extracted from soil. It is
reasonable to conclude that plants, despite lacking
hydrolytic capabilities themselves, may still obtain
the breakdown products of hydrolytic enzymes by
supplying labile carbohydrates to ectomycorrhizal
and ericoid mycorrhizal fungi with strong enzy-
matic capabilities. We note that arbuscular mycor-
rhizal fungi may provide plants access to organic
816 E. A. Hobbie and J. E. Hobbie
forms of phosphorus but lack the enzymatic capa-
bilities to degrade complex, N-containing polymers
(Chalot and Brun 1998). They appear adapted
primarily to uptake of soluble nutrients (Olsson
and others 2002).
Recent assessments of N cycling in soils have
suggested that the enzymatic degradation of com-
plex polymeric substances such as chitin and pro-
tein could largely control N availability in N-limited
systems (Read and Perez-Moreno 2003; Schimel
and Bennett 2004; Lindahl and others 2005). The
extensive enzymatic capabilities of some mycor-
rhizal fungi give plants access to breakdown prod-
ucts of complex polymers, such as chitin and
protein (Read and Perez-Moreno 2003), that can-
not be directly assimilated by either plant roots or
fungi. This access appears particularly important in
N-limited systems where mineralization is insuffi-
cient to support plant N demand (discussed in
Schimel and Bennett 2004). During decomposition,
the energy content of litter steadily declines as
compounds amenable to enzymatic attack are de-
graded by saprotrophic microbes. As the energy
content of litter declines, saprotrophic activity must
decrease (A
˚gren and Bosatta 1996). Because
mycorrhizal fungi have an energy source inde-
pendent from litter, that is, sugars from their host
plants, in the later stages of decomposition they can
successfully compete with free-living microbes for
organic N compounds (Lindahl and others 2007).
Soil Structure and Mycorrhizal Activity
Lindahl and others (2005) presented a conceptual
picture of the spatial relationships between hyphae
and soil particles in which enzymes are released from
the tips of hyphae into a matrix of secreted muco-
polysaccharides (Figure 1). This matrix holds en-
zymes close to the particles containing polymers of N
compounds, keeps secreted antibiotics nearby to
repress bacterial activity, and increases the proba-
bility of the hyphae taking up the primary products
of hydrolysis are amino acids and oligopeptides, be-
fore they diffuse away. Because the degradation
products are produced close to the hyphal tips,
mycorrhizal fungi can outcompete other soil mi-
crobes for the products. We point out that this sce-
nario localizes N mobilization near hyphal tips and
does not explicitly consider the potential role of roots
in N uptake. One argument against a large role for
roots in uptake is the nearly complete colonization
(95–100%) of fine roots by ectomycorrhizal fungi
(Hobbie and others 2001; Taylor and Alexander
2005), so that almost all soil-derived N must pass
through fungal hyphae prior to entering the plant.
In an alternative scenario, we assume that N is
equally available to fungi, bacteria, and roots, and
assume that uptake is proportional to surface area
of the soil organisms. Mycorrhizal fungi may have
up to 60 times more surface area available for up-
take than their associated fine roots (Simard and
others 2002). Ectomycorrhizal hyphal lengths of up
to 100 m cm
)3
(Coleman and others 2004), and
bacterial numbers of 3–5 ·10
8
cells cm
)3
(M.
Knorr, personal communication) have been re-
ported. Given these values, if fungal hyphae are
treated as 3 lm in diameter and bacteria are cyl-
inders 1 lm long by 0.5 lm in diameter, then the
surface area (in 1 cm
3
of soil) for bacteria and fungi
are comparable at 790 mm
2
for bacteria and at
940 mm
2
for fungal hyphae. Surface area for fine
roots is considerably less. Jackson and others
(1997) estimated 5–11 cm
2
of fine root area per
cm
2
of surface (forests and tundra) over a rooting
depth of 50 cm, or 10–55 mm
2
of fine roots per
cm
3
. Based on these calculations of relative surface
area, microbial uptake should dominate relative to
plant uptake. Therefore, the symbiosis between
plants and mycorrhizal fungi allows plants to
indirectly compete against free-living soil microbes
that would otherwise assimilate the vast majority of
available N. The independence of mycorrhizal fungi
from soil C sources allows them to pursue N-con-
taining molecules of low energy content that free-
living microbes cannot profitably use, or allows
them to adopt strategies (such as secretion of
mucopolysaccharides to restrict diffusion, Figure 1)
that would be energetically unfavorable for free-
living microbes.
Researchers have generally assumed that uptake
pathways can be quantified by adding
13
C- and
15
N-labeled amino acids to laboratory cultures or to
field plots. Although tracing isotopically labeled
amino acids added to soils does indicate possible
pathways, the actual pathways and rates of uptake
in the field remain difficult to determine. For
example, the concentrations of labeled amino acids
injected into soils are generally quite high
(1.2 mM, Na
¨sholm and others 1998; 2 mM, Lipson
and Monson 1998; 11 mM, McKane and others
2002) relative to ambient soil concentrations (20–
60 lM; Jones and others 2005a; 70–350 lM
assuming dry weight half of wet weight, Nordin
and others 2001). At these high concentrations of
added substrate, the actual mechanisms for uptake
may switch from high affinity to low affinity
transporters (Jones and Hodge 1999) or even rely
on passive diffusion.
An added complication is that concentrations of
amino acids measured in water extracted from soil
15
N and Nitrogen Cycle Through Mycorrhizal Fungi 817
may be very different from the amount available
close to bacteria, hyphae, or fine roots. Jones and
others (2005b) point out one potential problem:
the removal of roots from soil during sample
preparation will release high concentrations of
amino acids through breakage of fine roots con-
taining approximately 10 mM amino acids. The
authors also point out the need for studies of spatial
heterogeneity at small (soil micropatches) and large
(whole soil) scales. Given soil heterogeneity and
chronic N limitation of microbes, microbial uptake
of amino acids may reduce the concentrations
actually available to the microbes to low levels that
reflect a balance between diffusion from source
regions and uptake. Amino acids in micropores or
adsorbed to clays or organic matter may represent
source regions. For these reasons, concentrations in
a bulk extract do not reflect concentrations actually
available to microbes. Because
15
N addition levels
are often set at levels similar to reported soil con-
centrations (for example, Na
¨sholm and others
1998), we suspect that uptake rates estimated from
isotope additions are probably much higher than
those in undisturbed ecosystems.
To accurately assess soil concentrations of dif-
ferent potential N sources, new techniques are
needed to measure the fine-scale distribution of
labile organic N compounds. One attractive option
is to deploy soil sensors designed for specific com-
pound classes such as amino acids (Johnston and
others 2004). An alternate approach for quantify-
ing uptake pathways is to apply
13
C- and
15
N-la-
beled litter (for example, Bird and Torn 2006) and
trace the resulting label into microbial pools, al-
though this approach does not permit knowledge of
the exact form of N assimilated.
Isotopically labeled amino acids are commonly
added to soil but researchers have largely neglected
other potential N sources. For example, most fungi
can take up small peptides up to five amino acids in
size (Jennings 1995) yet only one study has applied
peptides in the field to study their uptake (Persson
and others 2003). In that study,
13
C- and
15
N-
labeled peptides derived from hydrolysis of algal cells
were added to small plots of the arbuscular mycor-
rhizal Deschampsia flexuosa, the ectomycorrhizal Picea
abies, and the ericoid mycorrhizal Vaccinium myrtillus.
In the injected solution, 90% of the N consisted of
peptide fragments greater than 700 Da (!110 Da per
amino acid) and 50% of the N consisted of peptide
fragments greater than 4,000 Da. Of the three spe-
cies tested, V. myrtillus was the only species in which
excess
13
C was detected in roots. Because most
peptides in the mix were larger than the five-amino
acid limit for peptide uptake (Jennings 1995), this
study cannot conclusively address whether oligo-
peptide uptake actually occurred. However, the ratio
of
13
C to
15
N detected in Vaccinium roots, 3.95, was
similar to that of the applied solution (4.68), there-
fore demonstrating considerable uptake of either
Figure 1. Possible mode of structure and function of mycorrhizal hyphae near particles of plant litter, humus, and
microbial necromass. The hyphal tip produces mucopolysaccharides that form a matrix connecting hyphae to substrates.
This matrix keeps secreted enzymes close to the hyphal tip while restricting diffusion of those enzymes and enzymatically
released nutrients. Secreted antibiotics may reduce competition by bacteria for assimilable compounds. Recalcitrant
polyphenolic substrates may be degraded by oxidizing enzymes to enable proteases and chitinases to release labile organic
compounds. Wall-bound enzymes may further process labile compounds before hyphal uptake. Figure modified from
Lindahl and others (2005).
818 E. A. Hobbie and J. E. Hobbie
individual amino acids or small oligopeptides derived
from the algal cell mix.
In contrast to these ambiguous results, conclu-
sive evidence that mycorrhizal fungi can use olig-
opeptides has come from both culture studies and
genetic approaches. Experiments have included
culturing plant–mycorrhizal symbioses on alanine
peptides (Abuzinadah and Read 1989), mycorrhizal
growth on alanine peptides and glutathione (a tri-
peptide) in pure culture (Abuzinadah and Read
1986), mycorrhizal growth on glycine peptides in
pure culture (Krznaric 2004, as cited in Hobbie and
Wallander 2006), and genetic characterization of
peptide transporters (Benjdia and others 2006).
Evidence that mycorrhizal fungi can degrade chitin
and thereby use this important N-containing
polymer as an N source has come from culture
studies of fungal growth on chitin (Kerley and Read
1995), expression of chitinolytic enzymes in the
field (Courty and others 2005), and genetic evi-
dence for chitinolytic enzymes such as N-acetyl-
hexosaminidase (Lindahl and Taylor 2004).
Assessing Mycorrhizal Species and Their
Functioning in Ecosystems
Researchers have developed many novel tools in
the last decade to understand both the distribution
and functioning of mycorrhizal fungi. These in-
clude the molecular identification of mycorrhizal
fungi in mycorrhizae (Horton and Bruns 2001) and
in the soil (Rosling and others 2003; Landeweert
and others 2005) and the rapid assessment of the
enzymatic capabilities of individual mycorrhizae
isolated from soil (Pritsch and others 2004; Courty
and others 2005).
In the following sections, we discuss the natural
abundance of N stable isotopes as a tool for assessing
mycorrhizal functioning in ecosystems. In this
method, d
15
N values are measured in plants,
mycorrhizal fungi, and soil pools (Hobbie and
Hobbie 2006). A major advantage of natural abun-
dance measures compared to other measures is that
they are not intrusive, and accordingly reflect pro-
cesses and rates under actual soil conditions.
THE FUNCTION OF MYCORRHIZAL SYMBIOSIS
AND NCYCLING:EVIDENCE FROM THE
NATURAL ABUNDANCE OF
15
N
Fractionation of N Isotopes During
Uptake or in Reactions
N isotopes in molecules will fractionate in inverse
proportion to the mass of the reacting species.
Thus, N in small molecules such as ammonium
(18 Da) may fractionate upon uptake into plants
and fungi; N in amino groups on amino acids may
fractionate in biochemical reactions such as trans-
amination (Macko and others 1986). Based on the
relative masses of ammonium and amino acids
(!110 Da), isotopes in intact amino acids should
fractionate about 2.5 times less than ammonium
during uptake [(110/18)
0.5
]. Whether potential
fractionation is expressed depends on the concen-
tration of substrate available for uptake or for
chemical reactions. When concentrations or supply
rates are very low, fractionation on uptake is not
expressed because all available N is taken up,
regardless of isotopic form (Fry 2006). In contrast,
when concentrations of substrate are high, then
fractionation is expressed in the product. In the N-
limited ecosystems discussed in this review, we
assume minimal fractionation on uptake because of
low substrate concentrations (Goericke and others
1994; Nadelhoffer and Fry 1994).
Studies of d
15
N Patterns in Cultures
of Mycorrhizal Symbionts
The concentration of available N in culture media
can influence patterns of fractionation against
15
N
on uptake. As detailed in Supplementary Appendix
1, mycorrhizal fungi or plants in culture are gen-
erally grown in liquid media at concentrations of
1.4–4.5 mM of available N. At these high levels,
isotopes in inorganic N are almost always fraction-
ated upon uptake into hyphae and plant roots in
culture (Emmerton and others 2001a,b; Ho
¨gberg
and others 1999; Henn and Chapela 2004; Schmidt
and others 2006). This is not true for isotopes in
organic N compounds because of the inverse rela-
tionship between molecular mass and fractionation.
For example, uptake of amino acids into mycor-
rhizal fungi in N-rich media did not fractionate
against
15
N, whereas uptake of ammonium did
fractionate (Emmerton and others 2001a). Ex-
pressed fractionation on uptake may also differ be-
tween different N forms if either diffusion rates or
assimilation rates differ. For example, nitrate dif-
fuses more quickly than ammonium through most
media and is also often assimilated more slowly
than ammonium. This combination allows greater
opportunities for nitrate to fractionate during up-
take than for ammonium despite the higher
molecular weight of nitrate (for example, see Sup-
plementary Appendix 1, Schmidt and others 2006).
In contrast to culture conditions, the concentra-
tion of substrates in the soils of N-limited forests
and tundra is likely to be very low resulting in
minimal fractionation. Similar minimal fraction-
15
N and Nitrogen Cycle Through Mycorrhizal Fungi 819
ation occurs when mycorrhizal or saprotrophic
fungi are grown on solid substrates such as agar
that restrict diffusion (Hobbie and others 2004) or if
N supply closely matches uptake (for example,
exponential growth, Hobbie and Colpaert 2003).
Data in Table 1from various ecosystems indicate
that the
15
N in the total soil, ammonium, and ni-
trate pools is very similar to that in nonmycorrhizal
plants. We therefore assume that nonmycorrhizal
plants take up N from the soil with little fraction-
ation against
15
N (Hobbie and Hobbie 2006).
Plants cultured as symbionts with ectomycor-
rhizal fungi differ in d
15
N values from plants or
fungi grown in pure culture. Relative to supplied
ammonium, ectomycorrhizal plants are generally
depleted in
15
N (lower d
15
N values), ectomycor-
rhizal fungi are enriched in
15
N (higher d
15
N val-
ues), and plants grown asymbiotically have similar
d
15
N values (Ho
¨gberg and others 1999; Kohzu and
others 2000; Schmidt and others 2006). However,
when nitrate is the N source, ectomycorrhizal
plants differ little in d
15
N relative to source N or
nonmycorrhizal plants (Ho
¨gberg and others 1999;
Schmidt and others 2006). A culture experiment
(Figure 2) illustrates the changes in plant d
15
N
relative to the nonmycorrhizal state when pine
seedlings were cultured with two species of
mycorrhizal fungi at low and high rates of supplied
ammonium nitrate (Hobbie and Colpaert 2003): (1)
d
15
N values were about 2&lower in pine needles
than in supplied N; (2) the
15
N depletion was
greater at low N than at high N availability; (3) the
15
N depletion correlated positively with fungal
biomass (Figure 3); and (4) the d
15
N values of the
fungi were 3–4&higher than the supplied N.
Field Measurements of
15
N Natural
Abundance
Across all terrestrial ecosystems, d
15
N values for
foliage range from )11 to 10&and for soil below
the surficial litter range from )2 to 10&(Handley
Table 1. Natural Abundance of
15
N in Plants, Soil, Inorganic N, and Mycorrhizal Fruiting Bodies in Various
Ecosystems
Ecosystem pool Sites
1 2 3 4 5a 5b
Plants
Nonmycorrhizal 2.9 nd nd 1.9 nd )1.8 ± 0.8
Arbuscular mycorrhizal )2.1 nd )2.3 ± 2.3 )2.9 nd nd
Ectomycorrhizal )4.0 )3.7 ± 0.6 )4.7 ± 1.7 )2.9
1
)4.1 ± 0.5 )6.4 ± 0.5
Ericoid mycorrhizal )4.8 )3.0 ± 0.2 )2.6 ± 2.1 )3.6 )7.6 ± 0.7 nd
Fungi
Ectomycorhizal 2–7 5.0 ± 3.8 4.2 ± 0.4 nd 7.5 ± 0.8 nd
Saprotrophic nd )1.3 ± 0.5 )1.8 ± 1.9 nd nd nd
Bulk organic soil 1.2 0.6 ± 0.3 )0.4 ± 1.3 4.1 0.5 ± 0.6 )0.7 ± 0.3
Mineral soil nd 6.0 ± 2.5 3.2 ± 1.3 nd 3.5 ± 0.5 nd
Ammonium 1.4 )0.7 ± 0.4 nd 4.5 nd bd
Nitrate 1.0 )0.1 ± 1.1 nd 9.5 nd nd
Coarse woody debris nd nd )0.3 ± 1.1 nd nd nd
Sites include: 1, acidic tussock tundra, Toolik Lake, Alaska, USA (Hobbie and Hobbie 2006); 2, marine boreal forest, Glacier Bay, Alaska, USA (Hobbie and others 1999a,
2000); 3, temperate coniferous forest, Washington, USA (Trudell and others 2003); 4, Australian heath (Schmidt and Stewart 1997); 5, Boreal forest/arctic tundra, Sweden
(listed, 5a, fellfield; 5b, heath values) (Michelsen and others 1996,1998). Some values are ±standard error. Nd, no data.
1
Designated as ectomycorrhizal or arbuscular mycorrhizal.
Figure 2. Growth rate and mycorrhizal colonization
influence d
15
N of ectomycorrhizal pine and ectomycor-
rhizal fungi. N supplied exponentially at rates of
3% day
)1
(low N) or 5% day
)1
(high N) to Pinus sylvestris
cultured with either Thelephora terrestris,Suillus luteus, or
without mycorrhizal symbionts. N was supplied as
ammonium nitrate at a d
15
N value of 1.4&and was the
limiting nutrient in all treatments. Data from Hobbie and
Colpaert (2003).
820 E. A. Hobbie and J. E. Hobbie
and others 1999a; Amundsen and others 2003;
Hobbie and others 2005; J. Craine unpublished).
The d
15
N of both foliage and soils is lower in early
successional environments, boreal forests, and
tundra than in temperate and tropical forests.
The key role in N-limited systems of mycorrhizal
fungi in influencing plant d
15
N patterns was first
proposed by Ho
¨gberg (1990) in studies of ectomy-
corrhizal and arbuscular mycorrhizal trees in Afri-
ca, and subsequently elaborated on in detailed field
studies of the d
15
N of plants and potential N sources
by Schmidt and Stewart (1997) in Australian
heathland, Hobbie and others (2000) in temperate
rainforest, and Hobbie and Hobbie (2006) in tundra
(Table 1). In these studies, it appears that ectomy-
corrhizal and ericoid mycorrhizal fungi have taken
up soil-derived N and created a
15
N-depleted group
of transfer compounds that is then passed on to the
host plants. The
15
N-enriched compounds remain
in the fungi. This process is the most plausible
mechanism leading to both
15
N-depleted plants and
15
N-enriched fungi.
Foliar d
15
N usually differs by mycorrhizal type
under the N-limited conditions prevalent in boreal,
arctic, and alpine ecosystems (Michelsen and others
1996,1998; Hobbie and others 2005), with ecto-
mycorrhizal and ericoid mycorrhizal plants lowest
in d
15
N, arbuscular mycorrhizal plants intermediate
in d
15
N, and nonmycorrhizal plants highest in d
15
N.
Similar patterns have been reported from tropical
savannah and monsoon forests (Schmidt and
Stewart 2003) and in a worldwide survey of plant
d
15
N patterns (J. Craine and others unpublished;
Table 2). Although arbuscular mycorrhizal plants
are only rarely as low in d
15
N as co-occurring
ectomycorrhizal plants, the lower d
15
N of arbuscu-
lar mycorrhizal plants than of nonmycorrhizal
plants suggests that arbuscular mycorrhizal fungi
also fractionate against
15
N when transferring N to
host plants. However, the extent to which the ar-
buscular mycorrhizal symbiosis alters plant d
15
N is
still unclear. Culture studies to date give variable
results (Michelsen and Sprent 1994; Handley and
others 1993,1999b; Azcon-Aquilar and others
1998; Wheeler and others 2000), but no study has
yet been done under conditions where fractionation
against
15
N on uptake can be ignored.
N isotope patterns in ectomycorrhizal fruiting
bodies have provided additional insight into the
role of mycorrhizal fungi in plant N supply. The
macroscopic fruiting bodies of many ectomycor-
rhizal fungi provide an easy means to sample spe-
cific species and thereby determine which species
are most involved in N cycling. The available
studies indicate that most ectomycorrhizal fruiting
bodies are enriched in
15
N relative to both co-
occurring saprotrophic fungi and ectomycorrhizal
plants (Michelsen and others 1998; Kohzu and
others 1999; Hobbie and others 1999a,2001,2005;
Henn and Chapela 2001). However, the fruiting
bodies of different ectomycorrhizal taxa vary con-
siderably in d
15
N (Taylor and others 2003; Trudell
and others 2004). In one study in boreal forests in
Alaska, ectomycorrhizal fungi of proteolytic taxa
were higher in d
15
N than taxa without such capa-
bilities (Lilleskov and others 2002), and that pat-
tern may hold as well in subalpine forests (Hobbie
and others 2005) and Swedish boreal forests
(Taylor and others 2003).
Additional evidence that ectomycorrhizal fungi
use organic N sources and that this function is af-
fected by N availability comes from the European
Forest Transect, a north-south transect from north-
ern Sweden to Italy, in which the proportion of
ectomycorrhizal fungal species able to use protein N
Table 2. Mean Foliar d
15
N Varies among Plants of
Different Mycorrhizal Type
Mycorrhizal type d
15
N(&)
Nonmycorrhizal 0.9 ± 0.2
Arbuscular mycorrhizal )1.1 ± 0.1
Ectomycorrhizal )2.3 ± 0.2
Ericoid mycorrhizal )5.0 ± 0.2
In a worldwide survey (J. Craine, unpublished), foliar d
15
N varied with tem-
perature, precipitation, N concentration, and mycorrhizal type. Because the dis-
tribution of mycorrhizal types varied with climate, foliar d
15
N for the four types
was here normalized to a common annual temperature (13.2!C), annual pre-
cipitation (751 mm y
)1
), and N concentration (1.58%).
Figure 3. Fungal biomass correlates strongly with foliar
d
15
N in mycorrhizal pine. R
2
= 0.88, P< 0.001. N sup-
plied exponentially at rates of 3% day
)1
(low N; empty
symbols) or 5% day
)1
(high N; filled symbols) to Pinus syl-
vestris cultured with either T. terrestris (squares) or S. luteus
(triangles). N was the limiting nutrient in both treat-
ments. Data from Hobbie and Colpaert (2003).
15
N and Nitrogen Cycle Through Mycorrhizal Fungi 821
in culture decreased markedly along the gradient of
increasing N availability (Taylor and others 2000). N
deposition is the primary cause of the change in N
availability. In the transect, A
˚heden in northern
Sweden has a deposition of less than 1 kg N ha
)1
y
)1
and d
15
N values of )6&for foliage and 6&for fungal
fruiting bodies. In contrast, a site in Germany has N
deposition of 10–20 kg N ha
)1
y
)1
,d
15
N values for
foliage of )2&, and d
15
N values for fungal fruiting
bodies of 2&(Taylor and others 2000).
MODEL OF FRACTIONATION OF NISOTOPES
WITHIN MYCORRHIZAL FUNGI FOLLOWED BY
TRANSFER OF
15
N-DEPLETED NTO PLANTS
Partitioning of N Between Plants
and Mycorrhizal Fungi
Both field and laboratory studies (Table 1and
Figures 2,4, and 5) are consistent; relative to sup-
plied N sources or soil N, mycorrhizal plants are
depleted in
15
N and mycorrhizal fungi are enriched
in
15
N. This pattern reflects the creation of
15
N-
depleted transfer compounds by ectomycorrhizal
fungi prior to transfer to plants (Hobbie and Col-
paert 2003). Glutamine and other amino acids have
been proposed as the likely transfer compounds for
many years (Smith and Smith 1990), and ammonia
has been recently proposed as an additional possi-
ble transfer compound in arbuscular mycorrhizal
symbioses (Jin and others 2005) and in ectomy-
corrhizal symbioses (Chalot and others 2006).
The distribution of N isotopes in plants and fungi
can be expressed by the following equations, in
which D
f
equals fractionation against
15
N during
creation of transfer compounds, T
r
equals the
transfer ratio or the proportion of N taken up by
fungi that is transferred to the host plants, and fis
the proportion of plant N supplied by fungi (Hobbie
and others 2000).
d15Nplant ¼d15 Navailable nitrogen #Df$1#Tr
ð Þ $ f
ð1Þ
If all plant N is supplied by the fungus (f= 1),
then the equation reduces to:
d15Nplant ¼d15 Navailable nitrogen #Df$1#Tr
ð Þ ð2Þ
The equation for the mycorrhizal fungus is:
15Nfungi ¼d15 Navailable nitrogen þDf$Trð3Þ
The exact mechanism of this fractionation is
unknown. A modeled value for D
f
of 8–10&gave a
good fit to data from field studies (Hobbie and
others 2000) and laboratory studies (Hobbie and
Colpaert 2003). This value is reasonable for frac-
tionation against
15
N during transfer of amino
groups by aminotransferases (Macko and others
1986). An additional 3&enrichment in
15
N during
formation of fungal fruiting bodies has also been
suggested from field studies (Hobbie and others
2005).
One problem in applying these equations to
field studies is the difficulty of determining the
d
15
N of the available N pool. The different forms
Figure 5. Foliar d
15
N reflects development of mycorrhi-
zae and N dynamics during primary succession. The d
15
N
of plants with standard errors (foliage, nitrogen = 5) and
individual fungi (fruiting bodies) along a primary suc-
cessional sequence at Lyman Glacier, Washington, USA
are shown. Mycorrhizal associate for plants is indicated
as: non, nonmycorrhizal; AM, arbuscular mycorrhizal;
ERM, ericoid mycorrhizal; and ECM, ectomycorrhizal.
Saprotrophic fungi are either Galerina (black triangles) or
Omphalina (black circles). Ectomycorrhizal (ECM) fungi
(clear circles) are all Laccaria. Data from Hobbie and others
(2005).
Figure 4. d
15
N of Picea sitchensis (Sitka spruce), organic
soil, ammonium, and ectomycorrhizal fruiting bodies in
various age forests in Glacier Bay, Alaska, USA (from
Hobbie and others 1999a,2000). Values are ±standard
error.
822 E. A. Hobbie and J. E. Hobbie
of N taken up by plants and mycorrhizal fungi
could include ammonium, nitrate, amino acids,
oligopeptides, and even amino sugars (Riemann
and Azam 2002; Roberts and others 2007). Al-
though the forms of N taken up are relatively
small inorganic and organic molecules, chitino-
lytic and proteolytic enzymes of some mycorrhi-
zal fungi could extend the sources of N to include
complex N-containing polymers. Determining the
d
15
N values of these different N sources poses
additional problems, as methods for isolating
amino acids, amino sugars, protein, and chitin for
isotopic analyses are not standardized and diffi-
cult to validate, and what is chemically available
(can be extracted) may not correspond with what
is biologically available. In addition, d
15
N signa-
tures of soil N pools generally increase with
depth, but knowing the location from within a
soil profile from which uptake is occurring
requires characterizing both the fungal commu-
nity by depth and the activity of that fungal
community.
For these reasons, studies determining the d
15
N
of available N have relied on easily measured N
pools, such as that of inorganic N or bulk N, and
have only occasionally attempted more ambitious
measures of the d
15
N of nucleic acids (Schwartz and
others 2007), hydrolyzed amino acids (Bol and
others 2004; Y. Yano personal communication),
dissolved organic N (Portl and others 2007), or
microbial biomass (Dijkstra and others 2006). An
alternative approach assumes that biological inte-
grators of the available N pool can be used by
determining the d
15
N of microbes (Hendricks and
others 2004) or of nonmycorrhizal plants (Hobbie
and Hobbie 2006). Values for the d
15
N of available
N can also be estimated or constrained by com-
paring those estimates against those of other pools
and developing simple analytical or computational
models of the resulting N isotope flux patterns (van
Dam and van Breemen 1995; Hobbie and others
1999b). Large challenges remain, but studies from
the last few years indicate that a wide variety of
different N forms should now be amenable to iso-
topic analysis.
Calculating the Proportion of Plant N
Supplied by Fungi (f)
Hobbie and Hobbie (2006) used a
15
N mass balance
approach with equations similar to (1)–(3) to
determine the proportion of plant N supplied by
fungi for the ectomycorrhizal and ericoid mycor-
rhizal tundra plants (see Table 1). Three simulta-
neous equations were set up as follows:
d15Navailable nitrogen ¼1#Tr
ð Þ $ d15Nfungi
þTr$d15Ntransfer
ð4Þ
d15Nplant ¼f$d15 Ntranfer þ1#fð Þ $ d15Navailable
ð5Þ
Df¼d15Navailable nitrogen #d15Ntransfer ð6Þ
In equation (5), plant N is either derived from
mycorrhizal transfer (f) or from direct plant uptake
(1 )f) from the soil. In these equations, d
15
N
available
N
,d
15
N
fungi
, and d
15
N
plant
are all known. This leaves
four unknowns (d
15
N
transfer
,T
r
,f, and D
f
) in three
simultaneous equations. Therefore, specifying any
of the four unknowns allows calculation of the
other three. For the Alaskan tundra, the estimated
values fit the two constraints of a D
f
of 8–10&and
an fof less than 100% for a transfer ratio T
r
of 33–
43%. The percentage of plant N entering via the
mycorrhizal fungi is 61–86% when values of 1–2&
for d
15
N
available N
are used in the calculation.
IMPLICATIONS OF LINKING NAND C
DYNAMICS TO
15
NPARTITIONING
Mycorrhizal Fungi During Primary
Succession
Primary successional sequences offer excellent
opportunities to study gradients of N availability
and mycorrhizal colonization and how N isotope
patterns change along those gradients. At Glacier
Bay, Alaska, glacial retreat since 1750 has pro-
duced a sequence of Sitka spruce (Picea sitchensis)
forests of various ages along the valley sides. Spruce
needles collected from sites 55 to 233 years old
declined in d
15
N with increasing site age whereas
that of the bulk soil, ammonium, and mycorrhizal
fungal fruiting bodies remained constant (Fig-
ure 4). This was accompanied by a shift from
dominance of N-fixing alder (Alnus) early in suc-
cession to spruce dominance in later succession.
Over time, the spruce shaded out alder and N
became limiting, as shown by declines in soil N
mineralization and N content of needles (Hobbie
and others 2000). Two scenarios could explain the
decline in spruce d
15
N. In one scenario, as N
became progressively limiting, fincreased as more
plant N came from mycorrhizal fungi as
15
N-
depleted compounds (Hobbie and others 2000).
Alternatively, mycorrhizal fungi may decrease the
proportion of assimilated N that they pass to their
plant hosts (transfer ratio, T
r
) with decreasing N
15
N and Nitrogen Cycle Through Mycorrhizal Fungi 823
availability. Various combinations of shifts in T
r
and
fcould produce similar d
15
N patterns in plants.
In a second study of primary succession, Hobbie
and others (2005) collected fruiting bodies of
ectomycorrhizal fungi, saprotrophic fungi, and fo-
liage from plants differing in mycorrhizal type
along a short (1 km) primary successional sequence
on a glacial forefront and adjacent secondary sub-
alpine forests in the Washington Cascades. Data on
mycorrhizal colonization and foliar d
15
N indicated
that plants of the youngest sites were probably
uncolonized by mycorrhizal fungi regardless of
mycorrhizal type, as nonmycorrhizal plants and
plants of the three main mycorrhizal types had
similar d
15
N values in very early succession before
ectomycorrhizal fruiting bodies appeared. In con-
trast, plants of different mycorrhizal type in later
succession differed widely in d
15
N (Figure 5), with
d
15
N of ectomycorrhizal and ericoid mycorrhizal
plants generally declining as succession proceeded
and d
15
N of nonmycorrhizal and arbuscular
mycorrhizal plants remaining constant. The earlier
fruiting of saprotrophic fungi than of ectomycor-
rhizal fungi reflected in Figure 5may also reflect
faster colonization in early succession of the glacial
forefront by saprotrophic fungi than mycorrhizal
fungi.
C Allocation to Mycorrhizal Fungi
The few estimates of C allocation to mycorrhizal
fungi in natural ecosystems all have methodo-
logical difficulties (Hobbie 2006). Although recent
work with in-growth cores suggests substantial
allocation to ectomycorrhizal fungi (Wallander
and others 2001; Clemmensen and others 2006;
Hendricks and others 2006), this approach has
yet to be validated against whole-ecosystem
budgets. Good estimates of allocation are possible
in short-term culture studies with seedlings; the
relevance of such studies to mycorrhizal alloca-
tion in mature trees in the field is unclear. A
review of available culture studies with seedlings
indicated that up to 20% of net primary pro-
duction was allocated to growth of extraradical
hyphae and the fungal portion of mycorrhizae
(Hobbie 2006). This allocation was positively
correlated to total belowground allocation (Fig-
ure 6). C allocation belowground correlates with
C allocation to mycorrhizal fungi, and C alloca-
tion to mycorrhizal fungi correlates with N allo-
cation by mycorrhizal fungi to plants and to N
isotope patterns in plants. It should therefore be
possible to estimate C allocation to mycorrhizal
fungi from d
15
N measurements on the plant–
mycorrhizal–soil system.
C Allocation to Fungi from Data on Foliar
d
15
N
Several lines of evidence suggest that patterns of C
allocation to mycorrhizal fungi could be estimated
from foliar d
15
N values. Most strikingly, culture
studies indicate that plant allocation to mycorrhizal
fungi, as deduced from mycelial biomass produced,
strongly correlates with lower foliar d
15
N values
(Figure 3). Other evidence suggesting a link be-
tween foliar d
15
N values and mycorrhizal C supply
includes declining foliar d
15
N with increasing N
limitation in field studies (Emmett and others 1998;
Hobbie and others 2000) and increased fruiting of
ectomycorrhizal fungi as N deposition decreases
(Wallenda and Kottke 1998).
In general, it appears that: (1) foliar d
15
N is
controlled by mycorrhizal fungi under N-limited
conditions, (2) relative belowground allocation in-
creases with increasing N limitation, and (3) foliar
d
15
N of ectomycorrhizal plants declines with
increasing N limitation. In addition, equations (1)–
(3) above quantify how the partitioning of N be-
tween mycorrhizal fungi and their plant hosts
influences d
15
N patterns in the symbiosis. The
equations can accordingly be rearranged to predict
N partitioning in the symbiosis from isotopic
parameters according to equation (7).
Tr¼1þd15Nplant #d15 Navailable nitrogen
!"
Dð7Þ
Figure 6. Allocation to ectomycorrhizal fungi is correlated
with total belowground allocation in culturestudies. Values
are expressed as a percentage relative to net primary pro-
duction. %NPP
fungi
= 0.473 ·%NPP
belowground
)14.6%,
R
2
= 0.57, n= 33, P< 0.001). Data from Hobbie (2006).
824 E. A. Hobbie and J. E. Hobbie
Mass Balance Calculations can Estimate
the Allocation of N and C to Plants
and to Mycorrhizal Fungi
The close coupling between N and C cycling in
terrestrial ecosystems (A
˚gren and Bosatta 1996)
suggested that C fluxes in the mycorrhizal symbi-
osis could be quantified from
15
N patterns. In
Hobbie and Hobbie (2006), our calculations based
on d
15
N values [equations (4)–(6)] gave us the
following quantities: (1) the fraction of total N
uptake by mycorrhizal fungi that is transferred to
plants, and (2) the fraction of plant N derived from
this transfer. Second, when the total net primary
production is known and stoichiometrically related
to plant N budgets, the quantity of N per year
entering the plant from mycorrhizal fungi as well as
the quantity of N per year remaining in the fungi
may be estimated. Third, the annual fungal N de-
mand can be extrapolated to annual C require-
ments by dividing by the growth efficiency (!50%)
and multiplying by the C:N of hyphae (10, Lodge
1987). Growth efficiency is generally estimated at
35–60%; in Supplementary Appendix 2 we sum-
marize studies that have estimated growth effi-
ciency for mycorrhizal fungi or plant–mycorrhizal
systems.
Given that transfer ratios (T
r
) between mycor-
rhizal fungi and plants can be calculated from
equation (7) above, the following equation can be
applied to estimate C demand of mycorrhizal fungi.
Equation (8) is derived in Supplementary Appen-
dix 3.
Cfungal = 1/Tr#1ð Þ $ Np$C=N$1=eðÞ:ð8Þ
In equation (8), T
r
is the transfer ratio for
mycorrhizal fungi, the proportion of N taken up by
fungi that is transferred to host plants; N
p
is the
yearly amount of plant N supplied by mycorrhizal
fungi; C/N is the C:N ratio of fungal hyphae; and e
is the efficiency with which plant C (sugars) is
metabolized to fungal C. Figure 7shows fungal
demand for C at variable T
r
for a plant N demand of
30 kg ha
)1
y
)1
.
The relationship between C demand of mycor-
rhizal fungi and transfer ratios shown in Figure 7
illustrates that C demand increases rapidly at low
transfer ratios for a given plant N demand. Such
low transfer ratios prevail at low N availability. The
restricted N availability in boreal and tundra eco-
systems therefore leads to low transfer ratios, low
plant N demands, low plant d
15
N values in ecto-
mycorrhizal and ericoid mycorrhizal plants, and a
high C allocation belowground to support fungal
symbionts.
To estimate C allocation to mycorrhizal fungi,
plant productivity measures are necessary. A few
studies have provided sufficient information for C
allocation to mycorrhizal fungi to be estimated. In
an Alaskan arctic tundra system, Hobbie and Hob-
bie (2006) used this approach and equations similar
to those above to conclude that between 61 and
86% of plant N was supplied via mycorrhizal
symbionts, and that between 8 and 17% of plant
net primary productivity was supplied to mycor-
rhizal symbionts to maintain the symbiosis. In a
mesocosm study of N-limited Douglas-fir (Pseud-
otsuga menziesii), foliar d
15
N values (!)4&) were
about 5&lower than the average d
15
N of the
probable plant N sources, litter and A horizon soil
(1.2&), 95–100% of root tips were colonized by
ectomycorrhizal fungi, and transfer ratios were
accordingly about 0.5 (Hobbie and others 2001). If
we assume that 100% of plant N was supplied by
mycorrhizal fungi, yearly N demand was 50 kg
ha
)1
, and the average transfer ratio was 0.50, then
yearly fungal demand of C was 1,000 kg ha
)1
.
One final example comes from studies along a
primary successional sequence. At Glacier Bay,
Alaska, Hobbie and others (1999b) modeled N
isotope patterns for fungal transfer ratios between
0.2 and 0.8. The two most N-limited sites at Glacier
Bay were dominated by P. sitchensis and Tsuga
heterophylla, two strongly ectomycorrhizal species.
If we assume that all N uptakes by these species first
passed through their fungal symbionts, then data
from Hobbie and others (2000) estimates a fungal
transfer ratio of between 0.2 and 0.5 for these two
Figure 7. C demand (C
fungal
) of mycorrhizal fungi in-
creases as the mycorrhizal transfer ratio (T
r
) for N de-
creases, according to equation (8). Plant N demand (N
p
)
is set to 30 kg ha
)1
y
)1
, fungal C:N is set to 10, and
microbial efficiency (e) is set to 0.5. T
r
is the proportion of
N taken up by fungi that is transferred to host plants. All
plant N is assumed to be supplied by mycorrhizal fungi.
15
N and Nitrogen Cycle Through Mycorrhizal Fungi 825
sites. Based on these values, we estimate that for
the annual plant N demand of 20 kg ha
)1
y
)1
at
these sites, between 400 and 1,600 kg C ha
)1
y
)1
was supplied to the fungal symbionts.
From theoretical principles, we suggest that
belowground C allocation is tightly coupled to
plant N supply when growth of mycorrhizal hy-
phae or fine roots is necessary for uptake, such as
when diffusion of available N is limited (Clarkson
1985; Ingestad and A
˚gren 1988). Under such con-
ditions, transfer ratios decline as soil N availability
declines because the fungal N requirement per unit
growth remains constant, whereas the amount of
available N declines. One way to decrease the N
requirement per unit surface area is to decrease the
diameter of the nutrient-absorbing organ. The
much smaller average diameter of fungal hyphae
(3 lm) compared to the smallest fine roots (about
100 lm) should accordingly favor a switch to
investment in fungal hyphae rather than fine roots
at low N availability, leading to increased values of
f. The coupling between belowground C allocation
to mycorrhizal fungi and N uptake will be partic-
ularly tight when the strong enzymatic capabilities
of many mycorrhizal fungi are used to liberate N
from N-containing polymers such as protein or
chitin prior to uptake.
CONCLUSIONS AND IMPLICATIONS
Based on the above examples and analyses, further
exploration of d
15
N patterns in N-limited systems is
likely to provide new insights into C and N cycling
in mycorrhizal symbioses. The Hobbie and Colpaert
(2003) model of N uptake by hyphae proposes that:
(1)
15
N is fractionated against during the formation
of amino acids for subsequent transfer to plants,
and (2) the transfer of
15
N-depleted amino acids to
plants is coupled to the retention of
15
N-enriched
compounds in the fungal mycelia. Recent sugges-
tions that ammonia could also function as a trans-
fer compound in arbuscular mycorrhizal and
ectomycorrhizal fungi can also be accommodated
within our conceptual model. This model explains
15
N distributions within the soil–mycorrhizal fun-
gus–plant system that include the consistently high
d
15
N of ectomycorrhizal fungi relative to host
plants, the low d
15
N values of ectomycorrhizal and
ericoid mycorrhizal plants, the lower d
15
N of these
plants at low N availability than at high N avail-
ability, and the large
15
N enrichment of bulk soil
relative to litter and foliage in systems dominated
by ectomycorrhizal fungi. The
15
N enrichment in
bulk soil has been attributed to long-term stabil-
ization of N derived from
15
N-enriched ectomy-
corrhizal fungi (Ho
¨gberg and others 1996; Lindahl
and others 2007). Quantitative analyses of isotopic
patterns in soil could accordingly be used to predict
relative contributions of mycorrhizal fungi versus
plant litter to soil N.
We have set forth equations showing how plant
d
15
N can correlate with C allocation to mycorrhizal
fungi. The challenge in the field is to determine
under what conditions such a correlation applies,
and to determine the needed level of knowledge
about d
15
N patterns for its application. For exam-
ple, the C cost to their host plants of different
species of ectomycorrhizal fungi undoubtedly dif-
fers (Bidartondo and others 2001), as does their
enzymatic capabilities to access recalcitrant organic
N pools. We know that C allocation to ectomycor-
rhizal fungi correlates with plant belowground
allocation in culture studies (Hobbie 2006) and that
C allocation to ectomycorrhizal fungi decreases
with increased N availability (Hobbie 2006) or N
deposition (Wallenda and Kottke 1998). These
observations suggest that estimates of large-scale
patterns of C allocation to mycorrhizal fungi across
gradients of climate, N availability, and N deposi-
tion may be possible by combining the above
equations with regional or global surveys of foliar
and soil d
15
N patterns.
Despite the strengths of this analytical approach
based on d
15
N patterns, many uncertainties remain
about the relative proportions of the different forms
of potentially available N and their d
15
N values. To
address this issue, natural abundance studies are
particularly needed to improve the characterization
of d
15
N among different organic N components in
the soil. For example, Y. Yano (unpublished) has
recently measured low d
15
N values for proteins in
arctic soils, which implies that the Hobbie and
Hobbie (2006) estimates of the percent plant N
supplied by mycorrhizal fungi are somewhat high.
These natural abundance studies should be aug-
mented by parallel tracer work in the field using
15
N- and
13
C-labeled substrates such as above-
ground and belowground litter.
Obtaining N from complex polymers such as
protein and chitin becomes increasingly important
as N availability declines and free-living hetero-
trophic microbes compete for N. Under such con-
ditions, degrading these complex polymers and
taking up the resulting oligopeptides, amino acids,
or amino sugars is a key function of mycorrhizal
fungi. Oligopeptide uptake should increase at low N
availability as it minimizes opportunities for diffu-
sion away and subsequent uptake by free-living
heterotrophic microbes. Expression of oligopeptide
transporter genes should also increase under such
826 E. A. Hobbie and J. E. Hobbie
conditions. Recent culture studies in the ectomy-
corrhizal fungus Hebeloma cylindrosporum indicate
that a low affinity peptide transporter (K
M
!100–
250 lM) was always present and a second peptide
transporter (K
M
!1.5 lM) appeared to be induced
at low N availability (Benjdia and others 2006).
Obtaining realistic estimates of N availability in N-
limited forests and tundra is quite difficult using
traditional methods because of the tight cycling
between hydrolytic enzymes and fungal uptake.
To assume that measures of mineralization
equate with N availability will probably underesti-
mate actual N availability when mycorrhizal fungi
supply N to plants. Schimel and Bennett (2004)
reviewed evidence, based on the total N needed for
plant growth, that most mineralization rates are
underestimates. Information presented in this re-
view and other recent papers (Read and Perez-
Moreno 2003; Ho
¨gberg and Read 2006; Lindahl
and others 2007) indicate that mycorrhizal fungi
use proteolytic and chitinolytic enzymes to degrade
complex N-containing polymers in the soil. Be-
cause labile organic N may dominate the pool of
assimilated N for mycorrhizal fungi, we therefore
conclude that mineralization is often a poor mea-
sure of N availability. In addition, because C from
roots and mycorrhizal fungi is an important energy
source fueling the activity of free-living microbes
(for example, the priming effect, Kuzyakov and
others 2000), altering this flux will alter the bal-
ance between immobilization and mineralization
in the free-living microbial community.
In cultures with ectomycorrhizal Pinus, mycor-
rhizal transfer ratios (T
r
) decreased as N availability
declined because fungi retained more of the system
N (Hobbie and Colpaert 2003). In the field, as N
availability declines, the amount of N captured for a
given hyphal investment in C (and N) should
similarly decline, and therefore the proportion of
assimilated fungal N available for transfer to plants
also declines. The low amount of N transferred by
mycorrhizal fungi to host plants relative to the
supplied C is one cause of the prevalence in arctic
and boreal ecosystems of N conservation strategies
(such as long needle lifespans) that lead to in-
creased N use efficiency.
The concepts and findings reviewed here inte-
grate laboratory studies of mycorrhizal fungi, their
ability to break down complex organic N, and the
fractionation against
15
N during creation of transfer
compounds with field observations of
15
N in soils,
mycorrhizal fungi, and host plants. Can use of the
large pool of organic N in soil by the symbiosis
between mycorrhizal fungi and host plants now be
quantified? Yes, but only in ecosystems where
much is known about the N isotopes of soil pools of
organic N and about plant growth. In other eco-
systems, patterns of d
15
N in soils, mycorrhizal
fungi, and plants can indicate that these fungi are
actively transferring N to their host plants.
ACKNOWLEDGMENTS
This work was supported by NSF OPP-0612598 and
NSF DEB-0614266. Comments on the manuscript
by Erik Lilleskov and two anonymous reviewers
are gratefully acknowledged.
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