This document summarizes a study that evaluated shifts in arbuscular mycorrhizal (AM) fungal communities along an anthropogenic nitrogen deposition gradient in coastal sage scrub vegetation in southern California. The researchers found that increasing nitrogen input was associated with displacement of larger-spored AM fungi species by proliferation of small-spored Glomus species. There was also a reduction in AM species richness and diversity, as well as decreases in spore abundance, root infection, and changes in spore production timing in more nitrogen-enriched sites. A fertilization experiment yielded similar results, indicating that nitrogen input likely explains the relationship between pollution and shifts in the AM communities.

1. 484
Ecological Applications, 10(2), 2000, pp. 484– 496
᭧ 2000 by the Ecological Society of America
SHIFTS IN ARBUSCULAR MYCORRHIZAL COMMUNITIES ALONG AN
ANTHROPOGENIC NITROGEN DEPOSITION GRADIENT
LOUISE M. EGERTON-WARBURTON AND EDITH B. ALLEN
Department of Botany and Plant Sciences, University of California, Riverside, California 92521-0124 USA
Abstract. We evaluated arbuscular mycorrhizal (AM) species diversity and abundance
in nine locations along an anthropogenic nitrogen deposition gradient in coastal sage scrub
(CSS) vegetation in southern California. The primary pollutants were nitrogen oxides de-
rived from vehicular emissions. Extractable soil N on the gradient ranged from 5 to 87 ␮g/
g during the summer months. For comparative purposes, we also assessed AM communities
in nitrogen-fertilized (60 kg N·haϪ1
·yrϪ1
) and unfertilized plots. Nitrogen enrichment in-
duced a shift in AM community composition. In particular, an increasing input of nitrogen
was associated with the displacement of the larger-spored species of Scutellospora and
Gigaspora (due to a failure to sporulate) with a concomitant proliferation of small-spored
Glomus species (e.g., Glomus aggregatum, Glomus leptotichum). A subsequent reduction
in species richness and diversity (as measured by Shannon–Wiener index) accompanied
eutrophication. Nitrogen enrichment also significantly reduced spore abundance, modified
the timing of AM spore production in the most eutrophied site, and reduced hyphal and
vesicular root infection. The fertilization experiment yielded similar patterns to those found
along the gradient, and hence nitrogen input most likely explains the relationship between
anthropogenic pollution and shifts in the AM communities. Such changes also indicated
that AM species were sensitive indicators of nitrogen enrichment. The CSS is currently
undergoing a conversion to Mediterranean annual grasslands, especially in the more urban
polluted areas, and the shifts in the mycorrhizal fungal community may facilitate grass
dominance in this system.
Key words: anthropogenic nitrogen deposition; arbuscular mycorrhizae; coastal sage scrub;
nitrogen enrichment; species diversity.
INTRODUCTION
Anthropogenic nitrogen deposition has been cited as
a causal factor in the decline of forest and grassland
ecosystems (e.g., Schulze 1989, Bobbink 1991). Apart
from the direct effect of nitrogenous emissions on
plants, the mechanism by which nitrogen influences the
plant community may be mediated by its effects on
mycorrhizal associations, or the symbiotic association
between plant roots and fungi (Smith and Read 1997).
While many studies have investigated the effects of
nitrogen enrichment on plants, less is known about the
effects on the fungal community. In this study, we in-
vestigated the effects of anthropogenic nitrogen en-
richment and experimental fertilization on arbuscular
mycorrhizal communities.
Chronic nitrogen deposition significantly alters the
mycorrhizal community. Most of the known effects of
anthropogenic nitrogen deposition on the mycorrhizal
community come from studies on the influence of am-
monia deposition (derived from intensive animal hus-
bandry) on the ectomycorrhizal (ECM) community in
forest ecosystems (reviewed in Wallenda and Kottke
1998). For the ECM community, there is a trend to-
wards a reduction in sporocarp production (Termor-
Manuscript received 17 August 1998; revised 15 March 1999;
accepted 13 April 1999; final version received 28 May 1999.
shuizen and Schaffers 1987, 1991, Arnolds 1988,
1991), and species diversity (Arnolds 1991, Dighton
and Jansen 1991), and shifts in species composition
and dominance with increasing eutrophication (Ru¨hl-
ing and Tyler 1991, Arnebrant and So¨derstro¨m 1992).
Nitrogen enrichment is also associated with a decrease
in ECM root infection (Dighton and Jansen 1991). Sim-
ilar changes also accompany experimental nitrogen fer-
tilization of forest communities (Menge and Grand
1978, Ohenoja 1978, Arnebrant and So¨derstro¨m 1992,
Termorshuizen 1993), but not always (Ohenoja 1988).
The effects of anthropogenic nitrogen deposition on
the arbuscular mycorrhizal (AM) community have re-
ceived little attention. In the only study to date, Heijne
et al. (1992) showed that nitrogen enrichment of heath-
lands using an artificial rain of ammonium sulfate did
not always result in a reduction in AM root infection.
On the contrary,variable mycorrhizal responses were
noted among species. Instead, the effects of nitrogen
enrichment on AM community dynamics have primar-
ily been concluded from fertilization studies. Empirical
field and glasshouse research indicates that nitrogen
fertilization can be associated with an increase (Heijne
et al. 1992, 1994) or decrease in root infection (Hayman
1982), a reduction in AM spore abundance and species
diversity (Hayman 1970, Johnson et al. 1991) and se-
lection for aggressive, possibly less effective, mutu-
alists (Johnson 1993).

2. April 2000 485MYCORRHIZAE AND NITROGEN DEPOSITION
TABLE 1. Coastal sage scrub locations assessed for abundance and diversity of AM species, and their respective land uses,
geographic coordinates, elevation, average annual rainfall, and soil pH and nitrate.
Site (abbreviation)
Primary
land use†
Latitude
(ЊN)
Longitude
(ЊE)
Eleva-
tion‡
(m)
Rain-
fall§
(mm) pH
Soil NO3
(␮g N/g)࿣
Santa Margarita Ecological Reserve (SMER)
Lake Skinner (SKIN)
Hemet (HEMT)
Motte Rimrock Reserve (MOTT)
Lake Mathews (MATH)
Waterman (WATER)
Box Springs Mountain (BOXS)
Mockingbird Canyon (MOCK)
Jurupa Hills (JURP)
ER
ER, WD
SP
ER
WD
SP
ER
WD
SP
33Њ29Ј
33Њ27Ј
33Њ43Ј
33Њ48Ј
33Њ51Ј
34Њ11Ј
33Њ58Ј
33Њ54Ј
34Њ03Ј
117Њ09Ј
117Њ02Ј
117Њ10Ј
117Њ15Ј
116Њ56Ј
117Њ20Ј
117Њ17Ј
117Њ20Ј
117Њ36Ј
338
317
600
550
467
340
800
340
350
357
275
291
272
314
325
287
244
265
5.8
6.1
5.9
5.5
5.9
5.5
6.0
6.3
5.3
2 (1–4)
4 (2–6)
6 (3–11)
7 (2–14)
11 (9–12)
17 (6–25)
18 (13–24)
28 (15–39)
57 (52–69)
Note: Sites are ranked from low to high nitrogen input based on soil nitrate concentrations recorded during peak atmospheric
nitrogen loads in the region.
† ER, ecological reserve; WD, water catchment district; PR, private property; SP, California state or county recreation park.
‡ Elevation data (expressed as meters above sea level) from USDS topographic maps for the San Bernardino quadrangle
(7.5Ј series) and Riverside East quadrangle (7.5Ј series).
§ Precipitation data for study locations, or closest locale, from National Climatic Data Center historical archives.
࿣ Mean soil NO3 concentrations (0–2 cm depth) with range in parentheses (Padgett et al. 1999).
The current study represents the first account of the
spatial and temporal patterns of AM fungi along an
anthropogenic nitrogen gradient in a shrubland com-
munity. Coastal sage scrub (CSS) was once the dom-
inant vegetation type along the southern California
coastal plain (Mooney 1977). However, much of the
CSS has been lost with extensive urban and agricultural
development or invaded by mediterranean annual
grasses (Westman 1981, Minnich and Dezzani 1998).
The remaining tracts are exposed to anthropogenic ni-
trogen deposition that in some locations exceeds 35 kg
N·haϪ1·yrϪ1 (Bytnerowicz and Fenn 1996). The primary
pollutants are nitrogen oxides (NOx) from vehicular
emissions. These are deposited on surfaces during sum-
mer and then infiltrate through the soil profile with the
onset of winter rainfall, where they are available for
plant uptake (Padgett et al. 1999). In this study, our
goals were to (1) document the abundance and diversity
of AM fungi along the nitrogen gradient, (2) assess
AM communities in nitrogen fertilized and nonfertil-
ized plots to determine if the application of fertilizer
yielded similar patterns as along the gradient, and (3)
use these data to test the hypothesis that an increasing
nitrogen input significantly alters AM community dy-
namics during the year.
METHODS
Study sites
This research was conducted at nine CSS commu-
nities in the Riverside–Perris Plain, Southern Califor-
nia, during 1995 and 1996 (Table 1). These commu-
nities primarily occur in water catchments or ecological
reserves, but for the most part represent remnants of
CSS in an increasingly urbanized region. The study
area is typified by a warm Mediterranean-type climate
where rainfall occurs from November to March, with
little or no rainfall from April to October. Measured
precipitation in the region ranges from 244 to 357 mm/
yr (Table 1), and mean annual temperatures for the
region range from 9ЊC (minima) to 26ЊC (maxima) (30
yr mean, Riverside Fire Station). The CSS community
in this area is commonly composed of the drought-
deciduous shrubs Artemisia californica Less. (nomen-
clature follows Hickman 1993), Encelia farinosa Tor-
rey & A. Gray, Eriogonum fasciculatum Benth., Salvia
apiana Jepson, and a number of native herbaceous pe-
rennial and annual species (Mooney 1977, Sawyer and
Keeler-Wolf 1995).
The soils on all sites were composed of sandy loams
or sandy clay loams derived from a weathered granite
or granodiorite except for the Santa Margarita Ecolog-
ical Reserve (SMER), where the sample sites were
sandy loams to loams that metamorphosed from a fine-
grained sandstone (USDA Soil Survey 1971). Soils
were slightly acidic (mean pH 5.8), contained signifi-
cant reserves of phosphorus (mean ϳ30 ␮g/g bicar-
bonate extractable) and potassium (200 ␮g/g), and dif-
fered primarily in the concentrations of extractable NO3
among sites (Table 1). NOx pollution can simultaneous-
ly result in both nitrogen enrichment and soil acidifi-
cation as a consequence of the hydrolysis of NOx. How-
ever, soil pH was not significantly correlated with NO3
concentrations along the gradient (r ϭ 0.039, P Ͼ 0.05;
see also Table 1), and atmospheric concentrations of
sulfur in the region are sufficiently low as to not cause
acidification events (Fenn and Bytnerowicz 1993;
Padgett et al. 1999). Therefore, the influence was one
of nitrogen enrichment on the mycorrhizal community,
and not pH.
The nine CSS sites constitute an anthropogenic ni-
trogen deposition gradient (Table 1). The gradient,
summarized here, is described in detail in Padgett et
al. (1999). The two lowest soil nitrogen sites, Santa
Margarita (SMER) and Lake Skinner (SKIN), are the
most distant from concentrated urban development and
are representative of intact CSS communities, although

3. 486 LOUISE M. EGERTON-WARBURTON AND EDITH B. ALLEN Ecological Applications
Vol. 10, No. 2
stands of exotic grasses occur in disturbed areas. The
high deposition sites, Box Springs Mountain (BOXS),
Mockingbird Canyon (MOCK), and Jurupa (JURP), are
surrounded by urbanization and are in the path of the
westerly winds that carry air pollution directly from
the Los Angeles Basin (Padgett et al. 1999). These sites
have expanses of exotic annual grasses in the genera
Bromus, Avena, and Schismus, and the exotic forbs
Erodium and Brassica (Allen et al. 1997), and existing
shrublands are sparse (Minnich and Dezzani 1998, L.
M. Egerton-Warburton personal observations). The re-
maining sites occur in a convergence zone where the
annual patterns of air flow result in these sites peri-
odically receiving both NOx deposits and cleaner air
from strong onshore breezes from the Pacific coast. The
vegetation on these moderate nitrogen sites include
both intact CSS communities and stands of exotic
grasses, the latter chiefly from past agricultural distur-
bances (Table 1). The Waterman Road site (WATR) is
the most northern of these sites and has intact CSS
vegetation surrounded by natural vegetation in the foot-
hills of the San Bernardino Mountains.
In southern California, dry deposition onto soil and
plant surfaces during summer represents the major
source of plant-available nitrogen; rainfall is only a
minor source of nitrogen input (Bytnerowicz and Fenn
1996; Padgett et al. 1999). The summer atmospheric
nitrate load (HNO3/NO3) typically exceeds 30 ␮g N/
m3 on a high deposition site (BOXS) in comparison
with 19–25 ␮g N/m3 on moderate nitrogen sites
(HEMT, MOTT) and ϳ14 ␮g N/m3 at a low nitrogen
site (SKIN); winter concentrations do not surpass 5 ␮g
N/m3 at any location (Padgett et al. 1999). As a con-
sequence, soil concentrations of nitrogen peak at the
end of summer (September/ October) and decline dur-
ing the growing season, where plant nitrogen uptake
and possibly leaching remove the available nitrogen.
The estimated turnover of nitrate on the gradient ranges
from 9.98 kg N·haϪ1·yrϪ1 at SKIN to 35 kg N·haϪ1·yrϪ1
at BOXS (Padgett et al. 1999). Deposition of NH4 from
atmospheric sources also occurs in the region (Byt-
nerowicz and Fenn 1996) and is part of the nitrogen
gradient (Padgett et al. 1999). Soil concentrations of
NH4 follow the same patterns of accumulation and de-
cline as NO3 except that the input of NH4 is small (ϳ1/
10 of NO3 input) and seasonal differences in soil NH4
concentrations are not as marked as those for NO3
(Padgett et al. 1999).
Sampling along the nitrogen deposition gradient
Sampling, recovery, and identification of AM spe-
cies.—In the present study, all estimates of AM pop-
ulations were based on direct observations of field col-
lections since greenhouse bait-plant assays yielded
only Glomus deserticola and Glomus occultum. Stutz
and Morton (1996) recommend the use of bait-plant
assays for assessment of mycorrhizal spore diversity,
but these changed both the species composition and the
density of spores in our samples. Instead, we examined
soils over 12 mo to detect sporulation of all species,
and cleaned spores of adhering debris using low in-
tensity sonication to assure accurate identification.
We selected remnant stands of CSS to sample at each
of the sites, as the vegetation varied from exotic grass-
land to shrubland in various proportions depending
upon the degree of urbanization and disturbance. All
sites were typical CSS except Hemet (HEMT), where
the CSS forms an intergrade with Adenostoma fasci-
culatum H.&A. (chamise) chaparral (Sawyer and Keel-
er-Wolfe 1995). A site of approximately one ha was
chosen at each site, and soils were collected from the
rhizospheres of different individual shrubs at each time.
In June, September, and December 1995, and March
and June 1996, soil samples were collected from under
the dripline of five randomly selected individuals of
Artemisia californica, Encelia farinosa, and Eriogon-
um fasciculatum (hereafter referred to by genus) at each
site except SMER and HEMT. At SMER, samples were
collected from under plants of Eriogonum, Artemisia,
and Salvia apiana, as Encelia farinosa does not occur
at this site. At HEMT, samples were collected from
under Eriogonum and Encelia; Artemisia was absent
from this locale. For each plant, the soil sample was a
composite formed by combining two adjacent soil cores
each 2.5 cm diameter and depth 5 cm; prior assessments
indicated that the highest density of AM spores could
be recovered from the top 5 cm of the soil profile.
Composite soil samples were placed in individual plas-
tic bags and stored atϪ20ЊC until extraction. At the
same time, soils were collected at the corresponding
depth for analysis of soil nitrogen and other soil nu-
trients (Padgett et al. 1999).
Five grams of air-dried soil per sample was processed
to recover AM spores using dry sieving and sucrose–
sodium hexametaphosphate centrifugation (Allen et al.
1979). The extracted sample was then filtered evenly
over individual gridded membranes and all spores pres-
ent on the membrane were counted. The large-spored
genera (100–250 ␮m), Acaulospora, Scutellospora,
and Gigaspora, were located on the membrane using
a dissecting microscope (40ϫ). Spores of these genera
were removed from the membrane, mounted in 1:1
polyvinyl alcohol (PVA): Melzer’s reagent and crushed
under a glass coverslip for identification to species. In
addition, a subsample of the small-spored Glomus spe-
cies (Ͻ100 ␮m) was collected from 30–40% of the
membrane area, mounted in PVA–Melzer’s reagent and
crushed under a coverslip for identification. Prepared
slides were examined using a light microscope (100–
400ϫ) equipped with Nomarski interference optics,
and spores identified (where possible) on the basis of
wall characters and in comparison with authenticated
samples. Spores that could not be identified to species
level were given a designation by genus and number.
A spore reference collection of permanent slides is held
in storage at the University of California, Riverside.

4. April 2000 487MYCORRHIZAE AND NITROGEN DEPOSITION
Data analysis.—Spore abundance was calculated as
the number of spores/g soil. Differences in abundance
were analyzed using a repeated measures two-way
analysis of variance (ANOVA) with spore abundance
as the dependent variable, and site and sampling time
as independent variables. Site and sampling times were
treated as fixed effects, since these variables were de-
liberately chosen to represent both certain conditions
(nitrogen deposition per site) and sampling times (sea-
son). In addition, spore abundance between host spe-
cies was analyzed by two-way ANOVA. Prior to all
ANOVA, spore abundance data were transformed using
log (1 ϩ x) function to satisfy normality (Zar 1984, St
John and Koske 1988). We used P Ͻ 0.05 as the cri-
terion for significance of effect and Fisher’s LSD test
to determine which mean values were significantly dif-
ferent from one another. Retransformed values are pre-
sented in the results and expressed as the arithmetic
mean and standard error.
Arbuscular mycorrhizal species richness (alpha di-
versity) was expressed as the average number of spe-
cies and determined by counting the number of species
whose spores were recovered from each site and sam-
pling time. These data complied with the assumptions
of ANOVA and were normally distributed, and thus
were not transformed prior to two-way ANOVA. Spe-
cies richness data were further analyzed between host
plant species. The relative abundance of AM species
for an individual site was also estimated. First, the
relative frequency of spores for each AM species over
the entire sampling period were summed. Then, the
relative abundance was calculated by dividing the
summed value for each AM species over the total num-
ber of spores of all AM species collected throughout
the sampling period. Differences in relative abundance
among sites was tested by contingency table analysis
and Tukey-type multiple comparison tests (Zar 1984).
Prior to analysis, the relative abundance data were
transformed using the formula, pЈ ϭ arcsin ͙p, where
p was the relative abundance of an individual AM spe-
cies (Zar 1984); retransformed data are presented in
the results. Differences in relative abundance were sta-
tistically examined only in those AM species that oc-
curred on at least three sites. Sites not containing an
individual species were omitted from analysis to pre-
vent the confounding effects of zero values on analyses.
Species diversity was quantified using the Shannon–
Wiener index (HЈ), where HЈ ϭ Ϫ⌺ ln(pi) pi, and pi
was the relative (proportional) abundance of AM spe-
cies for each site and i was the sampling time. Evenness
(E) was also calculated for each site as E ϭ HЈ/Hmax,
where Hmax ϭ ln S and S ϭ the total number of species
recovered. These data were analyzed using repeated
measures one-way ANOVA and Fisher’s LSD since the
repeated diversity measures were normally distributed
(Zar 1984, Magurran 1988). Species dominance mea-
sures were not calculated in the present study as the
AM population on each site was dominated by Glomus
aggregatum.
Univariate analyses indicated that differences exist-
ed among sites with respect to spore abundance and
AM species assemblages. Because these tests did not
explain relationships among sites, we used multivariate
analyses to group sites so that groupings reflected
changes in AM species composition with respect to soil
nitrogen, and identify interrelated suites of AM species
that represent changes in nitrogen input. We used ca-
nonical correspondence analysis (CCA) to directly in-
tegrate community (AM species) and environmental
data (soil nitrogen content) to analyze for and detect
anthropogenic stress-related changes in the AM com-
munity (ter Braak 1986). All CCA analyses utilized the
24 AM species so that all prospective indicator taxa
could influence the ordination; these data were arcsine
͙p transformed prior to CCA to normalize the data
set and downplay the influences of the more dominant
AM species. The environmental variables entered were
the soil nitrogen concentrations for each site and sam-
pling time. All environmental variables were standard-
ized to a mean of 0 and standard deviation of 1. CCA
ordination scores were scaled such that site scores were
weighted mean scores of AM species to take into ac-
count the possibility that the abundance of some AM
species may have fallen beyond their measured limits,
and thus focus the CCA so that sites scores were de-
rived from AM species composition. In addition, CCA
axes were neither rescaled nor detrended.
Root infection.—Fine root samples were recovered
from soil cores taken during the December and March
samplings. A subsample of roots from each site and
time were fixed in 90% (by volume) ethanol for 24 h
at room temperature and then stained to detect the pres-
ence of AM structures within the root using the method
of Koske and Gemma (1989). Mycorrhizal infection
was quantified as the percentage of root length colo-
nized by AM fungi using the methodology of Mc-
Conigle et al. (1990). Infection was expressed as the
percentage root length containing vesicles, arbuscules,
hyphae, or hyphal coils. No significant differences in
root infection could be detected between the December
and March samples (t-test; P Ͼ 0.05) and hence the
data were pooled for statistical analysis. Differences in
the percentage colonization by each structure and total
root infection between sites was analyzed using one-
way ANOVA. All data were transformed prior to AN-
OVA using the arcsine function (Zar 1984), with re-
transformed data presented in the results.
Root samples from low (SKIN), moderate (MOTT),
and high nitrogen sites (BOXS, JURP) were assessed
for the presence of extraradical hyphae using direct
immunofluorescence. While spores can be identified to
species level on the basis of wall morphology, a reliable
discrimination of hyphae among AM fungi cannot be
achieved readily by morphological criteria. In addition,
a failure to sporulate does not mean the fungus has

5. 488 LOUISE M. EGERTON-WARBURTON AND EDITH B. ALLEN Ecological Applications
Vol. 10, No. 2
disappeared from a site since a species may persist in
a vegetative (hyphal) but not reproductive state. Im-
munofluorescence permits the rapid identification of
hyphae to genus, but not species, level and indicates
which genera are present but not sporulating; to date,
there are no reliable tests to detect AM fungi at the
species level. However, since our study showed losses
of entire genera, detection to genus level was sufficient
for our purposes.
Polyclonal antibodies were raised against whole
spore fractions of Glomus deserticola, Acaulospora
laevis, Scutellospora calospora, or Gigaspora mar-
garita, and conjugated to fluoroscein isothiocyanate
(FITC) using the procedures described in Friese and
Allen (1991). For each AM species, spores were ex-
tracted from single-species cultures using sucrose flo-
tation, collected, and then surface-sterilized for 3 min
in 5% (by volume) sodium hypochlorite solution. Each
sample was rinsed three times in sterile deionized wa-
ter, resuspended in sterile phosphate-buffered saline
(pH 7) and sonicated to a suspension. Spore suspen-
sions were sent to Cocalico Biologicals Company
(Reamstown, Pennsylvania, USA) for the production
of antisera and subsequent FITC labelling. The titer
was evaluated 28 d postimmunization: spores of Glo-
mus deserticola, A. laevis, S. calospora, and Gigaspora
margarita were each placed into individual wells of a
test plate and a 200-␮L aliquot of the corresponding
FITC-labelled antisera was added to the well. Spores
were incubated for 24 h at room temperature and then
examined under fluorescent microscopy using a Zeiss
microscope with blue–purple excitation filter (450–490
nm), 510-nm beamsplitter, and 520-nm emission filter
(Carl Zeiss, Incorporated, Thornwood, New York).
These tests were replicated three times and scored as
positive if specific fluorescence could be detected when
compared with spores incubated in deionized water.
Because Scutellospora and Gigaspora are considered
to be closely related (Morton 1990), we evaluated
crossreactions using spores of S. calospora or Giga-
spora margarita as test material against the two anti-
sera. Crossreactions were not detected between Scutello-
spora and Gigaspora antisera and therefore all antisera
used in this study were genus specific.
To assess the presence or absence of each AM genus
on field-collected roots, washed and unfixed samples
of fine roots were incubated in individual FITC-la-
belled antisera for 12 h at room temperature. Root seg-
ments were then rinsed in water, mounted in PVA on
glass slides, and viewed using epifluorescence micros-
copy. Roots were scored for the presence or absence
of fluorescing hyphae after incubation in individual an-
tisera. Root segments incubated in deionized water
were included as controls.
Experimental nitrogen fertilization plots
Twenty plots, each 5 ϫ 5 m, were initiated at a low
N deposition site (SKIN), during January 1994. In a
randomized design, 10 plots received granulated
NH4NO3 fertilizer during the period between January
and March in 1994, 1995, and 1996 at a rate of 60 kg
N·haϪ1
·yrϪ1
as two 30-kg applications during each
growing season. An additional 10 plots were left un-
fertilized and constituted the control plots. As a base-
line for the experimental nitrogen fertilization studies,
we assessed the AM community during the first winter
and summer following the establishment of the plots.
Soil cores were collected from the dripline of five
plants each of Artemisia and Eriogonum in a nonfer-
tilized region adjacent to the experimental plots, and
analyzed for AM spore abundance and species com-
position. For the experimental study, soil samples were
collected from the dripline of five plants each of Ar-
temisia and Eriogonum in the fertilized and nonfertil-
ized plots following the same time frame as the nitrogen
gradient sampling with an additional sampling under-
taken in September 1996. Each soil sample was a com-
posite of two adjacent soil cores each 2.5 cm in di-
ameter and 5 cm in depth The recovery and identifi-
cation of spores followed the same methods as for the
descriptive study. Spore abundance (log [1 ϩ x ] trans-
formed) and species richness were analyzed using two-
way ANOVA, while the relative abundance of individ-
ual AM species (arcsine ͙p transformed) was com-
pared between fertilized and nonfertilized plots using
contingency analysis. Shannon–Wiener diversity (HЈ),
evenness (E), and Sorenson’s quantitative measure of
similarity (CN; Magurran 1988) were calculated for fer-
tilized and nonfertilized plots; HЈ and E were analyzed
between treatments using one-way ANOVA.
RESULTS
Nitrogen deposition gradient
Host plant species.—Host plant–AM species inter-
actions were not detected along the nitrogen gradient.
No significant differences in the relative abundance of
spores, species richness, and statistical measures of di-
versity and evenness were detected among host plant
species (P Ͼ 0.05). Subsequently, all analyses in our
study represent those undertaken on grouped samples
(n ϭ 15 per site and date). In contrast, other studies
indicate that the host plant species can influence AM
fungal communities, including those in soil nutrient
gradients (e.g., Johnson et al. 1992, Nelson and Allen
1993).
Spore abundance.—A significant temporal variation
in spore abundance was detected across the sampled
locations during the study (P Ͻ 0.001) (Fig. 1). Av-
eraged across all sites, the maximum spore abundance
was recorded during the September sampling and was
coincident with the completion of plant growth (Moon-
ey 1977). In contrast, the lowest spore abundance was
recorded in December. These similarities aside, a di-
chotomy existed among sites with respect to spore
abundance. Specifically, a significantly greater number

6. April 2000 489MYCORRHIZAE AND NITROGEN DEPOSITION
FIG. 1. Mean number of spores per gram of soil and sampling time for each site along the nitrogen gradient, and means
across all sites. Error bars indicate the standard error of the mean for each location and time. Site abbreviations correspond
to those listed in Table 1.
of spores per gram of soil were recovered from the
SMER, SKIN, MOTT, and MATH sites (low or mod-
erate nitrogen) than the HEMT, WATR, BOXS, MOCK,
and JURP sites (moderate or high nitrogen) (P Ͻ 0.001;
Fig. 1). In addition, the effect of sampling time on spore
abundance differed significantly among sites (P Ͻ
0.001): spore abundance peaked in September and was
lowest in December or June for all sites except JURP.
At JURP, maximum spore abundance occurred in June
1996 and was at a minimum in June 1995. These data
suggest that a shift in the pattern of spore production
may occur on a highly eutrophied site. However, any
generalizations regarding the impact of nitrogen input
on the timing of spore production remain limited due
to a lack of similar studies on the effects of eutrophi-
cation on the AM community.
Species composition.—Spores of 24 species of AM
fungi were identified throughout the 12-mo sampling
period, including five undescribed species of Glomus
and one undescribed species of Scutellospora (Table
2). In addition, all species were detected at each sample
date except Sclerocystis and Entrophospora; these gen-
era were detected in June and September 1995 but not
thereafter. In general, increasing nitrogen eutrophica-
tion was associated with a loss of AM species repre-
sentation, but especially the displacement of the larger-
spored genera Scutellospora and Gigaspora (Table 2).
On the low nitrogen sites large-spored species contrib-
uted to a significantly greater proportion of species
composition that in sites with higher soil nitrogen con-
tent (P Ͻ 0.05; ␹2). In addition, species were recovered
from the low nitrogen sites that were not found at any
of the other eutrophied sites, including Sclerocystis sp.
indet., Scutellospora sp. 1 (SMER), and Glomus sp. 1
(SKIN). In contrast, A. laevis was the only large-spored
species recovered from all sites, including the high ni-
trogen sites. This species is highly infective and ap-
parently less sensitive to fertilizer additions than either
Scutellospora or Gigaspora (Wilson and Tommerup
1992, Smith and Read 1997). In addition, Glomus lep-
totichum was detected only in moderate or high nitro-
gen sites.
Accompanying the loss of species representation
with eutrophication were significant shifts in the rel-
ative abundances of species (Table 2). Firstly, signif-
icant increases in the abundance of A. laevis, Glomus
aggregatum, and Glomus deserticola occurred in mod-
erate (MOTT, WATR) or high nitrogen sites (BOXS,
MOCK, JURP). In addition, a significantly greater pro-
portion of Glomus geosporum, Glomus claroideum,
Glomus occultum, or Glomus sp. 4 were recovered from
moderate or high nitrogen sites (HEMT, MOTT, WATR,
MOCK). Further, a high nitrogen input at BOXS was
associated with a significant increase in the abundance

7. 490 LOUISE M. EGERTON-WARBURTON AND EDITH B. ALLEN Ecological Applications
Vol. 10, No. 2
TABLE 2. Relative abundances of AM species (columns each total to 100%), indices of diversity, and results of contingency-
table Tukey multiple comparison tests for the nine assayed sites along the nitrogen gradient.
AM species SMER SKIN HEMT MOTT MATH
1) Entrophosphora sp. indet. 1.10
2) Sclerocystis sp. indet. 1.10 0.30
3) Gigaspora margarita Becker & Hall 5.4a 1.6b 0.6b 1.4b
4) Scutellospora scutata Walker & Diederichs 0.60 0.20
5) Scutellospora sp. 1 0.90
6) S. calospora (Nicol. & Gerd.) Walker & Sanders 2.9a 1.9a 2.5a 1.4a
7) Acaulospora elegans Trappe & Gerdermann 1.1a 0.4a
8) A. laevis Gerdermann & Trappe 2.4b 2.8b 1.7b 4.4a 2.2b
9) Glomus sp. 1 0.50
10) G. fasciculatum (Thaxter) Gerd. & Trappe
emend. Walker & Koske
1.7a 1.0a 1.9a
11) Glomus sp. 2 2.0b 1.8b 3.2b 5.6a
12) G. microcarpum Tul. & Tul. 4.9b 3.1b 3.1b 3.5b
13) G. aggregatum Schenck & Smith
emend. Koske
43.1b 39.9b 58.5b 40.4b 54.4b
14) G. etunicatum Becker & Gerd. 13.7a 14.4a 2.5b 3.0b 2.8b
15) G. deserticola Trappe, Bloss & Menge 1.8c 2.4b 4.1b 17.6a 5.5b
16) G. geosporum (Nicol. & Gerd.) Walker 3.2c 5.1b 2.8c 9.8a 8.2b
17) G. macrocarpum Tul. & Tul. 1.1b 2.2b 2.3b 0.6b 3.1b
18) G. mosseae (Nicol. & Gerd.) Gerd. & Trappe 2.9a 6.5a 3.1a 2.6a 4.5a
19) G. claroideum Schenck & Smith 4.8b 7.5b 13.6a 2.7c 2.3c
20) G. occultum Walker 2.3bc 4.5b 3.7b 4.0b 1.2c
21) G. leptotichum Schenck & Smith 6.1a 1.3b
22) Glomus sp. 3 0.9a 3.5a
23) Glomus sp. 4 1.0b 0.5b
24) Glomus sp. 5 1.1a 1.1a 1.6a
Total no. of species recovered per site 19 19 11 18 15
Total no. of large-spored species 7 4 2 4 5
Mean no. species (1 SE) per sample and date 5.1a
(0.3)
4.6a
(0.3)
2.6c
(0.1)
4.0b
(0.2)
4.0b
(0.2)
Mean diversity (HЈ) 0.766a 0.564a 0.269c 0.422b 0.579a
Maximum diversity (Hmax) 2.944 2.944 2.484 2.708 2.708
Evenness (E) 0.260a 0.192b 0.108d 0.156c 0.214b
Notes: Values represent the mean abundance of each AM species averaged over the five sample dates. Site abbreviations
correspond to Table 1.
* P Ͻ 0.05; ** P Ͻ 0.01; *** P Ͻ 0.001.
† NS ϭ not significant (P Ͼ 0.05). NA ϭ analysis not applicable (n Ͻ 3 sites); means within rows with the same superscript
letter do not differ significantly at P Ͻ 0.05 (Fisher’s LSD).
of Glomus microcarpum and Glomus macrocarpum.
Together, the spore and species abundance data indicate
that with increasing eutrophication, AM populations
were dominated by the proliferation of a few species
of Glomus.
Species richness and diversity.—The variation in
species richness (mean number of AM species per sam-
ple and date) differed significantly among sites (P Ͻ
0.05) (Table 2). Higher species richness was recorded
for SMER and SKIN (low N) than at MOTT, MATH,
WATR, and MOCK (moderate to high nitrogen). In
turn, these sites demonstrated greater species richness
than HEMT and the high nitrogen sites, BOXS and
JURP. In addition, species richness among sites varied
significantly with season (data not shown). Maximum
species richness occurred in December and was at a
minimum in September or June except for JURP; at
this site, maximum species richness occurred in June.
Statistical measures of diversity and evenness dif-
fered significantly among sites (Table 2). The AM com-
munities at SMER, SKIN, and MATH were signifi-
cantly more diverse than those at HEMT, BOXS, or
JURP (P Ͻ 0.05). The MOTT, WATR, and MOCK sites
were somewhat intermediate between these two groups.
The low evenness values across all sites indicated that
not all species were equally abundant within the as-
sessed AM populations. Nevertheless, the highest in-
dices were calculated for SMER, SKIN, and MATH;
and the lowest for HEMT, JURP, and BOXS. The
MOTT, WATR, and MOCK sites were again interme-
diate between these two groups with respect to even-
ness. These findings support the earlier observations
that nitrogen eutrophication significantly altered the di-
versity and distribution of AM species among sites.
CCA identified groupings of sites, groups of species
based on nitrogen sensitivity, and AM taxa that are
identifiers of eutrophication. CCA distinguished three
groups of sites in which the groupings reflected changes
in AM species assemblage along the gradient (data not
shown). The three groups were identified as follows:
group 1 SMER, SKIN; group 2 MOTT, WATR, MATH;
group 3 HEMT, MOCK, BOXS, JURP. These groups
correspond to the designated low, moderate or high
nitrogen sites respectively with the exception of HEMT.

8. April 2000 491MYCORRHIZAE AND NITROGEN DEPOSITION
TABLE 2. Extended.
WATR BOXS MOCK JURP
Signifi-
cance†
NA
NA
1.3b *
NA
NA
1.8a 2.3a NS
0.4a NS
6.6a 1.3b 4.5a 1.2b *
NA
NS
2.3b 2.6b 1.2b *
6.7a 2.1b 2.7b *
43.7b 66.2a 51.5b 74.1a **
4.7b 2.7b 8.9a 2.1b ***
11.9a 4.4b 1.1c 4.3b ***
5.4b 1.7c 10.1a 4.2b ***
1.0b 6.6a 0.6b 2.7b *
3.8a 4.4a 1.6a NS
5.1b 3.7bc 11.8a 1.6c ***
6.6a 0.7c 2.5bc 1.5c *
2.3b 0.5b 1.6b 2.7b *
2.4a 1.0a 1.5a NS
3.7a 2.4a NS
1.4a NS
15 14 14 12 *
4 1 2 1 *
3.3b
(0.2)
2.6c
(0.2)
3.4b
(0.2)
2.8c
(0.19)
*
0.385b 0.163c 0.315c 0.243c *
2.890 2.639 2.639 2.484 NA
0.133c 0.062d 0.119cd 0.097d ***
FIG. 2. Diagnostic canonical correspondence analysis (CCA) showing the dispersion of AM species coordinates in CCA
2 vs. CCA 1 (right-hand panel), and corresponding coordinates for soil nitrogen (left-hand panel); species numbers in the
right-hand panel correspond to those listed in Table 2. The AM species dispersion illustrates the nitrogen gradient: soil
nitrogen concentrations range from low (left-hand quadrants) to moderate or high (right-hand quadrants). The sizes of the
symbols for soil nitrogen are scaled relative to the magnitude of the soil nitrogen concentration: low (small circle), moderate
(large circle), or high (large filled circle) soil nitrogen. Soil nitrogen values are shown for five replicates per site.
Based on AM species assemblage (Table 2) and CCA,
HEMT was more similar to a high than moderate ni-
trogen sites (Table 1). These data suggest that the my-
corrhizal community of the CSS- chaparral transition
zone may differ in its response to nitrogen enrichment
than the CSS community and that even a small input
of nitrogenous pollutants exerts a strong, negative in-
fluence on the mycorrhizal community.
The plot of CCA 2 vs. CCA 1 illustrates the nitrogen
gradient and the corresponding dispersion of AM spe-
cies (Fig. 2). Species coordinates in the lower left-hand
quadrant of CCA space were associated with a negli-
gible input of nitrogen, and included Scutellospora sp.
1, Sclerocystis, and Entrophospora. In contrast, species
with coordinates in the lower right-hand quadrant of
CCA space were associated with the highest soil ni-
trogen concentrations throughout the sampling period
(up to 86 ␮g N/g). These nitrogen-tolerant species, Glo-
mus leptotichum, Glomus aggregatum, and Glomus mi-
crocarpum, may thus be useful indicators of eutrophi-
cation. The remaining AM species with coordinates in
both the upper left- and right-hand quadrants of CCA
space tolerate low (mean 16 ␮g N/g) or moderate (mean
27 ␮g N/g) nitrogen enrichment respectively. The up-
per left-hand quadrant contains species, such as Giga-
spora margarita and A. elegans, that persist with low
input of nitrogen, while AM species in the upper right-
hand quadrant (Glomus spp., Scutellospora scutata)
were associated with moderate soil nitrogen levels. We
also note three ‘‘borderline’’ species between low and
moderate nitrogen input: A. laevis, Glomus geosporum,

9. 492 LOUISE M. EGERTON-WARBURTON AND EDITH B. ALLEN Ecological Applications
Vol. 10, No. 2
FIG. 3. Mean percentage of hyphal, vesicular, and arbus-
cular infection within root samples collected from each site
along the nitrogen gradient. Site abbreviations correspond to
those listed in Table 1.
TABLE 3. Presence (●) or absence (⅙) of spores and immunofluorescently detectable extraradical hyphae in samples from
low-nitrogen (SKIN), moderate-nitrogen (MOTT), and high-nitrogen sites (BOXS, JURP).
AM genus
SKIN
Spores
Extraradical
hyphae
MOTT
Spores
Extraradical
hyphae
BOXS
Spores
Extraradical
hyphae
JURP
Spores
Extraradical
hyphae
Glomus
Acaulospora
Scutellospora
Gigaspora
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
⅙
⅙
●
●
●
●
●
●
⅙
⅙
●
●
●
⅙
Note: Site abbreviations correspond to those in Table 1.
and Glomus deserticola. Such species dispersion pat-
terns on the plot also confirm that a reduction in AM
species diversity accompanies an increasing nitrogen
input: the majority of taxa (15 of 24 species) were
present in the left- (low to moderate nitrogen) rather
than right-hand side of the plot (high nitrogen).
Root Infection.—Increasing nitrogen eutrophication
was accompanied by a decrease in the total mycorrhizal
infection within the root (Fig. 3). In particular, there
was a progressive and significant reduction in vesicular
and hyphal infection with increasing nitrogen input (P
Ͻ 0.05), intermediate and high nitrogen sites consis-
tently demonstrated lower percentages of vesicles and
hyphae than low nitrogen sites. Similar reductions in
hyphal and vesicle infection with nitrogen enrichment
have been recorded previously (e.g., Johnson 1993). In
contrast, arbuscular infection did not differ among lo-
cations (P Ͼ 0.05); low levels of arbuscules (Ͻ1% root
length infected) were detected at all sites along the
gradient. Since the arbuscular interface regulates the
directional transfer of carbon and nutrients between
host and mycobiont, our observations suggest that the
potential for nutrient and carbon transfer is not altered
by nitrogen enrichment. However, the relatively low
levels of arbuscular infection may denote limited nu-
trient transfer between symbiont and host, or that a
rapid turnover of these structures occurs in CSS plants.
The immunofluorescent assessments demonstrated
that all four AM genera were detected in the extra-
radical hyphae at SKIN and MOTT (Table 3); this find-
ing paralleled the AM species recovered as spores (Ta-
ble 2). Interestingly, samples from both high nitrogen
sites (JURP, BOXS) demonstrated the presence of im-
munofluorescently detectable Gigaspora and Scutel-
lospora hyphae at the root surface even when spores
were not detected in the associated soil samples. These
data suggest that both Scutellospora and Gigaspora
existed within the high nitrogen sites as extraradical
hyphae but were not actively sporulating.
Experimental nitrogen fertilization plots
At the initiation of the experimental fertilization
study, spore abundance averaged 29 Ϯ 3 spores/g soil
(mean Ϯ 1 SE) in winter, and 61 Ϯ 7 spores/g soil in
summer in the adjacent nonfertilized soils. Seventeen
species of AM fungi were identified in these samples
and these included the species listed in Table 4 with
the exception of Glomus sp. 2 and Glomus sp. 5.
The effects of nitrogen fertilization on spore abun-
dance in the CSS were marked. AM spores were sig-
nificantly more abundant in nonfertilized than fertilized
plots throughout the sampling period (P Ͻ 0.05) (Fig.
4). Both treatments, however, show maximum spore
abundance in September and a minimum during De-
cember.
Twenty species of AM fungi were recovered from
the plots (Table 4). Nineteen of these species were re-
covered from the nonfertilized plots compared with
only nine species from fertilized plots; this difference
was also reflected in a low index of similarity between
plots (CN ϭ 0.21). With the exception of A. laevis, the
fertilized plots lacked the presence of larger spored
species. In addition, a lower abundance of Glomus
geosporum was recovered from fertilized than nonfer-
tilized sites, while Glomus leptotichum was recovered
only from fertilized plots. The loss of AM species in
the fertilized plots was reflected in species richness and

10. April 2000 493MYCORRHIZAE AND NITROGEN DEPOSITION
TABLE 4. The relative abundance of AM species (columns
each total to 100%) in fertilized and nonfertilized plots at
SKIN.
AM species Fertilized
Nonfert-
ilized
Signif-
icance†
Sclerocystis sp. indent.
Gigaspora margarita
Scutellospora calospora
Acaulospora laevis
Glomus sp. 1
G. fasciculatum
Glomus sp. 2
G. microcarpum
G. aggregatum
G. etunicatum
G. deserticola
G. geosporum
G. macrocarpum
2.8
1.0
1.4
78.9
4.6
1.0
0.4
0.3
2.1
3.2
0.5
0.5
1.6
1.6
63.0
1.1
4.7
2.9
1.1
NA
NA
NA
NS
NA
NA
NS
NS
*
NS
NS
*
NA
G. mosseae
G. claroideum
G. occultum
G. leptotichum
Glomus sp. 3
Glomus sp. 4
Glomus sp. 5
5.8
3.5
1.9
1.4
5.1
2.7
4.3
1.4
1.4
NA
NS
NS
NA
NA
NA
NA
Total no. species recovered
per treatment
9 19 *
Total no. large-spored genera 1 4 *
Mean no. species (1 SE)
per sample and date
2.8
(0.3)
4.6
(0.4)
*
Mean diversity (HЈ)
Maximum diversity (Hmax)
Evenness (E)
0.796
2.197
0.362
1.548
2.944
0.526
*
NS
NS
† Significance: NA ϭ analysis not applicable as species
detected in only one treatment; NS ϭ means not significantly
different (P Ͼ 0.05); * ϭ means differ significantly at P Ͻ
0.05.
FIG. 4. Mean numbers of spores per gram soil, and sam-
pling time for nitrogen fertilized and nonfertilized plots at
Lake Skinner. Error bars indicate mean ϩ 1 SE.
diversity measures. More species were recovered from
nonfertilized than fertilized plots (P Ͻ 0.05), and di-
versity was significantly higher in the former than the
latter (P Ͻ 0.05). In addition, species richness peaked
in March in nonfertilized plots compared with Septem-
ber for fertilized plots. Evenness was higher in non-
fertilized than fertilized plots, however, this difference
was not statistically significant (P Ͼ 0.05).
DISCUSSION
We can draw three general conclusions about the
effects of anthropogenic nitrogen deposition on AM
communities in shrublands. First, the effects of an-
thropogenic nitrogen deposition and experimental ni-
trogen fertilization had similar consequences for AM
communities. Thus, nitrogen input, rather than other
environmental factors along the gradient, most likely
explains the causal relationship between anthropogenic
pollution and shifts in the AM community in the CSS.
This is an especially important consideration in south-
ern California, as ozone co-occurs with nitrogen oxides
(Environmental Protection Agency 1996) and could be
a part of the same gradient. However, at SKIN where
ozone is low, nitrogen fertilization alone caused similar
patterns of reduced species richness and loss of the
larger-spored species. Similarly, we can exclude soil
pH as a causal factor in the decline of the AM com-
munity since there was no correlation between soil pH
and concentrations of NO3.
Second, nitrogen enrichment significantly alters AM
species composition and richness, and markedly de-
creases the overall diversity of the AM community.
Nitrogen eutrophication exerted a strong selective pres-
sure for smaller spored Glomus species (Glomus lep-
totichum, Glomus aggregatum, Glomus geosporum),
and against species of Scutellospora and Gigaspora.
Because similar patterns of selection also occur in AM
communities after fertilization (Johnson 1993, and ref-
erences therein), these traits appear to represent gen-
eralized responses to nitrogen enrichment. Evidence
from the current study also indicates that the decline
of Scutellospora and Gigaspora was enforced by a cu-
mulative failure to sporulate and not the inability to
colonize roots, as the immunofluorescence assay
showed. One reason may be that spore production re-
quires large quantities of photosynthates. On the con-
trary, fertilization results in less carbohydrate being
allocated to the root/mycobiont owing to intense carbon
sink competition between the mycobiont and shoot
(Smith 1980). Because AM fungi differ in their re-
quirement for carbon (Douds and Schenck 1990), per-
haps Gigaspora and Scutellospora have a greater de-
mand for carbon than can be supplied by the host, or
alternatively, small-spored Glomus species more effi-
ciently utilize a scarce carbon resource. We can also
hypothesize that the energetic expenditure required to
produce large spores occurs at a greater cost to the
mycobiont than for small spores. However, such costs

11. 494 LOUISE M. EGERTON-WARBURTON AND EDITH B. ALLEN Ecological Applications
Vol. 10, No. 2
of reproduction have yet to be determined experimen-
tally for mycorrhizas.
Finally, nitrogen eutrophication reduces both the sur-
vival and/or reproduction of the mycobiont. Nitrogen
enrichment in the CSS reduces mycorrhizal infection
(growth, survival), and spore abundance (reproduction)
in a similar fashion as induced by nitrogen fertilization
alone, or nitrogen ϫ phosphorus interactions (e.g.,
Menge et al. 1978, Hayman 1982). These findings sup-
ply us with information on two points: (1) Root infec-
tion and sporulation requires carbon expenditure and a
decrease in both parameters indicates an overall re-
duction in carbon invested in the construction and
maintenance of the mycorrhizal association. (2) Mo-
lecular recognition and signalling mechanisms ex-
pressed by the mycobiont in concert with those in root
exudates or the rhizoplane act to elicit or suppress my-
corrhizal infection (Allen 1991, Anderson 1992). The
suppression of mycorrhizal infection in high fertility
soils may be linked to the abortion of appressoria at
the rhizoplane (Amijee et al. 1989). In our study, in-
fection was only determined at two points in the in-
fection process (December and March). As such, a
more intensive annual sampling regime that encom-
passes comparisons of the rate and phenology of in-
fection among CSS sites, particularly at the pre-infec-
tion stages, may better indicate the point(s) at which
eutrophication influences the infection process.
Such changes substantiate the hypothesis that an-
thropogenic nitrogen deposition exerts a significant
(negative) effect on AM community dynamics in the
CSS. We can also conclude that AM fungi are far more
sensitive indicators of nitrogen enrichment than the
plant community since marked shifts in the AM com-
munity were generated less than two years after the
commencement of nitrogen enrichment. This is prior
to any observable change in cover in the CSS plant
community (Allen et al. 1997).
The key issue is how these shifts affect functioning
at the local (rhizosphere) and ecosystem (CSS stand)
levels. At the local level, the observed shifts may alter
both the character and productivity of current and fu-
ture AM populations. Spore abundance is indicative of
initiation and perpetuation of the AM associations and
a decline may modify the demography of AM species
in the CSS. In the long term, the diminished recruitment
of spores may lead to local patterns of exclusion and
extinction of an individual species, in particular species
of Scutellospora and Gigaspora. Our study indicates
that these patterns are already established in high ni-
trogen sites or becoming prevalent in moderate nitro-
gen sites. Because of the strong nitrogen-based selec-
tion against sporulation of Scutellospora and Giga-
spora, their persistence is uncertain in increasingly eu-
trophied sites. Since the samples were collected from
rhizospheres of the appropriate CSS host species at
each of the sites, selection must have been determined
by high soil nitrogen rather than lack of a suitable host
plant. Opportunities for persistence and recruitment
will thus depend on a reduction of soil nitrogen levels
via the introduction of legislation to further reduce ve-
hicular emissions and ameliorative measures, such as
mulching, to immobilize inorganic nitrogen (Allen et
al. 1998, Zink and Allen 1998).
We can also hypothesize that nitrogen enrichment
has facilitated the loss of functional diversity in the
AM community. Altering, and in particular lowering,
mycorrhizal diversity can compromise plant commu-
nity performance (Van der Heijden et al. 1998). In the
CSS, nitrogen enrichment coincides with the conver-
sion from a species-rich AM community to one that is
dominated by small-spored mutualists that retain high
levels of fecundity and infectivity in nutrient-enriched
soils, or even increase in abundance with nitrogen en-
richment (e.g., Glomus leptotichum). Importantly, these
Glomus species are aggressive in disturbed or nutrient-
enriched environments but appear to be less effective
mycobionts. In symbiosis, they exert a net negative
carbon balance on the host that is reminiscent of par-
asitic, rather than mutualistic, associations (Johnson
1993).
At the ecosystem level, common convention has at-
tributed the decline of the CSS to a history of frequent
fires and the subsequent replacement of shrubs by an-
nual grasses (Minnich and Dezzani 1998). Nitrogen
enrichment further exacerbates the balance in favor of
grasses (Allen et al. 1997, 1998). Based on the current
study, we suggest that a loss of shrub species may also
be linked to the decline of the AM community with
nitrogen enrichment for the following reasons. CSS
species are facultatively mycorrhizal, yet the mycor-
rhizal population declines with increasing nitrogen in-
put and the most persistent AM associates are poten-
tially less effective mutualists. Although plant species
differ in the extent of their response to mycorrhizas,
the symbiosis is generally linked to positive benefits
for the host plant via increased rates of survival,
growth, and biomass production, and increased acqui-
sition of water, macro-, and micronutrients (Allen 1991,
Smith and Read 1997). In contrast, a less effective
mutualist can alter the dry matter allocation and nu-
trient status of the host (Johnson 1993), and host sus-
ceptibility to drought (Smith and Read 1997). Studies
are currently being conducted to determine the effects
of AM fungi from highly eutrophied CSS sites on host
survival, productivity, and nutrient status and the extent
to which these associations contribute to the decline of
the CSS.
The invasive species can also influence mycorrhizal
dynamics. Root exudates from Brassica can either de-
lay or prevent the germination of AM spores (Tom-
merup 1984), while the colonization of sites by Bromus
and Avena promotes the selection of small-spored Glo-
mus species, such as Glomus aggregatum (Allen et al.
1992, Nelson and Allen 1993). In addition, invasive
species demonstrate a superior survivorship and com-

12. April 2000 495MYCORRHIZAE AND NITROGEN DEPOSITION
petitiveness to CSS shrubs in nitrogen-enriched soils
(P. E. Padgett and E. B. Allen, unpublished data).
We posit that nitrogen enrichment may therefore fa-
cilitate a shift from shrub to grasslands by promoting
the displacement of mycorrhizal dependent CSS spe-
cies in favor of more competitive invasive species fol-
lowed by a successional replacement. We have initiated
correlative field and glasshouse studies to test this hy-
pothesis and further elucidate the impact of anthro-
pogenic nitrogen deposition on AM, and hence CSS,
community dynamics.
ACKNOWLEDGMENTS
We thank Sheila Kee and Tho Vo for assistance with soil
sampling and spore extraction, and Pam Padgett for generous
access to soil N data. This research was supported by com-
petitive grants from the National Science Foundation (DEB-
9408079 and DEB-9526564) and U.S. Department of Agri-
culture (NRI 95–37101–1700) to E. B. Allen, and in part by
a Fulbright Post-Doctoral Fellowship from the Australian–
American Education Foundation to L. Egerton-Warburton.
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