American Journal of Botany 99(10): 1691–1701. 2012.

ARBUTUS MENZIESII (ERICACEAE) FACILITATES REGENERATION

DYNAMICS IN MIXED EVERGREEN FORESTS BY PROMOTING
MYCORRHIZAL FUNGAL DIVERSITY AND HOST CONNECTIVITY1

PETER G. KENNEDY2,6, DYLAN P. SMITH2,3, TOM R. HORTON4, AND RANDY J. MOLINA5

2Department of Biology, Lewis and Clark College, 0615 S.W. Palatine Hill Rd., Portland, Oregon 97219 USA; 3Department of
Biology, Stanford University 371 Serra Mall, Stanford, California 94305 USA; 4Department of Environmental and Forest
Biology, SUNY College of Environmental Science and Forestry, Syracuse, New York 13210 USA; and 5Department of Forest
Ecosystems and Society, Oregon State University, Corvallis, Oregon 97331 USA

• Premise of study: In the mixed evergreen forests in the western United States, Arbutus menziesii is able to quickly resprout
following disturbance and, as such, act as a nurse tree during forest regeneration. The mechanism for this nurse tree effect has
frequently been ascribed to mycorrhizal fungi, but no detailed molecular-based studies of the mycorrhizal fungal communities
associated with A. menziesii roots have yet been conducted.
• Methods: We examined the structure of the mycorrhizal fungal communities associated with A. menziesii in varying forest
types and seasons and assessed the potential for common mycelial networks between A. menziesii and Pinaceae hosts, particu-
larly Pseudotsuga menziesii. Study sites were located in the Klamath-Siskyou region in southern Oregon, United States. Mo-
lecular approaches were used to identify the mycorrhizal fungi (ITS rDNA) and plant hosts (trnL cDNA).
• Key results: Arbutus menziesii hosts a highly diverse mycorrhizal fungal community with similar composition to communities
found on other angiosperm and Pinaceae hosts. Phylogenetic analyses of the mycorrhizal genus Piloderma revealed that host
species and geographic location had little effect on fungal taxon relatedness. Multihost fungal taxa were significantly more
frequent and abundant than single-host fungal taxa, and there was high potential for the formation of common mycelial net-
works with P. menziesii.
• Conclusions: Our results suggest A. menziesii is a major hub of mycorrhizal fungal diversity and connectivity in mixed ever-
green forests and plays an important role in forest regeneration by enhancing belowground resilience to disturbance.

Key words: Arbutus menziesii; common mycelial networks; Ericaceae; forest regeneration; host specificity; Klamath-Siski-
you; mycorrhizal fungi; Pinaceae; Pseudotsuga menziesii.

Some ectomycorrhizal (ECM) fungal species are well doc- (Perry et al., 1989; Molina et al., 1992), and recent experimen-
umented to associate with a wide range of host plant species, tal studies isolating the effects of these networks have con-
while others appear to be restricted to specific host genera or sistently demonstrated their significance in forest regeneration
families (Molina et al., 1992). A lack of host specificity among (Booth, 2004; Teste and Simard, 2008; Booth and Hoeksema,
many ECM fungi has significant ecological implications as it 2010).
allows individuals of the same or different plant species to be While high potential for interspecific mycelial networks
linked by shared fungi that form common mycelial networks among different Pinaceae species (Horton and Bruns, 1998;
(Simard et al., 2012). These networks are functionally impor- Cullings et al., 2000) and between Pinaceae and angiosperms
tant during seedling establishment; young plants benefit via (Simard et al., 1997; Horton et al., 1999; Kennedy et al., 2003;
access to extensive mycelial networks supported by already Nara and Hogetsu, 2004) has been demonstrated, many temper-
established individuals (Van der Heijden and Horton, 2009). In ate forests have plant species in the family Ericaceae growing
this manner, common mycelial networks play key roles in eco- close to Pinaceae and angiosperm ECM hosts. Some of these
system recovery from natural and anthropogenic disturbances ericaceous plants, particularly members of the genera Arcto-
staphylos and Arbutus, form mycorrhizas with the same fungi
associated with ECM host species (Zak 1974, 1976; Molina and
1 Manuscript received 9 June 2012; revision accepted 24 August 2012. Trappe, 1982; Acsai and Largent, 1983; Smith et al., 1995;
The authors gratefully acknowledge the assistance of David Perry and Molina et al., 1997; Massicotte et al., 1999; Hagerman et al.,
Marty Main in identifying the study area and providing assistance with the 2001; Krpata et al., 2007). Mycorrhizas formed by Arbutus and
field sampling of study 1. Two anonymous reviewers provided constructive Arctostaphylos resemble ectomycorrhizas in that they colonize
comments on a previous version of this manuscript. Funding was provided short feeder roots, often branching repeatedly, and develop a
by the Student Academic Affairs Board at Lewis & Clark College to D.P.S., mantle of mycelium on the surface and an epidermal Hartig net
a MSA Martin-Baker Award and the National Science Foundation (DEB# as seen with other ECM angiosperms. They differ from ecto-
1020735 and 0742868) to P.G.K. and by the U. S. Forest Service to T.R.H.
and R.J.M.
mycorrhizas, however, in having intracellular fungal penetra-
6 Author for correspondence (e-mail: pkennedy@lclark.edu); 0615 S.W. tion of the epidermal cells as seen in most members of the
Palatine Hill Rd., Portland, OR 97219; phone: 503-768-7509; fax: 503- Ericaceae. Zak (1974, 1976) called these symbioses “ecten-
768-7568 domycorrhizas” due to the intracellular fungal growth, while
Molina and Trappe (1982) referred to them as “arbutoid” my-
doi:10.3732/ajb.1200277 corrhizas (sensu Harley, 1969) and considered them a subtype

American Journal of Botany 99(10): 1691–1701, 2012; http://www.amjbot.org/ © 2012 Botanical Society of America
1691
 15372197, 2012, 10, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.3732/ajb.1200277 by Cochrane Mexico, Wiley Online Library on [21/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1692 AMERICAN JOURNAL OF BOTANY [Vol. 99

of ectomycorrhizas. Peterson et al. (2003) formally described MATERIALS AND METHODS

and classified them as arbutoid mycorrhizas.
Common mycelial networks between ericaceous and other We conducted two independent studies to address our research questions.
hosts have long been suspected based on patterns from sporo- The first study (note they are not presented in chronological order) was a gen-
carp occurrence in pure stands, culture syntheses, seedling eral survey of the mycorrhizal fungal communities associated with A. menziesii
establishment, and soil transfer experiments (Molina and Trappe, in varying forest types and at different time periods. The second was a more
focused study looking at the potential for common mycelial networks between
1982; Acsai and Largent, 1983; Perry et al., 1989; Amaranthus Arbutus menziesii Pursh. and Pinaceae hosts, particularly Psuedotsuga men-
and Perry, 1994). For example, in the western United States, ziesii (Mirb.) Franco. Both were conducted at nearby locations in southern
Amaranthus et al. (1990) and Horton et al. (1999) documented Oregon, United States and used similar molecular approaches to identify my-
preferential establishment of Pseudotsuga menziesii seedlings corrhizal fungi and plant hosts.
in areas adjacent to, respectively, Arbutus menziesii and Arcto-
staphylos glandulosa. Horton et al. (1999) demonstrated the Site location and sampling: Study 1—Study 1 was conducted in the mixed
presence of many of the same mycorrhizal fungal species on evergreen forests of the city of Ashland, Oregon watershed (42°10′52N,
both P. menziesii and A. glandulosa roots within the same soil 122°43′85W, 1082 m a.s.l.). The climate is characterized by cool wet winters and
moderately hot dry summers (Appendix S1, see Supplemental Data with the
cores. In a Mediterranean ecosystem, Richard et al. (2009) doc- online version of this article). Within the watershed, a ~1-km2 area was chosen
umented a similar pattern of improved Quercus ilex seedling based on host species composition. One ~1800-m2 site was identified where
establishment in areas with Arbutus unedo and also confirmed A. menziesii was the only ECM host species present (hereafter referred to as the
that Q. ilex and A. unedo hosted mutually compatible mycor- pure forest). One other area (~6000 m2), consisting of A. menziesii individuals
rhizal fungal species. intermixed with other ECM hosts, was also identified (hereafter referred to as the
Mycorrhizal fungal links between arbutoid and ECM hosts mixed forest). In the mixed forest, P. menziesii and Pinus ponderosa were equally
dominant, with a minimal presence of Quercus kelloggii (>5%). The areas were
may be particularly important in regard to ecosystem response separated by ~0.5 km. The age of A. menziesii individuals in both areas was simi-
to fire, which is the dominant disturbance regime in many parts lar, although all individuals in the pure forest had multiple stems due to resprout-
of the western United States and the Mediterranean (Halofsky ing after the 1959 fire (M. Main, Small Woodland Services, Inc., personal
et al., 2011; Silva et al., 2011). Unlike Pinaceae ECM hosts, communication). Soils in both forests were similar, consisting of decomposed
Arbutus and Arctostaphylos species have the ability to resprout granitic soil types with depth to bedrock of ~1 m (Badura and Jahn, 1977).
directly following fire and other disturbances. Their persistent In early January 2011, one ~900-m2 site in the pure forest and one ~3000-m2
site in the mixed forest were sampled. A second site of equal size in each forest
root systems and resprouting stems can provide a means to sus- located >75 m from the first sample site was also sampled in early June 2011
tain “a reservoir of ectomycorrhizal fungi, thereby maintaining (N = 4). Although the two sites within each forest were spatially separated from
[the] diversity and activity of the fungi needed for establish- another by distances well beyond those observed for spatial autocorrelation in
ment of forest regeneration” (Molina and Trappe, 1982, p. 506). ECM fungal communities (Lilleskov et al., 2004; Peay et al., 2010), they are ef-
Indeed, Arbutus menziesii has been noted as a nurse tree for fectively within forest replicates rather than true replicates of pure and mixed
P. menziesii in southwestern Oregon (Molina and Trappe, 1982; forest. The two sampling times were chosen to correspond with significant differ-
ences in the environmental conditions present in the study area. At each site, 30
Amaranthus et al., 1990), and the ability of ericaceous plants A. menziesii individuals were randomly selected for sampling (N = 120). From
and members of the Pinaceae to participate in common myce- each tree, a 15 × 15 × 15 cm soil sample (hereafter referred to as a core) was col-
lial networks of ECM symbioses may have important ecologi- lected 0.75 m from the base of the trunk. Cores were bagged individually and
cal and management implications (Perry et al., 1989; Molina transported to the laboratory within 48 h of collection. Cores were searched for
et al., 1992). A. menziesii roots (typically much smaller than Pinaceae roots) for no more than
In this study, we sought to better understand the role of the 5 min to facilitate the processing of 60 cores per sampling period. Selected roots
were rinsed with tap water to remove adhering soil, cut into 5-cm fragments, and
ericaceous tree, A. menziesii, in the belowground dynamics inspected with a dissecting microscope at 10× magnification. The first eight my-
of mixed evergreen forests of the Klamath-Siskiyou region corrhizal root tips encountered per core were removed, with no morphotyping
of the western United States. Although A. menziesii is a typi- done on any samples. The first eight colonized root tips from Pinaceae hosts were
cal component of the midcanopy present in these diverse for- also removed from 15 randomly selected cores from the June mixed-forest samples.
ests, particularly on drier, nonserpentine soils (Whittaker, During the January sampling, six mixed-forest cores were misprocessed and
1960), no systematic field study of the mycorrhizal fungi as- therefore not included in the final analyses (final N = 114).
sociated with A. menziesii roots has been conducted. Given
the potential for ericaceous and Pinaceae hosts to share my- DNA extraction and amplification: Study 1—Total genomic DNA was ex-
tracted from colonized root tips using the REDExtract-N-Amp PCR kit (Sigma-
corrhizal fungi and the nurse tree effects as noted, we ex- Aldrich, St. Louis, Missouri, USA). Sampled root tips were individually placed
pected the mycorrhizal fungal communities associated with directly into 10 µL of extraction solution and heated at 65°C for 10 min and
A. menziesii would have many fungal taxa in common with 95°C for 10 min. Following that incubation, 30 µL of neutralization solution was
other mycorrhizal host species. We also took advantage of a added to each sample, which was then stored at −20°C. The internal transcribed
unique study area to examine the effect of forest composi- spacer (ITS) region was amplified from root tip samples using the primer pair
tion on the A. menziesii-associated mycorrhizal communi- ITS1F/ITS4 (Gardes and Bruns, 1993) and a 54°C annealing temperature (see
Gardes and Bruns, 1996 for additional details about PCR conditions). PCR suc-
ties. A major fire burned through one of the two study areas cess was assessed by electrophoresis on 1.5% agarose gels. Samples yielding
in 1959, creating patches of monodominant A. menziesii in- multiple amplicons were reamplified using the phylum-specific primer pairs
termixed with patches where A. menziesii remained present ITS1F/ITS4B (Gardes and Bruns, 1993) or ITS1F/ITS4A (Larena et al., 1999).
along with other ECM hosts. We addressed three research All amplicons with single products were purified using ExoSap-IT (USB Corp.,
questions: (1) What is the composition and diversity of my- Cleveland, Ohio, USA) and sequenced in either the forward or both directions at
corrhizal fungi on colonized root tips of A. menziesii? (2) Is the University of Arizona GATC sequencing facility (N = 909).
the mycorrhizal fungal community associated with A. men-
Sequence analysis and mycorrhizal taxon identification: Study 1—Se-
ziesii influenced by forest type or season of sampling? (3) quence chromatograms were visually assessed and edited using the program
Is there potential for common mycelial networks between Sequencher v4.10 (Gene Codes, Ann Arbor, Michigan, USA). All samples
A. menziesii and Pinaceae hosts, particularly P. menziesii, in were assembled at ≥97% sequence similarity, a cut-off widely used to designate
mixed forest settings? ECM operational taxonomic units (OTUs) at the species level (Peay et al., 2008).
 15372197, 2012, 10, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.3732/ajb.1200277 by Cochrane Mexico, Wiley Online Library on [21/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
October 2012] KENNEDY ET AL.—MYCORRHIZAL COMMUNITIES AND MIXED EVERGREEN FORESTS 1693

To identify the mycorrhizal fungal taxa present, representative sequences from the remaining eight were harvested on 22 May. Following harvesting, the sam-
each unique contig were assessed using the ITS pipeline program (Nilsson ples were transported to the laboratory and stored at 4°C during processing. All
et al., 2009) and the UNITE database (Koljalg et al., 2005) (Appendix S2, see mycorrhizal root tips collected were morphotyped separately for each soil sam-
online Supplemental Data). When either or both databases gave query matches ple and host species. Morphotypes from each host were grouped by soil sample
≥97%, taxa were named in accordance with the most informative match at the with no attempt made to match morphotypes across soil samples. Arbutus men-
genus level or above (e.g., Tuber sp. 1). When both databases gave query ziesii and P. menziesii mycorrhizal root tips were relatively easy to identify
matches ≤97%, taxa were named at the lowest level shared between the two based, respectively, on their small, tripartite arbutoid and larger pinnate mor-
databases (e.g., Sphaerosporella [UNITE] vs. Genebea [GenBank] was desig- photypes. The host genus from any individual, unbranched root tips that were
nated at Pyronemataceae sp. 1). For some samples, the top GenBank match was not easy to categorize was analyzed with molecular techniques (see below). All
not well defined taxonomically (e.g., uncultured soil fungus); in these cases, the root tips were sorted, morphotyped, and lyophilized within 3 wk of harvest.
query taxon was named based on more specifically defined samples within the
top 15 GenBank matches. A representative sequence from each mycorrhizal Molecular identification of fungi and plants: Study 2—DNA was extracted
taxon (including those present on conifers only) is present in GenBank as from one to three root tips per sample as described by Gardes and Bruns (1996).
accessions JQ393029–JQ393154. Dimethyl sulfoxide (DMSO, 5% v/v) was added, and an annealing temperature
of 53°C was used for all PCR amplifications. Several root tips were extracted
Host species identification: Study 1—Due to the presence of additional together only when they were part of a single morphologically uniform cluster.
mycorrhizal hosts at the mixed forest sites, molecular host analyses were con- When available, at least two samples from each morphotype were processed.
ducted to confirm root identity. For all mycorrhizal root samples containing DNA was also extracted from small pieces of voucher leaf material by the same
OTUs that were unique to the mixed forest, the plant plastid trnL cpDNA re- method. The PCR reagents, protocols, and cycling parameters followed those
gion was amplified from root tips using the trnL_c-trnL_d primer pair (Taberlet of Gardes and Bruns (1996). Mycorrhizal fungi were identified on PCR ampli-
et al., 1991) under conditions previously described (Kennedy et al., 2011). To fication using ITS1F/ITS4B or ITS1F/ITS4 as primer pairs. If a DNA extrac-
confirm that A. menzeisii was the only mycorrhizal host in the pure forest, a tion for a sample did not yield a clean PCR product, a second extraction from
subset of root samples from the pure forest sites was also checked. Successful the sample was processed. Extraction was repeated until two clean PCR prod-
PCR products were digested with the restriction enzyme DpnII, and the result- ucts were obtained for each morphotype from each core or until the morphotype
ing restriction fragment length polymorphism (RFLP) banding patterns were sample was used up in processing. We used the restriction enzymes AluI, DpnII,
scored by eye on 1.5% agarose gels. Banding patterns clearly distinguished and HinfI to generate RFLP banding patterns with a 100-bp ladder as a refer-
A. menziesii, P. menziesii, and P. pondersosa. ence (New England Biolabs, Ipswich, Massachusetts, USA). Samples yielding
the same banding pattern across all three enzymes were considered a unique
species (an RFLP type) (Kårén et al., 1997). Amplified and restricted DNA
Statistical and phylogenetic analyses: Study 1—The program EstimateS products were run in 3% agarose gels, stained with ethidium bromide, and digi-
(Colwell, 2005) was used to assess mycorrhizal fungal community diversity. tally photographed for storage (UVP Laboratory Products, Upland, California,
Because mycorrhizal fungi can differentially influence local root tip abundance, USA). Digital copies of the gels were analyzed by eye to group similar RFLP
analyses were based on soil core presence rather than root tip abundance within types together. RFLP patterns from a single enzyme that appeared similar from
cores. Minimum estimates of fungal taxa richness were obtained using the different gels were run again in adjacent lanes for better matching comparisons.
Chao2 estimator, and taxa diversity was calculated using the Shannon–Wiener To avoid overrepresenting richness, RFLPs were generated with PCR product
index. To account for unequal final sample sizes, we also calculated estimates from ITS1F/ITS4 reactions for all types that yielded RFLPs with ITS1F/ ITS4B,
of richness and diversity using rarefaction to 21 cores (the lowest number of and checked against the ITS1F/ITS4B RFLP data. A representative sample of
samples across forests and seasons). The statistical effects of forest type and each unique RFLP type was reamplified for sequencing and processed in the
season on mycorrhizal assemblage structure were examined using a two-way same way as study 1. Host plant identification of all root tips was also con-
ANOSIM in the program Primer v5 (Clarke and Gorley, 2001). Bray–Curtis firmed using the methods described. In most cases, the original plant identifica-
similarity values were calculated from a square-root transformed core by taxon tion was confirmed. A few exceptions occurred where a P. menziesii root tip
matrix. A similar comparison was made using a MRPP analysis under the was actually P. ponderosa.
default settings in the program PC-ORD v6 (McCune and Mefford, 2011). For
both the ANOSIM and MRRP analyses, the data were analyzed twice, once
with all data included, and once with all singletons and doubletons removed. To Statistical analysis: Study 2—To examine whether multi-host mycorrhizal
determine whether fungal taxa were significantly associated with forest type or fungal taxa (i.e., those found on both A. menziesii and Pinaceae hosts) were
season, indicator species analyses were conducted in PC-ORD. more frequent or abundant than single-host fungal taxa, two Kruskal–Wallis
As noted already, the sampling design of this study does not have true replica- tests were conducted. Taxon frequency was assessed using number of cores;
tion of forest type level. We have chosen to analyze the data in a way that allows taxon abundance was assessed using number of root tips. Both tests were run in
us to examine differences between the two types of forests (by using the sampling the program SPSS v19 (IBM, Armonk, New York, USA) and considered sig-
in each forest at each time as replicates), but we stress that the results presented nificant at P < 0.05.
should be interpreted cautiously. In particular, while the close spatial proximity of
the two forest types may limit differences based on environmental variation, it is
not possible to inferentially separate effects of forest type (i.e., pure vs. mixed) RESULTS
from that of other factors that may have varied between the two locations.
The effect of host species on mycorrhizal fungal taxa relatedness was exam- Study 1— Mycorrhizal root tips were present in 113 of the
ined by combining 11 Piloderma ITS sequences from A. menziesii root samples 114 cores analyzed. Of the total root tips sampled from all
in this study with 27 Piloderma sequences collected from the GenBank data- cores, 88% (909/1032) were sequenced, and 60% of these
base from a diverse range of host species and sampling locations. All sequences
were aligned with the program MUSCLE (Edgar 2004) and spot-checked by
(545/909) yielded a clean sequence >350 bp with at least one
eye in the program MacClade v4 (Maddison and Maddison, 1999). Nonparsi- primer pair. In all, 109 cores yielded at least one clean sequence.
moniously informative sequences ends were trimmed, and a Bayesian analysis Of the 537 root tips with mycorrhizal sequences, 495 (92%)
was run in Mr. Bayes v3 (Huelsenbeck and Ronquist, 2001) for five million were present on A. menziesii and 42 (8%) on Pinaceae roots.
generations (burn-in value = 5000). Sequencing success varied somewhat by forest type, with the
mixed and pure forests yielding an average of 3.7 and 4.9 se-
Site location and sampling: Study 2—Study 2 was conducted in a mixed quences/core, respectively (combined average = 4.4/core).
evergreen forest in Applegate Valley, Oregon, ~45 km northwest of site 1 Identification of mycorrhizal host by trnL plastid amplification
(42°19′00″N, 123°14′00″W, 390 m a.s.l.). In May 1998, 23 mature A. menziesii was successful for 82% (178/218) of samples from the mixed
individuals were selected nonrandomly for sampling based on abundant
P. menziesii saplings (~1–1.5 m tall) beneath their crowns and a lack of other
forest. Nine putatively A. menziesii samples were found to be
ECM hosts in the immediate vicinity. One soil sample was removed near each from Pinaceae roots and removed from the A. menziesii-only
A. menziesii individual among the P. menziesii saplings using a 5-cm diameter analyses. All of the 54 samples checked from the pure forest
soil corer to a depth of 15 cm. Fifteen samples were harvested on 7 May, and were confirmed to belong to A. menziesii.
 15372197, 2012, 10, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.3732/ajb.1200277 by Cochrane Mexico, Wiley Online Library on [21/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1694 AMERICAN JOURNAL OF BOTANY [Vol. 99

A total of 126 unique mycorrhizal fungal taxa were encoun- single host (Fig. 3). Additionally, from the 26 taxa present on
tered, with 103 present on A. menziesii exclusively, 17 on both multiple hosts, 50% (13) occurred on both A. menziesii and a
A. menziesii and Pineaceae hosts, and six present exclusively on Pinaceae host within at least one soil core.
Pinaceae hosts. Members of the genera Cenococcum, Piloderma,
Wilcoxina, and the family Corticiaceae were the five most fre-
quently occurring fungal taxa, but the majority of taxa (82/120, DISCUSSION
68%) were present in only one or two cores (Fig. 1A). While
individual taxa were typically rare, many mycorrhizal fungal We found that A. menziesii hosts a diverse mycorrhizal
lineages were represented by multiple taxa. For example, fungal community. The high fungal taxa richness observed in
Inocybe, Cortinarius, Piloderma, Tomentella, Thelephoraceae, both studies, as well as the inability to saturate the taxa accumu-
Sebacina, and Russula all had at least five taxa represented lation curves, is similar to that previously documented on a
(Fig. 1B). Uncorrected and rarified Chao 2 taxa richness esti- range of angiosperm (Ishida et al., 2007; Morris et al., 2008;
mates were notably higher than observed richness at each sam- Bahram et al., 2011) and Pinaceae ECM hosts (Horton and
pling and for the entire data set, indicating many more fungal Bruns, 2001; Lilleskov et al., 2004; Cox et al., 2010). Along
taxa were likely to be identified with additional sampling effort with parallels in taxa richness, there was also considerable
(Table 1). Mycorrhizal fungal richness and diversity varied overlap in the major lineages of mycorrhizal fungi on A. men-
between the four samples, being generally higher in the pure ziesii and other ECM hosts. For example, the high representa-
forest and at the January sampling (Table 1). tion of taxa in the genera Cenococcum, Wilcoxina, Piloderma,
Forest type (i.e., pure vs. mixed) and season (i.e., January vs. Thelephora, Tomentella, Inocybe, and Cortinarius in both the
June) both had significant effects on the structure of mycor- pure and mixed forest of study 1 indicates that the mycorrhizal
rhizal fungal communities of A. menziesii, though with varying fungal community associated with A. menziesii has a similar
levels of significance. ANOSIM and MRPP analyses indicated phylogenetic structure to those of co-occurring Pinaceae hosts
that the difference between the pure and mixed forest (ANO- (Horton and Bruns, 1998; Cline et al., 2005; Dunham et al.,
SIM R = 0.034, P = 0.012; MRPP A = 0.002, P = 0.026) was 2007). At the same time, the presence of hypogeous fruiting
greater than by season (ANOSIM R = 0.027, P = 0.026; MRPP genera such as Gilkeya, Genea, Genebea, and Tuber is similar
A = 0.001, P = 0.071). The significant effects of both variables to that seen in western North American Quercus and Cercocar-
remained present when singletons and doubletons were ex- pus ECM communities (Smith et al., 2007; Morris et al., 2008;
cluded from the analyses (data not shown). Indicator species McDonald et al., 2010). Taken together, these data suggest that
analysis showed that two taxa, Helotiales 1 and Gilkeya 1, were A. menziesii acts as a generalist host to ECM fungi commonly
significantly indicative of the mixed forest (both P = 0.02), but associated with both Pinaceae and angiosperm host species. As
no taxa were significantly indicative of the pure forest (online such, A. menziesii likely plays an important role in maintaining
Appendix S3). With regard to season, Piloderma 3 was signifi- a diverse mycorhizal fungal community in the mixed evergreen
cantly indicative of the January communities (P = 0.02), but no forests of western North America.
taxa were significant in the June communities. The ANOSIM and MRPP analyses indicated that the mycor-
Several mycorrhizal fungal taxa were present on both rhizal fungal communities associated with A. menziesii were sig-
A. menziesii and Pinaceae hosts (Table 2). Across the full data set nificantly affected by both season and forest type. These effects
(i.e., the four sites sampled), 17 taxa were present on A. men- were seen on analyses of both the whole data set and with single-
ziesii and Pinaceae hosts, and six of these taxa were present on tons and doubletons removed, indicating these patterns were not
both types of hosts within the same core. A phylogenetic analy- driven primarily by differences among rare taxa. Given the ef-
sis of the genus Piloderma, one of the more diverse and abun- fects observed in the community-level analyses, it was surprising
dant genera encountered, showed little support for an influence that only a few individual fungal taxa were significantly affected
of host species on phylogenetic relatedness among taxa (Fig. 2). by either factor. Apparently smaller but cumulative differences in
Specifically, the Piloderma taxa associated with A. menziesii taxon abundance between samples were responsible for the ob-
were typically closely related to congeneric taxa found on served shifts in mycorrhizal fungal community structure. Similar
different host plants and at geographically distant sampling temporal differences in ECM fungal abundance have been ob-
locations. served in other studies of both Pinaceae (Koide et al., 2007) and
angiosperm hosts (Malajczuk and Hingston, 1981). Although the
Study 2— In the 23 cores, 86 total mycorrhizal fungal taxa causes of shifts in mycorrhizal fungal community structure over
were found. Clean sequences were obtained for 43 taxa, while intraannual time scales are not fully understood, they likely relate
43 were designated as unique RFLP types only. In general, to differing tolerances of individual ECM fungi to a range of en-
there was high overlap in the lineages of mycorrhizal fungal vironmental conditions (Erland and Taylor, 2002). In our study
taxa present in this study and those encountered in study 1 system, the strongly seasonal climate suggests differences in
(Table 2). For example, a member of the genus Cenococcum moisture and temperature levels are likely to be particularly im-
was the most frequent encountered taxon, and Inocybe was the portant driving factors.
most diverse genus in both studies. Rare taxa were also common, In addition to being temporally dynamic, the mycorrhizal
with half of the 86 taxa represented by a single root tip sample. fungal community structure of A. menziesii also varied spatially.
Of the taxa present on two or more root tips, 58% (25) were While spatial differences have been correlated with a wide
encountered on both A. menziesii and P. menziesii, 21% (9) on range of abiotic factors (Erland and Taylor, 2002), our data sug-
P. menziesii only, 19% (8) on A. menziesii only, 2% (1) on both gest that the presence or absence of other ECM hosts may be an
A. menziesii and P. ponderosa. Kruskal–Wallis tests revealed equally important explanatory variable. Overall fungal lineage
that mycorrhizal fungal taxa present on multiple hosts were sig- diversity was similar in the pure and mixed forest, but there were
nificantly more frequent (χ2 = 7.51, df = 1, P = 0.006) and abun- notable shifts in abundances within specific taxonomic groups
dant (χ2 = 4.90, df = 1, P = 0.027) than taxa associated with a (online Appendix S4). For example, the mycorrhizal fungal
 15372197, 2012, 10, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.3732/ajb.1200277 by Cochrane Mexico, Wiley Online Library on [21/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
October 2012] KENNEDY ET AL.—MYCORRHIZAL COMMUNITIES AND MIXED EVERGREEN FORESTS 1695

Fig. 1. (A) Rank–frequency plot of the 20 most frequently occurring mycorrhizal fungal taxa associated with Arbutus menziesii in study 1 (total num-
ber of cores analyzed = 114). Inset: Rank–frequency of all 120 mycorrhizal fungal taxa associated with A. menziesii. (B) Rank–abundance plot of the
number of individual taxa within each mycorrhizal fungal lineage identified in study 1.

communities in the pure forest had many more unique Corti- examined by Massicotte et al. (1994) using fungal spore inocu-
narius, Piloderma, and Tomentella/Thelephoraceae taxa than in lations. The latter study found that a number of ECM fungal
the mixed forest (8 vs. 4, 4 vs. 1, and 9 vs. 4, respectively). In species failed to form ectomycorrhizas after inoculation of cer-
contrast, the number of unique Inocybe taxa in the mixed forest tain host species when those host species were grown alone in a
was four times higher than in the pure forest (8 vs. 2). The im- pot; however, if another compatible host species was also grown
portance of neighboring plants on mycorrhizal composition in the same pot with the incompatible host, then the fungus formed
was highlighted by Molina et al. (1992) and experimentally ectomycorrhizas with both hosts. Those authors hypothesized
 15372197, 2012, 10, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.3732/ajb.1200277 by Cochrane Mexico, Wiley Online Library on [21/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1696 AMERICAN JOURNAL OF BOTANY [Vol. 99

TABLE 1. Taxa richness and diversity of mycorrhizal fungal communities associated with Arbutus menziesii in study 1. Taxa estimated and diversity
calculated the using Chao2 and Shannon–Wiener indices in EstimateS. Rarefaction values based on 21 samples are indicated parentheses.

Forest type Pure forest Mixed forest Total

Season January June January June

Samples 30 30 21 28 109
Taxa observed 55 49 39 44 120
Taxa estimated 127 (93) 84 (80) 94 72 (72) 209
Taxa diversity 3.78 (3.61) 3.68 (3.51) 3.53 3.58 (3.43) 4.34

that neighborhood effects were driven by host-specific spore A. menziesii roots in the pure forest, both of which belonged to
germination triggered by the compatible host followed by sec- the same Rhizopogon lineages experimentally examined by
ondary host colonization once ECM fungi are growing in the Molina et al. (1997) (our present study’s Rhizopogon sp. 1 =
mycelial stage. Recent support for this kind of neighborhood R. salebrosus and Rhizopogon sp. 2 = an unidentified taxon in
effect was also observed by Hubert and Gehring (2008) and the subsection Rhizopogon [Rusca et al., 2006], which is the
Bahram et al. (2011), who both found evidence of changes in same subsection as R. occidentalis). In light of these results, it
ECM community structure between areas where the root sys- seems likely that prior to the 1959 fire, those two Rhizopogon
tems of co-occurring ECM host species overlapped compared taxa germinated by spore with Pinaceae hosts and subsequently
to where present alone. colonized A. menziesii via mycelium. After the Pinaceae hosts
Our data on the genus Rhizopogon provides further insight were removed by the disturbance, A. menziesii appears to have
into the specificity patterns of A. menziesii mycorrhizal com- maintained associations with both taxa for over 50 yr. Addi-
munities. Molina et al. (1997) found that R. occidentalis, R. el- tional evidence for the persistence of Rhizopogon with A. men-
lenae, and R. subcaerulescens (= R. salebrosus) did not colonize ziesii was the observation of a Pterospora andromeda individual
A. menziesii from spores when it was the only host species pres- in the pure forest (P. Kennedy and D. Smith, personal observa-
ent, but did colonize A. menziesii secondarily when grown in tions). Pterospora andromeda is a mycoheterotrophic plant
the same pot with Pinus ponderosa. From that result, the au- known only to associate with Rhizopogon species in the subsec-
thors questioned whether A. menziesii would maintain an asso- tion Amylopogon, of which R. salebrosus is a member (Bidart-
ciation with Rhizopogon species when other hosts were not ondo and Bruns 2001).
present. In our study, we found two Rhizopogon species on Interestingly, Molina and Trappe (1982) found that the
P. menziesii-specific Rhizopogon species, R. vinicolor, was able
TABLE 2. Mycorrhizal fungal taxa present on Arbutus menziesii and to successfully colonize A. menziesii in a pure culture synthesis
Pinaceae hosts in both studies. Note the taxon naming for each study experiment. In a follow up to that study, however, when inocu-
was done separately, so, for example, Cenococcum 1 in study 1 is not lum was applied via spores, A. menziesii did not host R. vini-
necessarily the identical ITS genotype of Cenococcum 1 in study 2. color, either alone or when grown in the same pots with
Study 1 Study 2 P. menziesii (Molina et al., 1997). Our data from study 2 cor-
roborate this latter result. We found that R. villosulus, another
On both hosts: On both hosts:
On both hosts same core On both hosts same core
P. menziesii-specific species, was present on P. menziesii, but
not on A. menziesii. Unlike the pure culture synthesis results,
Cenococcum 1 Cenococcum 1 Atheliaceae 1 Atheliaceae 1 our findings suggest that P. menziesii-specific Rhizopogon species
Cortinarius 11 Cortinarius 8 Cenococcum 1 Cenococcum 1 are in fact host-specific under natural conditions (Molina and
Cortinarius 8 Genabea 1 Clavulinaceae 1 Continarius 2 Trappe, 1994). As noted by Duddridge (1986), the presence of
Gauitieria 1 Gilkeya 1 Continarius 2 Inocybe 02
Genabea 1 Hygrophorus 1 Cortinarius 4 Inocybe 03
glucose in the pure culture synthesis method may have allowed
Gilkeya 1 Russula 1 Inocybe 01 Piloderma 2 P. menziesii-specific Rhizopogon species to colonize A. menziesii
Hebeloma 1 Inocybe 02 Thelephoraceae 2 under laboratory conditions. Collectively, these data suggest
Hebeloma 2 Inocybe 03 Tomentella 2 the generalist nature of A. menziesii mycorrhizal communities
Hygrophorus 1 Inocybe 05 Unkn. RFLP 01 represents a gradient that includes (1) many species that can
Hygrophorus 2 Piloderma 1 Unkn. RFLP 05 germinate from spores with A. menziesii (Massicotte et al., 1999),
Inocybe 11 Piloderma 2 Unkn.RFLP 08 (2) some species that can colonize A. menziesii via mycelium
Piloderma 3 Thelephoraceae 1 Unkn. RFLP 19
Russula 1 Thelephoraceae 2 Unkn. RFLP 26 but need a different host to induce spore germination (Molina
Sebacina 3 Thelephoraceae 4 et al., 1997), and (3) few species that do not associate with
Tomentella 3 Thelephoraceae 5 A. menziesii at either the spore or mycelial stages (Molina et al.,
Tomentella 8 Tomentella 1 1997).
Tricholoma 3 Tomentella 2 Both of our studies indicated that a diverse array of mycor-
Unkn. RFLP 01 rhizal fungi associate with A. menziesii and co-occurring
Unkn. RFLP 05
Unkn. RFLP 06 Pinaceae hosts. In study 2, which was specifically designed to
Unkn. RFLP 08 examine host specificity, we found that multi-host mycorrhizal
Unkn. RFLP 19 fungal taxa were nearly twice as abundant as single-host fungal
Unkn. RFLP 26 taxa. This level is comparable to other studies that have also
Unkn. RFLP 27 encountered a high abundance of multi-host fungal taxa in mixed
Unkn. RFLP 30 species forests (Horton and Bruns, 1998; Kennedy et al., 2003;
Unkn. RFLP 32
Ishida et al., 2007; Tedersoo et al., 2008; but see Smith et al., 2009).
Note: Unkn. = unknown. Although multi-host fungal taxa were less abundant in study 1,
 15372197, 2012, 10, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.3732/ajb.1200277 by Cochrane Mexico, Wiley Online Library on [21/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
October 2012] KENNEDY ET AL.—MYCORRHIZAL COMMUNITIES AND MIXED EVERGREEN FORESTS 1697

Fig. 2. A Bayesian phylogenetic reconstruction of 27 mycorrhizal fungal taxa from the genus Piloderma based on ITS rDNA sequences. Nodes are
labeled with posterior probabilities. Piloderma taxa from study 1 that are associated with Arbutus menziesii are in boldface; see the methods for details
about the selection of the other 16 taxa. Each taxon is labeled with mycorrhizal host, geographic location, and GenBank accession number in parentheses.
In two cases, sequences were obtained from fruit bodies instead of mycorrhizal root tips. A mycorrhizal root sample belonging to the family Atheliaceae
from Pinus densiflora was used as the outgroup.

the smaller sample size and high numbers of singletons and provide support for earlier work in mixed evergreen forests in-
doubletons suggest that more sampling may have revealed ad- dicating that A. menziesii can act as an important source of my-
ditional fungal taxa that colonized both A. menziesii and co- corrhizal inoculum for later-seral species (Amaranthus and Perry,
occurring Pinaceae hosts. These results of low specificity 1989; Molina et al., 1992). For example, Borchers and Perry
 15372197, 2012, 10, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.3732/ajb.1200277 by Cochrane Mexico, Wiley Online Library on [21/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1698 AMERICAN JOURNAL OF BOTANY [Vol. 99

Fig. 3. (A) Rank–frequency and (B) rank–abundance plots of multi-host and single-host mycorrhizal fungal taxa in study 2. Multiple-host taxa are
defined as those taxa found on both host species and single-host taxa as those found on only one of the two host species. The ordering of mycorrhizal fungal
taxa with identical abundances or occurrences is represented randomly.

(1990) showed that ECM fungal diversity on P. menziesii seed- considerably among species and locations (Dahlberg and Sten-
lings was enhanced when planted in soils collected under lid, 1994; Guidot et al., 2001; Redecker et al., 2001; Kretzer
A. menziesii relative to under other ECM host species or from et al., 2004; Beiler et al., 2010), most are larger than the diam-
recent forest clearings. eter of our soil cores, which suggests that, within each core, we
Our data also indicate that A. menziesii and co-occurring co- sampled the same fungal genotype. These networks, which
nifers are highly likely to be linked into common mycelial net- benefit connected individuals by allowing for greater access to
works. In both studies, we found numerous examples of both nutrient and water resources (Simard et al., 1997; Teste and
types of hosts being colonized by the same fungal taxon within Simard, 2008; Booth and Hoeksema, 2010; Simard et al., 2012),
a soil core (Table 2). While the size of fungal genets varies along with the potential of resident fungal inoculum from already
 15372197, 2012, 10, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.3732/ajb.1200277 by Cochrane Mexico, Wiley Online Library on [21/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
October 2012] KENNEDY ET AL.—MYCORRHIZAL COMMUNITIES AND MIXED EVERGREEN FORESTS 1699

established mycorrhizas to initiate associations between ECM BOOTH, M. G. 2004. Mycorrhizal networks mediate overstorey–understo-
fungi and secondary hosts, suggest a direct belowground mech- rey competition in a temperate forest. Ecology Letters 7: 538–546.
anism for the preferential establishment of P. menziesii seed- BOOTH, M. G., AND J. D. HOEKSEMA. 2010. Mycorrhizal networks counter-
lings around A. menziesii following disturbance (Amaranthus et act competitive effects of canopy trees on seedling survival. Ecology
91: 2294–2302.
al., 1990). Similar mechanisms of facilitation have also been BORCHERS, S. L., AND D. A. PERRY. 1990. Growth and ectomycorrhiza for-
noted between members of the closely related genus Arcto- mation of Douglas-fir seedlings grown in soils collected at different
staphylos and conifers (Danielson, 1984; Hagerman et al., 2001; distances from pioneering hardwoods in southwest Oregon clear-cuts.
Visser, 1995; Horton et al., 1999). Canadian Journal of Forest Research-Revue Canadienne De Recherche
In conclusion, our data demonstrate that A. menziesii hosts a Forestiere 20: 712–721.
wide number of mycorrhizal fungal taxa, many of which are CLARKE, K. R., AND R. N. GORLEY. 2001. PRIMER v5: User manual/
shared with co-occurring host species. As such, it appears that tutorial. PRIMER-E, Plymouth, UK.
A. menziesii is best thought of as a significant hub rather than a CLINE, E., J. AMMIRATI, AND R. EDMONDS. 2005. Does proximity to mature
terminal node for mycorrhizal fungal diversity and connectivity trees influence ectomycorrhizal fungus communities of Douglas-fir
in mixed evergreen forests (Southworth et al., 2005). This posi- seedlings? New Phytologist 166: 993–1009.
COLWELL, R. 2005. EstimateS: Statistical estimation of species richness and
tion is particularly important in light of disturbances caused by
shared species from samples, version 7.5. User’s guide and application
fire, which are a frequent part of the landscape A. menziesii in- published at website http://purl.oclc.org/estimates.
habits, particularly in the Klamath-Siskiyou region (Halofsky COURTY, P. E., K. PRITSCH, M. SCHLOTER, A. HARTMANN, AND J.
et al., 2011). The ability of A. menziesii to resprout from surviving GARBAYE. 2005. Activity profiling of ectomycorrhiza communities in
root systems immediately after fire, likely maintains the mycor- two forest soils using multiple enzymatic tests. New Phytologist 167:
rhizal fungal inoculum to support later colonizing species in the 309–319.
family Pinaceae and thus plays a key role in maintaining the COX, F., N. BARSOUM, E. A. LILLESKOV, AND M. I. BIDARTONDO. 2010. Nitrogen
below- and aboveground diversity in these ecosystems (Molina availability is a primary determinant of conifer mycorrhizas across com-
and Trappe, 1982; Amaranthus and Perry, 1989). Future studies plex environmental gradients. Ecology Letters 13: 1103–1113.
with better replication at the forest-type level will help clarify CULLINGS, K. W., D. VOGLER, V. PARKER, AND S. FINLEY. 2000. Ectomy-
the role of neighborhood effects on mycorrhizal community corrhizal specificity patterns in a mixed Pinus contorta and Picea
engelmannii forest in Yellowstone National Park. Applied and Envir-
structure. In addition, examining the composition of the mycor- onmental Microbiology 66: 4988–4991.
rhizal communities on other Arbutus species will help broaden DAHLBERG, A., AND J. STENLID. 1994. Size, distribution and biomass of gen-
our understanding of the ecological role of arbutoid host spe- ets in populations of Suillus bovinus (L., Fr) Roussel revealed by so-
cies in mixed evergreen forests. Finally, examining the func- matic incompatibility. New Phytologist 128: 225–234.
tioning of multihost fungal taxa on A. menziesii and other hosts DANIELSON, R. M. 1984. Ectomycorrhizal associations in Jack pine stands in
using enzyme assays (Courty et al., 2005) and other methods northeastern Alberta. Canadian Journal of Botany-Revue Canadienne
will help illuminate how ECM host specificity patterns affect De Botanique 62: 932–939.
nutrient cycling in mixed forest settings. DUDDRIDGE, J. A. 1986. The development and ultrastructure of ectomycor-
rhizas. 4. Compatible and incompatible interactions between Suillus gre-
villei (Klotzsch) Sing. and a number of ectomycorrhizal hosts in vitro in
LITERATURE CITED the presence of exogenous carbohydrate. New Phytologist 103: 465–471.
DUNHAM, S. M., K. H. LARSSON, AND J. W. SPATAFORA. 2007. Species rich-
ACSAI, J., AND D. L. LARGENT. 1983. Mycorrhizae of Arbutus menziesii ness and community composition of mat-forming ectomycorrhizal
Pursh and Arctostaphylos manzanita Parry in Northern California. fungi in old- and second-growth Douglas-fir forests of the HJ Andrews
Mycotaxon 16: 519–536. Experimental Forest, Oregon, USA. Mycorrhiza 17: 633–645.
AMARANTHUS, M. P., R. J. MOLINA, AND D. A. PERRY. 1990. Soil organisms, EDGAR, R. 2004. MUSCLE: Multiple sequence alignment with high accu-
root growth, and forest regeneration. In Forestry on the Frontier— racy and high throughput. Nucleic acids research 32: 1792–1797.
Proceedings of the National Society of American Foresters National ERLAND, S., AND A. F. S. TAYLOR. 2002. Diversity of ecto-mycorrhizal fun-
Convention, 89–93, 1989, Society of American Foresters, Spokane, gal communities in relation to the abiotic environment. In M. G. A. van
Washington. der Heijden and I. R. Sanders [eds.], Mycorrhizal ecology, 163–200.
AMARANTHUS, M. P., AND D. A. PERRY. 1989. Interaction effects of vegeta- Springer, Berlin, Germany.
tion type and Pacific madrone soil inocula on survival, growth, and GARDES, M., AND T. D. BRUNS. 1993. ITS Primers with enhanced specific-
mycorrhiza formation of Douglas-fir. Canadian Journal of Forest ity for Basidiomycetes—Application to the identification of mycor-
Research-Revue Canadienne De Recherche Forestiere 19: 550–556. rhizae and rusts. Molecular Ecology 2: 113–118.
AMARANTHUS, M. P., AND D. A. PERRY. 1994. The functioning of ectomy- GARDES, M., AND T. D. BRUNS. 1996. Community structure of ectomyc-
corrhizal fungi in the field: Linkages in space and time. Plant and Soil orrhizal fungi in a Pinus muricata forest: Above- and below-ground
159: 133–140. views. Canadian Journal of Botany-Revue Canadienne De Botanique
BADURA, G., AND P. JAHN. 1977. Soil resource inventory for the Rogue River 74: 1572–1583.
National Forest. U.S. Forest Service, Portland, Oregon, USA. GUIDOT, A., J. C. DEBAUD, AND R. MARMEISSE. 2001. Correspondence
BAHRAM, M., S. POLME, U. KOLJALG, AND L. TEDERSOO. 2011. A single between genet diversity and spatial distribution of above- and be-
European aspen (Populus tremula) tree individual may potentially har- low-ground populations of the ectomycorrhizal fungus Hebeloma cy-
bour dozens of Cenococcum geophilum ITS genotypes and hundreds lindrosporum. Molecular Ecology 10: 1121–1131.
of species of ectomycorrhizal fungi. FEMS Microbiology Ecology 75: HAGERMAN, S., S. SAKAKIBARA, AND D. M. DURALL. 2001. The potential
313–320. for woody understory plants to provide refuge for ectomycorrhizal inocu-
BEILER, K. J., D. M. DURALL, S. W. SIMARD, S. A. MAXWELL, AND A. M. lum at an interior Douglas-fir forest after clear-cut logging. Canadian
KRETZER. 2010. Architecture of the wood-wide web: Rhizopogon Journal of Forest Research-Revue Canadienne De Recherche
spp. genets link multiple Douglas-fir cohorts. New Phytologist 185: Forestiere 31: 711–721.
543–553. HALOFSKY, J. E., D. C. DONATO, D. E. HIBBS, J. L. CAMPBELL, M. D. CANNON, J.
BIDARTONDO, M. I., AND T. D. BRUNS. 2001. Extreme specificity in epipara- B. FONTAINE, J. R. THOMPSON, ET AL. 2011. Mixed-severity fire regimes:
sitic Monotropoideae (Ericaceae): Widespread phylogenetic and geo- Lessons and hypotheses from the Klamath-Siskiyou region. Ecosphere
graphical structure. Molecular Ecology 10: 2285–2295. 2: Article 40.
 15372197, 2012, 10, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.3732/ajb.1200277 by Cochrane Mexico, Wiley Online Library on [21/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1700 AMERICAN JOURNAL OF BOTANY [Vol. 99

HARLEY, J. L. 1969. Biology of mycorrhiza. Lean, London, UK. MCCUNE, B., AND M. MEFFORD. 2011. PC-ORD. Multivariate analysis of eco-
HORTON, T. R., AND T. D. BRUNS. 1998. Multiple-host fungi are the most logical data, version 6. MJM Software, Gleneden Beach, Oregon, USA.
frequent and abundant ectomycorrhizal types in a mixed stand of MCDONALD, K. R., J. PENNELL, J. L. FRANK, AND D. SOUTHWORTH. 2010.
Douglas fir (Pseudotsuga menziesii) and bishop pine (Pinus muri- Ectomycorrhizas of Cercocarpus ledifolius (Rosaceae). American
cata). New Phytologist 139: 331–339. Journal of Botany 97: 1867–1872.
HORTON, T. R., AND T. D. BRUNS. 2001. The molecular revolution in ecto- MOLINA, R. J., H. B. MASSICOTTE, AND J. M. TRAPPE. 1992. Specificity
mycorrhizal ecology: Peeking into the black-box. Molecular Ecology phenomena in mycorrhizal symbioses: Community-ecological conse-
10: 1855–1871. quences and practical applications. In M. F. Allen [ed.], Mycorrhizal
HORTON, T. R., T. D. BRUNS, AND V. PARKER. 1999. Ectomycorrhizal fungi functioning, an integrative plant–fungal process, 357–423. Chapman
associated with Arctostaphylos contribute to Pseudotsuga menziesii and Hall, New York, New York, USA.
establishment. Canadian Journal of Botany-Revue Canadienne De MOLINA, R. J., J. E. SMITH, D. MCKAY, AND L. MELVILLE. 1997. Biology of the
Botanique 77: 93–102. ectomycorrhizal genus, Rhizopogon 3. Influence of co-cultured conifer
HUBERT, N. A., AND C. A. GEHRING. 2008. Neighboring trees affect ec- species on mycorrhizal specificity with the arbutoid hosts Arctostaphylos
tomycorrhizal fungal community composition in a woodland-forest uva-ursi and Arbutus menziesii. New Phytologist 137: 519–528.
ecotone. Mycorrhiza 18: 363–374. MOLINA, R. J., AND J. M. TRAPPE. 1982. Lack of mycorrhizal specificity by
HUELSENBECK, J., AND F. RONQUIST. 2001. MRBAYES: Bayesian inference the ericaceous hosts Arbutus menziesii and Arctostaphylos uva-ursi.
of phylogenetic trees. Bioinformatics 17: 754–755. New Phytologist 90: 495–509.
ISHIDA, T. A., K. NARA, AND T. HOGETSU. 2007. Host effects on ectomycor- MOLINA, R. J., AND J. M. TRAPPE. 1994. Biology of the ectomycorrhizal
rhizal fungal communities: Insight from eight host species in mixed genus, Rhizopogon 1. Host associations, host-specificity and pure cul-
conifer–broadleaf forests. New Phytologist 174: 430–440. ture syntheses. New Phytologist 126: 653–675.
KAREN, O., N. HOGBERG, A. DAHLBERG, L. JONSSON, AND J. NYLUND. 1997. MORRIS, M. H., M. E. SMITH, D. M. RIZZO, M. REJMANEK, AND C. S. BLEDSOE.
Inter- and intraspecific variation in the ITS region of rDNA of ec- 2008. Contrasting ectomycorrhizal fungal communities on the roots
tomycorrhizal fungi in Fennoscandia as detected by endonuclease of co-occurring oaks (Quercus spp.) in a California woodland. New
analysis. New Phytologist 136: 313–325. Phytologist 178: 167–176.
KENNEDY, P. G., R. GARIBAY-ORIJEL, L. M. HIGGINS, AND R. ANGELES- NARA, K., AND T. HOGETSU. 2004. Ectomycorrhizal fungi on established
ARGUIZ. 2011. Ectomycorrhizal fungi in Mexican Alnus forests sup- shrubs facilitate subsequent seedling establishment of successional
port the host co-migration hypothesis and continental-scale patterns in plant species. Ecology 85: 1700–1707.
phylogeography. Mycorrhiza 21: 559–568. NILSSON, R. H., G. BOK, M. RYBERG, E. KRISTIANSSON, AND N.
KENNEDY, P. G., A. D. IZZO, AND T. D. BRUNS. 2003. There is high po- HALLENBERG. 2009. A software pipeline for processing and iden-
tential for the formation of common mycorrhizal networks between tification of fungal ITS sequences. Source Code for Biology and
understorey and canopy trees in a mixed evergreen forest. Journal of Medicine 4: 1–6.
Ecology 91: 1071–1080. PEAY, K. G., P. G. KENNEDY, AND T. D. BRUNS. 2008. Fungal commu-
KOIDE, R. T., D. L. SHUMWAY, B. XU, AND J. N. SHARDA. 2007. On tem- nity ecology: A hybrid beast with a molecular master. BioScience 58:
poral partitioning of a community of ectomycorrhizal fungi. New 799–810.
Phytologist 174: 420–429. PEAY, K. G., P. G. KENNEDY, S. DAVIES, S. TAN, AND T. D. BRUNS. 2010.
KOLJALG, U., K. H. LARSSON, K. ABARENKOV, R. H. NILSSON, I. J. ALEXANDER, Potential link between plant and fungal distributions in a dipterocarp
U. EBERHARDT, S. ERLAND, ET AL. 2005. UNITE: A database provid- rainforest: Community phylogenetic structure of tropical ectomyc-
ing web-based methods for the molecular identification of ectomycor- orrhizal fungi across a plant and soil ecotone. New Phytologist 185:
rhizal fungi. New Phytologist 166: 1063–1068. 529–542.
KRETZER, A. M., S. M. DUNHAM, R. J. MOLINA, AND J. W. SPATAFORA. 2004. PERRY, D. A., M. P. AMARANTHUS, J. G. BORCHERS, S. L. BORCHERS, AND
Microsatellite markers reveal the below ground distribution of genets R. E. BRAINERD. 1989. Bootstrapping in ecosystems. BioScience 39:
in two species of Rhizopogon forming tuberculate ectomycorrhizas on 230–237.
Douglas fir. New Phytologist 161: 313–320. PETERSON, R. L., H. B. MASSICOTTE, AND L. H. MELVILLE. 2003. Mycorrhizas:
KRPATA, D., O. MUEHLMANN, R. KUHNERT, H. LADURNER, F. GOEBL, AND U. Anatomy and cell biology. CABI, Guelph, Ontario, Canada.
PEINTNER. 2007. High diversity of ectomycorrhizal fungi associated REDECKER, D., T. SZARO, R. BOWMAN, AND T. D. BRUNS. 2001. Small gen-
with Arctostaphylos uva-ursi in subalpine and alpine zones: Potential ets of Lactarius xanthogalactus, Russula cremoricolor and Amanita
inoculum for afforestation. Forest Ecology and Management 250: francheti in late-stage ectomycorrhizal successions. Molecular
167–175. Ecology 10: 1025–1034.
LARENA, I., O. SALAZAR, V. GONZÁLEZ, M. C. JULIÁN, AND V. RUBIO. 1999 RICHARD, F., M. SELOSSE, AND M. GARDES. 2009. Facilitated establish-
Design of a primer for ribosomal DNA internal transcribed spacer ment of Quercus ilex in shrub-dominated communities within a
with enhanced specificity for ascomycetes. Journal of Biotechnology Mediterranean ecosystem: Do mycorrhizal partners matter? FEMS
75: 187–194. Microbiology Ecology 68: 14–24.
LILLESKOV, E. A., T. D. BRUNS, T. R. HORTON, D. L. TAYLOR, AND P. RUSCA, T., P. G. KENNEDY, AND T. D. BRUNS. 2006. The effect of different
GROGAN. 2004. Detection of forest stand-level spatial structure in pine hosts on the sampling of Rhizopogon spore banks in five Eastern
ectomycorrhizal fungal communities. FEMS Microbiology Ecology Sierra Nevada forests. New Phytologist 170: 551–560.
49: 319–332. SILVA, J. S., P. VAZ, F. MOREIRA, F. CATRY, AND F. C. REGO. 2011. Wildfires
MADDISON, D. R., AND W. P. MADDISON. 1999. MacClade 4. Sinauer, as a major driver of landscape dynamics in three fire-prone areas of
Sunderland, Massachusetts, USA. Portugal. Landscape and Urban Planning 101: 349–358.
MALAJCZUK, N., AND F. J. HINGSTON. 1981. Ectomycorrhizae associated SIMARD, S. W., K. J. BEILER, M. A. BINGHAM, J. R. DESLIPPE, L. J. PHILIP, AND
with Jarrah. Australian Journal of Botany 29: 453–462. F. P. TESTE. 2012. Mycorrhizal networks: Mechanisms, ecology, and
MASSICOTTE, H. B., R. J. MOLINA, D. L. LUOMA, AND J. E. SMITH. 1994. modelling. Fungal Biology Reviews 26: 39–60.
Biology of the ectomycorrhizal genus, Rhizopogon 2. Patterns of host– SIMARD, S. W., D. A. PERRY, M. D. JONES, D. D. MYROLD, D. M. DURALL,
fungus specificity following spore inoculation of diverse hosts grown AND R. J. MOLINA. 1997. Net transfer of carbon between ectomycor-
in monoculture and dual culture. New Phytologist 126: 677–690. rhizal tree species in the field. Nature 388: 579–582.
MASSICOTTE, H. B., R. J. MOLINA, L. TACKABERRY, J. E. SMITH, AND M. P. SMITH, J. E., R. J. MOLINA, AND D. A. PERRY. 1995. Occurrence of ectomy-
AMARANTHUS. 1999. Diversity and host specificity of ectomycor- corrhizas on ericaceous and coniferous seedlings grown in soils from
rhizal fungi retrieved from three adjacent forest sites by five host spe- the Oregon Coast Range. New Phytologist 129: 73–81.
cies. Canadian Journal of Botany-Revue Canadienne De Botanique SMITH, M. E., G. W. DOUHAN, A. K. FREMIER, AND D. M. RIZZO. 2009. Are true
77: 1053–1076. multihost fungi the exception or the rule? Dominant ectomycorrhizal
 15372197, 2012, 10, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.3732/ajb.1200277 by Cochrane Mexico, Wiley Online Library on [21/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
October 2012] KENNEDY ET AL.—MYCORRHIZAL COMMUNITIES AND MIXED EVERGREEN FORESTS 1701

fungi on Pinus sabiniana differ from those on co-occurring Quercus TESTE, F. P., AND S. W. SIMARD. 2008. Mycorrhizal networks and distance
species. New Phytologist 182: 295–299. from mature trees alter patterns of competition and facilitation in dry
SMITH, M. E., J. M. TRAPPE, AND D. M. RIZZO. 2007. Genea, Genabea Douglas-fir forests. Oecologia 158: 193–203.
and Gilkeya gen. nov.: Ascomata and ectomycorrhiza formation in a VAN DER HEIJDEN, M. G. A., AND T. R. HORTON. 2009. Socialism in soil?
Quercus woodland. Mycologia 98: 699–716. The importance of mycorrhizal fungal networks for facilitation in
SOUTHWORTH, D., X. HE, W. SWENSON, C. S. BLEDSOE, AND W. HORWATH. natural ecosystems. Journal of Ecology 97: 1139–1150.
2005. Application of network theory to potential mycorrhizal net- VISSER, S. 1995. Ectomycorrhizal fungal succession in Jack pine stands
works. Mycorrhiza 15: 589–595. following wildfire. New Phytologist 129: 389–401.
TABERLET, P., L. GIELLY, G. PAUTOU, AND J. BOUVET. 1991. Universal WHITTAKER, R. H. 1960. Vegetation of the Siskiyou Mountains, Oregon
primers for amplification of 3 noncoding regions of chloroplast DNA. and California. Ecological Monographs 30: 279–338.
Plant Molecular Biology 17: 1105–1109. ZAK, B. 1974. Ectendomycorrhiza of Pacific madrone (Arbutus
TEDERSOO, L., T. JAIRUS, B. M. HORTON, K. ABARENKOV, T. SUVI, I. SAAR, AND menziesii). Transactions of the British Mycological Society 62:
U. KOLJALG. 2008. Strong host preference of ectomycorrhizal fungi 202–205.
in a Tasmanian wet sclerophyll forest as revealed by DNA barcoding ZAK, B. 1976. Pure culture synthesis of Pacific madrone ectendomycor-
and taxon-specific primers. New Phytologist 180: 479–490. rhizae. Mycologia 68: 362–369.