Ectomycorrhizal diversity on Betula papyrifera and Pseudotsuga menziesii seedlings grown in the greenhouse or outplanted in single-species and mixed plots in southern British Columbia

Abstract

The inoculum potential and diversity of the ectomycorrhizal fungal community usually decrease on a site following logging. The objective of this study was to determine if planting a mixture of tree species following logging retains a higher diversity of ectomycorrhizal fungi than planting a single species. Paper birch (Betula papyrifera Marsh.) and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) were planted either alone or in mixture and in different proportions and densities on each of three sites in the southern interior of British Columbia. Ectomycorrbizal types were characterized following detailed morphological examination on 12 seedlings per plot at 4, 16, and 28 months following out planting (field bioassay). Thelephora, E-strain, Rhizpogon (for Douglas-fir only), Mycelium radicis atrovirens, and Cenococcum mycorrhizae were the most abundant. The Thelephora mycorrhizae decreased in dominance over the sampling period from 82 to 41% of ectomycorrhizae on birch and from 26 to 15% on Douglas-fir. Rhizopogon mycorrhizae remained consistently abundant an Douglas-fir roots (36% of mycorrhizae at 4 months to 37% at 28 months). By 28 mouths, 91% of birch and 56% of Douglas-fir mycorrhizae were types common to the two species. This has important implications for possible nutrient of carbon transfer between the two species. At 16 (P = 0.068) and 28 months (P = 0.088) following outplanting, the evenness of the ectomycorrhizal community on Douglas-fir root systems was higher in mixed than in single-species plots. Richness (number of ectomycorrhizal types) and Simpson's diversity index per seedling were not affected by tree mixture treatments. Planting density did not affect richness, evenness, or diversity. The ectomycorrhizae that developed an Douglas-fir or birch seedlings grown in the greenhouse in soils from these sites were very similar to those that developed in the field. This demonstrates that greenhouse bioassays can be used to predict which types of ectomycorrbizae will form on seedlings grown on disturbed sites, at least for the first few years following outplanting.

Full text

Ectomycorrhizal diversity on Betula papyrifera
and Pseudotsuga menziesii seedlings grown in
the greenhouse or outplanted in single-species
and mixed plots in southern British Columbia
Melanie D. Jones, D.M. Durall, S.M.K. Harniman, Debbie C. Classen, and
Suzanne W. Simard
Abstract: The inoculum potential and diversity of the ectomycorrhizal fungal community usually decrease on a site following
logging. The objective of this study was to determine if planting a mixture of tree species following logging retains a higher
diversity of ectomycorrhizal fungi than planting a single species. Paper birch (Betula papyrifera Marsh.) and Douglas-fir
(Pseudotsuga menziesii (Mirb.) Franco) were planted either alone or in mixture and in different proportions and densities on
each of three sites in the southern interior of British Columbia. Ectomycorrhizal types were characterized following detailed
morphological examination on 12 seedlings per plot at 4, 16, and 28 months following outplanting (field bioassay).
Thelephora, E-strain, Rhizopogon (for Douglas-fir only), Mycelium radicis atrovirens, and Cenococcum mycorrhizae were the
most abundant. The Thelephora mycorrhizae decreased in dominance over the sampling period from 82 to 41% of
ectomycorrhizae on birch and from 26 to 15% on Douglas-fir. Rhizopogon mycorrhizae remained consistently abundant on
Douglas-fir roots (36% of mycorrhizae at 4 months to 37% at 28 months). By 28 months, 91% of birch and 56% of
Douglas-fir mycorrhizae were types common to the two species. This has important implications for possible nutrient or
carbon transfer between the two species. At 16 (P=0.068) and 28 months (P=0.088) following outplanting, the evenness of
the ectomycorrhizal community on Douglas-fir root systems was higher in mixed than in single-species plots. Richness
(number of ectomycorrhizal types) and Simpson’s diversity index per seedling were not affected by tree mixture treatments.
Planting density did not affect richness, evenness, or diversity. The ectomycorrhizae that developed on Douglas-fir or birch
seedlings grown in the greenhouse in soils from these sites were very similar to those that developed in the field. This
demonstrates that greenhouse bioassays can be used to predict which types of ectomycorrhizae will form on seedlings grown
on disturbed sites, at least for the first few years following outplanting.
Résumé : Habituellement, le potentiel d’inoculum et la diversité des champignons ectomycorhiziens sur un site diminuent
suite à la récolte. L’objectif de cette étude consistait à déterminer si la plantation d’un mélange d’espèces d’arbres suite à la
coupe permettrait de maintenir une plus grande diversité de champignons ectomycorhiziens que la plantation d’une seule
espèce. Le bouleau à papier (Betula papyrifera Marsh.) et le douglas taxifolié (Pseudotsuga menziesii (Mirb.) Franco) furent
plantés seuls ou en mélange, dans différentes proportions et densités, dans trois sites situés dans la zone continentale au sud
de la Colombie-Britannique. Dans le cas de l’essai au champ, les types d’ectomycorhizes furent caractérisés après un examen
morphologique détaillé de 12 semis par parcelles, 4, 16 et 28 mois après la plantation. Les champignons mycorhiziens
Thelephora, la race E, Rhizopogon (sur le douglas taxifolié uniquement), Mycelium radicis atrovirens et Cenococcum étaient
les plus abondants. La dominance de Thelephora a diminué au cours de la période d’échantillonnage de 82 à 41% des
ectomycorhizes chez le bouleau et de 26 à 15% chez le douglas taxifolié. Rhizopogon est demeuré aussi abondant chez le
douglas taxifolié (de 36% des ectomycorhizes à 4 mois jusqu’à 37% à 28 mois). Vers le 28emois, 91% des mycorhizes chez
le bouleau et 56% chez le douglas taxifolié étaient communes aux deux espèces. Cela a d’importantes implications dans le
transfert potentiel des nutriments et du carbone entre les deux espèces. À 16 (P= 0,068) et 28 (P= 0,088) mois après la
plantation, l’homogénéité de la population d’ectomycorhizes associée au système racinaire du douglas taxifolié était plus
grande dans les parcelles où il y avait un mélange d’espèces que dans celles où il n’y avait qu’une seule espèce. La richesse
(nombre de types d’ectomycorhizes) et l’indice de diversité de Simpson par semis n’étaient pas affectés par le mélange des
espèces d’arbre. La densité de la plantation n’a pas affecté la richesse, l’homogénéité ou la diversité. Les ectomycorhizes qui
se sont développés sur les semis de douglas taxifolié ou de bouleau cultivés en serres dans des sols provenant de ces sites
étaient très semblables à ceux qui se sont développés sur le terrain. Cela démontre qu’on peut faire des essais en serres pour
Received January 21, 1997. Accepted August 19, 1997.
M.D. Jones,1D.M. Durall, S.M.K. Harniman, and D.C. Classen. Department of Biology, Okanagan University College, Kelowna,
BC V1V 1V7, Canada.
S.W. Simard. British Columbia Ministry of Forests, Research Branch, Kamloops Region, 515 Columbia Street, Kamloops, BC V2C 2T7,
Canada.
1Author to whom all correspondence should be addressed. e-mail: mjones@okanagan.bc.ca
Can. J. For. Res. 27: 1872–1889 (1997)
1872
© 1997 NRC Canada

prédire quel type d’ectomycorhizes se formeront sur les semis cultivés sur des sites perturbés, au moins pour les quelques
premières années après la plantation.
[Traduit par la Rédaction]
Introduction
The removal of trees from a site causes changes in the ectomy-
corrhizal fungal community. In particular, there is a loss of
fungal species richness, and the inoculum potential can de-
crease (Schoenberger and Perry 1982; Parke et al. 1984; Pilz
and Perry 1984; Amaranthus et al. 1987). Some change in the
ectomycorrhizal fungal community is unavoidable because the
soil environment changes following logging, and the ectomy-
corrhizal hosts will change in species composition and age.
Nevertheless, it is important to maintain the quantity and di-
versity of ectomycorrhizal inoculum on a site (Amaranthus
and Perry 1994; Allen et al. 1995). Ectomycorrhizae are im-
portant nutrient- and water-absorbing structures of most conif-
erous trees, but species of ectomycorrhizal fungi differ in their
ability to transport water (Parke et al. 1983; Dosskey et al.
1990), break down organic forms of nutrients (Abuzinadah and
Read 1986), absorb mineral nutrients (Burgess et al. 1993),
and protect nursery seedlings from pathogens (Chakravarty
and Unestam 1987). Some of these effects may be due to dif-
ferences in the microbial communities in the mycorrhi-
zosphere of ectomycorrhizae formed by different fungi (Li
et al. 1992). Different ectomycorrhizal fungi also preferen-
tially colonize different types of substrates, including mineral
soil (Malajczuk and Hingston 1981), organic soil (Harvey
et al. 1997), decaying wood, and different ages of trees or parts
of the root system (Deacon and Fleming 1992). Each tree can
form associations with many species of ectomycorrhizal fungi.
Thus, for trees to maximize their exploitation of the heteroge-
neous soil resources on a site, it is important that the diversity
of ectomycorrhizal fungi be maintained following logging
(Amaranthus and Perry 1994).
There is increasing interest among foresters in planting
mixtures of conifers and hardwoods, as a change from planting
pure conifer stands. In British Columbia, this is especially true
for the Interior Cedar–Hemlock biogeoclimatic zone, where
mortality in plantations of Douglas-fir (Pseudotsuga menziesii
(Mirb.) Franco) is frequently unacceptable due to infection by
the root pathogen Armillaria ostoyae (Romagnesi) Herink
(Morrison et al. 1991). Hardwoods such as paper birch (Betula
papyrifera Marsh.) are less susceptible to Armillaria root rot.
Other incentives for interplanting Betula spp. and Douglas-fir
include improvement to the nitrogen and pH status of the soil
(Miller 1984; Troedsson 1985; Van Cleve et al. 1986; Bradley
and Fyles 1995) and increases in structural diversity of the
resulting forest (Simard and Vyse 1994).
This study is part of a larger study to investigate the above-
and belowground interactions of paper birch and Douglas-fir
in mixed and pure stands. The objective of this part of the study
was to determine the effect of planting density and diversity
on the ectomycorrhizal fungal community of paper birch and
Douglas-fir. Our hypothesis was that increased density or di-
versity of tree species would result in an increase in the diver-
sity of ectomycorrhizae associated with either tree species. In
addition, we used seedlings planted in field soil in the green-
house to (i) detect differences in inoculum across the site prior
to outplanting and (ii) determine whether the differences
found in mixed plots depended on the continued presence of a
living tree or whether they were caused by changes to the soil.
In a greenhouse bioassay, some of the chemical and biological
changes caused by the planting of a particular tree species are
still present, but mycorrhizal inoculum is no longer connected
to a living tree. We hypothesized that both the presence of a
living tree and the changes to soil chemistry and biology would
influence the types of ectomycorrhizae formed.
We are aware of only one previous study that has compared
the diversity of ectomycorrhizae in mixed and monoculture
plantations. In 25- to 30-year-old plantations, Heslin et al.
(1992) found one more ectomycorrhizal type on Sitka spruce
(Picea sitchensis (Bong.) Carrière) roots in plots where the
spruce had been interplanted with Japanese larch (Larix lep-
tolepis (Sieb. & Zucc.) Gord.) than in plots where only Sitka
spruce had been planted. The number of ectomycorrhizal fungi
that produced epigeous sporocarps was much higher in the
mixed than in the single-species plots. Also relevant is the
work of Bills et al. (1986) where sporocarps of mycorrhizal
fungi were enumerated in second-growth red spruce (Picea
rubens Sarg.) and in mixed conifer–hardwood stands. In that
study, sporocarp species richness was substantially higher in
the mixed stands. We have chosen to quantify treatment effects
on ectomycorrhizal diversity by examining the root tips di-
rectly because sporocarp numbers are generally poor predic-
tors of the relative abundance of different fungal species on
root tips (Gardes and Bruns 1996).
Materials and methods
Study sites
The study was performed on three sites ranging inelevation from 650
to 750 m in the Interior Cedar–Hemlock biogeoclimatic zone (ICH)
of the southern interior of British Columbia. Two sites, Adams Lake
(51°28′N, 119°30′W) and Malakwa (50°58′N, 118°44′W), are lo-
cated in the Thompson Moist Warm ICH (ICHmw3) variant (Lloyd
et al. 1990) and have Humo–Ferric Podzol soils (Canada Soil Survey
Committee 1978). The Hidden Lake (50°34′N, 118°50′W) site is lo-
cated in the Shuswap Moist Warm ICH (ICHmw2) variant and has a
Dystric Brunisol soil. The dominant tree species at all sites were
P. menziesii,B. papyrifera, western redcedar (Thuja plicata Donn ex
D. Don), and western hemlock (Tsuga heterophylla (Raf.) Sarg.),
with western redcedar (typically arbuscular mycorrhizal) the most
abundant tree species at Hidden Lake and Malakwa.
The sites were clear-cut in 1978 (Hidden Lake), 1987 (Adams
Lake), and 1988 (Malakwa). The Hidden Lake and Adam Lake sites
were planted with Douglas-fir, Hidden Lake in 1983 following
bunching and burning, and Adams Lake in 1988 following broadcast
burning. The Malakwa site was not burned and was seeded with do-
mestic grasses and clover and planted with Douglas-fir and Engel-
mann ×white spruce (Picea engelmannii ×glauca) in 1989. Paper
birch, trembling aspen (Populus tremuloides Michx.), and black cot-
tonwood (Populus trichocarpa Torr. & Gray), all of which are ec-
tomycorrhizal species, also regenerated naturally on these sites. In the
fall of 1991, all three sites were destumped with an excavator, most
Jones et al. 1873
© 1997 NRC Canada

of the soil shaken off the root systems, and the stumps and slash
pushed to the edges of the cut blocks. This procedure also removed
the young planted Douglas-fir and spruce and the aboveground por-
tions of the hardwoods at all three sites. The hardwoods, along with
raspberry (Rubus ideaus L.), falsebox (Paxistema myrsinites (Pursh)
Raf.), black huckleberry (Vaccinium membranaceum Dougl.), and
thimbleberry (Rubus parviflorus Nutt.), have resprouted on the sites
since 1991.
The larger study, of which this is a part, consists of three addition
series experiments where paper birch was interplanted with Douglas-
fir, western larch (Larix occidentalis Nutt.), or western redcedar at
varying densities and proportions. Study plots,40 ×40 m, were estab-
lished in the three cut blocks in the fall of 1991. Seedlings were
planted in the spring of 1992. The mycorrhizal study was conducted
on the paper birch (EP) and Douglas-fir (DF) plots only and only on
the following seven treatments: 0EP/1600DF, 800EP/800DF,
1600EP/0DF, 0EP/3200DF, 3200EP/0DF, 800EP/400DF, and
3200EP/1600DF (stems per hectare of paper birch/Douglas-fir). One
replicate plot of each treatment was established on each of the three
sites (7 treatments ×3 replicates =21 plots) between 10 and 140 m
away from adjacent uncut forest.
The ectomycorrhizal fungal community in the treatment plots was
investigated in two ways. Firstly, containerized seedlings (interior
Douglas-fir seed lot 1053, stock type PSB 415B, age 1 +0; paper
birch seed lot 2756, stock type PSB 615A, age 1 +0) produced by
Pacific Regeneration Technologies Inc., Vernon, B.C., were out-
planted onto the sites in the treatments described above (field bioas-
say). The root systems of 200 seedlings of each species were
examined for mycorrhizal colonization under a dissecting scope prior
to outplanting. Secondly, paper birch and Douglas-fir seedlings (the
same seed lots as above) were grown in a greenhouse in soils collected
from the field plots (greenhouse bioassay). The ectomycorrhizae
formed by both groups of seedlings were characterized, based on
detailed examination of their morphology, and counted.
Field bioassay
In September 1992, 1993, and 1994, roots were sampled from
12 Douglas-fir and (or) 12 birch growing in the border rows of each
treatment plot (i.e., 24 seedlings were sampled in each mixed plot and
12 in each single-species plot; 12 seedlings ×5 treatments ×3sites=
180 seedlings of each species). At these sampling dates, seedlings had
been growing in the field for 4 (1992), 16 (1993) or 28 months (1994).
In 1992, entire root systems were removed. In subsequentyears, roots
were sampled by loosening the soil at the base of the trees and then
sampling only those roots that could be traced to the main stem.
Usually only one or two long lateral roots (up to 1 m) were sampled
from the birch. Douglas-fir roots were generally shorter, so more
major laterals were sampled from these trees. The roots were moist-
ened and stored at 4°C until examination (up to 4 months).
The roots were washed over a 2-mm sieve and cut into lengths of
approximately 2 cm. Only “new” roots extending out from the plug
were examined. The 2-cm fragments were selected randomly, and
every live ectomycorrhizal tip per fragment was examined, until 200
mycorrhizae per seedling (150 in 1992) had been categorized. Thus,
the maximum number of mycorrhizae characterized was 12 000 per
site per species per year (5 treatments for each tree species ×12 seed-
lings ×200 live ectomycorrhizae) in 1993 and1994 and 9000 in1992.
The actual counts in 1993 and 1994 ranged from 10 610 to 12 000
because, occasionally, there were fewer than 200 ectomycorrhizae per
sample. In 1992, the actual counts ranged from 4307 to 6753 per
species per site for two reasons. Firstly, there was significant mortal-
ity in the first growing season (no significant difference between treat-
ments), so 12 seedlings of each species could not be sampled from
every plot. Secondly, there were often fewer than 150 live ectomycor-
rhizae on the roots which extended from the root plug. After 3 years
of growth, seedling survival was 90%; the major causes of mortality
were summer drought and frost.
Greenhouse bioassays
Twelve soil samples (collected with a spade from the top 10 cm) were
collected randomly from each treatment plot in September or October
1991, 1992, and 1993 (the purpose of the 1991 bioassays was to
determine whether differences existed among treatment plots prior to
outplanting). Surface organic layers, where present, were excluded
from the samples. The soils were stored at 4°C for less than 2 weeks,
at which time each sample was mixed and distributed into two 4 ×
21 cm Leach tubes (Ray Leach Cone-tainer Single Cell System, sup-
plied by Stuewe and Sons, Corvallis, Oregon). The soils were not
sieved, so they contained roots of regenerating plants, including ec-
tomycorrhizal roots which could act as inocula. One Leach tube was
planted with three birch seeds and the other with three Douglas-fir
seeds. The birch seeds had been surfaced sterilized in 10% H2O2and
rinsed in sterile distilled water. The Douglas-fir seeds had been sur-
face sterilized and scarified by shaking in 35% H2O2for 15 min fol-
lowed by 3% H2O2for 5 h. Birch and Douglas-fir seeds were also
planted in autoclaved field soil to quantify infection by greenhouse
contaminants. Seedlings were watered daily by overhead misting, but
were not fertilized. Following 14 weeks of growth in the Okanagan
University College greenhouse, Kelowna, B.C., the seedlings were
harvested and the root systems stored at 4°C. All live ectomycorrhizal
root tips, up to a maximum of 200 per container (actual counts aver-
aged across all containers per site ranged from 30 to 100 mycorrhizae
per container for birch and from 2 to 60 for Douglas-fir) were exam-
ined and categorized. Each unique mycorrhizal “type” was fully char-
acterized as described below.
Characterization of mycorrhizal types
A number of characteristics were used to categorize the tips into
consistently recognizable morphological types (Ingleby et al. 1990;
Goodman et al. 1996). Colour and texture of mycorrhizal tips, colour
and abundance of external hyphae, and the presence and colour of
hyphal strands were described while examining root tips under a
stereomicroscope at 40×. The appearance of the inner and outer man-
tle surface (felt or net prosenchyma; net, irregular interlocking, ir-
regular noninterlocking, or regular synenchyma (as per Ingleby et al.
1990)), the size of hyphal elements in the mantle, the diameter, sur-
face texture, and frequency of clamps on external hyphae, and the
presence and type of cystidia were described under 400 or 1000×. The
descriptions were compared with other published descriptions of ec-
tomycorrhizae (e.g., Agerer 1987–1995; Ingleby et al. 1990).
Data analysis
Treatment effects on the percentage of live ectomycorrhizae classified
as specific ectomycorrhizal types (for each type comprising over 5%
of the ectomycorrhizae for at least one harvest) were analyzed in two
ways: by one-factor analysis of variance (ANOVA) among the five
treatments and by one-factor planned contrasts for the effects of plant-
ing density or tree species mixture. Data for birch and Douglas-fir
were analyzed separately. For planned contrasts, the 800/400,
800/800, 1600/0, and 0/1600 plots were classified as low-density
treatments and the 3200/0, 0/3200, and 3200/1600 plots as high-density
treatments. For the determination of species mixture effects, the data
from the 800/400, 800/800, and 3200/1600 plots were contrasted with
data from the 1600/0 and 3200/0 or 0/1600 and 0/3200 plots. Data
were transformed according to Steel and Torrie (1980, pp. 233–236).
Analyses on transformed data are mentioned in the text.
Richness, Simpson’s (=1−Σp
i
2
, where piis the proportion of live
ectomycorrhizae of type i; Krebs 1989), and Shannon–Weiner (Krebs
1989, p. 361) diversity indices and Simpson’s evenness index were
calculated using the AID Programs (W.S. Overton, unpublished). Ec-
tomycorrhizal richness is the number of different morphological types
found on Douglas-fir or birch seedlings. Simpson’s evenness index
was calculated as
Can. J. For. Res. Vol. 27, 1997
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© 1997 NRC Canada

Evenness=Simpson’s diversityindex
1−C−1
where Cis the number of morphological types. Each index was cal-
culated both for individual seedlings (based on, for example, 200
mycorrhizae per seedling) and for whole plots (the data treated as one
sample of 2400 mycorrhizae). Because Shannon’s diversity index
was less sensitive to the treatments than was Simpson’s index, these
data are not included.
Two-factor ANOVAs were performed on richness, evenness, and
diversity indices to test for treatment and host species effects. For the
individual seedling data, the mean value for the 12 seedlings per plot
was used for the ANOVA. Thus, for both the seedling and whole-plot
data, the sites were the replicates (N=3). Planned contrasts were also
performed to test for density (low versus high) or host mixture (single
species versus mixed) effects on the indices. The grouping of treat-
ments was as described above except that, for analysis of the bioassay
results, the treatments were combined into three groups: mixed
(800/400, 800/800, 3200/1600), birch only (1600/0, 3200/0), and
Douglas-fir only (0/1600, 0/3200). Generally, the planned contrasts
were for two factors: tree species and density, or tree species and host
mixture. When there was an interaction between tree species and
either density or mixture, separate one-factor planned contrasts were
performed for each species.
To test for similarities between the mycorrhizal types on bioassay
and field-grown seedlings, simple linear regressions were performed
between the percentage of mycorrhizae of a specific type on the two
types of seedlings. Each of the most common mycorrhizal types was
tested separately, with data consisting of means from each of the three
sites for 1992 and 1993 (the only years where both greenhouse and
field bioassays were performed; df =5 for each regression). In addi-
tion, one regression was performed using the six data points above
from each of the six most common ectomycorrhizal types (df =35).
Results
Field bioassay
Only mycorrhizae that appeared to be alive (roots with intact
tips, mantles where individual hyphal elements could be rec-
ognized) were characterized. Significant numbers of dead root
tips were observed only in 1992, at the end of the first growing
season (Table 1). In subsequent years, less than 1% of root tips
examined were classified as dead. No mantles were observed
on the roots of seedlings prior to outplanting, and mycorrhizal
colonization was lower at the end of the first growing season
(Table 1) than in subsequent years. In 1993 and 1994, more
than 95% of the live root tips examined were mycorrhizal, with
no differences among sites (data not shown).
Paper birch
Nine different ectomycorrhizal types were described on the
birch roots collected from the field in 1992, 4 months follow-
ing outplanting. This increased to 20 types in 1993 and 26 in 1994
(Table 2). A Thelephora-like mycorrhiza (OUC240, Table 2)
was the mos t common type, although its abundance dec reased with
time (Fig. 1a): 61–98% (range from 5 treatments ×3sites=
15 plots) of the live ectomycorrhizae examined per plot in
1992 and 37–76% in 1993. In 1994, the frequency of this my-
corrhizal type continued to decrease at Adams Lake and Hid-
den Lake where it represented 36–43% of the ectomycorrhizae
characterized per plot, but not at Malakwa where its abundance
was 53–68%. Mycelium radicis atrovirens Melin (MRA,
OUC170), E-strain (OUC060, OUC061), and Cenococcum
(OUC030) mycorrhizae (Table 2) were also prevalent. The
MRA and Cenococcum types were found in virtually every
plot in every year (data not shown) and both increased in fre-
quency between 1992 and 1994 (Fig. 1a). Sectioning of MRA
mycorrhizae confirmed that a Hartig net was present and that
neither intracellular penetration nor colonization beyond the
cortex occurred.
The proportion of specific ectomycorrhizal types on birch
differed at the three sites. For example, in 1992, E-strain type I
mycorrhizae were common in every plot at Hidden Lake, but
were never observed in the Adams Lake samples. Although
E-strain I mycorrhizae became common at the other sites in
subsequent years, their abundance remained highest at Hidden
Lake. Likewise, Cenococcum mycorrhizae were most preva-
lent at Adams Lake and least common at Hidden Lake through-
out the sampling period. Thelephora mycorrhizae remained
more prevalent at Malakwa than at the other two sites.
The treatments affected the frequency of occurrence of some
of the major ectomycorrhizal types by 1994. The E-strain II
mycorrhizae were more common in mixed (9.6 ±1.7% of my-
corrhizae) than in monoculture plots (3.8 ±1.2%) (P=0.03,
planned contrast). MRA mycorrhizae were more common in
high-density (26.0 ±1.9%) than in low-density plots (16.3 ±
2.5%) (P=0.02).
Douglas-fir
The number of ectomycorrhizal types on outplanted Douglas-
fir increased from nine in 1992 to 25 in 1993 and 32 in 1994
(Table 2). The ectomycorrhizal types on Douglas-fir roots
were generally more diverse than those on birch root systems.
There were three or four codominant types: a Rhizopogon-like
type, the Thelephora type, and the two E-strain types (Fig. 1b).
In addition to these three types, Cenococcum mycorrhizae
were also found in every Douglas-fir plot in 1993 and 1994,
albeit at low frequencies.
As with birch, the relative number of Thelephora mycor-
rhizae decreased with time, except at Malakwa (Fig. 1b). The
number of Rhizopogon-like mycorrhizae remained reasonably
consistent over time; the number of E-strain mycorrhizae in-
creased at Malakwa and Adams Lake between 1992 and 1993.
By 1994, the Rhizopogon-like and E-strain types were the most
abundant mycorrhizae at Hidden Lake and Adams Lake
whereas Thelephora and Rhizopogon-like mycorrhizae re-
mained the most abundant types at Malakwa. Neither density
Site
Adams Lake Malakwa Hidden Lake P*
Paper birch
% dead†4.2±1.4a3.1±1.6a1.9±1.1a0.2
% mycorrhizal‡59±5a79±3b82±2b0.002
Douglas-fir
% dead†13.7±4.2b5.1±1.7ab 4.6±0.8a0.05
% mycorrhizal‡56±17a58±13a78±5a0.5
Note: Values are means ±SE; N=5 plots. Values within a row followed
by the same lowercase letters are not significantly different (P< 0.05).
*One-factor ANOVA.
†Percentage of the total number of tips examined per plot.
‡Percentage of the live root tips examined per plot.
Table 1. Percent mycorrhizal colonization and percentage of dead
root tips of paper birch or Douglas-fir seedlings grown at different
densities in mixed or single-species plots in 1992.
Jones et al. 1875
© 1997 NRC Canada

Macroscopic description Mantle type(s) Emanating hyphae Mycelial strands Cystidia Year/host*
Amphinema-like
OUC 020
Light brown to orange
mycorrhiza, rough, with
loose yellowish mycelial
strands and fine yellow to
white emanating hyphae
Outer: felt prosenchyma,
3–4 µm wide
Inner: net synenchyma,
3–4 µm wide
4µm wide; very
abundant loosely
formed clamps;
hyaline; smooth to
finely verrucose; stains
yellow in 10% KOH
Loosely organized;
only 3 or 4
hyphae wide;
abundant clamps
Absent 91G/B
92F/B+D
92G/B+D
93F/B+D
93G/D
94F/D
OUC 021
Light brown mycorrhiza;
rough, with abundant
yellow and brown
emanating hyphae
Outer: Felt prosenchyma,
2µm wide
Inner: Net synenchyma,
4µm wide
2–3 µm wide; hyaline;
smooth; abundant
clamps
Absent Absent 94F/B+D
Cenococcum
OUC 030
Black mycorrhiza; grainy
and shiny with straight
dark brown emanating
hyphae; strands absent
Outer: net synenchyma;
thick-walled radially
arranged isodiametric
cells, 4–5 µm wide ×
10–20 µm long
Inner: net synenchyma to
noninterlocking irregular
synenchyma, 3–6 µm
wide
3–4 µm wide; smooth; no
clamps
Absent Absent 91G/B
92F/B+D
92G/B+D
93F/B+D
93G/B+D
94F/B+D
Basidiomycotina
OUC 040
White mycorrhiza; rough;
abundant white emanating
hyphae and mycelial
strands
Outer: felt prosenchyma
Inner: net synenchyma or
irregular synenchyma
3–4 µm wide; frequent
clamps; slightly
verrucose
Compact; white;
1 mm wide
Absent 93F/B+D
93G/B+D
Basidiomycotina
OUC 050
Light orange – rust
mycorrhiza with abundant
light emanating hyphae
and strands
Outer: net prosenchyma,
3µm wide
Inner: net or irregular
synenchyma, 2.5 µm
wide
4µm wide; very
abundant clamps;
smooth to finely
verrucose
Compact; white to
orange
Absent 93F/B+D
93G/B+D
94F/D
E-strain I
OUC 060
Light brownish – orange
mycorrhiza with mantle
becoming lighter towards
apex; smooth; rare
emanating hyphae
Outer: felt prosenchyma,
3–8 µm wide
Inner: net prosenchyma,
1.5–2.5 µm wide
4–7 µm wide; no clamps;
smooth to verrucose;
pinched in at septa
Absent Absent 91G/B
92F/B+D
92G/B+D
93F/B+D
93G/B+D
94F/B+D
E-strain II
OUC 061
Dark brown – orange
mycorrhiza with mantle
becoming lighter towards
apex; rough texture; rare,
golden brown emanating
hyphae
Outer: net synenchyma,
4–12 µm wide
Inner: irregular
interlocking
synenchyma, 4 µm wide
4–10 µm wide; no
clamps; golden brown;
verrucose
Absent Absent 91G/B+D
92F/B+D
92G/B+D
93F/B+D
93G/B+D
94F/B+D
Table 2. Morphological characteristics of 43 different ectomycorrhizal types observed on paper birch and Douglas-fir grown in a greenhouse
or in the field in southern British Columbia.
Can. J. For. Res. Vol. 27, 1997
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© 1997 NRC Canada

Macroscopic description Mantle type(s) Emanating hyphae Mycelial strands Cystidia Year/host*
OUC 062
Dark reddish brown
mycorrhiza; rough; rare
emanating hyphae
Outer: felt or net
prosenchyma, 3–6 µm
wide
Inner: none distinguishable
5µm wide; no clamps;
verrucose
Absent Absent 93G/B+D
94F/B+D
OUC 063
Brown to orange
mycorrhiza; rough; rare
golden emanating hyphae
Outer: locking irregular
synenchyma, 7–8 µm
wide
Inner: net or irregular
synenchyma, 9 µm wide
2–2.5 µm wide; smooth
to lightly verrucose
Absent Absent 94F/B
Basidiomycotina
OUC 072
Black–brown mycorrhiza;
smooth; rare emanating
hyphae
Outer: felt prosenchyma,
2–3 µm wide
Inner: irregular
nonlocking synenchyma,
10–20 µm wide
2–3 µm wide; clamps
common
Absent Absent 93F/B+D
Hebeloma-like I
OUC 080
Light brown – orange
mycorrhiza; moderate to
abundant fine white
hyphae
Outer: felt or net
prosenchyma, 2–3 µm
wide
Inner: net synenchyma,
1–2 µm wide
2–3 µm wide; very
frequent clamps;
abundant verrucose
ornamentation; hyaline
Absent Absent 91G/B
92G/D
93F/B
94F/B+D
Hebeloma-like II
OUC 084
Tan or light tan; smooth and
swollen; rare white
hyphae on younger
mycorrhizas
Outer: felt prosenchyma
to net synenchyma,
3–4 µm wide, often
arranged in parallel
Inner: net synenchyma,
2–3 µm wide
2–3 µm wide; rare
clamps; smooth
Absent Absent 93G/B
94F/B+D
Basidiomycotina
OUC 095
Clear/translucent mycorrhiza
with very rare hyaline,
emanating hyphae;
smooth and shiny
Outer: felt prosenchyma,
2µm wide
Inner: net prosenchyma,
not readily visible
2µm wide; frequent
clamps; hyaline,
smooth; distinct
“banding” inside
hyphae
Absent Absent 93F/B+D
93G/B+D
94F/B+D
OUC 100
White mycorrhiza; smooth;
rare hyaline, emanating
hyphae
Outer: net prosenchyma,
1.5–2 µm wide
Inner: net or
noninterlocking irregular
synenchyma, 1–2 µm
wide
1–1.5 µm wide; no
clamps; contents
granular
Absent Absent 92G/D
93F/B
94F/B+D
OUC 105
Long, straight mycorrhiza;
grey–brown in colour;
emanating hyphae rare
Outer: felt prosenchyma,
2–5 µm wide
Inner: net synenchyma,
2–4 µm wide
1–2 µm wide; no clamps;
smooth
Absent Absent 93G/B+D
93F/B+D
Table 2 (continued).
Jones et al. 1877
© 1997 NRC Canada

Macroscopic description Mantle type(s) Emanating hyphae Mycelial strands Cystidia Year/host*
OUC 115
White, shiny mycorrhizas;
no emanating hyphae
apparent
Outer: noninterlocking
irregular synenchyma,
4–20 µm wide
Inner: regular
synenchyma, 4–20 µm
wide
Absent Absent Absent 93F/D
OUC 120
Black, rough mycorrhiza;
rare emanating hyphae
Outer: felt prosenchyma,
2.5–3 µm wide; thick
walled
Inner: net synenchyma,
2µm wide; thick walled
2.5–3 µm wide; no
clamps; brown; smooth
Absent Short; awl shaped;
40–60 µmin
length
92F/B
93F/B+D
93G/B+D
94F/D
Basidiomycotina
OUC 130
Tan or grayish mycorrhiza;
smooth and milky in
appearance; rare white
emanating hyphae
Outer: felt prosenchyma,
6–10 µm wide
Inner: net prosenchyma,
4–5 µm wide
4µm wide; frequent
clamps; smooth
Absent Absent 92G/B+D
93F/B+D
93G/B+D
94F/B+D
Lactarius-like I
OUC 148
Light brown or white
mycorrhiza; smooth, no
emanating hyphae visible
Outer: net prosenchyma,
2.5 µm wide
Inner: net synenchyma,
3µm wide; laticifers
containing yellow latex
frequently observed
Absent Absent Absent 92F/B
92G/B+D
93F/B+D
93G/B+D
94F/B+D
Lactarius-like II
OUC 150
Light brown – grey
mycorrhiza; smooth; no
apparent emanating
hyphae
Outer: irregular locking
synenchyma, 2–4 µm
wide; “jigsaw puzzle”
pattern
Inner: net synenchyma,
1–2 µm wide
Absent Absent Absent 94F/B+D
MRA
OUC 170
Black–brown or grey
mycorrhiza; very narrow
(<0.5 mm) with rough
texture; rare to common
emanating hyphae
Outer: felt prosenchyma,
3µm wide
Inner: net synenchyma,
2µm wide
2µm wide; no clamps;
smooth to finely
verrucose
Absent Absent 91G/B+D
92F/B+D
92G/B+D
93F/B+D
93G/B+D
94F/B+D
Paxillus-like
OUC 180
Silver–white mycorrhiza;
smooth with frequent
white emanating hyphae
and mycelial strands
Outer: net prosenchyma
Inner: net synenchyma
2.5–5 µm in diameter;
abundant clamps
Differentiated but
loosely organized
Absent 93F/B+D
93G/B+D
Table 2 (continued).
Can. J. For. Res. Vol. 27, 1997
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Macroscopic description Mantle type(s) Emanating hyphae Mycelial strands Cystidia Year/host*
Basidiomycotina
OUC 190
Black, rough, swollen
mycorrhiza; rare
emanating hyphae
Outer: net or irregular
synenchyma, 4 µm wide
Inner: outer and inner are
not distinguishable
2.5 µm wide; very
frequent clamps;
brown; smooth to
verrucose
Absent Absent 94F/B+D
Basidiomycotina
OUC 194
Grey–brown mycorrhiza;
smooth; rare emanating
hyphae
Outer: net prosenchyma
Inner: net synenchyma to
irregular nonlocking
synenchyma
4µm wide; frequent
clamps; smooth;
hyaline
Absent Absent 93F/D
Rhizopogon-like
OUC 210
White and brown–black
mycorrhiza; abundant and
matted emanating hyphae
form a tubercle around
mycorrhizas; strands
common; found only on
Douglas-fir; sandy debris
common
Outer: net prosenchyma,
2µm wide
Inner: net synenchyma,
2µm wide when visible
2µm wide; no clamps;
smooth; densely packed
Loosely organized;
hyphae 2 µm
wide
Absent 91G/D
92F/D
92G/D
93F/D
93G/D
94F/D
OUC 211
Grey to dark brown
mycorrhiza; crusted and
clumped together;
abundant emanating
hyphae; reddish brown
strands; thick crust around
tips
Outer: felt prosenchyma,
if any, 2–3 µm wide
Inner: not visible
1.5–2 µm wide; no
clamps; smooth; dark
brown
Compact; 100 µm
in diameter
Absent 94F/D
Russula-like I
OUC 220
Creamy tan mycorrhiza;
velvety texture; emanating
hyphae absent
Outer: net prosenchyma or
synenchyma, 3 µm wide
Inner: not visible
Absent Absent Abundant;
10–15 µmin
length; 3–5 µm
wide; bottle
shaped; multilobed
93F/B+D
93G/B+D
94F/D
Russula-like II
OUC 228
Black – dark brown
mycorrhiza; swollen;
emanating hyphae very
rare
Outer: net prosenchyma,
5µm wide
Inner: net synenchyma,
4µm wide
Rare; 5 µm wide; smooth
to lightly verrucose
Absent Very distinct;
70–100 µmin
length; 5–7 µm
wide; verrucose;
clamps observed
at some septa
94F/B
OUC 230
Light brown mycorrhiza
covered with abundant
white hyphae and strands
Outer: felt prosenchyma,
3µm wide
Inner: net synenchyma,
not readily visible
3µm wide; crystalline
ornamentation; no
clamps
Compact and
frayed-looking
with crystalline
ornamentation on
the hyphae
Absent 93F/D
93G/D
94F/D
Table 2 (continued).
Jones et al. 1879
© 1997 NRC Canada

Macroscopic description Mantle type(s) Emanating hyphae Mycelial strands Cystidia Year/host*
Thelephora-like
OUC 240
Light tan to dark brown
mycorrhiza; smooth and
swollen; emanating
hyphae rare to common
Outer: net prosenchyma,
4–9 µm wide
Inner: net synenchyma,
2–3 µm wide
2–3 µm wide; frequent
clamps; smooth
Absent Common; 50–60 µm
in length; 2 µm
wide; rounded tip;
clamps on basal
septa
91G/B+D
92F/B+D
92G/B+D
93F/B+D
93G/B+D
94F/B+D
OUC 241
White–yellow mycorrhiza;
smooth; no emanating
hyphae
Outer: net synenchyma,
6–10 µm wide
Inner: none observed
Absent Absent Common;
100–150 µmin
length; 2–3 µm
wide; rounded
tips; basal clamps
94F/B+D
Basidiomycotina
OUC 250
Black – dark brown
mycorrhiza; smooth and
swollen; emanating
hyphae may be rare or
common
Outer: felt prosenchyma,
4µm wide
Inner: nonlocking or
regular synenchyma,
5–6 µm wide
2–3 µm wide; abundant
clamps; smooth to
lightly verrucose; thick
walled; dark brown
Absent Absent 92F/D
93F/B
94F/B+D
Basidiomycotina
OUC 251
Black mycorrhiza; very
grainy and swollen;
abundant emanating
hyphae
Outer: nonlocking or
regular synenchyma,
10 µm wide; cells
rounded
Inner: not visible
3–4 µm wide; abundant
clamps; dark brown;
smooth
Absent Absent 93F/D
94F/B+D
OUC 252
Black mycorrhiza; rough
with abundant brown
emanating hyphae
Outer: nonlocking
synenchyma, 12–20 µm
wide
Inner: net synenchyma,
2µm wide
2µm wide; no clamps;
smooth
Absent Absent 93F/D
94F/B
Tuber-like
OUC 260
Light to medium brown
mycorrhiza; smooth to
slightly grainy; very rare,
fine emanating hyphae
Outer: irregular locking
synenchyma, 5–7 µm
wide
Inner: not observed
2.5 µm wide; smooth Absent Common; 60–90 µm
in length; 1.5 µm
in diameter;
sharp, awl-like tips
91G/B
92F/B+D
92G/B
93F/B+D
93G/B+D
94F/B+D
OUC 300
Black – dark brown
mycorrhiza; rough
texture; frequent black
emanating hyphae
Outer: felt prosenchyma,
3–4 µm wide
Inner: net prosenchyma,
3µm wide
2–4 µm wide; rare clamp
connections; hyaline;
smooth
Absent Absent 94F/D
Table 2 (continued).
Can. J. For. Res. Vol. 27, 1997
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nor diversity of host species significantly affected (P> 0.05)
the abundance of the six most common mycorrhizal types on
Douglas-fir.
Comparison of birch and Douglas-fir
Birch and Douglas-fir shared five of the six most common
mycorrhizal types. Rhizopogon-like mycorrhizae were ob-
served only on Douglas-fir. None of the major types was ex-
clusive to birch. In the 1994 samples, 91% of the birch and
56% of the Douglas-fir mycorrhizal root tips were classified as
types common to the two species.
Diversity of ectomycorrhizae
The diversity of ectomycorrhizae was examined by calculating
Simpson’s diversity values, as well as the two components of
diversity: richness and evenness. The average richness
(number of ectomycorrhizal types) per seedling increased be-
tween 1992 and 1993 for both species (Figs. 2aand 2b), with
little further increase in 1994. There were more types per
Douglas-fir than per birch seedling in 1992 (P=0.03 for two-
factor planned contrast), but with little difference in the later
years (P> 0.4). The same was true for the number of mycor-
rhizal types observed per plot (Table 3). Significant effects of tree
species mixture or density (data not shown) on mycorrhizal
Macroscopic description Mantle type(s) Emanating hyphae Mycelial strands Cystidia Year/host*
OUC 350
Dark brown – rust
mycorrhiza; rough
texture; abundant
brown–red emanating
hyphae; brown–red strands
Outer: irregular
nonlocking synenchyma
Inner: not visible
2–3 µm in diameter;
brown; no clamps
Absent Absent 93F/D
Basidiomycotina
OUC 360
Tan–brown mycorrhiza;
smooth with frequent
emanating hyphae
Outer: net synenchyma,
4–6 µm wide
Inner: not visible
1.5–2 µm wide; frequent
clamps; smooth
Absent Absent 93F/D
94F/D
OUC 370
Tan mycorrhiza; swollen and
smooth; rare emanating
hyphae
Outer: net prosenchyma,
1.5 µm wide
Inner: net prosenchyma,
1.5–2 µm wide
3µm wide; no clamps;
smooth or verrucose
Absent Absent 94F/B+D
OUC 380
Tan or brown mycorrhiza;
rough texture; rare
emanating hyphae; dark
brown strands
Outer: felt prosenchyma,
2–4 µm wide
Inner: not visible
3µm wide; no clamps;
golden brown; smooth
Absent Absent 94F/B+D
OUC 390
Black or dark brown
mycorrhiza, covered with
white “fluff”; abundant
emanating hyphae
Outer: felt prosenchyma,
2µm wide
Inner: felt or net
prosenchyma, 1 µm wide
1–3 µm wide; no clamps;
smooth
Loosely organized Absent 94F/D
OUC 400
Dark brown mycorrhiza;
rough and grooved
appearance; no emanating
hyphae
Outer: net prosenchyma,
2–3 µm wide
Inner: nonlocking
synenchyma, 5–6 µm
wide
4–5 µm wide; clamps
common; smooth;
straight
Absent Absent 94F/B+D
OUC 410
Black or brown mycorrhiza;
rough texture; rare black
emanating hyphae
Outer: net synenchyma,
4µm wide
Inner: irregular locking
synenchyma, 4–5 µm
wide
7µm wide; no clamps;
smooth; clear
Absent Common;
200–250 µmin
length; 3–4 µm
wide; hyaline and
smooth with
rounded tips and
no clamps
94F/B
*91, 1991; 92, 1992; 93, 1993; 94, 1994; G, greenhouse bioassay; F, field bioassay; B, paper birch; D, Douglas-fir.
Table 2 (concluded).
Jones et al. 1881
© 1997 NRC Canada

richness were not observed at the seedling (Fig. 2, P> 0.4) or
whole-plot level (Table 3, P> 0.6).
As the Thelephora mycorrhizae became less abundant on
field-grown birch (Fig. 1a), average evenness per seedling in-
creased (Fig. 2c). A similar, but less pronounced pattern oc-
curred in Douglas-fir (Fig. 2d). Evenness was higher in
Douglas-fir than in birch during the first year of the experiment
(seedlings, P=0.03; whole plot, P=0.02) when Thelephora
mycorrhizae dominated the birch samples, but the difference
was no longer apparent over time (Fig. 2; Table 3). Evenness
per Douglas-fir seedling was higher in mixed plots than in
single-species plots in 1993 (P=0.07, one-factor planned con-
trast) and 1994 (P=0.09). Evenness per seedling (P> 0.1,
two-factor planned contrasts, data not shown) and per plot (P>
0.4) was not affected by planting density.
Because evenness and richness increased with time, diver-
sity, as calculated by the Simpson’s index, also increased be-
tween 1992 and 1994, especially for birch (Figs. 2eand 2f;
Table 3). Ectomycorrhizal diversity was higher in Douglas-fir
than in birch in 1992 (P=0.008) only. The Simpson’s diver-
sity index per seedling was not significantly affected by tree
diversity (P> 0.1) or density (P> 0.1, data not shown). Nei-
ther the density nor mixture treatments affected diversity at the
whole-plot level (P> 0.4, two-factor planned contrasts; Table 3).
Fig. 1. Proportions of the six most common mycorrhizal types on roots of (a) paper birch and (b) Douglas-fir sampled from three field sites in
the fall of 1992, 1993, and 1994. Percentages are based on observations of 4300–6700 live mycorrhizal tips per species per site in 1992,
10 600 – 12 000 in 1993, and 11 000 – 12 000 in 1994.
Can. J. For. Res. Vol. 27, 1997
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Greenhouse seedlings
Birch
Thelephora,Cenococcum, and MRA mycorrhizae occurred in
abundance on the birch seedlings in 1991 (Fig. 3a). Only the
MRA type differed, but not significantly so, among treatments
(P=0.15 for a one-factor ANOVA on square root transformed
data; P> 0.3 for the other fungi). The frequency of occurrence
of MRA was higher in the 800/400, 800/800, 3200/1600, and
1600/0 treatments (>13%) than in the others (<3%). This pat-
tern was not repeated in 1992, and there were no treatment
effects in subsequent years. No mycorrhizae were present on
seedlings grown in soil collected from the field sites and then
sterilized.
The greenhouse bioassays predicted the general composi-
tion of the ectomycorrhizal community that developed on
field-grown birch seedlings (r2=0.84, P< 0.0001, df =29: 5
types ×2 years ×3 sites, simple regression of the data plotted
in Figs. 1aand 3afor 1992 and 1993). For example, Thele-
phora mycorrhizae were the most abundant type on both
greenhouse- and field-grown seedlings. The ability of the
greenhouse bioassay to predict changes in the frequency of
individual types among sites and between the two years varied.
It was poor for Thelephora mycorrhizae (r2=0.082, P=0.6,
df =5: 3 sites ×2 years) but better for E-strain I (r2=0.63, P=
0.06) and MRA (r2=0.71, P=0.04). MRA mycorrhizae were
more common in the bioassays than in the field.
Douglas-fir
The types of mycorrhizae on Douglas-fir seedlings grown in
the greenhouse (Fig. 3b) were similar to those that developed
in the field (r2=0.51, P=0.0001, df =35: 6 types ×2years×
3 sites; Thelephora:r2=0.69, P=0.04, df =5; E-strain I: r2=
0.50, P=0.1; MRA: r2=0.83, P=0.01; Rhizopogon:r2=0.58,
P=0.08). There was no correlation between the proportion of
Cenococcum mycorrhizae formed in the greenhouse and in the
field (r2=0.079, P=0.6). In general, Rhizopogon mycorrhizae
were less common and MRA mycorrhizae more common on
greenhouse than on field seedlings.
Douglas-fir seedlings produced fewer roots (Fig. 3) than
did birch seedlings when grown in the greenhouse. Therefore,
data were less suitable for statistical analyses of treatment ef-
fects on individual mycorrhizal types. Nevertheless, by 1993,
when numbers were high enough to analyze, there was no
Richness Evenness Diversity
1992
Birch
Malakwa 3.4±0.5 0.27±0.06 0.20±0.05
Hidden Lake 4.4±0.2 0.47±0.07 0.36±0.05
Adams Lake 4.0±0.4 0.44±0.08 0.33±0.07
Douglas-fir
Malakwa 4.6±0.4 0.73±0.04 0.57±0.05
Hidden Lake 5.2±0.5 0.76±0.03 0.61±0.03
Adams Lake 5.4±0.7 0.85±0.01 0.67±0.02
1993
Birch
Malakwa 9.2±0.7 0.64±0.04 0.56±0.04
Hidden Lake 9.0±0.7 0.50±0.12 0.66±0.09
Adams Lake 9.6±1.1 0.80±0.02 0.71±0.02
Douglas-fir
Malakwa 12.2±0.4 0.83±0.03 0.76±0.03
Hidden Lake 8.6±1.0 0.71±0.04 0.61±0.02
Adams Lake 10.0±1.2 0.72±0.03 0.65±0.03
1994
Birch
Malakwa 7.0±0.9 0.67±0.04 0.57±0.02
Hidden Lake 10.8±0.8 0.85±0.03 0.75±0.01
Adams Lake 10.8±1.2 0.88±0.01 0.79±0.02
Douglas-fir
Malakwa 11.4±1.5 0.81±0.03 0.74±0.03
Hidden Lake 8.8±0.4 0.80±0.03 0.71±0.03
Adams Lake 12.2±1.1 0.85±0.02 0.78±0.02
Note: Values are means ±SE of the five treatment plots at each site. A
two-factor ANOVA did not detect differences between treatments (P> 0.2),
but did detect species effects in 1992 (P=0.02 for richness, evenness, and
diversity). There were significant site ×species interactions in 1993
(diversity, P=0.02; evenness, P=0.06) and in 1994 (richness,P=0.03;
evenness, P=0.01; diversity, P=0.001).
Table 3. Number of mycorrhizal types (richness) and their
evenness and Simpson’s diversity index, per plot, from paper birch
and Douglas-fir roots collected from three sites in 1992, 1993, and
1994.
Fig. 2. Richness (number of morphological types of
ectomycorrhizae), evenness (equitability), and Simpson’s diversity
index (=1−Σp
i
2
, where piis the proportion of live ectomycorrhizae
of type i) per seedling for the ectomycorrhizal communities of (a,c,
e) paper birch and (b,d,f) Douglas-fir planted in mixed (solid bars)
or single-species plots (stippled bars) for 4 (1992), 16 (1993), or
28 months (1994). Values are means ±1 SE; N=3 sites. Richness
(P=0.03 for two-factor planned contrast), evenness (P=0.03), and
diversity (P=0.008) were higher for Douglas-fir than for birch
seedlings in 1992 (P> 0.1 for 1993 and 1994). Planting treatments
did not affect richness or diversity; evenness was higher for
Douglas-fir in mixed than in single-species plots in 1993 (P=0.07)
and 1994 (P=0.09).
Jones et al. 1883
© 1997 NRC Canada

indication of a treatment effect on the proportion of different
ectomycorrhizal types formed by Douglas-fir in the green-
house (P> 0.3, one-factor ANOVA, data not shown).
Diversity of ectomycorrhizae
Neither richness, Simpson’s diversity index, nor evenness
per seedling was affected by host mixture or density treatment
in either year (P> 0.05, two-factor planned contrasts, data not
shown); however, evenness (P=0.07, two-factor planned con-
trast) and diversity (P=0.04) were higher, when calculated per
plot, for seedlings grown in soil collected from birch plots in
1993 (Fig. 4) than for seedlings grown in soil from Douglas-fir
or from mixed plots. The abundances of the most common
mycorrhizal types were not individually affected.
Comparison of field and greenhouse seedlings
In 1992 and 1993, the number of types detected on greenhouse-
and field-grown birch seedlings was similar: nine types in
1992 (Table 2) and 17 in the greenhouse versus 20 in the field
in 1993. Only 19 types were found on greenhouse-grown
Douglas-fir in 1993, compared with 25 on field-grown plants.
Of the 10 mycorrhizal types observed in the field but not in the
Fig. 3. Proportions of the six most common mycorrhizal types on roots of (a) paper birch and (b) Douglas-fir seedlings grown in a greenhouse
in soils collected from three sites in the fall of 1991, 1992, and 1993. Percentages are based on observations of at least 8600 live mycorrhizal
tips per site for birch and 140 (Malakwa) to 660 (Hidden Lake) for Douglas-fir in 1991, at least 3000 per site for birch and 240 (Malakwa) to
1102 (Hidden Lake) for Douglas-fir in 1992, and 5600 (Adams Lake) to 7700 (Malakwa) for birch and 3200 (Malakwa) to 5300 (Adams
Lake) for Douglas-fir in 1993.
Can. J. For. Res. Vol. 27, 1997
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© 1997 NRC Canada

greenhouse (Table 2), all except three were found on only one
field-grown seedling. In total, types that did not develop on
greenhouse-grown seedlings represented 1.7% of the mycor-
rhizal roots on field-grown Douglas-fir and 0.1% on field-
grown birch.
Discussion
The types of ectomycorrhizae formed by field-grown birch and
Douglas-fir changed with time and differed among sites. Plant-
ing mixtures of these two species increased the evenness of
ectomycorrhizal types present on the root systems of Douglas-
fir 16 and 28 months after outplanting. In contrast, planting
density did not significantly affect ectomycorrhizal diversity
on these two species. In addition, the greenhouse bioassay pre-
dicted the types of ectomycorrhizae that developed on field-
grown seedlings during the first few years following
outplanting.
Changes with time
The main changes in the types of ectomycorrhizae associated
with field seedlings over the first 3 years following outplanting
were a decrease in the prevalence of Thelephora mycorrhizae
and an increase in the prevalence of one type of E-strain my-
corrhiza and of MRA with birch (Fig. 1). In addition, the
number of types of ectomycorrhizae increased over the 3 years
(Table 2). It is common to find that the number of types of
ectomycorrhizae associated with planted seedlings increases
(Dahlberg and Stenström 1991) and changes over the first
10 years following outplanting (Last et al. 1984; Danielson
1991). A change in ectomycorrhizae with time also occurs in
forests developing under more natural situations, including
following fire (Visser 1995).
The increase in ectomycorrhizal richness also occurred in
greenhouse seedlings between 1991 and 1993. The low rich-
ness in 1991 was likely because many of the ectomycorrhizal
roots originally on the site were removed in 1991 during des-
tumping. Soils collected in 1992 and 1993 contained inoculum
from increasing numbers of ectomycorrhizal fungi because the
samples included live roots from the regenerating birch and
Douglas-fir as well as other ectomycorrhizal plants that regen-
erated naturally on the sites.
Colonization by Thelephora is usually attributed to nursery
inoculum, and the proportion of Thelephora mycorrhizae com-
monly decreases during the first few years following out-
planting (Danielson 1991; Villeneuve et al. 1991; Berch and
Roth 1993; Richter and Bruhn 1993). It is possible that Thele-
phora was associated with the seedlings when they came from
the nursery. Although no ectomycorrhizae were observed on
seedlings of each tree species from the same batch as those
planted, nursery mycorrhizae frequently have very thin man-
tles or lack a mantle altogether and may have been missed at
the low magnification used (Roth and Berch 1992). The de-
crease of Thelephora mycorrhizae in the 2 years following
outplanting would be consistent with the nursery as the source
of Thelephora. Nevertheless, the possibility that colonization
occurred from field inoculum cannot be disregarded. Thele-
phora mycorrhizae were common in the greenhouse bioassay,
and the lack of mycorrhizae in control plants grown in steril-
ized soil indicates that Thelephora inoculum was present in the
field soil. The decline in abundance of Thelephora mycorrhi-
zae over time could have been due to a lower long-term com-
petitive ability on these sites compared with fungi such as
Rhizopogon, E-strain, Cenococcum, and MRA.
In contrast with our results, Richter and Bruhn (1993) ob-
served a decrease in the number of mycorrhizae from which
ectendomycorrhizal fungi, such as E-strain, were isolated dur-
ing the 4 years following outplanting of red pine (Pinus resi-
nosa Ait.) into hardwood sites, and they cited similar results
by other workers. Danielson (1991) also found a reduction in
the dominance of E-strain fungi on spruce 4 years following
outplanting. It may be that the E-strain fungi in these other
studies were greenhouse contaminants (Simard et al. 1997a)
and poorly suited to the specific field sites. Successional pat-
terns of ectomycorrhizae following outplanting tend to be site
specific (Chu-Chou and Grace 1990). Danielson and Pruden
(1989) disproved the idea that E-strain fungi are dominant only
on newly planted seedlings; however, they attributed their re-
sults to the fact that they were studying root systems of white
spruce (Pinus glauca (Moench) Voss) planted in alkaline prai-
rie soils where the normal succession of ectomycorrhizae may
have been prohibited.
Ecology of “pioneer” fungi
In this study, all of the mycorrhizae comprising 5% or more of
the root system of either species in the field (Fig. 1) were ones
Fig. 4. Richness, evenness, and Simpson’s diversity index per plot
for the ectomycorrhizal communities of (a,c,e) paper birch and (b,
d,f) Douglas-fir grown in a greenhouse in soils collected 4 (1992)
or 16 months (1993) from mixed birch–Douglas-fir plots (solid
bars), plots planted with only birch (stippled bars), or plots planted
with only Douglas-fir (cross-hatched bars). Values are means ±
1 SE; N=3 sites. Results of two-factor (tree species and mixture
treatment) planned contrasts: 1992 richness, evenness, and
diversity, P> 0.3; 1993 richness, P=0.5; 1993 evenness, P=0.07;
1993 diversity, P=0.04.
Jones et al. 1885
© 1997 NRC Canada

that commonly are found in nurseries and in the first few years
following outplanting (Mason et al. 1987; Chu-Chou and
Grace 1990; Danielson 1991; Hunt 1991; Richter and Bruhn
1993). Thelephora,Cenococcum, MRA, Rhizopogon, and E-
strain mycorrhizae are commonly formed on containerized
nursery seedlings and in substrates, such as mine spoil, which
lack ectomycorrhizal roots (Danielson and Pruden 1989;
Danielson 1991), indicating that the fungi forming these my-
corrhizae can disperse easily. In this study, all these mycorrhizae
also were found on the greenhouse seedlings, indicating that
the fungi involved can colonize roots via mycelia which is not
already attached to living roots. This also characterizes mycor-
rhizal fungi designated as “early stage” by Mason et al. (1982).
Newton (1992) preferred the term “pioneer” fungi because
they can be found on any age of tree, but agreed that a key
characteristic is their ability to colonize roots from small
amounts of isolated mycelia or from spores. E-strain and
Thelephora spp. were categorized as early stage fungi and
Cenococcum and MRA as “multistage” fungi in the natural jack
pine (Pinus banksiana Lamb.) stands studied by Visser (1995).
The mycorrhizae that occurred at frequencies of between 1
and 4% in the second year following outplanting included
types resembling those formed by Lactarius spp., Rus-
sula spp., Laccaria spp., and Tomentella spp. (Table 2). Lac-
tarius spp. and Russula spp. are “late-stage” fungi according
to Mason et al. (1982) and Visser (1995) because they form
mycorrhizae from mycelial inoculum only under sterile con-
ditions, or when already attached to another seedling (Fleming
1984; Fox 1986). Inoculum for these fungi could have been
provided by other ectomycorrhizal species on the site, such as
trembling aspen or black cottonwood. If there was a depend-
ence on inoculum from living roots, the higher density plant-
ings might be expected to have a higher number of
mycorrhizae formed by these late-stage fungi; however, the
frequencies of these fungi did not differ between treatments.
In addition, these fungi also were present in the bioassays, so
in this system, they appear to colonize roots from a small
amount of isolated inoculum.
Site effects
Although this experiment was not designed to test differences
among the sites, obvious differences in the ectomycorrhizal
communities were present. The most striking was the lower diver-
sity of ectomycorrhizae on birch roots at Malakwa (Table 3). The
abundance of Thelephora mycorrhizae remained higher on
both birch and Douglas-fir roots at Malakwa than at the other
two sites. The sites are similar with respect to soil type and
texture, parent material, and original overstory tree species.
Simard et al. (1997b) found Thelephora mycorrhizae on
Douglas-fir seedlings planted in intact forests at Adams Lake
but not at Malakwa. This suggests that the type of inoculum
present in the original forest stands may have differed. In ad-
dition, the three sites had different logging and site preparation
histories. At Malakwa, grass and clover seed were applied in
1989 and again in 1992, and these arbuscular mycorrhizal
plants have become more common there than at the other sites.
Ectomycorrhizal development can be inhibited at sites with an
abundance of arbuscular mycorrhizal plants (Ellis and Pen-
nington 1992; Richter and Bruhn 1993; Amaranthus and Perry
1994). Malakwa was the only site not to be burned following
logging, but this is not likely to be the cause of differences
among the sites. The bunching-and-burning treatment at Hid-
den Lake would have had only localized effects on the soil.
Diversity of ectomycorrhizae
The lowest number of ectomycorrhizal types occurred in the
first year when outplanted seedlings were becoming estab-
lished and were suffering mortality from drought and frost;
more than 40 types were observed 28 months after out-
planting. There can be 100 species of ectomycorrhizal fungi in
a typical Douglas-fir stand (Allen et al. 1995), and Douglas-fir
can associate with 2000 mycobionts across its range (Trappe
1977). Bruns (1995) concluded that resource partitioning is
likely responsible for some of the ectomycorrhizal diversity on
a single tree species, although disturbance or density-dependent
processes may also be important.
Spatial variability in roots (age, physiology) and soil re-
sources (concentrations and forms of nutrients, pH, aeration,
moisture) is enormous. Because the 5000 species of ectomy-
corrhizal fungi differ considerably in their ability to transport
water, break down organic nutrients, protect roots against
pathogens, and colonize specific types of soils or other sub-
strates, the tree is likely to gain physiological versatility by
being associated with a high diversity of ectomycorrhizal
fungi. The differences among fungi mean that on a site with a
high diversity of mycorrhizal fungi the nutrient-absorbing part
of the mycorrhiza (the hyphae) can vary for different soil mi-
crosites through which the root system grows. It is important
to begin to investigate the level of physiological diversity and
redundancy among ectomycorrhizae in a root system.
Diversity indices are typically calculated based on species
and comprise two components: species richness and evenness.
The use of evenness in calculating the diversity of ectomycor-
rhizal fungi is not appropriate when fungal individuals are not
distinguished (Bruns (1995). In this study, we used detailed
microscopic examination to classify mycorrhizae into mor-
phological types, and these morphological types were used to
calculate richness, evenness, and diversity indices. Since we
were studying ectomycorrhizal diversity and not the diversity
of ectomycorrhizal fungi, the calculation of evenness is appro-
priate in this case. Mycorrhizal root tips were the units of clas-
sification because we were interested in nutrient uptake and
exchange, and the individual root tip is the organ by which the
plant ultimately receives nutrients.
The use of morphological types may overestimate or, more
likely, underestimate the number of ectomycorrhizal fungal
species. Two different developmental stages of mycorrhizae
formed by the same fungal species can be categorized as two
different types. It is even more likely that ectomycorrhizae
formed by closely related fungi cannot be distinguished. Nev-
ertheless, this approach is much more accurate than the count-
ing of sporocarps to determine the relative abundance of
different ectomycorrhizal fungi (Gardes and Bruns 1996). It is
a goal of our research program and others to use molecular
techniques to confirm and refine the separation of mycorrhizae
based on morphological characteristics, but this will require
many years. In the meantime, the current approach has pro-
duced valuable information on the effect of various silvicultu-
ral practices (Dahlberg and Stenström 1991; Kernaghan et al.
1995; Jones et al. 1996; Simard et al. 1997a) and length of
primary (Helm et al. 1996) or secondary succession (Visser
1995) on ectomycorrhizal diversity.
Can. J. For. Res. Vol. 27, 1997
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Treatment effects on evenness
An increase in evenness implies a decrease in the abundance
of the dominant ectomycorrhizal types and (or) an increase in
the abundance of less common ones. In our study, the effect of
mixing birch and Douglas-fir was to increase the abundance
of minor, but common types on Douglas-fir. In particular,
Cenococcum and MRA types were more common, but not
significantly so (P=0.1), in mixed than in single-species plots
in 1993. We did not observe a decrease in the abundance of
dominant types such as Thelephora or Rhizopogon on
Douglas-fir in mixed versus single-species plots, although Si-
mard et al. (1997a) observed a decrease in the abundance of
Rhizopogon mycorrhizae on Douglas-fir grown in mixture
with birch in Adams Lake soil and of Thelephora mycorrhizae
when seedlings were grown in contact with the roots of mature
trees (Simard et al. 1997c).
There are two factors that may have contributed to the
higher evenness of ectomycorrhizae found on Douglas-fir
seedlings when outplanted in mixture with birch than when
planted alone. Firstly, ectomycorrhizal fungi vary in their de-
gree of host specificity (Molina et al. 1992), but this host range
often can be expanded when the fungus is already mycorrhizal
with a primary host (Massicotte et al. 1994; Smith et al. 1995).
Secondly, birch trees modify the soil in which they grow, both
chemically and biologically, and this may alter the relative
ability of different ectomycorrhizal fungi to grow and to colo-
nize Douglas-fir.
The appearance of a new mycorrhizal type on one of the
species growing in the mixed plots would have been the best
evidence of a specificity phenomenon, but neither birch nor
Douglas-fir seedlings formed a greater number of mycorrhizal
types when grown in mixture than when grown in pure plots
(Fig. 2). In addition, there were no ectomycorrhizal types
unique to birch or Douglas-fir seedlings grown in mixed rather
than single-species plots (data not shown). In contrast, Simard
et al. (1997a) found that Douglas-fir grown in Adams Lake
soil in a greenhouse formed Tuber mycorrhizae only when
growing together with birch. Most of the common mycorrhizal
types observed in this study were formed by fungi with broad
host ranges (Molina et al. 1992; Rhizopogon is an exception).
Nevertheless, specificity phenomena may influence ectomy-
corrhizal evenness. A fungus may colonize a secondary host
to a greater extent (e.g., Douglas-fir) only when grown with a
primary one (e.g., paper birch) because it obtains more carbon
from the primary host than from the secondary one (Massicotte
et al. 1994). For example, if a fungus obtained more carbon
when mycorrhizal with birch than with Douglas-fir, this could
result in the increase in abundance of that mycorrhizal type on
Douglas-fir when the two tree species are grown in mixture.
Birch may have caused increased ectomycorrhizal even-
ness on Douglas-fir by changing the chemical or biological
status of the soil in ways that favoured an increase in abun-
dance of some less common mycorrhizal types. The higher
evenness observed when seedlings were grown in soil from
birch plots than in soil from Douglas-fir or mixed plots (Fig. 4)
supports this idea. Soil in which birch has grown typically has
higher pH, total and extractable N, exchangeable Ca and Mg,
and amount of C available for microorganisms, but lower or-
ganic matter content, than conifer soil (Troedsson 1985;
Van Cleve et al. 1986; Liljelund 1988; Bradley and Fyles
1995). The bacterial community differs in the vicinity of hard-
wood roots (Amaranthus et al. 1990; Borchers and Perry
1990), and specific bacteria can affect the formation of mycor-
rhizae by different ectomycorrhizal fungi (Duponnois et al.
1993; Fitter and Garbaye 1994; Garbaye 1994). The amount
of inoculum of specific ectomycorrhizal fungi likely differed
between the mixed and single-species plots. For example, both
Cenococcum and MRA mycorrhizae were more common on
birch than on Douglas-fir, so the inoculum levels of these fun-
gal types would be higher where birch had grown. Other green-
house bioassay studies have found that Douglas-fir forms more
Cenococcum mycorrhizae when grown in soil collected under
hardwoods than from areas some distance away (Borchers and
Perry 1990).
Sharing of mycorrhizal fungi by tree species
In the present study, 91% of the birch and 56% of the Douglas-
fir mycorrhizal roots examined from the field formed the same
mycorrhizal types. A high potential exists for the direct trans-
port of nutrients or carbon through hyphal linkages between
these two tree species. Recent studies on carbon transfer be-
tween paper birch and Douglas-fir at Adams Lake (Simard
et al. 1997c) have settled the question of whether net transfer
can occur from one plant species to the other and whether the
amounts involved are ecologically significantly (Newman
1988; Miller and Allen 1992). In the field site at Adams Lake,
the net transfer of isotopically labelled carbon from birch to
Douglas-fir was equivalent to 4% of the labelled carbon fixed
by the birch and 7% of that fixed by Douglas-fir. Thus the
presence of a shared mycorrhizal community would be ex-
pected to reduce the negative effects of competition between
the two species. Reduced interspecific competition occurred
when inoculated versus noninoculated Douglas-fir and pon-
derosa pine (Pinus ponderosa Dougl. ex P. & C. Laws.) were
grown together in sterilized soils (Perry et al. 1989).
Comparison of greenhouse and field bioassays
The greenhouse bioassays served two functions. The first was
to test for differences in mycorrhizal inoculum across the sites
prior to the installation of the treatments. The greenhouse bio-
assay of soils collected in 1991 did not reveal any differences
in inoculum across the sites; however, the low numbers of
roots produced made statistical evaluation difficult. The sec-
ond purpose was to test for treatment effects on mycorrhiza
formation by birch or Douglas-fir, even on plots where the
species being tested had not been planted. Greenhouse bioas-
says allowed us to detect effects that were due to changes in
biological and chemical conditions in the soil caused by birch
or Douglas-fir (see above). Thus, for example, we were able to
determine that Douglas-fir seedlings formed a greater even-
ness of mycorrhizal types when grown in soils collected from
monoculture birch plots than from mixed or monoculture
Douglas-fir plots.
There are several criticisms that apply to the use of green-
house bioassays for prediction of ectomycorrhizal communi-
ties in the field. Firstly, because the soil is typically disturbed
during collection, and because connections between ectomy-
corrhizal hyphae and living roots are severed, some fungi are
less likely to form mycorrhizae in greenhouse bioassays than
in the field (Deacon and Fleming 1992; Newton 1992). In
addition, seedlings tend to form mycorrhizae with different
fungal species than mature trees. Lastly, contamination by
Jones et al. 1887
© 1997 NRC Canada

spores of ectomycorrhizal fungi that are present in the green-
house, or which enter through the greenhouse windows, occurs
frequently. Nevertheless, in this study, where the greenhouse
was free of mycorrhizal contamination, there was remarkable
agreement with respect to the proportion and abundance of the
major types of fungi between bioassay and field seedlings over
the first 2 years following outplanting (Figs. 1 and 2). Mycor-
rhizal types observed on field seedlings, but not on bioassay
seedlings, comprised only 1.7% of tips examined on field-
grown Douglas-fir and 0.1% on birch. This indicates that most
of the mycorrhizae found on field seedlings were formed by
fungi that can colonize without connections to living roots of
other trees. The results are not surprising given that the green-
house and field bioassay seedlings were similar in age and that
both were growing on recently disturbed soils.
We conclude that, in the absence of contaminants, green-
house bioassays can predict the major ectomycorrhizal fungi
that colonize young seedlings on clearcuts during the first few
years of outplanting. For the reasons outlined above, greenhouse
bioassays would not be expected to predict the proportion of
ectomycorrhizal types found on mature trees, or on seedlings
planted in less disturbed environments.
Acknowledgments
This study was funded by the Hardwood Management Subpro-
gram of the Canada – British Columbia Partnership Agreement
on Forest Resource Development (1991–1995). Technical as-
sistance by Kerry McLean is gratefully acknowledged. We are
grateful to Drs. Shannon Berch, Tom Bruns, and Bob Danielson
and to three anonymous reviewers for comments on an earlier
version of this paper.
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