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Role of mycorrhizal networks and tree proximity inectomycorrhizal colonization of planted seedlings

Francois P. TESTEa,*, Suzanne W. SIMARDb, Daniel M. DURALLc

aDepartment of Renewable Resources, University of Alberta, Edmonton, AB, Canada T6G 2H1bDepartment of Forest Sciences, University of British Columbia, Vancouver, BC, Canada V6T 1Z4cBiology and Physical Geography Unit, University of British Columbia Okanagan, Kelowna, BC, Canada V1V 1V7

a r t i c l e i n f o

Article history:

Received 21 August 2008

Revision received 24 October 2008

Accepted 13 November 2008

Published online 18 January 2009

Corresponding editor:

John W.G. Cairney

Keywords:

Colonization

Distance effect

Diversity

Douglas-fir

Ectomycorrhiza

Fungal community

Mycorrhizal network

Residual tree

* Corresponding author. Tel.: þ1 780 492 315E-mail address: teste@ales.ualberta.ca (F.

1754-5048/$ – see front matter ª 2008 Elsevidoi:10.1016/j.funeco.2008.11.003

a b s t r a c t

The importance of mycorrhizal network (MN)-mediated colonization under field conditions

between trees and seedlings was investigated. We also determined the combined influ-

ences of inoculum source and distance from trees on the ectomycorrhizal (EM) community

of seedlings. On six sites, we established trenched plots around 24 residual Pseudotsuga

menziesii var. glauca trees and then planted seedlings at four distances (0.5, 1.0, 2.5, and

5.0 m) from the tree into four mesh treatments that served to restrict inoculum access (i.e.,

planted into mesh bags with 0.5, 35, 250 mm pores or directly into soil). Ectomycorrhizal

communities were identified after two growing seasons using morphological and molec-

ular techniques. Mesh treatments had no effect on seedling mycorrhizal colonization,

richness, or diversity, suggesting that MN-mediated colonization, was not an essential

mechanism by which EM communities were perpetuated to seedlings. Instead, wind-borne

and soil inoculum played an important role in seedling colonization. The potential for MNs

to form in these forests was not dismissed, however, because trees and seedlings shared

83 % of the abundant EM. Seedlings furthest from trees had a simpler EM community

composition and reduced EM richness and diversity compared to seedlings in closer

proximity.

ª 2008 Elsevier Ltd and The British Mycological Society. All rights reserved.

Introduction inoculum potential or perhaps directly by a MN (Borchers &

Mycorrhizal networks (MNs) are fungal hyphae that connect

the roots of at least two plants, assisting the spread of

mycorrhizal fungi and potentially providing a conduit for

interplant resource transfer (Simard & Durall 2004). Estab-

lishing seedlings are colonized by propagules of ectomycor-

rhizal (EM) fungi (spores, sclerotia, hyphae of excised EM root

tips) or when their roots contact mycelium of an established

plant (i.e., MN-mediated). Established trees can facilitate

seedling EM colonization indirectly by contributing to the soil

4; fax: þ1 780 492 1767.P. Teste).er Ltd and The British My

Perry 1990; Berman & Bledsoe 1998; Dickie et al. 2002).

Mycorrhizal network-mediated colonization of seedlings by

established vegetation was first demonstrated in the field by

Fleming (1983, 1984), and has been suggested as the most

important method of colonization in forests (Robertson 1954;

Newton 1992). This is supported by studies showing that

seedlings planted near mature trees usually have higher EM

fungal species richness and diversity than those in the open

(Kranabetter & Wylie 1998; Durall et al. 1999; Cline et al. 2005).

Distinct shifts in EM community composition (i.e., species

cological Society. All rights reserved.

22 F.P. Teste et al.

occurrence and abundance) have also occurred when seed-

lings have regenerated near trees compared to far away

(Dickie et al. 2002; Outerbridge & Trofymow 2004; Dickie &

Reich 2005).

None of these field studies have clearly shown, however,

that MN-mediated colonization is the dominant mechanism

for EM fungal spread. Newman (1988) suggested that MN-

mediated colonization of seedlings would be faster, greater,

and more species-rich than seedling colonization in the

absence of a MN. However, these hypotheses have yet to be

carefully tested. Separating colonization by MNs from that by

EM soil inoculum or wind-dispersed spores is needed to

determine the ecological importance of MN-mediated

colonization.

The importance of MNs to plant and fungal ecology, and

their role in ecosystem recovery following disturbance, is

gaining increasing attention (Taylor 2006). The likelihood that

MNs form in communities dominated by EM plants appears

high given low host specificity of most EM fungal species

(Molina et al. 1992). Although there have been numerous

studies of EM fungal specificity and function, controversy still

remains over whether MNs can physically connect tree roots

of the same or different species (Selosse et al. 2006). This is

likely due to the artificial conditions of laboratory studies

(Brownlee et al. 1983; Read et al. 1985; Finlay & Read 1986; Wu

et al. 2001) or to a lack of direct observations of MNs in the field

(Simard & Durall 2004; Booth 2004; McGuire 2007). However,

recent studies using molecular techniques (Kennedy et al.

2003; Lian et al. 2006) are providing strong evidence that MNs

occur in the field.

We define mycorrhizal network potential as a measure of

the likelihood that a MN occurs between two plants. If two

plants growing in close proximity share the same EM taxa,

then there is a strong likelihood that a MN exists between

them, hence demonstrating high MN potential. In this study,

we used morphological and molecular techniques to compare

EM fungi on residual trees and seedlings in order to determine

the MN potential between these plants in the field.

The objectives of this study were firstly, to determine the

relative importance of MN-mediated colonization versus

other modes of seedling colonization, secondly, to determine

the degree of EM similarity between Douglas-fir trees and

seedlings in the field, and thirdly, to determine how restricting

the inoculum source and varying the proximity to conspecific

residual trees affect the EM colonization of planted seedlings.

The following hypotheses were tested: (i) MN-mediated colo-

nization is the dominant mechanism fostering similar EM

communities between residual trees and seedlings; (ii) there is

a distance at which the EM community of planted seedlings is

most similar to residual trees; (iii) there is a high degree of EM

community similarity between seedlings and conspecific

trees; thus, they share some of the same EM taxa; and (iv) EM

status (colonization, richness, and diversity levels) of seed-

lings is greatest where there is access to all inoculum sources.

The use of mesh barriers in field experiments has been

advocated to control and limit MN connections between

plants (Robinson & Fitter 1999). In this study we used mesh

bags to investigate the potential effects of MNs originating

from mature trees on EM colonization of planted seedlings.

We did not sterilize the soil inside the mesh bags, nor did we

remove any excised tree roots from the mesh bags after

installation because our objective was to determine whether

either MNs of existing trees or soil inoculum (including

excised tree roots) was the dominant mechanism by which

planted seedlings are colonized under typical reforestation

procedures. The possible side effects related to inserting the

mesh bags in this study are comparable to the soil distur-

bances that tree planters create when planting seedlings.

Material and methods

Site description

The study was conducted within the Thompson Dry, Cool

Interior Douglas-fir (IDF) biogeoclimatic variant (IDFdk2) 25–

50 km north of Kamloops, British Columbia, Canada (spanning

50�510N, 120�250W to 50�560N, 120�180W). This area is near the

northern-most limit of interior Douglas-fir (Pseudotsuga men-

ziesii var. glauca) in North America, where precipitation ranges

from 300 to 750 mm annually (Meidinger & Pojar 1991). The six

study sites had loamy Gray Luvisolic soils (Soil Classification

Working Group 1998) and tree canopies dominated by interior

Douglas-fir. Understory vegetation was dominated by arbus-

cular mycorrhizal (AM) pinegrass (Calamagrostis rubescens),

nodding onion (Allium cernuum), white hawkweed (Hieracium

albiflorum), and several minor EM shrubs and herbs (Hagerman

et al. 2001).

Experimental design

Six study sites were selected in spring 2004. All sites had been

logged 1–11 years previously using variable retention har-

vesting, where a low density of pole-sized Douglas-fir trees

(see Table S1, Supplementary for characteristics) were

retained throughout. On each site, residual Douglas-fir trees at

least 30 m apart were numbered and then four were randomly

selected and assigned to one of four mesh treatments, where

seedlings were planted into bags with 0.5 mm, 35 mm or 250 mm

pore sizes, or directly into soil. Douglas-fir seedlings were then

planted at four randomly selected distances (0.5 m, 1.0 m,

2.5 m, 5.0 m) from the residual tree either directly into the soil

or into a mesh-bag (15 cm diameter and 35 cm deep) con-

taining carefully excavated soil. We minimized disturbance to

the MN by carefully excavating the soil in three distinct soil

layers (intact forest floor, mineral A horizon, and some of the

mineral B horizon) then placed these layers back in the same

order. Prior to planting the seedlings in the ground of the no

mesh treatment, the surrounding soil was disturbed to ensure

initial soil disturbance was consistent across all mesh treat-

ments. The four distance locations were offset from each

other to avoid spatial interdependence. These treatments

were organized in a split plot design with six replicate sites,

where the whole plot effect was mesh pore size and the split

plot effect was distance from the residual tree. The interior

Douglas-fir seedlings (Seedlot # 42309, British Columbia

Ministry of Forest and Range Tree Seed Center, Surrey, BC,

Canada) that were planted had been commercially grown in

a nursery for one year in 512A styroblocks� (Beaver Plastics,

Edmonton, AB, Canada). Douglas-fir seedlings were grown in

Role of mycorrhizal networks and tree proximity 23

containers for one year prior to planting, were non-mycor-

rhizal at the time of planting (based on a random subsample of

10 and determined using the methods described below), and

ranged in height from 13 to 30 cm.

The purpose of the mesh treatments was to assess the

capacity for MNs originating from residual trees to actively

colonize nearby seedlings, thus determining the importance

of MN-mediated colonization. Seedlings planted in a 0.5 mm

mesh bag could be colonized by wind-borne or soil propa-

gules, but not by the tree’s MN because the pores were too

small for hyphal penetration, preventing the formation of

mycorrhizal hyphal connections with other generalist EM

plants (Teste et al. 2006). Seedlings planted in 35 and 250 mm

mesh bags allowed us to distinguish MN colonization by

individual hyphae from that by rhizomorphs. All mesh bags

were made out of sturdy plain-weave nylon (Plastok Ltd.,

Birkenhead, UK), had a volume of 6185 cm3, and the 0.5, 35,

and 250 mm mesh had an estimated percent open pore space

of 13.3 %, 34.0 %, and 54.1 %, respectively. Seedlings planted

directly into soil could potentially be colonized by all inoc-

ulum sources, including short-distance hyphal connections

with ‘contact’ exploration type EM (Agerer 2001). The occur-

rence of short-distance MN connections was considerably

reduced in the 250 mm mesh treatment because the thickness

(w0.32 mm) of the nylon mesh was greater than the typical

length (0–0.25 mm) of the extraradical hyphae or cystidia of

EM ‘contact’ exploration types found in this study. The

purpose of the four distance treatments was to assess the

spatial influence of residual trees on EM communities of

planted seedlings.

A wedge-shaped area (35 m2) was trenched around each

tree in a southern direction to a soil depth of 50–70 cm to

exclude the influence of surrounding tree roots. Trenches

were lined with heavy polyethylene and refilled with exca-

vated soil. All trees within a 2 m radius of the sides of the

wedge-shaped areas and on the north side of the residual

trees were felled. All vegetation in the wedge-shaped area was

clipped regularly throughout the first growing season and at

the beginning of the second growing season to minimize

shading effects on seedlings.

Sampling of residual tree ectomycorrhizas

From August 26 to 30, 2005, soil cores (10 cm diameter, 20 cm

deep) were collected in four cardinal directions (North, South,

West, and East) approximately 2 cm from the outer rim of the

mesh bags. For seedlings planted directly into the soil (i.e.,

without mesh bags), soil cores were taken from the same

distance away from each seedling, but only after the seedling

had first been carefully excavated. Samples were placed in

plastic bags and stored at 4 �C until further processing. All

samples were processed within 4 months of field sampling.

Sampling of seedling ectomycorrhizas

Seedlings were harvested immediately after soils were

sampled for residual tree EM (14 months after planting). After

clipping off the shoots, root systems with some surrounding

soil were placed in plastic bags and stored at 4 �C until

further processing. All samples were processed within 6

months of field sampling (without any visible root tip

deterioration).

Morphotyping and molecular identificationof ectomycorrhizas

Roots of residual trees and seedlings were carefully washed

under running tap water and then cut into approximately

2 cm pieces. All root fragments were placed in a baking dish

containing distilled water and thoroughly mixed. We

randomly subsampled and counted up to 200 residual tree and

300 seedling EM root tips. Gross morphology of EM roots and

rhizomorphs was described using a stereomicroscope, while

the mantle, cystidia, emanating hyphae, and Hartig net were

described using a compound microscope under 400 or 1000�magnification. Morphological descriptions were made with

reference primarily to Goodman et al. (1996) and Hagerman

et al. (2001) and to a minor extent to Ingleby et al. (1990) and

Agerer (1985–1998). After morphotyping, root systems were

dried and weighed.

DNA extraction and PCR amplification of the ITS region of

fungal nuclear rDNA was conducted on at least one (typically

two or three) subsample (typically 5 EM tips) per morphotype

per host type. Extraction and isolation of DNA, sequencing

and nucleotide Basic Local Alignment Search Tool (BLAST)

searches followed the methods outlined in Twieg et al. (2007).

Primer pairs used in PCR amplifications were: ITS1-F and ITS4;

NSI1 and NLC2 (Martin & Rygiewicz 2005). Samples that were

not successfully amplified or sequenced were given an EM

taxon name based on morphotyping data. Out of the 154 root

tips extracted, 89 % yielded DNA for BLAST searches.

Statistical analyses

All statistical analyses were carried out using the R statistical

environment for statistical computing and graphics

(R Development Core Team 2007). Multivariate community

data, univariate analyses, and linear mixed-effects model

fitting were performed in R with the vegan (Oksanen et al.

2006), stats (R Development Core Team 2007), and nlme (Pin-

heiro et al. 2007) packages, respectively. The split plot design

was analysed using a linear mixed-effects model (Pinheiro &

Bates 2000), where sites were used as blocks and set as the

random factor. The whole plot and split plot fixed factors were

the mesh and distance treatments, respectively. Planted

seedlings representing the mesh and distance treatment

combinations on each site were the experimental units (n¼ 6).

For the residual tree analyses, the experimental units were the

sum of the four core subsamples.

Relative abundance (RA) of each EM taxa was calculated as

the abundance of the taxon divided by the sum of abundances

of all EM taxa in an experimental unit (McCune & Grace 2002).

Percent EM colonization was calculated following Teste et al.

(2006). Ectomycorrhizal richness on residual trees was rela-

tivized based on total number of EM root tips at each distance

since it is known that richness decreases with fewer root tips

sampled (Taylor 2002). We calculated EM taxa richness and

Shannon diversity index (H0) using functions, specnumber and

diversity, respectively. The H0 emphasizes species richness

because it is weighted towards rare species (Magurran 2004).

24 F.P. Teste et al.

When EM taxa were found in all mesh treatments except the

0.5 mm mesh, we determined if this exclusion was due to MN-

mediated colonization or chance using a randomization test

(Monte Carlo simulation) based on resampled presence/

absence data. Contingency tables were constructed for pres-

ence and absence of EM taxa grouped by mesh treatments and

analysed with Pearson’s Chi-square test and Monte Carlo

simulated P-values. The lme function (method set to restricted

maximum likelihood) was used to fit the linear mixed-effects

models. Variance partitioning and significance (P< 0.05) of the

fixed effects (mesh and distance treatments) were determined

with the functions, anova and summary of the fitted lme

objects.

Results

Ectomycorrhizal community composition and structure

A total of 30 228 residual tree and 23 387 seedling-EM root tips

were observed and counted. A total of 36 EM taxa were found

on either residual tree or seedling root systems (Fig 1). Residual

trees hosted 32 EM taxa and seedlings hosted 26 EM taxa, with

22 taxa in common (Fig 1). Wilcoxina rehmii dominated EM

communities on both trees and seedlings; mycorrhizas

formed by Rhizopogon rudus, Cenococcum geophilum, Rhizopogon

vinicolor and Amphinema byssoides were also abundant (Fig 1).

Fig 1 – Relative abundance of resid

Ectomycorrhizal community similarity

Trees and seedlings shared 61 % of all EM taxa found in this

study (Fig 1) and 83 % of those taxa had a relative abundance

greater than 5 % (Fig 1). The NMS ordination method showed

a high degree of overlap between the residual tree and seed-

ling EM communities (Fig 2). Community analyses based on

abundance and presence–absence of EM taxa also showed

high similarity between the residual trees and seedlings

(Table 1).

Mesh effects on seedling ectomycorrhizal community

Mesh treatment had little effect on the seedling EM

communities (Table 1). Similarly, seedling EM colonization,

richness and diversity were unaffected by the mesh treat-

ments (data not shown). The interaction between mesh and

distance was tested as a fixed effect but was not significant

for the univariate responses presented above. Only 50 % of

all EM taxa encountered were found on seedlings in the

0.5 mm mesh as opposed to an average of 77 % on seedlings

having partial or full access (35, 250 mm, and no mesh) to

a MN (Fig 3), but this trend was statistically weak (X2¼ 6.5,

P¼ 0.09). Furthermore, four EM taxa (Inocybe sp.4, Lactarius

deterrimus, Russula brevipes and Tuber sp.) were excluded by

the 0.5 mm mesh (i.e., absent from the 0.5 mm mesh but

present at least once in the other mesh treatments).

ual tree and seedling EM taxa.

Fig 2 – NMS ordination of residual tree and seedling EM

fungal communities. Sørensen distance measure was

used; R2 [ 0.66. Data from 23 tree and seedling plots

(instead of 24) were used since one residual tree’s crown

snapped off due to strong winds that occurred between the

2004 and 2005 growing season. NMS ordinations were

performed using the metaMDS function to visualize the

similarity between residual tree and seedling EM

communities at the whole plot level.

Role of mycorrhizal networks and tree proximity 25

However, the randomization tests indicated that these

events likely occurred by chance (Inocybe sp.4 P¼ 0.74; L.

deterrimus P¼ 0.61; R. brevipes P¼ 0.35; and Tuber sp.

P¼ 0.63).

Table 1 – Summary of multivariate community analyses showi(Pseudotsugamenziesii var. glauca) tree and seedling EM commu

Communityanalysis method

Grouping entity Data matrix ty

Mantel test Tree vs seedling Presence–absenc

Abundance

ANOSIM Tree vs seedling Presence–absenc

Abundance

MRBP Mesh treatment levels Presence–absenc

Abundance

MRBP Distance treatment levels Presence–absenc

Presence–absenc

Presence–absenc

Presence–absenc

Abundance

Mantel tests and Analysis of Similarity (ANOSIM) were carried out with th

the similarity between tree and seedling EM communities was significant.

community data, we carried out Blocked Multi-Response Permutation Pro

the function, mrpp (with blocks set as the strata). The Mantel r and ANO

complete dissimilarity; a value of 0 indicates a completely random groupi

ANOSIM R associated P-values evaluates how likely is an observed dissimi

group agreement (A) is a statistic used to test the hypothesis of no differen

from 0 to 1 where a value of 1 indicates that two or more groups are com

observed difference due to chance (McCune & Grace 2002). Only statis

comparisons. Non-significant (P< 0.05) multiple comparison tests are no

Distance effects on the residual tree and seedlingectomycorrhizal communities

Distance treatments influenced the composition of both the

tree and seedling EM communities (Table 1). Seedlings

furthest from residual trees had a simpler EM community

composition, mainly dominated by W. rehmii, compared to

seedlings in closer proximity (Fig 4). Ectomycorrhizal coloni-

zation of root samples from residual trees was high but not

affected by distance from the bole (Table 2). However, both EM

richness and diversity in samples from residual trees

decreased with distance (Table 2). Similar to residual trees, EM

colonization of planted seedlings was unaffected by distance

from residual trees (Table 2). However, seedling EM richness

and diversity were greatest at 0.5 m from the residual tree bole

and decreased with distance (Table 2).

Discussion

MN-mediated colonization

Our first hypothesis, that MN-mediated colonization is the

dominant mechanism fostering continuity of the EM

community (Newton 1992), was rejected. Even though many

EM species co-occurred on trees and seedlings on our sites

(see below), we found that wind-borne and soil EM inoculum

already present in mesh bags (could include chlamydospores,

sclerotia, hyphal fragments, severed tree EM tips, and dis-

rupted network hyphae) was the most important inoculum

source for newly colonized seedlings. This is similar to

McGuire’s (2007) observations in an EM monodominant rain

forest, but differs from Simard et al. (1997) and Onguene &

ng degree of similarity between residual interior Douglas-firnities and treatment effect differences

pe Similaritymeasure

Multiplecomparisons

Statistic P-value

e Chao – r¼ 0.629 <0.001

Chao – r¼ 0.305 <0.001

e Sørensen – R¼ 0.156 <0.001

Sørensen – R¼ 0.244 <0.001

e Chao – A¼ 0.133 0.065

Chao – A¼ 0.021 0.003

e Chao – A¼ 0.100 <0.001

e Chao 0.5 m vs 2.5 m A¼ 0.062 <0.001

e Chao 0.5 m vs 5.0 m A¼ 0.162 <0.001

e Chao 1.0 m vs 5.0 m A¼ 0.115 <0.001

Chao – A¼ 0.035 <0.001

e functions, mantel and anosim, respectively, to determine whether

To test for mesh and distance treatment effects with the multivariate

cedures (MRBP) following the guidance of McCune & Grace (2002) with

SIM R statistics range from �1 to þ1 where: a value of �1 indicates

ng; and þ1 indicates a completely similar grouping. The Mantel r and

larity or similarity due to chance. The MRBP chance-corrected within-

ce between two or more groups and represents the effect size (ranges

pletely different). The associated P-value evaluate how likely is an

tics with a value > 0.1 and P< 0.05, were considered for multiple

t included.

0

5

10

15

20

25

30

35

40

45

50W

ilcoxin

a r

ehm

ii

Rhiz

opogon v

inic

olo

r

Rhiz

opogon r

udus

Cenococcum

geophilum

Am

phin

em

a b

yssoid

es

Thele

phora terrestris

Russula

brevip

es

Russula

adusta

Tom

entella s

ubcla

vig

era

Sebacin

a incrustans

Russula

nig

ric

ans

Inocybe s

p.4

Suillu

s lakei

Tuber s

p.

Inocybe s

p.1

Lactariu

s r

ubrilacteus

Pseudotom

entella tris

tis

Lactariu

s d

eterrim

us

Tom

entella s

p.

Rhiz

opogon r

ogersii

Inocybe s

p.2

Russula

xeram

pelina

Tom

entella s

ubtestacea

Inocybe s

p.3

Piloderm

a fallax

LB

O

Relative ab

un

dan

ce (%

)

MN access

MN restricted

* * ** * ** ** * * *

*

Fig 3 – Relative abundance of EM taxa found on seedlings having access to the MN (35 mm, 250 mm, and no mesh) or where

access was restricted (0.5 mm mesh). * denotes relative abundance [ 0 for MN-restricted access.

Fig 4 – Relative abundance of common (>1 %) EM taxa found on Pseudotsuga menziesii var. glauca trees (T) and seedlings (S)

growing at the four distances from P. menziesii var. glauca residual trees.

26 F.P. Teste et al.

Table 2 – Distance effects on EM status of Pseudotsugamenziesii var. glauca mature trees and seedlings. Valuesare means with 95 % confidence intervals (inparantheses). Statistically significant distance treatmenteffects detected by a Bonferroni multiple comparison testare designated by different letters (P < 0.05)

Distance from tree (m)

0.5 1.0 2.5 5.0

Tree responses

Colonization

(%)

99.5 (12.1) a 99.2 (11.8) a 86.0 (11.5) a 78.2 (12.4) a

Richness 9.4 (3.8) ab 11.3 (3.7) a 6.6 (3.6) ab 3.4 (3.9) b

Diversity 1.4 (0.2) a 1.2 (0.2) ab 0.8 (0.2) bc 0.3 (0.2) c

Seedling responses

Colonization

(%)

84.0 (7.0) a 85.9 (6.9) a 81.1 (6.9) a 77.4 (7.1) a

Richness 5.4 (0.6) a 4.8 (0.6) a 3.9 (0.6) a 3.5 (0.6) a

Diversity 1.1 (0.1) a 1.0 (0.1) a 0.8 (0.1) ab 0.7 (0.1) b

Role of mycorrhizal networks and tree proximity 27

Kuyper (2002) in an undisturbed mixed Douglas-fir temperate

forest and in mixed (AM and EM) rain forests, respectively,

where undisrupted MNs were more important colonizers of

understory seedlings than other inoculum sources. Never-

theless, this study does not discount that MN-mediated

colonization does occur simultaneously with the other

mechanisms (see below) or may be important in other forest

ecosystems (e.g., sand dune forests) or on primary succession

sites (e.g., volcanic deserts, mine tailings).

Lateral extent of EM communities

Seedling EM richness and diversity increased with proximity

to trees, suggesting that nearby residual trees acted as EM

refugia and facilitated the spread of EM fungi to establishing

seedlings. Our second hypothesis, that there is a distance

where the EM community of seedlings is most similar to trees,

was supported. Indeed, seedlings closest to the residual trees

had the most similar EM communities compared to those at

further distances. The proximity pattern observed in this

study was also noted in other studies assessing the effect of

distance from live trees on seedling EM status (Dickie et al.

2002; Outerbridge & Trofymow 2004). In contrast to Dickie et al.

(2005) and Hagerman et al. (1999), we found no clear relation-

ship between percent EM colonization in planted seedlings

and distance to the closest live tree. We suspect that resilient

soil propagules and wind-dispersed spores of rapidly growing

EM fungal species, such as W. rehmii, R. vinicolor, and Thele-

phora terrestris, accounted for the high EM colonization levels

on our most distant seedlings.

Ectomycorrhiza taxa richness and diversity of the residual

trees also significantly decreased with distance, correspond-

ing with our estimated decline in root density. This decline in

tree root density with distance was likely responsible for the

decline in seedling EM richness and diversity since seedlings

simply had access to less inoculum at the furthest distances.

Patterns of declining residual tree root and EM root tip

densities with distance (data not shown) are consistent with

estimates based on coarse-root growth rates of interior

Douglas-fir in the IDF biogeoclimatic zone (Richardson 2000).

As expected, this general pattern has also been found in other

Douglas-fir forests (Outerbridge & Trofymow 2004; Cline et al.

2005; Luoma et al. 2006) as well as other coniferous and

hardwood forests (Durall et al. 1999; Hagerman et al. 1999;

Kranabetter et al. 1999; Dickie et al. 2002; Dickie & Reich 2005).

The AM herbaceous plants that dominated the understory

vegetation on our sites may also have negatively affected the

EM fungal communities on seedlings furthest away from

mature trees, where EM similarity and EM status were lowest

and AM plant abundance highest (Teste & Simard unpub-

lished data). Invasion or increases of AM plants have been

shown to reduce EM fungal abundance and change EM species

composition in the dry pinyon pine forests of the USA (Has-

kins & Gehring 2004). Dominance of early post-disturbance

plant communities by AM herbs is common in IDF forests

such as ours (Stark et al. 2006). Other studies show that below-

ground competition with AM plants is an important factor

reducing EM seedling recruitment and establishment in dry

forest ecosystems (Simard et al. 2003; McHugh & Gehring

2006).

Potential establishment of MNs

The Douglas-fir trees and seedlings in this study shared most

of the observed EM taxa, and community analyses supported

our third hypothesis that there is a high degree of EM

community similartiy between seedlings and conspecific

trees. As seedling root systems were destructively sampled in

the field, we often noticed an abundance of fungal mycelium

on both sides of the mesh bags (apparently originating from

the seedling on one side and from the nearby tree’s root

system on the other). These results provide good evidence

that the potential for MNs to form in these dry forests was

high. These results agree with other studies demonstrating

that high EM similarity exists between mature trees and

surrounding seedlings (Simard et al. 1997; Jonsson et al. 1999;

Matsuda & Hijii 2004; Cline et al. 2005) and between different

species of woody plants (Simard et al. 1997; Horton & Bruns

1998; Horton et al. 1999; Kennedy et al. 2003; Dickie et al. 2004).

Convincing arguments for high MN potential to exist

between EM hosts of the same or different species have been

offered by Molina et al. (1992). Their discussion involved EM

species specificity patterns and multiple-host fungal ranges as

the norm rather than the exception. Their argument relied on

the assumption that plant hosts sharing the same EM fungal

species would be connected via a MN. We agree with Kennedy

et al. (2003), however, that their concept would be strength-

ened by identification of the same EM fungal genet on multiple

host plants; the availability of more advanced molecular

techniques allows us to provide more convincing evidence of

the occurrence of MN connections in the field. Lian et al. (2006)

found the same Tricholoma matsutake genets connecting Pinus

densiflora trees in a field study in Japan.

EM inoculum potential and community composition

Given the complexity of the design and its lack of balance

(uneven number of replicates per treatment combination), we

were not able to directly test for mesh and distance interac-

tions at the community level (multivariate responses).

28 F.P. Teste et al.

However, following the advice of McCune and Grace (2002), we

visualized interactions using joint plot overlays on an ordi-

nation diagram. In all cases, treatments were patterned as

perpendicular vectors indicating independence and non-

interaction. Seedlings with access to all sources of inoculum,

including MNs, soil propagules and spores, and that were also

in close proximity to trees, tended to have the greatest EM

colonization, richness, diversity, and abundance; thus, we

accepted our fourth hypothesis that EM status is greatest

where there is access to all inoculum sources. Of these inoc-

ulum sources, wind-dispersed EM fungal spores may not have

been the dominant colonization vectors because (i) epigeous

and hypogeous sporocarps were infrequent on the six sites

during the two-year study period, which is consistent with

previous studies showing a paucity of fruitbodies in recent

clearcuts (Durall et al. 2006), and (ii) the immediate range of

spatial dispersal of spores is limited to a few meters (Burnett

2003). Since Rhizopogon species were abundant on root tips,

especially on seedlings that could not form MNs, Rhizopogon

may have colonized seedlings from a viable and tenacious

spore bank and/or from disrupted network hyphae in the

soil (KjØller & Bruns 2003). The most abundant EM taxon,

W. rehmii, likely colonized seedlings via soil propagules

(chlamydospores, sclerotia, hyphal fragments, severed tree

EM tips, and disrupted network hyphae) and to a lesser extent

via undisrupted MNs (Mikola 1988), if they occurred on our

harvested sites.

We were surprised that W. rehmii was the most abundant

EM taxon on both residual trees and seedlings. Typically,

Wilcoxina occurs as a greenhouse bioassay contaminant and is

completely absent from mature trees (Cline et al. 2005). Yet,

Mikola (1988) noted that this species can be a rapid colonizer

of new root tips under field conditions. It is possible that the

high abundance of Wilcoxina was an artifact of the small soil

disturbances caused during mesh bag insertions. However,

Wilcoxina has been noted as an abundant member of the

woody understory plant community in similar Interior

Douglas-fir forests (Hagerman et al. 2001). Thus, it is likely that

W. rehmii mycelia are shared among multiple woody plant

species, and the fungus is an aggressive colonizer of interior

Douglas-fir roots. Given that planted seedlings were non-

mycorrhizal at the time of planting, we conclude that the

ectendomycorrhizal W. rehmii is a naturally occurring and

abundant member of these dry coniferous forests.

Some of the most abundant EM taxa, including R. rudus,

C. geophilum, R. vinicolor, and A. byssoides were present on both

the residual trees and seedlings. T. terrestris, by contrast, was

abundant only on seedlings furthest away from residual trees,

a pattern also observed by Kranabetter & Wylie (1998), Durall

et al. (1999), and Cline et al. (2005).

Conclusions

We present evidence that MN-mediated colonization is not

the dominant mechanism promoting continuity of the EM

community from mature trees to nearby establishing seed-

lings in recently harvested sites. Our results instead suggest

that wind-borne or soil EM inoculum (e.g., chlamydospores,

sclerotia, hyphal fragments, severed tree EM tips, and

disrupted network hyphae) were the main vectors responsible

for initial EM colonization of seedlings. Given that EM simi-

larity was high, we show there is still potential for MNs to

form between established trees and seedlings in these forests

after initial colonization events. At forest harvest, retaining

residual trees at sufficient spatial distribution to maintain

a diverse EM community appears important for recovery of

the EM fungal community following disturbance. This may be

especially important where EM hosts are surrounded by AM

vegetation, as the case in our study. Mature tree root zones

extend several meters beyond the tree crown, ensuring

seedling colonization and continuity of the EM fungal

community both spatially and through time.

Acknowledgements

We acknowledge David Huggard, Shannon Berch, Melanie

Jones and Robert Guy for their thoughtful comments at the

early stages of the study. We are very grateful to Amanda

Schoonmaker for her help with the field work. We are indebted

to Gordon Leschyson for renting his CanDig� mini-excavator

and Rocky Hudson at High Country Cold Storage Ltd. for the

nursery-grown seedlings. We thank Bill Clarke, Lenka Kudrna,

and MaryAnn for help with the molecular work in the labo-

ratory. We also thank Brendan Twieg for his insightful advice

during the morphotyping, molecular and community data

analyses. Finally we thank all the anonymous reviewers for

their comments on various drafts of the manuscript. Funding

was provided by a FSP of Forest Investment Innovation of

British Columbia grant, a Canadian Foundation for Innovation

grant, and a Natural Sciences and Engineering Research

Council (NSERC) Discovery Grant (DG) to SWS, an NSERC PGS

scholarship to FPT, and an NSERC DG to DMD. We declare that

the experiments comply with the current laws of the country

in which they were performed.

Supplementary material

Supplementary data associated with this article can be found,

in the online version, at doi:10.1016/j.funeco.2008.11.003.

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