• ectomycorrhizal fungi communities;
  • green-tree retention;
  • ITS-RFLP analysis;
  • Pseudotsuga menziesii (Douglas-fir);
  • rRNA genes;
  • species diversity


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  • • 
    The influence of mature trees on colonization of Douglas-fir (Pseudotsuga menziesii) seedlings by ectomycorrhizal fungi (EMF) is not well understood. Here, the EMF communities of seedlings planted near and far from trees are compared with each other, with EMF of seedlings potted in field soils and with EMF of mature trees.
  • • 
    Seedlings were planted within 6 m, or beyond 16 m, from residual Douglas-fir trees in recently harvested green-tree retention units in Washington State, USA, or potted in soils gathered from near each residual tree. Mature tree roots were sampled by partly excavating the root system. The EMF communities were assessed by polymerase chain reaction–restriction fragment length polymorphism and sequence analysis of ribosomal RNA genes.
  • • 
    Seedlings near trees had higher species richness and diversity of EMF communities compared with seedlings far from trees. The EMF communities of seedlings near trees were more similar to those of mature trees, while seedlings far from trees were more similar to glasshouse seedlings.
  • • 
    By enhancing the EMF diversity of seedlings, residual trees may maintain or accelerate the re-establishment of mycorrhizal communities associated with mature forests.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Concern over the low structural diversity and reduced species richness of managed Douglas-fir forests in the Pacific North-west of the USA has led to the proposal that clearcutting be replaced by retention forestry (Kohm & Franklin, 1997). Retention forestry, also known as green-tree retention, requires some proportion of the mature trees to be protected during harvesting. Residual trees may be evenly spaced (dispersed retention) or clumped to create tree ‘islands’ or protect sensitive areas by creating buffer zones, for example along stream banks (aggregated retention). Residual trees have been demonstrated to reduce erosion, moderate the effects of wind and solar exposure, and increase diversity in forests recovering from harvesting by retaining some forest-dependent plant and animal species (Perry et al., 1989a; North et al., 1996). Retention of green trees, snags, and logs is now mandated by private (WFPA, 1995) and public (FEMAT, 1993; WFPB, 1995) forest management plans in the Pacific North-west. The effectiveness of retention forestry in maintaining ectomycorrhizal fungal (EMF) diversity, however, has not been fully evaluated.

Forest harvesting affects EMF by changing the age structure and species composition of the forest overstory trees and understory plants which serve as their hosts and by modifying the physical, chemical and biological components of their soil habitat. Some EMF taxa appear to be primarily associated with mature forests, while other EMF taxa may persist as resistant soil propagules until disturbances such as fire or harvesting provide the opportunity for colonization of seedling roots (Visser, 1995; Taylor & Bruns, 1999; Horton & Bruns, 2001; Jones et al., 2002, 2003).

Several researchers have reported declines in diversity for EMF after forest harvesting. Byrd et al. (2000) found lower species richness and significant changes in EMF community composition for 8-yr-old lodgepole pine (Pinus contorta) clearcuts in Wyoming, when compared with unharvested sites. In a mixed subalpine fir (Abies lasiocarpa) and Engelmann spruce (Picea engelmannii) forest in Canada, Hagerman et al. (1999a) also observed declines in diversity of EMF in soil cores after clearcutting, but declines were less severe within 2 m of the forest edge. Similarly, Stockdale (2000) observed a 23% decline in EMF taxa, distinguished morphologically, after thinning of a late-successional Douglas-fir forest in Oregon, while soil cores from within the dripline of residual trees showed no decline in species richness. Impacts of forest clearcutting and thinning on mycorrhizal communities could result from loss of inoculation potential, decreases in inputs of carbon from host plants, changes to the soil environment, or a combination of these factors (Jones et al., 2003).

Several recent studies have documented edge effects on EMF diversity of seedlings planted in clearcuts adjacent to unharvested forest. Engelmann spruce (nonmycorrhizal) bioassay seedlings planted within 2 m of forest edges had higher EMF species richness than seedlings planted 16 m into clearcuts (Hagerman et al., 1999b). By contrast, proximity to the forest edge had no effect on previously colonized Engelmann spruce seedlings planted in mineral soil exposed by mechanical mounding at the same site (Jones et al., 2002). Previously colonized western hemlock (Tsuga heterophylla) and lodgepole pine seedlings had decreased EMF species richness per seedling when planted beyond 7 m from the forest edge into clearcuts (Durall et al., 1998). The same was true for naturally regenerated western hemlock seedlings near the edge of mature forests (Kranabetter & Wylie, 1998). When seedlings from the forest were transplanted into clearcuts, EMF diversity declined owing to the loss of several taxa which were apparently unable to persist in forest openings (Kranabetter & Friesen, 2002). Changes in EMF species composition probably are at least partly caused by inoculation by resistant soil propagules rather than hyphal connections. Nevertheless, it is not clear from these studies whether changes in the soil environment or loss of hyphal linkages are responsible for the lower EMF species richness of seedlings in clearcuts (Jones et al., 2003).

The few studies that have examined the mycorrhizal diversity of seedlings growing near isolated trees have found effects similar to those conferred by forest edges. Proximity to mature paper birch (Betula papyrifera) trees resulted in higher mycorrhizal diversity of paper birch seedlings both in clearcuts and in mixed conifer forests (Kranabetter, 1999). Dickie et al. (2002) demonstrated that red oak (Quercus rubra) seedlings growing near harvested, stump-sprouting (living) oak trees had greater mycorrhizal colonization and diversity than seedlings near dead oak stumps. Hyphal linkages to mature trees may be critical for maintaining some EMF taxa on seedlings, possibly owing to high demand for photosynthate (Fleming, 1983, 1984). Soil trenches dug around Douglas-fir seedlings in 90- to 120-yr-old Douglas-fir/paper birch forests resulted in decreases in EMF species richness, and taxa forming rhizomorphs decreased sharply in abundance (Simard et al., 1997).

The objective of this study was to assess the spatial influence of residual trees on EMF communities of Douglas-fir seedlings. The following hypotheses were tested: (1) the EMF community of seedlings growing near isolated mature trees will be more diverse than that of seedlings far from trees; (2) EMF taxa associated with mature trees will be more common on seedlings near mature trees, and (3) EMF taxa able to colonize seedlings from soil propagules (i.e. those present on glasshouse seedlings) will be more common on seedlings far from mature trees.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Site characteristics

This study took place in recently harvested second growth Douglas-fir forests in the foothills of the western Cascade mountain range in Washington state, USA, approximately 50 km south-east of the city of Seattle. Two sites, ‘Beatles’ (47°20.970′ N, 121°49.895′ W) and ‘Imagine’ (47°23.956′ N, 121°48.938′ W) were on public land managed by the City of Seattle Cedar River watershed while one site, ‘Green River’ (47°18.719′ N, 121°42.609′ W) was owned and managed by the Plum Creek Timber Company. The sites were between 7 km and 13 km from each other, and have been described in detail elsewhere (Cline, 2004). Sites were selected based on the presence of mature Douglas-fir trees sufficiently isolated to allow seedlings to be planted in circular bands within 6 m from the center tree and beyond 16 m from the center tree, without approaching within 16 m of neighboring trees. Site characteristics and attributes of each center tree are presented in Table 1.

Table 1.  Site harvest dates, elevation, soil characteristics, and Douglas-fir (Pseudotsuga menziesii) center tree attributes
 Site Green RiverImagineBeatles
  1. Soil pH and moisture are means ± 1 SE averaged from monthly growing season soil cores, with four replicates per sample date and a total of 36 cores for Green River and 40 cores for Beatles and Imagine, respectively. Soil moisture was calculated as per cent dry weight. Within rows, means with the same letter are not significantly different by Tukey's honestly significant difference test (α= 0.05). DBH, diameter at breast height.

Year of harvest199619941992
Elevation (m)808457488
Soil pH4.22 ± 0.075a3.87 ± 0.045b3.99 ± 0.066b
Soil moisture (%)  76 ± 6.6  91 ± 8.4  92 ± 8.1
Center tree attributes
Age (y)447269
Height (m)344442
DBH (cm)43.466.362.5

Before harvesting, forests at the sites were dominated by Douglas-fir, but with young and mature western hemlock also common at Beatles and Imagine. Other tree species present at or near the sites were western red cedar (Thuja plicata Donn. ex D. Don), Pacific silver fir (Abies amabilis Dougl. ex Forbes), grand fir (Abies grandis (Dougl.) Lindl.), Sitka spruce (Picea sitchensis (Bong.) Carr.), vine maple (Acer circinatum Pursh), red alder (Alnus rubra Bong.), black cottonwood (Populus trichocarpa T. & G.) and Sitka willow (Salix sitchensis Sanson). Green River (808 m elevation) was harvested in 1996, Imagine (457 m) was harvested in 1994, and Beatles (488 m) was harvested in 1992.

Experimental design

In spring of 1998, mycorrhizal nursery-grown bare root 2–1 Douglas-fir seedlings (provided by Plum Creek Timber Co. from their nursery) were planted within circular plots centered upon a single mature Douglas-fir tree at each of the three sites. Around each tree, 40 seedlings were planted in a circular plot within 6 m of the base of the tree and 40 seedlings were planted 16–30 m from the base of the tree. Each seedling was planted at least 1 m from the nearest seedling, and previously established understory vegetation was cleared to provide space as needed.

In spring of 1998, 16 seedlings were also potted in soil from each site. Soil grab samples from 0 to 40 cm depth were merged from four randomly selected locations 20–30 m from the center of each plot to provide a composite sample from each site. Seedlings were grown in a sheltered outdoor area at the University of Washington glasshouse maintained by the Biology department. Water was provided through wicking from below (to avoid leaching of nutrients) by immersing pots in 2 cm of water, in separate saucers to minimize transfer of EMF spores from seedling to seedling. No fertilizers were provided; seedlings were limited to the nutrients present in the soils at sampling.

Sampling of mycorrhizas

Douglas-fir seedlings  In spring of 1998, a subsample of nursery seedlings was taken to determine the initial mycorrhizal composition of seedlings (n = 8). Seedlings planted < 6 m and > 16 m from center trees were sampled in fall of 1998, spring of 1999, autumn of 1999, and spring of 2000, with replicate seedlings selected randomly from each plot at each site (2 plots × 3 sites × 8 replicates = 48 seedlings per sample date). In spring of 2000, unusually high seedling mortality caused by mountain beavers denning near the plot area at Green River forced reduction of the number of replicates to four. Glasshouse seedlings were sampled in the autumn of 1998, spring of 1999 and autumn of 1999, with four replicates for soil from each of the three sites. Nursery seedlings were small enough that all mycorrhizal root tips could be examined within a reasonable period. For field and glasshouse seedlings, roots were subsampled and 100 root tips were examined from each seedling. Seedlings were carefully excavated to obtain the entire root system. In the laboratory, seedling roots were soaked in distilled water then gently teased apart to break up soil aggregates. Roots were rinsed then placed in shallow trays over a 1-cm grid in random orientations. The root tip closest to each of the 100 evenly spaced grid points was removed for further analysis.

Mature tree roots  The EMF communities of mature Douglas-fir trees were assessed by partly excavating the root system of each center tree. Owing to the high degree of soil disturbance involved, excavation could not be performed until the final seedling sample date in spring of 2000, to avoid severing potential hyphal connections between seedlings and the center tree. Four random bearings were taken at each center tree. Along each bearing, a major root (> 20 cm diameter) was excavated outwards from the base of each center tree to a distance of 2 m. At this distance, a grab sample of smaller branch roots was taken and returned to the laboratory for analysis. Where necessary to obtain sufficient quantities of fine roots, the excavation was extended up to 4 m from the base of the tree. Mature roots were prepared as described above for field seedlings.

Morphological and molecular identification of EMF taxa

Morphological analysis  Root tips were viewed with a dissecting microscope and sorted into categories of inactive or necrotic, nonmycorrhizal, and mycorrhizal. Root tips lacking a visible mantle were presumed to be mycorrhizal and sampled for molecular analysis unless abundant root hairs were present. Root tips were considered inactive or necrotic if the entire root tip was shrunken and desiccated or if the root cortex was partly or completely decayed. Mycorrhizal root tips were sorted into broadly defined morphotypes based on morphological characteristics, including branching structure and shape, mantle color and texture, and emanating hyphae and rhizomorphs based on described methods (Ingleby et al., 1990; Agerer, 1991; Goodman et al., 2000). Root tips from each morphotype for each seedling or each root sample were counted and lyophilized to prepare for long-term storage and/or DNA extraction.

DNA extraction  DNA was extracted from mycorrhizal root tips using the method described by Gardes & Bruns (1993) with the following modifications: the volume of cetyltrimethylammonium bromide (CTAB) lysis buffer was reduced from 300 µl to 40 µl, liquid nitrogen was used instead of a dry ice bath, autoclaved plastic micropestles were used (Sigma, St Louis MO, USA), and the DNA extract was resuspended in TE (10 mm Tris, 1 mm ethylenediaminetetraacetic acid (EDTA), pH 8.0). Extracted DNA samples were stored at −40°C for future analysis.

Restriction fragment length polymorphism (RFLP) analysis  Initially, for each seedling or mature root sample, up to four randomly selected root tips from each morphotype for each sample were screened by internal transcribed spacer (ITS)-RFLP analysis (108 RFLPs total) followed by ITS sequence analysis for a randomly selected subsample. Because root tips of the same morphotype from each sample were found to be identical, in later sampling, a single root tip was randomly selected for molecular analysis for every morphotype from each seedling or root sample. In total, over 24 000 root tips were examined for this study, of which 11 856 were active mycorrhizal root tips. A total of 767 root tips were selected for DNA extraction and RFLP analysis. To generate RFLPs, polymerase chain reaction (PCR) was used to amplify the internal transcribed spacers of the nuclear ribosomal RNA gene, using the fungus-specific primer pair ITS-1F (Gardes & Bruns, 1993) and ITS-4 (White et al., 1990). The final reaction mixture consisted of a 1 : 1000 dilution of the DNA extract, 200 µm each of dATP, dTTP, dCTP, dGTP, 200 nm of each primer, 3 mm MgCl2, 0.5 mg ml−1 of sterile bovine serum albumin, 0.05 units µl−1 of Taq polymerase (various suppliers), and the standard PCR buffer as supplied by the manufacturer. The volume of the final reaction mixture was 25 µl, overlaid with a single drop of mineral oil (Sigma). Sterile water was used as a negative control, and DNA from Tricholoma saponaceum was used as a positive control for each run of PCR. Amplification was performed with a denaturation of 95°C for 35 s, an annealing temperature of 55°C for 55 s, and an increasing extension period of 72°C for 45 s plus 4 s per cycle over 36 cycles.

Amplified products were digested with the restriction enzymes HpaII, CfoI, and RsaI, separated on 2% agarose gels, stained with ethidium bromide, and digitally photographed for band analysis using the gelcompar ii software package (Applied Maths, Inc., Austin TX, USA). Gel images were processed to eliminate distortion and bending of the gel, and band sizes were calibrated by comparison with a standard DNA ladder. Cluster analysis was performed by gelcompar ii using upgma with the fuzzy dice procedure, with a band position tolerance of 1%. Results were examined and adjusted by hand as needed. Ambiguous RFLP clusters were further evaluated by sequencing of the ITS rRNA gene for up to 10 samples from each RFLP pattern.

DNA sequence analysis  For each EMF taxon, as defined by distinct RFLP patterns based on cluster analysis, sequences were determined that were used to identify closely related known taxa based on sequence homology. Sequencing of the ITS rRNA gene using the primer pair ITS-1F and ITS-4 was followed by sequencing of approximately 360 bp of the mitochondrial large subunit rRNA gene using primer pair ML5/ML6 described by White et al. (1990) and/or approximately 650 bp of the nuclear large subunit rRNA gene using the primer pair LR0R/LR16 (Moncalvo et al., 2000), as needed to obtain a reliable identification. Closely related sequences were identified using the National Center for Biotechnology Information web-based blast search engine (Altschul et al., 1997). phylip version 3.6a3 (Felsenstein, 2002) was used to generate neighbor-joining, parsimony, and maximum-likelihood trees to examine the phylogenetic placement of unidentified EMF taxa with reference to published sequences obtained through blast searches as well as sequences obtained from EMF taxa fruiting at the study sites (Cline, 2004). Sequence homology greater than or equal to 98% for the ITS region and 99% for the nLSU rRNA gene were considered sufficient to assign tentative species-level designations to unidentified EMF taxa (Table 2). Sequences from sporocarps and mycorrhizal root tips collected during the course of this study are available from GenBank under the accession numbers AY356323, AY750156AY750169 and AY751555AY751568.

Table 2.  Closest related blast sequences and relative abundance (%) for ectomycorrhizal fungal (EMF) taxa on Douglas-fir (Pseudotsuga menziesii) seedlings and trees
Closest matching blast sequence(s)rRNA type% SimilarityAccession numberSourceConsensus EMF taxonNursery seedlingsaGlasshouse seedlingsb> 16 m seedlingsc< 6 m seedlingsdMature treese
  • For each tree root sample or seedling (except nursery seedlings), a subsample of 100 root tips was examined. Columns sum to 100%. aNursery-grown seedlings were sampled before planting in spring 1998. bGlasshouse seedlings were potted in field soils and sampled 1998–99. Seedlings were planted in spring 1998 c> 16 m or d< 6 m from mature trees and sampled 1998–2000. eMature tree roots were excavated spring 2000. Data are means of relative abundance as per cent of total mycorrhizal root tips per tree root sample or seedling.

  • *

    Partial sequence only.

  • Could not be distinguished from R. vinicolor II.

Tuber borchiiITS 94%AF106890blastTuber I59.05 7.06 7.94 5.29 
Rhizopogon rudusITS 98%AF377107blastRhizopogon rudus31.7310.5514.8911.03 
Aleuria aurantiaITS 96%*AF072090blastAscomycota I 6.33    
Hebeloma cavipesITS 99%AF124670blastHebeloma cavipes 2.37 5.01 0.66 0.47 
Wilcoxina rehmiiITS 99%AF266708blastWilcoxina rehmii 0.5219.67 0.13 0.60 
Tomentella ellisiiITS 94%AF272913blastTomentella ellisii gp. 27.26 1.65 1.96 
Thelephora terrestrisITS 99%AY750163Sporocarp #EC181.C128Thelephora terrestris 16.38 2.39 0.52 
Rhizopogon vinicolorITS 98%AF263933blastRhizopogon vinicolor I  4.79 9.77 6.67 
RFLP only    Rhizopogon spp.  4.7731.3020.55 
Cenococcum geophilumITS 99%AY112935blastCenococcum geophilum  1.94 3.84 2.2019.48
Rhizopogon parksiiITS100%AF058314blastRhizopogon parksii  1.47 4.60 4.83 
Phialophora finlandiaITS100%AJ534704blastPhialophora finlandia  0.98 0.72 0.03 1.53
Pseudotomentella tristisITS 99%AF274772blastPseudotomentella tristis  0.11 6.57 7.3211.67
Rhizopogon villosulusLSU100%AF071464blastRhizopogon villosulus   2.49  
Boletus zelleriITS100%AY750158Sporocarp #EC176.C41Boletus zelleri   2.13 6.20 3.14
Athelia neuhoffiiITS 89%*U85798blastAtheliaceae II   1.32 0.08 
Clavulina cristataLSU 99%AY586648blastClavulina cristata   1.11 1.13 8.29
Tomentella sublilacinaITS 99%AF323111blastTomentella sublilacina   0.99 2.78 
RFLP only   blastRussula spp.   0.98 0.48 5.56
Amphinema byssoidesLSU 96%AY568626blastAtheliaceae I   0.91 0.13 
Tomentella stuposaITS 99%AY010277blastTomentella stuposa   0.84 1.97 2.01
Pseudotomentella nigraITS 99%*AF274770blastPseudotomentella nigra   0.82 0.57 
Tomentellopsis sp.ITS 95%AJ410774blastTomentellopsis I   0.64  
Tylospora asterophoraLSU 96%AY463480blastTylospora I   0.37 2.09 2.14
Amphinema byssoidesLSU 97%AF291288blastAmphinema I   0.36 0.06 
Tylospora asterophoraLSU100%AF325323blastTylospora asterophora   0.36 1.25 
RFLP only    Tomentella spp.   0.36 0.39 
Tomentella lateritiaITS 93%*AJ534912blastTomentella II   0.30 0.84 1.33
Piloderma fallaxITS 99%AY010281blastPiloderma fallax   0.20 0.14 
Melanogaster macrosporusITS 96%AJ555526blastMelanogaster I   0.19 1.52 
Inocybe pudicaLSU 94%AY038323blastInocybe II   0.19 0.17 
Russula sphagnophilaLSU 98%AF506464blastRussula sphagnophila gp.    0.18 0.08
Tomentella botryoidesLSU 98%AY586717blastTomentella I   0.09  
Amphinema byssoidesLSU 99%AF291288blastAmphinema byssoides   0.06 0.57 
Inocybe sierraensisLSU 97%AY239025blastInocybe sierraensis gp.   0.05  1.14
Dermocybe cinnamomeaITS 99%AY750159Sporocarp #EC177.C56Dermocybe cinnamomea   0.03 0.10 0.54
Russula nigricansITS 99%AF418607blastRussula nigricans   0.0310.98 7.50
Inocybe flocculosaLSU 96%AY380375blastInocybe I   0.02 0.06 
Russula bicolorITS 99%AY750161sporocarp #EC179.C83Russula bicolor    1.19 2.06
Lactarius mitissimusITS 97%AF157412blastLactarius I    1.11 0.64
Russula chloroidesITS 98%AF418604blastRussula chloroides    0.98 9.54
Laccaria laccataITS 95%AF204814blastLaccaria I    0.98 
Amanita muscariaLSU 99%AF097367blastAmanita muscaria    0.72 0.72
Sebacina sp.LSU 99%AF440647blastSebacina I    0.62 0.78
Truncocolumella citrinaITS 98%L54097blastTruncocolumella citrina    0.61 8.86
Russula xerampelina gp.ITS100%AY750164Sporocarp #EC182.C130Russula xerampelina gp.    0.16 
Inocybe sindoniaLSU 96%AY380393blastInocybe sindonia gp.    0.14 
Peziza limneaLSU 99%AF335147blastPeziza limnea    0.14 
Piloderma byssinumITS 98%AY010279blastPiloderma byssinum    0.11 4.17
Macowanites americanusITS100% S. L. MillerMacowanites americanus    0.10 5.50
Inocybe praetervisaLSU 97%AY038322blastInocybe praetervisa gp.    0.07 
Russula adustaITS 98%AY061652blastRussula adusta     2.56
RFLP only    Unidentified   0.50 0.02 0.85

Statistical analyses

The EMF taxon abundance, measured as the proportion of root tips of an EMF taxon for a sample, was divided by total abundance of active EMF root tips per sample to give relative abundance. Because one EMF individual is likely to colonize multiple root tips within its region of growth, numbers of seedlings or root samples in which each EMF taxon occurred were used for calculations of species richness and diversity indices, rather than EMF root tip abundance. Any EMF that could not be unambiguously assigned to a taxon (i.e. Rhizopogon spp., Tomentella spp., Russula spp.) were omitted from calculations of species richness and diversity indices. For the purpose of comparison, EMF associated with mature tree roots, seedlings < 6 m or > 16 m from the center tree, and observed in the glasshouse bioassay were considered as separate communities despite occurring in the same general area. This is consistent with other studies of mycorrhizal communities (Taylor & Bruns, 1999).

The number of EMF taxa observed when sampling a community was adjusted by rarefaction for comparison with communities having a smaller sample size of observed EMF. Rarefaction was calculated using the correction proposed by Simberloff (1971) for the method described by Sanders (1968) using a web-based calculator (J. Brzustowski, This web-based calculator was also used to estimate species richness by the method proposed by Chao (1984). Jackknife estimates of species richness were obtained using the PC-ord software package (McCune & Mefford, 1997). The Shannon–Weaver diversity index was calculated as:

  • H′ = −Σ pi log10 pi

(pi was the number of seedlings or soil samples containing each individual EMF taxon, divided by the total number of individual observations of EMF; Magurran, 1988). Differences between diversity indices were tested using t-tests as described by Magurran (1988). Simpson's reciprocal diversity index was calculated as 1/D, with:

  • D = Σ n(n − 1)/N(N − 1)

(n is the number of seedlings or soil samples containing each individual EMF taxa; N is the total number of individual observations of EMF). The Morista–Horn similarity index was calculated as:

  • image

(an and bn are the number of EMF root tips for individual taxa from community a and b; aN and bN are the total number of EMF root tips for each community; Magurran, 1988).

Type III multifactor anova (glm procedure) was used to test for differences between groups with date and site as fixed factors, and comparisons of taxon frequencies were performed using contingency tables (spss for Windows 10.0.5; SPSS Inc., Chicago, IL, USA). Effectiveness of sampling effort was evaluated using ‘species area’ curves (in this case, ‘species-sample unit’ curves) calculated by PC-ord. For detrended correspondence analysis (DCA), PC-ord was used to identify outliers and to standardize relative abundance data using the arcsine square-root transformation. The only taxon that qualified as an outlier (> 3 SD) was ‘Rhizopogon spp.’ an artificial grouping of Rhizopogon mycorrhizas which could not be definitively assigned to a species group. This group was excluded from the ordination analysis, but exclusion did not substantially change the result.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

From 1998 to 2000, 52 taxa were observed on Douglas fir seedlings and mature trees (Table 2). Two taxa within the Atheliaceae could not be resolved to genus and were designated as ‘Atheliaceae I’ and ‘Atheliaceae II.’ Sequence homology placed another taxon in the Ascomycota, with an Aleuria species as the closest matching sequence but the sequences were not similar enough to place it definitively within this genus, therefore the taxon was designated as ‘Ascomycota I.’ Phylogenetic analysis revealed the presence of two clades within the genus Wilcoxina, with 72% bootstrap support (data not shown). One of the clades was Wilcoxina rehmii; it was not clear whether the other clade was a separate Wilcoxina species or represented population level variability of W. rehmii. Because it was not possible to distinguish between the two clades on the basis of RFLPs with the endonucleases used in this study, they were combined as a single taxon and designated as ‘Wilcoxina rehmii’ in this analysis (Table 2).

Within the genus Rhizopogon, while several putative species-level taxa could be distinguished by RFLPs as well as by sequence, some collections could only be identified to the genus level with RFLP analysis, and it was not feasible to sequence each individually because of the large number of samples. Where possible, these were assigned to taxa within the genus Rhizopogon based on morphological characters. The remainder are designated as ‘Rhizopogon spp.’ in Table 2. Most of these had RFLP patterns consistent with the Rhizopogon rudus found on nursery seedlings which persisted after outplanting, but morphological characters and RFLP patterns could not be used to exclude other Rhizopogon species. Within the genera Russula and Tomentella it was also not possible to distinguish among taxa for all collections, therefore ‘Russula spp.’, and ‘Tomentella spp.’ are included as categories in Table 2. Root tip samples in these categories were excluded from calculations of diversity indices.

With the exception of the nursery seedlings, all the treatment groups were undersampled, based on species area curves relating the number of taxa observed to the number of samples (Fig. 1). In particular, the mature tree roots would have required a much higher number of samples to adequately reflect the EMF community, as the species area curve had not begun to level off.


Figure 1. ‘Species-area’ curves showing increase in number of observed ectomycorrhizal fungal (EMF) taxa with increased sampling effort of Douglas-fir (Pseudotsuga menziesii) seedlings and trees. Seedlings were planted in spring 1998 < 6 m (open circles) or > 16 m (closed circles) from mature trees and sampled 1998–2000, glasshouse seedlings (open squares) were potted in field soils in spring of 1998 and sampled from 1998 to 1999, and mature tree roots (closed squares) were excavated spring 2000. Each subsample consisted of 100 root tips.

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EMF of nursery and glasshouse seedlings

Douglas-fir nursery seedlings were mycorrhizal with a total of five EMF taxa at the time of planting, based on a subsample of eight seedlings, with a total of 2612 mycorrhizal root tips. Owing to the small sample size, species richness estimates could not be calculated. The Shannon–Weaver diversity index (H′) was 0.63 while the Simpson diversity index (1/D) was 4.41 (Table 3). These low values reflect both the low number of taxa and the low evenness of the EMF community on nursery seedlings. Tuber I and Rhizopogon rudus together constituted more than 90% of mycorrhizal root tips (Table 2).

Table 3.  Species richness and diversity of Douglas fir (Pseudotsuga menziesii) seedling and tree ectomycorrhizal fungal (EMF) communities
 Nursery seedlingsGlasshouse seedlings> 16 m seedlings< 6 m seedlingsMature trees
  • For each tree root sample or seedling (except nursery seedlings), a subsample of 100 root tips was examined. Any EMF that could not be unambiguously identified to taxon were omitted from calculations of diversity and species richness. Nursery-grown seedlings were sampled before planting in spring 1998, and all mycorrhizal roots were examined; ND, could not be determined. Glasshouse seedlings were potted in field soils and sampled 1998–99. Seedlings were planted > 16 m or < 6 m from mature trees and sampled 1998–2000. Mature tree roots were excavated spring 2000.

  • *

    Data are means ± 1 SE.

  • First-order estimate.

  • §

    Second-order estimate.

Samples   8 34  84  84 12
Mycorrhizal root tips261249536654745339
Taxa observed   5 11  34  43 20
Taxa per sample*   3.1 ± 0.1  2.0 ± 0.2   3.3 ± 0.1   4.1 ± 0.2  3.6 ± 0.4
Estimated species richness
Chao-1ND 15.5  46.1  53.3 27.1
Chao-2§ND 15.5  46.1  57.1 62.7
Jackknife-1ND 13.7  45.9  56.8 30.7
Jackknife-2§ND 15.3  51.4  63.2 35.5
Diversity index
Shannon–Weaver (H′)   0.63  0.92   1.14   1.19  1.19
Simpson 1/D   4.4  8.2  10.9  15.1 15.1

Nursery fungi persisted on the planted seedlings both in the glasshouse and in the field, with the exception of an ascomycete related to the genus Aleuria, which was not detected after planting (Table 2). The nursery dominant Tuber I decreased in abundance after planting, both under glasshouse and field conditions. It appeared that Rhizopogon rudus decreased in abundance after planting both in the glasshouse and at the field sites, although owing to the large number of unidentified Rhizopogon spp. it was impossible to rule out the possibility that R. rudus increased in abundance on field seedlings. Abundance of Hebeloma cavipes increased in the glasshouse bioassay but decreased for field seedlings. W. rehmii was rarely detected on field seedlings, but proliferated dramatically on glasshouse seedlings after planting.

While four of the five nursery EMF taxa persisted after seedlings were potted in soils from the field sites and grown in the glasshouse, seven new taxa colonized the seedlings (Table 2). The observed species richness of 11 taxa was estimated to represent an actual species richness of between 13.7 and 15.5 taxa (Table 3). The Shannon–Weaver and Simpson diversity indices were substantially higher for glasshouse seedlings than for nursery seedlings (Table 3). Members of the Thelephoraceae predominated, with 44% of mycorrhizal tips, while Rhizopogon spp. were also common, with at least three Rhizopogon species together constituting 22% of mycorrhizal tips (Table 2). Most of the remaining taxa were ascomycetes, with four taxa making up 30% of mycorrhizal tips, including Tuber I from the nursery and the widespread generalist Cenococcum geophilum (Table 2).

At the time of sampling, 42% of the EMF on root tips of glasshouse seedlings were nursery EMF taxa. The remaining root tips were colonized either from propagules present in the field soils, or from airborne spores deposited in the glasshouse. The EMF taxa that colonized glasshouse bioassay seedlings also colonized field seedlings, but the pattern of relative abundance was different. In general, Rhizopogon species were much more abundant on field seedlings compared with glasshouse seedlings, while Thelephora terrestris and a species in the Tomentella ellisii group were less common on field seedlings.

EMF of excavated mature tree roots

A total of 20 EMF taxa were observed on mature tree roots. Estimates of the actual species richness ranged from a low of 27.1 ± 4.9 for the Chao-1 estimate to 62.7 ± 22 for the Chao-2 estimate, with the first- and second-order jacknife estimates intermediate (Table 3). Both observed and estimated taxon richness were substantially higher than for nursery and glasshouse seedlings, but generally lower than for field seedlings (Table 3). The lower species richness was not surprising considering that mature tree roots were collected on only one sample date, while seedling mycorrhizas were collected over a 2-yr period. When compared with field seedling mycorrhizas from spring 2000 only, the EMF community on mature trees had a higher species richness than either < 6 m or > 16 m seedlings. While an equal number of root tips (1200) were examined for each treatment group (< 6 m seedlings, > 16 m seedlings and mature tree), the mature trees had a lower proportion of viable mycorrhizal root tips, and therefore a smaller total sample size, yet the observed species richness was higher at 20 than the 16 taxa observed for > 16 m seedlings and 18 taxa observed for < 6 m seedlings. The EMF community on mature trees had greater evenness than that of the < 6 m and > 16 m seedlings when compared for that sample date only, based on the longer tail of the rank-abundance curve (Fig. 2b). The rank-abundance curves for all three treatment groups approximated a log-linear relationship, but the mycorrhizal community on mature trees was more even, as the slope was significantly less than that of the < 6 m and > 16 m seedlings, based on nonoverlap of 95% confidence intervals.


Figure 2. Rank-abundance curves for ectomycorrhizal fungal (EMF) taxa of Douglas-fir (Pseudotsuga menziesii) seedlings and trees. Slopes (B) of best fit lines (log10(abundance) rank−1) are given for each. (a) Seedlings were planted in spring 1998 < 6 m (open circles; B = −0.054) or > 16 m (closed circles; B = −0.066) from mature trees and sampled 1998–2000, glasshouse seedlings (open squares; B = −0.183) were potted in field soils spring 1998 and sampled from 1998 to 1999, and mature tree roots (closed squares; B = −0.074) were excavated spring 2000. (b) EMF rank abundance spring 2000 only for < 6 m seedlings (open circles; B = −0.109), 16 m seedlings (closed circles; B = −0.103) and mature trees (closed squares; B = −0.074).

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Diversity indices reflected the greater evenness of the EMF communities of the mature trees, in that both indices were higher for mature trees than for > 16 m seedlings even over the entire sampling period, despite the substantially lower observed species richness (20 taxa compared with 34 taxa). The Simpson diversity index, which gives more weight to evenness than the Shannon–Weaver index, was much higher for the mature tree than for > 16 m seedlings; with 15.1 compared with 10.9, and was identical to the value of 15.1 calculated for < 6 m seedlings (Table 3).

The genus Rhizopogon was not detected on mature tree roots (Table 2). Instead, mature tree roots were dominated by several taxa in the genus Russula, with a combined relative abundance of 27.2 ± 10.4%, and by Cenococcum geophilum, with 19.5 ± 5.1%. In addition, Pseudotomentella tristis, Truncocolumella citrina, Clavulina cristata and Macowanites americanus each had relative abundances greater than 5% (Table 2). Of these, P. tristis and C. cristata were relatively evenly split between < 6 m and > 16 m field seedlings, while T. citrina, M. americanus, and most of the Russula taxa (particularly Russula bicolor and Russula chloroides) were found only on < 6 m seedlings.

EMF of field seedlings

Seedlings > 16 m from the center tree were observed to form mycorrhizas with 34 EMF taxa (Table 3). The species area curve was leveling off but had not reached a plateau after sampling 8400 root tips on 84 seedlings from 1998 to 2000 (Fig. 1). Estimates placed the actual number of taxa between 45.9 and 51.4 (Table 3). The Chao-2 estimate has been shown to be less biased than the jackknife method for samples with low frequencies (Chao, 1984, 1987), therefore the estimate of 46.1 ± 7.2 taxa was probably the most reliable. The Shannon–Weaver and Simpson diversity indices, at 1.14 and 10.9, respectively, were higher than for glasshouse seedlings, but lower than for mature tree roots and seedlings planted < 6 m of mature trees (Table 3). The rank abundance curve was nearly log-linear, with slightly higher abundance of the most dominant taxa (Fig. 2a), which were members of the genus Rhizopogon (Table 2).

On seedlings < 6 m from mature trees, 43 EMF taxa were observed (Table 3). The same number of root tips were examined for < 6 m seedlings as for > 16 m seedlings and the species area curve did not reach a plateau (Fig. 1). The slope was steeper than for the > 16 m seedling EMF community and was similar to the curve for mature tree roots (Fig. 1). Rarefaction to a sample size of 223 EMF individuals observed (the number of EMF individuals observed for > 16 m seedlings) yielded an estimate of 40.0 (SD = 1.5) EMF taxa on < 6 m seedlings, which was still substantially higher than 34, the number of observed taxa for > 16 m seedlings. Estimates of the actual species richness for the EMF community of < 6 m seedlings were higher than those for > 16 m seedlings, and the Shannon–Weaver diversity index was significantly higher at 1.19 compared with 1.14 for > 16 m seedlings (two-tailed t(102) = 2.85, P < 0.01) (Table 3). The higher diversity of EMF communities of seedlings < 6 m from center trees than of seedlings > 16 m from center trees appeared to result from the presence of a greater number of infrequent taxa, based on the longer tail of the rank-abundance curve (Fig. 2a). The EMF community was significantly more even for < 6 m seedlings than for > 16 m seedlings, as the slope of the rank-abundance curve was significantly steeper for > 16 m seedlings (Fig. 2a), based on nonoverlap of 95% confidence intervals for the slopes.

Five genera were observed to have an abundance greater than 5%, for < 6 m and > 16 m seedlings considered as a group. Of these, Rhizopogon species were more common on > 16 m seedlings, with a relative abundance of 63.1 ± 3.0% on > 16 m seedlings but only 43.1 ± 3.3% on < 6 m seedlings. The nursery taxon Tuber I was also more common on > 16 m seedlings, while Russula and Tomentella were more common on < 6 m seedlings, and the abundance of Pseudotomentella species was similar on < 6 m and > 16 m seedlings. Contingency tables using frequency rather than abundance provided an additional approach to detecting differences between < 6 m and > 16 m seedling mycorrhizal communities. Six taxa occurred at significantly greater frequencies than expected on < 6 m seedlings compared with > 16 m seedlings: R. nigricans, Tylospora I, Tomentella stuposa, Tomentella sublilacina, Boletus zelleri and Melanogaster I (Table 4). There were no taxa that occurred at significantly greater frequencies than expected on > 16 m seedlings.

Table 4.  Results of contingency tables showing ectomycorrhizal fungal (EMF) taxa occurring at frequencies higher than expected on < 6 m seedlings compared with > 16 m seedlings
EMF taxaPearson χ2P-value
  • Seedlings were planted in spring 1998 < 6 m or > 16 m from mature Douglas fir (Pseudotsuga menziesii) trees. Seedlings were excavated in autumn and spring from 1998 to 2000. For each seedling, 100 root tips were examined. Pearson χ2 values are shown for all EMF taxa with P-values < 0.05. No EMF taxa were present at higher than expected frequencies on > 16 m seedlings.

  • *

    P-value was determined using Fisher's exact test because expected cell values were less than 5.

Russula nigricans18.3890.000
All Russula spp.17.1110.000
Tylospora I 4.7670.029
All Tylospora spp. 6.9080.009
Tomentella stuposa 7.8800.005
Tomentella sublilacina 5.7530.034*
All Tomentella spp.13.1250.000
Boletus zelleri 7.9540.005
Melanogaster I 6.8050.009

The higher diversity and evenness of the mycorrhizal community for < 6 m seedlings appeared to be the result of taxa shared with mature trees. Eight of the taxa found on < 6 m seedlings but not on > 16 m seedlings were also found on roots from the mature tree, including R. bicolor, R. chloroides and M. americanus (a member of the Russulaceae), Lactarius I, Amanita muscaria, T. citrina, and Piloderma byssinum. By contrast, only one of the taxa found on > 16 m seedlings but not on < 6 m seedlings was shared with the mature tree, a species in the Inocybe sierraensis group (Table 2). Taxa found on mature tree roots accounted for 38.8 ± 3.7% of < 6 m seedling root tips, but only 17.0 ± 2.6% of > 16 m seedling mycorrhizas (Fig. 3a). By contrast, > 16 m seedlings hosted a greater proportion of EMF observed in the glasshouse bioassay. Glasshouse EMF taxa accounted for 84% of mycorrhizal tips for > 16 m seedlings, but only 61% of mycorrhizal tips for < 6 m seedlings (Fig. 3a). Nursery EMF taxa also appeared to persist more readily on > 16 m seedlings (Fig. 3a).


Figure 3. Similarity of ectomycorrhizal fungal (EMF) communities of Douglas-fir (Pseudotsuga menziesii) seedlings < 6 m (tinted columns) and > 16 m (open columns) from trees to EMF of mature trees and glasshouse and nursery seedlings. (a) Proportion of EMF taxa shared with mature trees, glasshouse seedlings potted in field soils, and standard bare-root nursery seedlings. Values are means of relative abundance ± SE of shared EMF taxa. Groups (< 6 m, > 16 m) with different letters are significantly different by multifactor type III anova (α = 0.05) with date and site as fixed factors. (b) Morista–Horn similarity index (Cmh) comparing < 6 m seedlings with > 16 m seedlings based on similarity to mature trees, glasshouse, and nursery seedlings.

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The Morista–Horn similarity index, which takes into account the similarity of the distribution of the EMF community, reinforced these findings. The < 6 m seedlings had mycorrhizal communities with greater similarity than > 16 m seedlings to the mycorrhizal communities of the mature tree (Fig. 3b). By contrast, > 16 m seedlings had greater similarity than < 6 m seedlings to mycorrhizal communities of glasshouse bioassay seedlings and nursery seedlings (Fig. 3b).

It was interesting to note that EMF taxa forming epigeous fleshy sporocarps (e.g. Agaricales) were significantly more common for < 6 m seedlings than for > 16 m seedlings, reflecting a closer similarity to mature tree EMF (Fig. 4a). By contrast, taxa forming hypogeous fleshy sporocarps (e.g. Rhizopogon, Tuber) were significantly more common for > 16 m seedlings than for < 6 m seedlings (Fig. 4a), while nontruffle-forming ascomycetes and resupinates were not significantly different in abundance for < 6 m and > 16 m seedlings (data not shown). Nevertheless, while proximity to mature trees appeared to have an important effect on mycorrhizal communities based on substantial shifts in similarity indices, the < 6 m and > 16 m seedling mycorrhizal communities were still more similar to each other (Morista–Horn similarity index = 0.85) than to any other group. Partly owing to the high abundance of Rhizopogon spp., taxa forming rhizomorphs were significantly more common for < 6 m and > 16 m seedlings than for mature trees roots and for glasshouse and nursery seedlings (Fig. 4b).


Figure 4. Per cent relative abundance of ectomycorrhizal fungal (EMF) functional groups from Douglas-fir (Pseudotsuga menziesii) seedlings and trees. (a) Epigeous (tinted columns) includes taxa forming fleshy above-ground sporocarps (e.g. Agaricales); hypogeous (open columns) includes taxa forming fleshy below-ground sporocarps (e.g. Tuber, Rhizopogon). Sporocarp categories do not sum to 100% because resupinates and nontruffle-forming Ascomycota are not shown. (b) Per cent relative abundance of EMF taxa forming rhizomorphs. Seedlings were planted < 6 m or > 16 m from mature trees. Glasshouse seedlings were potted in field soils. Values are means ± 1 SE; EMF communities with different letters are significantly different by Tukey's HSD (α = 0.05).

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Ordination of EMF communities

Ordination of EMF communities using DCA showed clear segregation of field seedling EMF communities from mature tree EMF communities with lower scores on the primary axis, and from glasshouse and nursery seedling EMF communities with higher scores on the primary axis (Fig. 5a). The primary axis had an eigenvalue of 0.551 and explained more than half of the variance in the data (r2 = 0.56). The secondary axis had an eigenvalue of 0.262 and explained 16% of the variance in the data; the two axes together explained 72% of the variance.


Figure 5. Detrended correspondence analysis (DCA) of ectomycorrhizal fungal (EMF) communities on Douglas-fir (Pseudotsuga menziesii) seedlings and trees. Groups consist of seedlings planted < 6 m and > 16 m from mature trees, glasshouse seedlings, mature tree roots, and nursery seedlings. A DCA was used to analyse arcsin square-root transformed relative abundance of EMF taxa. Outliers (> 3 SD) were excluded from the analysis. (a) All groups, with rare taxa downweighted. Samples are labeled by site (GR, Green River; IM, Imagine; BE, Beatles). (b) < 6 m and > 16 m seedlings only. Plots are labeled by site and season and year of sampling (F, autumn; S, spring). Lines are vectors showing temporal progression for each plot.

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When analysed separately, it was clear that < 6 m and > 16 m seedling EMF communities were only partly segregated along the primary axis, with seedlings < 6 m from trees generally scoring higher (Fig. 5b). The data were weakly structured. The primary axis had an eigenvalue of 0.412 and explained 33.3% of variation. The secondary axis had an eigenvalue of 0.291 and explained only 4.5% of the variation in the data. Vectors tracing temporal progression from spring to fall and from year to year revealed no clear pattern of change over time (Fig. 5b).


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

EMF of nursery and glasshouse seedlings

As expected, the taxa colonizing seedlings in the nursery and glasshouse treatments were EMF which have been shown to readily colonize seedlings from spores or soil propagules (Bledsoe & Tennyson, 1982; Castellano et al., 1985; Berch & Roth, 1993; Massicotte et al., 1994; Parlade et al., 1996; Horton et al., 1998; Baar et al., 1999; Taylor & Bruns, 1999). In mature bishop pine (Pinus muricata) forests in California, Taylor & Bruns (1999) found colonization of bioassay seedlings to occur primarily by Rhizopogon spp., T. sublilacina, Tuber spp. and Wilcoxina spp., similar to the genera we observed in our bioassay.

The nursery taxon R. rudus persisted on glasshouse seedlings potted in field soils. However, W. rehmii matching those from the nursery occurred at a much higher abundance on glasshouse seedlings than on field seedlings. We cannot rule out the possibility that at least some of the colonization by Wilcoxina occurred because of spore deposition in the glasshouse, since Wilcoxina is a common glasshouse contaminant. Wilcoxina may be a poor competitor, flourishing only in the absence of the diverse fungal inoculum present in the field. Also, the relatively poor mycorrhizal development of the potted seedlings may have allowed the ectendomycorrhizal Wilcoxina to be detected more frequently by molecular methods. On field seedlings, Wilcoxina could have been present as a secondary symbiont of tips colonized by ectomycorrhizal taxa forming a well-developed mantle, causing the molecular signal to be obscured during PCR amplification because of the more prevalent DNA from the ectomycorrhizal symbiont.

Baar et al. (1999) found that both Rhizopogon spp. and Wilcoxina spp. established readily from resistant soil propagules (i.e. spores) on bishop pine bioassay seedlings. However, Rhizopogon spp. were more common on bioassay seedlings than field seedlings while Wilcoxina spp. were less common, in contrast to what we observed. It should be noted that our glasshouse treatment differed from a bioassay in that seedlings were previously colonized by nursery EMF taxa. The higher relative abundance of Rhizopogon spp. on field seedlings compared with seedlings potted in field soils in our study was intriguing considering the ease with which Rhizopogon spp. colonize roots from spores (Castellano et al., 1985; Parlade et al., 1996; Baar et al., 1999; Kjoller & Bruns, 2003). Rhizopogon spp. may have been favored under dry field conditions (Parke et al., 1983). Glasshouse seedlings differed from field seedlings in that they were irrigated and therefore probably experienced little water stress.

Thelephora terrestris was absent from nursery seedlings, but was common on glasshouse seedlings potted in field soils. Because T. terrestris is a common glasshouse contaminant, colonization of glasshouse seedlings could have occurred either from spores present in field soils or from local airborne spores. Roth & Berch (1992) found that Douglas fir nursery seedlings were dominated by T. terrestris, however, Castellano & Molina (1989) found that presence of T. terrestris varied from nursery to nursery. In our study, there was a trend of higher abundance for T. terrestris on seedlings > 16 m from trees than on seedlings < 6 m from trees. Other studies have shown that T. terrestris decreased in abundance on seedlings near forest edges compared with seedlings further into clearcuts (Durall et al., 1998; Kranabetter & Wylie, 1998; Kranabetter, 1999; Jones et al., 2002). Simard et al. (1997) observed a sixfold increase in Thelephora on trenched Douglas-fir seedlings, implying that forest edge effects on T. terrestris abundance may be caused by inability to outcompete EMF taxa linked to mature trees. Kranabetter & Friesen (2002) found that T. terrestris was able to replace EMF taxa from the forest when western hemlock seedlings were transplanted into forest openings.

EMF of excavated mature tree roots

The genera Tylospora (particularly Tylospora I), Tomentella (particularly Tomentella sublilacina and Tomentella stuposa), Russula (particularly R. nigricans), and Boletus zelleri occurred more frequently on seedlings < 6 m from mature trees than on seedlings > 16 m from trees. These EMF taxa were also common on mature tree roots, with the exception of T. sublilacina. From this pattern it appears that the predominance of T. sublilacina on seedlings < 6 m from trees was probably caused by differences in the soil environment, while the other four taxa may have colonized seedlings directly from mature tree roots. Boletus zelleri was the only EMF taxon forming long rhizomorphs, which would make it a particularly compelling candidate for dependence on hyphal linkages to mature trees.

Boletus zelleri was more frequent and more abundant on seedlings planted in microsites with buried wood compared with sites lacking buried wood, while other EMF taxa did not appear to be influenced by the amount of buried wood (Cline, 2004). Other studies have observed differences in EMF species composition when wood and soil substrates are compared (Kropp, 1982; Goodman & Trofymow, 1998; Smith et al., 2000; Tedersoo et al., 2003). Buried soil wood may be particularly important during periods of drought because of its ability to retain moisture.

EMF of field seedlings

Thirty-four EMF taxa were observed on Douglas-fir seedlings planted > 16 m from mature trees, while 43 taxa occurred on seedlings planted within 6 m of mature trees, using sequence and RFLP analysis. We know of no comparable studies for Douglas-fir. However, these data fall within the range of morphotypes reported for seedlings near forest edges or on isolated trees in studies of other tree species. Naturally regenerated paper birch seedlings near paper birch trees had 47 morphotypes (Kranabetter, 1999), while lodgepole pine, white spruce and subalpine fir seedlings planted near forest edges averaged 52 morphotypes per tree species (Kranabetter et al., 1999). Kranabetter & Wylie (1998) found 44 morphotypes on naturally regenerated western hemlock seedlings in 4-yr-old forest openings near forest edges. Only 25 morphotypes were observed on forest seedlings transplanted into clearcuts, while forest seedlings transplanted into the forest had 38 morphotypes (Kranabetter & Friesen, 2002). Simard et al. (1997) found that trenching reduced the number of morphotypes observed from 17 to 9 for Douglas fir seedlings growing in mature mixed forests of Douglas fir and paper birch. By comparison, Roth & Berch (1992) observed 33 morphotypes on Douglas-fir seedlings 1 yr after outplanting into clearcuts on Vancouver Island.

We found that the EMF community was more species rich for seedlings planted < 6 m compared with > 16 m from mature trees. Diversity indices and rank abundance curves reflected the greater evenness of the EMF community of seedlings < 6 m from trees, although the rank abundance curves for each seedling type were very similar by spring of 2000, 2 yr after planting. Studies of seedlings (Hagerman et al., 1999b) and ectomycorrhizal sporocarp production (Sparks, 2003) near forest edges provide evidence that 16 m is sufficient to isolate seedlings from mature trees. Nevertheless, we are not able to rule out the possibility that roots or, more likely, mycelial strands connected to mature tree roots could have grown out to reach the > 16 m seedlings by the end of the study period.

The relative magnitude of the effect of proximity to isolated mature Douglas fir trees on EMF diversity of Douglas fir seedlings was comparable to that observed in several studies of seedlings growing near mature trees (Kranabetter & Wylie, 1998; Hagerman et al., 1999b; Kranabetter, 1999). The effect was less extreme than the nearly twofold increase in EMF species richness observed for red oak seedlings growing near oak trees (Dickie et al., 2002) and for untrenched Douglas-fir seedlings compared with trenched seedlings growing in mixed Douglas-fir forests (Simard et al., 1997). By contrast, Durall et al. (1998) found only slight decreases in EMF species richness of western hemlock and lodgepole pine seedlings with distance from the forest edge, while Jones et al. (2002) found no differences in species richness with distance for previously colonized Engelmann spruce seedlings planted in mineral soil exposed by mounding. Jones et al. (2003) proposed that forest edge effects on EMF species richness were minimal for previously colonized (i.e. standard nursery) seedlings, but we detected effects of proximity to trees despite the fact that our seedlings were previously colonized. Our seedlings retained their nursery EMF taxa after planting, but significant differences in EMF species richness and diversity developed among seedlings < 6 m compared with > 16 m from residual trees.

Other mycorrhizal trees and shrubs at the site could have provided an additional reservoir of EMF inoculum, although we did not study this. Western hemlock advance regeneration was common at our sites (Cline, 2004). There appears to be some overlap between western hemlock and Douglas fir EMF communities (Roth & Berch, 1992; Smith et al., 1995), a topic that deserves further study. In other western USA forests, Horton & Bruns (1998) showed that a substantial proportion of EMF taxa were shared on Douglas-fir and bishop pine, while Arctostaphylos shrubs have been shown to provide a refuge for EMF taxa which colonize Douglas-fir seedlings after harvesting (Horton et al., 1999; Hagerman et al., 2001).

Field seedling EMF communities were dominated by Rhizopogon spp., which occurred on over half of all mycorrhizal root tips. This appeared to be partly due to the ability of the nursery taxon R. rudus to persist and colonize new roots on seedlings after planting. This was not surprising, since Castellano & Trappe (1985) found that inoculated Rhizopogon vinicolor persisted and colonized new roots on Douglas-fir seedlings after outplanting in clearcuts. Members of the genus Rhizopogon appear to establish well-distributed and persistent spore banks (Kjoller & Bruns, 2003), even in mature forests where they are not common on roots (Taylor & Bruns, 1999). Like Taylor & Bruns (1999), we found Rhizopogon spp. to be absent from mature tree roots but abundant on glasshouse bioassay seedlings.

For the genus as a whole, Rhizopogon spp. were present on 63% of root tips for seedlings > 16 m from mature trees but only on 43% for seedlings < 6 m from trees. The dominance of Rhizopogon spp. was responsible for the significantly higher proportion of taxa forming rhizomorphs on field seedlings than on nursery and glasshouse seedlings and on mature roots. The long rhizomorphs produced by Rhizopogon are thought to be adapted for long-distance transport (Agerer, 2001), but may also play a role in competing with other EMF taxa for the colonization of new roots. Simard et al. (1997) found that R. vinicolor was 20 times more abundant on untrenched Douglas-fir seedlings than on trenched seedlings, and suggested that R. vinicolor benefited from hyphal linkages to mature trees. This did not appear to be the case in our study, since seedlings > 16 m from trees had higher abundance of Rhizopogon spp. than seedlings < 6 m from trees.

Several of the EMF taxa found more frequently on seedlings < 6 m from trees have been reported to be favored by high soil nitrogen conditions, including T. sublilacina and Tylospora fibrillosa (Taylor et al., 2000; Lilleskov et al., 2002), and Russula spp. (Avis et al., 2003). Nevertheless, it seems unlikely that nitrogen would be more available near trees. In Douglas fir forests near our study sites, Barg & Edmonds (1999) found no differences in soil nitrogen dynamics at 6 m compared with 1 m from isolated mature trees. Nitrogen is often more available in the first few years after forest harvesting. Parsons et al. (1994) detected increases in soil nitrate in experimentally created gaps of 15 or more trees in lodgepole pine forests. Also, only some of the EMF taxa occurring more frequently < 6 m from trees were associated with high nitrogen substrates. For example, T. stuposa was reported by Tedersoo et al. (2003) to predominate in coarse woody debris, which usually has a high carbon to nitrogen ratio.

Ordination of EMF communities

The EMF communities of seedlings < 6 m from and > 16 m from trees were not entirely segregated when all EMF communities were included in the ordination. Nevertheless, EMF communities of seedlings < 6 m from trees had generally lower scores on the primary axis, closer to the scores for mature tree roots (Fig. 5a). The secondary axis appeared to be associated with site differences for all field samples. The EMF of mature trees had the greatest separation by site along axis 2, with low scores for Imagine, high scores for Green River and intermediate scores for Beatles. The EMF of seedlings < 6 m from mature trees followed the same pattern, while the EMF of seedlings > 16 m from mature trees had the relative position of Beatles and Imagine reversed. Glasshouse seedling EMF clustered tightly together, indicating that the site of origin of the soil in which the seedling was planted had relatively little effect on the EMF community.

Ordination of only the field seedling EMF communities revealed an effect of proximity to mature trees which was related to the primary axis. When proximity was coded as a quantitative factor (< 6 m = 1, > 16 m = 0), the treatment effect accounted for 25.5% of variation along axis 1 (r = 0.505). The secondary axis again appeared to be related to site. Imagine was associated with high values, Green River was intermediate (but highly variable) and Beatles was associated with low values along axis 2. It was notable that samples from seedlings < 6 m from trees at Green River assorted closely with samples from seedlings > 16 m from trees at the same site. This was responsible for most of the overlap between EMF communities of the two groups of seedlings. In general, differences among sites were greater than differences between seedlings < 6 m compared with > 16 m from mature trees at each site.

Ordination revealed a pattern of association between the < 6 m seedling EMF community and the mature tree community. The EMF community for seedlings > 16 m from trees was more closely associated with communities of glasshouse and nursery seedlings. It is not clear whether this similarity is a result of the direct influence of mature trees on colonization of seedlings, or a secondary effect of trees on the soil environment which facilitates the colonization or persistence of taxa associated with mature forest soils.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Residual trees play a dual role in recently harvested forests. First, by retaining a unique EMF community on their root systems, and second, by influencing the species composition and enhancing the diversity of EMF communities on seedlings adjacent to the trees. We have also shown for these seedlings that proximity to trees increases colonization levels and root to shoot ratios, while generally inhibiting stem growth (Cline, 2004). The long-term impact of residual trees on seedling survival and growth is difficult to predict, but it appears likely that residual trees would have lasting impacts on mycorrhizal diversity at the landscape level (i.e. at wider spatial scales) as stands recover from the effects of harvesting.

Differences in the mycorrhizal communities of seedlings as a result of proximity to mature trees could have important functional consequences not just for host plants but for the ecosystem as a whole as harvested forests regenerate. High mycorrhizal diversity has been proposed to increase plant productivity (van der Heijden et al., 1998; Jonsson et al., 2001) and potentially influence plant responses to environmental fluctuations or disturbance (Bledsoe, 1986; Jones et al., 2003). In addition to promoting EMF biodiversity, presence of residual trees influenced the species composition of mycorrhizal communities, which could impact ecosystem-level processes. For example, taxa producing fleshy epigeous sporocarps were significantly more abundant on seedlings < 6 m from trees. If the balance between epigeous and hypogeous sporocarp production were to be influenced over the longer term, this could have important consequences for mycophagous animals.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

We are grateful to Robert Cline, Amy LaBarge, Georgia Murray, Grace Sparks and Teresa Turk for assistance in the field. We thank the City of Seattle Cedar River Watershed and the Plum Creek Timber Company for permission to work in their forests. Financial support for this project was provided by the College of Forest Resources Ecosystem Sciences Division, Sigma Xi, the Puget Sound Mycology Society and the Stuntz Foundation. Doctor P. Brandon Matheny provided sequence analysis of Inocybe, and Dr Steven Miller provided sequence analysis of Russula and Macowanites.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
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