Supplementary material
Ectomycorrhizal fungal exoenzyme activity differs on seedlings planted in forests versus clearcuts
Trees: Structure and Function Special Issue “Root-Nagoya 2014”
Jennifer K.M. Walker*, Valerie Ward, Melanie D. Jones
University of British Columbia, Okanagan Campus
Biology Department, Science Building
1177 Research Road, Kelowna
BC V1V 1V7, Canada
*Corresponding author current address:
University of Western Sydney
Hawkesbury Institute for the Environment
Locked Bag 1797, Penrith
NSW 2751, Australia
email: , phone: +61 2 4570 1643, fax: +61 2 4570 1103
Table S1 Enzyme names used in this experiment, their assay substrate, and the component on which they act in soils.
Enzyme name / Assay substrate / Acts onβ-xylosidase / 4-MU1 ß-D-xylopyranoside / hemicellulose
β-glucuronidase / 4-MU-ß-D D-glucuronide hydrate / hemicellulose
cellobiohydrolase / 4-MU ß-D-cellobioside / cellulose
β-glucosidase / 4-MU ß-D-glucopyranoside / cellulose
N-acetylglucosaminidase / 4-MU N-acetyl-ß-glucosaminide / chitin
leucine aminopeptidase / L-leucine 7-AMC2 / proteins and peptides
acid phosphatase / 4-MU phosphate free acid / organically bound P
laccase / ABTS3 / phenolics (e.g. lignin)
14-methylumbelliferone
27-aminomethylcoumarin
3Diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)
Table S2 Relative abundance1 of ectomycorrhizal fungal a) OTUs and b) representative species (collectively) detected in forest and clearcut plots on seedlings sampled specifically for exoenzyme assays2.
a)
Best INSD and/or UNITE3 OTU identity / Relative abundance in forest plots / Relative abundance in clearcut plots /Tylospora asterophora 1 / 0.14 / 0.04
Tylospora fibrillosa 1 / 0.12 / 0.02
Amphinema byssoides 1 / 0.1 / 0.19
Pseudotomentella sp. / 0.06 / 0.01
Wilcoxina sp. 1 / 0.04 / 0.11
T. fibrillosa 2 / 0.03 / 0
A. byssoides 2 / 0.03 / 0.02
Atheliaceae sp. 2 / 0.03 / 0
Russula sp. 2 / 0.03 / 0
Pseudotomentella tristis / 0.02 / 0
Sebacina sp. / 0.02 / 0.01
Russula sp. 1 / 0.02 / 0
Cenococcum geophilum 3 / 0.02 / 0
Cortinarius idahoensis / 0.02 / 0
Thelephora terrestris 1 / 0.01 / 0.28
T. terrestris 2 / 0.01 / 0.07
Thelephoraceae sp. / 0.01 / 0
Tomentella sp. / 0.01 / 0
T. asterophora 2 / 0.01 / 0.02
Tylospora sp. / 0.01 / 0
Piloderma fallax / 0.01 / 0
Piloderma byssinum / 0.01 / 0
Piloderma sp. 1 / 0.01 / 0
Piloderma sp. 2 / 0.01 / 0
Piloderma sp. 3 / 0.01 / 0
Atheliaceae sp. 1 / 0.01 / 0
Atheliaceae sp. 3 / 0.01 / 0
Trichophaea hybrida / 0.01 / 0.04
Laccaria sp. 1 / 0.01 / 0.01
Laccaria sp. 2 / 0.01 / 0
Russula sp. / 0.01 / 0
C. geophilum 1 / 0.01 / 0.02
C. geophilum 2 / 0.01 / 0
C. geophilum 4 / 0.01 / 0
C. geophilum 5 / 0.01 / 0
Hydnotrya sp. / 0.01 / 0.01
Cortinarius sp. / 0.01 / 0
Cortinarius salor / 0.01 / 0
Cortinarius obtusus / 0.01 / 0
Cortinarius casimiri / 0.01 / 0
Thelephora sp. / 0 / 0.04
Pseudotomentella nigra / 0 / 0.01
T. fibrillosa 3 / 0 / 0.01
Amphinema sp. / 0 / 0.01
Wilcoxina sp. 2 / 0 / 0.01
Wilcoxina rehmii / 0 / 0.01
Cortinarius croceus / 0 / 0.01
Lyophyllum sp. / 0 / 0.01
Meliniomyces bicolor / 0 / 0.02
b) 4
Best collective identity / Relative abundance in forest plots / Relative abundance in clearcut plots /Tylospora asterophora / 0.20 / 0.05
Tylospora fibrillosa / 0.17 / 0.03
Amphinema byssoides / 0.14 / 0.24
Cenococcum geophilum / 0.09 / 0.03
Pseudotomentella tristis / 0.07 / 0.01
Russula spp. / 0.07 / 0
Cortinarius spp. / 0.07 / 0.01
Wilcoxina spp. / 0.06 / 0.15
Piloderma spp. / 0.04 / 0
Laccaria spp. / 0.03 / 0.01
Thelephora terrestris / 0.01 / 0.39
Trichophaea hybrida / 0.01 / 0.05
1 Relative abundance was calculated by dividing the number of ectomycorrhizae from each taxon per seedling by the total number of ectomycorrhizae counted on that seedling
2 For results of a detailed study on the ectomycorrhizal fungal community on spruce roots in forests, clearcuts and microsites, see Walker and Jones (2013).
3 INSD = International Nucleotide Sequence Database; UNITE = User-friendly Nordic ITS Ectomycorrhiza Database
4 Relative abundance of dominant ECM fungi ( 3% of ectomycorrhizae in either forest or clearcut plots) colonizing spruce roots, after combining OTUs whose best database identities matched to the same species or genus.
Table S3 Multivariate ANOVA of soil chemical (abiotic) properties among microsites1, plot-level treatments2 and blocks from soils surrounding seedlings harvested for enzyme assays. The non-independent soil properties included total carbon, total and mineralizeable nitrogen (N), available phosphorus (P), ammonium and nitrate, and pH. Mineralizeable N, available P, ammonium and nitrate were cube-root transformed in order to meet assumptions of normality for analyses.
Degrees of freedom (DF) / Pillai’s Trace / Approximate F (F-statistic) / Number DF (model) / Density DF (error) / Pr (>F)(p-value)
Block / 2 / 0.81166 / 1.3660 / 14 / 28 / 0.23342
Plot / 1 / 0.61726 / 2.9951 / 7 / 13 / 0.04178*
Microsite / 2 / 1.66081 / 9.7930 / 14 / 28 / 2.25e-07***
Plot X Microsite / 2 / 0.39526 / 0.4926 / 14 / 28 / 0.91775
Residuals / 19
1The three microsites are control mineral soil, mineral soil under downed wood, and decayed wood.
2The two plot-level treatments are mature forest and clearcut.
Table S4 Growth measurements of seedlings harvested for enzyme assays between forest and clearcut plots and among microsites. Values represent raw means (SD) for seedling dry weight (Seedling d wt) and root to shoot ratio (Root:Shoot). P-values in the table correspond to univariate statistical tests performed on log-transformed data. N = 5 seedling samples per microsite type per forest plot and 10 seedlings samples per microsite type per clearcut plot.
Plot or Microsite / Seedling d wt (g) / Root:ShootForest / 0.018 (0.010) / 0.69 (0.39)
Clearcut / 0.019 (0.010) / 0.78 (0.43)
P (plot) / 0.2 / 0.2
Control / 0.018 (0.012) / 0.81 (0.56)
Downed / 0.018 (0.009) / 0.67 (0.36)
Decayed / 0.021 (0.013) / 0.77 (0.29)
P (microsite) / 0.3 / 0.2
Table S5 Multivariate ANOVA of ectomycorrhizal enzyme activity profiles (i.e. the overall activity of all eight enzymes1 from all ectomycorrhizae per seedling) among microsites, plot-level treatments2 and blocks3. All data were cube-root transformed in order to meet assumptions of normality for analyses.
Degrees of freedom (DF) / Pillai’s Trace / Approximate F (F-statistic) / Number DF (model) / Density DF (error) / Pr (>F)(p-value)
Block / 2 / 0.21496 / 1.8214 / 16 / 242 / 0.0291*
Plot / 1 / 0.59456 / 21.997 / 8 / 120 / <2e-16***
Microsite / 2 / 0.13613 / 1.1047 / 16 / 242 / 0.3511
Plot X Microsite / 2 / 0.06984 / 0.5472 / 16 / 242 / 0.9195
Residuals / 127
1 ß-glucuronidase, ß-xylosidase, cellobiohydrolase, ß-glucosidase, N-acetylglucosaminidase, acid phosphatase, leucine aminopeptidase and laccase.
2The two plot-level treatments were mature forest and clearcut.
3The three blocks differed in elevation.
Table S6 Multivariate anova analyses of dominant ECM fungal species exoenzyme profiles in a) forest plots1 and b) clearcut plots2. Enzyme profiles represent the combined activity of ß-glucuronidase, ß-xylosidase, cellobiohydrolase, ß-glucosidase, acid phosphatase, N-acetylglucosaminidase, leucine aminopeptidase and laccase. Replication was at the level of seedling, and only those ECM fungal species that occurred on more than one root tip on at least three seedlings and in all microsite types in the forest or the clearcut were used to generate this data.
a)
Degrees of freedom (DF) / Pillai’s Trace / Approximate F (F-statistic) / Number DF (model) / Density DF (error) / Pr (>F)(p-value)
Taxon / 4 / 2.72866 / 4.8291 / 32 / 72 / 1.514e-08***
Microsite / 2 / 0.65686 / 0.9781 / 16 / 32 / 0.5008
Taxon X Microsite / 8 / 1.95904 / 0.8918 / 64 / 176 / 0.6972
Residuals / 22
1 The five dominant ECM fungal species in forest plots included Amphinema byssoides, Tylospora fibrillosa, Tylospora asterophora, Cortinarius sp., and Cenococcum geophilum.
b)
Degrees of freedom (DF) / Pillai’s Trace / Approximate F (F-statistic) / Number DF (model) / Density DF (error) / Pr (>F)(p-value)
Taxon / 2 / 1.4694 / 14.8849 / 16 / 86 / 2e-16***
Microsite / 2 / 0.3396 / 1.0993 / 16 / 86 / 0.3687
Taxon X Microsite / 4 / 0.6478 / 1.0870 / 32 / 180 / 0.3547
Residuals / 49
2 The three dominant ECM fungal species in clearcut plots included A. byssoides, Thelephora terrestris, and Wilcoxina sp.