New Phytologist Supporting Information
Article title: Mycorrhizal associations determine phosphorus cycling in deciduous forest soils
Authors: Anna Rosling1*, Meghan G. Midgley2, Tanya Cheeke2, 3, Hector Urbina1, Petra
Fransson3, Richard P. Phillips2
Article acceptance date: Click here to enter a date.
The following Supporting Information is available for this article:
Fig. S1 Map of plot locations
Fig. S2 PCA showing difference between AM and ECM dominated plots, and soil layers.
Fig. S3a and b Total soil enzymatic activity vs. ergosterol and MBC.
Fig. S4 Microbial Biomass Carbon (MBC) and Ergosterol
Fig. S5 PCA of fungal community structure of top-soil from four AM and ECM dominated plots.
Fig. S6 Seasonal dynamics in total Po and Pi as well as MBC and P
Table S1 Seasonal average soil P contents (mg P kg-1 dry soil) by mycorrhizal association and soil
depth.Seasonal average soil P contents (mg P kg-1 dry soil) by mycorrhizal association and soil
depth.
Table S2 Average soil P contents normalized by soil ergosterol (mg P mg-1 ergosterol).
Table S3 Average relative abundance of the ten most common orders of ectomycorrhizal fungi.
Table S4 Estimated seasonal average size of P pool (kg ha-1).
Methods S1 In situ soil solution P
Methods S2 Sequential P extraction
Methods S3 Soil enzymatic activity
Methods S4 Ergosterol extraction
1
Methods S5 Fungal community sequence analysis
Fig. S1 Overview of plot locations in the Indiana University’s Moores Creek Research and
Teaching Preserve, AM plots in Dark grey and ECM plots in light grey.
2
Fig. S2 PCA (Principle components analysis) ordination plot of total Pi, total Po, P sorption, MB
C:P, enzyme activity (AP), ergosterol, soil moisture (SM), organic matter content (OM) and pH,
showing the difference in soil chemistry between AM and ECM dominated plots. Symbols are
circles and squares to represent AM and ECM mycorrhizal association, respectively, and filled
and open circles to represent 0-5 cm soil layer and 5-15 cm soil layer, respectively. MRPP
analysis showed significant (A=0.053, p=0.000007) separation between soil layers.
3
a)
140
total phosphatase activity /kg soil
120
100
80
60
40
20
0
0
b)
2
4
6
Ergosterol mg/kg soil
8
10
140
total phosphatase activity / kg soil
120
100
80
60
40
20
0
0
500
1000
1500
2000
2500
MBC mg / kg soil
3000
3500
4000
4500
Fig. S3 Total phosphatase activity in soil is strongly correlated to a) total extracted ergosterol
(r2=0.61); and less so to b) Microbial Biomass Carbon (MBC) in ECM dominated plots in light
grey (r2=0.43) and in AM dominated plots in dark grey (r2=0.04).
4
12
Ergosterol mg/kg soil
10
8
6
4
2
0
0
500
1000
1500
MBC mg / kg soil
2000
2500
Fig. S4 Microbial Biomass Carbon (MBC) and Ergosterol (used as a proxy for biomass of fungi in
Dicarya) extracted from samples collected in May and September were more strongly
correlated (r2=0.86) in ECM dominated plots (in light grey) compared to AM dominated stands
(r2=0.54) (in dark grey).
5
Fig. S5 PCA (Principle components analysis) Ordination analysis of fungal community structure
of top 0-5 cm soils in four randomly selected plots from each mycorrhizal treatment (AM and
ECM) sampled in May (MAY) and September (SEPT).
6
a)
70
mgPi/kg soil
60
50
40
30
20
10
0
May
b)
July
Sept
Nov
160
140
mgPo/kg soil
120
100
80
60
40
20
0
May
July
Sept
Nov
May
July
Sept
Nov
c) 160
140
MB C:P
120
100
80
60
40
20
0
7
mgMBC/kg soil
cd)
1800
1600
1400
1200
1000
800
600
400
200
0
*
*
May
e)
*
July
Sept
July
Sept
Nov
90
80
mg MBP/kg soil
70
60
50
40
30
20
10
0
May
Nov
Fig. S6 Seasonal dynamics for ECM stands (in light grey) and AM stands (in dark grey) of a) total
organic P; b) total inorganic P; c) MB C:P; d) MBC and e) MBP. Values are weighted average
across soil layers with SEM as error bars (n=7). We found no significant effect of mycorrhizal
association on total Pi (P= 0.8777) and MBP (P =0.9728), but there was a significant effect of
mycorrhizal association on total Po (P =0.0273) and MBC (P =0.0031). Sample points when
mycorrhizal associations were significant different are indicated by *.
8
AM
P pools
Total P M, L, MxL
Total Pi L
Total Po M, L, MxL
MBP M, L, MxL
Inorganic P
Resin Pi L, MxL
Bicarbonate Pi L
Hydroxide Pi L
Acid Pi M, L, MxL
Organic P
Bicarb Po M, L, MxL
Hydroxide Po M, L, MxL
0-5 cm
158±7 a
47±4 a
104±7 a
31±3 a
ECM
5-15 cm
118±8 b
29±3 b
86±8 a
10±2 b
0-5 cm
155±7 a
52±5 a
93±8 a
38±5 a
5-15 cm
86±10 c
25±2 b
59±10 b
5±1 c
2.6±0.4 a
4.4±0.4 a
31±3 a
9±0.7 a
1.1±0.1 b
1.4±0.2 b
22±3 b
4±0.5 b
4.5±0.8 a
5.2±0.6 a
30±3 ab
12±1.8 a
0.7±0.1 b
1.1±0.2 b
21±2 b
2±0.2 c
8.2±1.3 b
99±6.9 a
7.7±1.1 b
82±8.0 a
24.7±2.3 a
78±6.1 a
10.7±1.1 b
52±10.0 b
Table S1 Seasonal average soil P contents in operational pools determined using a partial
Hedley fractionation (mg P kg-1 dry soil) and Microbial biomass P (MBP) for AM and ECM
dominated plots and further separated by soil depth. Seasonal average soil P contents in
operational pools determined using a partial Hedley fractionation (mg P kg-1 dry soil) and
Microbial biomass P (MBP) for AM and ECM dominated plots and further separated by soil
depth.
M Main effect of mycorrhizal association.
L Main effect of soil layer.
MxL Interaction effect for mycorrhizal association x soil layer.
For each P pool different letters indicate that values are significantly different based on Tukey ttest.
9
AM
P pools
Total Pi erg-1 M, L
Total Po erg-1 M, L
MBP erg-1 M
Inorganic P
Resin Pi erg-1 M, L
Bicarbonate Pi erg-1 M
Hydroxide Pi erg-1 M, L
Acid Pi erg-1 M
Organic P
Hydroxide Po erg-1 M, L
Enzyme activity
AP erg-1 M, L, MxL
PD erg-1 M
ECM
0-5 cm
44±3 ab
87±4 b
29±1 a
5-15 cm
65±3 a
182±14 a
30±2 a
0-5 cm
10±0.4 c
16±0.5 c
7±0.4 b
5-15 cm
31±1.6 b
60±4 b
5±0.4 b
2.0±0.1 a
4.2±0.2 a
30±2 a
8±0.3 a
2.6±0.1 a
2.8±0.2 ab
50±3 a
9±0.4 a
0.5±0.02 b
1.1±0.1 b
7±0.3 b
1.9±0.1 b
0.9±0.04 b
1.7±0.2 b
26±1 a
2±0.1 b
85±4 b
180±14 a
15±0.4 c
56±4 b
24±1 b
5±0.1 a
52±2 a
5±0.2 a
9±0.2 c
3±0.1 a
44±1 a
3±0.1 a
Table S2 Average soil P contents normalized by the amount of fungi in the Dicarya, estimated as
extracted soil ergosterol (mg P mg-1 ergosterol). Based on data from May and September
samples and presented for AM and ECM dominated plots, further separated by soil depth.
M Main effect of mycorrhizal association.
L Main effect of soil layer.
MxL Interaction effect for mycorrhizal association x soil layer.
For each P pool different letters indicate that values are significantly different based on Tukey ttest.
10
AM
ECM
May
Dothideomycetes;Hysteriales;Gloniaceae (Cenococcum)
6.6
9.9
May
0.1
Pezizomycetes;Pezizales;Tuberaceae (Tuber)
1.7
0
0.2
0.1
Agaricomycetes;Agaricales;Amanitaceae (Amanita)
1.8
0
0.1
0.1
Agaricomycetes;Agaricales;Cortinariaceae (Cortinarius)
2.6
0.3
0
0
Agaricomycetes;Agaricales;Hydnangiaceae;Laccaria
0.3
0.1
0
0
Agaricomycetes;Agaricales;Inocybaceae (Inocybe)
2.7
0
0.3
0.2
Agaricomycetes;Agaricales;Tricholomataceae (Tricholoma)
4.0
0.1
0
0
Agaricomycetes;Russulales;Russulaceae (Lactarius, Russula)
5.5
1.3
5.6
0.6
Agaricomycetes;Sebacinales;Sebacinaceae (Sebacina)
2.7
0.2
0.1
0.1
10.7
0.2
0.5
0.3
Other
1.4
0
1.1
0.4
Total
40
12
8
2
Agaricomycetes;Thelephorales;Thelephoraceae (Thelephora)
Sept
Sept
0.2
Table S3 Average relative abundance of the ten most common orders of ectomycorrhizal fungi
observed in the study compared by ECM and AM plots as well as time of sampling. Less
abundant orders of ECM fungi are summarized in Other. Total average relative abundance is
given as the end.
11
P pools
Total Pi T
Total Po M, T
MBP T
Inorganic P
Resin Pi T, MxT
Bicarbonate Pi T
Hydroxide Pi T
Acid Pi T
Organic P
Bicarbonate Po M, MxT
Hydroxide Po M, T
AM 0-15 cm
62.7±6 a
161.1±13 a
32.6±3 a
2.8±0.4 a
4.2±0.4 a
45.8±5 a
10.1±1 a
14.1±2 b
158.7±12 a
ECM 0-15 cm
59.7±4 a
121.5±17 b
32.3±3 a
3.2±0.5 a
4.2±0.4 a
43.6±4 a
8.7±1 a
26.6±2 a
110.2±15 b
Table S4 Estimated seasonal average size of P pool (kg ha-1) presented by mycorrhizal
association. Values are calculated from soil P contents in operational pools and bulk soil density
of 0-5 cm and 5-15 cm soil layers separately. Only plots with measurements from both soil
layers were used.
M Main effect of mycorrhizal association.
T Main effect of sampling time.
MxT Interaction effect for mycorrhizal association x sampling time.
For each P pool different letters indicate that values are significantly different based on
Students t-test.
12
Methods S1 In situ soil solution P
Blank anion membrane sheets (Maltz Sales, Foxboro, MA, US) were cut into 2 x 3 cm strips and
charged in 0.5M NaHCO3 at a minimum of 10 mL solution per strip for at least 15 minutes.
Starting in mid-April 2012, five charged AEM strips were deployed in each plot following a
stratified random structure. Strips were inserted at an angle of approximately 45 into the
upper 5 cm soil layer and incubated in the field for 2 - 3 weeks. Upon collection, the five
replicate AEMs were pooled in a zip lock bag and new strips were deployed at new locations in
the plot. Following collection, pooled AEM strips were rinsed in ddH2O, transferred to a 50 mL
centrifugation tube and adsorbed P was extracted by vertical shaking for 4 h in 25 mL of 0.5M
HCl (Shaw & DeForest, 2013). Extracts were frozen until analyzed.
Methods S2 Sequential P extraction
Resin P was extracted by horizontal shaking of soil and a charged AEM strip for 4h in 30 mL of
ddH2O. The AEM strip was then rinsed in ddH2O, transferred to a new centrifuge tube and
extracted as for soil solution P (above). Remaining soil slurry was centrifuged at 385 g for 10
minutes before discarding the supernatant. The pellet was re-suspended in 30 mL 0.5 M
NaHCO3 and Bicarbonate P was extracted by horizontal shaking for 16h before centrifugation at
385 g for 10 minutes. Extracted Bicarbonate P was filtered through a pre-rinsed Whatman No. 1
filter paper. Excess supernatant was discarded and the pellet was re-suspended in 30 mL 0.1 M
NaOH and followed by 30 mL of 1.0 M HCl in the same manner as Bicarbonate P to collect
Hydroxide P and Acid P, respectively. Total phosphorus Ptot in the Bicarbonate P and Hydroxide
P extracts was determined after digesting a portion of each extract via persulfate digestion
(Rowland & Haygarth, 1997) and organic P (Po) was calculated as Ptot - Pi. Rowland and
Haygarth’s (1997) method was originally designed for soil solutions, which have more neutral
pH and lower P concentrations than Bicarbonate or Hydroxide extracts. To adapt this method,
we diluted our solutions with an extract to water ratio of 1:3 for Bicarbonate extracts and 1:19
for Hydroxide extracts. We added 300 µL of 12N HCl to Bicarbonate solutions to ensure they
were acidic during digestion. Controls were treated in the same manner to determine digestion
13
efficiency. Unfortunately all extracts from May were consumed in establishing the digest
procedure and no data on Bicarbonate Po are thus available for this time point. Extracts were
stored at -20°C until analyzed. We quantified P in all extracts using the ammonium molybdateascorbic acid method on a Lachat QuikChem 8000 Flow Injection Analyzer (Lachat Instruments,
Loveland, CO, USA) (Shaw & DeForest, 2013). In situ soil solution P, Resin P, Acid Pi, and Ptot in
digested samples were quantified using 0.5M HCl as carrier and in standards. Upon defrosting,
organic matter precipitated in Hydroxide P extracts, the supernatant was acidified by dilution
1:4 with 2M HCl and the extraction solution was diluted in the same way and used as carrier
and in standards. Bicarbonate P samples were acidified by adding 20 μL of 12N HCl to 5 mL of
extract and incubated over night at 4°C before analysis. The acidification releases CO2 from
bicarbonate over the course of 24h, but may also hydrolyze some labile Po. Thus inorganic P in
bicarbonate extracts may be slightly overestimated. However, we are confident that hydrolysis
reactions under the weak acid were minor since significantly more P was detected in the
samples after complete digestion. For these samples as well as the P adsorption extracts,
ddH2O was used as carrier and in standards. Measured P content is reported as mg P kg-1 dry
soil after correcting field moist soil weights by their dry weight equivalent.
Methods S3 Soil enzymatic activityMethods S3 Soil enzymatic activity
Soil (~1.75 g fresh weight) was suspended in 100 mL of sodium acetate buffer (pH 5.0) and
aliquots were dispensed into black 96-well microplates for reaction with 4-methylumbelliferone
(MUB)-labeled substrates. Potential AP and PD activity was measured colorimetrically by adding
MUB-phosphate substrates to soil slurries at saturating conditions. Eight replicates were run for
each soil sample (sample solution and substrate). Additionally, blank wells (buffer), sample
controls (buffer and soil solution), reference standards (MUB standard and buffer), quench
standard wells (MUB standard and soil solution), and negative control wells (substrate and
buffer) were run to control for quenching by the substrate and buffer alone. Samples were
incubated in the dark at 23°C for 2 h for AP and 5 h for PD. At the end of incubation, 20 μL of
1 M NaOH was added to each well, raising the pH to improve fluorescence (German et al.,
2011). Fluorescence was measured on a plate reader using 365 nm excitation and 450 nm
14
emission filters. Potential enzyme activity rates for each sample were calculated as μmol MUB
g−1 dry soil h−1. We excluded any outliers (values more than two standard deviations from the
mean) within the 8 replicates for each assay.
Methods S4 Ergosterol extractionMethods S4 Ergosterol extraction
Ergosterol extractions were performed on freeze-dried and ground-up soils sampled in May and
September, stored in the dark prior to extraction (Salmanowicz & Nylund, 1988; Nylund &
Wallander, 1992). After extraction, samples where filtered through a 0.5 mL Teflon syringe filter
(Millex LCR-4; Millipore, Billerica, MA, USA), and chromatographically analyzed using highperformance liquid chromatograph (HPLC) with an attach C18 reverse-phase column (Nova-Pak,
3.9x150mm, Waters, Milford, CT, USA) preceded by a C18 reverse-phase guard column (NovaPak, 3.9x20mm, Waters), using methanol as mobile-phase and UV detector operating at 282 nm
measuring the absorbance of the elution.
Methods S5 Fungal community sequence analysis
After quantification by Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies, CA, USA) on a
TECAN F500 microplate reader aliquots of 35 ng of each sample were pooled up together for
Ion Torrent sequencing technology (Life Technologies, California, USA), at Uppsala Genome
Center (Uppsala, Sweden). The Torrent Suite 4.0.4 software delivered a de-multiplexing dataset
of 1 002 075 sequences with a Q ≥20 per base from the 16 samples. Following a more stringent
quality filtering of fastq files (Q ≥25 in sequence average, minimum length 150 bp) using
mothur v1.33.3 (Schloss et al., 2009) we retrieved 634,103 sequences. The software ITSx v 1.0.9
(Bengtsson-Palme et al., 2013) was applied to extract the ITS2 region retrieving those longer
than 80 bp produced a final dataset of 607,132 total sequences after removing non-fungal
sequences. This ITS2 dataset was de novo clustered using the software TSC (Jiang et al., 2012)
with the following parameters: data type (–r 454, since Ion Torrent and 454 LifeSciences
sequencing technology has similar sequencing errors patterns (Yang et al., 2013)), clustering
algorism single linkage (-s sl, as recommended for ITS locus in fungi (Lindahl et al., 2013)) at
0.97 sequence similarity (considered a suitable cutoff for species delimitation in fungi (Blaalid et
15
al., 2013)) and cutoff for high and low abundant sequences (–c 500). This produced 37,665
Molecular Operational Taxonomic Units (MOTUs). Singleton and doubleton were removed
resulting in 8,558 MOTUs (corresponding to 98.3 % of the fungal sequences). The longest
sequence was retrieved from each MOTU into a representative sequence dataset using QIIME
v1.8.0 (Caporaso et al., 2010) script reprentative_set.py. MOTUs were assigned taxonomy using
BLASTn 2.2.29+ with the following parameters, e-value = 1e-10 and word size = 7 (Madden,
2002) against the ITS2 region of the 2015-03-02_UNITE + INDS database (Koljalg et al., 2013). To
account for difference in sequence depth among samples, the MOTU table was rarefied to
6,833 reads per sample using the script single_rarefaction.py in Qiime. This rendered a total of
3683 MOTUs being analysed in this study, representative sequences of these have been
submitted to GenBank and are available under Accession nr: KT194384 - KT198041. PCA were
performed using the package ampvis (http://madsalbertsen.github.io/ampvis/) and phyloseq
(McMurdie & Holmes, 2013) in R v 3.0.2. Adonis statistical test and species diversity estimates
were computed in Qiime. The final taxonomic assignments were corrected relying on 97, 90, 85,
80, 75 and 70 % of sequence identity for assigning MOTUs with names of a species, genus,
family, order, class, or phylum, respectively (Tedersoo et al., 2014). Ectomycorrhizal life
strategy was assigned to fungal taxa based on available information at genus level (Tedersoo et
al., 2010). In the absence of a resolved phylogeny of the Helotiales and the difficulty of
distinguishing between saprotrophic and symbiotic species in this group none of these MOTUs
were classified as ecotmycorrhizal.
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