1
Van der Heijden et al.
2
Supporting Information
3
4
Experimental microcosms: Using closed growth chambers and sterile conditions we
5
established novel experimental microcosms (Figure S2; see van der Heijden and Wagg (2013)
6
and Wagg et al. (2014) for further details). To avoid outside greenhouse-borne microbial
7
contamination, incoming air was filtered through a hydrophobic filter with a pore size of 0.2
8
μm (Millex®-FG50; Millipore Corporation, Billerica, USA) and water was filtered through a
9
hydrophilic filter with a 0.22 μm pore size (Millex®-GP50; Millipore Corporation, Billerica,
10
USA). Microcosms were assembled, inoculated, and planted within a laminar flow hood. All
11
parts used for the microcosms were sterilized by autoclaving for 30 min at 121 °C, with the
12
exception of the Plexiglas tops and the PVC microcosm bottoms. The bottom and top of the
13
microcosms were sterilized by submersing in 0.5% sodium hypochlorite for 20–30 minutes,
14
then in 70% Ethanol with a few drops of Tween 20 for a few minutes and air-dried within the
15
Laminar flow hood. Each microcosm, which had a diameter of 23.5 cm and a depth of 12 cm
16
was filled with 6.45 kg sieved and autoclaved (110°C for 2h) dune sand.
17
18
P and N fertilization: The microcosms were maintained in a greenhouse, watered weekly
19
and randomized every 2-4 weeks. A modified low N and P Hoagland nutrient solution
20
(Hoagland and Arnon, 1950) was supplied at regular time-intervals and a total of 39.7 mg N
21
kg soil-1 and 4.2 mg P kg soil-1 (or 45.3 kg N ha-1 year-1 and 4.8 kg P ha-1 year-1) was added to
22
each microcosm. To increase the relative abundance of
23
atom% enriched) was mixed with the soil prior to the establishment of the microcosms and a
24
total of 0.806 mg 15N-K15NO3 and 0.366 mg 15N-15NH415NO3 (both 98 atom% enriched) was
25
supplied with the nutrient solution during the experiment. By doing this we enhanced the 15N
15
N, 0.017 mg
15
N (K15NO3, 98
1
26
concentration of plant available soil nitrogen to 1036 ‰ (the average δ15N of plant N in
27
microcosms without a symbiont). This δ15N concentration contrasts with the δ15N
28
concentration of atmospheric nitrogen, which is assumed to be 0 ‰. Subsequently, by using
29
the difference in
30
nitrogen we calculated the amount of biological nitrogen fixation.
31
Quality control of treatments: It is difficult to test the ecological function of microbes
32
because non-inoculation treatments get easily contaminated (Read, 2002). At final harvest we
33
examined the microcosms for the purity of the microbial inoculation treatments. For the AM
34
fungi treatment we quantified in all microcosms the fungal colonization of plant roots and the
35
hyphal length in the soil (Table S1). Root colonization by AM fungi varied from 0% in all
36
non-mycorrhizal microcosms (control and rhizobia treatments) to 71.8% and 83.0% in
37
microcosms with only AM fungi or with AM fungi and rhizobia, respectively. Similarly,
38
hyphal length varied from 0.25 meter in control and rhizobia microcosms and up to 18.6
39
meter in microcosms inoculated with AM fungi. Hyphal length in the non-mycorrhizal
40
microcosms possibly reflect non-mycorrhizal structures or dead hyphae, which were already
41
present in the sterilized soil. We screened the roots for nodules in all microcosms and nodules
42
were only observed in microcosms where rhizobia had been added (Table S1). In summary,
43
neither AM fungi nor rhizobia colonization was observed in microcosms to which these had
44
not been added, demonstrating that we successfully manipulated the AM fungi and rhizobia in
45
the microcosms without the contamination of AM fungi or rhizobia from the outside during
46
the entire course of the experiment (16.5 months). Although we minimized the exposure of
47
the microcosms to possible environmental contaminations by working in a laminar flow hood,
48
we cannot exclude that bacteria and fungi other than AM fungi and rhizobia could have made
49
it into the microcosms (e.g. endophytic bacteria and fungi colonizing plant seeds).
15
N concentration between plant available soil nitrogen and atmospheric
50
2
51
References
52
Van der Heijden MGA, Wagg C. (2013). Soil microbial diversity and agro-ecosystem
53
functioning. Plant Soil 363:1–5.
54
Hoagland DR, Arnon DI. (1950). The water-culture method for growing plants without soil.
55
Calif Agric Exp Stn Circ 347:1–32.
56
Read DJ. (2002). Towards Ecological Relevance - Progress and Pitfalls in the Path Towards
57
an Understanding of Mycorrhizal Functions in Nature. In:Mycorrhizal Ecology, Van der
58
Heijden, MGA & Sanders, IR (eds) Ecological Studies Vol. 157, Springer Berlin Heidelberg,
59
pp. 3–29.
60
Wagg C, Bender SF, Widmer F, van der Heijden MGA. (2014). Soil biodiversity and soil
61
community composition determine ecosystem multifunctionality. Proc Natl Acad Sci U S A
62
111:5266–70.
63
3
64
65
Figure S1: Photographic impression of the nutrient poor dune grassland reference site at the
66
Noordhollands Duinreservaat at Egmond Binnen (the Netherlands).
4
67
68
Figure S2: Experimental microcosms. Drawing (a) and photograph (b, c) of the gnotobiotic
69
microcosms in which plants were grown under controlled conditions without microbial
70
contamination from the outside. The photograph in (c) illustrates the experimental grassland
71
plant community after 5 months of growth in a microcosm containing AMF and rhizobia.
72
Note that incoming pressured air was filtered through a 0.2 μm hydrophobic filter (right
73
arrow), while water was filtered through a 0.22 μm hydrophilic filter (left arrow).
74
5
75
76
Figure S3: Productivity of microcosms by harvests. Total shoot biomass in microcosms
77
after the first harvest (5.5 months), the second harvest (11 months) and the third harvest (16.5
78
months). Microcosms contained no plant symbionts (C), only rhizobia (R), only AM fungi
79
(M) or microcosms contained both symbionts (MR). Bars represent means (n=9; ± sem) and
80
statistical details are given in Table S3.
81
6
82
83
Figure S4: Plant survival. The survival of the plant species by functional group was
84
determined at the third harvest in microcosms containing no plant symbionts (C), only
85
rhizobia (R), only AM fungi (M) or both symbionts (MR). Bars represent means (n=9; ± sem)
86
and statistic details of functional groups are given in Table S1.
87
7
88
89
Figure S5: Correspondence between symbiont presence and biomass production. The
90
correspondence analysis (CCA) was constrained for the variables AM fungi * rhizobia to
91
quantify their influence on the variation in biomass production of the individual plant species.
92
Microcosms (“sample scores”) containing no plant symbionts (C), only rhizobia (R), only AM
93
fungi (M) or both symbionts (MR) are depicted with triangles and the Eigenvectors of the
94
individual plants species (“species scores”) reported with arrows (color-coded by functional
95
plant groups): Luzula campestris (Luc), Anthoxanthum odoratum (Ano), Festuca ovina (Feo),
96
Koeleria macrantha (Kom), Carlina vulgaris (Cav), Senecio jacobaea (Sej), Achillea
97
millefolium (Acm), Hieracium pilosella (Hip), Plantago lanceolata (Pll), Trifolium repens
98
(Trr) and Lotus corniculatus (Loc).
99
8
100
101
Figure S6: Plant nutrient acquisition by functional groups. P content (a) and mineral N
102
content (b) in plants (colored by functional group) of the grassland microcosms containing no
103
plant symbionts (C), only rhizobia (R), only AM fungi (M), both symbionts (MR). The
104
nutrient content refers to the overall nutrient uptake in the microcosms (sum of the three
105
harvests). Bars represent means (n=9; ± sem) and letters (colored by functional group)
106
indicate statistical significance; treatments not sharing a letter differ at P<0.05 (Tukey’s
107
HSD). Statistic details are reported in Table S1.
108
9
109
Supplementary Dataset Legends
110
Supplementary Data 1: Productivity. The text file “Supplementary_data_1_productivity
111
.txt” contains the raw biomass recordings [in mg] of each species at each harvest and from
112
each microcosm. The file also contains the biomass of the combined root systems at the third
113
harvest.
114
Supplementary Data 2: Plant survival. The text file “Supplementary_data_2_plant_survival
115
.txt” contains the raw survival recordings [in %] of each species from each microcosm at the
116
third harvest.
117
Supplementary Data 3: Nutrients. The text file “Supplementary_data_3_nutrients.txt”
118
contains the raw P and N measurements [in mg] of plants of each functional group from each
119
microcosm. The file contains the nutrient measures of the first and second harvests
120
(combined) and of the third harvest. The file also contains the nutrient analyses of the
121
combined root systems at the third harvest.
122
Supplementary
123
“Supplementary_data_4_seedling_establishment.txt” comprises the shoot biomass recordings
124
[in mg] of the planted seedlings in each microcosm.
125
Supplementary Data 5: R code. The zip file “Supplementary_data_5_R_code.txt” contains
126
all custom R scripts and functions utilized for the statistical examination of the data and
127
plotting of the figures.
Data
4:
Seedling
establishment.
The
text
file
128
129
10
130
131
132
133
134
135
136
137
138
139
Supplementary Table S1: AM fungal colonization levels, hyphal length, root length, occurrence of root nodules, plant diversity, plant P
content, plant N content, plant productivity, plant survival of three plant functional groups and the total plant community; and productivity of
four legume seedlings and five non-legume seedlings (mean (± se)) in nutrient poor grassland microcosms where the presence and composition
of plant symbionts was manipulated. Plant productivity of each plant species is reported in Table S21. Microcosms contained no plant symbionts
(C), only rhizobia (R), only AM fungi (M), or microcosms contained both symbionts (MR). Variables were untransformed (§), natural log
transformed (∞) or ranked (†) to meet the requirements to perform the analysis. F-values and significance levels for a two-way analysis of
variance with rhizobia (R) and AM fungi (M) as separate factors and their interaction term (R*M) are shown (ns not significant, * P < 0.05, ** P
< 0.01, *** P < 0.001). #Hyphae in the non-mycorrhizal treatments represent non-mycorrhizal hyphae or dead hyphae that were already present
in the soil before starting the experiment.
Variable
Root colonization by AM fungi [%]§
Hyphal length [m g soil-1]#†
Root length [m g soil-1]†
Root nodules
Plant productivity1 [g]
Total including roots∞
Grasses (Shoots)†
Herbs (Shoots)†
Legumes (Shoots)†
Roots†
C
Mean (±se)
0.00 (0.00)
0.25 (0.05)
0.55 (0.06)
Absent
R
Mean
0.00
0.37
0.48
Present
21.71 (1.58)
12.15 (0.50)
0.13 (0.01)
0.06 (0.00)
9.37 (1.21)
19.40
11.51
0.12
0.05
7.71
(±se)
(0.00)
(0.08)
(0.04)
M
Mean (±se)
71.78 (3.10)
18.67 (1.12)
0.28 (0.01)
Absent
MR
Mean
83.00
16.52
0.25
Present
R
F
(±se)
(1.77)
(0.93)
(0.01)
0.017
2.348
ns
(0.98)
(0.61)
(0.00)
(0.00)
(0.53)
19.69 (0.48)
1.59 (0.21)
7.76 (0.25)
0.78 (0.10)
9.56 (0.26)
22.21
1.32
7.96
2.40
10.53
(0.98)
(0.08)
(0.28)
(0.21)
(0.63)
0.011
0.738
0.099
7.878
0.000
ns
ns
ns
ns
**
ns
M
F
9,882
110.356
77.871
R*M
F
**
***
***
0.311
98.801
103.425
151.017
7.888
ns
***
***
***
**
4.579
0.355
*
4.786
0.094
2.270
10.151
3.206
*
ns
ns
ns
**
ns
Plant diversity (H)§
1.22 (0.05)
1.21 (0.06)
1.35 (0.05)
1.43 (0.06)
0.370
ns
10.829
**
0.758
ns
Plant survival [%]
Total†
Grasses†
Herbs†
35.67 (2.29)
85.25 (2.92)
6.91 (2.67)
34.47 (2.58)
77.11 (5.09)
7.49 (1.73)
84.40 (0.93)
89.33 (2.57)
82.73 (2.27)
85.23 (1.88)
87.64 (2.24)
81.84 (3.12)
0.133
1.121
0.214
ns
97.127
3.634
99.722
***
0.093
0.307
0.001
ns
ns
ns
ns
***
ns
ns
11
Legumes†
8.39 (2.77)
16.67 (3.40)
78.72 (1.47)
88.89 (3.92)
7.527
**
129.871
***
0.000
ns
55.951
99.787
119.181
209.168
48.776
***
1.678
0.973
3.560
4.784
0.565
ns
48.911
97.020
106.629
159.115
133.388
***
Plant P content [mg]
Total including roots§
Grasses (Shoots)†
Herbs (Shoots)†
Legumes (Shoots)†
Roots†
11.69 (0.80)
8.38 (0.52)
0.05 (0.00)
0.02 (0.00)
3.25 (0.59)
11.39
8.65
0.04
0.02
2.68
(0.66)
(0.62)
(0.00)
(0.00)
(0.14)
15.42 (0.54)
1.05 (0.13)
7.29 (0.17)
0.65 (0.09)
6.43 (0.41)
16.69
0.90
7.57
1.83
6.40
(0.31) 0.641
(0.08) 0.186
(0.23) 0.223
(0.14) 25.443
(0.33) 0.894
ns
Plant N content [mg]
Total including roots†
Grasses (Shoots)†
Herbs (Shoots)†
Legumes (Shoots)†
Roots†
211.79 (5.18)
167.65 (4.37)
2.35 (0.18)
1.05 (0.06)
40.74 (2.16)
207.90
165.02
2.31
1.18
39.39
(5.38)
(5.33)
(0.13)
(0.11)
(1.14)
211.22 (3.20)
20.28 (2.33)
99.67 (2.60)
10.42 (1.49)
80.85 (0.96)
292.49
18.10
106.20
57.23
110.96
(8.85) 41.483
(1.37) 0.237
(2.11) 1.467
(4.90) 14.557
(5.62) 2.033
***
8.01 (0.97)
5.57 (1.27)
22.77 (2.72)
4.91 (0.36)
4.46
4.29
20.66
4.32
(1.18)
(1.47)
(3.07)
(0.78)
4.16 (0.64)
3.23 (0.52)
30.86 (3.98)
9.18 (1.48)
3.72
2.54
23.90
7.16
(0.39)
(0.41)
(2.58)
(1.21)
5.344
2.053
2.091
2.213
*
7.83 (0.38)
4.90 (0.54)
2.79 (0.34)
3.53 (0.36)
9.96
5.99
2.82
5.13
(0.84)
(0.51)
(0.37)
(0.34)
9.64 (1.12)
2.46 (0.49)
1.50 (0.34)
2.02 (0.37)
57.13
70.98
33.96
76.17
(9.47)
(16.8)
(7.29)
(21.8)
25.192
68.902
35.205
94.277
Seedling establishment [mg]
Non Legumes: (Shoot productivity)
Festuca ovina†
Koeleria macrantha∞
Plantago lanceolata§
Senecio jacobaea∞
Legumes: (Shoot productivity)
Lotus corniculatus†
Trifolium repens†
Trifolium arvense†
Trifolium dubium†
ns
ns
***
ns
ns
ns
***
ns
ns
ns
ns
***
***
***
***
4.624
3.450
3.267
9.936
21.640
0.838
1.963
1.030
***
***
***
***
***
***
***
***
*
ns
ns
**
***
ns
ns
ns
50.247
0.004
1.966
6.208
10.292
2.898
0.111
0.597
0.018
4.964
37.491
36.190
32.690
ns
ns
*
ns
***
ns
ns
*
**
ns
ns
ns
ns
*
***
***
***
140
12
141
142
143
144
145
146
147
Supplementary Table S2: Plant productivity of each plant species (mean (± se)) in nutrient poor grassland microcosms where the presence and
composition of plant symbionts was manipulated. Microcosms contained no plant symbionts (C), only rhizobia (R), only AM fungi (M), or
microcosms contained both symbionts (MR). Variables were untransformed (§), natural log transformed (∞) or ranked (†) to meet the
requirements to perform the analysis. F-values and significance levels for a two-way analysis of variance with rhizobia (R) and AM fungi (M) as
separate factors and their interaction term (R*M) are shown (ns not significant, * P < 0.05, ** P < 0.01, *** P < 0.001). Letters report significant
differences in pairwise comparisons (Tukey’s HSD) between treatments (treatments differing at P < 0.05 are marked with different letters).
Plant productivity [mg]
Grasses
Anthoxanthum odoratum†
Festuca ovina†
Koeleria macrantha†
Luzula campestris†
C
mean (±se)
5040.44
1875.67
1334.00
3898.56
R
mean (±se)
R*M
F
(227.24)
(30.36)
(23.69)
(43.99)
B
B
B
B
739.67
289.89
238.00
49.33
(88.03)
(34.41)
(8.49)
(11.28)
B
B
B
B
0.003ns
0.452ns
1.147ns
0.133ns
77.073***
98.063***
31.024***
97.245***
0.253ns
0.135ns
0.007ns
0.182ns
(0.65)
(1.99)
(1.29)
(4.43)
(1.51)
A
B
A
A
A
242.44
56.56
384.11
6513.00
559.00
(22.27)
(5.50)
(32.09)
(264.91)
(93.79)
B
A
B
B
B
224.11
46.11
422.11
6846.67
420.22
(57.99)
(5.57)
(47.52)
(359.93)
(76.09)
B
AB
B
B
B
0.705ns
5.265*
1.807ns
0.473ns
0.919ns
101.457***
6.726*
102.566***
102.584***
100.359***
0.349ns
0.003ns
0.118ns
1.564ns
0.345ns
36.44 (4.12)
17.89 (0.92)
A
A
B
B
1847.44 (217.20) C
553.56 (36.13) C
5.479*
9.266**
149.632***
134.628***
12.064**
3.079ns
A
AB
A
A
A
7.44
33.89
12.44
54.67
7.67
A
A
38.67 (2.92)
16.56 (1.08)
M
F
923.33
333.22
216.22
113.67
(1.60)
(6.06)
(1.82)
(5.43)
(2.75)
Legumes
Lotus corniculatus†
Trifolium repens†
R
F
A
A
A
A
A
A
A
A
8.78
45.22
10.44
59.33
9.78
MR
mean (±se)
(606.79)
(205.31)
(174.71)
(945.02)
(920.46)
(308.47)
(311.92)
(649.98)
Herbs
Achillea millefolium†
Carlina vulgaris∞
Hieracium pilosella†
Plantago lanceolata†
Senecio jacobaea†
M
mean (±se)
4293.44
1669.11
1183.44
4365.33
522.56 (58.94)
260.78 (53.23)
148
13
149
150
151
152
153
154
Supplementary Table S3: Statistics (F-ratios, degrees of freedom and significance level) of repeated measures analysis of variance for the
effects of Block (B), AM fungi (M), Rhizobia (R), Harvest (H), the AM fungi × Harvest interaction term (M x H), the Rhizobia × Harvest
interaction term (R × H) and the AMF × Rhizobia × Harvest interaction term (M × R × H) for shoot biomass data of Anthoxanthum odoratum
(Ao), Luzula campestris (Lu) Festuca ovina (Fo), Koeleria macrantha (Km), Lotus corniculatus (Lc), Trifolium repens (Tr), Hieracium pilosella
(Hp), Plantago lanceolata (Pl), Achillea millefolium (Am), Senecio jacobaea (Sj) and Carlina vulgaris (Cv) and total above ground shoot
biomass (Sh). The degrees of freedom are the same for each plant species and only shown for the first plant species (Ao).
155
156
157
Plant species
Source of variation Ao
Lu
Fo
Km
Lc
Tr
Hp
Pl
Am
Sj
Cv
Sh
B
F8,24=4.9**
1.2ns
0.7ns
1.1ns
1.5ns
1.0ns
1.1ns
0.2ns
1.1ns
1.1ns
0.6ns
1.8ns
M
F1,24=71.3*** 148.9*** 51.4***
33.7***
1422.0*** 742.5*** 228.7*** 2736*** 230***
394***
3.3ns
8.2**
R
F1,24=0.0ns
0.47ns
0.4ns
1.7ns
24.0***
13.3**
0.1ns
0.0ns
0.8ns
3.3ns
4.3*
2.4ns
H
F2,64=423.0*** 443.0*** 494.8*** 287.9*** 198.0*** 340.8*** 216.0*** 199.9*** 242.4*** 175.0*** 1731*** 1790***
M×R
F1,64=0.2ns
0.2ns
0.0ns
0.2ns
36.0***
8.5**
0.5 ns
0.2 ns
0.5ns
2.2ns
0.0ns
5.2*
M×H
F2,64=265.5*** 105.9*** 171.5*** 83.8***
79.9***
101.0*** 12.7***
373.3*** 16.8ns
15.2***
1.5ns
230.9***
R×H
F2,64=2.8ns
1.6ns
0.6ns
3.3*
2.8ns
30.4***
1.1 ns
0.7ns
0.3ns
1.4ns
1.1ns
0.4ns
R×M×H
F2,64=0.33ns
0.8ns
0.0ns
1.2ns
3.9*
35.6***
0.4 ns
0.0ns
0.1ns
2.3ns
0.0ns
3.4*
____________________________________________________________________________________________________________________
ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
158
159
160
161
162
163
164
165
166
14