Van der Heijden et al.

Supporting Information

Experimental microcosms: Using closed growth chambers and sterile conditions we

established novel experimental microcosms (Figure S2; see van der Heijden and Wagg (2013)

and Wagg et al. (2014) for further details). To avoid outside greenhouse-borne microbial

contamination, incoming air was filtered through a hydrophobic filter with a pore size of 0.2

μm (Millex®-FG50; Millipore Corporation, Billerica, USA) and water was filtered through a

hydrophilic filter with a 0.22 μm pore size (Millex®-GP50; Millipore Corporation, Billerica,

USA). Microcosms were assembled, inoculated, and planted within a laminar flow hood. All

parts used for the microcosms were sterilized by autoclaving for 30 min at 121 °C, with the

exception of the Plexiglas tops and the PVC microcosm bottoms. The bottom and top of the

microcosms were sterilized by submersing in 0.5% sodium hypochlorite for 20–30 minutes,

then in 70% Ethanol with a few drops of Tween 20 for a few minutes and air-dried within the

Laminar flow hood. Each microcosm, which had a diameter of 23.5 cm and a depth of 12 cm

was filled with 6.45 kg sieved and autoclaved (110°C for 2h) dune sand.

P and N fertilization: The microcosms were maintained in a greenhouse, watered weekly

and randomized every 2-4 weeks. A modified low N and P Hoagland nutrient solution

(Hoagland and Arnon, 1950) was supplied at regular time-intervals and a total of 39.7 mg N

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

each microcosm. To increase the relative abundance of 15N, 0.017 mg 15N (K15NO3, 98 atom%

enriched) was mixed with the soil prior to the establishment of the microcosms and a total of

0.806 mg 15N-K15NO3 and 0.366 mg 15N-15NH415NO3 (both 98 atom% enriched) was supplied

with the nutrient solution during the experiment. By doing this we enhanced the 15N

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concentration of plant available soil nitrogen to 1036 ‰ (the average δ15N of plant N in

microcosms without a symbiont). This δ15N concentration contrasts with the δ15N

concentration of atmospheric nitrogen, which is assumed to be 0 ‰. Subsequently, by using

the difference in 15N concentration between plant available soil nitrogen and atmospheric

nitrogen we calculated the amount of biological nitrogen fixation.

Quality control of treatments: It is difficult to test the ecological function of microbes

because non-inoculation treatments get easily contaminated (Read, 2002). At final harvest we

examined the microcosms for the purity of the microbial inoculation treatments. For the AM

fungi treatment we quantified in all microcosms the fungal colonization of plant roots and the

hyphal length in the soil (Table S1). Root colonization by AM fungi varied from 0% in all

non-mycorrhizal microcosms (control and rhizobia treatments) to 71.8% and 83.0% in

microcosms with only AM fungi or with AM fungi and rhizobia, respectively. Similarly,

hyphal length varied from 0.25 meter in control and rhizobia microcosms and up to 18.6

meter in microcosms inoculated with AM fungi. Hyphal length in the non-mycorrhizal

microcosms possibly reflect non-mycorrhizal structures or dead hyphae, which were already

present in the sterilized soil. We screened the roots for nodules in all microcosms and nodules

were only observed in microcosms where rhizobia had been added (Table S1). In summary,

neither AM fungi nor rhizobia colonization was observed in microcosms to which these had

not been added, demonstrating that we successfully manipulated the AM fungi and rhizobia in

the microcosms without the contamination of AM fungi or rhizobia from the outside during

the entire course of the experiment (16.5 months). Although we minimized the exposure of

the microcosms to possible environmental contaminations by working in a laminar flow hood,

we cannot exclude that bacteria and fungi other than AM fungi and rhizobia could have made

it into the microcosms (e.g. endophytic bacteria and fungi colonizing plant seeds).

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References

Van der Heijden MGA, Wagg C. (2013). Soil microbial diversity and agro-ecosystem

functioning. Plant Soil 363:1–5.

Hoagland DR, Arnon DI. (1950). The water-culture method for growing plants without soil.

Calif Agric Exp Stn Circ 347:1–32.

Read DJ. (2002). Towards Ecological Relevance - Progress and Pitfalls in the Path Towards

an Understanding of Mycorrhizal Functions in Nature. In:Mycorrhizal Ecology, Van der

Heijden, MGA & Sanders, IR (eds) Ecological Studies Vol. 157, Springer Berlin Heidelberg,

pp. 3–29.

Wagg C, Bender SF, Widmer F, van der Heijden MGA. (2014). Soil biodiversity and soil

community composition determine ecosystem multifunctionality. Proc Natl Acad Sci U S A

111:5266–70.

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Figure S1: Photographic impression of the nutrient poor dune grassland reference site at the

Noordhollands Duinreservaat at Egmond Binnen (the Netherlands).

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Figure S2: Experimental microcosms. Drawing (a) and photograph (b, c) of the gnotobiotic

microcosms in which plants were grown under controlled conditions without microbial

contamination from the outside. The photograph in (c) illustrates the experimental grassland

plant community after 5 months of growth in a microcosm containing AMF and rhizobia.

Note that incoming pressured air was filtered through a 0.2 μm hydrophobic filter (right

arrow), while water was filtered through a 0.22 μm hydrophilic filter (left arrow).

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Figure S3: Productivity of microcosms by harvests. Total shoot biomass in microcosms

after the first harvest (5.5 months), the second harvest (11 months) and the third harvest (16.5

months). Microcosms contained no plant symbionts (C), only rhizobia (R), only AM fungi

(M) or microcosms contained both symbionts (MR). Bars represent means (n=9; ± sem) and

statistical details are given in Table S3.

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Figure S4: Plant survival. The survival of the plant species by functional group was

determined at the third harvest in microcosms containing no plant symbionts (C), only

rhizobia (R), only AM fungi (M) or both symbionts (MR). Bars represent means (n=9; ± sem)

and statistic details of functional groups are given in Table S1.

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Figure S5: Correspondence between symbiont presence and biomass production. The

correspondence analysis (CCA) was constrained for the variables AM fungi * rhizobia to

quantify their influence on the variation in biomass production of the individual plant species.

Microcosms (“sample scores”) containing no plant symbionts (C), only rhizobia (R), only AM

fungi (M) or both symbionts (MR) are depicted with triangles and the Eigenvectors of the

individual plants species (“species scores”) reported with arrows (color-coded by functional

plant groups): Luzula campestris (Luc), Anthoxanthum odoratum (Ano), Festuca ovina (Feo),

Koeleria macrantha (Kom), Carlina vulgaris (Cav), Senecio jacobaea (Sej), Achillea

millefolium (Acm), Hieracium pilosella (Hip), Plantago lanceolata (Pll), Trifolium repens

(Trr) and Lotus corniculatus (Loc).

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Figure S6: Plant nutrient acquisition by functional groups. P content (a) and mineral N

content (b) in plants (colored by functional group) of the grassland microcosms containing no

plant symbionts (C), only rhizobia (R), only AM fungi (M), both symbionts (MR). The

nutrient content refers to the overall nutrient uptake in the microcosms (sum of the three

harvests). Bars represent means (n=9; ± sem) and letters (colored by functional group)

indicate statistical significance; treatments not sharing a letter differ at P<0.05 (Tukey’s

HSD). Statistic details are reported in Table S1.

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Supplementary Dataset Legends

Supplementary Data 1: Productivity. The text file

“Supplementary_data_1_productivity .txt” contains the raw biomass recordings [in mg] of

each species at each harvest and from each microcosm. The file also contains the biomass of

the combined root systems at the third harvest.

Supplementary Data 2: Plant survival. The text file “Supplementary_data_2_plant_survival

.txt” contains the raw survival recordings [in %] of each species from each microcosm at the

third harvest.

Supplementary Data 3: Nutrients. The text file “Supplementary_data_3_nutrients.txt”

contains the raw P and N measurements [in mg] of plants of each functional group from each

microcosm. The file contains the nutrient measures of the first and second harvests

(combined) and of the third harvest. The file also contains the nutrient analyses of the

combined root systems at the third harvest.

Supplementary Data 4: Seedling establishment. The text file

“Supplementary_data_4_seedling_establishment.txt” comprises the shoot biomass recordings

[in mg] of the planted seedlings in each microcosm.

Supplementary Data 5: R code. The zip file “Supplementary_data_5_R_code.txt” contains

all custom R scripts and functions utilized for the statistical examination of the data and

plotting of the figures.

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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 S2 1. 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.

C R M MR R M R*MVariable Mean (±se) Mean (±se) Mean (±se) Mean (±se) F F FRoot colonization by AM fungi [%]§ 0.00 (0.00) 0.00 (0.00) 71.78 (3.10) 83.00 (1.77) 9,882 **

Hyphal length [m g soil-1]#† 0.25 (0.05) 0.37 (0.08) 18.67 (1.12) 16.52 (0.93) 0.017 ns 110.356 *** 4.579 *

Root length [m g soil-1]† 0.55 (0.06) 0.48 (0.04) 0.28 (0.01) 0.25 (0.01) 2.348 ns 77.871 *** 0.355 ns

Root nodules Absent Present Absent Present

Plant productivity 1 [g]Total including roots∞ 21.71 (1.58) 19.40 (0.98) 19.69 (0.48) 22.21 (0.98) 0.011 ns 0.311 ns 4.786 *

Grasses (Shoots)† 12.15 (0.50) 11.51 (0.61) 1.59 (0.21) 1.32 (0.08) 0.738 ns 98.801 *** 0.094 ns

Herbs (Shoots)† 0.13 (0.01) 0.12 (0.00) 7.76 (0.25) 7.96 (0.28) 0.099 ns 103.425 *** 2.270 ns

Legumes (Shoots)† 0.06 (0.00) 0.05 (0.00) 0.78 (0.10) 2.40 (0.21) 7.878 ** 151.017 *** 10.151 **

Roots† 9.37 (1.21) 7.71 (0.53) 9.56 (0.26) 10.53 (0.63) 0.000 ns 7.888 ** 3.206 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† 35.67 (2.29) 34.47 (2.58) 84.40 (0.93) 85.23 (1.88) 0.133 ns 97.127 *** 0.093 ns

Grasses† 85.25 (2.92) 77.11 (5.09) 89.33 (2.57) 87.64 (2.24) 1.121 ns 3.634 ns 0.307 ns

Herbs† 6.91 (2.67) 7.49 (1.73) 82.73 (2.27) 81.84 (3.12) 0.214 ns 99.722 *** 0.001 ns

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130131132133134135136137138139

Legumes† 8.39 (2.77) 16.67 (3.40) 78.72 (1.47) 88.89 (3.92) 7.527 ** 129.871 *** 0.000 ns

Plant P content [mg]Total including roots§ 11.69 (0.80) 11.39 (0.66) 15.42 (0.54) 16.69 (0.31) 0.641 ns 55.951 *** 1.678 ns

Grasses (Shoots)† 8.38 (0.52) 8.65 (0.62) 1.05 (0.13) 0.90 (0.08) 0.186 ns 99.787 *** 0.973 ns

Herbs (Shoots)† 0.05 (0.00) 0.04 (0.00) 7.29 (0.17) 7.57 (0.23) 0.223 ns 119.181 *** 3.560 ns

Legumes (Shoots)† 0.02 (0.00) 0.02 (0.00) 0.65 (0.09) 1.83 (0.14)25.44

3***

209.168***

4.784*

Roots† 3.25 (0.59) 2.68 (0.14) 6.43 (0.41) 6.40 (0.33) 0.894 ns 48.776 *** 0.565 ns

Plant N content [mg]

Total including roots† 211.79 (5.18) 207.90 (5.38) 211.22 (3.20) 292.49 (8.85)41.48

3***

48.911***

50.247***

Grasses (Shoots)† 167.65 (4.37) 165.02 (5.33) 20.28 (2.33) 18.10 (1.37) 0.237 ns 97.020 *** 0.004 ns

Herbs (Shoots)† 2.35 (0.18) 2.31 (0.13) 99.67 (2.60) 106.20 (2.11) 1.467 ns 106.629 *** 1.966 ns

Legumes (Shoots)† 1.05 (0.06) 1.18 (0.11) 10.42 (1.49) 57.23 (4.90)14.55

7***

159.115***

6.208*

Roots† 40.74 (2.16) 39.39 (1.14) 80.85 (0.96) 110.96 (5.62) 2.033 ns 133.388 *** 10.292 **

Seedling establishment [mg]Non Legumes: (Shoot productivity)Festuca ovina† 8.01 (0.97) 4.46 (1.18) 4.16 (0.64) 3.72 (0.39) 5.344 * 4.624 * 2.898 ns

Koeleria macrantha∞ 5.57 (1.27) 4.29 (1.47) 3.23 (0.52) 2.54 (0.41) 2.053 ns 3.450 ns 0.111 ns

Plantago lanceolata§ 22.77 (2.72) 20.66 (3.07) 30.86 (3.98) 23.90 (2.58) 2.091 ns 3.267 ns 0.597 ns

Senecio jacobaea∞ 4.91 (0.36) 4.32 (0.78) 9.18 (1.48) 7.16 (1.21) 2.213 ns 9.936 ** 0.018 ns

Legumes: (Shoot productivity)

Lotus corniculatus† 7.83 (0.38) 9.96 (0.84) 9.64 (1.12) 57.13 (9.47)25.19

2***

21.640***

4.964*

Trifolium repens† 4.90 (0.54) 5.99 (0.51) 2.46 (0.49) 70.98 (16.8)68.90

2***

0.838ns

37.491***

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Trifolium arvense† 2.79 (0.34) 2.82 (0.37) 1.50 (0.34) 33.96 (7.29)35.20

5***

1.963ns

36.190***

Trifolium dubium† 3.53 (0.36) 5.13 (0.34) 2.02 (0.37) 76.17 (21.8)94.27

7***

1.030ns

32.690***

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).

C R M MR R M R*MPlant productivity [mg] mean (±se) mean (±se) mean (±se) mean (±se) F F F

GrassesAnthoxanthum odoratum† 5040.44 (920.46) A 4293.44 (606.79) A 923.33 (227.24) B 739.67 (88.03) B 0.003ns 77.073*** 0.253ns

Festuca ovina† 1875.67 (308.47) A 1669.11 (205.31) A 333.22 (30.36) B 289.89 (34.41) B 0.452ns 98.063*** 0.135ns

Koeleria macrantha† 1334.00 (311.92) A 1183.44 (174.71) A 216.22 (23.69) B 238.00 (8.49) B 1.147ns 31.024*** 0.007ns

Luzula campestris† 3898.56 (649.98) A 4365.33 (945.02) A 113.67 (43.99) B 49.33 (11.28) B 0.133ns 97.245*** 0.182ns

HerbsAchillea millefolium† 8.78 (1.60) A 7.44 (0.65) A 242.44 (22.27) B 224.11 (57.99) B 0.705ns 101.457*** 0.349ns

Carlina vulgaris∞ 45.22 (6.06) AB 33.89 (1.99) B 56.56 (5.50) A 46.11 (5.57) AB 5.265* 6.726* 0.003ns

Hieracium pilosella† 10.44 (1.82) A 12.44 (1.29) A 384.11 (32.09) B 422.11 (47.52) B 1.807ns 102.566*** 0.118ns

Plantago lanceolata† 59.33 (5.43) A 54.67 (4.43) A 6513.00 (264.91) B 6846.67 (359.93) B 0.473ns 102.584*** 1.564ns

Senecio jacobaea† 9.78 (2.75) A 7.67 (1.51) A 559.00 (93.79) B 420.22 (76.09) B 0.919ns 100.359*** 0.345ns

LegumesLotus corniculatus† 38.67 (2.92) A 36.44 (4.12) A 522.56 (58.94) B 1847.44 (217.20) C 5.479* 149.632*** 12.064**

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Trifolium repens† 16.56 (1.08) A 17.89 (0.92) A 260.78 (53.23) B 553.56 (36.13) C 9.266** 134.628*** 3.079ns

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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).

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.

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