Impact of land use on arbuscular myco rrhizal fungal...

Title: Impact of land use on arbuscular mycorrhizal fungal communities in rural 1

Canada 2

Running Title: Land use and AM fungi in Canadian ecozones 3

4

Mulan Dai1, Luke D. Bainard1, Chantal Hamel1*, Yantai Gan1, Derek Lynch2 5

1Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada, P.O. Box 6

1030, 1 Airport Road, Swift Current, Saskatchewan, Canada S9H 3X2 7

2 Dept. Plant and Animal Sciences, Faculty of Agriculture, Dalhousie University, 50 Pictou Road, 8

Truro NS, Canada B2N 5E3 9

*Address correspondence to Chantal Hamel, [email protected]

AEM Accepts, published online ahead of print on 30 August 2013Appl. Environ. Microbiol. doi:10.1128/AEM.01333-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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Abstract. The influence of land use on soil bio-resources is largely unknown. We examined the 10

communities of arbuscular mycorrhizal (AM) fungi in wheat-growing cropland, natural areas and 11

semi-natural roadsides. We sampled the Canadian Prairie extensively (317 sites), and sampled 20 12

sites in the Atlantic maritime ecozone for comparison. The proportion of the different AM fungal 13

tasa in the communities found at these sites varied with land use type and ecozones, based on 14

pyrosequencing of 18S rDNA amplicons, but the lists of AM fungal taxa obtained from the 15

different land use types and ecozones were very similar. In the Prairie, Glomeraceae was the 16

most abundant and diverse family of Glomeromycota, followed by Claroideoglomeraceae, but in 17

the Atlantic maritime Claroideoglomeraceae was most abundant. In the Prairie, species richness 18

and Shannon’s diversity were highest in roadsides, whereas cropland had a higher degree of 19

species richness than roadsides in the Atlantic maritime. The frequency of occurrence of the 20

different AM fungal taxa in croplands in the Prairie and Atlantic maritime ecozones was highly 21

correlated, but the AM fungal communities in these ecozones had different structures. We 22

conclude that the AM fungal resources of soils are resilient to disturbance and that the richness 23

of AM fungi under cropland management has been maintained, despite evidence of a structural 24

shift imposed by this type of land use. Roadsides in the Canadian Prairie are a good repository 25

for the conservation of AM fungal diversity. 26

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INTRODUCTION 31

Arbuscular mycorrhizal (AM) fungi are a ubiquitous group of obligate biotrophic fungi that play 32

a key role in the functioning and sustainability of agroecosystems (1). These mutualistic fungi 33

associate with the roots of the majority of agricultural plants and have shown the potential to 34

increase crop productivity. The primary function of the symbiosis involves the transfer of 35

photosynthetic carbon from the host plant to the fungal symbiont in exchange for increased 36

uptake of phosphate and ammonium as well as other essential mineral nutrients (2, 3). AM fungi 37

also provide other functional benefits to the host plant including protection from abiotic and 38

biotic stresses (1). For example, there is evidence that these mutualistic fungi can increase the 39

fitness of their host plant in harsh environments (4) including under low soil fertility (5, 6, 7), 40

drought (8, 9), and salinity (10). AM fungi are also involved in other ecological processes that 41

are critical in agroecosystems such as maintaining soil structure and stability (11) and the cycling 42

of major elements such as carbon, phosphorus, and nitrogen (2). The beneficial effects and 43

ecological services provided by AM fungi reveals their importance in the efficient functioning 44

and sustainability of agroecosystems. 45

AM fungi share a long history of co-evolution with plants in various ecosystems resulting in 46

their adaptation to specific natural areas (12, 13). In these areas, highly mutualistic plant-AM 47

fungal pairs are stabilized by a positive feedback loop through which mutual rewards in the form 48

of soil nutrients and carbon are preferentially given by AM fungi and host plants to their 49

symbiotic partners (14). Highly mutualistic plant-AM fungal pairs improve the performance of 50

an ecosystem, in particular the efficiency of nutrient cycling, plant productivity, and the survival 51

of AM fungi. Unfortunately, land management practices often impact the stability and 52


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performance of the AM symbiosis, resulting in potential consequences on the overall 53

productivity and sustainability of agroecosystems. 54

Annual cropping practices deeply transform the plant cover and soil conditions from their 55

natural state through the use of heavy machinery and the application of fertilizers and pesticides. 56

As a result, conventional agricultural practices have an impact on AM fungal communities. 57

Monoculture cropping deprives AM fungal taxa that have low compatibility with the crop plant 58

from host support, and subsequently reduces AM fungal diversity (15, 16). While non-host crops 59

(e.g. canola, rape seed) and fallow treatments deprive all AM fungi of an appropriate host plant 60

(17). Soil tillage and the termination of annual crops cause intense disturbance to AM fungal 61

networks and have a negative impact on extraradical hyphal density and AM root colonization of 62

subsequent crops (18, 12). Fertilization is known to strongly impact the composition, growth and 63

function of AM fungi (18, 19, 20, 21). Overall, agricultural practices have been reported to 64

reduce the diversity and abundance of AM fungi to varying degrees depending on the intensity of 65

crop management (22, 23, 24). 66

The objective of this study was to evaluate the impact of annual crop production on AM 67

fungal communities in rural Canada by comparing the relative abundance and composition of 68

AM fungi in annually cropped land, semi-natural roadsides, and natural areas. Our survey most 69

intensely examined cropland across the edapho-climatic zones of the rural Prairie Provinces 70

where 81.5 % of all Canadian croplands are located (25), but we also included some cropland on 71

podzolic soils of the Atlantic maritime ecozone. This study provides detailed information on the 72

composition and diversity of indigenous AM fungi in these prime agricultural regions and 73

allowed us to test the hypothesis that crop production influences the relative abundance and 74

composition of AM fungal communities in the landscape of rural Canada. We also predicted that 75


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roadsides are a repository for the conservation of AM fungal diversity in areas of intensive crop 76

production. 77

78

MATERIALS AND METHODS 79

Description of the surveyed areas. Soil and root samples were taken from cropland (176 fields 80

of spring wheat or durum wheat [Triticum aestivum L. or Triticum durum L.]), adjacent 81

roadsides (117 sites), and natural areas (24 sites). In order to provide a good coverage of the AM 82

fungal community of the vast area that is the Canadian Prairie at a key stage of wheat 83

development, i.e. at heading, sampling was performed over two years, in 2009 and 2010. Pairing 84

of close-by sampling areas of cropland, roadside, or natural area was performed where possible 85

to ensure that comparisons made between land use types were not biased by climate or soil 86

conditions. Seventy two percent of the croplands were paired; 11.2% were paired with natural 87

areas and 60.8% with roadsides. The distance between paired cropland and roadsides areas was 88

less than 100 m, and the distance between paired cropland and natural area usually less than 100 89

m and never exceeded 1 km. Cropland soil at several locations was covered by wheat plants up 90

to the dirt roads and in absence of roadside or natural area. Natural areas are scarce and to 91

provide power for multivariate analyses of community structure, four unpaired samples were 92

also taken from pristine natural areas, i.e. two sites on municipal land and two sites in protected 93

parks. 94

Sampling efforts were mainly focused on covering all the edapho-climatic zones of the 95

Canadian Prairie ecozone (26), known as the Brown, Dark Brown, Black and Gray soil zones 96

(27). Samples were also taken from paired cropland and roadsides at 10 locations in the Atlantic 97

maritime ecozone (26) in Nova Scotia. In the Canadian Prairie, samples came from just above 98


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the U.S.A. border to the boreal forest over an area spanning approximately1450 km between 99

Beaverlodge, Alberta to the west, and Brandon, Manitoba to the east, but the majority of samples 100

were collected in the province of Saskatchewan. 101

In the Atlantic maritime ecozone, samples were all taken from soils classified as Podzol (27). 102

The croplands under organic production in this ecozone were sometimes weedy. Frequent use of 103

perennial hay crops with complex plant compositions and the application of animal manure are 104

characteristics of the cropping systems sampled in the Atlantic maritime ecozone. 105

Croplands located on Brown, Dark Brown and Black Chernozems of the Prairie ecozone may 106

have over 100 years of agricultural history, while croplands on Gray Luvisol and Dark Gray 107

Chernozems may have been broken for crop production from natural forest in more recent times 108

(28). All cropping systems in the Prairie province are based on the production of wheat, which is 109

often grown in crop rotation systems. The most common rotation crops are canola, pea, lentil, 110

barley and flax. Organic systems are fertilized with plow down green manure crops in occasional 111

semi-fallow years. Ninety nine of the sampling sites in cropland were conventionally managed, 112

thus normally untilled, and 77 were under organic management. Roadsides were a buffering area 113

between the production field and the road. They were typically gravelly, shaped as a ditch, and 114

colonized by the invasive forage grass species crested wheatgrass (Agropyron cristatum [L.] 115

Gaertn) and bromegrass (Bromus inermis Leyss.) that were seeded on the edges of newly 116

constructed roads, and by several competitive species. Some of these species have wide 117

adaptation, but many are only found in certain edapho-climatic zones (Table 1S). The vegetation 118

of most roadsides is cut and harvested as forage in July. Natural areas were areas spared from 119

agriculture by their inconvenient location in the landscape, which is most often related to 120

topography. Natural areas were usually small virgin patches, but some were also very large (Fig. 121


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1). The natural areas of different edapho-climatic zones had very distinct plant communities in 122

contrast to roadsides which were dominated by the same invasive forage grasses throughout the 123

Prairie (Table 1S). 124

Soil sampling and processing of samples. At each cropland site, composite samples were 125

randomly taken to a depth of 7.5 cm over a sampling area about 25 m2 in size that were deemed 126

representative of the field, based on topography. Thirty cores were taken with a soil probe 127

directly on the row and pooled in a bag. Field edges and accesses were avoided. Stones and stiff 128

roots prevented the use of the soil probe in most roadsides and natural areas and composite 129

samples from these areas consisted of six soil columns cut out from the top 7.5 cm soil layer with 130

a shovel. Care was taken to collect all roadside samples of the Prairie Provinces from stands or 131

patches of bromegrass. Soil samples were kept on ice in a cooler during transportation. Sampling 132

sites located outside of a 320 km radius from the laboratory in Swift Current were sampled by 133

collaborators and sent by rapid courier. Soil samples were homogenized and freed from stones 134

by sieving through a 2-mm mesh sieve in the laboratory. Samples were stored at -23°C prior to 135

DNA extraction. 136

Molecular analysis. Metagenomic DNA was extracted from 0.5-g soil samples drawn from each 137

site using an UltraClean Soil DNA isolation kit (catalogue No. 12800-100, Mo Bio Laboratories, 138

Inc.) according to the maximum yields protocol of the manufacturer, and stored at -20°C. The 139

fusion primers constructed with primers AMV4.5NF/AMDGR, adaptors and Multiplex Identifier 140

(MID) (Table 2S) based on the 454 sequencing technical bulletin No. 013-2009 of Roche, were 141

used to amplify AM fungal 18S rDNA. Primers AMV4.5NF/AMDGR were previously used 142

successfully in several studies (29, 30) to amplify sequences from environmental samples of all 143


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four AM fungal orders including Diversisporales, Glomerales, Archaeosporales and 144

Paraglomerales, as well as other fungi of the Ascomycota, Basidiomycota, and Chytridiomycota. 145

Each soil DNA sample was diluted (1:20) and amplified separately with the AM fungal 146

primer set AMV4.5NF/AMDGR. In order to decrease variations in the PCR process, samples 147

were amplified in triplicates (31) using the fusion primer set in a PCR reaction with 10 μL 148

volume per subsample. Platinum PCR SuperMix (catalogue No. 11306-016, Invitrogen) was 149

used in the PCR reactions. The final concentration of the reagent mix per 10 μL volume was 150

0.0165 U μL-1Taq DNA polymerase, 1.24 mM MgCl2, 16.5 mM Tris-HCl (pH 8.4), 41.25 151

mMKCl, 165 μM (each) dNTP, and 0.2 μM (each) primer. Thermal cycling was conducted in a 152

Veriti 96-well fast Thermal Cycler (Applied Biosystems) with the following conditions: 10 min 153

of denaturation at 95°C for the first step; 35 cycles of 30 s of denaturation at 94°C, 30 s of 154

annealing at 55°C, and 1 min of elongation at 72°C; followed by 9 min of final elongation at 155

74°C. The three PCR products were pooled and purified by ChargeSwitch® PCR Clean-Up kit 156

(Cat. No. CS12000, InvitrogenTM). Purified PCR amplicons were normalized at 25 ng μL-1 with 157

a Savant DNA 120 SpeedVac concentrator (Thermo scientific®). The concentration of purified 158

amplicons was measured using a Nano Drop 1000 spectrophotometer (Thermo scientific). All 159

amplicons from each sample were barcoded with one of sixteen Roche’s multiplex identifiers 160

(MIDs). The tagged samples were pooled and sent for pyrosequencing, which was performed 161

under contract at NRC Plant Biotechnology Institute (NRC-PBI, Saskatoon, Saskatchewan, 162

Canada). 163

Bioinformatic and phylogenetic analysis. Pyrosequencing reads containing ambiguous 164

nucleotides (average score of quality ≥ 30) (32), a single nucleotide mismatch with the PCR 165

primer, or which were of atypical length (<230 bp or >250 bp) were removed from the dataset 166


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using Mothur version 1.15.0 (33). Sequences belonging to other groups than the Glomeromycota 167

were identified by comparison with the Silva eukaryotic reference for 18S rDNA sequences 168

(http://www.arb-silva.de/) and AM fungal reference sequences obtained from GenBank. All 169

unique sequences were filtered for reduced computational complexity using Mothur. The average 170

length of the cleaned sequences was 241 bp. The clean AM fungal sequences were aligned with 171

each other using MUSCLE (34) and alignments were clustered based on 97 % similarity into 172

operational taxonomic units (OTU) using the furthest neighbor algorithm with Mothur. In 173

addition, Shannon diversity index (H’), ACE estimator index, and OTU richness were calculated 174

with Mothur. Taxonomic assignment was performed by comparing a representative sequence of 175

each OTU to GenBank non-redundant nucleotide database (35). 176

A representative sequence of each AM fungal OTU analyzed in this study were deposited in 177

GenBank under access numbers PRJNA198741 and are listed in Table 3S. Representative OTU 178

sequences and AM fungal reference sequences from GenBank were aligned using MUSCLE (34), 179

and neighbor-joining phylogenetic reconstruction (36) was used to build the phylogenetic tree in 180

MEGA 5 (37). Default parameters were used except that bootstrap replication was set at 1000 181

with the Kimura 2-parameter model (38). The nomenclature used here was proposed by 182

Redecker et al (39). The abundance of each OTU in a sample was expressed as the number of 183

reads of that OTU relative to all fungal reads in that sample. 184

Statistical analysis. Comparison of AM fungal communities between paired samples of 185

cropland and adjacent roadsides or natural areas was made by subjecting the paired-site data to 186

Student’s t-test using R. The effect of land use type on ACE estimator and Shannon’s H’, was 187

tested by ANOVA (40) for all three biomes, and the significance of the differences between land 188


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use type means was assessed post hoc using Duncan’s test, with the package “Agricolae” in R 189

(41). 190

The hypothesis that soil in different land use types and ecozones had distinct AM fungal 191

community structures was tested by subjecting the relative AM fungal OTU abundance data 192

from both, paired and unpaired sites to multi-response permutation procedures (MRPP) in PC-193

ORD v.6 (42). Non-metric multidimensional scaling (NMS) analysis (43) was used in PC-ORD 194

to visualize the whole data. The two most informative dimensions of a 3-dimensional solution 195

explaining 59.9% of the variance were used to construct the NMS ordination graph. 196

The “gplots” (44) and “RColorBrewer” packages (45) were used in R to plot a heatmap 197

showing the proportion of each OTU making up the AM fungal communities found in cropland, 198

roadsides and natural areas. Spearman correlation analysis was used in JMP v. 3.2.6 to evaluate 199

the relationship between the relative abundance of AM fungal OTUs and their frequency of 200

occurrence. 201

202

RESULTS 203

Molecular analysis of AM fungi. The pyrosequencing platform produced an average of 4213 204

reads per sample after cleaning. Among land use types, the average reads per sample were 5411 205

for roadsides, 3641 for cropland, and 2318 for natural areas. The primers used in this study are 206

not specific to AM fungi and a large proportion of the reads belonged to other fungal phyla. The 207

taxonomic distribution and proportion of all sequences obtained in this study are shown in Figure 208

2. Sequences belonging to the Ascomycota accounted for the highest proportion of reads across 209

all three land use types (> 55 %). Glomeromycota sequences represented the second largest 210

phylum (13.9-16.7 %) except in natural areas where the proportion of sequences of 211


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Basidiomycota was higher (17.8 %). Paired comparisons of land use type in the Prairie revealed 212

higher relative abundance of AM fungal reads in roadsides and natural areas than cropland (P < 213

0.0001) (Table 1). In contrast, there was no significant difference in the relative abundance of 214

AM fungal reads in cropland and roadside samples from the Atlantic maritime region (P = 0.11). 215

In total, we detected 122 OTUs based on 97 % similarity (Fig. 1S). In the Prairie ecozone (n = 216

317), we detected 120 AM fungal OTUs. The majority of the OTUs and reads belonged to the 217

Glomeraceae (78 OTUs and 47.6 % of AM fungal reads) and Claroideoglomeraceae (30 OTUs 218

and 36.9 % of AM fungal reads). The remaining OTUs belonged to the Diversisporaceae (9 219

OTUs and 6.6 % of AM fungal reads), Gigasporaceae (1 OTU), Archaeosporaceae (1 OTU), and 220

Paraglomeraceae (1 OTU) (Figure 2S). 221

Seventy two AM fungal OTUs were detected in the Atlantic maritime samples (n = 20). The 222

majority of the OTUs represented the Glomeraceae (37 OTUs) and Claroideoglomeraceae (26 223

OTUs), but reads of Claroideoglomeraceae (49.7 % of all AM fungal reads) were more abundant 224

than reads of Glomeraceae (39.3 % of all AM fungal reads). The remaining OTUs belonged to 225

the Diversisporaceae (6 OTUs, 6.7 % of the reads), Gigasporaceae (2 OTUs, 0.8 % of the read), 226

and Paraglomeraceae (Fig. 3). Paraglomeraceae was represented only by OTU2, but this OTU 227

was abundant in the Atlantic maritime ecozone, accounting for 3.4 % of all AM fungal reads (Fig. 228

3). The Archaeosporal OUT, which shared 99% similarity with A. trappei (OTU1) and was rare 229

in the Prairie, was undetected in the Atlantic maritime ecozone. 230

Known reference sequences of Glomeromycota retrieved from GenBank yielded good 231

matches (≥ 95 % similarity) with 52 of the AM fungal OTUs. The remaining 70 OTUs yielded 232

better matches with “unknown” or “uncultured” Glomeromycota sequences and had a lower 233

level of similarity (90-95 %) with known reference sequences. The sequence comparison and the 234


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phylogenetic analysis (Fig. 2S) document the taxonomic identity of the AM fungal OTUs from 235

this study. Several of the dominant OTUs shared high levels of similarity with known reference 236

sequences. In particular, OTU9, OTU27, OTU30, OTU57, OTU61, OTU109 were among the 237

most frequent OTUs corresponding to identified AM fungi (Fig. 4 and Fig. 1S). Glomus 238

iranicum is likely abundant in the Prairie. OTU109 ranked seventh for read abundance and 239

shared 97% similarity with G. iranicum. The most dominant OTU (OTU119) and numerous 240

other OTUs had G. iranicum as closest known match, though their level of similarity was often 241

low (Fig. 4). 242

Effect of land use on AM fungal richness and diversity. Land use type had a significant effect 243

on the richness and diversity of AM fungi in the Prairie (Table 1). Roadsides had higher OTU 244

richness (P < 0.0001), ACE richness estimate (P < 0.0001), and Shannon’s H (P < 0.0001) 245

compared to cropland, but the diversity and richness of AM fungi in cropland and natural areas 246

were similar (Table 1). Natural areas were similar to cropland in having lower levels of richness 247

and diversity than roadsides (Table 1). In the Atlantic maritime, the ACE richness estimates (P = 248

0.11) and Shannon’s H’ (P = 0.06) of cropland and roadsides were similar, but a higher AM 249

fungal OTU richness (P = 0.006) was found in cropland than roadsides. Rarefaction analysis was 250

used to compare the AM fungal richness observed in the different land use types (Fig. 5). This 251

analysis revealed a poor coverage of the AM fungal diversity in the natural areas and highest 252

richness and coverage in roadside of the Prairie. Cropland and roadside curves of Atlantic 253

maritime were also short because of poor sample number, but AM fungal OTUs in cropland was 254

richer than roadsides. 255

Effect of land use on the distribution and community structure of AM fungi. There was 256

considerable overlap of the OTUs detected in the different land use types. In the Prairie, 53 257


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OTUs accounting for 79.1 % of all AM fungal reads were found in soil under all three land use 258

types (Fig. 6. There were only 16 OTUs (0.52 % of reads) solely detected in roadsides, 7 OTUs 259

(0.13 % reads) solely detected in cropland, and only 1 OTU (0.016 % of reads) unique to natural 260

areas. The AM fungal taxa frequently encountered in natural areas were also frequently 261

encountered in roadside and cropland soil of the Prairie and to a lesser extent in the cropland of 262

Atlantic maritime, as shown by correlation analysis (Table 2). However, the abundance of these 263

AM fungal taxa varied with land use type. This effect was evident in the top ten most abundant 264

OTUs, which accounted for 52.6 % of the total AM fungal reads (Fig. 7 and Fig. 1S). Some 265

OTUs were more abundantly distributed in cropland such as OTU61, OTU19, OTU57, OTU27, 266

and OTU86. Two OTUs closely related to Funneliformis mosseae, OTU57 and OTU61, 267

accounted for 8 % and 15.8 % respectively of all AM fungal reads in cropland (Fig. 5 and Fig. 268

2S). In contrast, other abundant AM fungal OTUs (OTU9, OTU30, OTU119, OTU28, and 269

OTU109) were more common in the stable ecosystems of the roadsides or natural areas. For 270

example, OTU30 accounted for 18.7 % of all AM fungal reads from natural areas, 6 % from 271

roadsides, and only 0.67 % of cropland. OTU87 was 98 % similar to Glomus indicum (Fig. 2S). 272

This taxon, which is reported for the first time in the Canadian Prairie, mostly occurred in 273

roadsides. 274

There was considerable overlap of the OTUs detected in the Prairie and Atlantic maritime. 275

Only two OTUs were unique to the Atlantic maritime (OTU23, 98% similarity with 276

Scutellospora calospora, and OTU39, 93% similarity with Claroideoglomus lamellosum) and 277

together, they accounted for only 0.91 % of the AM fungal reads. Six of the ten most dominant 278

OTUs in the Prairie were also the most dominant in the Atlantic maritime ecozone (Fig. 5). The 279

OTUs corresponding to sequences of F. mosseae (OTU61 and OTU57) were only abundant in 280


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croplands in the Atlantic maritime ecozone (Fig. 5b).In cropland, these OTUs accounted for 281

12.5 % and 11.8 % of all AM fungal OTUs, whereas in roadside they were almost absent. 282

The analysis of AM fungal communities revealed different AM fungal communities in soil 283

under different types of land use (P = 0.000001) (Fig. 8). In the Prairie, the structural difference 284

in the AM fungal communities in cropland and natural areas non-significant (Table 1). Distinct 285

AM fungal community structures were detected in roadsides and croplands in both ecozones 286

(Table 1). The frequency of occurrence of OTUs in croplands in the two ecozones were highly 287

correlated (Table 2), but the structure of their AM fungal communities was different (P = 0.015) 288

(Fig. 8). The communities in roadside in the two ecozones were different (P = 0.00048). 289

290

DISCUSSION 291

In this study, we examined the influence of land use on AM fungi through a large survey 292

conducted in two contrasting ecozones of rural Canada: the Prairie and Atlantic maritime. We 293

found community overlap in natural areas, roadsides, and cropland in both ecozones. Dominant 294

AM fungal taxa were generally dominant in all zones, but not under all types of land use. We 295

found that crop production has a homogenizing effect on AM fungal communities, which 296

appeared to be similar in both ecozones. Contrary to our prediction, we found no evidence of a 297

negative effect of crop production on the taxonomic diversity of AM fungi in cropland and only 298

a weak influence on community structure, using natural areas as a reference point. 299

We found that AM fungi in cropland and natural areas are similar in diversity and richness, 300

but their relative abundance is lower in cropland. A mitigated effect of agriculture on AM fungal 301

diversity concurs with recent reports on the resilience of the soil microbiota (49), but the 302

possibility that the large areas of cropland neighboring natural areas modify the AM fungal 303


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communities of the natural areas used as reference cannot be ruled out. As predicted, we found 304

more diverse AM fungal communities in roadsides than in cropland of the Prairie. 305

Roadsides of the Prairie region were revealed as an important repository for AM fungal 306

diversity. Roadsides are spatially and temporally heterogeneous environments offering a wide 307

range of niches, which favours diversity (46, 47). Roadsides offer a large diversity of adapted 308

hosts with different phenologies, the stability of a perennial cover, and gradients of soil moisture, 309

which is the dominant factor determining ecosystem processes in the Prairie (48). The abundance 310

of spatial and temporal niches in Prairie roadsides may explain the higher abundance and much 311

higher level of AM fungal diversity in this land use type compared to cropland which are 312

homogenous across the landscape, and natural areas which are homogeneous within a site. The 313

intensity of agriculture is already very high in the Prairie region and is expected to increase with 314

increasing demand for food and biofuel crops such as canola, and areas in native Prairie 315

remnants are expected to shrink further. Our results suggest that AM fungal diversity can be 316

conserved in Prairie roadsides. 317

A different picture of AM fungal communities in the Atlantic maritime landscape emerged 318

from the analysis of the limited number samples taken on podzolic soils in this ecozone. 319

Cropland in the Atlantic maritime hosted richer AM fungal communities than roadsides, 320

disproving both of our hypotheses of a negative effect of agriculture on AM fungal communities 321

and of a role for roadside in the conservation of AM fungal diversity in this ecozone. However, 322

these results suggested that AM fungal diversity can be increased by certain cropping practices 323

as reported earlier (50, 16). All cropland in the Atlantic maritime were on mixed farms under 324

organic management that included mixed perennial hay crops in their crop rotation system. 325


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Diversity of AM fungal communities in the Canadian landscape. The influence of land use 326

on the communities of AM fungi in the Canadian landscape is largely unknown. We were 327

surprised to find similar levels of AM fungal diversity in natural areas and adjacent croplands of 328

the Prairie. Numerous studies have reported negative effects of crop production on AM fungi (51, 329

52, 53, 54). Soil tillage has a negative impact on AM hyphal network biomass and infectivity (55, 330

56) and on the richness of AM fungal communities (57, 58, 59). But in the Canadian Prairie, 331

soils are rarely tilled (25) as no-till is a soil water conservation practice (60, 61). Prairie soils 332

were commonly tilled two decades ago, but tillage was shallow (10 cm) (62, 60) and rarely 333

practiced in the fall. Therefore, the influence of tillage on AM fungal communities is likely to be 334

minimal in the Prairie. In the Atlantic maritime, where soil tillage is commonly practiced and 335

more intensive, AM fungal species richness was higher in cropland than roadsides (Table 1), 336

suggesting that tillage is not the main driver of the AM fungal diversity in croplands. The 337

addition of nitrogen fertilizer to soil decreases soil pH and can affect AM fungal richness in 338

cropland (63, 64). But Prairie soils are naturally well buffered and fertilizers are usually applied 339

parsimoniously since water availability rather than soil fertility normally limits yield. For the 340

same reason, the level of soil P fertility, which influences AM fungal activity and development 341

(17, 65, 12, 66, 67, 68, 69) may have been similar in cropland and natural areas. 342

A few generalist AM fungal taxa were very abundant. Generalist AM fungi are predicted to 343

be pioneer species with high dispersal capacity (70). If we accept high read abundance and 344

frequency as evidence of high dispersal capacity, two AM fungal taxa corresponding to F. 345

mosseae correspond to this description. These two OTUs, OTU61 and OTU57, had very high 346

frequency and abundance in cropland. F.mosseae is known as a cosmopolitan species (71) that is 347

particularly successful in croplands throughout the world (72). Although we cannot infer the 348


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identity of OTU61 and OTU57 based on the high levels of similarity with known species due to 349

the short length of our amplicons, we know from the morphological examination of trap cultures 350

from croplands made by us (unpublished) and by others (73) that F. mosseae is common in 351

cultivated Prairie soils. The abundance of these two F. mosseae-like OTUs was a striking 352

characteristic of cropland in the Atlantic maritime, as these were very rare in adjacent roadsides 353

in this ecozone (Fig. 5b). However, these OTUs were dominant throughout the Prairie suggesting 354

that the AM fungi they represent are favoured by crop production and dry environments, which 355

agrees with previous reports of the preference of F. mosseae for semiarid rather than mesic 356

environments (74). The influence of these F. mosseae-like taxa on crop productivity is unknown. 357

The relative similarity of the AM fungal communities of cropland in contrasting ecozones was 358

surprising. The uniformity of plant cover within cropland may have a homogenizing effect on 359

AM fungal communities. We based this hypothesis on published evidence of the selective 360

influence of plants on extraradical AM fungal growth (14) and on the structure of AM fungal 361

communities in soil (75, 76). Host plants have a large influence in shaping AM fungal 362

communities in soils (77, 78). The obvious difference between the environments created by the 363

different land use types was plant cover. In both ecozones, all croplands were planted with wheat 364

the year of sampling. Although some cropping systems include a phase with a perennial crop, as 365

was the case on the cropland in the Atlantic maritime, croplands are normally under the influence 366

of plants with short annual life cycles and they are usually devoid of living plants during most of 367

the year at these latitude. 368

Within group variation may explain the low AM fungal species richness and diversity index 369

found in natural areas. ‘Natural areas’ was a heterogeneous group. Different plant cover spanned 370

in forms from mixed grass vegetation in the semiarid zone, to poplar-willow groves in the 371


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Parkland, up to the spruce forest in the Boreal forest, but plant diversity was relatively low in any 372

given natural area. The AM fungal communities associated with these different plant covers may 373

also be variable. 374

In contrast to the vegetation of natural areas which was distinct in the edapho-climatic zones 375

of the Prairie, the vegetation of roadsides was relatively similar everywhere, as they were 376

initially planted with the same persistent grass species, but their plant cover was often diverse 377

within a sampling site. Roadside stands included several weedy species adapted to specific 378

edapho-climatic zones, but also plant species with different adaptations to soil moisture which 379

grew at different elevations in the roadside. The main role of roadsides is to drain water off the 380

roads. The temporal diversity created by the phenology of the plant species making up the 381

vegetation cover in roadsides is compounded by the practice of mowing. The heterogeneity of 382

the roadside environment favours diversity. The roadside environment also differ from other 383

types of land use in being less limited by water, a factor that may benefit AM fungal growth (74). 384

Our observation suggests that cropland is a type of land use conducive to the maintenance of 385

healthy AM fungal communities in the Atlantic maritime ecozone, unless OTU61 and OTU57 386

represent generalist taxa that are harmful. The AM fungal species that preferentially associate 387

with crop plants may outcompete less compatible species, which could disappear over time (24). 388

The fact that all but one AM fungal OTU found in natural areas was also found in cropland and 389

roadsides (Fig. 7) suggests that little of the indigenous AM fungal taxa have been lost due to 390

human activity, at least in the Prairie ecozone. However, the land was not all broken at the same 391

time in the Prairie, and it is possible that certain AM fungal taxa extirpated from lands with a 392

long history of crop production still remain in lands that were broken in more recent times. 393


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AM fungal resource in the Canadian Prairie. Our survey of the AM fungal communities of 394

the Canadian Prairie was extensive. It constitutes the first attempt to understand the AM fungal 395

resource hosted in this globally important wheat producing region (79). This survey and dataset 396

constitutes a baseline that allows tracking changes in AM fungal resource that can be caused by 397

the fortuitous introduction of invasive species through the practice of AM inoculation (70), to 398

other anthropological activities, or to climate change. Our survey spanned over a dry and a wet 399

year on the Prairie, which should be conducive to the detection of AM fungal taxa with different 400

life-history traits and to the capture of diversity. 401

Overall, the diversity and abundance of the Glomeraceae, represented by four genera, is 402

revealed as a feature of the AM fungal communities of the Prairie soils, as compared to the 403

Atlantic maritime where Claroideoglomeraceae was more abundant. Claroideoglomeraceae, 404

represented by three genera, was second to Glomeraceae in term of diversity and abundance in 405

Prairie soils. This family was particularly abundant in roadsides and natural areas. The scarcity 406

of Paraglomus also appears as a characteristic of the AM fungal communities of Prairie soils as 407

compared to the Atlantic maritime ecozone, where Paraglomus was a dominant OTU. 408

Our study reports for the first time the presence of AM fungi related to Glomus indicum in the 409

Canadian Prairie. We recently reported AM fungi related to Glomus iranicum in cultivated fields 410

of the Prairie ecozone (80) and now confirm the importance of this group. The large number of 411

OTUs clustering with G. iranicum in phylogenetic analysis and the relative abundance of certain 412

of these OTU (eg.: OTU109 is the 7th most abundant OTU), support that G. iranicum-related 413

taxa are well adapted to the Prairie ecozone. This concurs with the initial discovery of this taxa in 414

the semiarid wheat-growing region of southwestern Iran (81). We report for the first time the 415

presence of Archaeosporaceae in the Prairie based on the detection of an OTU sharing 99 % 416


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similarity with Archaeospora trappei. Our observation of one Paraglomeraceae sharing 97 % of 417

similarity with Paraglomus brasilianum concurs with the recent report of the presence of 418

Paraglomus in the Canadian Prairie (82), and our observation of a rare sequence clustering with 419

Scutellospora concurs with the earlier report of Scutellospora calospora in Prairie cropland (83). 420

These earlier reports were also based on short sequences of the 18S rRNA gene of insufficient 421

length for species identification, highlighting the need for AM fungal surveys based on 422

morphology or for better molecular methods to further improve our understanding of the 423

diversity of these important fungi in the Prairie. 424

425

CONCLUSIONS 426

From this extensive survey of the AM fungal diversity contained in soils of the Canadian Prairie 427

landscape and in twenty soils of the Atlantic maritime ecozone, we can conclude that land use 428

type influences the abundance of a set of AM fungal taxa largely shared by cropland, roadsides 429

and natural areas of the Prairie and the Atlantic maritime ecozones. The attributes of the plant 430

cover and the soil moisture availability level associated with land use appears as the most likely 431

factors shaping the structure of AM fungal communities in the Canadian landscape. Roadsides 432

bear a high degree of heterogeneity offering soil moisture and multiple niches for the 433

conservation of AM fungal diversity in the Prairie region. We found no evidence of a negative 434

impact of crop production on the diversity of AM fungal communities in our survey, although it 435

does influence the structure of these communities. 436

437

ACKNOWLEDGEMENT 438


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This study was funded by the Organic Science Cluster project A, activity 2 (project No. 04340) 439

to C. Hamel and the Ministry of Education Ph.D. Scholarship granted by the Chinese Ministry of 440

Education to M. Dai. We thank Xiaohong Yang and Zhiqing Zhou for support of M. Dai study in 441

Canada. The assistance of Kira Kotilla, Herny Jenzen, Newton Lupwayi, Sukhdev Malhi, Guy 442

Lafond, Reynald Lemke, Cynthia Grant, numerous seed growers and organic growers of 443

Saskatchewan with the provision of samples and sampling sites; of Dallas Thomas with 444

bioinformatics, Marc St. Arnaud with navigation in the landscape, and the technical assistance of 445

Keith Hanson and Elijah Atuku are gratefully acknowledged. 446

447

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82. Ellouze W, Hamel C, Vujanovic V, Gan YT, Bouzid S, St-Arnaud M. 2013. Chickpea 663

genotypes shape the soil microbiome and affect the establishment of the subsequent durum 664

wheat crop in the semiarid North American Great Plains. Soil Biol. Biochem. 63:129-141. 665

83. Ma WK, Siciliano SD, Germida JJ. 2005. A PCR-DGGE method for detecting arbuscular 666

mycorrhizal fungi in cultivated soils. Soil Biol. Biochem. 37:1589-1597. 667

668


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TABLE 1 Effect of land use type on the relative abundance, richness, diversity and community structure of AM fungi in the Prairie and

Atlantic maritime ecozones of Canada. Paired univariate comparisons of land use types in each of the Prairie and Atlantic maritime ecozones

were analyzed using Student’s t-test and multivariate comparisons, by multi-response permutation procedure (MRPP). The significance of land

use type overall effects on univariate data was analyzed by ANOVA whereas effects on community structure were tested by MRPP. Means are

presented with standard errors. Letters beside values indicate a significant difference according to Duncan’s test.

Ecozones Land use

type n

AM fungal

OTUs

Relative

abundance % ACE Richness

Shannon’s

H

AM fungal

community

Prairie provinces Cropland 107 90 15.3±1 16.1±2 7.7±0.7 1.6±0.09

Roadside 107 110 16.7±1.4 33.3±3.1 14.1±0.9 2.3±0.07

Paired samples t-test = <.0001 <.0001 <.0001 <.0001 MRPP = < .000001

Prairie provinces Cropland 20 71 11.6±2.0 23.0±3.6 7.7±1.2 1.6±0.20

Natural area 20 58 15.2±2.4 19.7±7.2 7.8±1.0 1.9±0.20

Paired samples t-test = <.0001 0.17 0.93 0.33 MRPP = 0.38

Atlantic maritime Cropland 10 55 12.0±1.2 29.5±5.2 15.1±1.2 2.4±0.10

Roadside 10 46 10.2±3.7 19.4±2.6 9.7±1.3 2.1±0.13

Paired samples t-test = 0.11 0.11 0.006 0.06 MRPP = < .00017

All observations Cropland 176 104 13.7±0.8 18.6±1.6 b 8.3±0.53 b 1.6±0.07 b

Roadside 117 112 16.0±1.3 34.3±3.2 a 13.8±0.79 a 2.3±0.07 a

Natural area 24 58 15.2±2.4 19.8±6 b 7.8±0.92 b 1.9±0.14 b

ANOVA= 0.29 <.0001 <.0001 <.0001 MRPP = < .000001


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TABLE 2 Coefficients of correlation associated with the relationship between the

frequency of occurrence of AM fungal taxa in soil under different land use in the

Prairie and the Atlantic maritime ecozones, according to Spearman correlation

analysis (P < 0.01; N = 122).

Atlantic

maritime

cropland

Atlantic

maritime

roadside

Prairie

cropland

Prairie

roadside

Prairie

natural

areas

Atlantic maritime cropland

1 0.5574 0.8629 0.7452 0.7456

Atlantic maritime roadside

0.5574 1 0.5494 0.7072 0.5756

Prairie cropland

0.8629 0.5494 1 0.7873 0.8403

Prairie roadside

0.7452 0.7072 0.7873 1 0.8300

Prairie natural areas

0.7456 0.5756 0.8403 0.8300 1

671

672


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FIG 1 (A) Typical field and roadside areas at the fringe of the Boreal forest; (B) typical natural 673

vegetation in the semiarid southwest of the Canadian Prairie; (C) satellite view showing the 674

organization of the landscape in the Canadian Prairie and the importance of roadsides as 675

repository of biodiversity in the zone of intense utilization of land for the production of crops. 676

Legal land units, called quarter sections, are distinguishable when they bear different vegetation. 677

The grid road system is emphasized in an overlay. The arrow indicates a deep coulee on the edge 678

of which native prairie remnants could be found. Satellite photo provided by Kim Hodge, SGIC 679

Ortho-Photography Project 2008–2011, GC/AAFC. © Her Majesty the Queen in right of Canada 680

2009, as represented by the Minister of Agriculture and Agri-Food Canada. All rights reserved. 681

FIG 2 Proportional abundance of the Glomeromycota, Ascomycota, Basidiomycota, other 682

Eukaryotes, and non-identified fungi in cropland, roadsides and natural areas of the Canadian 683

Prairie, as represented by the number of reads belonging to these groups that were obtained from 684

454 pyrosequencing of amplicons. 685

FIG 3 Importance of the AM fungal families represented in the AM fungal communities in the 686

rural landscape of the Canadian Prairie and Atlantic Maritime. 687

FI G 4 Heat map showing the OTU profiles of the AM fungal communities in Prairie croplands, 688

roadsides and natural areas, based on the number of OTU reads relative to all AM fungal OTU 689

reads obtained under a land use type. The 70 OTUs with highest number of reads are shown and 690

the 50 rare OTUs without visible variation among different land use type were omitted. The 691

OTU identification numbers are listed at the right of the heat map, with the identity of each AM 692

fungal taxa, according to the closest match with a known species in Genbank, and their level of 693

similarity. 694


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FIG 5 Rarefaction curves of AM fungal OTUs calculated from the clean read data obtained from 695

cropland, roadside and natural areas of the Canadian Prairie and Atlantic maritime ecozones. 696

FIG 6 Venn diagram comparing the diversity of AM fungal OTUs shared by, or found only in 697

cropland (N = 166), roadside (N = 107), and natural areas (N = 24) of the Canadian Prairie. 698

Gamma diversity is 120. 699

FIG 7 Relative abundance of the dominant AM fungal OTUs in the (A) Canadian Prairie and (B) 700

Atlantic maritime sites under different types of land use, based on the number of OTU reads 701

relative to all AM fungal reads for each type of land use. 702

FIG 8 Nonmetric multidimensional (NMS) ordination showing the relationship between the AM 703

fungal communities as observed under different land use types in different ecozones (MRPP: P < 704

0.000001, A = 0.091) . Symbols are the mean (± 1 SE) ordination coordinates of samples from 705

each land use type in the Prairie (Pr) and Atlantic maritime (At) ecozones. 706


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RoadsideCropland

AC

B


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Cropland

13.9% 65.4% 11% 4.4%5.3%

16 7% 56.8% 11.3% 5.0% 10.2%

Roadside

16.7%

Natural area15.2% 58.8% 17.8% 4.5%

3.7%

0 10 20 30 40 50 60 70 80 90 100Sequence reads proportion %

Glomeromycota Ascomycota Basidomycota Other Eukaryotes Non-identified fungi


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60%)

30

40

50

60

f AM

fung

al re

ads

(% Prairie

Atlantic Maritime

0

10

20

Abun

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Natural area

Cropland Roadside OTU Neastest known taxa

83103

9775777170

Glomus indicumGlomus iranicumGlomus iranicumGlomus iranicumGlomus iranicumSeptoglomus constrictumGlomus iranicum

94% GU059539.194% HM153423.194% HM153424.192% HM153423.192% HM153423.191% AM946956.191% JQ864324 1

Similarity

708956

12048

10820444595

107

Glomus iranicumGlomus indicumGlomus clarumGlomus iranicumDiversispora spurcaGlomus iranicumClaroideoglomus sp. 1Entrophospora nevadensisEntrophospora nevadensisGlomus iranicumSeptoglomus constrictum

91% JQ864324.196% GU059539.199% AJ505619.195% HM153424.190% FR686954.196% HM153424.196% JX301667.199% FN397100.197% FN397100.195% HM153423.198% AM946956 1107

49101

915394929926

100

Septoglomus constrictumRhizophagus irregularisGlomus iranicumGlomus iranicumGlomus viscosumGlomus iranicumGlomus iranicumGlomus iranicumEntrophospora infrequensGlomus iranicum

98% AM946956.188% JQ864338.197% HM153424.193% JQ864324.197% AJ505813.195% HM153424.192% HM153423.195% HM153423.196% FR865453.195% HM153424.100

55

1010

1515

Relative abundance (%)Relative abundance (%)

0

5

10

15

Relative abundance (%)

6885982487541032

212

Funneliformis mosseaeGlomus indicumGlomus iranicumClaroideoglomus lamellosumGlomus indicumSeptoglomus constrictumClaroideoglomus lamellosumClaroideoglomus lamellosumParaglomus laccatumClaroideoglomus lamellosum

95% FR750227.194% JQ864332.192% HM153424.193% FR773151.1 98% GU059537.197% AJ534309.192% FR750221.1 95% FR773151.1 98% HE613466.193% FR750221.1

000

125921

12260964163802234

Claroideoglomus lamellosumFunneliformis mosseaeClaroideoglomus sp.1Glomus iranicumFunneliformis mosseaeGlomus iranicumOtospora bareaeFunneliformis mosseaeGlomus indicumClaroideoglomus sp. 1Glomus iranicum

93% FR750221.1 96% FR715930.195% JX301666.194% JQ864324.195% U96142.192% JQ864324.194% JQ864341.195% FR750227.193% GU059536.194% JX301666.1 92% JQ864324 134

93114

5550698816

116121

Glomus iranicumGlomus iranicumGlomus hoiSeptoglomus constrictumRhizophagus irregularisGlomus iranicumGlomus indicumClaroideoglomus sp. 1Glomus macrocarpumGlomus iranicumGl i i

92% JQ864324.192% HM153424.199% AJ854087.197% AJ534309.199% FJ009612.192% JQ864324.198% GU059542.194% JX301666.197% FR772325.192% HM153424.1

37106

461886272857

10917

Glomus iranicumGlomus iranicumOtospora bareaeClaroideoglomus sp. 1 Glomus indicumClaroideoglomus lamellosumClaroideoglomus lamellosumFunneliformis mosseaeGlomus iranicumClaroideoglomus sp. 1

92% HM153423.199% HM153423.191% JQ864341.196% JX301666.195% GU059539.197% FR773151.1 94% FR773151.1 98% HE578143.197% HM153424.197% JX301666.1

9611930

119

g pClaroideoglomus lamellosumFunneliformis mosseaeClaroideoglomus sp. 1 Claroideoglomus lamellosumGlomus iranicum

95% FR750221.1 97% FR750227.196% JX301667.197% FR773151.1 94% HM153424.1


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120

OTU

s

Prairie roadsidePrairie cropland

Prairie 80

100

of A

M fu

ngal

O natural area

40

60

Atlantic maritime cropland

Num

ber o

0

20

Atlantic maritime roadside

Number of AM fungal sequences

0 20000 40000 60000 80000 100000 120000


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Cropland

7

532

1

39

Roadside

Natural area

12

16


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OTU28 Claroideoglomus lamellosum 94%


OTU19 Claroideoglomus sp.1 96%

OTU9 Claroideoglomus lamellosum 95%Roadside

Natural

Cropland

OTU119 Glomus iranicum 94%


OTU86 Glomus indicum 95%


OTU61 Funneliformis mosseae 97%


A 0 10 20

%

Relative abundance (% AM fungal reads)


OTU2 Paraglomus laccatum 98%





OTU19 Claroideoglomus sp.1 96%

B 0 10 20


OTU48 Diversispora spurca 90%

OTU46 Otospora bareae 91%

OTU2 Paraglomus laccatum 98%

Relative abundance (% AM fungal reads)

Roadside

Cropland


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Cropland (At)25)

p ( )Roadside (At)Cropland (Pr)Roadside (Pr)Natural area (Pr)

Axi

s 3

( r2

= 0.

2

Axis 2 ( r2 = 0.21)


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