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Arbuscular mycorrhizal (AM) fungi are a ubiquitous group of obligate biotrophic fungi that play a key role in the functioning and sustainability of agroecosystems (1). These mutualistic fungi associate with the roots of the majority of agricultural plants and have shown the potential to increase crop productivity. The primary function of the symbiosis involves the transfer of photosynthetic carbon from the host plant to the fungal symbiont in exchange for increased uptake of phosphate and ammonium as well as other essential mineral nutrients (2, 3). AM fungi also provide other functional benefits to the host plant, including protection from abiotic and biotic stresses (1). For example, there is evidence that these mutualistic fungi can increase the fitness of their host plant in harsh environments (4), including under low soil fertility (5–7), drought (8, 9), and salinity (10). AM fungi are also involved in other ecological processes that are critical in agroecosystems, such as maintaining soil structure and stability (11) and the cycling of major elements, such as carbon, phosphorus, and nitrogen (2). The beneficial effects and ecological services provided by AM fungi reveal their importance in the efficient functioning and sustainability of agroecosystems.
AM fungi share a long history of coevolution with plants in various ecosystems, resulting in their adaptation to specific natural areas (12, 13). In these areas, highly mutualistic plant-AM fungal pairs are stabilized by a positive-feedback loop through which mutual rewards in the form of soil nutrients and carbon are preferentially given by AM fungi and host plants to their symbiotic partners (14). Highly mutualistic plant-AM fungal pairs improve the performance of an ecosystem, in particular, the efficiency of nutrient cycling, plant productivity, and the survival of AM fungi. Unfortunately, land management practices often impact the stability and performance of the AM symbiosis, resulting in potential consequences to the overall productivity and sustainability of agroecosystems.
Annual cropping practices deeply transform the plant cover and soil conditions from their natural state through the use of heavy machinery and the application of fertilizers and pesticides. As a result, conventional agricultural practices have an impact on AM fungal communities. Monoculture cropping deprives AM fungal taxa that have low compatibility with the crop plant from host support and subsequently reduces AM fungal diversity (15, 16), while nonhost crops (e.g., canola, rape seed) and fallow treatments deprive all AM fungi of an appropriate host plant (17). Soil tillage and the termination of annual crops can cause intense disturbance to AM fungal networks and have a negative impact on extraradical hyphal density and the AM root colonization of subsequent crops (12, 18). Fertilization is known to strongly impact the composition, growth, and function of AM fungi (18–21). Overall, agricultural practices have been reported to reduce the diversity and abundance of AM fungi to various degrees, depending on the intensity of crop management (22–24).
The objective of this study was to evaluate the impact of annual crop production on AM fungal communities in rural Canada by comparing the relative abundances and compositions of AM fungi in annually cropped land, seminatural areas along roads, and natural areas. Our survey most intensely examined cropland across the edaphoclimatic zones of the rural prairie provinces, where 81.5% of all Canadian croplands are located (25), but we also included some cropland on podzolic soils of the Atlantic maritime ecozone. This study provides detailed information on the composition and diversity of indigenous AM fungi in these prime agricultural regions and allowed us to test the hypothesis that crop production influences the relative abundances and compositions of AM fungal communities in the landscape of rural Canada. We also predicted that roadsides are a repository for the conservation of AM fungal diversity in areas of intensive crop production.
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MATERIALS AND METHODS
Description of the surveyed areas.Soil and root samples were taken from cropland (176 fields of spring wheat or durum wheat [Triticum aestivum L. or Triticum durum L.]), their adjacent roadsides (117 sites), and natural areas (24 sites). In order to provide good coverage of the AM fungal community of the vast area that is the Canadian prairie at a key stage of wheat development, i.e., at heading, sampling was performed over 2 years, in 2009 and 2010. Pairing of close-by sampling areas of cropland, roadside, or natural area was performed where possible to ensure that comparisons made between land use types were not biased by climate or soil conditions. Seventy-two percent of the croplands were paired; 11.2% were paired with natural areas and 60.8% with roadsides. The distance between paired cropland and roadside areas was less than 100 m, and the distance between paired cropland and natural areas was usually less than 100 m and never exceeded 1 km. Cropland soil at several locations was covered by wheat plants up to the dirt roads and in the absence of roadside or natural areas. Natural areas are scarce, and to provide power for multivariate analyses of community structure, four unpaired samples were also taken from pristine natural areas, i.e., two sites on municipal land and two sites in protected parks.
Sampling efforts were focused mainly on covering all the edaphoclimatic zones of the Canadian prairie ecozone (26), known as the brown, dark brown, black, and gray soil zones (27). Samples were also taken from paired cropland and roadsides at 10 locations in the Atlantic maritime ecozone (26) in Nova Scotia. In the Canadian prairie, samples came from just above the U.S. border to the boreal forest over an area spanning approximately1,450 km between Beaverlodge, Alberta, to the west, and Brandon, Manitoba, to the east, but the majority of samples were collected in the province of Saskatchewan.
In the Atlantic maritime ecozone, all samples were taken from soils classified as podzol (27). The croplands under organic production in this ecozone were sometimes weedy. Frequent use of perennial hay crops with complex plant compositions and the application of animal manure are characteristics of the cropping systems sampled in the Atlantic maritime ecozone.
Croplands located on brown, dark brown, and black chernozems of the prairie ecozone may have over 100 years of agricultural history, while croplands on gray luvisol and dark gray chernozems may have been broken for crop production from natural forest in more recent times (28). All cropping systems in the prairie province are based on the production of wheat, which is often grown in crop rotation systems. The most common rotation crops are canola, pea, lentil, barley, and flax. Organic systems are fertilized with plow-down green-manure crops in occasional semifallow years. Ninety-nine of the sampling sites in cropland were conventionally managed, thus, normally untilled, and 77 were under organic management. Roadsides were a buffering area between the production field and the road. They were typically gravelly, shaped as a ditch, and colonized by the invasive forage grass species crested wheatgrass (Agropyron cristatum [L.] Gaertn) and bromegrass (Bromus inermis Leyss.), which were seeded on the edges of newly constructed roads, and by several competitive species. Some of these species have wide adaptation, but many are found only in certain edaphoclimatic zones (see Table S1 in the supplemental material). The vegetation of most roadsides is cut and harvested as forage in July. Natural areas were areas spared from agriculture by their inconvenient location in the landscape, which is most often related to topography. Natural areas were usually small virgin patches, but some were also very large (Fig. 1). The natural areas of different edaphoclimatic zones had very distinct plant communities, in contrast to roadsides, which were dominated by the same invasive forage grasses throughout the prairie (Table S1).
(A) Typical field and roadside areas at the fringe of the boreal forest; (B) typical natural vegetation in the semiarid southwest of the Canadian prairie; (C) satellite view showing the organization of the landscape in the Canadian prairie and the importance of roadsides as a repository of biodiversity in the zone of intense utilization of land for the production of crops. Legal land units, called quarter sections, are distinguishable when they bear different vegetation. The grid road system is emphasized in an overlay. The arrow indicates a deep coulee on the edge of which native prairie remnants could be found. Satellite photo provided by Kim Hodge (SGIC Ortho-Photography Project 2008–2011, GC/AAFC) with the permission of Her Majesty the Queen in right of Canada 2009, as represented by the Minister of Agriculture and Agri-Food Canada.
Soil sampling and processing of samples.At each cropland site, composite samples were randomly taken to a depth of 7.5 cm over a sampling area about 25 m2 in size that was deemed representative of the field, based on topography. Thirty cores were taken with a soil probe directly on the row and pooled in a bag. Field edges and accesses were avoided. Stones and stiff roots prevented the use of the soil probe in most areas along roads and in natural areas, and composite samples from these areas consisted of six soil columns cut out from the top 7.5-cm soil layer with a shovel. Care was taken to collect all roadside samples of the prairie provinces from stands or patches of bromegrass. Soil samples were kept on ice in a cooler during transportation. Sampling sites located outside a 320-km radius from the laboratory in Swift Current were sampled by collaborators and sent by rapid courier. Soil samples were homogenized and freed from stones by sieving them through a 2-mm mesh sieve in the laboratory. Samples were stored at −23°C prior to DNA extraction.
Molecular analysis.Metagenomic DNA was extracted from 0.5-g soil samples drawn from each site using an UltraClean soil DNA isolation kit (catalogue no. 12800-100; Mo Bio Laboratories, Inc.) according to the maximum-yield protocol of the manufacturer, and samples were stored at −20°C. The fusion primers constructed with primers AMV4.5NF/AMDGR, adaptors, and the multiplex identifier (MID) (see Table S2 in the supplemental material) based on those in a 454 sequencing technical bulletin of Roche (29) were used to amplify the AM fungal 18S rRNA gene (rDNA). Primers AMV4.5NF/AMDGR were previously used successfully in several studies (30, 31) to amplify sequences from environmental samples of all four AM fungal orders, namely, the Diversisporales, Glomerales, Archaeosporales, and Paraglomerales, as well as other fungi of the Ascomycota, Basidiomycota, and Chytridiomycota.
Each soil DNA sample was diluted (1:20) and amplified separately with the AM fungal primer set AMV4.5NF/AMDGR. In order to decrease variations in the PCR process, samples were amplified in triplicate (32) using the fusion primer set in a PCR with a 10-μl volume per subsample. Platinum PCR SuperMix (catalogue no. 11306-016; Invitrogen) was used in the PCRs. The final concentration of the reagent mix per 10-μl volume was 0.0165 U μl−1Taq DNA polymerase, 1.24 mM MgCl2, 16.5 mM Tris-HCl (pH 8.4), 41.25 mM KCl, 165 μM (each) deoxynucleoside triphosphate (dNTP), and 0.2 μM (each) primer. Thermal cycling was conducted in a Veriti 96-well fast thermal cycler (Applied Biosystems) with the following conditions: 10 min of denaturation at 95°C for the first step; 35 cycles of 30 s of denaturation at 94°C, 30 s of annealing at 55°C, and 1 min of elongation at 72°C; and 9 min of final elongation at 74°C. The three PCR products were pooled and purified with a ChargeSwitch PCR clean-up kit (catalogue no. CS12000; Invitrogen). Purified PCR amplicons were normalized at 25 ng μl−1 with a Savant DNA 120 SpeedVac concentrator (Thermo Scientific). The concentration of purified amplicons was measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific). All amplicons from each sample were barcoded with one of Roche's 16 multiplex identifiers (MIDs). The tagged samples were pooled and sent for pyrosequencing, which was performed under contract at the NRC Plant Biotechnology Institute (NRC-PBI, Saskatoon, Saskatchewan, Canada).
Bioinformatic and phylogenetic analysis.Pyrosequencing reads containing ambiguous nucleotides (average quality score of ≥30) (33) or a single nucleotide mismatch with the PCR primer or reads that were of atypical length (<230 bp or >250 bp) were removed from the data set using Mothur version 1.15.0 (34). Sequences belonging to groups other than the Glomeromycota were identified by comparison with the Silva eukaryotic reference for 18S rDNA sequences (http://www.arb-silva.de/) and AM fungal reference sequences obtained from GenBank. All unique sequences were filtered for reduced computational complexity using Mothur. The average length of the cleaned sequences was 241 bp. The clean AM fungal sequences were aligned with each other using MUSCLE (35), and alignments were clustered based on 97% similarity into operational taxonomic units (OTUs) using the furthest-neighbor algorithm with Mothur. In addition, the Shannon diversity index (H′), abundance coverage estimator (ACE) index, and OTU richness were calculated with Mothur. Taxonomic assignment was performed by comparing a representative sequence of each OTU to sequences in the GenBank nonredundant nucleotide database (36).
Representative OTU sequences and AM fungal reference sequences from GenBank were aligned using MUSCLE (35), and neighbor-joining phylogenetic reconstruction (37) was used to build the phylogenetic tree in MEGA 5 (38). Default parameters were used except that bootstrap replication was set at 1,000 with the Kimura 2-parameter model (39). The nomenclature used here was proposed by Redecker et al. (40). The abundance of each OTU in a sample is expressed as the number of reads of that OTU relative to all fungal reads in that sample.
Statistical analysis.Comparison of AM fungal communities between paired samples of cropland and adjacent roadsides or natural areas was made by subjecting the paired-site data to Student's t test using R. The effect of land use type on the ACE index and Shannon's H′ was tested by analysis of variance (ANOVA) (41) for all three biomes, and the significance of the differences between land use type means was assessed post hoc using Duncan's test with the Agricolae package in R (42).
The hypothesis that soils in different land use types and ecozones have distinct AM fungal community structures was tested by subjecting the relative AM fungal OTU abundance data from both paired and unpaired sites to multiresponse permutation procedures (MRPP) in PC-ORD v. 6 (43). Nonmetric multidimensional scaling (NMS) analysis (44) was used in PC-ORD to visualize the whole data. The two most informative dimensions of a 3-dimensional solution explaining 59.9% of the variance were used to construct the NMS ordination graph.
The gplots (45) and RColorBrewer packages (46) were used in R to plot a heatmap showing the proportion of each OTU making up the AM fungal communities found in cropland, along roadsides, and in natural areas. Spearman correlation analysis was used in JMP v. 3.2.6 to evaluate the relationship between the relative abundances of AM fungal OTUs and their frequencies of occurrence.
Nucleotide sequence accession number.A representative sequence of each AM fungal OTU analyzed in this study was deposited in GenBank under accession numbers KF620139 to KF620260 (OTUs 1 to 122), and their sequences are listed in Table S3 in the supplemental material.
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Molecular analysis of AM fungi.The pyrosequencing platform produced an average of 4,213 reads per sample after cleaning. Among land use types, the average numbers of reads per sample were 5,411 for roadsides, 3,641 for cropland, and 2,318 for natural areas. The primers used in this study are not specific to AM fungi, and a large proportion of the reads belonged to other fungal phyla. The taxonomic distribution and proportion of all sequences obtained in this study are shown in Fig. 2. Sequences belonging to the Ascomycota accounted for the highest proportion of reads across all three land use types (>55%). Glomeromycota sequences represented the second-largest phylum (13.9 to 16.7%), except in natural areas, where the proportion of sequences of Basidiomycota was higher (17.8%). Paired comparisons of land use type in the prairie revealed higher relative abundances of AM fungal reads from roadsides and natural areas than from cropland (P < 0.0001) (Table 1). In contrast, there was no significant difference in the relative abundances of AM fungal reads in cropland and roadside samples from the Atlantic maritime region (P = 0.11).
Proportional abundances of the Glomeromycota, Ascomycota, Basidiomycota, other eukaryotes, and nonidentified fungi in soils of croplands, roadsides, and natural areas of the Canadian prairie, as represented by the numbers of reads belonging to these groups, which were obtained from 454 pyrosequencing of amplicons.
In total, we detected 122 OTUs based on 97% similarity (see Fig. S1 in the supplemental material). In the prairie ecozone (n = 317), we detected 120 AM fungal OTUs. The majority of the OTUs and reads belonged to the Glomeraceae (78 OTUs and 47.6% of AM fungal reads) and Claroideoglomeraceae (30 OTUs and 36.9% of AM fungal reads). The remaining OTUs belonged to the Diversisporaceae (9 OTUs and 6.6% of AM fungal reads), Gigasporaceae (1 OTU), Archaeosporaceae (1 OTU), and Paraglomeraceae (1 OTU) (Fig. S2).
Seventy-two AM fungal OTUs were detected in the Atlantic maritime samples (n = 20). The majority of the OTUs represented the Glomeraceae (37 OTUs) and Claroideoglomeraceae (26 OTUs), but reads of Claroideoglomeraceae (49.7% of all AM fungal reads) were more abundant than reads of Glomeraceae (39.3% of all AM fungal reads). The remaining OTUs belonged to the Diversisporaceae (6 OTUs, 6.7% of the reads), Gigasporaceae (2 OTUs, 0.8% of the read), and Paraglomeraceae (Fig. 3). Paraglomeraceae was represented only by OTU2, but this OTU was abundant in the Atlantic maritime ecozone, accounting for 3.4% of all AM fungal reads (Fig. 3). The archaeosporal OTU, which shared 99% similarity with Archaeospora trappei (OTU1) and was rare in the prairie, was undetected in the Atlantic maritime ecozone.
Importance of the AM fungal families represented in the AM fungal communities in the rural landscape of the Canadian prairie and Atlantic maritime ecozones.
Known reference sequences of Glomeromycota retrieved from GenBank yielded good matches (≥95% similarity) with 52 of the AM fungal OTUs. The remaining 70 OTUs yielded better matches with unknown or uncultured Glomeromycota sequences and had a lower level of similarity (90 to 95%) with known reference sequences. The sequence comparison and the phylogenetic analysis (see Fig. S2 in the supplemental material) document the taxonomic identities of the AM fungal OTUs from this study. Several of the dominant OTUs shared high levels of similarity with known reference sequences. In particular, OTU9, OTU27, OTU30, OTU57, OTU61, and OTU109 were among the most frequent OTUs corresponding to identified AM fungi (Fig. 4 and Fig. S1). Glomus iranicum is likely abundant in the prairie. OTU109 ranked seventh for read abundance and shared 97% similarity with G. iranicum. The most dominant OTU (OTU119) and numerous other OTUs had G. iranicum as the closest known match, though their level of similarity was often low (Fig. 4).
Heat map showing the OTU profiles of the AM fungal communities in soils from prairie croplands, roadsides, and natural areas, based on the number of OTU reads relative to all AM fungal OTU reads obtained for a land use type. The 70 OTUs with the highest numbers of reads are shown, and the 50 rare OTUs without visible variation among different land use types were omitted. The OTU identification numbers are listed at the right of the heat map along with the percent identity of each AM fungal taxon to its closest match to a known species in GenBank.
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 Canadaa
Effect of land use on AM fungal richness and diversity.Land use type had a significant effect on the richness and diversity of AM fungi in the prairie (Table 1). Roadsides had higher OTU richness (P < 0.0001), ACE richness (P < 0.0001), and Shannon's H (P < 0.0001) than cropland, but the levels of diversity and richness of AM fungi in cropland and natural areas were similar (Table 1). Natural areas were similar to cropland in having lower levels of richness and diversity than roadsides (Table 1). In the Atlantic maritime, the ACE richness (P = 0.11) and Shannon's H′ (P = 0.06) values for cropland and roadsides were similar, but a higher AM fungal OTU richness (P = 0.006) was found in cropland than along roadsides. Rarefaction analysis was used to compare the levels of AM fungal richness observed in the different land use types (Fig. 5). This analysis revealed a poor coverage of the AM fungal diversity in the natural areas and highest richness and coverage in samples from roadsides of the prairie. Cropland and roadside curves of the Atlantic maritime region were also short because of poor sample number, but AM fungal OTUs in cropland were richer than in roadside samples.
Rarefaction curves of AM fungal OTUs calculated from the clean read data obtained from croplands, roadsides, and natural areas of the Canadian prairie and Atlantic maritime ecozones.
Effect of land use on the distribution and community structure of AM fungi.There was considerable overlap of the OTUs detected in the different land use types. In the prairie, 53 OTUs accounting for 79.1% of all AM fungal reads were found in soil under all three land use types (Fig. 6). There were only 16 OTUs (0.52% of reads) solely detected along roadsides, 7 OTUs (0.13% of reads) solely detected in cropland, and only 1 OTU (0.016% of reads) unique to natural areas. The AM fungal taxa frequently encountered in natural areas were also frequently encountered in roadside and cropland soil of the prairie and to a lesser extent in the cropland of the Atlantic maritime region, as shown by correlation analysis (Table 2). However, the abundances of these AM fungal taxa varied with land use type. This effect was evident in the top 10 most abundant OTUs, which accounted for 52.6% of the total AM fungal reads (Fig. 7; see also Fig. S1 in the supplemental material). Some OTUs, such as OTU61, OTU19, OTU57, OTU27, and OTU86, were more abundantly distributed in cropland. Two OTUs closely related to Funneliformis mosseae, OTU57 and OTU61, accounted for 8% and 15.8%, respectively, of all AM fungal reads in cropland (Fig. 7; see also Fig. S2). In contrast, other abundant AM fungal OTUs (OTU9, OTU30, OTU119, OTU28, and OTU109) were more common in the stable ecosystems of the roadsides or natural areas. For example, OTU30 accounted for 18.7% of all AM fungal reads from natural areas, 6% from roadsides, and only 0.67% from cropland. OTU87 was 98% similar to Glomus indicum (Fig. S2). This taxon, which is reported for the first time to occur in the Canadian prairie, mostly occurred along roads.
Venn diagram comparing the numbers of AM fungal OTUs shared by, or found only in, croplands (n = 166), roadsides (n = 107), and natural areas (n = 24) of the Canadian prairie. The gamma diversity score is 120.
Relative abundances of the dominant AM fungal OTUs in the Canadian prairie (A) and Atlantic maritime (B) sites under different types of land use, based on the number of OTU reads relative to all AM fungal reads for each type of land use. OTUs are identified by the name and the percent identity of their closest match to a known species in GenBank.
There was considerable overlap of the OTUs detected in the prairie and Atlantic maritime ecozones. Only two OTUs were unique to the Atlantic maritime region (OTU23, 98% similarity with Scutellospora calospora, and OTU39, 93% similarity with Claroideoglomus lamellosum), and together, they accounted for only 0.91% of the AM fungal reads. Six of the 10 most dominant OTUs in the prairie were also the most dominant in the Atlantic maritime ecozone (Fig. 7). The OTUs corresponding to sequences of F. mosseae (OTU61 and OTU57) were abundant only in croplands in the Atlantic maritime ecozone (Fig. 7A). In cropland, these OTUs accounted for 12.5% and 11.8% of all AM fungal OTUs, whereas in roadside soil, they were almost absent.
The analysis of AM fungal communities revealed different AM fungal communities in soil under different types of land use (P = 0.000001) (Fig. 8). In the prairie, the structural difference in the AM fungal communities in cropland and natural areas was nonsignificant (Table 1). Distinct AM fungal community structures were detected in roadside and in cropland samples in both ecozones (Table 1). The frequencies of occurrence of OTUs in croplands in the two ecozones were highly correlated (Table 2), but the structures of their AM fungal communities were different (P = 0.015) (Fig. 8). The communities in roadside soil in the two ecozones were different (P = 0.00048).
Nonmetric multidimensional (NMS) ordination showing the relationship between the AM fungal communities as observed under different land use types in different ecozones (MRPP, P < 0.000001; A = 0.091). Symbols are the mean (±1 standard error) ordination coordinates of samples from each land use type in the prairie (Pr) and Atlantic maritime (At) ecozones.
Coefficients of correlationa associated with the relationship between the frequencies of occurrence of AM fungal taxa in soils under different types of land use in the prairie and the Atlantic maritime ecozones
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In this study, we examined the influence of land use on AM fungi through a large survey conducted in two contrasting ecozones of rural Canada: the prairie and Atlantic maritime region. We found community overlap in natural areas, roadsides, and croplands from both ecozones. Dominant AM fungal taxa were generally dominant in all zones but not under all types of land use. We found that crop production has a homogenizing effect on AM fungal communities, which appeared to be similar in the two ecozones. Contrary to our prediction, we found no evidence of a negative effect of crop production on the taxonomic diversity of AM fungi in cropland and only a weak influence on community structure, with the diversity of natural areas as a reference point.
We found that AM fungi in cropland and natural areas are similar in diversity and richness but that their relative abundance in cropland is lower. A mitigated effect of agriculture on AM fungal diversity concurs with recent reports on the resilience of the soil microbiota (47), but the possibility that the large areas of cropland neighboring natural areas modify the AM fungal communities of the natural areas used as reference cannot be ruled out. As predicted, we found more diverse AM fungal communities in roadside soils than in cropland soils of the prairie.
Roadsides of the prairie region were revealed as an important repository for AM fungal diversity. Roadsides are spatially and temporally heterogeneous environments offering a wide range of niches, which favors diversity (48, 49). Roadsides offer a large diversity of adapted hosts with different phenologies, the stability of a perennial cover, and gradients of soil moisture, which is the dominant factor determining ecosystem processes in the prairie (50). The abundance of spatial and temporal niches in prairie roadsides may explain the higher abundance and much higher level of AM fungal diversity in this land use type than in cropland, which is homogenous across the landscape, and in natural areas, which are homogeneous within a site. The intensity of agriculture is already very high in the prairie region and is expected to increase with increasing demand for food and biofuel crops, such as canola, and remnant areas of native prairies are expected to shrink further. Our results suggest that AM fungal diversity can be conserved in prairie roadsides.
A different picture of AM fungal communities in the Atlantic maritime landscape emerged from the analysis of the limited number of samples taken on podzolic soils in this ecozone. Cropland in the Atlantic maritime hosted richer AM fungal communities than roadsides, disproving both of our hypotheses of a negative effect of agriculture on AM fungal communities and of a role for roadsides in the conservation of AM fungal diversity in this ecozone. However, these results suggested that AM fungal diversity can be increased by certain cropping practices, as reported earlier (16, 51). All cropland in the Atlantic maritime ecozone were on mixed farms under organic management that included mixed perennial hay crops in their crop rotation system.
Diversity of AM fungal communities in the Canadian landscape.The influence of land use on the communities of AM fungi in the Canadian landscape is largely unknown. We were surprised to find similar levels of AM fungal diversity in natural areas and adjacent croplands of the prairie. Numerous studies have reported negative effects of crop production on AM fungi (52–55). Soil tillage has a negative impact on AM hyphal network biomass and infectivity (56, 57) and on the richness of AM fungal communities (58–60), but in the Canadian prairie, soils are rarely tilled (25), as not tilling is a soil water conservation practice (61, 62). Prairie soils were commonly tilled 2 decades ago, but tillage was shallow (10 cm) (61, 63) and rarely practiced in the fall. Therefore, the influence of tillage on AM fungal communities is likely to be minimal in the prairie. In the Atlantic maritime ecozone, where soil tillage is commonly practiced and more intensive, AM fungal species richness was higher in cropland than in roadsides (Table 1), suggesting that tillage is not the main driver of the AM fungal diversity in croplands. The addition of nitrogen fertilizer to soil decreases soil pH and can affect AM fungal richness in cropland (64, 65), but prairie soils are naturally well buffered, and fertilizers are usually applied parsimoniously since water availability rather than soil fertility normally limits yield. For the same reason, the levels of soil P fertility, which influence AM fungal activity and development (12, 17, 66–70), may have been similar in cropland and natural areas.
A few generalist AM fungal taxa were very abundant. Generalist AM fungi are predicted to be pioneer species with a high dispersal capacity (71). If we accept high read abundance and frequency as evidence of high dispersal capacity, two AM fungal taxa corresponding to F. mosseae correspond to this description. These two OTUs, OTU61 and OTU57, had very high frequencies and abundances in cropland. F. mosseae is known as a cosmopolitan species (72) that is particularly successful in croplands throughout the world (73). Although we cannot infer the identities of OTU61 and OTU57 based on their high levels of similarity with known species, due to the short lengths of our amplicons, we know from the morphological examination of trap cultures from croplands made by us (unpublished) and by others (74) that F. mosseae is common in cultivated prairie soils. The abundance of these two F. mosseae-like OTUs was a striking characteristic of cropland in the Atlantic maritime ecozone, as these were very rare in adjacent roadside soils in this ecozone (Fig. 7). However, these OTUs were dominant throughout the prairie, suggesting that the AM fungi that they represent are favored by crop production and dry environments, which agrees with previous reports of the preference of F. mosseae for semiarid rather than mesic environments (75). The influence of these F. mosseae-like taxa on crop productivity is unknown.
The relative similarity of the AM fungal communities of croplands in contrasting ecozones was surprising. The uniformity of plant cover within croplands may have a homogenizing effect on AM fungal communities. We based this hypothesis on published evidence of the selective influence of plants on extraradical AM fungal growth (14) and on the structure of AM fungal communities in soil (76, 77). Host plants have a large influence in shaping AM fungal communities in soils (78, 79). The obvious difference between the environments created by the different land use types was plant cover. In both ecozones, all croplands were planted with wheat the year of sampling. Although some cropping systems include a phase with a perennial crop, as was the case on the cropland in the Atlantic maritime ecozone, croplands are normally under the influence of plants with short annual life cycles, and they are usually devoid of living plants during most of the year at these latitudes.
Within-group variation may explain the low AM fungal species richness and diversity index found in natural areas. Natural areas were a heterogeneous group. The forms of different plant covers went from mixed-grass vegetation in the semiarid zone to poplar and willow groves in the parkland to spruce forest in the boreal forest, but plant diversity was relatively low in any given natural area. The AM fungal communities associated with these different plant covers may also be variable.
In contrast to the vegetation of natural areas, which was distinct in the edaphoclimatic zones of the prairie, the vegetation of roadsides was relatively the same everywhere, as they were initially planted with the same persistent grass species, but their plant cover was often diverse within a sampling site. Roadside stands included several weedy species adapted to specific edaphoclimatic zones but also plant species with different adaptations to soil moisture, which grew at different elevations of the roadside. The main role of roadsides is to drain water off the roads. The temporal diversity created by the phenology of the plant species making up the vegetation cover in roadsides is compounded by the practice of mowing. The heterogeneity of the roadside environment favors diversity. The roadside environment also differs from other types of land use in being less limited by water, a factor that may benefit AM fungal growth (75).
Our observation suggests that cropland is a type of land use conducive to the maintenance of healthy AM fungal communities in the Atlantic maritime ecozone, unless OTU61 and OTU57 represent generalist taxa that are harmful. The AM fungal species that preferentially associate with crop plants may outcompete less compatible species, which may disappear over time (24). The fact that all but one AM fungal OTU found in natural areas was also found in cropland and roadside soils (Fig. 6) suggests that few of the indigenous AM fungal taxa have been lost due to human activity, at least in the prairie ecozone. However, the land was not all broken at the same time in the prairie, and it is possible that certain AM fungal taxa extirpated from lands with a long history of crop production still remain in lands that were broken in more recent times.
AM fungal resource in the Canadian prairie.Our survey of the AM fungal communities of the Canadian prairie was extensive. It constitutes the first attempt to understand the AM fungal resource hosted in this globally important wheat-producing region (80). This survey and data set constitute a baseline that allows us to track changes in AM fungal resources that can be caused by the fortuitous introduction of invasive species through the practice of AM fungal inoculation (71), by other anthropological activities, or by climate change. Our survey spanned a dry and a wet year on the prairie, which should be conducive to the detection of AM fungal taxa with different life history traits and to the capture of diversity.
Overall, the diversity and abundance of the Glomeraceae, represented by four genera, is revealed as a feature of the AM fungal communities of the prairie soils, compared to the Atlantic maritime soils, where Claroideoglomeraceae were more abundant. Claroideoglomeraceae, represented by three genera, were second to Glomeraceae in terms of diversity and abundance in prairie soils. This family was particularly abundant in roadsides and natural areas. The scarcity of Paraglomus also appears as a characteristic of the AM fungal communities of prairie soils compared to those of the Atlantic maritime ecozone, where Paraglomus was a dominant OTU.
Our study reports for the first time the presence of AM fungi related to Glomus indicum in the Canadian prairie. We recently reported AM fungi related to Glomus iranicum in cultivated fields of the prairie ecozone (81) and now confirm the importance of this group. The large number of OTUs clustering with G. iranicum in the phylogenetic analysis and the relative abundances of certain of these OTUs (e.g., OTU109 is the 7th-most-abundant OTU), support the idea that G. iranicum-related taxa are well adapted to the prairie ecozone. This concurs with the initial discovery of these taxa in the semiarid wheat-growing region of southwestern Iran (82). We report for the first time the presence of Archaeosporaceae in the prairie based on the detection of an OTU sharing 99% similarity with Archaeospora trappei. Our observation of one member of the family Paraglomeraceae sharing 97% of similarity with Paraglomus brasilianum concurs with the recent report of the presence of Paraglomus in the Canadian prairie (83), and our observation of a rare sequence clustering with Scutellospora concurs with the earlier report of Scutellospora calospora in prairie cropland (84). These earlier reports were also based on short sequences of the 18S rRNA genes, which are of insufficient length for species identification, highlighting the need for AM fungal surveys based on morphology or for better molecular methods to further improve our understanding of the diversity of these important fungi in the prairie.
Conclusions.From this extensive survey of the AM fungal diversity contained in soils of the Canadian prairie landscape and in 20 soils of the Atlantic maritime ecozone, we can conclude that land use type influences the abundance of a set of AM fungal taxa largely shared by cropland, roadsides, and natural areas of the prairie and the Atlantic maritime ecozones. The attributes of the plant cover and the soil moisture availability level associated with land use appears as the most likely factors shaping the structure of AM fungal communities in the Canadian landscape. Roadsides bear a high degree of heterogeneity and offer soil moisture and multiple niches for the conservation of AM fungal diversity in the prairie region. We found no evidence of a negative impact of crop production on the diversity of AM fungal communities in our survey, although it does influence the structures of these communities.
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This study was funded by the Organic Science Cluster, project A, activity 2 (project no. 04340 grant to C. Hamel) and a Ministry of Education Ph.D. scholarship granted by the Chinese Ministry of Education to M. Dai. We thank Xiaohong Yang and Zhiqing Zhou for support of M. Dai's study in Canada.
We gratefully acknowledge the assistance of Kira Kotilla, Henry Jenzen, Newton Lupwayi, Sukhdev Malhi, Guy Lafond, Reynald Lemke, Cynthia Grant, numerous seed growers, and organic growers of Saskatchewan for the provision of samples and sampling sites, Dallas Thomas for bioinformatics assistance, Marc St. Arnaud for navigation in the landscape, and Keith Hanson and Elijah Atuku for technical assistance.
- Reminder: study for your unit 2 test, which will take place Thursday.
- Review materials are posted April 5th.
- Topics this week: population density, population pyramids, population distribution, Canada's Aboriginal population in the 21st Century
- Stages of population change, p. 192-193.
- Intro to Canada's Aboriginal population in the 21st Century: p. 196-200
- Note, part 1: Canada's Aboriginal populations
Block B: Lab 105 activites
1. Please complete the activity by entering the data into the table. Once complete, please print your work and add it to your notes.
2. Please complete the activity by adding data to the chart and creating your own population pyramids. You may wish to use MS Excel or other software to create the graphs . Once complete, please print your work and add it to your notes
Population Pyramids:Demographics graph activity _cgc1d.doc
These Rates & Pyramids activities are to be completed by the end of class on Friday, April 13th. You will have an additional half period on Friday to complete these tasks.