Responses of arbuscular mycorrhizal fungal communities to soil core transplantation across Saskatchewan prairie climatic regions

Abstract: No information exists on the susceptibility of arbuscular mycorrhizal fungal (AMF) communities in Canadian prairie agriculture soils to climate change. An experiment was initiated in mid-May 2011 in which replicated soil cores were transplanted reciprocally from four cultivated prairie sites in Saskatchewan, Canada, representing different regional climatic zones ranging from semiarid to subhumid regional climates, such that replicated (n = 4) soil cores from each site were present at all sites. Field pea was grown in all cores and at harvest in early-September 2011; soil samples were collected to analyze the changes of AMF communities over the cropping season. A total of 82 operational taxonomic units belonging to eight AMF genera were identified using 18S rRNA gene pyrosequencing. When soils were transplanted to new environments, the relative abundance of AMF changed considerably. Typically, Shannon diversity declined when soil cores were transplanted to new environments. We present evidence that with the altered climatic conditions following transplantation of soil cores, the relative abundance of AMF was significantly altered, and some taxa were enhanced, suppressed, or disappeared in the home-away soils compared with home-site soils. This study implies that the future climate change effects on AMF may impact specific phylogenetic taxa differently, such that rare species or those with low abundance may increase or decrease with unknown consequences. Understanding the potential responses of AMF communities to soil–climate interactions is important when considering the impacts of climate change on soil microbial communities.


Introduction
Arbuscular mycorrhizal fungi (AMF) of the phylum Glomeromycota are ubiquitous and abundant soil microorganisms. They establish symbioses with most species of land plants and occur in almost every terrestrial ecosystem (Smith and Read 1997). They play a crucial role in the functioning and sustainability of agroecosystems (Gianinazzi et al. 2010). They have been studied for decades because of their several important roles including nutrient and carbon cycling (Perry et al. 1990;Drigo et al. 2010), plant health (Song et al. 2011), pathogen protection and resistance (Azcón-Aguilar and Barea 1997; Jung et al. 2012), plant fitness in polluted environments (Kaldorf et al. 1999), soil health (Rillig 2004), water uptake (Augé 2001), and plant productivity (Hamel and Strullu 2006).
Recent reports predict that climatic conditions in the prairies will change. It has been suggested that there will be an increase in the frequency of droughts, particularly in the semiarid regions of the prairie provinces, reducing crop yields in these locations (Mkhabela et al. 2011;Bonsal et al. 2012;Dumanski et al. 2015). Little is known regarding the impact of changing climate on the diversity of AMF in prairie soils. However, several studies examined the phylogenetic diversity of AMF communities in different climatic regions in Chernozemic soils of western Canadian cropped and natural ecosystems. For example, Yang et al. (2010) identified some specific AMF sequences, namely Glomus viscosum, Funneliformis mosseae, and Glomus hoi, which were dominant under sufficient soil moisture conditions. The Black Chernozem soils harbored the most diversified AMF communities in cropped soils compared with Brown and Dark Brown soils (Dai et al. 2012). Furthermore, Bainard et al. (2014) demonstrated that the abundance of individual AMF genus such as Funneliformis were correlated differently with soil phosphate levels, in particular, populations of Rhizophagus were significantly shifted to a dominance of Funneliformis taxa with increasing phosphate levels under wheat cultivation. Similarly, a positive correlation was observed between the abundance of Funneliformis operational taxonomic units (OTUs) and soil pH under the semiarid prairie agroecosystem. A better understanding of the impacts of climate change on AMF communities is vital for predicting the long-term impact on sustainable agriculture.
Transplanting undisturbed soils across multiple ecosystems presents an approach to examine the responses of microbial community dynamics and plant-microbe interactions to climate change conditions. For example, Bottomley et al. (2006) found that the reciprocally transplanted soil cores between two high elevation sites had different fungal and bacterial community composition compared with the site of origin and suggested that this might be due to the temperature and soil moisture differences. Other studies showed a small change in atmospheric or soil temperature altered soil mineralization and decomposition processes (Parton et al. 2007;Adair et al. 2008;John et al. 2011) that eventually affected bacterial or AMF communities (Heinemeyer and Fitter 2004). About 20 yr after soil transplantation between two sites approximately 1000 km apart, phylogenetic analysis revealed that bacteria, fungi, and nitrogen (N)-cycling genes were clustered based on the site factors rather than soil characteristics . Moreover, a long-term soil transplant study over three agricultural research stations across warm, cold, and subtropical zones revealed a differential pattern of 16S rRNA bacterial genes and fluctuated OTUs richness along with the cooling and warming conditions (Liang et al. 2015).
Soil transplantation studies are a useful way to evaluate the responses of microbial communities to changing climate conditions (Waldrop and Firestone 2006;Lazzaro et al. 2011;Vanhala et al. 2011;Zumsteg et al. 2013;Zhao et al. 2014). However, the responses of AMF communities to changing climatic conditions remain poorly understood, particularly in agricultural ecosystems of Canada. Thus, this study was initiated to examine potential responses of Canadian prairie AMF communities to changing climatic conditions. We established a soil transplant experiment at four different cultivated sites representing different regional climatic areas ranging from semiarid to subhumid, in Saskatchewan. Our research addresses two hypotheses: (1) soil core transplantation into different climates will alter the abundance and diversity of AMF communities and (2) different AMF phylogenetic groups will show differential sensitivity to soil-climate interactions.
The 30 yr  annual mean temperature and precipitation varied between 1.3 and 4.3°C and 357.4 and 395.8 mm across the four study sites, respectively. The historical weather data for annual and crop season average (May to September) temperature, precipitation, soil classification, and major soil physicochemical properties at each site are described in Tables 1 and 2. The regional data were obtained from Environment Canada (http://climate. weather.gc.ca) and the AAFC metrological databank.

Soil core establishment and experimental design
In May (15)(16)(17)(18)(19)(20)(21)(22) 2011, 16 open-ended aluminum soil cores (20 cm diameter) were pushed into the soil to a depth of 37 cm using a truck-mounted hydraulic press at each of the sites. The undisturbed soil cores were subsequently extracted manually, and four cores from each site were transplanted (randomly) at the home (original) site. The remaining 12 cores from each site were transported to each of the three other sites where four cores from each site were transplanted in two rows 45 cm apart using a completely randomized design (Fig. 1B).

Initial soil sampling
Prior to seeding into the cores, composite soil samples were collected from each site from the 0 to 15 cm depth (approximately 100 g core −1 ) in May 2011 using a JMC Backsaver N-2 soil core (Clements Associates, Inc., Newton, IA, USA). Samples were stored in plastic bags and maintained at −20°C. Soil pH and electrical conductivity (EC) (1:2 soil:water suspensions) (Sparks et al. 1996), soil organic carbon (SOC) (dry combustion using LECO analyzer) (Wang and Anderson 1998), and soil organic matter (SOM) (Walkley and Black 1934) were performed in the Central Soil Science Laboratory at the College of Agriculture and Bioresources, University of Saskatchewan, and other soil chemical analyses (mineral nutrients) were performed at the ALS Environmental Laboratory, Saskatoon, Canada, and summarized in Table 2.

Seeding in soil cores
Field pea (Pisum sativum L. 'CDC Meadow') was seeded into the soil cores (Fig. 1C) as the pea plant showed good  Barry (2013). (B) Experimental layout of the transplanted soil cores at Swift Current site. (C) Field pea growing inside the soil core during the study season. arbuscular mycorrhizal root colonization (Jin et al. 2013a), inoculated with rhizobia at the recommended rate (equivalent to 3 mL kg −1 seed N-Prove® containing Rhizobium leguminosarum bv. viceae, 5.0 × 10 8 viable cells g −1 inoculant, Novozymes BioAg, Saskatoon, SK, Canada) was hand seeded into the soil cores at a depth of 4 cm and thinned to three pea seedlings per core on emergence. Weeds were controlled by hand three to four times during growing seasons. At seeding, 650 mL of water were applied to each core. Seeding and harvesting in 2011 were performed on 9 June and 10 Sept. at the SW site, 8 June and 7 Sept. at the OL site, 6 June and 5 Sept. at the ST site, and 7 June and 9 Sept. at the MF site.
AMF trap culture with field core soil samples At maturity, field pea was harvested by hand, and soil samples (ca. 200 g) were retrieved from each core to 15 cm using an alcohol-sterilized soil probe. The seasonal decomposition of fine roots prevented collection of consistent root samples from the different soil cores for AMF community analyses. Consequently, we elected to use an AMF trap culture with field pea as the host plant grown in the soils collected at harvest for this purpose. Field moist soils were passed through a 4 mm sieve and then mixed (1:1 w/w) with sterilized (autoclaved three times at 120°C for 2 h) sand (crystalline silica in the form of quartz, fine-grained particle size: 0.13-0.20 mm, Microcrystalline Silica CAS, Unimin Corp., Portage, WI, USA), and 400 g of each soil-sand mix were placed in replicated (n = 4) 750 mL pots lined with plastic. Surface-disinfected pea seeds (two plants per pot) were seeded in each pot. The trap cultures were maintained in a growth chamber with ambient day/night temperatures of 24/18°C with 16 h day lengths for 8 wk.
At 8 wk, roots were harvested, thoroughly rinsed in tap water, and then washed with deionized water to remove any residue soil particles and debris and blotted dry. The cleaned roots were immediately immersed in liquid N and preserved at −80°C until molecular analysis.
DNA extraction, polymerase chain reaction library preparation, and 454 pyrosequencing platform DNA was extracted from the AMF trap root using the Qiagen Plant DNeasy kit (QIAGEN, Mississauga, ON, Canada) according to the manufacturer's recommended protocol. About 100 mg of freeze-dried pea root tissue was placed in a 2 mL screw-top micro-centrifuge tube with 5 mm ceramic beads and pulverized to a powder using a Precellys® 24 tissue homogenizer (Bertin Technologies, Rockville, MD, USA). Pure genomic root DNA was eluted in Tris-EDTA buffer for further analysis.
A nested polymerase chain reaction (PCR) protocol was used to amplify an ∼800 bp partial fragment of AMF 18S rRNA gene for 454 pyrosequencing (Jin et al. 2013b;Bainard et al. 2014). The universal eukaryotic primers NS1 and NS4 (White et al. 1990) were used in the first round of PCR, followed by AMF-specific primer pair AML1 and AML2 (Lee et al. 2008). The forward primer (AML1) and reverse primer (AML2) also included tags CS1 and CS2 (Fluidigm Corp., San Francisco, CA, USA) that were anchors in a third PCR reaction adding Titanium multiplex identifiers (MIDs) and Lib-L adaptors sequences (Supplementary Table S1 1 ).
The first PCR conditions were as follows: initial denaturing step at 95°C for 15 min; 30 cycles at 95°C for 30 s; 50°C for 30 s; 72°C for 1 min 30 s; and a final extension step at 72°C for 3 min, with a 5 μL reaction volume including 1 μL of 1/10 diluted DNA template, 1 mmol L −1 dNTPs, 0.4 μmol L −1 each primer (NS1 and NS4) and FastStart High Fidelity (Roche, 04 738 292 001). About 5 mL of the reaction mixture in the second round of PCR included FastStart High Fidelity, 1 μL of 1/10 diluted first PCR product, and 0.4 μmol L −1 of each primer (AML1-CS1F and AML2-CS2R). The conditions for the second round of PCR were as follows: initial denaturing step at 95°C for 15 min; 33 cycles at 95°C for 30 s; 60°C for 30 s; 72°C for 1 min 30 s; and the final extension step at 72°C for 5 min.
The third PCR was performed to incorporate 10 nt MIDs (Titanium Lib-L forward-MDs-CS1 and Titanium Lib-L reverse adaptor-CS2) and contained 0.5 μL of PCR, 1 μL of 2 μmol L −1 barcodes, 0.5 μL of dimethyl sulfoxide, 0.1 μL FastStart High Fidelity, and 0.2 μL of 10 mmol L −1 dNTP. The third PCR conditions were initial denaturing step at 95°C for 10 min; 15 cycles at 95°C for 15 s; 60°C for 30 s; 72°C for 1 min; and a final extension step at 72°C for 3 min.
All final PCR products were run on 2% agarose gel and quantified using PicoGreen®. Samples were combined into pools of 64 samples based on their MIDs. Each pool was purified with three AMPure XP (Agencourt/ Beckman Coulter, Life Sciences Division, Indianapolis, IN, USA) protocols (ratio 0.5), and quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). The samples were run on an Agilent 2100 Bioanalyzer using a high-sensitivity DNA kit to confirm the size and quality of amplicons. Finally, unidirectional sequencing was performed in half region runs for each pool on a GS-FLX+ system (454 Life Sciences, Branford, CT, USA/Roche Applied Science, Penzberg, Germany) at McGill University, Montréal and Génome Québec Innovation Centre (https://genomequebec.com), Quebec, Canada.

Bioinformatics of AMF sequences
The raw pyrosequencing reads were processed using MOTHUR pipeline version 1.31 (Schloss et al. 2009) to clean ambiguous nucleotides (average score of quality <30) (Huse et al. 2010). The excessively long and short homopolymers, low-quality, and chimeric sequences were removed from the dataset. The average 700-800 bp long 18S rRNA gene sequences were targeted for downstream analysis. The clean sequences were aligned against Silva eukaryotic reference sequences (http:// arb-silva.de/). The reference sequence files were downloaded from public UCHIME domain (Edgar et al. 2011). Non-Glomeromycota sequences were also removed from the dataset. The representative sequences clustered into OTUs, based on 97% similarity, were performed using CD-HIT Suite (http://cd-hit.org).

Phylogenetic analyses of AMF OTUs
For taxonomic identification, the representative sequences of each AMF OTU were compared with GenBank reference sequences using BLAST search against NCBI nucleotide collection database (http:// blast.ncbi.nlm.nih.gov/Blast.cgi). The OTUs and GenBank reference sequences (97% or above similarity) were aligned using ClustalW (Tamura et al. 2013). The neighbor-joining phylogenetic reconstruction (Saitou and Nei 1987) was used to build a phylogenetic tree using MEGA version 6. The tree was constructed using 82 OTUs and 45 GenBank reference sequences to group into representative AMF genera and species. The bootstrap replication method was set at a confidence level of 1000 with the Kimura 2-parameter model (Hall 2004). The systematics of AMF taxa was used as the classification of Schüßler and Walker (2010) and identification of some Glomus spp. (Oehl et al. 2015).

Shannon diversity index of AMF communities
Species diversity in an ecosystem can be estimated using a variety of indices (Bonilla-Rosso et al. 2012). Our study focused on the effect of the environmental change on species abundance, diversity, and composition including low (rare)-abundant species. The Shannon diversity index (SDI: H) accounts for both abundance and evenness/equitability (EH) of the OTUs belonging to specific phylogenetic taxa and sensitive to rare species present in the samples (Morris et al. 2014). The SDI formula refers to the proportion of species (i) relative to the total number of species (pi) and then multiplied by the natural logarithm of this proportion (lnpi) (DeJong 1975;Brodie et al. 2003). The resulting value is summed across species and multiplied by −1. The list file (number of observed OTUs) generated after clustering OTUs in MOTHUR bioinformatic pipeline was used as the total number of species (pi) to formulate SDI.

Data analysis
For the estimation of percent relative abundance (PRA) of AMF sequences, the number of absolute sequence reads (ASR) present in each sample OTU was divided by the total number of ASR from the total OTUs in that sample and multiplied by 100 (Amend et al. 2010). Linear mixed models of analysis of variance using PROC MIXED in SAS (version 2.0.5) was applied to obtain P values for the two fixed effects (soil and site) on the PRA of eight AMF genera (Funneliformis, Glomus, Rhizophagus, Septoglomus, Diversispora, Claroideoglomus, Archaeospora, and Paraglomus), three dominant OTUs (OTU65, OTU5, and OTU42), and Shannon diversity index of AMF communities. Replication and randomization of soil cores within environments and their interactions considered as random effects. Soil, site, and soil × site interactions were the fixed effects, and this model separated random errors (type III) from the two fixed effects. Prior to statistical analysis, PRA data were subjected to a normality test to ascertain skewness and kurtosis and were transformed (i.e., square root and arcsine) when required. The original (home-site) soil core was compared with the home-away soil cores (same soil transplanted into other three sites) and mean separation was performed using Tukey's honestly significant difference test at P ≤ 0.05.
Permutational multivariate analysis of variance (PerMANOVA) using PC-ORD version 6.0 (McCune and Mefford 1999) was applied to test the significance of soil and site (categorical variables/predictors) on the relative abundance and composition of AMF taxa. To visualize the difference in AMF communities among transplanted soil cores across the four sites, a distance matrix of AMF in various soil cores was ordinated using the principal coordinate analysis (PCoA). The Bray-Curtis distance algorithm and "slow and thorough" autopilot mode were used for this analysis in PC-ORD version 6.0. A biplot PCoA analysis was performed with the whole data set (soil, climate/site, and AMF) as the soil-climate variables were included in the second matrix, and relative abundance of eight AMF genera were arranged in the main matrix to identify the magnitude of the changes in AMF and environmental variables. The two most informative dimensions (axes 1 and 2) of the three-dimensional solution were used to construct the PCoA graph.

Submission of nucleotide sequences
The sequences representing 82 AMF OTUs analyzed in this study was deposited in the GenBank submission portal (https://submit.ncbi.nlm.nih.gov/subs/) under the accession numbers SUB6280397 (MN428685-MN428766). The OTUs are also listed in the Supplementary Table S4 1 .

Climate and soil variation across the study sites
According to historical  weather data, the ST Dark Brown and MF Black soil zones experienced relatively high amounts of precipitation and low temperatures (Table 1). In comparison, SW Brown and OL Dark Brown sites were drier and warmer. The average temperature and precipitation during the study period (May to September) were considerably increased across the four study sites compared with the last 30 yr cropping season average temperature and precipitation (Table 1). The MF site had higher SOM and SOC compared with SW, OL, and ST (Table 2). The soil pH was relatively lower in ST compared with SW, OL, and MF.

Pyrosequencing and identification of AMF communities
The DNA extractions from replicated 64 pea trap root samples were amplified successfully using nested PCR protocols. The universal fungal (NS1/NS4) and AM fungal (AML1/AML2) specific primer pairs were deemed to have good mycorrhizae specificity with an average of less than 19% of the sequences from non-Glomeromycota (data not shown). A total of 19 799 AMF sequence reads were obtained from 18S rRNA pyrosequencing platform grouped into 82 OTUs representing eight AMF genera (Fig. 2). For the identification of these OTUs, phylogenetic analyses with 45 GenBank AMF reference sequences were performed. Forty-seven OTUs clustered to species belonging to Glomeraceae family (27 OTUs to Glomus sp., 12 OTUs to Funneliformis sp., four OTUs to Septoglomus sp., and four OTUs to Rhizophagus sp.), 12 OTUs to Paraglomus sp., 11 OTUs to Claroideoglomus sp., 11 OTUs to Archaeospora sp., and one OTU to Diversispora sp. (Figs. 2 and 3).
Transplanting soil cores into the four different climate sites caused a substantial shift in the AMF communities (Fig. 3). For example, Archaeospora was detected in the MF soil at the original MF site, but it was not detected when the MF soil was transplanted to SW and OL sites. Similarly, both Archaeospora and Septoglomus were found in the ST soil but were not detected when ST soil was transplanted to SW. In contrast, some taxa that were not detected at the site of origin were detected when soil cores were transplanted to other sites. Diversispora, for example, was undetectable in SW soil at the original SW site but was detected in the SW soil transplanted at the OL site. Similarly, Septoglomus was absent in OL soil at the original OL site and was only detectable in the OL soil transplanted to the ST site.
An inconsistent shift in the PRA of the dominant AMF taxa (e.g., Funneliformis, Glomus, Claroideoglomus, and Paraglomus) was observed when home-site (original) soil cores were transplanted to new climatic ecoregions (Fig. 3). The genus Funneliformis in SW soil (32% PRA) at semiarid SW site shifted when SW soil was transplanted to subhumid MF and ST sites, accounting for 52% and 68% of the taxa, respectively. Similarly, a substantial increase of taxa belonging to the genus Glomus was detected when SW soil (6% PRA) was transplanted to the other three sites, increasing at OL (24%), ST (18%), and MF (14%). In contrast, a drastic reduction of the genus Rhizophagus was detected when MF (15% PRA) soil was transplanted to ST (3%), OL (4%), and SW (11%) sites. Similarly, the genus Claroideoglomus in SW soil at the SW site accounted for 53% of the taxa; when SW soil core was transplanted to ST and MF sites, the proportion of Claroideoglomus was reduced to 32% for both sites. The Paraglomus community shifted greatly both in the original and new environments. For instance, Paraglomus was 8% in MF soil at the site of origin (MF) but was Fig. 2. Phylogenetic analysis of 82 arbuscular mycorrhizal fungi operational taxonomic units (OTUs). The OTUs are clustered into eight genera of the Glomeromycota, namely Funneliformis sp., Septoglomus sp., Rhizophagus sp., Glomus sp., Diversispora sp., Claroideoglomus sp., Archaeospora sp., and Paraglomus sp. Phylogenetic relationships were obtained by neighbor-joining analysis (Kimura 2 parameter model) of AMF 18S rRNA gene pyrosequencing. Forty-five GenBank reference sequences with accession number within parenthesis are indicated by the black boxes and the OTUs by the red boxes. Tree branches contain the statistical distance frequency values which are greater than 30. undetectable in MF soil transplanted to the ST site, and accounted for only 1% of the taxa at the OL site. However, the abundance of Paraglomus was enhanced from 8% to 13% in MF soil at the SW site.
PerMANOVA analysis revealed that the soil (P < 0.0001) and local climate (site) (P = 0.0030) had a significant impact on the composition of the AMF communities, and the effect of soil was more pronounced than site and soil-site interactions on the composition of AMF taxa (Supplementary Table S3 1 ). These effects were also supported by the PCoA analysis (Fig. 4). The ordination plot revealed that there was a clear difference in AMF abundance among the soils (distance measured between the same soils at different sites and different soils at their home-sites) in response to soil-climate interactions. The soils (either same or different soil cores) had no distinct clusters and were scattered along with PCoA axes 1 and 2 which explained 55% and 32% variation, respectively. The greatest dissimilarity in AMF community abundance and composition along with the axis 1 was exhibited between SW soils followed by OL, ST, and MF soils. Relatively higher dispersion of AMF among the four different soil types (home-site cores) along with the axes 1 and 2 was plotted. The PCoA biplot ordination clearly showed that the soil (pH and EC) and climate (temperature and precipitation) variables had a strong influence on the changes in AMF taxa across the soils and sites (Fig. 4). The direction and length of the arrows (variables) revealed that changes in AMF were increased with the increasing geographical temperature and SOM along with semiarid (SW and OL) to subhumid (ST and MF) regions. The scaling of AMF (genera) points within the PCoA plot showed that the relative abundance of certain taxa responded strongly to certain soil and site conditions. For example, Claroideoglomus was dominant in the higher temperature region (i.e., SW site), Septoglomus, Paraglomus, and Rhizophagus were associated with higher SOM sites (i.e., MF and ST), and Funneliformis was frequently distributed regardless of soil and site characteristics (Figs. 3 and 4).

AMF (OTUs) abundance
Three of the dominant OTUs, namely OTU65, OTU5, and OTU42 shared above 98% similarity with GenBank reference sequences for Funneliformis (accession No. MH629488.1), Dominikia (accession No. GU059535.1), and Claroideoglomus (accession No. KX879058.1), respectively, and were frequently distributed across the soils and sites (Fig. 5). The relative abundance of these three OTUs was significantly changed in response to soil transplantation across the semiarid to subhumid regions (Table 3). The PRA of OTU65 (Funneliformis sp.) was significantly (P ≤ 0.05) increased when original (home-site) soil core was transplanted to a new environment (Fig. 5A). In contrast, the PRA of OTU42 (Claroideoglomus sp.) was significantly (P ≤ 0.05) reduced when the home-site soil was exposed to new climatic regions regardless of site characteristics (Fig. 5C). Interestingly, the shift in abundance of OTU5 (Dominikia sp.) was less predictable and depended on which soil type was moved to which climatic region, specifically, dry soils (i.e., SW and OL) moved to wetter sites (i.e., ST and MF) had a significant (P ≤ 0.05) increase of the relative abundance of OTU5 (Fig. 5B). In contrast, moving wetter soils (i.e., ST and MF) to the dry sites (i.e., SW and OL) had a drastic reduction of the abundance of OTU5 (Fig. 5B).

AMF community diversity
The impact of soil and regional climates on AMF community diversity was assessed using Shannon's diversity index (H). According to PROC MIXED, the diversity of AMF communities across the semiarid to subhumid prairie regions were significant in response to soil type (P < 0.0001), site climate (P = 0.0032), and their interactions (P < 0.0001) ( Table 3). The Shannon diversity indices showed that MF (H = 3.8) soil had the greatest diversity of AMF taxa, whereas ST (H = 2.8) and OL (H = 2.6) soils harbored a moderate diversity, and a relatively low diversity was observed in SW (H = 2.5) soil (Fig. 6).
Transplanting soils to new environments had a significant impact on AMF diversity. The MF soil (H = 3.8) at the MF site (home-site) had higher diversity index than the other three soils (SW, OL, and ST), but the levels of diversity were significantly (P ≤ 0.05) reduced when MF soil was transplanted to other sites (Fig. 6). For instance, the diversity in MF soil at SW site was reduced to less than half (from 3.8 to 1.7) compared with MF soil at the MF site. Similarly, diversity levels were nearly double (from 1.4 to 2.5) in SW soil at SW site compared with the SW soil at MF site (Fig. 6).
When the soils were transplanted to a new climate zone from the site of origin (home-site), the diversity index was significantly (P ≤ 0.05) reduced (Fig. 6). This trend was consistent for all four home-site soils transplanted to the other three sites. However, OL soil at SW and MF sites, SW soil at ST and MF sites, and MF soil at OL and SW sites had no significant changes in diversity.

Discussion
The 30 yr average  temperature and precipitation varied between 14.3 and 15.4°C and 47.9 and 53.0 mm across the study sites, respectively (Table 1). Both SW and OL represent semiarid climates, with lower annual precipitation and higher temperatures than the subhumid sites (ST and MF). In the 2011 study year, both SW and ST experienced higher than average precipitation and temperature, whereas MF experienced a relatively dry growing season. However, SW had 1.4°C higher temperature and 17.7 mm lower precipitation than MF site during the cropping season. Table 3. P values from the PROC MIXED analysis of variance (SAS) for the effects (fixed) of soil and site on the relative abundance a of eight arbuscular mycorrhizal fungi (AMF) genera, three dominant operational taxonomic units (OTUs), and Shannon diversity index of AMF communities. Note: Fixed effects of soil and site were extracted by separating random effects due to replicates within environments and their interactions with fixed effects.
a Relative abundance termed as percent relative abundance of the sequences (18S rRNA) of the eight AMF genera distributed across the soils and sites.
b Effect: "Soil" refers to the different soil cores used in this study transplanted to the four sites and reflects the impact of differing soil characteristics.
"Site" refers to the experimental sites to which the different soil cores were transplanted and reflects the soil cores exposed to different local climates.
c Shannon diversity (combination of abundance and evenness) was determined through the MOTHUR bioinformatics pipeline using the observed AMF OTUs (list file).
These four experimental sites provided a suitable range of soil and environmental conditions to examine the responses of the AMF communities to the differences within relatively short distances of prairie agroecological zones. Others have reported that small changes in atmospheric or soil temperature and moisture can influence microbial processes such as SOM decomposition (Adair et al. 2008;Parton et al. 2007). In the current study, SOM increased along the ∼450 km apart reflecting the wetter and cooler environments at both MF and ST compared with other the two more southerly sites (i.e., SW and OL) (Les Fuller 2010; Anderson and Cerkowniak 2010) (Table 1). Soil fertility, which is largely influenced by the turnover of organic matter decomposition and mineralization, has been linked to levels of AMF root colonization (Smith and Read 1997). The results of this study showed that the AMF abundance and diversity was higher in the cooler and wetter sites (MF and ST) compared with the drier and warmer sites (SW and OL).
Our pyrosequencing data are consistent with the previous reports of AMF diversity of prairie AMF communities. For instance, Dai et al. (2012) identified 33 dominant AMF OTUs using pyrosequencing, representing Funneliformis, Rhizophagus, Claroideoglomus, and Diversispora from 76 wheat fields across Dark Brown, Black, Dark Gray Chernozems, and Gray Luvisol in the Canadian prairies. Similarly, the 51 AMF OTUs in pea roots using MiSeq-Illumina were classified into four genera, namely Rhizophagus, Glomus, Claroideoglomus, and Funneliformis from the four experimental sites representing Brown, Dark Brown, and Black Chernozems (Islam et al. 2014). Others used denaturing gradient gel electrophoresis (DGGE) (Ma et al. 2005) and spore morphology (Talukdar and Germida 1993) analyses to reveal a higher abundance of Glomus, Acaulospora, Gigaspora, and Scutellospora in prairie soils, but in this study, the latter three genera were not detected.
Consistent with our hypotheses, soil transplantation significantly altered the abundance, composition, and diversity of AMF community, and some AMF groups (Archaeospora, Diversispora, Paraglomus, Rhizophagus, and Septoglomus) showed strong sensitivity to soil transplantation (Fig. 3), although, these AMF taxa were either low in abundance or undetectable in some soils among the transplanted soil cores. The differences in AMF taxa due to exposure to different environments suggest that the AMF were adapted to the home environment and transplanting the soil cores ultimately caused a significant shift of AMF communities, presumably due to conditions that either stressed or promoted some taxa over others. Gai et al. (2012) also found that soil transplantation induced a significant shift in AMF communities under contrasting geographical elevation gradients and the AMF diversity (species richness) and root colonization decreased with increasing altitude. Other reports showed that soil transplantation from north to south boreal forest zones (Vanhala et al. 2011) and California oak to grassland ecosystems (Waldrop and Firestone 2006) resulted in a loss of microbial biomass, changes in the composition of fungal and bacterial communities, and altered functionality in response to warmer climatic conditions. However, inconsistent responses of soil fungal diversity and composition to environmental elevation frequently have been reported (Lugo et al. 2008;Margesin et al. 2009).
In this study, three AMF taxa, namely Funneliformis (12 OTUs), Glomus (27 OTUs), and Claroideoglomus (11 OTUs), were abundant at all study sites. Of these taxa, the PRA of Funneliformis and Glomus were increased by more than double and Claroideoglomus decreased by nearly double in the SW (warm and dry site) soil cores transplanted into MF and ST (wet and cool) sites (Fig. 3). This AMF shift indicates that the transplanted AMF communities either experienced stress or acclimated and adapted to the new environment. It is likely that edaphic factors, such as rapid fluctuation of soil moisture levels due to differences in temperature gradient, affected the AMF communities. Numerous reports revealed a significant alteration in soil microbial communities due to soil moisture changes (Schimel et al. 1999;Chen et al. 2007;Williams 2007). Moreover, the differences in the abundance of AMF genera in the transplanted soils strongly suggest that the observed alteration was driven by the regional climate gradients associated with the latitudinal locations.
Taxa with low abundance such as Archaeospora, Diversispora, Paraglomus, Rhizophagus, and Septoglomus were detected in home climates but were undetectable when transplanted to other climates (Fig. 3) resulting in a significant reduction in Shannon diversity indices in the soils transplanted to other sites (Fig. 6). The reduction in diversity indices in the soil cores transplanted into the new environment, regardless of whether the sites were warmer or cooler, was observed for all four soils used in this study. This indicates that the shift in AMF likely reflects a "home-site" advantage rather than climatic preference based on moisture availability or warming condition, signifying the potential local adaptation impact on AMF communities under environmental stress gradient conditions (Thrall et al. 2007;Yang et al. 2018). However, little is known about the adaptation strategy of AMF communities under multiple environmental gradient interactions (Johnson et al. 2013).
The above changes in abundance and diversity of AMF taxa were not consistent across the four experimental sites. We observed an interesting shift in the AMF taxa with low abundance. Some taxa were present in the transplanted soils but were not detected in the soils at the original site suggesting that the home-site advantage may not be preferable to all AMF taxa (Fig. 3). Here, a distinct pattern emerges in which other inherent plant-AMF interaction factors such as AMF-soil compatibility for effective plant-AMF symbiosis may be involved along with the above-mentioned local-site adaptation (Helgason and Fitter 2009;Herrera-Peraza et al. 2011;Yang et al. 2016). The AMF taxa "presence versus absence" is apparently due to spore germination and hyphal growth or spore dormancy under modified soil environmental condition or could reflect potential movement of AMF spores and hyphae (water-or air-borne inoculum) into the transplanted cores. Some AMF species, particularly lower abundance or rare species, exhibit a strong dependency on explicit environmental conditions (Van der Heijden et al. 1998;Klironomos 2000Klironomos , 2003Macel et al. 2007).
The OTU22 belonging to Diversispora sp. was detected only in the three soil cores out of 16 (Fig. 3). We anticipated that the lower detectability of this species might be partially associated with the higher number of PCR cycles (63 cycles in two round PCR), required to eliminate incorrect size fragments using universal fungal (NS1 and NS4) and AMF (AML1 and AML2) primer sets. A previous study harvested AMF amplicons with 50 cycles with the similar primers under pyrosequencing platform (Bainard et al. 2014). Several studies recommend minimizing the number of PCR cycles in DNA library preparation to limit the possibility of formation of artifacts, chimeras, single-stranded DNA molecules and lowering the chance of amplification of the rare or low-abundant sequences (Qiu et al. 2001;Bonnet et al. 2002;Schmidt et al. 2013). In contrast, the effect of cycle numbers had a minor importance in the microbial community analysis from environmental samples even at a high number of PCR cycles (Lueders and Friedrich 2003;Acinas et al. 2005), and no effects on the Shannon diversity indices were observed with higher cycle numbers (Sipos et al. 2007). This single crop season data demonstrates the usefulness of the soil transplantation technique as a tool to investigate the impact of soil-climate alterations on AMF communities in cropping ecosystem across Saskatchewan. We did find an indication from the PCoA analysis that the changes in AMF communities are susceptible to two major factors, SOM and atmospheric temperature followed by soil pH and precipitation (Fig. 4). This is also apparent in three dominant OTUs, which showed a preferential shift based on site and soil factors (Fig. 5). For example, Funneliformis (OTU65) responded positively when exposed to new environments, no matter if it was within a dry or wet climate (Fig. 5A). The observed abundance of Funneliformis was consistent with several other studies where Funneliformis, specifically F. mosseae, was found abundantly distributed across the prairie regions and the world, regardless of climatic variations (Avio et al. 2009;Rosendahl et al. 2009;Dai et al. 2013). An opposite trend was observed in Claroideoglomus (OTU42) that responded negatively to new environments (Fig. 5C). However, the genus Dominikia (OTU5) exhibited strong site selectivity. For example, dry soils (i.e., SW and OL) moved to wetter sites (i.e., ST and MF) significantly increased relative abundance and wet soils moved to dry sites significantly decreased relative abundance of OTU5 (Fig. 5B). This result confirmed a preferential response of different AMF clades to certain environmental conditions. A previous study on prairie cropping systems also showed that the specific group of AMF shifted along with the successive fluctuation of soil chemical properties during the cropping season (Bainard et al. 2014). Responses to changes in soil-climate variables may vary between phylogenetic identities and growth habit of AMF (fast and slow growers), and thus certain AMF may have adapted contrasting sites to maximize their fitness in "warm or cold" and "dry or wet" environments.

Conclusion
In conclusion, drivers of changes in AMF communities in the soil transplants are not clearly understood. Additional research is required to provide a better picture of AMF community shifts and subsequent functionality under long-term climate change scenarios. In this study, we did not exclude the possibility that some of the observed shifts in AMF communities may be a transient response to the disturbance during excavation and reinstallation of soil core transplantation to the contrasting environmental sites. Furthermore, differences in root colonization detected using AMF trap culture versus field root culture were not compared. Nevertheless, the observed structural changes of AMF communities in this soil transplant study suggest soil microbiomes, especially prairie AMF resources, will change under future climate.