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Arbuscular mycorrhizal fungi composition in soils of switchgrass harvested for bioenergy under 1
alternate nitrogen management 2
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Alden C. Dirks,a#* Randall D. Jackson
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aDepartment of Agronomy, University of Wisconsin-Madison, Madison, Wisconsin, USA
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Running Head: AMF metabarcoding of switchgrass agroecosystems 7
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#Address correspondence to Alden C. Dirks, adirks@umich.edu 9
*Present address: Department of Ecology and Evolutionary Biology, University of Michigan, 10
Ann Arbor, Michigan, USA 11
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Keywords 13
amplicon sequence variants; DADA2; Glomeromycotina; fungal metabarcoding; PacBio Sequel 14
SMRT Sequencing 15
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Abstract 17
Learning more about the biodiversity and composition of arbuscular mycorrhizal fungi (AMF) 18
under alternative agricultural management scenarios may be important to the sustainable 19
intensification of switchgrass grown as a bioenergy crop. Using PacBio single-molecule 20
sequencing and taxonomic resolution to the level of amplicon sequence variant (ASV), we 21
assessed the effects of nitrogen amendment on AMF associating with switchgrass and explored 22
relationships between AMF and switchgrass yield across three sites of varying productivity in 23
AEM Accepted Manuscript Posted Online 24 July 2020Appl. Environ. Microbiol. doi:10.1128/AEM.00880-20Copyright © 2020 Dirks and Jackson.This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
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Wisconsin. Nitrogen amendment had little effect on AMF diversity metrics or community 24
composition. While AMF ASV diversity was not correlated with switchgrass yield, AMF family 25
richness and switchgrass yield had a strong, positive relationship at one of our three sites. Each 26
of our sites was dominated by unique ASVs of the species Paraglomus brasilianum, indicating 27
regional segregation of AMF at the intraspecific level. Our molecular biodiversity survey 28
identified putative core members of the switchgrass microbiome as well as novel clades of AMF, 29
especially in the order Paraglomerales and the genus Nanoglomus. Furthermore, our phylogenies 30
unite the unknown, cosmopolitan, soil-inhabiting clade GS24 with Pervetustaceae, an enigmatic 31
family prevalent in stressful environments. Future studies should isolate and characterize the 32
novel genetic diversity found in switchgrass agroecosystems and explore potential yield benefits 33
of AMF richness. 34
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Importance 36
We assessed the different species of beneficial fungi living in agricultural fields of switchgrass, a 37
large grass grown for biofuels, using high-resolution DNA sequencing. Contrary to our 38
expectations, the fungi were not greatly affected by fertilization. However, we found a positive 39
relationship between plant productivity and the number of families of beneficial fungi at one site. 40
Furthermore, we sequenced many species that could not be identified with existing reference 41
databases. One group of fungi was highlighted in an earlier study for being widely distributed but 42
of unknown taxonomy. We discovered that this group belonged to a family called 43
Pervetustaceae, which may benefit switchgrass in stressful environments. To produce higher 44
yielding switchgrass in a more sustainable manner, it could help to study these undescribed fungi 45
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and the ways in which they may contribute to greater switchgrass yield in the absence of 46
fertilization. 47
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Introduction 49
Arbuscular mycorrhizal fungi (AMF) are ubiquitous soil-dwelling organisms from the 50
phylum Mucoromycota, subphylum Glomeromycotina, that form symbiotic relationships with 51
upwards of 80% of vascular plant families (1–2). AMF can confer many benefits to their hosts 52
including pathogen protection, drought tolerance, and water acquisition, but principal among 53
them is the uptake of mineral nutrients. The AMF mycelium mines nitrogen (N), phosphorus (P), 54
and other nutrients from the soil and transfers them to host plants via intraradical structures 55
called arbuscules. In exchange, host plants share photosynthetically fixed carbon (C) with the 56
AMF (3–4). In recent years, global interest in sustainable agricultural intensification has brought 57
AMF to the forefront as natural biofertilizers to offset synthetic fertilizer application. However, 58
knowledge gaps concerning the effects of agricultural management (e.g., N fertilization) on 59
AMF hinder farmers’ ability to leverage “mycorrhizal technology” for sustainability and 60
productivity goals (5–7). 61
To better understand the effects of N addition on AMF biodiversity and composition, we 62
assayed the mycorrhizal communities associating with switchgrass (Panicum virgatum L.) in N-63
amended and unamended stands in Wisconsin. Switchgrass – a warm-season plant endemic to 64
the tallgrass prairies of North America – is cultivated as a cellulosic biofuel feedstock and forage 65
crop for cattle (8). Switchgrass agroecosystems can provide a number of important ecosystem 66
services such as soil and nutrient retention that make it an attractive option for low-carbon-67
economy agricultural landscapes (9). However, switchgrass cultivation is hampered in part by 68
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the highly context-dependent relationship between N addition and plant growth (10). While a 69
global meta-analysis reported that N fertilization significantly increases switchgrass growth (11), 70
N amendment did not increase switchgrass aboveground net primary productivity compared to 71
unamended plots in a two-year agronomic trial in Wisconsin (12). Similar results were found by 72
Jach-Smith and Jackson (13), Ruan et al. (14), Wang et al. (15), and Emery et al. (16). Duran et 73
al. (12) hypothesized that this variation in switchgrass response is a function of microbial N 74
immobilization. Since switchgrass is highly mycorrhizal-dependent (17) and AMF are sensitive 75
to soil stoichiometry and agricultural intensification (18–20), increasing our knowledge of AMF 76
in these agroecosystems could help explain variation in switchgrass response to N addition (6). 77
While switchgrass generally benefits from N supplied by AMF (21), exogenous N inputs 78
can decrease the abundance and functioning of AMF, potentially negating their symbiotic 79
benefits (22). Jach-Smith and Jackson (23) found that N amendment decreased AMF root 80
colonization of switchgrass and allocation of resources to nutrient-transfer structures. In a 81
follow-up study, Jach-Smith and Jackson (24) showed that N addition effectively replaced N that 82
otherwise would have been supplied to switchgrass by AMF. Conversely, in a microcosm 83
experiment, elevating N did not influence mycorrhizal-mediated N uptake and transfer to 84
switchgrass (25). Moreover, Emery et al. (26) found that N amendment did not affect AMF root 85
colonization of switchgrass or extraradical hyphae, but it slightly decreased AMF operational 86
taxonomic unit richness and Shannon diversity index. Considering these conflicting findings, it is 87
difficult to discern a general pattern in the effects of N addition on AMF associating with 88
switchgrass (27). 89
Part of the uncertainty around the effects of N addition on AMF and switchgrass 90
productivity could stem from the limited taxonomic resolution of traditional methodologies used 91
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to characterize AMF, which fail to detect certain clades while masking the extensive functional 92
diversity present among AMF after over 500 million years of evolution (28). For example, AMF 93
staining procedures to measure root colonization and nutrient-transfer structures do not detect 94
AMF in the order Paraglomerales, which have weakly staining or completely non-staining 95
tissues (29–30). Furthermore, morphological metrics have no inherent meaning for plant 96
performance given that AMF species exist along a symbiotic spectrum ranging from mutualism 97
to parasitism (31–32). In fact, alternative strains within a given AMF species can result in 98
completely different outcomes for plant performance (33–35). In addition, the 16:1ω5cis fatty 99
acid biomarker commonly used to measure AMF abundance in soils (36–37) is only present at 100
miniscule quantities or is completely absent from species in Paraglomeraceae and 101
Gigasporaceae (38). On the other hand, fatty acid indicators for these two families (e.g., 102
16:1ω7cis, 18:1ω9cis, 20:1ω9cis) are typically treated as biomarkers for saprotrophic fungi and 103
gram-negative bacteria, conflating the abundance of multiple guilds (38–40). Finally, AMF 104
metabarcoding of the small subunit ribosomal DNA (rDNA) gene region with short-read 105
sequencing platforms does not discriminate AMF to the species level (27, 41–42). This problem 106
is compounded when operational taxonomic units are clustered at 97% similarity – a 107
conservative threshold and arbitrary proxy for species-level delimitation of filamentous fungi 108
(43–44). 109
In the context of a long-term, replicated bioenergy cropping systems experiment, we 110
conducted a metabarcoding study of AMF using PacBio Sequel Single Molecule, Real-Time 111
(SMRT) Sequencing (Note S1) and characterized the communities to the level of amplicon 112
sequence variant (ASV) for a high-resolution investigation of the effects of N addition on AMF 113
composition (41). PacBio offers comparatively long sequence reads spanning conserved and 114
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highly variable regions of rDNA for an unprecedented opportunity to investigate AMF at a fine 115
taxonomic resolution (42). ASVs are exact, error-free sequences and thus serve as biologically 116
meaningful representatives of AMF genotypes, revealing intraspecific variation and strains of 117
interest for bioprospecting (9, 43). To our knowledge, this study is the first to employ PacBio 118
Sequel SMRT Sequencing with ASVs in the analysis of AMF communities and one of only a 119
handful to use PacBio for AMF metabarcoding (18, 41, 45–47). We asked the following 120
questions: Does long-term N amendment affect AMF ASV diversity or community structure in 121
Wisconsin switchgrass agroecosystems? Does AMF diversity explain variation in switchgrass 122
yield? We hypothesized that N amendment would decrease AMF diversity (27, 48) and that 123
AMF diversity would be correlated with switchgrass yield because of its positive linkage to 124
ecosystem functioning (3). 125
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Methods 127
Sites and experimental design 128
The US Department of Energy’s Great Lakes Bioenergy Research Center designed a 129
Marginal Lands Experiment to test the efficacy of growing perennial biofuel feedstock crops on 130
abandoned agricultural fields or land deemed unsuitable for high-productivity agriculture. This 131
study focused on three Wisconsin sites: Rhinelander in northern Wisconsin, Hancock in the 132
central part of the state, and Oregon in the south (Fig. S1). The coordinates, soil texture, and 133
mean soil physicochemical properties of the three sites are presented in Table S1; for a 134
comprehensive assessment of the soils at each site, see Kasmerchak and Schaetzl (49), and for 135
information on the ecology and land-use history of each site, see 136
https://lter.kbs.msu.edu/research/long-term-experiments/marginal-land-experiment/. 137
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Monoculture plots of switchgrass (Panicum virgatum var. “Cave-in-Rock”) measuring 19.5 x 138
12.2 m were sown with seed in 2013. In 2015, N amendments were randomly applied to the plots 139
in a paired experimental design with half receiving no N amendment (control) and the other half 140
receiving 56 kg ha-1
N (amended) applied annually as granular ammonium nitrate. Thus, the 141
experimental units in this study were the paired half plots (19.5 x 6.1 m). Rhinelander and 142
Oregon each consisted of four replicates per treatment while Hancock consisted of three 143
replicates per treatment (n = 11). 144
Soil sampling and switchgrass yield 145
In August 2018, a sliding hammer soil corer fitted with a 5-cm diameter head was used to 146
extract soil cores to a depth of 15 cm. Three soil cores spaced 1.8 m from the edge of the plot 147
and 1 m from each other were collected in every experimental unit (Fig. S1). The cores were 148
placed into Whirl-Pak® bags and transported in an iced cooler to a -20 ˚C freezer. Within 8 h, 149
the soil samples were transferred to a -80 ˚C freezer for storage prior to sieving. The soil samples 150
were thawed in a refrigerator at 4 ˚C overnight and manually disintegrated through a 2-mm sieve. 151
The sieved soil was returned to the Whirl-Pak® bags and preserved at -80 ˚C until DNA 152
extraction. Switchgrass yields were determined in November 2018 by harvesting switchgrass 153
with a combine, leaving 15.25-cm residual stubble. Following switchgrass harvest, soil cores 154
from each experimental unit were taken to a depth of 25 cm and analyzed for pH, phosphorus 155
levels, and cation exchange capacity by the University of Wisconsin Soil and Forage Laboratory. 156
Preserved soil samples from August were analyzed with a Flash EA 1112 Flash Combustion 157
Analyzer for total carbon and nitrogen determination. 158
DNA extraction, amplification, and sequencing 159
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DNA was extracted from approximately 250 mg of soil from each sample with Qiagen 160
DNeasy PowerSoil Kit (Qiagen catalog #12888-100) according to the manufacturer’s 161
instructions. DNA concentration was measured with a Thermo Scientific NanoDrop 1000 162
Spectrophotometer to determine dilution ratios for PCR. Extracts were amplified using the AMF-163
specific wobble primers wSSUmCf (5’-TAT YGY TCT TNA ACG AGG AAT C-3’) and 164
wLSUmBr (5’-AAC ACT CGC AYA YAT GYT AGA-3’), which span an rDNA fragment 165
consisting of the partial small subunit locus (pSSU), whole internal transcribed spacer region 166
including the 5.8S locus (ITS), and partial large subunit locus (pLSU; 41, 50). Dried, 12-nmol 167
RxnReady® Primer Pools (forward and reverse primers premixed) were manufactured by 168
Integrated DNA Technologies according to PacBio Sequel specifications, namely HPLC 169
purification and the addition of a 5’ NH4-C6 block and universal sequence: PacBio-specific 170
wSSUmCf (/5AmMC6/ GCA GTC GAA CAT GTA GCT GAC TCA GGT CAC TAT YGY 171
TCT TNA ACG AGG AAT C) and PacBio-specific wLSUmBr (/5AmMC6/ TGG ATC ACT 172
TGT GCA AGC ATC ACA TCG TAG AAC ACT CGC AYA YAT GYT AGA). For more 173
information on the primer-specific requirements for successful PacBio sequencing, see 174
https://www.pacb.com/wp-content/uploads/2015/09/Procedure-and-Checklist-Preparing-175
SMRTbell-Libraries-PacB-Barcoded-Universal-Primers.pdf. 176
PCR reactions were prepared using the Phusion High-Fidelity PCR Kit from New 177
England Biolabs (NEB catalog #E0553L) in 25-µL reactions: 1 µL of template DNA at a 178
concentration of 0.1-40 ng µL-1
, 2.5 µL 10 µM primers, 0.5 µL 10 mM dNTPs, 0.25 µL Phusion 179
DNA Polymerase, 5 µL 5X Phusion HF Buffer, and 15.75 µL water. PCR reactions were run on 180
an Eppendorf Mastercycler® pro S thermal cycler using the parameters specified by Schlaeppi et 181
al. (41): 2 min initial denaturation at 98 °C, 40 cycles of 10-s denaturation at 98 °C, 30-s 182
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annealing at 60 °C, and 1-min elongation at 72°C, with a final elongation of 10 min at 72 ˚C. Of 183
the 66 soil samples, 48 (at least two samples per experimental unit) successfully amplified and 184
were selected for sequencing. Samples were submitted to University of Wisconsin 185
Biotechnology Center and purified using AMPure XP beads, ligated with universal hairpin 186
adapters for circular sequencing, and barcoded with sample-specific primers. The 48 samples 187
were pooled based on molarity and sequenced on one PacBio Sequel SMRT Cell with Sequel 188
Sequencing Kit v3.0 chemistry. 189
Bioinformatics 190
PacBio Sequel subreads were demultiplexed and assembled into circular consensus 191
sequences (CCS) with SMRT Analysis v6.0.0.47841 (lima v1.7.0, pbccs v3.4.1). Sequences with 192
fewer than five passes and a minimum predicted accuracy < 0.9 were removed, resulting in a pre-193
filtering error rate comparable to Illumina MiSeq (41). CCS BAM files were converted to 194
FASTQ format and their quality scores changed to the conventional 0-41 system using BBMap 195
v38.50 (51). CCS were clustered into ASVs with DADA2 v1.14 (52–53) according to the 196
workflow and recommendations of the “DADA2 + PacBio” tutorial (53; available at 197
https://benjjneb.github.io/LRASManuscript/LRASms_fecal.html) on a MacBook Pro (early 2011 198
model) using R v3.6.0 (54). The resulting pSSU-ITS-pLSU ASVs were separated by rDNA locus 199
using ITSx v1.1.2 (55) implemented in the R package rITSx v0.0.3 (56). A multi-locus 200
phylogenetic tree of complete ITS and partial LSU sequences was constructed with Tree-based 201
Alignment Selector (T-BAS) v2.1 on DeCIFR from North Carolina State University’s Center for 202
Integrated Fungal Research (57; accessible via https://decifr.hpc.ncsu.edu/) using auto values of 203
the MAFFT algorithm, the GTRGAMMA substitution model, 100 bootstrap replicates, and 204
denovo RAxML placement. ASVs with ambiguous rDNA loci according to ITSx or falling 205
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outside of Glomeromycotina according to the kingdom-wide phylogenetic tree were excluded 206
from further analysis. Preliminary taxonomic assignments were made using DADA2’s 207
implementation of the naive Bayesian classifier method from Wang et al. (58) and the UNITE 208
v8.0 ITS fungal taxonomic reference (59–60). 209
Two phylogenetic trees specific to AMF were subsequently constructed – one covering 210
the entire Glomeromycotina subphylum and the other limited to the order Paraglomerales – to 211
assign taxonomy and resolve undescribed clades. We manually assembled an LSU rDNA 212
reference database representing 4 of 4 orders, 12 of 12 families, 40 of 41 genera, and 100 of 334 213
species currently described in Glomeromycotina (61–62), including NCBI-designated type-214
material sequences from 14 species and rediscovered, undesignated type-material sequences 215
from 48 other species (Note S2). A further 38 species were represented by non-type-material 216
sequences. In total, 436 ASVs, 282 AMF reference sequences, and two type-material sequences 217
from Mucoromycotina as an outgroup were used in constructing the phylogenetic trees. We 218
aligned our sequences using the E-INS-i iterative refinement method in MAFFT v7 (63) and 219
conducted phylogenetic analysis with RAxML v8.2.11 (64) using the GTRGMMA substitution 220
model and 100 bootstrap replicates. The best-fitting phylogenetic trees were visualized and 221
annotated in iTOL v4 (65). 222
Data analysis 223
All analyses and statistical tests were conducted in R v3.6.0. Agronomic and soil data 224
were averaged across replicates within each experimental unit and ASV abundance was summed 225
across replicates within each experimental unit. For each experimental unit, we calculated the 226
following metrics of AMF alpha biodiversity using vegan v2.5.5 (66) or picante v1.8 (67): 227
richness, Shannon diversity index, Simpson’s diversity index, Pielou’s evenness index, Faith’s 228
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phylogenetic diversity index, and phylogenetic mean pairwise distance (68). We tested the effect 229
of N amendment on switchgrass yield and soil variables using random-intercept linear mixed 230
effects models with lme4 v1.1.21 (69), specifying N treatment and site as interacting fixed 231
effects and paired plot as a random grouping effect. To test for correlations between switchgrass 232
yield and soil variables, we fit separate random-intercept linear mixed effects models for each 233
site to avoid issues of multicollinearity, specifying the soil variables and N treatment as fixed 234
effects and paired plot as a random grouping effect. To determine effects of N amendment on 235
AMF community composition, we first visualized ASV UniFrac beta diversity with non-metric 236
multidimensional scaling (NMDS) ordination using phyloseq v1.28.0 (70). We then conducted a 237
permutational multivariate analysis of variance (PERMANOVA) on UniFrac dissimilarity with 238
vegan, specifying N treatment as the predictor and constraining permutations by site. We also 239
tested for correlations between dissimilarity matrices of AMF community and abiotic soil 240
variables using Mantel tests with vegan, again constraining permutations by site. Finally, we 241
conducted an indicator species analysis on sites and N treatment using indicspecies v1.7.8 (71). 242
We visually checked assumptions of normally distributed errors and homogeneity of 243
variance and transformed variables when appropriate to meet model assumptions. For linear 244
mixed effects models, we used lmerTest v3.1.0 (72) to calculate type III ANOVA p values via 245
Satterthwaite’s degrees of freedom method. Statistical significance was determined by 246
comparison to the Bonferroni critical value of p < 0.002. For post-hoc analyses, we used lsmeans 247
v2.30.0 (73) to obtain least-square means and pairwise linear contrast Tukey-adjusted p values 248
via Kenward-Roger degrees of freedom method. We used MuMIn v1.43.15 (74) to calculate 249
model conditional coefficients of determination (Rc2). For the Mantel tests, statistical 250
significance was set to the Bonferroni critical value of p < 0.008. 251
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Data availability 252
Raw CCS reads are accessible via National Center for Biotechnology Information 253
(NCBI) under the Sequence Read Archive (SRA) BioProject accession number PRJNA590305 254
(BioSamples SAMN13324198-SAMN13324245). Amplicon sequence variants are available 255
through NCBI under GenBank accession numbers MT765295-MT765730. Reproducible 256
bioinformatics scripts and R markdown documents are available at 257
https://github.com/aldendirks/amf_metabarcoding. 258
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Results 260
Taxonomy and phylogeny of ASVs recovered from switchgrass agroecosystems 261
A total of 190,700 high-quality, non-chimeric amplicons clustered into 436 ASVs (for a 262
complete breakdown of the number of sequences remaining after each bioinformatics processing 263
step, see Table S2). Rarefaction curves of ASV accumulation versus sample size reached a clear 264
asymptote for every experimental unit, indicating relatively complete sampling of AMF diversity 265
(Fig. S2). According to UNITE taxonomic assignment, the 436 ASVs were spread across 266
Glomeromycotina, representing all four orders in the subphylum. In addition, 7 of 12 families, 13 267
of 41 genera, and 21 of 334 species that are currently described in Glomeromycotina were 268
recovered. However, almost half of the ASVs (211, 48.4%) could not be assigned to a species 269
hypothesis (Fig. 1) and more than a quarter (119, 27.3%) could not be assigned to the level of 270
genus. While all ASVs were placed in Glomeromycotina, a few (24, 5.5%) were unassignable 271
even at the order level. Manual taxonomic assignment according to phylogeny resulted in the 272
representation of all four orders, 9 of 12 families, 20 of 41 genera, and 17 of 334 species 273
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currently described in Glomeromycotina, as well as an additional 36 well-supported clades of 274
unknown specific identity (Figs. S3–S4). 275
Comparing UNITE taxonomic assignments to our Glomeromycotina phylogenetic tree 276
(Figs. S3–S4) indicated that certain clades, while described in the literature, were not represented 277
in the UNITE database and thus taxonomic assignment of related ASVs consistently failed. Of 278
the 24 ASVs that could not be assigned to any order, 18 were resolved to a Glomerales clade 279
encompassing the recently described species Nanoglomus plukenetiae (75) and the remaining six 280
to the Pervetustaceae clade in Paraglomerales (29). Furthermore, eight ASVs were closely 281
related to described species in Paraglomeraceae or Pervetustaceae in Paraglomerales but were 282
not assigned to any family. Of the 119 ASVs that were not assigned a generic rank, 44 belonged 283
to the clade encompassing Nanoglomus plukenetiae. This non-exhaustive exploration of failures 284
in taxonomic assignment showcases both the biological (e.g., myriad undescribed taxa in the 285
Nanoglomus plukenetiae sensu lato clade) and artificial (e.g., incomplete data curation of 286
Paraglomerales) issues that hinder accurate identification of AMF environmental sequences 287
using even curated reference databases. 288
A few clades stood out for their high sequence abundance or consistent presence across 289
the experimental units, which suggests a role in the core microbiome of switchgrass (Fig. 2; 76). 290
The previously mentioned clade encompassing Nanoglomus plukenetiae included ASVs with 291
some of the greatest absolute abundances (ASVs 1, 4, and 9). While no ASV belonging to this 292
clade occurred at more than one site, representatives of the Nanoglomus plukenetiae sensu lato 293
clade were present in both N-amended and control plots at all three sites (Figs. S3–S4). In total, 294
64 of the 436 ASVs (~15%) belonged to this diverse but poorly understood clade, which may 295
represent various undescribed species in the genus Nanoglomus or new genera within 296
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Glomeraceae. Within Paraglomerales, 73 ASVs (~17%) had high LSU sequence similarity to 297
Paraglomus brasilianum and 51 more (~12%) to Paraglomus laccatum. Since ASVs belonging 298
to these species also occurred at all three sites (Figs. S3–S4), Paraglomus spp. appear to be 299
important members of the core switchgrass microbiome as well. Of the 436 ASVs recovered in 300
this study, only three ASVs occurred at all three sites, two of which belonged to Paraglomus 301
brasilianum (ASVs 8 and 27); the last – ASV 78 – belonged to Microdominikia litorea, another 302
potential member of the core switchgrass microbiome (Fig. 2). 303
In addition to the Nanoglomus plukenetiae sensu lato clade, some of the less-abundant 304
clades identified in this study might be undescribed lineages of AMF. Our phylogenetic analysis 305
showed that the unknown, worldwide, soil-inhabiting clade deemed GS24 (77) corresponds to 306
the family Pervetustaceae in Paraglomerales, which contains one species, Pervetustus simplex, 307
described in 2017 from the deserts of Oman (29). We recovered seven ASVs in three clades 308
belonging to this enigmatic family, two of which were well-supported (Fig. 3). These ASVs were 309
most prevalent in the sandy soils of our Hancock site, indicating they may confer some degree of 310
stress tolerance to switchgrass under water-limiting or nutrient-poor conditions (76). Finally, two 311
ASVs – ASVs 187 and 427 – were phylogenetically resolved as basal to the rest of 312
Paraglomeraceae, suggesting a new genus or a new family in Paraglomerales might be 313
necessary to accommodate this newfound genetic diversity (Fig. 3). 314
Effects of nitrogen amendment on switchgrass yield and soil variables 315
The effect of N amendment on square-root-transformed switchgrass yield depended on 316
site (site × N interaction, F2,8 = 14.04, p = 0.002; Fig. 4). At Rhinelander, N amendment 317
enhanced switchgrass yield compared to unamended control plots (t8 = 5.93, p < 0.01) but had no 318
significant effect at Oregon (t8 = -1.52, p = 0.66) or Hancock (t8 = 1.21, p = 0.82). Log-319
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transformed total P, total N, C:N ratio, ASV Faith’s phylogenetic index, species richness, and 320
square-root-transformed family richness differed significantly by site (Fig. 5) but no soil 321
variables were significantly affected by nitrogen amendment (Table 1). PERMANOVA results 322
indicated ASV UniFrac community dissimilarity was not significantly affected by N treatment 323
(F1,20 = 0.23276, p > 0.80; Fig. 6). However, community dissimilarity was significantly 324
correlated with dissimilarity in soil total N (r = 0.12, p = 0.004). 325
Relationship between soil variables and switchgrass yield, and indicator taxa 326
No metric of AMF ASV diversity was a significant predictor of switchgrass yield at any 327
site in linear mixed effects regression models including N treatment. At the family level, AMF 328
richness was a marginally significant predictor of yield at Oregon (F1,4.92 = 21.3, p = 0.006, Rc2 = 329
0.88); every additional family represented in the AMF community at Oregon was associated with 330
an increase in 0.61 Mg ha-1
of switchgrass yield (Fig. 7). There was also a significant 331
relationship between switchgrass yield and cation exchange capacity at Hancock (F1,3 = 332
157.3865, p = 0.001). Holding N addition constant, every unit increase in cation exchange 333
capacity was associated with an increase in 0.38 Mg ha-1
of switchgrass. 334
No taxa were indicators of N treatment, whether analyzed at the level of ASV, species, 335
genus, family, or order. A total of 13 ASVs were indicators of site, occurring exclusively at their 336
respective sites and in 50% or more of plots at that site. Three Paraglomus brasilianum ASVs 337
(ASVs 5, 18, and 114) and two Microdominikia litorea ASVs (ASVs 98 and 139) were 338
indicators of Hancock (p < 0.02). Three Paraglomus brasilianum ASVs (ASVs 6, 12, and 65) 339
were also indicators of Rhinelander, as well as one Nanoglomus plukenetiae sensu lato ASV 340
(ASV 1; p ≤ 0.02). Two Paraglomus laccatum ASVs (ASVs 41 and 63), one Scutellospora 341
calospora ASV (ASV 149), and an unknown Rhizoglomus species (ASV 186) were indicators of 342
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Oregon (p ≤ 0.03). At the familial level, Pervetustaceae was an indicator at Hancock, occurring 343
in half of the plots and almost exclusively at this site (p < 0.05). 344
345
Discussion 346
N addition did not affect AMF ASV diversity or composition 347
Contrary to our expectations, we found no effect of N addition on AMF ASV diversity or 348
community composition. No consensus exists on how N enrichment affects AMF diversity 349
despite the considerable amount of research this topic has received (27). It is difficult to compare 350
the effects of N addition across studies given the different methodologies employed to assess 351
AMF diversity, in particular metabarcoding studies versus spore-based studies. We observed that 352
the most frequently recovered ASVs from switchgrass soils were primarily those with small 353
spores (e.g., AMF belonging to the genera Dominikia, Nanoglomus, Microkamienskia, 354
Paraglomus, and Rhizoglomus) or species that sporulate rarely or only seasonally (e.g., 355
Acaulospora spp.), which would have made morphology-based detection and classification 356
extremely difficult, even for expert taxonomists (78). In addition, we detected numerous 357
undescribed lineages of AMF of unknown taxonomic diversity – for example, it is unclear how 358
many species were represented in the Nanoglomus plukenetiae sensu lato clade detected in this 359
study – that would have made accurate morphotyping possible only after extensive taxonomic 360
research concerning the undescribed genetic diversity found in switchgrass agroecosystems. 361
In addition to limitations in AMF taxonomy, a robust predictive framework concerning 362
the effects of N addition on AMF is elusive considering the number of variables that might play 363
a role in the context dependency of AMF response to N addition. These include plant community 364
diversity, host photosynthetic pathway, time of the year, initial diversity of the AMF community, 365
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and baseline soil nutrients (27, 79). Given that Antoninka et al. (48) observed decreased AMF 366
diversity in monocultures but not polycultures, and that Egerton-Warburton et al. (80) reported 367
decreased diversity of AMF communities associated with warm-season grasses but not cool-368
season grasses, we expected N addition to negatively affect AMF diversity in the context of 369
switchgrass monocultures. Theories of soil resource stoichiometry predict changes in AMF under 370
varying ratios of available N and P (19). The N application rate used in this study had no 371
significant effect on soil N or P at any site, so the fact that we did not observe changes in ASV 372
diversity with N amendment may be consistent with resource stoichiometry theory. Although our 373
rate of N amendment matched estimates for switchgrass harvest removal (15), this is only about 374
half of the recommended N application rate (81–82). At higher application rates of N, shifts in 375
soil resource stoichiometry could be more apparent with subsequent effects on AMF diversity 376
(83). Finally, in a metanalysis of the effects of N addition on AMF Shannon diversity, short-term 377
(< 5 years) fertilization studies had no effect on AMF diversity (83). We sampled AMF 378
communities after four years of N addition, which may not have been sufficient time to observe 379
consistent changes in AMF. 380
Previous studies have reported shifts in AMF community composition without changes in 381
alpha diversity (83). N enrichment can affect AMF community structure via competitive effects 382
where AMF compete with each other for host C, or selective effects, where AMF species have 383
different sensitivities to agricultural inputs (79). N amendment had no discernable effect on AMF 384
community structure or on soil N in the timeframe of our study, but the correlation between 385
AMF community structure and soil N within sites suggests competitive or selective effects were 386
at play independent of N treatment. Treseder et al. (84) found that AMF with extensive 387
extraradical hyphae (e.g., Gigasporacae) had low tolerance of elevated soil N, but AMF with 388
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greater allocation to intraradical structures (e.g., Glomus spp.) were more prevalent in N enriched 389
environments. We recovered relatively few Gigasporaceae AMF and little functional 390
information exists on other taxa prevalent in our study (e.g., Paraglomerales), limiting our ability 391
to determine whether community structure in relation to soil N was a function of morphology 392
Positive correlation trend between yield and AMF family richness at most productive site 393
We found no universal relationships between AMF metrics and switchgrass yield at the 394
ASV level. Indeed, improved taxonomic resolution may not allow one to detect relationships 395
between AMF and plant productivity if fine-scale taxonomic groupings of AMF are not good 396
proxies for functional trait differences. However, at the family level in our most productive site 397
(Oregon), we detected a relationship between switchgrass yield and AMF family richness. Along 398
with N treatment and plot, these variables could account for 88% of the variation in switchgrass 399
yield. In another bioenergy study on productive soils, Emery et al. (85) did not observe a 400
correlation between switchgrass yield and AMF diversity, but they did not analyze AMF 401
diversity at the family level. According to Chagnon et al. (86), AMF functional traits are shared 402
at the family level. Thus, with greater richness of AMF families, switchgrass may have 403
benefitted from greater diversity as a result of AMF functional complementarity (e.g., P 404
acquisition, plant pathogen protection, and abiotic stress tolerance, among others). This result is 405
consistent with a meta-analysis by Yang et al. (87), which found that plant performance was 406
positively promoted by AMF family richness but not species richness or phylogenetic diversity. 407
In our study, all experimental units at Oregon contained ASVs from Glomeraceae and 408
Paraglomeraceae. Interestingly, only the two most productive experimental units contained 409
ASVs from Pervetustaceae, a recently described family with worldwide occurrence for which 410
little functional information exists. 411
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Future research should explore the generality of these results and the potential yield 412
benefits of actively augmenting AMF family richness via spore inoculations. House and Bever 413
(88) demonstrated that the establishment of Andropogon gerardii – a tallgrass prairie plant 414
closely related to switchgrass – benefited from inoculation with seven regionally adapted AMF 415
morphotypes, suggesting a small number of diverse AMF species is sufficient to observe 416
beneficial effects of AMF inoculation on plant establishment. Accordingly, inoculations of 417
switchgrass agroecosystems that include regionally adapted AMF from multiple families might 418
result in benefits not only to switchgrass yield but also to switchgrass establishment. These 419
benefits could make switchgrass a more viable crop for farmers to grow as establishment and 420
early-season productivity are agronomic impediments to its widespread cultivation (15, 89). In 421
inoculation experiments, we recommend investigators start with the putative core AMF of the 422
switchgrass microbiome and supplement the microbiome with representatives from the families 423
present in the highest yielding plots of Oregon. 424
Switchgrass soil microbiome dominated by poorly studied taxa 425
Our understanding of AMF is biased towards those that are most prevalent in disturbed 426
habitats (90). Concerted efforts to understand uncultured fungi in less disturbed environments 427
could benefit perennial agriculture as a whole given that these agroecosystems might harbor 428
AMF communities that are more similar to non-agricultural ecosystems. In addition to the 429
numerous undescribed clades identified in this study, little is known about the ecology of the 430
putative core AMF of the switchgrass microbiome. Microdominikia litorea was described in 431
2018 in association with Xanthium spinosum, a spiny plant growing on Mediterranean sand 432
dunes (91). Comparing our ASVs of Microdominikia litorea to environmental sequences in 433
GenBank, only two isolates from grasslands of northern China were similar (KF836940, 434
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KF936961; 100% query cover, > 97.5% identical). Nanoglomus plukenetiae was only recently 435
described from the Amazonian lowlands of Peru in association with the Inca nut, Plukenetia 436
volubilis (75). Deposited sequences from an unpublished switchgrass study at the Konza Prairie 437
Biological Station near Manhattan, Kansas were > 99% identical to our Glomeraceae sp. 1 438
subclade of the N. plukenetiae sensu lato group (JX276891), providing further evidence that 439
these AMF may be core members of the switchgrass microbiome. Finally, Gosling et al. (92) 440
discussed the lack of ecological information about Paraglomerales as inspiration for their survey 441
of this order across England. They found that Paraglomerales AMF were widely dispersed across 442
agroecosystems but were more common in organically managed soils than conventionally 443
managed soils. We expect that the widespread use of primers that are unbiased in their 444
amplification of AMF rDNA (50) and resolution to the level of ASV (53) should promote greater 445
inclusion and tractability of previously overlooked clades. 446
AMF biogeography characterized by high beta diversity 447
Similar to previous work (e.g., 93), we documented high ASV beta diversity among our 448
three sites. It is unclear to what extent AMF distribution is shaped by dispersal limitation versus 449
environmental filtering. In our study, evidence for the former was the greater number of shared 450
ASVs between the two closest sites (Oregon and Hancock) compared to the more distant 451
adjacent sites (Hancock and Rhinelander), and the complete lack of shared ASVs between the 452
sites at opposite ends of Wisconsin. However, the within-site correlation between community 453
dissimilarity and soil N suggests environmental filtering was also important in shaping AMF 454
structure, and closer sites may have been more similar in soil variables important in filtering 455
AMF. Using restriction fragment length polymorphism of AMF SSU rDNA, Hazard et al. (94) 456
found that local environment, and not geographical distance, determined distribution of AMF in 457
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Ireland. If the rate of evolution in a population is greater than the rate of dispersal, the use of 458
ASVs may provide fine enough taxonomic resolution to observe the evolution of population 459
structure distinct from gene flow. Indeed, we do not know the time scales over which an AMF 460
genotype can disperse 350 km, the approximate distance between our furthest sites, versus the 461
rate of evolutionary change in ITS rDNA that would result in different ASVs. More importantly, 462
it remains to be determined if the genotypes represented by rDNA ASVs have genetic 463
differences in other regions of the genome with concomitant impacts on ecosystem functioning 464
and switchgrass performance. 465
Conclusions 466
Four years of nitrogen addition at levels meant to replace harvested plant-tissue N had no 467
consistent effect on AMF composition or diversity. Across three Wisconsin sites, different 468
factors were associated with switchgrass yield highlighting the context dependency of 469
switchgrass production for bioenergy. The ASVs elucidated in this study theoretically represent 470
exact, living genotypes present in switchgrass soils. In the absence of taxonomic consensus about 471
AMF species delimitations and, therefore, the full extent of intraspecific functional diversity 472
(95), ASVs serve as consistent sequence identifiers for future studies seeking to understand AMF 473
functioning and biogeography as well as for bioprospecting of regionally adapted AMF inocula. 474
To build a more predictive “mycorrhizal technology” for sustainable agricultural intensification 475
of switchgrass (6), future research should evaluate more directly how specific AMF genotypes 476
affect ecosystem functioning via soil biodiversity manipulation experiments (83). 477
478
Acknowledgments 479
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The authors wish to thank Dr. Maggie Phillips, Dr. Gary Oates, Dr. Harry Read, and Dr. 480
Gregg Sanford for technical guidance and logistical support. Thank you to research staff Jimmy 481
Sustachek and Mark Walsh for help with fieldwork. ACD is grateful to Dr. Timothy James for 482
tutelage on phylogenetic analyses and useful feedback on the manuscript. This material is based 483
upon work supported in part by the University of Wisconsin-Madison Center for Integrated 484
Agricultural Systems Mini-Grant and the Great Lakes Bioenergy Research Center, U.S. 485
Department of Energy, Office of Science, Office of Biological and Environmental Research 486
under Award Numbers DE-SC0018409 and DE-FC02-07ER64494. 487
488
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751
Figures 752
Figure 1. Summary of taxonomic distribution of ASVs according to the UNITE ITS fungal 753
database by site and nitrogen treatment. N- refers to unamended experimental units (0 kg N ha-1
) 754
and N+ refers to the amended experimental units (56 kg N ha-1
). 755
756
Figure 2. Ternary plot showing the site distributions for 53 AMF species-level clades determined 757
by LSU rDNA phylogenies. Taxa present with > 4% relative abundance at all three sites are 758
labelled with their name. The size of points corresponds to the total sequence abundance for that 759
taxon across all three sites. Most taxa are only present at one site or rarely present at the others. 760
As a result, most points are positioned at the tips of the ternary plot. The number of unique ASVs 761
for a given site and the number of shared ASVs between two sites are indicated along the axes on 762
the outside of the ternary plot. Three ASVs – ASVs 8, 27, and 78 – were found at all three sites. 763
764
Figure 3. Phylogenetic tree of Paraglomerales reference taxa and recovered ASVs, as well as 765
representatives of the previously unknown clade GS24 (77) with Archaeospora spainiae 766
(Archaeosporales) as the outgroup. Site occurrence is indicated by the filled circles on the 767
outside of the tree. “H” refers to Hancock (inside circles), “O” to Oregon (middle circles), and 768
“R” to Rhinelander (outside circles). Bootstrap support ≥ 0.75 is indicated by grey dots on the 769
branches with the size of the dots corresponding to relative bootstrap support. Teal text indicates 770
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ASVs or reference taxa with a clear taxonomic affinity, while orange text indicates ASVs that 771
could not be assigned to a species name based on available phylogenetic information. 772
773
Figure 4. Boxplots of switchgrass yield at three sites under N-addition (amended) or no N-774
addition (control) treatments. The boxplots show the minimum, median, maximum, and 775
interquartile range of the data. The letters above the boxplots indicate statistical difference of 776
switchgrass yield between sites, with different letters representing statistically significant 777
differences (p < 0.05). The horizontal bar above the boxplots indicates statistical difference of 778
switchgrass yield between treatments within sites; two asterisks denote a significant difference (p 779
< 0.01) in switchgrass yield between amended and control plots at Rhinelander. 780
781
Figure 5. Boxplots of soil variables at three sites under N-addition (amended) or no N-addition 782
(control) treatments. The boxplots show the minimum, median, maximum, and interquartile 783
range of the data. The letters above the boxplots indicate statistical difference of soil variables 784
between sites, with different letters representing statistically significant differences (p < 0.05). 785
No soil variables differed significantly between the two N treatments within a site. 786
787
Figure 6. Non-metric multidimensional scaling (NMDS) of UniFrac beta dissimilarity. Each 788
point corresponds to the AMF community of an experimental unit. The size of points represents 789
switchgrass yield, the shape of points corresponds to N treatment, and the color of points 790
indicates site. Paired experimental units are linked by lines. 791
792
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Figure 7. Scatterplot showing the relationship between AMF family richness and N treatment on 793
switchgrass yield at Oregon. Orange points correspond to experimental units under no N 794
amendment (control) and teal points correspond to experimental units under N amendment 795
(amended). The lines correspond to best-fitting linear regressions. The gray shading around the 796
regression lines is the 95% confidence intervals of the regressions. 797
798
Tables 799
Table 1. Linear mixed effects regression models type III ANOVA resultsa 800
Site Nitrogen Site × Nitrogen
Response Variable F(2,8) p F(1,8) p F(2,8) p
Square-root-transformed switchgrass yield (Mg/ha) 33.39 <0.0001 10.10 0.01 14.04 0.002
pH 6.11 0.03 0.04 0.85 0.29 0.76
Cation exchange capacity (cmolc/kg) 8.68 0.01 0.34 0.58 0.05 0.95
Log-transformed total P (ppm) 40.63 <0.0001 0.26 0.63 2.11 0.18
Total C (%) 4.85 0.04 4.21 0.07 1.80 0.23
Total N (%) 17.08 0.001 2.39 0.16 0.01 0.99
C:N ratio 27.01 0.0003 2.89 0.13 3.53 0.08
ASV richness 8.67 0.01 0.35 0.57 2.44 0.15
ASV Shannon diversity index 3.68 0.07 1.32 0.28 0.96 0.42
ASV Simpson’s diversity index 3.90 0.07 0.54 0.48 0.09 0.91
ASV Pielou's evenness index 0.62 0.56 0.59 0.47 0.04 0.96
ASV Faith’s phylogenetic diversity index 20.97 0.0007 0.99 0.35 1.32 0.32
ASV phylogenetic mean pairwise distance 3.88 0.07 1.88 0.21 3.11 0.10
Species richness 14.65 0.002 1.62 0.24 1.09 0.38
Species Shannon diversity index 3.01 0.11 5.82 0.04 3.44 0.08
Species Simpson’s diversity index 1.29 0.33 2.32 0.17 3.33 0.09
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Species Pielou’s evenness index 0.09 0.92 4.32 0.07 3.44 0.08
Genus richness 10.81 0.005 1.18 0.31 2.12 0.18
Genus Shannon diversity index 0.86 0.46 1.23 0.30 3.58 0.08
Genus Simpson’s diversity index 0.24 0.79 0.29 0.60 3.83 0.07
Genus Pielou’s evenness index 0.21 0.82 0.44 0.53 0.63 0.56
Family richness 14.68 0.002 1.02 0.34 2.22 0.17
Family Shannon diversity index 0.89 0.45 0.24 0.64 3.56 0.08
Family Simpson’s diversity index 0.30 0.75 0.004 0.95 3.25 0.09
Family Pielou’s evenness index 0.26 0.78 0.28 0.61 1.49 0.28
aSignificant variables at the Bonferroni-corrected threshold of p < 0.002 are bolded. 801
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SpeciesAcaulospora minuta
Acaulospora spinosa
Cetraspora gilmorei
Claroideoglomus claroideum
Diversispora eburnea
Diversispora epigaea
Dominikia difficilevidera
Dominikia iranica
Funneliformis mosseae
Gigaspora rosea
Glomus compressum
Glomus indicum
Glomus macrocarpum
Palaeospora spainiae
Paraglomus brasilianum
Paraglomus laccatum
Paraglomus occultum
Rhizophagus irregularis
Scutellospora alterata
Scutellospora heterogama
Septoglomus viscosum
Unknown
40000
30000
20000
10000
0
Nu
mb
er
of R
ea
ds
Site and Nitrogen Treatment
N+N- N- N+ N- N+
Hancock Oregon Rhinelander
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10000
20000
30000
40000
50000
Paraglomus brasilianum
Microdominikia litorea
Paraglomus
laccatum
Glomeraceae sp. 1
(Nangoloms plukenetiae sensu lato)
Oregon(201 unique ASVs)
Hancock(142 unique ASVs)
Rhinelander(78 unique ASVs)
11 s
hare
d A
SV
s
1 shared ASV
0 sh
are
d A
SV
s
Three Universal ASVs
ASV 8 - Paraglomus brasilianum
ASV 27 - Paraglomus brasilianum
ASV 78 - Microdominikia litorea
Number of Reads
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A
B
C
A A
B
A A
B
A A
B
A A
B
A A
B
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ControlAmended
N Treatment
SiteHancockOregonRhinelander
Low
Medium
High
Yield
0.00
-0.25
-0.40 -0.20 0.00 0.20 0.40
NMDS1
NM
DS
2
Stress = 0.06
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Rc
2 = 0.88
N Treatment
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