Construction of the TRF reference database
Samples for cloning were chosen based on the diversity of terminal restriction fragments (TRFs)
produced in the MboI digest. A subset of samples from each field site or soil inoculum source was
combined and a single clone library constructed per site (field experiment) or soil inoculum source
(greenhouse experiment). The samples were chosen such that all unique TRFs of a site/soil source were
represented in the subset. For the field experiment, ten samples were selected from each site and the
PCR amplicons pooled for cloning. For the greenhouse experiment, six samples were selected from each
site and their PCR amplicons combined into a single cloning reaction per site. The chosen samples were
separately reamplified using their respective PCR protocols and untagged AML1-AML2 primers. The
pooled PCR amplicons were separated by electrophoresis on a 1.5% agarose gel and purified from
excised bands using the Promega Wizard SV Gel and PCR clean-up system. The purified PCR products
were ligated into the pGEM-T Easy Vector and cloned into JM109 High Efficiency Competent Cells using
the pGEM-T Easy Vector System. The transformed cells were plated onto LB/Amp plates and grown
overnight at 37°C. 50-60 colonies of recombinant cells, per library (= site or soil source) were isolated
and plated onto new LB/Amp plates and grown overnight at 37C. Colonies were then propagated in
liquid LB/Amp overnight at room temperature to produce plasmid template for RFLP analysis.
The inserts of 3.2 ul of frozen cell culture, using 5U of Taq polymerase, were re-amplified in 40ul
PCR reactions with 1X reaction buffer, 2 mM MgCl, 400 uM of each dNTP and 0.4 uM of the M13F-M13R
primers using the following thermal cycling program: 94°C for 7 min, followed by 24 cycles of 94°C for 35
s, 55°C for 45 s, and 72°C for 3 min, ended by a final extension period of 72°C for 3 min. The M13 PCR
product of each clone was digested with MboI and the restriction fragments visualized on a 1.5%
agarose gel. Each unique pattern was given an identifier and clones were scored accordingly. Twenty
four clones were chosen based on their RFLP patterns per site or soil source from each experiment, for
sequencing, totaling in 288 clones (=24*5 + 24*7). Multiple clones of unique banding patterns were
sequenced from each site or soil source within experiments. The M13 PCR products from clones
selected for sequencing were purified using the EZNA(96) Omega PCR purification kit. The purified PCR
products were sent for unidirectional sequencing with the AML2 primer to the Georgia Genomics
Facility. The sequence electropherograms were corrected, trimmed, and compared against the public
databases, using the BLASTN similarity search algorithm. Glomeromycota sequence was assigned to
virtual taxa (VT) using BLAST by comparison to the MaarjAM database (Öpik et al. 2010). Only 4 of the
288 sequenced clones (1.3%) resulted in non-AMF sequences. 35 unique VT of AMF were identified
among the 288 sequenced clones. A total of 96 clones were chosen for bidirectional sequencing with the
vector primers T7 promoter and SP6 based upon their VT assignment, in order to include the entire PCR
amplicon. M13 PCR product from all different VT from each site or soil source were bidirectionally
sequenced at least once. Also included were the three clones of non-AMF origin.
Full length sequences were aligned, corrected, and trimmed so the fragment was flanked by the
AML1 and AML2 primers. Three samples did not yield usable sequences. The program Trifle (Junier et al.
2008) was used to perform in silico digests of the final 93 clone sequences with enzymes readily
available through New England Biolabs. Suitable enzymes were those differentiating among VT by the
produced terminal fragments (TRF). Three suitable enzymes were identified; MboI, HinfI and TfiI.
Double end-labeled PCR products of the primers AML1-FAM and AML2-NED of each of the 96 clones
were digested with these three enzymes and submitted for fragment analysis on an ABI3730xl with the
internal size standard ROX1000. A few clones did not yield readable TRF peaks for all six combinations of
forward and reverse fragments produced by the three enzymes. The combination of MboI and TfiI
yielded the best discrimination among VT. We detected 37 unique TRF patterns among the 86 clones
for which we had full length sequences and complete TRFLP profiles. From these empirical fragment
patterns, a combined TRFLP reference database was created for the AMF taxa present across all sites
(Supplemental Table 1 and Supplemental Figure 1 for a methods flowchart). VT represented by multiple
TRF patterns were entered into the database twice (Supplemental Table 1).
The virtual taxa identified in MaarjAM do not necessarily line up to currently recognized AMF
species – some VT include multiple species, while some species include multiple VT. In addition, the
phylogenetic relationships between VT are unclear, since these distinctions were made using only a
portion of the 18S rRNA gene region. Krüger et al. (2012) provided a better resolved phylogeny of the
AMF using consensus sequences across the full length SSU region from 76 vouchered species. In order to
measure phylogenetic community structure more accurately, we used the RAxML Evolutionary
Placement Algorithm (Berger et al. 2011) to determine the most likely placement of each of our 800 bp
cloned sequences on the reference tree created, using Krüger et al. (2012)’s sequence alignment. Using
this approach, our cloned sequences represented 17 distinct AMF species. 14 of these matches were to
named species, while three were to clades determined by Krüger et al. (2012) to represent as-yetunnamed species. In this case, clones matching to the same species occasionally produced different
TRFLP patterns. When creating the TRFLP database, species were entered multiple times as necessary to
represent all possible TRFLP patterns (see Supplemental Table 2).
References
Berger SA, Krompass D, Stamatakis A (2011) Performance, accuracy, and web server for evolutionary
placement of short sequence reads under maximum likelihood. Systematic Biology 60, 291-302.
Junier P, Junier T, Witzel K (2008) TRiFLe, a Program for In Silico Terminal Restriction Fragment Length
Polymorphism Analysis with User-Defined Sequence Sets. Applied and Environmental
Microbiology 74, 6452-5456.
Krüger M, Krüger C, Walker C, Stockinger H, Schüßler A (2012) Phylogenetic reference data for
systematics and phylotaxonomy of arbuscular mycorrhizal fungi from phylum to species level.
New Phytologist 193, 970-984.
Öpik M, Vanatoa A, Vanatoa E, et al. (2010) The online database MaarjAM reveals global and
ecosystemic distribution patterns in arbuscular mycorrhizal fungi (Glomeromycota). New
Phytologist 188, 223-241.
# of
Virtual
MboF
MboR
TfilF
TfilR
clones
Taxon
36
520
50
61
405
1
48
178
109
359
52
2
44
520
50
61
688
1
44
520
50
760
748
1
193
520
109
61
52
6
56
317
109
61
52
2
56
421
109
61
52
1
56
418
50
61
688
1
74
178
109
61
52
2
61
660
50
61
271
3
62
370
50
61
271
1
222
178
50
410
271
4
222
178
50
496
271
4
222
178
109
710
52
1
136
178
50
359
271
2
166
520
50
410
271
1
239
660
109
61
52
5
238
222
50
760
748
1
84
495
50
359
405
1
113
178
50
359
405
14
115
178
50
61
405
1
204
317
50
760
748
2
159
317
50
496
271
1
204
131
50
410
358
1
204
317
50
410
358
5
214
317
50
359
405
1
219
495
50
410
358
1
219
660
50
359
358
3
69
178
50
359
358
2
74
178
50
410
358
4
63
178
50
359
748
1
64
178
50
330
271
1
326
178
50
760
748
5
222
50
61
52
1
222
50
61
688
1
222
50
61
688
2
Species as identified by
RAxML EPA
Acaulospora cavernata
Acaulospora longula
Acaulospora spinosa
Acaulospora spinosa
Claroideoglomus claroideum
Claroideoglomus etunicatum
Claroideoglomus etunicatum
Claroideoglomus species
Claroideoglomus species
Diversispora epigaea
Diversispora species
Glomus indicum
Glomus indicum
Glomus indicum
Glomus iranicum
Glomus iranicum
Paraglomus brasilianum
Paraglomus occultum
Rhizophagus clarus
Rhizophagus irregularis
Rhizophagus irregularis
Rhizophagus irregularis
Rhizophagus species
Rhizophagus species
Rhizophagus species
Rhizophagus species
Rhizophagus species
Rhizophagus species
Sclerocystis sinuosa
Sclerocystis sinuosa
Septoglomus africanum
Septoglomus africanum
Septoglomus africanum
non-AM fungus
non-AM fungus
plant
Table S1. TRFLP reference database. Each unique observed terminal restriction fragment pattern
detected among 86 bidirectionally sequenced clones is listed, with the number of clones producing that
pattern, the best matching virtual taxa as determined by BLAST search against the MaarjAM database
for that sequence group, and phylogenetic species assignment to the Krüger et al. (2012) reference tree.
Table S2. Linear models of AMF richness and phylogenetic diversity in the field experiment with highly
leveraged P.pumila populations excluded.
A. Uninvaded P. pumila population (Vermillion River Observatory) excluded
# of Species
A. Uninvaded P. pumila population
(Vermillion River Observatory)
excluded
Site History
Host Population History
Host Population History2
Site History*Pop History
A. petiolata Removal Treatment
Site History*Removal Trt
Pop History*Removal Trt
Pop History2*Removal Trt
Site History*Pop History*Removal Trt
Initial Seedling Size
Total Fluorescence
B. Longest invaded P. pumila
population (Black Rock Forest)
excluded
Site History
Host Population History
Host Population History2
Site History*Pop History
A. petiolata Removal Treatment
Site History*Removal Trt
Pop History*Removal Trt
Pop History2*Removal Trt
Site History*Pop History*Removal Trt
Initial Seedling Size
Total Fluorescence
Faith's PD
F
0.14
4.60
P
0.716
0.042
F
0.03
3.21
P
0.873
0.085
1.12
1.39
0.31
0.07
7.71
0.30
0.25
0.58
0.79
0.010
1.05
3.03
0.09
0.23
12.05
0.315
0.094
0.773
0.633
0.002
0.31
1.66
0.42
23.82
0.578
0.209
0.522
<.0001
0.74 0.398
3.43 0.076
3.63 0.068
33.13 <.0001
2.03
0.167
1.03
0.320
0.19
0.664
0.61
0.442
3.62
0.069
0.13
0.720
2.90
0.102
1.47
0.236
0.00
0.979
0.64
0.432
20.81 <0.0001
4.17 0.052
0.60 0.445
0.18 0.672
0.87 0.360
4.67 0.041
0.07 0.794
6.29 0.019
3.70 0.066
0.33 0.574
5.73 0.025
28.97 <.0001
Fig. S1. Flowchart of the methodological procedure for the creation of the TRFLP Database
Fig. S2 Species richness (A,B) or phylogenetic diversity (C,D) of arbuscular mycorrhizal fungi in Pilea
pumila roots plotted against the P. pumila population’s history of coexistence with A. petiolata in the
field experiment, with either the uninvaded population (VRO, A,C) or longest invaded population (BRF,
B, D) excluded . Each symbol is the average of all replicates in a given host population*A. petiolata
removal treatment combination (average of five field sites with a mean of 3 replicates per site, for a
total of ~15 replicates per population). Solid symbols and solid line = A. petiolata at ambient levels;
Open symbols and dashed line = all A. petiolata individuals weeded out of the 1 m2 study plots.