SUPPORTING INFORMATION
Trifolium species associate with a similar richness of soil-borne mutualists in their introduced and native ranges.
Kevin J. McGinn; Wim H. van der Putten; Richard P. Duncan; Natasha Shelby; Carolin Weser; Philip E. Hulme
Appendix S1: Field site details
Table 1. Root nodule sampling locations for each Trifolium species (A = T. arvense, C = T. campestre, D = T. dubium, O = T. ornithopodioides, P = T. pratense,
R = T. repens, S = T. striatum) in New Zealand (NZ) and the United Kingdom (UK). Sampling for each species was conducted over a comparable geographic
area in both ranges.
Species Country
A
A
UK
UK
A
UK
A
A
C
C
C
C
C
D
D
D
D
D
O
O
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
Site
Locality
no.
1
Bournemouth
2
Gosport: Browndown
Devon: Off Ilsham Marine Drive, near
3
Torquay
4
Swansea marina
5
Kenfig
1
Gosport: Browndown
2
Gower: near Whiteford Burrows
3
Devon: near Chudleigh
4
Devon: headland south of Berryhead
5
Kenfig: near Sharkham Point
1
West Knighton
2
Bournemouth
3
Devon: near Chudleigh
4
Gower: Port Eynon
5
Gower: Crofty
1
Bournemouth
2
Gosport
Latitude
Longitude
50.719495
50.792363
-1.857508
-1.193203
50.458359
-3.491381
51.614266
51.515509
50.793469
51.619075
50.614183
50.382074
51.506086
50.686890
50.720274
50.614117
51.544036
51.639117
50.720299
50.792120
-3.930945
-3.727773
-1.194668
-4.255334
-3.623614
-3.499986
-3.743090
-2.390173
-1.849621
-3.623618
-4.211099
-4.136976
-1.849496
-1.180596
O
O
O
P
P
P
P
P
R
R
R
R
R
S
S
S
S
S
A
A
A
A
A
C
C
C
C
C
D
D
D
D
D
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
Gower: Port Eynon
Gower: Loughour Estuary
Kenfig: old road near Sharkham point
West Knighton roadside bank
Devon: Berryhead
Gower: Pennard Burrows
Kenfig
Gower: Crofty
West Knighton
Bournemouth
Devon: near Chudleigh
Gower: Port Eynon
Swansea: Gorseinon park
Bournemouth
Gosport: Browndown
Devon: headland south of Berryhead
Gower: Broadpool
Gower: Pennard Burrows
Pope Street, Addington
Kaitorete Spit
HW 1 Bridge
Wakanui Beach Road, Wakanui
Seadown Rd, Timaru
Streeters Road
Western Valley Road
Big Hill Road, Banks Peninsula
Wakanui School Road, Wakanui
Seadown Rd, Timaru
Bridleway, Governors Bay
Lake Forsyth
HW 1 Bridge
Wakanui Beach Road, Wakanui
Caroline Bay, Timaru
51.543854
51.630818
51.506815
50.687200
50.394077
51.577112
51.515440
51.638975
50.686878
50.720274
50.614043
51.543870
51.673771
50.719368
50.793469
50.382116
51.597861
51.575920
172.609618
172.698799
172.232281
171.863984
171.286188
172.625468
172.795569
173.064849
171.814292
171.284508
172.651952
172.723295
172.231962
171.864058
171.251068
-4.211267
-4.134735
-3.740627
-2.381096
-3.492447
-4.090455
-3.727951
-4.136470
-2.390506
-1.849621
-3.623618
-4.211144
-4.037835
-1.858172
-1.194668
-3.499871
-4.151891
-4.091192
-43.536464
-43.701749
-43.646740
-44.026936
-44.265423
-43.736258
-43.747027
-43.701749
-43.976931
-44.270326
-43.632160
-43.809611
-43.646715
-44.026713
-44.389271
P
NZ
1
P
P
P
P
R
NZ
NZ
NZ
NZ
NZ
2
3
4
5
1
R
NZ
2
R
R
R
NZ
NZ
NZ
3
4
5
S
NZ
1
S
S
S
S
NZ
NZ
NZ
NZ
2
3
4
5
O
NZ
1
O
O
O
O
NZ
NZ
NZ
NZ
2
3
4
5
Roadside, Governors Bay Teddington
Road, Teddington
Days Road, Springston
McCrorys Road, Pendarves
Waipopo Road, Seadown
Withells Rd, Ealing
Pope Street, Addington
Chorlton Road, Okains Bay, Banks
Peninsula
Corbetts Road, Wakanui
Caroline Bay, Timaru
Seadown Rd, Seadown
Heathcote Quarry Track at Major
Hornbrook Road, Mount Pleasant
Wakanui Beach road
Western Valley Road
Big Hill Road, Banks Peninsula
Shaw Street/Domain Ave, Temuka
Roadside, Governors Bay Teddington
Road, Teddington
Lincoln University campus
Lake Forsyth
Caroline Bay, Timaru
Nancy Ave, Mariehau
172.655803
-43.651580
172.437424
171.987335
171.290495
171.417599
172.609757
-43.645590
-43.885801
-44.288008
-44.044594
-43.536279
173.047200
-43.704497
171.895358
171.250939
171.284746
-44.001981
-44.389192
-44.269265
172.717020
-43.571704
171.864023
172.795645
173.064903
171.295862
-44.026671
-43.747033
-43.701631
-44.249368
172.656328
-43.651840
172.468438
172.723000
171.251316
172.638089
-43.643517
-43.809847
-44.389350
-43.500066
Table 2. Root sampling locations for Trifolium species (A = T. arvense; F = T. fragiferum; R = T. repens) in New Zealand (NZ) and the United Kingdom (UK) for
analysis of associated communities of arbuscular mycorrhizal fungi. The three species differ slightly in habitat preferences: T. arvense is adapted to dry,
infertile, sandy soils, T. fragiferum to waterlogged and saline soils and T. repens is less habitat-restricted, but does not perform well in dry soils (Burdon,
1983; Knight, 1985; Townsend, 1985; see footnote); we aimed to sample a range of these habitats in order to detect a variety of AMF taxa. The final column
states the number of root samples from separate plants at each site used for the analysis (63 root samples in total). The distance between sampling sites in
NZ was 42 km, whereas in the UK, it was 198 km.
Species Range Site
Location
number
Latitude
Longitude
Habitat / soil description
A
A
NZ
NZ
n1
n2
Pope Street, Christchurch
Little River, Banks Peninsula
A
A
F
NZ
NZ
NZ
n6
n7
n11
Kaitorete Spit, Banks Peninsula
Birdlings Flat, Banks Peninsula
Motukarara, Banks Peninsula
F
F
R
R
NZ
NZ
NZ
NZ
n12
n14
n1
n2
Lake Forsyth, Banks Peninsula
Streeters Road, Banks Peninsula
Pope Street, Christchurch
Little River, Banks Peninsula
R
R
R
NZ
NZ
NZ
n3
n4
n5
Bossu Road, Banks Peninsula
Chorlton Road, Banks Peninsula
Okains Bay, Banks Peninsula
A
A
A
F
F
F
UK
UK
UK
UK
UK
UK
u1
u20
u22
u25
u29
u3
Bournemouth, England
Pennard Burrows, Gower, Wales
Kenfig, Gower, Wales
Crofty, Gower, Wales
Pwll, Llanelli, Wales
Stanpit Marsh, Christchurch, England
-43.536548 172.609818 Waste ground; well-drained, rocky soil
-43.769456 172.790641 Edge of car park; well-drained,
gravelly soil
-43.825509 172.698964 Dunes; sandy soil
-43.815543 172.698964 Dry grassy road verge; clayey soil
-43.729049 172.584264 Amenity grassland; damp, organic rich
loam
-43.809746 172.723392 Marsh; silty soil
-43.736169 172.625697 Grassland; gravelly, moist loam
-43.536548 172.609818 Waste ground; well-drained, rocky soil
-43.769456 172.790641 Edge of car park; well-drained,
gravelly soil
-43.802360 172.841276 Grassland; clayey soil
-43.675339 173.044438 Grassland; clayey, gravelly soil
-43.704496 173.047237 Road verge near beach; well-drained,
sandy soil
50.719495
-1.857508 Road verge; well-drained, sandy soil
51.576022
-4.091374 Dunes; organic rich sandy soil
51.515509
-3.727773 Dunes; sandy soil
51.639580
-4.137559 Marsh; silty, organic rich soil
51.684829
-4.202671 Damp grassy verge; organic rich loam
50.730127
-1.762038 Marsh; wet sandy soil
Soil
pH
5.65
5.41
No. of
root
samples
4
3
5.61
5.21
4.77
3
3
2
5.09
6.28
7.44
5.8
3
3
4
3
5.55
5.79
5.55
3
2
3
5.36
5.24
6.48
7.05
7.26
5.3
3
3
3
3
3
3
R
UK
u19
Port Eynon, Gower, Wales
51.543870
R
R
UK
UK
u26
u8
Gorseinon, Swansea, Wales
West Knighton, Dorset, England
51.673771
50.686878
-4.211144 Car park near beach; well-drained
sandy soil
-4.037835 Grassland; organic rich, coal deposits
-2.390506 Arable field hedgerow; clay soil
7.07
3
6.81
4.84
3
3
1
Burdon J.J. (1983) Biological Flora of the British Isles. Trifolium repens L. Journal of Ecology, 71, 307–330.
Knight W.E. (1985) Miscellaneous Annual Clovers. Clover Science and Technology (ed. by N. Taylor), pp. 547–562. American Society of Agronomy, Inc., Wisconsin, USA.,
Wisconsin, USA.
Townsend C.E. (1985) Miscellaneous Perennial Clovers. Clover Science and Technology (ed. by N.L. Taylor), pp. 563–578. American Society of Agronomy, Inc., Wisconsin,
USA.
Figure 1. Locations in New Zealand from which
Trifolium roots were sampled for mycorrhizal
analysis. Site labels correspond to species and
site number codes in Table 2.
6
Figure 2. Locations in the United
Kingdom from which Trifolium roots
were sampled for mycorrhizal analysis.
Site labels correspond to species and
site number codes in Table 2.
7
Figure 3. Locations in New
Zealand from which Trifolium
root nodules were sampled for
rhizobia isolation. Site labels
correspond to species and site
number codes in Table 1.
8
1
Figure 4. Locations in the United
Kingdom from which Trifolium
root nodules were sampled for
rhizobia isolation. Site labels
correspond to species and site
number codes in Table 1.
9
Appendix S2: Lab methods and sample sizes
Table 1. Primer nucleotide sequences, the genes and target organisms they amplify and references for each primer.
Primer
Sequence (from 5' to 3')
Gene amplified and target
organisms
Repetitive intergenic
sequences (all bacteria)
ERIC1R
ATGTAAGCTCCTGGGGATTCAC
ERIC2
AAGTAAGTGACTGGGGTGAGCG
Y5
ATGCGKTTYARRGGMCTNGATCT
Repetitive intergenic
sequences (all bacteria)
nodD (rhizobia)
Y6
27f
CGCAWCCANATRTTYCCNGGRTC
AGAGTTTGATCMTGGCTCAG
nodD (rhizobia)
16S (all bacteria)
1492r
LR1
GRTACCTTGTTACGACTT
GCATATCAATAAGCGGAGGA
16S (all bacteria)
LSU (all fungi)
FLR2
FAM-FLR3
GTCGTTTAAAGCCATTACGT
GTTGAAAGGGAAACGRTTRAAG
LSU (all fungi)
LSU (Class Glomeromycota)
NED-FLR4
ATTACGTCAACATCCTTA
LSU (Class Glomeromycota)
References
Versalovic J., Schneider M., De Bruijn F.J., & Lupiski J.R. (1994) Genomic
fingerprinting of bacteria using repetitive sequence-based polymerase chain
reaction. Methods in Molecular and Cellular Biology, 5, 25–40.
See row above
Zézé A., Mutch L., & Young J. (2001) Direct amplification of nodD from
community DNA reveals the genetic diversity of Rhizobium leguminosarum in
soil. Environmental microbiology, 3, 363–370.
See row above
Lane D. (1991) 16S/23S rRNA sequencing. Nucleic Acid Techniques in Bacterial
Systematics (ed. by E. Stackebrandt and M. Goodfellow), pp. 115–175. John
Wiley and Sons, New York,
See row above
Van Tuinen D., Jacquot E., Zhao B., Gollotte A., & Gianinazzi-Pearson V. (1998)
Characterization of root colonization profiles by a microcosm community of
arbuscular mycorrhizal fungi using 25S rDNA‐targeted nested PCR. Molecular
Ecology, 7, 879–887.
Trouvelot S., van Tuinen D., Hijri M., & Gianinazzi-Pearson V. (1999)
Visualization of ribosomal DNA loci in spore interphasic nuclei of glomalean
fungi by fluorescence in situ hybridization. Mycorrhiza, 8, 203–206.
See row above
Gollotte A., van Tuinen D., & Atkinson D. (2004) Diversity of arbuscular
mycorrhizal fungi colonising roots of the grass species Agrostis capillaris and
Lolium perenne in a field experiment. Mycorrhiza, 14, 111–117.
See row above
10
Table 2. PCR protocols for each primer pair.
Primer pair
ERIC1R /
ERIC2
PCR protocol
Each 25 µl PCR reaction contained: 16.7 µl DNA-free water, 2.5 µl 10x PCR buffer,
1.6 µl dNTPs (concentration of 5 mM/µl), 1 µl of each primer (concentration of 25
µM/µl), 0.2 µl Taq polymerase (5 U/μl, HotStarTaq Plus, Qiagen) and 2 µl of
template DNA. Thermal cycling conditions: 94° C for 3 min followed by 35 cycles of
94° C for 1 min, 52° C for 1 min, 72° C for 1 min, with a final extension of 7 min at
72° C and held at 10° C.
Y5 / Y6
Each 25 µl PCR reaction contained: 18.7 µl DNA-free water, 2.5 µl 10x PCR buffer,
1 µl dNTP mix (concentration of 5 mM/µl), 0.8 µl of each primer (concentration of
10 µM/µl), 0.2 µl Taq polymerase (5 U/μl, HotStarTaq Plus, Qiagen) and 1 µl of
template DNA. Thermal cycling conditions: 95° C for 2 min followed by 25 cycles of
95° C for 1 min, 59° C for 1 min, 72° C for 2 min, with a final extension of 5 min at
72° C and held at 10° C.
27f / 1492r
Each 25 µl reaction contained: 18.3 µl DNA-free water, 2.5 µl 10x PCR buffer, 2 µl
dNTPs (concentration of 5 mM/µl), 0.5 µl of each primer (concentration of 10
µM/µl), 0.2 µl Taq polymerase (5 U/μl, HotStarTaq Plus, Qiagen) and 1 µl of
template DNA. Thermal cycling conditions were: 95° C for 5 min followed by 35
cycles of 95° C for 30 s, 57° C for 40 s, 72° C for 1 m, with a final extension of 10
min at 72° C and held at 10° C.
LR1 / FLR2
Each reaction contained: 12.75 μl milli-Q water, 2.5 μl 10x Fast Start High Fidelity
Reaction Buffer (Roche Diagnostics), 1 µl MgCl2 (25 mM), 2.5 μl of each primer
(concentration of 10 μM), 2.5 μl dNTP mix (concentration of 2 mM each), 0.125 μl
BLOTTO (10% w/v fat-free milk powder), 0.125 μl Taq polymerase (5 U/μl,
HotStarTaq Plus, Qiagen) and 1 μl template DNA. Thermal cycling conditions: 5
min at 95o C, then 35 cycles of 30 s at 94o C, 40 s at 58o C and 70 s at 72o C,
followed by 7 min at 72o C before cooling.
FAM-FLR3 /
NED-FLR4
Each reaction contained: 11.88 μl milli-Q water, 2.5 μl Fast Start High Fidelity
Reaction Buffer with MgCl2 (Roche Diagnostics) (10 μM), 1µl MgCl2 (25 mM), 1.8 μl
of primer FAM-FLR3 (10 μM), 2.5 μl of primer NED-FLR4 (10 μM), 0.7 μl of primer
FLR3 (10 μM), 2.5 μl dNTP mix (10 μM), 0.125 μL Taq polymerase (5 U/μl,
HotStarTaq Plus, Qiagen) and 2 μl 1:100 parts diluted PCR product from the first
reaction. Thermal cycling conditions: 5 min at 95o C, then 27 cycles of 30 s at 94o C,
40 s at 54o C and 60 s at 72o C, followed by 15 min at 72o C before cooling.
11
Table 3. Number of rhizobia isolates used for rep-PCR fingerprinting per Trifolium species in each
range (NZ = New Zealand, UK = United Kingdom) and the number of different field sites from which
the isolates originated.
Trifolium
species
T. arvense
T. campestre
T. dubium
T. pratense
T. striatum
Number of
NZ isolates
10
12
7
10
9
Number of
NZ field sites
4
5
4
4
4
Number of
UK isolates
10
13
13
13
13
Number of
UK field sites
5
5
5
5
5
Table 4. Number of rhizobia isolates used for nodD phylogenetic analyses per Trifolium species from
each range (NZ = New Zealand, UK = United Kingdom) and the number of field sites from which the
isolates originated.
Trifolium species
T. arvense
T. campestre
T. dubium
T. ornithopodioides
T. pratense
T. repens
T. striatum
Number of
NZ isolates
7
12
5
8
8
8
6
Number of
NZ field sites
4
5
4
4
4
4
3
Number of
UK isolates
9
13
11
12
14
11
7
Number of
UK field sites
5
5
5
5
5
5
4
12
Table 5. Details for rhizobia isolates used for rep-PCR fingerprinting per Trifolium species (A = T.
arvense, C = T. campestre, D = T. dubium, P = T. pratense, S = T. striatum) in each range (New Zealand
or United Kingdom). Field site numbers for each species correspond to Table 1. A maximum of three
nodules were sampled from each species, per field site, with each nodule originating from separate
plants; the ‘nodule no.’ column indicates which nodule per species/site the isolate originated from.
Isolate
reference
pu11
pu12
pu21
pu22
pu23
pu31
pu32
pu33
pu41
pu42
pu43
pu51
pu52
pn11
pn12
pn13
pn21
pn22
pn23
pn31
pn32
pn33
pn42
au11
au12
au13
au22
au31
au32
au33
au41
au42
au52
an11
an12
an13
an21
an22
an31
an32
an51
an52
an53
cu11
Species Range
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
C
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
UK
Field
site
1
1
2
2
2
3
3
3
4
4
4
5
5
1
1
1
2
2
2
3
3
3
4
1
1
1
2
3
3
3
4
4
5
1
1
1
2
2
3
3
5
5
5
1
Nodule
no.
1
2
1
2
3
1
2
3
1
2
3
1
2
1
2
3
1
2
3
1
2
3
2
1
2
3
2
1
2
3
1
2
2
1
2
3
1
2
1
2
1
2
3
1
13
cu12
cu13
cu21
cu22
cu23
cu31
cu32
cu33
cu41
cu43
cu51
cu52
cn13
cn21
cn22
cn23
cn31
cn32
cn41
cn42
cn43
cn51
cn52
cn53
dn13
dn21
dn22
dn23
dn41
dn42
dn52
du11
du12
du13
du21
du23
du31
du32
du33
du41
du42
du43
du51
du53
sn11
sn13
sn31
sn32
sn33
sn41
sn42
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
S
S
S
S
S
S
S
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
NZ
NZ
NZ
NZ
NZ
NZ
NZ
1
1
2
2
2
3
3
3
4
4
5
5
1
2
2
2
3
3
4
4
4
5
5
5
1
2
2
2
4
4
5
1
1
1
2
2
3
3
3
4
4
4
5
5
1
1
3
3
3
4
4
2
3
1
2
3
1
2
3
1
3
1
2
3
1
2
3
1
2
1
2
3
1
2
3
3
1
2
3
1
2
2
1
2
3
1
3
1
2
3
1
2
3
1
3
1
3
1
2
3
1
2
14
sn43
sn51
su11
su12
su13
su21
su22
su23
su32
su33
su41
su42
su43
su51
su53
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
NZ
NZ
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
UK
4
5
1
1
1
2
2
2
3
3
4
4
4
5
5
3
1
1
2
3
1
2
3
2
3
1
2
3
1
3
15
Table 6. Details for rhizobia isolates included in the nodD phylogeny for each Trifolium species (A = T.
arvense, C = T. campestre, D = T. dubium, O = T. ornithopodioides, P = T. pratense, R = T. repens, S =
T. striatum) from each range (New Zealand or United Kingdom). Field site numbers per species
correspond to Table 1. A maximum of three nodules were sampled from each species, per field site,
with each nodule originating from separate plants; the ‘nodule no.’ column indicates which nodule
per species/site the isolate originated from.
Isolate
reference
a11n
a11u
a12u
a13n
a13u
a21n
a22u
a31u
a32n
a33u
a41u
a42u
a51n
a52n
a52u
a53n
c11u
c12u
c13n
c13u
c21n
c21u
c22n
c22u
c23n
c31n
c31u
c32n
c32u
c33n
c33u
c41n
c41u
c42u
c43n
c43u
c51n
c51u
c52n
c52u
c53n
d11u
d12u
Species Range Field site
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
D
D
NZ
UK
UK
NZ
UK
NZ
UK
UK
NZ
UK
UK
UK
NZ
NZ
UK
NZ
UK
UK
NZ
UK
NZ
UK
NZ
UK
NZ
NZ
UK
NZ
UK
NZ
UK
NZ
UK
UK
NZ
UK
NZ
UK
NZ
UK
NZ
UK
UK
1
1
1
1
1
2
2
3
3
3
4
4
5
5
5
5
1
1
1
1
2
2
2
2
2
3
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
1
1
Nodule
no.
1
1
2
3
3
1
2
1
2
3
1
2
1
2
2
3
1
2
3
3
1
1
2
2
3
1
1
2
2
3
3
1
1
2
3
3
1
1
2
2
3
1
2
GenBank
accession no.
KT429304
KT429305
KT429306
KT429307
KT429308
KT429309
KT429310
KT429311
KT429312
KT429313
KT429314
KT429315
KT429384
KT429316
KT429317
KT429318
KT429319
KT429320
KT429321
KT429322
KT429323
KT429324
KT429325
KT429326
KT429327
KT429328
KT429329
KT429330
KT429331
KT429332
KT429333
KT429334
KT429335
KT429336
KT429337
KT429338
KT429339
KT429340
KT429341
KT429342
KT429343
KT429344
KT429345
16
d13n
d13u
d21n
d21u
d23n
d23u
d31u
d32u
d33u
d41u
d42n
d43u
d51u
d53n
o11n
o11u
o12u
o13u
o21n
o21u
o22n
o23u
o31n
o31u
o32n
o32u
o33n
o33u
o41u
o42n
o42u
o43n
o52u
o53u
p11u
p12n
p12u
p13n
p13u
p21u
p22n
p22u
p23n
p23u
p31n
p31u
p32n
p32u
p33n
p33u
p41u
D
D
D
D
D
D
D
D
D
D
D
D
D
D
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
NZ
UK
NZ
UK
NZ
UK
UK
UK
UK
UK
NZ
UK
UK
NZ
NZ
UK
UK
UK
NZ
UK
NZ
UK
NZ
UK
NZ
UK
NZ
UK
UK
NZ
UK
NZ
UK
UK
UK
NZ
UK
NZ
UK
UK
NZ
UK
NZ
UK
NZ
UK
NZ
UK
NZ
UK
UK
1
1
2
2
2
2
3
3
3
4
4
4
5
5
1
1
1
1
2
2
2
2
3
3
3
3
3
3
4
4
4
4
5
5
1
1
1
1
1
2
2
2
2
2
3
3
3
3
3
3
4
3
3
1
1
3
3
1
2
3
1
2
3
1
3
1
1
2
3
1
1
2
3
1
1
2
2
3
3
1
2
2
3
2
3
1
2
2
3
3
1
2
2
3
3
1
1
2
2
3
3
1
KT429346
KT429347
KT429348
KT429349
KT429350
KT429351
KT429385
KT429386
KT429387
KT429388
KT429352
KT429389
KT429390
KT429353
KT429354
KT429391
KT429392
KT429393
KT429355
KT429394
KT429356
KT429395
KT429357
KT429396
KT429358
KT429397
KT429359
KT429398
KT429399
KT429360
KT429400
KT429361
KT429401
KT429402
KT429403
KT429362
KT429404
KT429363
KT429405
KT429406
KT429364
KT429407
KT429365
KT429408
KT429366
KT429409
KT429367
KT429410
KT429368
KT429411
KT429412
17
p42n
p42u
p43u
p51u
p53u
r11u
r12u
r13u
r21n
r21u
r23n
r23u
r31n
r31u
r32n
r33u
r41n
r41u
r42n
r42u
r43n
r52n
r52u
r53u
s11n
s13n
s22u
s32n
s32u
s41n
s41u
s42n
s42u
s43n
s43u
s51u
s53u
P
P
P
P
P
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
S
S
S
S
S
S
S
S
S
S
S
S
S
NZ
UK
UK
UK
UK
UK
UK
UK
NZ
UK
NZ
UK
NZ
UK
NZ
UK
NZ
UK
NZ
UK
NZ
NZ
UK
UK
NZ
NZ
UK
NZ
UK
NZ
UK
NZ
UK
NZ
UK
UK
UK
4
4
4
5
5
1
1
1
2
2
2
2
3
3
3
3
4
4
4
4
4
5
5
5
1
1
2
3
3
4
4
4
4
4
4
5
5
2
2
3
1
3
1
2
3
1
1
3
3
1
1
2
3
1
1
2
2
3
2
2
3
1
3
2
2
2
1
1
2
2
3
3
1
3
KT429369
KT429413
KT429414
KT429415
KT429416
KT429417
KT429418
KT429419
KT429370
KT429420
KT429371
KT429421
KT429372
KT429422
KT429373
KT429423
KT429374
KT429424
KT429375
KT429425
KT429376
KT429377
KT429426
KT429427
KT429378
KT429379
KT429428
KT429380
KT429429
KT429381
KT429430
KT429382
KT429431
KT429383
KT429432
KT429433
KT429434
Table 7. Number of root samples and field sites used for TRFLP analysis of mycorrhizal communities
associated with Trifolium species in New Zealand (NZ) and the United Kingdom (UK).
Trifolium
species
T. arvense
T. arvense
T. fragiferum
T. fragiferum
T. repens
T. repens
Range
NZ
UK
NZ
UK
NZ
UK
Number of
samples
13
9
8
9
15
9
Number
of sites
4
3
3
3
5
3
18
Appendix S3: Additional results
Figure 1. The number of root samples in which each arbuscular mycorrhizal fungi operational
taxonomic unit (OTU) was detected in New Zealand (NZ) and the United Kingdom (UK).
19
Table 1. Results of GenBank BLAST searches (conducted in August 2014) showing the closest
taxonomic matches to 16S sequences obtained from Trifolium root-nodule bacterial isolates in New
Zealand (NZ) and the United Kingdom (UK) that failed to amplify with rhizobia-specific nodD primers.
Sequences from seven isolates (s31u, o52n, r22n, r32u, o13n, d11n, c12n) were of insufficient
quality for identification. Although this showed that isolates a23u, a43u and o23n were Rhizobium
leguminosarum bv. trifolii, they were not included in rhizobia rep-PCR or phylogenetic analyses.
Isolate
ref
Range
c11n
r13n
a42n
p51n
d43n
r51n
d52u
s12n
NZ
NZ
NZ
NZ
NZ
NZ
UK
NZ
p53n
d12n
a23n
o12n
s53n
p52n
s22n
a21u
a53u
c53u
r51u
o53n
s52n
NZ
NZ
NZ
NZ
NZ
NZ
NZ
UK
UK
UK
UK
NZ
NZ
d32n
r53n
a43n
d31n
d33n
d51n
r11n
s23n
s52u
o51n
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
UK
NZ
p43n
NZ
p41n
o23n
a23u
a43u
s21n
NZ
NZ
UK
UK
NZ
GenBank
accession of
closest match
KC243291.1
JQ977301.1
AB681415.1
FN397515.1
AY660698.1
KJ184943.1
HM566574.1
FR799426.1/
EU810854.1
AY660698.1
AB495351.1
KJ152098.1
HQ224646.1
GU188915.1
HM099662.1
JX195144.1
KC236770.1
KC236770.1
AJ315076.1
AB576894.1
KF941208.1
HE716932.1/
NR_102966.1
JN853238.1
JN853218.1
EU853188.1
HG940537.1
JX035940.1
EU853188.1
FJ225193.1
JQ080603.1
JX266407.1
KF555635.1/
JQ782497.1
KF555635.1/
JQ782497.1
KF817809.1
KF749031.1
KF748994.1
KF748994.1
JQ977536.1
Closest match taxa
Arthrobacter sp.
Arthrobacter sp.
Bacillus psychrosaccharolyticus
Bacillus simplex
Bacillus sp.
Bacillus sp.
Bacillus sp.
Bacillus sp. / Actinobacterium
Sequence
similarity
(%)
100
99
99
100
99
100
92
93/93
Overlap in
sequences
(%)
100
98
99
99
99
100
100
99/99
Bacillus sp. / Bacillus psychrosaccharolyticus
Duganella sp.
Enterobacter sp.
Erwinia billingiae
Erwinia billingiae
Erwinia sp.
Paenibacillus sp.
Paenibacillus sp.
Paenibacillus sp.
Paenibacillus sp.
Paenibacillus sp.
Pantoea agglomerans
Pantoea agglomerans / Pantoea vagans
98/98
99
100
99
99
100
99
99
99
99
99
99
94/94
100/100
100
93
99
100
100
100
99
99
99
100
100
100/100
Pantoea sp.
Pantoea sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp. / Pseudomonas fluorescens
93
99
99
99
99
100
100
99
99
100/100
100
100
100
99
100
100
100
100
100
100/100
Pseudomonas sp. / Pseudomonas fluorescens
100/99
100/100
Rahnella aquatilis
Rhizobium leguminosarum bv. trifolii
Rhizobium leguminosarum bv. trifolii
Rhizobium leguminosarum bv. trifolii
Yonghaparkia
99
100
100
100
99
100
100
100
100
99
20