1
Supplemental Results and Discussion.
2
Descriptions of the two Atheliaceae species.
3
Although morphologically similar to Amphinema byssoides, Amphinema sp.
4
SH029298.06FU (hereafter Amphinema sp.) was identifiable via sequence analysis as a
5
unique but closely related species (Supplemental Fig. 2). ITS sequence similarity was
6
consistently at 99-100% between our sequences and those characterized as Amphinema sp.,
7
versus 95-96% between our isolates and Amphinema byssoides, which is close but below the
8
typical intraspecific similarity threshold of ~97%, and well below the estimate for A.
9
byssoides of 99.7% intraspecific similarity in the ITS (Supplemental Table 3 of Nilsson et
10
al. 2008). Like A. byssoides, Amphinema sp. was characterized by a plectenchymatous
11
mantle with many emanating hyphae covered by small protuberances, giving the hyphae a
12
warty appearance (Supplemental Fig. 3a). “H” anastomoses between the emanating hyphae,
13
clamp connections, and somewhat loose rhizomorphs were commonly present.
14
Atheliaceae sp. SH029299.06FU (hereafter Atheliaceae sp.) was distinct from Amphinema
15
sp. and A. byssoides. Sequence similarity between Atheliaceae sp. and Amphinema sp. was
16
86-87%. As with Amphinema sp., the mantle was plectenchymatous and characterized by
17
profuse emanating hyphae, H anastomoses, and clamp connections. However, the hyphae of
18
Atheliaceae sp. were smooth (Supplemental Fig. 3b) and rhizomorphs were not observed.
19
Thelephora terrestris was also readily distinguishable, due to its DNA sequence and distinct
20
morphotype (Agerer 1987-2008).
21
22
23
Discussion regarding species identification.
24
Our results suggest that species in nursery settings identified as Amphinema byssoides might
25
represent at least two related taxa. Amphinema sp. is very close to A. byssoides both
26
morphologically and molecularly, and hence likely to be lumped with A. byssoides. It
27
appears that Amphinema sp. has been lumped with A. byssoides in sequencing studies of
28
EcMF on nursery seedlings (Kernaghan et al. 2003; Stenström et al. 2013). In fact, without
29
additional study, it is unclear whether they are conspecific or sister species. Based on these
30
studies and ours Amphinema sp. appears to be present in nurseries in both North America
31
(on P. glauca) and Europe (on P. abies). This pair of taxa might be best described as the A.
32
byssoides complex until species identities are clearly resolved.
33
Atheliaceae sp. should be readily distinguished in greenhouse surveys. Nevertheless in some
34
studies the Atheliaceae species in the present study have been lumped, e.g., Flykt et al.
35
(2008) identified one variant of their morphotype 4 (EU427328) as a strain of Amphinema
36
byssoides, although it has 99% similarity to Atheliaceae sp. and only 85% similarity to A.
37
byssoides. It is important to distinguish these species given their phylogenetic and possible
38
functional divergence. What is needed is a phylogenetic and taxonomic treatment of
39
Amphinema and related Atheliaceae that will permit easier communication about taxonomy
40
and function of these related species.
41
42
References
43
Agerer R (1987-2008) Color Atlas of Ectomycorrhizae. Einhorn-Verlag. Schwäbisch-
44
Gmünd, Germany.
45
Flykt E, Timonen S, Pennanen T (2008) Variation of ectomycorrhizal colonization in
46
Norway spruce seedlings in Finnish forest nurseries. Silva Fenn 42:571-585
47
48
Kernaghan G, Widden P, Bergeron Y, Légaré S, Par D (2003) Biotic and abiotic factors
49
affecting ectomycorrhizal diversity in boreal mixed-woods Oikos. 102:3:497-504
50
Nilsson RH, Kristiansson E, Ryberg M, Hallenberg N, Larsson K-H (2008) Intraspecific
51
ITS variability in the kingdom Fungi as expressed in the international sequence databases
52
and its implications for molecular species identification. Evol Bioinformatics 4:193-201
53
Stenström E, Ndobe NE, Jonsson M, Stenlid J, Menkis A (2014) Root-associated fungi of
54
healthy-looking Pinus sylvestris and Picea abies seedlings in Swedish forest nurseries.
55
Scand J For Res 29:12-21
56
57
58
Supplemental Figures
59
a
c
60
61
62
63
64
65
66
b
d
d
Supplemental Figure 1. a) examination of containerized seedlings for EcMF morphotypes;
b) brown mycelium of T. terrestris at base of container; c) white mycelium; d) white
mycelium (left) and yellow mycelium (right) of the Atheliaceae species at base of container.
Tylospora fibrillosa SH060014.06FU
Atheliaceae sp.SH060016.06FU
Atheliaceae sp. SH060015.06FU
Tylospora sp. SH022138.06FU|
Tylospora sp. SH029300.06FU
Amphinema sp. 7 JN943925
Amphinema sp. 3 JN943898
Amphinema sp. SH438147.06FU
Amphinema sp. SH015020.06FU
Amphinema sp. SH015021.06FU
Amphinema sp. SH015026.06FU
Amphinema sp. SH015023.06FU
Atheliaceae sp.SH029299.06FU
Basidiomycete EL100 AY010285
o21-1-2a
o12-1-1
n21-1-1
Amphinema sp. 5 JN943911
Amphinema sp. 5 JN943909
o24-4-1
n25-1-1
Amphinema sp. 1 JN943919.1
Amphinema byssoides AY219839.1
o16-4-1
Amphinema sp. 1 JN943927
o20-2-2
Amphinema sp. SH029298.06FU
n7-3-2
n22-3-2
Amphinema sp. SH029304.06FU
Amphinema sp. 4 JN943895.1
Amphinema sp. SH029305.06FU
Amphinema sp. 2 JN943915.1
Amphinema sp. SH029302.06FU
Amphinema sp. SH025002.06FU
Amphinema sp. SH025001.06FU
Amphinema sp. SH029303.06FU
Amphinema byssoides JQ711816.1
Amphinema byssoides GU550106
Amphinema byssoides GQ162810.1
Amphinema byssoides JN943932
Amphinema byssoides AY838271.1
Amphinema byssoides JN943908.1
Amphinema sp. O48 AJ534707.1
Amphinema byssoides JX907809.1
Amphinema byssoides SH029296.06FU
Amphinema sp. SH020869.06FU
Amphinema sp. SH020867.06FU
Amphinema sp. SH020868.06FU
0.1
67
68
69
70
71
72
73
Atheliaceae sp.
SH029299.06FU
Amphinema sp.
SH029298.06FU
Amphinema
byssoides
SH029296.06FU
Supplemental Figure 2. Neighbor joining phylogenetic tree of Atheliaceae including study
isolates. Reference or representative sequences for UNITE species hypotheses are indicated
by name as indicated in the database followed by SHXXXXXX.06FU; other sequences are
indicated by their taxonomic ID in GenBank followed by their GenBank accession number;
sequences bolded and preceded by “o” or “n” represent selected sequences of root tips from
the present study, indicating root tips from old or new parts of the root system, respectively.
74
75
76
77
78
79
80
a
b
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
Supplemental Figure 3. Hyphal characteristics of a) Amphinema sp.SH029298.06FU and b)
Atheliaceae sp. SH029299.06FU. Note the warty hyphal surface of the former, and smooth
hyphal surface of the latter. Scale is the same in both images.
4
N concentration (%)
a
3
2
1
0
Slopes differ, p = 0.0009
Slopes differ (p = 0.0005)
b
Mn concentration (%)
0.08
0.06
0.04
b
Slopes differ, p = 0.0005
0.02
B concentration (%)
c
0.004
0.002
Slopes differ, p = 0.0024
0.000
0.0
0.5
1.0
1.5
2.0
Amphinema root tips (log (1 + %))
104
105
106
107
Supplemental Figure 4. Slope comparison of regression lines for concentration of three
foliar nutrients as a function of Amphinema sp. root tip relative abundance. Solid symbols =
unfertilized, open symbols = fertilized.
108
109
Supplemental Tables
110
111
Supplemental Table 1. Concentration and forms of elements present in Scott’s Champion
Fertilizer mix used to fertilize seedlings.
Element
Form
Nitrogen
Ammonical N
8.70%
Nitrate N
12.30%
Phosphorus
Phosphate
8.00%
Potassium
Soluable potash
18.00%
Magnesium
Water soluble
0.15%
Boron
Concentration
0.0262%
Copper
Water soluble
0.0262%
Iron
Chelated
0.1050%
Manganese
Water soluble
0.0105%
Molybdenum
Water soluble
0.0525%
112
113
114
115
Supplemental Table 2. Percentage of the sampled white spruce seedling root tips colonized by the five ectomycorrhizal fungal species
or non-mycorrhizal, as determined by both morphotyping and sequence analysis.
Amphinema sp.
SH029298.06FU
Atheliaceae sp.
SH029299.06FU
Thelephora
terrestris
Sphaerosporella
brunnea
Boletus
variipes
Nonmycorrhizal
1
0
49
47
0
0
4
2
0
46
42
0
0
12
3
0
0
87
0
0
13
4
53
0
48
0
0
0
5
6
0
77
0
0
18
6
15
0
59
0
0
27
7
0
0
86
0
0
14
8
0
0
75
0
0
25
9
22
0
26
0
0
53
10
44
0
50
0
0
7
11
48
0
0
50
0
2
12
50
0
46
0
0
5
13
35
0
26
0
0
39
14
0
0
90
0
0
10
15
80
0
0
0
0
20
16
4
0
89
0
0
8
17
0
66
31
0
0
3
18
48
0
15
0
35
2
19
0
36
46
0
0
18
20
27
59
0
0
0
14
21
50
0
46
0
0
4
Seedling
Fertilized
Unfertilized
116
117
118
Supplemental Table 3. Mean ± SE foliar nutrient concentrations by fertilization treatment, and foliar nutrient deficiency thresholds
119
from Fisher & Binkley (2000) and references therein. Nutrient concentrations that are at or below published nutrient limitation
120
thresholds are indicated in bold.
Treatment
n
N
P
K
Ca
Mg
S
Mn
Fe
Cu
B
(g/kg)
121
Unfertilized
11
Fertilized
10
Deficiency
threshold
1 nd = no data
Al
Zn
Na
(mg/kg)
9.4 +
1.0
2.3 +
0.2
5.1 +
0.5
4.6 +
0.3
1.3 +
0.05
0.7 +
0.07
0.5 +
0.03
0.2 +
0.02
3.0 +
0.3
26 +
2.7
65.5 +
8.2
32.8 +
4.1
13.5 +
1.5
26.6
+ 2.5
2.8 +
0.2
4.4 +
0.5
5.5 +
0.5
1.6 +
0.1
0.8 +
0.03
0.5 +
0.05
0.2 +
0.01
3.3 +
0.2
27 +
3.8
60.9 +
5.2
51.7 +
6.6
12.2 +
1.4
10.512.5
1-1.4
2.5-3.0
1.0-1.5
0.5-0.8
nd1
0.025
.0.05
3
12
nd
15
nd
122
123
124
125
Supplemental Table 4. Average ± SE for seedling biomass and allocation, and p value for effect of
fertilization treatment.
Needle
(g)
Stem
(g)
Roots
(g)
Root:shoot
ratio
Fertilized
1.09 + 0.10
1.16 + 0.18
1.69 + 0.17
0.86 + 0.15
Aboveground
biomass
(g)
2.25 + 0..26
Unfertilized
0.91 + 0.08
0.72 + 0.07
1.27 + 0.15
0.77 + 0.05
1.63 + 0.15
2.90 + 0.29
0.05
0.003
0.01
0.1
0.01
0.02
Treatment
P value
126
127
128
129
130
Total biomass
(g)
3.94 + 0.36