Supplemental Results and Discussion.

Descriptions of the two Atheliaceae species.

Although morphologically similar to Amphinema byssoides, Amphinema sp. SH029298.06FU (hereafter Amphinema sp.) was identifiable via sequence analysis as a unique but closely related species (Supplemental Fig. 2). ITS sequence similarity was consistently at 99-100% between our sequences and those characterized as Amphinema sp., versus 95-96% between our isolates and Amphinema byssoides, which is close but below the typical intraspecific similarity threshold of ~97%, and well below the estimate for A. byssoides of 99.7% intraspecific similarity in the ITS (Supplemental Table 3 of Nilsson et al. 2008). Like A. byssoides, Amphinema sp. was characterized by a plectenchymatous mantle with many emanating hyphae covered by small protuberances, giving the hyphae a warty appearance (Supplemental Fig. 3a). “H” anastomoses between the emanating hyphae, clamp connections, and somewhat loose rhizomorphs were commonly present.

Atheliaceae sp. SH029299.06FU (hereafter Atheliaceae sp.) was distinct from Amphinema sp. and A. byssoides. Sequence similarity between Atheliaceae sp. and Amphinema sp. was 86-87%. As with Amphinema sp., the mantle was plectenchymatous and characterized by profuse emanating hyphae, H anastomoses, and clamp connections. However, the hyphae of Atheliaceae sp. were smooth (Supplemental Fig. 3b) and rhizomorphs were not observed. Thelephora terrestris was also readily distinguishable, due to its DNA sequence and distinct morphotype (Agerer 1987-2008).

Discussion regarding species identification.

Our results suggest that species in nursery settings identified as Amphinema byssoides might represent at least two related taxa. Amphinema sp. is very close to A. byssoides both morphologically and molecularly, and hence likely to be lumped with A. byssoides. It appears that Amphinema sp. has been lumped with A. byssoides in sequencing studies of EcMF on nursery seedlings (Kernaghan et al. 2003; Stenström et al. 2013). In fact, without additional study, it is unclear whether they are conspecific or sister species. Based on these studies and ours Amphinema sp. appears to be present in nurseries in both North America (on P. glauca) and Europe (on P. abies). This pair of taxa might be best described as the A. byssoides complex until species identities are clearly resolved.

Atheliaceae sp. should be readily distinguished in greenhouse surveys. Nevertheless in some studies the Atheliaceae species in the present study have been lumped, e.g., Flykt et al. (2008) identified one variant of their morphotype 4 (EU427328) as a strain of Amphinema byssoides, although it has 99% similarity to Atheliaceae sp. and only 85% similarity to A. byssoides. It is important to distinguish these species given their phylogenetic and possible functional divergence. What is needed is a phylogenetic and taxonomic treatment of Amphinema and related Atheliaceae that will permit easier communication about taxonomy and function of these related species.

References

Agerer R (1987-2008) Color Atlas of Ectomycorrhizae. Einhorn-Verlag. Schwäbisch-Gmünd, Germany.

Flykt E, Timonen S, Pennanen T (2008) Variation of ectomycorrhizal colonization in Norway spruce seedlings in Finnish forest nurseries. Silva Fenn 42:571-585

Kernaghan G, Widden P, Bergeron Y, Légaré S, Par D (2003) Biotic and abiotic factors affecting ectomycorrhizal diversity inboreal mixed-woods Oikos. 102:3:497-504

Nilsson RH, Kristiansson E, Ryberg M, Hallenberg N, Larsson K-H (2008) Intraspecific ITS variability in the kingdom Fungi as expressed in the international sequence databases and its implications for molecular species identification. Evol Bioinformatics 4:193-201

Stenström E, Ndobe NE, Jonsson M, Stenlid J, Menkis A (2014) Root-associated fungi of healthy-looking Pinus sylvestris and Picea abies seedlings in Swedish forest nurseries.Scand JFor Res 29:12-21

Supplemental Figures

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.

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.

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.


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.

Supplemental Tables

Supplemental Table 1.Concentration and forms of elements present in Scott’s Champion Fertilizer mix used to fertilize seedlings.

Element / Form / Concentration
Nitrogen / Ammonical N / 8.70%
Nitrate N / 12.30%
Phosphorus / Phosphate / 8.00%
Potassium / Soluable potash / 18.00%
Magnesium / Water soluble / 0.15%
Boron / 0.0262%
Copper / Water soluble / 0.0262%
Iron / Chelated / 0.1050%
Manganese / Water soluble / 0.0105%
Molybdenum / Water soluble / 0.0525%

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.

Seedling / Amphinema sp. SH029298.06FU / Atheliaceae sp. SH029299.06FU / Thelephora terrestris / Sphaerosporella brunnea / Boletus variipes / Non-mycorrhizal
Fertilized / 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
Unfertilized / 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

Supplemental Table 3. Mean ± SE foliar nutrient concentrations by fertilization treatment, and foliar nutrient deficiency thresholds from Fisher & Binkley (2000) and references therein. Nutrient concentrations that are at or below published nutrient limitation thresholds are indicated in bold.

Treatment / n / N / P / K / Ca / Mg / S / Mn / Fe / Cu / B / Al / Zn / Na
(g/kg) / (mg/kg)
Unfertilized / 11 / 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
Fertilized / 10 / 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
Deficiency threshold / 10.5-12.5 / 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

1nd = no data

Supplemental Table 4.Average ± SE for seedling biomass and allocation, and p value for effect of fertilization treatment.

Treatment / Needle
(g) / Stem
(g) / Roots
(g) / Root:shoot ratio / Aboveground biomass
(g) / Total biomass (g)
Fertilized / 1.09 + 0.10 / 1.16 + 0.18 / 1.69 + 0.17 / 0.86 + 0.15 / 2.25 + 0..26 / 3.94 + 0.36
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
P value / 0.05 / 0.003 / 0.01 / 0.1 / 0.01 / 0.02