Supporting Information
A.
Temperature (°C)
30
25
20
15
10
5
0
Max Temp
Rainfall (cm)
B.
Min Temp
9
8
7
6
5
4
3
2
1
0
Rain (cm)
Figure S1.Weather data during the study period measured at the University of Georgia’s San
Luis Weather Station. (A) The daily maximum and minimum temperature are relatively stable,
while (B) the daily rainfall totals indicate the transition from dry to wet seasons during the study
period of February through July, 2011.
Appendix S1. Detailed description of genetic barcoding of seedling-associated fungi.
Total DNA was extracted from protocorm samples using a CTAB protocol (Doyle & Doyle
1990). We amplified the fungal nuclear ribosomal internal transcribed spacer (ITS) with primers
ITS1F/ITS4 (White et al. 1990), ITS1OF/ITS4OF (Taylor & McCormick 2008) or the Tulasnella
specific combination ITS1/ITS4-TUL (Taylor & McCormick 2008). We also attempted to
amplify the mitochondrial ribosomal large subunit (mtLSU) with primers ML5/ML6 (White et
al. 1990). Reactions contained 1X ThermoPol Reaction Buffer (NEB; New England Biolabs,
Ipswich, MA), 1.0 mM MgCl2 (Sigma-Aldrich, St. Louis, MO), 0.25 µM each dNTP (NEB), 0.1
µM each primer, 0.0125 mg Bovine Serum Albumin (BSA; NEB), 10-20 ng DNA, and 0.5 units
NEB Taq DNA polymerase in 15 µL reactions. Reactions included denaturation for 5min at
94°C followed by 35 cycles of denaturation at 94°C for 30s, annealing at 55°C or 53°C for 30s,
and extension at 72°C for 45s, with a final extension at 72°C for 10min. We attempted molecular
cloning when multiple PCR product bands were visualized on an agarose gel or sequencing
failed. PCR products were ligated in the pDrive cloning vector (Qiagen, Valencia, California,
USA) and transformed into StrataGene XL-10 Gold Ultracompetent cells (StrataGene, Agilent
Technologies, Santa Clara, CA) for blue-white screening. Cloned DNA was re-amplified using
the original primers. Products were purified by ExoSAP-IT (Sigma-Aldrich) and sequenced with
BIGDYE v 3.1 (ABI; Applied Biosystems, Foster City, CA) on an ABI3730 sequencer at the
University of Georgia Genomics Facility (University of Georgia, Athens, GA). Sequences were
edited and assembled in Sequencher 4.9 (GeneCodes, Ann Arbor, MI). We used NCBI BLAST
searches to identify fungi by sequence similarity. Sequences were deposited in GenBank
(accession numbers JX999046 - JX999054).
2
Appendix S2. Detailed description of conditional inference trees.
Conditional inference trees are a type of permutation tree useful for identifying ecological
patterns when variables may interact hierarchically. The algorithm first tests the global null
hypothesis that all predictor variables are independent of each other and the response variable. If
this hypothesis is rejected, the algorithm tests a partial null hypothesis that single predictor
variables are not associated with the response variable in order to identify the predictor with the
greatest association with the response variable as indicated by P-values. At this point, the data
are split into two nodes based on this predictor variable and the process repeats until no splits are
possible by the test criteria. The minimum split criterion in our analysis was 0.95, which
corresponds to a univariate P-value of 0.05. A minimum of 20 observations were required to
split an internal node was and a minimum of seven were required for terminal nodes. Since
conditional inference trees are non-parametric, results are presented with non-transformed data.
Since transformation can reduce variance and influence splitting in conditional inference trees,
we repeated analyses using log-transformed branch diameter, square root-transformed openness,
and arc-sine square root-transformed proportion of colonized root sections, which improved
normality did not produce qualitative differences that would alter interpretations. Canopy
openness and branch diameter are significantly collinear in both the seed pack analysis (n = 240,
r = -0.26, P = 0.004) and the adult mycorrhizal analysis (n = 51, r = -0.336, P = 0.016), so only
canopy openness is considered. While there was some overlap in host tree species among study
sites, the distribution of tree species was not fully independent of study site. The study site ALO
in particular comprised several different large remnant tree species. However, we included site
and tree species as possible predictors since both study site and the random effect of tree were
significant in the ordinal analysis and these variables may interact hierarchically (Table 1).
3
4
Table S1. A high diversity of seedling-associated fungi was identified through genetic barcoding and BLAST searches.
Accession
Seed
Study
Seed
Host tree
Fungal affiliation
Role
Nearest taxon
Match
e-
%
source
site
pack
JX999048
ALO
EFR
018
Inga
B Tremellales
P
Uncultured Tremellales
GU993525
1e-149 92
JX999049
ALO
EFR
018
Inga
A Hysteriales
S
Glonium pusillum
EU55134
0
99
JX999050
REF
EFR
022
Sapium
A Pleosporales
OA
Pyrenocheta
HM208715
0
91
JX999051
REF
CAB
040
Sapium
A Hypocreales
OA
Fusarium oxysporum
FJ466709
0
100
JX999054
ALO
CAB
043
Sapium
A Pleosporales
OA
Pleosporales sp.
HM855222
0
100
JX999052
ALO
CAB
043
Sapium
B Tremellales
E
Bullera mrakii
AF314973
0
100
JX999053
ALO
CAB
043
Sapium
B Septobasidiales
P
Septobasidium pallidum
HM209415
0
94
JX999046
ALO
ALO
065
Ehretia
B Cantharellales
OM
Uncultured
HQ687894
0
88
value
Ceratobasidium
JX999047
ALO
ALO
067
Nectandra
A Pleosporales
OA
Uncultured Ascomycete
EU489902
0
100
R084VSA†
REF
ALO
084
Erythrina
B Cantharellales
OM
Cantharellus
AD001574
2e-78
97
cinnabarinus
5
Note: Sequences are ITS except for one mtLSU sequence (†). The GenBank accession number is given for each sequence except
R084VSA, which is too short for submission to GenBank and is listed below. Seed source, study site, seed packet, and host tree
species (by genus) are listed for each sequence. Fungal affiliation shows the inferred major division of fungi (A=Ascomycete,
B=Basidiomycete) and order. Role shows the possible ecological nature of the association based on the inferred taxonomy (E = Plant
Endophyte, OA = Orchid Associate, OM = Orchid Mycorrhiza, P = Pathogen, S = Saprotroph). The nearest taxon name and matching
accession number for each the nearest GenBank match are listed with the corresponding e-value and % identity to the sequence.
R084VSA:
ATGCTTGTAGAGACAGTGGGCCCATCGTTACTTTATTCATGCAGGACCGTATTTAGCGGACAATGAATTTTGCTACCTTT
GGATCCTTATAGTTAAGACCGCCATTGACCAGATTTTCAKTCGGTAATATTAAACTCACCTTCTTTATATTAATGGCATT
GGGCAAAATTCGACACCTAT