SUPPORTING INFORMATION
TABLE S1. Geographic locations of 11 sampled study populations.
Pop.
Region
Locality
N
Sampling dates mASL
Habitat type
ERP
Guanacaste
Rincon de la Vieja
6
July, 2009
975
Primary forest
MIR
Guanacaste
Miravalles
11
February, 2010
892
Pasture
UMV N. Tilarán
Monteverde
11
July, 2009
1233
Roadside
N. Tilarán
Monteverde
16
July, 2009;
1350
Pasture
EMV
July, 2011
ETT
N. Tilarán
Monteverde
6
July, 2009
1412
Roadside
ALO
N. Tilarán
San Luis
16
July, 2009;
1071
Pasture
1096
Pasture
1069
Pasture
July, 2011
CAB
N. Tilarán
San Luis
10
January, 2009;
July, 2009;
July, 2011
EFR
N. Tilarán
San Luis
11
July, 2009;
July, 2011
ECE
S. Tilarán
Cedral
13
March, 2010
1312
Roadside
ESR
S. Tilarán
San Ramon
7
March, 2011
1123
Roadside
ECH
Talamanca
Chirropó
12
July, 2010
1503
Roadside
Note: N = the number of plants yielding at least one fungal sequence; mASL = elevation in
meters above sea level.
APPENDIX S1. Assessing sampling sufficiency.
We bootstrapped sequences from the best sampled populations with contrasting levels of
diversity in each group (Tulasnellaceae = ALO, MIR; Sebacinales = ALO, EFR). We resampled
populations two to the maximum number of sequences/population, 1000 times without
replacement, in order to estimate standard error π at each sample size with pegas (Paradis 2010)
in R v.2.15.1 (R Core Development Team 2010). We also produced sample based rarefaction
curves for haplotype and phylogenetic richness per population with picante (Kembel et al. 2010)
in R (R Core Development Team 2010). Because some haplotypes that were distinguishable
across ITS were indistinguishable across 5.8S, phylogenetic diversity analyses only considered
phylogenetically distinguishable haplotypes. The curves show that mycorrhizal diversity within
the best sampled populations asymptotes rapidly and that phylogenetic richness asymptotes
much more rapidly than haplotype richness (Figure S1). Populations with ≥ 5 sequences should
provide robust estimates of mycorrhizal diversity for both taxa. We might find more haplotypes
within each of these groups by sampling more populations, but new haplotypes are unlikely to
add substantial phylogenetic diversity. Thus, we adequately sampled most populations.
a.
b.
15
Phylogenetically distinguishable haplotype richness
Phylogenetic richness
Richness
10
5
0
1
2
3
4
5
6
7
8
9
10
11
Sites
c.
d.
0.25
ALO
EFR
Nucleotide Diversity
0.20
0.15
0.10
0.05
0.00
-0.05
0
2
4
6
8
10
12
14
Number Samples
FIGURE S1. Assessing sampling sufficiency with resampling procedures. Curves are shown for
Tulasnellaceae (a-b) and Sebacinales (c-d). These curves show resampling of ITS nucleotide
diversity (π) in the two best sampled populations (a,c) and the haplotype and phylogenetic
richness across populations (b,d).
APPENDIX S2. Detailed Summary of Fungal Identification.
Here, we detail the total taxonomic diversity of fungi associating with the roots of
Epidendrum firmum, including taxa that are not known to form mycorrhizal symbioses with
orchids. Where possible, we assess taxonomic congruence between the ITS and mtLSU loci.
The top BLAST match to the GenBank database was recorded for each sequence (Table
S2). Searches for additional matches were performed in cases where the top match did not
provide a clear indication of taxonomy. We identified 25 basidiomycete MOTUs from 260
sequences spanning the 5.8S region and at least five additional MOTUs from 43 mtLSU
sequences (Figure 2). Orchid-mycorrhizal taxa include Cantharellales (Tulasnellaceae and
Ceratobasidiaceae), Sebacinales, Thelephorales, Russulales and Boletales. The mtLSU
confirmed the symbioses with Tulasnellaceae, including 30 plants (six in addition to ITS
sequences; Figure 2) in nine populations, where one plant associated with >1 mtLSU MOTU.
Most plants for which both Tulasnellaceae ITS and mtLSU sequences are available (22/24
plants; 92%) yielded matching MOTUs for both loci. Although we did not obtain mtLSU
sequences from plants yielding ITS MOTU A or B, these MOTUs are well-supported by 5.8S
(Figure S2). We did not obtain mtLSU sequences for Sebacinales, possibly due to primer
inefficiency. Other basidiomycetes are unlikely to form mycorrhizas, but two Trechisporales
MOTUs were sampled from five sites may frequently form an unknown symbiotic association
(Figure 2).
We identified 20 MOTUs from 62 ascomycete sequences (Figure 2). Potential orchid
mycorrhizal ascomycetes include the Helotiales, but the relationship with most ascomycetes is
unclear as they could be generalist pathogens or saprotrophs that opportunistically invade the
root velamen without forming mycorrhizas (Herrera et al. 2010). Many of the ascomycetes are
similar to endophytes of Bletilla spp. in southwest China (Tao et al. 2008) and orchids of La
Reunion Island (Martos et al. 2012). Relatively frequent associates include the Hypocreales,
Capnodiales and Pleosporales (Figure 2). Fusarium spp. (Hypocreales) can induce germination
in Cypripedium spp. seeds (Vujanovic et al. 2000). One plant associated with four Capnodiales
MOTUs and another associated with two (Figure 2), suggesting this is a non-specific and
opportunistic association. A single MOTU similar to Nigrospora sp. (Trichosphaeriales)
occurred in seven plants from five sites and may represent a specialized parasite (Figure 2).
Multiclavula vernalis - U66439
Multiclavula corynoides - U66440
Uncultured Tulasnellaceae from Cypripedium parviflorum (Orchidaceae) - DQ925545
Uncultured Tulasnellaceae from Cypripedium japonicum (Orchidaceae) - DQ925645
Epulorhiza sp. from Pe-QS-0-1 (Orchidaceae) - GU166408
Uncultured Tulasnellaceae from Cypripedium debile (Orchidaceae) - DQ925509
Uncultured Tulasnellaceae from Cypripedium montanum (Orchidaceae) - DQ925513
1000 Uncultured Tulasnellaceae fromCypripedium macranthon var. rebunense(Orchidaceae) - DQ925528
Uncultured Tulasnellaceae fromCypripedium macranthon var. rebunense(Orchidaceae) - DQ925527
Uncultured Tulasnellaceae from Cypripedium reginae (Orchidaceae) - DQ925524
Uncultured Cantharellales from Riccardia sp. (Aneuraceae) - DQ368717
Uncultured Tulasnellaceae fromCypripedium macranthon var. rebunense(Orchidaceae) - DQ925603
Uncultured Tulasnellaceae from Cypripedium montanum (Orchidaceae) - DQ925572
509
Uncultured Tulasnellaceae from Cypripedium guttatum (Orchidaceae) - DQ925577
Uncultured Tulasnellaceae from Cypripedium reginae (Orchidaceae) - DQ925590
Uncultured Tulasnellaceae from Cypripedium montanum (Orchidaceae) - DQ925601
Uncultured Tulasnellaceae from Cypripedium montanum (Orchidaceae) - DQ925602
Uncultured Tulasnellaceae from Cypripedium parviflorum (Orchidaceae) - DQ925550
924
Uncultured Tulasnellaceae fromCypripedium macranthon var. rebunense(Orchidaceae) - DQ925549
Uncultured Tulasnellaceae from Cypripedium fasciculatum (Orchidaceae) - DQ925610
848
Uncultured Tulasnellaceae from Cypripedium fasciculatum (Orchidaceae) - DQ925637
656Uncultured Tulasnellaceae from Cypripedium fasciculatum (Orchidaceae) - DQ925638
Uncultured Tulasnellaceae from Cypripedium fasciculatum (Orchidaceae) - DQ925620
Uncultured Tulasnellaceae from Cypripedium californicum (Orchidaceae) - DQ925636
Uncultured Tulasnellaceae from Cypripedium reginae (Orchidaceae) - DQ925615
Uncultured Tulasnellaceae from Cypripedium fasciculatum (Orchidaceae) - DQ925625
Uncultured Tulasnellaceae from Cypripedium reginae (Orchidaceae) - DQ925640
Uncultured Tulasnellaceae from Cypripedium debile (Orchidaceae) - DQ925505
912Uncultured Tulasnellaceae from Cypripedium debile (Orchidaceae) - DQ925506
729
Symbiont of E. firmum - Talamanca - ECH - OTU6 - JX998961
Uncultured Tulasnellaceae from Orchidaceae - JF69150 3
Uncultured Cantharellales from Orchidaceae - HM451571
1000 675 Uncultured Cantharellales from Orchidaceae - HM451854
668
730 Uncultured Tulasnella from Stelis hallii (Orchidaceae) - DQ178118
Uncultured Tulasnellaceae from Orchidaceae - JF69099 4
551 Uncultured Tulasnellaceae from Orchidaceae - JF69125 9
540
Tulasnella tomaculum - AY373296
Tulasnella violea - AY373293
Uncultured Cantharellales from Orchidaceae - HM451713
Uncultured Cantharellales from Orchidaceae - HM451605
Tulasnella violea - DQ520097
593 Symbiont of E. firmum - Tilaran - ETT - OTU1 - JX998860
573
981Symbiont of E. firmum - Tilaran - ETT - OTU1 - JX998892
709
Symbiont of E. firmum - Tilaran - EFR - OTU1 - JX998908
990
Symbiont of E. firmum - Tilaran - ECE - OTU0 - JX998872
754 Symbiont of E. firmum - Tilaran - ECE - OTU0 - KC440181
633 Symbiont of E. firmum - Talamanca - ECH - OTU0 - JX998888
Symbiont of E. firmum - Guanacaste - MIR - OTU0 - JX998901
Symbiont of E. firmum - Tilaran - EFR - OTU0 - JX998907
Symbiont of E. firmum - Talamanca - ECH - OTU0 - JX998864
Symbiont of E. firmum - Talamanca - ECH - OTU0 - JX998882
Symbiont of E. firmum - Talamanca - ECH - OTU0 - JX998878
Symbiont of E. firmum - Guanacaste - MIR - OTU0 - JX998904
Symbiont of E. firmum - Guanacaste - MIR - OTU0 - JX998900
Symbiont of E. firmum - Talamanca - ECH - OTU0 - JX998960
862
Tulasnella sp. from Tipularia discolor (Orchidaceae) - AY373316
Uncultured Tulasnellaceae from Orchidaceae - JF69104 5
998 Tulasnella sp. from CI-QS (Orchidaceae) - GU166416
518Uncultured Tulasnellaceae from Orchidaceae - JF69110 3
Uncultured Tulasnellaceae from Orchidaceae - JF69151 1
Uncultured Tulasnellaceae from Orchidaceae - JF69137 1
Uncultured Tulasnellaceae from Cypripedium californicum (Orchidaceae) - DQ925493
Uncultured Tulasnellaceae from Cypripedium californicum (Orchidaceae) - DQ925499
691 Uncultured Tulasnellaceae fromCypripedium californicum (Orchidaceae) - DQ925498
670
Uncultured Tulasnellaceae from Cypripedium californicum (Orchidaceae) - DQ925497
Uncultured Cantharellales from Orchidaceae - HM451639
635
Uncultured Cantharellales from Orchidaceae - HM451638
999
Uncultured Cantharellales from Orchidaceae - HM451577
779 Uncultured Cantharellales from Orchidaceae - HM451649
Uncultured Cantharellales from Orchidaceae - HM451789
Uncultured Cantharellales from Orchidaceae - HM451651
650
Uncultured Cantharellales from Orchidaceae - HM451648
Uncultured Cantharellales from Orchidaceae - HM451755
Uncultured Tulasnellaceae from Orchidaceae - JF69104 0
Uncultured Cantharellales from Orchidaceae - HM451552
673
Uncultured Cantharellales from Orchidaceae - HM451644
Tulasnella sp. from Tipularia discolor (Orchidaceae) - AY373300
Uncultured Tulasnellaceae from Orchidaceae - JF69136 8
992
Tulasnella sp. from Tipularia discolor (Orchidaceae) - AY373304
Uncultured Cantharellales from Orchidaceae - HM451733
Tulasnella sp. from Tipularia discolor (Orchidaceae) - AY373315
Uncultured Tulasnellaceae from Orchidaceae - JF69104 7
813 Uncultured Cantharellales from Orchidaceae - HM451505
730 Uncultured Cantharellales from Orchidaceae - HM451538
630
Uncultured Cantharellales from Orchidaceae - HM451504
Uncultured Cantharellales from Orchidaceae - HM451518
Uncultured Cantharellales from Orchidaceae - HM451740
Uncultured Cantharellales from Orchidaceae - HM451688
Tulasnella violea - AY373303
Tulasnella albida - AY373294
Tulasnella pruinosa - AY373295
Uncultured Cantharellales from Orchidaceae - HM451507
Uncultured Tulasnellaceae from Orchidaceae - JF69150 5
Tulasnella asymmetrica from Thelymitra luteocilium (Orchidaceae) - DQ388046
Tulasnella asymmetrica from Thelymitra luteocilium (Orchidaceae) - DQ388047
Tulasnella sp. from Pv-QS-0-2 (Orchidaceae) - GU166405
Uncultured Tulasnellaceae from Orchidaceae - JF69099 7
Tulasnella sp. from CI-QS (Orchidaceae) - GU166417
Uncultured Tulasnellaceae from Orchidaceae - JF69131 7
Uncultured Cantharellales from Orchidaceae - HM451566
Uncultured Tulasnella from Stelis superbiens (Orchidaceae) - DQ178074
Symbiont of E. firmum - Guanacaste - MIR - OTU2 - JX998966
Symbiont of E. firmum - Guanacaste - MIR - OTU2 - JX998898
Uncultured Tulasnellaceae from Orchidaceae - JF69103 0
Symbiont of E. firmum - Guanacaste - MIR - OTU2 - JX998997
Uncultured Cantharellales from Orchidaceae - HM451877
639
Uncultured Cantharellales from Orchidaceae - HM451879
Uncultured Cantharellales from Orchidaceae - HM451739
Uncultured Cantharellales from Orchidaceae - HM451878
Symbiont of E. firmum - Guanacaste - MIR - OTU2 - JX998895
Uncultured Tulasnella sp. From Cryptothallus mirabilis (Aneuraceae) - AY192472
635
Uncultured Tulasnellaceae from Orchidaceae - JF69133 7
995
Tulasnella irregularis from C3-DT-TC-2 (Orchidaceae) - GU166423
Uncultured Cantharellales from Orchidaceae - HM451508
Uncultured Cantharellales from Orchidaceae - HM451716
509 Uncultured Tulasnellaceae from Orchidaceae - JF69113 2
618
Uncultured Tulasnellaceae from Orchidaceae - JF69140 4
Symbiont of E. firmum - Tilaran - UMV - OTU3 - JX998928
953 Symbiont of E. firmum - Tilaran - ALO - OTU3 - JX998849
Symbiont of E. firmum - Tilaran - ECE - OTU3 - JX998876
Uncultured Tulasnellaceae from Orchidaceae - JF69106 0
Uncultured Cantharellales from Orchidaceae - HM451584
Uncultured Tulasnellaceae from Orchidaceae - JF69151 7
Uncultured Tulasnellaceae from Orchidaceae - JF69127 0
Uncultured Cantharellales from Orchidaceae - HM451512
Tulasnella calospora from Dcr-QS-02 (Orchidaceae) -GU166412
Symbiont of E. firmum - Guanacaste - ERP - OTU4 - JX998857
Symbiont of E. firmum - Guanacaste - ERP - OTU4 - JX998947
Symbiont of E. firmum - Tilaran - ECE - OTU4 - JX998929
Symbiont of E. firmum - Tilaran - EMV - OTU4 - JX998999
Symbiont of E. firmum - Tilaran - EMV - OTU4 - JX998989
Symbiont of E. firmum - Guanacaste - ERP - OTU4 - JX998858
Symbiont of E. firmum - Tilaran - ETT - OTU4 - JX998861
Symbiont of E. firmum - Tilaran - CAB - OTU4 - JX998943
Symbiont of E. firmum - Tilaran - CAB - OTU4 - JX998910
Symbiont of E. firmum - Tilaran - ECE - OTU4 - JX998956
Symbiont of E. firmum - Tilaran - ECE - OTU4 - JX998953
Symbiont of E. firmum - Tilaran - ALO - OTU4 - JX998976
Tulasnella danica - AY373297
Symbiont of E. firmum - Tilaran - ALO - OTU4 - JX998998
Uncultured Tulasnellaceae from Orchidaceae - JF69144 0
Tulasnella calospora from Pch-SM-TC-1 (Orchidaceae) - GU166404
Tulasnella calospora from Acianthus exsertus (Orchidaceae) - DQ388041
Uncultured Tulasnellaceae from Orchidaceae - JF69128 0
Uncultured Tulasnellaceae from Orchidaceae - JF69111 5
Symbiont of E. firmum - Tilaran - ESR - OTU4 - JX998994
Symbiont of E. firmum - Tilaran - ESR - OTU4 - JX998965
Symbiont of E. firmum - Tilaran - EMV - OTU4 - JX999000
Symbiont of E. firmum - Tilaran - EMV - OTU4 - JX999002
Symbiont of E. firmum - Tilaran - EMV - OTU4 - JX999003
Symbiont of E. firmum - Guanacaste - MIR - OTU4 - JX998967
Symbiont of E. firmum - Tilaran - UMV - OTU4 - JX998919
Symbiont of E. firmum - Guanacaste - ERP - OTU4 - JX998948
Symbiont of E. firmum - Tilaran - UMV - OTU4 - JX998916
Symbiont of E. firmum - Tilaran - EFR - OTU4 - JX998934
Symbiont of E. firmum - Tilaran - UMV - OTU4 - JX998921
Symbiont of E. firmum - Tilaran - ESR - OTU4 - JX998931
Symbiont of E. firmum - Tilaran - ESR - OTU4 - JX998930
Symbiont of E. firmum - Tilaran - ESR - OTU4 - JX998995
Symbiont of E. firmum - Tilaran - EMV - OTU4 - JX998992
Symbiont of E. firmum - Tilaran - EMV - OTU4 - JX998991
Symbiont of E. firmum - Tilaran - ECE - OTU4 - JX998870
Symbiont of E. firmum - Tilaran - EMV - OTU4 - JX998936
Symbiont of E. firmum - Tilaran - ETT - OTU4 - JX998894
Symbiont of E. firmum - Tilaran - ECE - OTU4 - JX998868
Tulasnella calospora from DCr-QS-01 (Orchidaceae) - GU166419
Symbiont of E. firmum - Tilaran - ALO - OTU4 - JX99898 0
Symbiont of E. firmum - Tilaran - ALO - OTU4 - JX99897 8
Symbiont of E. firmum - Tilaran - CAB - OTU4 - JX998970
Tulasnella sp. from Goodyera pubescens (Orchidaceae) - AY373275
Tulasnella sp. from Goodyera pubescens (Orchidaceae) - AY373270
Symbiont of E. firmum - Talamanca - ECH - OTU4 - JX998879
Symbiont of E. firmum - Tilaran - ALO - OTU4 - JX998985
Symbiont of E. firmum - Tilaran - ALO - OTU4 - JX998852
Symbiont of E. firmum - Tilaran - ALO - OTU4 - JX998853
Symbiont of E. firmum - Tilaran - ALO - OTU4 - JX998975
Symbiont of E. firmum - Tilaran - EMV - OTU4 - JX998937
Symbiont of E. firmum - Tilaran - EMV - OTU4 - JX998935
Symbiont of E. firmum - Tilaran - EMV - OTU4 - JX998942
Symbiont of E. firmum - Tilaran - EMV - OTU4 - JX998939
Symbiont of E. firmum - Tilaran - ALO - OTU4 - JX998983
Symbiont of E. firmum - Tilaran - ALO - OTU4 - JX998941
Tulasnella calospora from Paphiopedilum barbatum (Orchidaceae) - HM450046
Tulasnella bifrons - AY373290
Tulasnella calospora from PS-AT-0-1 (Orchidaceae) - GU166422
Tulasnella calospora from PS-AT-0-2 (Orchidaceae) - GU166429
717
Uncultured Cantharellales from Orchidaceae - HM451500
933
Tulasnella calospora - FJ755217
Uncultured Tulasnella from Andean forest - GU732322
Uncultured Cantharellales from Orchidaceae - HM451766
Uncultured Cantharellales from Orchidaceae - HM451522
Tulasnella calosporafrom Cymbidium sinense (Orchidaceae) - EF393622
Tulasnella calospora - AY373298
Tulasnella diliquescens - AY373291
Uncultured Cantharellales from Orchidaceae - HM451723
Uncultured Tulasnella from Stelis superbiens (Orchidaceae) - DQ178101
Uncultured Tulasnella from Stelis superbiens (Orchidaceae) - DQ178105
MOTU A
MOTU B
MOTU C
MOTU D
MOTU E
MOTU F
Samples from
Guanacaste
Tilarán
Talamanca
0.2
FIGURE S2. Phylogeny of Tulasnellaceae based on 5.8S. Values at nodes represent ≥50%
bootstrap support out of 1000 iterations. Epidendrum firmum symbionts are listed by mountain
range, population, MOTU and GenBank accession number. Shaded bars represent samples from
each mountain range.
Auricularia auricula-judae - FJ462752
Auricularia auricula-judae - EU520237
Auricularia polytricha - FJ792587
Sebacina calcea - AJ427408
577
882
Sebacina sp. from Quercus douglasii (Fagaceae) - DQ974769
Symbiont of E. firmum - Tilaran - EFR - OTU19 - JX998775
Sebacina mycorrhiza from Neottia nidus-avis (Orchidaceae) - AF440658
700
Sebacina epigaea - EU819537
958 Sebacina incrustans - EU819442
Uncultured Sebacinaceae from Pleurothallis lilijae (Orchidaceae) - EU107987
Uncultured Sebacinales from Pinus sylvestris forest soil - FJ475810
Uncultured Sebacinales from Cavendishia bracteata (Ericaceae) - HM230851
Uncultured Sebacinales from Orchidaceae - JF691281
Uncultured Sebacinaceae fromStelis superbiens (Orchidaceae) - EU107986
670
Symbiont of E. firmum - Tilaran - ALO - OTU13 - JX998829
825
Symbiont of E. firmum - Guanacaste - MIR - OTU13 - JX998836
983
Uncultured Sebacina mycobiont of Phleum pratense (Poaceae) - EU910901
993fungal sp. from Dactylorhiza incarnata (Orchidaceae) - AM697891
MOTU G
MOTU H
Uncultured mycorrhizal fungus of Dactylorhiza incarnata (Orchidaceae) - AJ549120
Uncultured Sebacinales Ceratandra grandiflora(Orchidaceae) - FJ788822
Sebacina vermifera (Oberw.) P. Roberts isolate K228 - EU625993
Sebacina
vermifera (Oberw.) P. Roberts isolate K224 - EU625991
502
Sebacina vermifera from Eriochilus scaber (Ericaceae) - FN663136
Epacris pulchella (Ericaceae) root associated fungus - AY627837
Uncultured Sebacinales from Orchidaceae - HM451799
Uncultured Sebacina from Veratrum album (Melanthiaceae) - HQ154305
683
Uncultured Sebacinales from Pterygodium catholicum (Orchidaceae) - FJ788816
Uncultured Sebacina mycobiont of Riccardia palmata (Aneuraceae) - EU909229
648Uncultured Sebacina from Thuidium tamariscinum (Thuidiaceae) - HQ154340
Uncultured Sebacinales from Hieracium murorum (Asteraceae) - FJ556816
Uncultured Sebacinales from Orchidaceae - HM451829
Uncultured Sebacina from Pseudotsuga menziesii forest soil - HM488520
Uncultured Sebacinales from Orchidaceae - JF691443
Uncultured Sebacinales from Orchidaceae - HM451823
Uncultured Sebacinales from Orchidaceae - HM451794
Uncultured Sebacinales from Orchidaceae - HM451817
Uncultured Sebacinales from Cavendishia bracteata (Ericaceae) - HM230849
Uncultured fungus from peatlands - AM260833
Uncultured fungus from peatlands - AM260832
Uncultured Sebacinales from Disterigma sp. (Ericaceae) - FN663809
Uncultured mycorrhiza from Disterigma microphyllum (Ericaceae) - EU625929
Uncultured Sebacinales from Gaultheria erecta (Ericaceae) - HM230858
501Uncultured mycorrhiza from Disterigma alaternoides (Ericaceae) - EU625982
Uncultured mycorrhiza from Disterigma alaternoides (Ericaceae) - EU625983
Uncultured mycorrhiza from Disterigma microphyllum (Ericaceae) - EU625936
Uncultured Sebacinales from Vaccinium poasanum (Ericaceae) - FN663820
Uncultured Sebacinales from Vaccinium poasanum (Ericaceae) - FN663801
654
Uncultured Sebacinales from Cavendishia nobilis (Ericaceae) - FN663693
Uncultured mycorrhiza from Ceratostema oellgaardii (Ericaceae) - EU625950
Uncultured Sebacinales from Ceratostema reginaldii (Ericaceae) - FN663631
Uncultured Sebacinales from Disterigma alaternoides (Ericaceae) - FN663645
Uncultured fungus from peatlands - AM260834
Uncultured Sebacinales from Orchidaceae - HM451820
Uncultured Sebacinaceae from Pleurothallis lilijae (Orchidaceae) - EU107991
Uncultured Sebacinaceae from Epipactis palustris (Orchidaceae) - AY634132
Sebacina vermifera mycorrhiza from Caladenia tesselata (Orchidaceae) - DQ983816
527
Uncultured Sebacinaceae from Caladenia formosa (Orchidaceae) - AY330695
Uncultured Sebacinales from Orchidaceae - HM451809
665 Uncultured Sebacinales from Orchidaceae - HM451811
826 Uncultured Sebacinales from Pterygodium catholicum (Orchidaceae) - FJ788824
Uncultured Sebacinales from Orchidaceae - JF691222
Uncultured Sebacinales of Pterygodium acutifolium (Orchidaceae) - FJ788825
Uncultured Sebacinales of Pterygodium acutifolium (Orchidaceae) - FJ788826
Uncultured Sebacinales from Orchidaceae - HM451801
Symbiont of E. firmum - Tilaran - ALO - OTU17 - JX998815
Symbiont of E. firmum - Tilaran - ALO - OTU17 - JX998844
Symbiont of E. firmum - Tilaran - ALO - OTU17 - JX998831
Symbiont of E. firmum - Talamanca - ECH - OTU17 - JX998777
Symbiont of E. firmum - Tilaran - EFR - OTU17 - KC140566
Symbiont of E. firmum - Tilaran - CAB - OTU17 - JX998779
Symbiont of E. firmum - Guanacaste - MIR - OTU17 - JX998801
Symbiont of E. firmum - Tilaran - EMV - OTU17 - JX998819
Symbiont of E. firmum - Tilaran - ALO - OTU17 - JX998840
Symbiont of E. firmum - Tilaran - ALO - OTU17 - JX998813
Symbiont of E. firmum - Tilaran - CAB - OTU17 - JX998781
Symbiont of E. firmum - Tilaran - ALO - OTU17 - JX998842
Symbiont of E. firmum - Tilaran - UMV - OTU17 - JX998825
Symbiont of E. firmum - Guanacaste - MIR - OTU14 - JX998837
Symbiont of E. firmum - Talamanca - ECH - OTU14 - JX998834
Symbiont of E. firmum - Tilaran - UMV - OTU16 - JX998828
Symbiont of E. firmum - Tilaran - ETT - OTU16 - KC140565
Symbiont of E. firmum - Guanacaste - MIR - OTU16 - JX998798
Symbiont of E. firmum - Talamanca - ECH - OTU16 - JX998789
Symbiont of E. firmum - Tilaran - CAB - OTU16 - JX998778
Symbiont of E. firmum - Talamanca - ECH - OTU16 - JX998774
Symbiont of E. firmum - Tilaran - ECE - OTU16 - JX998782
Symbiont of E. firmum - Talamanca - ECH - OTU16 - JX998833
Symbiont of E. firmum - Tilaran - UMV - OTU17 - JX998827
Symbiont of E. firmum - Guanacaste - MIR - OTU17 - JX998797
Symbiont of E. firmum - Tilaran - EMV - OTU17 - JX998816
Symbiont of E. firmum - Tilaran - EFR - OTU17 - JX998839
Symbiont of E. firmum - Tilaran - UMV - OTU17 - JX998826
Symbiont of E. firmum - Tilaran - ETT - OTU17 - JX998795
Symbiont of E. firmum - Tilaran - ALO - OTU17 - JX998814
Symbiont of E. firmum - Tilaran - UMV - OTU17 - JX998848
Symbiont of E. firmum - Guanacaste - ERP - OTU17 - JX998824
Symbiont of E. firmum - Tilaran - ETT - OTU17 - JX998793
Symbiont of E. firmum - Tilaran - CAB - OTU17 - JX998812
Symbiont of E. firmum - Tilaran - ALO - OTU17 - JX998843
Symbiont of E. firmum - Guanacaste - ERP - OTU17 - JX998823
Symbiont of E. firmum - Tilaran - ETT - OTU17 - JX998792
Symbiont of E. firmum - Tilaran - EFR - OTU17 - JX998805
Symbiont of E. firmum - Tilaran - EFR - OTU17 - JX998838
Symbiont of E. firmum - Tilaran - EFR - OTU17 - JX998810
Symbiont of E. firmum - Tilaran - EMV - OTU17 - JX998822
Symbiont of E. firmum - Tilaran - ECE - OTU17 - JX998784
Symbiont of E. firmum - Tilaran - EMV - OTU17 - JX998820
Symbiont of E. firmum - Tilaran - ETT - OTU17 - JX998796
Symbiont of E. firmum - Tilaran - ALO - OTU17 - JX998776
Symbiont of E. firmum - Tilaran - ECE - OTU17 - JX998788
Symbiont of E. firmum - Tilaran - ALO - OTU17 - JX998830
Symbiont of E. firmum - Tilaran - EMV - OTU17 - JX998818
Symbiont of E. firmum - Tilaran - ECE - OTU17 - JX998787
Symbiont of E. firmum - Guanacaste - MIR - OTU17 - JX998800
Symbiont of E. firmum - Tilaran - UMV - OTU17 - JX998847
Symbiont of E. firmum - Tilaran - EMV - OTU17 - JX998817
Symbiont of E. firmum - Tilaran - ECE - OTU17 - JX998785
Symbiont of E. firmum - Tilaran - ALO - OTU17 - JX998845
Symbiont of E. firmum - Tilaran - EMV - OTU17 - JX998791
Symbiont of E. firmum - Tilaran - EMV - OTU15 - JX998835
Symbiont of E. firmum - Tilaran - EFR - OTU17 - JX998809
Symbiont of E. firmum - Tilaran - CAB - OTU17 - JX998780
Symbiont of E. firmum - Tilaran - CAB - OTU17 - JX998832
Symbiont of E. firmum - Tilaran - ALO - OTU17 - JX998841
Symbiont of E. firmum - Tilaran - EFR - OTU17 - JX998804
621
Symbiont of E. firmum - Talamanca - ECH - OTU17 - JX998790
Symbiont of E. firmum - Tilaran - EFR - OTU17 - JX998808
Symbiont of E. firmum - Guanacaste - MIR - OTU17 - JX998803
Symbiont of E. firmum - Tilaran - ECE - OTU17 - JX998783
Symbiont of E. firmum - Tilaran - ECE - OTU17 - JX998786
Symbiont of E. firmum - Guanacaste - MIR - OTU17 - JX998799
Symbiont of E. firmum - Tilaran - UMV - OTU17 - JX998846
MOTU J
Samples from
Guanacaste
Tilarán
Talamanca
MOTU I
MOTU K
MOTU L
0.09
FIGURE S3. Phylogeny of Sebacinales based on 5.8S. Values at nodes represent ≥50% bootstrap
support out of 1000 iterations. Epidendrum firmum symbionts are listed by mountain range,
population, MOTU and GenBank accession number. Shaded bars represent samples from each
mountain range.
Sistotrema sp. - AF345558
Ceratobasidium goodyerae-repentis - AF345556
Ceratobasidium sp. - AY293252
857
830
884
1000Tulasnella deliquescens - AY382798
Uncultured Tulasnellaceae of Cypripedium reginae (Orchidaceae) - EF370068
Uncultured Tulasnellaceae of Cypripedium californicum (Orchidaceae) -EF370080
Tulasnellaceae of Cypripedium calceolus (Orchidaceae) - AY578249
526Uncultured
Uncultured Tulasnellaceae of Cypripedium calceolus (Orchidaceae) - EF370071
Uncultured Tulasnellaceae of Cypripedium montanum (Orchidaceae) - AY578247
Uncultured Tulasnellaceae of Cypripedium macranthon var. rebunense (Orchidaceae) - EF370075
Uncultured Tulasnellaceae of Cypripedium fasciculatum (Orchidaceae) - AY578241
667Uncultured
Tulasnellaceae of Cypripedium reginae (Orchidaceae) - EF370069
Tulasnella cystidiophora - AY856080
Mycorrhiza of Neuwiedia veratrifolia (Orchidaceae) - AF490613
543 Uncultured Tulasnellaceae of Cypripedium candidum (Orchidaceae) - AY578251
963Uncultured Tulasenllaceae of Cypripedium macranthon var. rebunense (Orchidaceae) - EF370077
Tulasnella violea - AY382814
885 Epulorhiza albertensis of Dactylorhiza majalis (Orchidaceae) - AF345563
Tulasnella tomaculum - AY382812
608Tulasnella violea - AY382813
581Tulasnella eichleriana - AY382799
Symbiont of E. firmum - Guanacaste - MIR - OTU30 - JX999019
598Tulasnella pruinosa - AF518724
965
Tulasnella pruinosa from Dactylorhiza majalis (Orchidaceae) - AF345561
Symbiont of E. firmum - Tilarán - CAB - OTU30 - JX999017
irregularis - AF345560
957Tulasnella
Tulasnella irregularis - AD001656
Tulasnella albida - AY382804
764
Symbiont of E. firmum - Guanacaste - MIR - OTU30 - JX999020
Tulasnella violea - AY382815
Symbiont of E. firmum - Guanacaste - MIR - OTU30 - JX999018
Symbiont of E. firmum - Tilarán - EMV - OTU28 - JX999012
Symbiont
of E. firmum - Tilarán - UMV -OTU29 - JX999013
945 Symbiont of
E. firmum - Tilarán - ALO - OTU29 - JX999015
977Symbiont of E.
firmum - Tilarán - UMV -OTU29 - JX999014
Symbiont of E. firmum - Tilarán - ESR - OTU31 - JX999039
Symbiont of E. firmum - Guanacaste - RIN - OTU31 - JX999035
Symbiont of E. firmum - Tilarán - ESR - OTU31 - JX999042
Symbiont of E. firmum - Tilarán - ALO - OTU31 - JX999045
Symbiont of E. firmum - Tilarán - ALO - OTU31 - JX999044
Symbiont of E. firmum - Guanacaste - RIN - OTU31 - JX999036
Mycorrhiza of Neuwiedia veratrifolia (Orchidaceae) - AF490609
Symbiont of E. firmum - Tilarán - ESR - OTU31 - JX999041
Symbiont of E. firmum - Guanacaste - RIN - OTU31 - JX999034
Symbiont of E. firmum - Tilarán - ECE - OTU31 - JX999026
Symbiont of E. firmum - Tilarán - ETT - OTU31 - JX999043
Symbiont of E. firmum - Talamanca - ECH - OTU31 - JX999037
Symbiont of E. firmum - Tilarán - ETT - OTU31 - JX999030
Symbiont of E. firmum - Tilarán - CAB - OTU31 - JX999025
Symbiont of E. firmum - Tilarán - UMV -OTU31 - JX999028
Symbiont of E. firmum - Tilarán - UMV -OTU31 - JX999027
Uncultured Tulasnella of Paphiopedilum armeniacum (Orchidaceae) - FJ786686
Symbiont of E. firmum - Tilarán - ESR - OTU31 - JX999040
Symbiont of E. firmum - Guanacaste - RIN - OTU31 - JX999032
Symbiont of E. firmum - Tilarán - ETT - OTU31 - JX999029
Symbiont of E. firmum - Tilarán - ECE - OTU31 - JX999023
Symbiont of E. firmum - Talamanca - ECH - OTU31 - JX999038
Mycorrhiza of Neuwiedia veratrifolia (Orchidaceae) - AF490612
Tulasnella danica - AY382805
Symbiont of E. firmum - Tilarán - ECE - OTU31 -T014ECE20R1
Tulasnella calospora - AY382801
Symbiont of E. firmum - Tilarán - ECE - OTU31 - JX999022
MOTU C & D
Unmatched (E)
MOTU E
MOTU F
(some C)
Samples from
Guanacaste
Tilarán
Talamanca
0.1
FIGURE S4. Phylogeny showing Tulasnellaceae based on mtLSU. Values at nodes represent
≥50% bootstrap support out of 1000 iterations. Tip labels include mountain range, population,
mtLSU MOTU and GenBank accession number. Bars show corresponding MOTU labels from
ITS. Sequences from plants yielding ITS MOTUs C and D were not clearly differentiated by
mtLSU. One mtLSU sequence was obtained from a plant for which no ITS sequence was be
obtained, but this sequence is similar MOTU E. Plants yielding the most common ITS MOTU, F,
also yielded the most common mtLSU MOTU. This large clade also contained one sequence
from a plant that only yielded MOTU C in ITS analysis. This plant probably was colonized by
multiple mycorrhizal taxa. Sample T014ECE20R1 yielded a sequence that was too short for a
GenBank accession number, but is listed here:
TATATTAATGGCATTGGGCAAAATTCGCTTTCTATACAAAGATATTATTCGTCGCAG
AAAGCTGTGTTTTAAGTAAACAGTCGGGGCCTCCGATTCTGTGCCACCACTTTATAT
TATAAATAATATTCAGTGGTTCTTCATATCGTCTAACTTATGAGGATAATTTGCCGAG
A
TABLE S3. The diversity of Tulasnellaceae and Sebacinales based on ITS. N = the number of
sequences in the analysis (number of plants contributing sequences, if different), π = rarefied
nucleotide diversity, NH = the number of haplotypes (number of phylogenetically distinguishable
haplotypes, if different) and SESMPD = the standardized effect size of mean phylogenetic
distance, where negative values indicate phylogenetic clustering and positive values indicate
phylogenetic evenness (* indicates P > 0.05 based on 999 random draws from the sample pool).
NA indicates no analysis was possible (e.g. a single phylogenetically distinguishable taxon was
present and SESMPD meaningless). Sebacinales sequences were not detected in ESR and the
association between one plant and Sebacinales subgroup A in EFR was excluded from analysis.
Tulasnellaceae
N
π
ERP
4
MIR
Sebacinales
SESMPD N
π
0.033 2
-1.278
2
0.004 2 (1)
NA
8 (6)
0.228 6 (4)
-0.326
8 (6)
0.041 7 (5)
0.956
Mean
6 (5)
0.131 4 (3)
-0.802
5 (4)
0.023 4.5 (3)
NA
Total
12 (10)
0.263 8 (5)
-0.693
10 (8)
0.034 7 (5)
1.001
NH
NH
SESMPD
Guanacaste
Northern Tilarán
UMV
4
0.057 2
-1.236
7 (6)
0.007 3 (2)
-0.654
EMV
12
0.064 4 (3)
-1.815*
8 (5)
0.013 6 (4)
-0.868
ETT
5
0.195 3
0.037
5 (4)
0.006 4 (2)
-0.788*
ALO
11
0.074 4
-2.321*
13 (10)
0.039 8 (4)
1.397
CAB
3
0.044 2
-1.367
6 (5)
0.006 4 (3)
-0.914*
EFR
3
0.203 3
-0.045
8
0.005 2 (1)
NA
Mean
6.3
0.106 3 (2.8)
-1.125
7.8 (6.3) 0.013 4.5 (2.7) -0.365
Total
38
0.132 13 (7)
-1.404
47 (38)
0.016 10 (16)
0.148
Southern Tilarán
ECE
8
0.172 6 (4)
-0.881
7 (5)
0.010 7 (2)
-0.571
ESR
5
0.030 2 (1)
NA
-
-
-
-
Mean
6.5
0.101 4 (2.5)
NA
-
-
-
-
Total
13
0.145 8 (4)
-0.784
-
-
-
-
Tilarán mean 6.4
0.105 3.3 (2.7) -1.090
7.7 (6.1) 0.013 5 (2.4)
-0.400
Tilarán total
51
0.141 15 (7)
-1.427
54 (43)
0.017 18
0.156
7 (6)
0.173 4 (3)
1.511
6 (5)
0.009 5 (4)
-0.633
Talamanca
ECH
Species Level
Mean
6.4 (6.1) 0.116 3.5(2.9)
-0.702
7.0 (5.6) 0.014 4.9 (2.9) -0.180
Total
70 (67)
NA
70 (56)
0.202 21 (10)
0.018 23 (14)
NA