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Basic and Applied Ecology ] (]]]]) ]]]–]]] www.elsevier.de/baae
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9 Guilds of mycorrhizal fungi and their relation to trees, ericads, orchids
and liverworts in a neotropical mountain rain forest
11
Ingrid Kottkea,Ã, Ingeborg Hauga, Sabrina Setaroa, Juan Pablo Suarezc, Michael Weißa,
´
13 b b a
Markus Preußing , Martin Nebel , Franz Oberwinkler
15 a
Eberhard-Karls-Universita Tu
¨t ¨bingen, Spezielle Botanik, Mykologie und Botanischer Garten, Auf der Morgenstelle 1, D-72076 Tu ¨-
bingen, Germany
17 b
Staatliches Museum fu Naturkunde Stuttgart, Rosenstein 1, D-70191 Stuttgart, Germany
¨r
c
´
Centro de Biologıa Celular y Molecular, Universidad Te´cnica Particular de Loja, San Cayetano Alto s/n C.P. 11 01 608, Loja,
F
19 Ecuador
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21
Received 9 March 2006; accepted 5 March 2007
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23
25
Abstract
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27
Mycorrhizas of vascular plants and mycorrhiza-like associations of liverworts and hornworts are integral parts of
terrestrial ecosystems, but have rarely been studied in tropical mountain rain forests. The tropical mountain rain forest
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29
´
area of the Reserva Biologica San Francisco in South Ecuador situated on the eastern slope of the Cordillera El
Consuelo is exceptionally rich in tree species, ericads and orchids, but also in liverworts. Previous light and electron
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31
microscopical studies revealed that tree roots are well colonized by structurally diverse Glomeromycota, and that
epiphytic, pleurothallid orchids form mycorrhizas with members of the Tulasnellales and the Sebacinales
33
(Basidiomycota). Sebacinales also occurred in mycorrhizas of hemiepiphytic ericads and Tulasnellas were found in
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liverworts belonging to the Aneuraceae. On the basis of these findings, we hypothesized that symbiotic fungi with a
35
broad host range created shared guilds or even fungal networks between different plant species and plant families. To
test this hypothesis, molecular phylogenetic studies of the fungi associated with roots and thalli were carried out using
37
sequences of the nuclear rDNA coding for the small subunit rRNA (nucSSU) of Glomeromycota and the large subunit
R
rRNA (nucLSU) of Basidiomycota. Sequence analyses showed that Sebacinales and Tulasnellas were only shared
39
within but not between ericads and orchids or between liverworts and orchids, respectively. Regarding arbuscular-
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mycorrhiza-forming trees, however, 18 out of 33 Glomus sequence types were shared by two–four tree species
41
belonging to distinct families. Nearly all investigated trees shared one sequence type with another tree individual. Host
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range and potential shared guilds appeared to be restricted to the plant family level for Basidiomycota, but were
43
covering diverse plant families in case of Glomeromycota. Given that the sequence types as defined here correspond to
C
fungal species, our findings indicate potential fungal networks between trees.
45
¨ ¨
r 2007 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.
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47
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Zusammenfassung
49
Die Mykorrhizen der Gefaßpflanzen und ahnliche Symbiosen von Lebermoosen und Hornmoosen sind wesentliche,
¨ ¨
¨
funktionale Bestandteile terrestrischer Okosysteme, wurden bisher in tropischen Bergregenwaldern aber kaum
¨
51
ÃCorresponding author. Tel.: +49 7071 2976688; fax: +49 7071 295534.
53 E-mail address: Ingrid.Kottke@uni-tuebingen.de (I. Kottke).
55 ¨ ¨
1439-1791/$ - see front matter r 2007 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.
doi:10.1016/j.baae.2007.03.007
Please cite this article as: Kottke, I., et al. Guilds of mycorrhizal fungi and their relation to trees, ericads, orchids and liverworts in a neotropical
mountain rain forest. Basic and Applied Ecology (2007), doi:10.1016/j.baae.2007.03.007

2. BAAE : 50160
ARTICLE IN PRESS
2 I. Kottke et al. / Basic and Applied Ecology ] (]]]]) ]]]–]]]
1 ´
untersucht. Das Gebiet der Reserva Biologica San Francisco im tropischen Bergregenwald von Sudecuador, am
¨
Osthang der Cordillera El Consuelo gelegen, zeichnet sich durch eine sehr hohe Artenzahl bei Baumen, Ericaceen und
¨
3 Orchideen, aber auch bei Lebermoosen aus. Vorausgehende licht- und elektronenmikroskopische Untersuchungen
hatten gezeigt, dass die arbuskularen Mykorrhizen der Baume von strukturell unterschiedlichen Glomeromyceten
¨ ¨
5 gebildet wurden. Epiphytische, pleurothallide Orchideen waren von Tulasnella und Vertretern der Sebacinales
(Basidiomycota) mykorrhiziert, wobei Sebacinales auch an den Ericaceen und Tulasnellales auch an Lebermoosen der
7 Familie Aneuraceae auftraten. Aus diesen Beobachtungen ergab sich die Frage, ob die symbiotischen Pilze ein breites
Wirtsspektrum haben und moglicherweise pilzliche Netzwerke zwischen unterschiedlichen Pflanzen bilden konnen.
¨ ¨
9 Molekularphylogenetische Untersuchungen der Mykobionten unter Verwendung der nucSSU der Glomeromyceten
und der nucLSU der Basidiomyceten ergaben ein differenziertes Bild. Identische oder nahezu identische Sequenzen der
11 Basidiomyceten wurden nur innerhalb der Familien aber nicht zwischen Orchideen und Ericaceen oder Orchideen und
Aneuraceen gefunden. Bei den Glomeromyceten hingegen kamen 18 von 33 Sequenztypen der Formgattung Glomus in
13 zwei bis vier verschiedenen Baumarten unterschiedlicher Familien vor. Nahezu alle untersuchten Baume hatten einen
¨
gemeinsamen Sequenztyp mit einem anderen Baumindividuum. Pilzliche Netzwerke von Glomeromyceten zwischen
15 Baumen unterschiedlicher Familienzugehorigkeit waren demnach moglich, vorausgesetzt die hier definierten
¨ ¨ ¨ ¨
Sequenztypen entsprechen gemeinsamen Pilzarten.
17 ¨ ¨
r 2007 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.
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19 Keywords: Glomus; Sebacinales; Tulasnella; Andean clade of Ericaceae; Aneuraceae; pleurothallid orchids; nucSSU; nucLSU; fungal
´
networks; Reserva Biologica San Francisco
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23 fungal species, also support plant diversity and produc-
Introduction
25
Mycorrhizas of vascular plants and mycorrhiza-like
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tivity (Read, 1998).
Recent studies in the species-rich Andean rain forest
59
associations of liverworts and hornworts are integral revealed the co-occurrence of many mycorrhizal types 61
27 within short local distances, including some previously
parts of terrestrial ecosystems. Mycorrhizal fungi do not
only improve the nutrient status and thereby the fitness unknown ones (Beck, Kottke, & Oberwinkler, 2005; 63
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29 Haug et al., 2004; Haug, Weiß, Homeier, Oberwinkler,
of plant individuals (Smith & Read, 1997) but also
influence richness and composition of plant commu- & Kottke, 2005). Light microscopical investigations 65
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31 showed arbuscular mycorrhizas in 112 tree species from
nities (Hartnett & Wilson, 1999). Distinct fungal guilds
appear to be present in important plant ecosystems, 53 families on mineral as well as pure organic soils 67
33 (Kottke, Beck, Oberwinkler, Homeier, & Neill, 2004). A
such as Basidiomycota in ectomycorrhiza-dominated
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forests, Ascomycota in ericoid-mycorrhiza-dominated new type of mycorrhizas formed by Sebacinales 69
35 (Basidiomycota) with members of the hemiepiphytic
heathlands and Glomeromycota in arbuscular-mycor-
rhiza-dominated grasslands (Francis & Read, 1994; Andean clade ericads was documented by ultrastructur- 71
37 al characters and molecular identification (Setaro,
Kottke, 2002) and tropical ecosystems (Alexander &
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Lee, 2005). Experiments showed higher competitiveness Oberwinkler, & Kottke, 2006a; Setaro, Weiß, Ober- 73
39 winkler, & Kottke, 2006b). Transmission electron
of mycorrhizal versus non-mycorrhizal plants in arbus-
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cular-mycorrhiza-forming plant communities (Grime, microscopical studies proved Sebacinales and Tulasnella 75
41 (Basidiomycota) in epiphytic orchids of the subtribe
Mackey, Hillier, & Read, 1987), and revealed a positive
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correlation between diversity of arbuscular mycorrhizal ´
Pleurothallidinae (Suarez et al., 2006), and Tulasnella in 77
43 the thalloid liverwort Aneura pinguis (M. Preußing,
fungi and plant species richness (Van der Heijden et al.,
C
1998). Plants, on the other hand, may regulate the unpublished). We hypothesized that fungal networks 79
45 might be formed by Glomeromycota between distinct
community structure and diversity of mycorrhizal fungi
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by unspecific or specific binding (Johnson, lJdo, tree species of different families, and that identical 81
47 Sebacinales or Tulasnella species might link epiphytic
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Genney, Anderson, & Alexander, 2005). Fungi with a
broad host range could even establish functional orchids with hemiepiphytic ericads or with liverworts of 83
49 the Aneuraceae, via mycorrhizal associations. Even if no
mycorrhizal networks to improve nutrient exploitation
from soil resources, yield interplant carbon transfer, functional, physical fungal networks between epiphytic 85
51 and terrestrial plants could be expected, a broad host
facilitate seedling establishment and influence interplant
competition (Simard & Durall, 2004). A high number of range and common fungal guilds would improve the 87
53 chances of long-term maintenance of both the fungi and
fungal species with differences in functional compat-
ibility could, by an additive beneficial effect of each the plants, in such heterogeneous ecosystem. Further 89
55 ‘‘ploughing up the wood wide web’’ (Helgason, Daniell,
Please cite this article as: Kottke, I., et al. Guilds of mycorrhizal fungi and their relation to trees, ericads, orchids and liverworts in a neotropical
mountain rain forest. Basic and Applied Ecology (2007), doi:10.1016/j.baae.2007.03.007

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ARTICLE IN PRESS
I. Kottke et al. / Basic and Applied Ecology ] (]]]]) ]]]–]]] 3
1 Husband, Fitter, & Young, 1998) in order to identify the ier). The trees were found within 15 plots of 400 m2 each, 57
associated fungi was, therefore, challenging (Fig. 1). on a mountain ridge along the altidudinal gradient
3 The identification of the mycorrhiza-forming fungi in between 1920 and 2100 m a.s.l. and on the steep slope of 59
the forest could not be done by sampling spores or the close ravine. Additional root samples were collected
5 fruiting bodies, because these methods would have only from four indigenous tree species in a nearby afforesta- 61
yielded a very narrow spectrum of the fungal commu- tion area and from three indigenous tree species in a
7 nities (Husband, Herre, Turner, Gallery, & Young, nursery. Five cups with three fine roots (1 cm in length) 63
2002; Sanders, 2004a). Instead, rDNA sequencing of the were collected from each tree individual. Humus grown
9 associated fungi from the plant material was carried out. roots were sampled of 11 ericads in the pristine forest on 65
The sequence types (ribosomal genotypes), at the the mountain ridge within or close to the tree plots,
11 present stage of knowledge, can rarely be related ´
along road sides and in the bushy paramo. Ten cups 67
precisely to either morphological or biological species. containing one mycorrhiza (0.5 cm in length) were
13 However, the amount of information derived from the collected from each ericad individual. Roots of four 69
sequences of the ribosomal genes appeared to be pleurothallid orchid species were removed from stand-
15 meaningful in previous ecological studies on arbuscular ing tree stems along the mountain ridge close to the tree 71
mycorrhizas (Helgason et al., 2002; Husband et al., plots and sometimes 1–2 m away from the sampled
17 2002; Sanders, 2004b) as well as on mycorrhiza-forming ericads or Aneuraceae. One–four roots (1–2 cm in 73
Basidiomycota (Bidartondo, Bruns, Weiß, Sergio, & ´ length) per orchid individual that had close contact to
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19 Read, 2003; Bidartondo, Burghardt, Gebauer, Bruns, & the substrate were sampled in separate cups. Three 75
Read, 2004; Weiß, Selosse, Rexer, Urban, & Oberwink- species of Aneuraceae (liverworts) were collected from
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21 ler, 2004). rotten wood or, less frequently, from wet rocks. One 77
In order to test the hypothesis that fungal networks or slice of the thallus (0.5 cm thick) was sampled from each
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23 at least common fungal guilds could potentially be liverwort individual. Details of the sampled individuals 79
formed in the tropical mountain rain forest between are presented in Appendix A, Supplementary Table 1.
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25 trees of diverse families, between ericads and orchids, or All the roots and thalli were cleaned under tap water the 81
between orchids and the aneuracean liverworts (Fig. 1), same day. The velamen was afterwards removed from
27 we sequenced fungal rDNA directly from the mycor- orchid roots. Orchid roots and thalli of the liverworts 83
rhizas of 21 tree species, 11 species of the Ericaceae and were controlled for hyphal colonization by light micro-
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29 four species of the Orchidaceae. We also sequenced the scopy from sections. Tree and ericad roots were 85
fungal rDNA from the mycorrhizal-like associations of controlled for mycorrhization using standard staining
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31 three aneuracean liverwort species. Most sampling was methods (Haug et al., 2004; Setaro et al., 2006a). All the 87
carried out within an area of about 12 ha. The DNA further proceeded samples were highly colonized by
33 sequences of the fungi suspected to form shared guilds, mycorrhizal fungi. Roots and liverworts slices were 89
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the Glomeromycota, the Sebacinales and the Tulasnel- dried in 1.5 ml tubes and kept on silica gel for DNA
35 lales, were analyzed by molecular phylogenetic methods. isolation. 91
Identical or closely related sequences were identified in
37 order to evaluate the potential of fungal networks or 93
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fungal guilds within and between the plant families.
39 Processing of fungal DNA sequences 95
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41 DNA was isolated from the dried mycorrhizal 97
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Materials and methods samples using the DNAeasy Plant Mini Kit (Qiagen,
43 Hilden, Germany). One–four cups per plant individual 99
C
Site and sampling were processed, the numbers of cups yielding sequences
45 are given in Appendix A, Supplementary Table 1. Part 101
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The investigations were carried out in South Ecuador of the nuclear rDNA coding for the small subunit rRNA
47 during 2001–2005. Study sites lay in a tropical mountain (nucSSU) of the arbuscular fungi of the tree mycor- 103
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rain forest area on the eastern slope of the Cordillera El rhizas, and part of the large subunit rRNA (nucLSU) of
49 Consuelo at 1850–2300 m a.s.l. and in the nearby bushy the putative Sebacinales and Tulasnellales in orchids, 105
´
paramo at 2700–3000 m a.s.l. The pristine forest is ericads and liverworts were amplified by the polymerase
51 exceptionally rich in tree species, ericads and orchids, chain reaction (PCR). Primers and PCR design are given 107
and also in number of liverworts (Homeier, Dalitz, & in Appendix A, Supplementary Tables 2 and 3, the
53 Breckle, 2002; Parolly, Kurschner, Schafer-Verwimp, &
¨ ¨ targeted plants are given in Supplementary Table 1 (see 109
Gradstein, 2004). Appendix A, Supplementary Table 1). For detailed
55 Roots were sampled from 17 different tree species in information see Appendix A: Processing of fungal 111
the pristine forest (identified and marked by J. Home- sequences.
Please cite this article as: Kottke, I., et al. Guilds of mycorrhizal fungi and their relation to trees, ericads, orchids and liverworts in a neotropical
mountain rain forest. Basic and Applied Ecology (2007), doi:10.1016/j.baae.2007.03.007

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1 57
Stelis
3 59
5 Stelis 61
7 63
Cavendishia
9 65
11 Sebacina Tulasnella Aneura 67
13 Aneura 69
15 Sebacina Tulasnella
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17 73
Cavendishia Glomus
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19 roots 75
V. Uhle-Schneider
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21 Fig. 1. Scheme of the plants and their mycorrhizas assumed to form common fungal guilds or networks in the tropical mountain 77
rain forest of South Ecuador. Diverse trees as indicated by different leaves form mycorrhizas with Glomeromycota. The potential
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fungal network as found on the basis of Glomus sequences is indicated by spores and hyphal connections in the humus layer. The
23 79
liana-like Cavendishia forms ectendomycorrhizas with Sebacinales, the epiphytic orchid Stelis forms endomycorrhizas with
25
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Sebacinales and Tulasnellas, and the thalloid liverwort Aneura growing on a rotten stem forms mycorrhiza-like associations with
Tulasnellas. The mycorrhizal types of ericads (left), orchids (middle) and Aneuraceae (right) are displayed by sections in which 81
hyphae were stained by methyl blue. No common fungal guilds were found among the ericads and the epiphytic orchids (suspected
27 for Sebacinales) and among the epiphytic orchids and the Aneuraceae (suspected for Tulasnellas). 83
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29 85
of more than 99%; A. pinguis and Riccardia smaragdina
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31 Phylogenetic analysis in clade 2, A. pinguis and Riccardia sp. in clade 6. All the 87
mycobiont sequences are new to science.
33 Sequence similarities were determined using the 89
EC
BLAST sequence similarity search tool (Altschul et al.,
35 1997) provided by NCBI (www.ncbi.nlm.nih.gov). 91
Sequence alignments were done with MAFFT and Sebacinales mycobionts in ericads and orchids
37 subsequent analyses were conducted with PAUP using 93
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the BIONJ algorithm. For details see Appendix A: All the sebacinalean sequences obtained from the
39 Phylogenetic analysis. ericad and orchid mycorrhizas are new to science and 95
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can be assigned to Sebacinales (Fig. 3) as defined by
41 Weiß et al. (2004). The order Sebacinales so far 97
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contains, amongst others, Sebacina vermifera isolates
43 Results from Australian orchids (Warcup, 1988; Warcup & 99
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Talbot, 1967; Weiß et al., 2004) and fungal sequences of
45 Tulasnella mycobionts in orchids and liverworts mycorrhizas of Canadian Gaultheria shallon (Ericaceae; 101
N
Berch, Allen, & Berbee, 2002; Fig. 3). Neither sebaci-
47 The phylogenetic analysis of the nucLSU sequences of nalean mycobionts of the epiphytic pleurothallid orchids 103
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Tulasnella yielded eight clades of epiphytic orchid of Ecuador and the terrestrial orchids of Australia, nor
49 mycobionts and nine clades of Aneura and Riccardia sebacinalean mycobionts of ericads from Ecuador and 105
mycobionts (Fig. 2). Tulasnella sequences were not Canada showed identical sequences. The mycobionts of
51 shared between orchid and liverwort species. However, orchids and ericads of the study site clustered in separate 107
an analysis of proportional differences between se- clades. Sebacinales sequences from both orchid and
53 quences of the nucLSU D1/D2 regions showed that ericad mycorrhizas were not included in shared well- 109
different orchid species shared sequences with simila- supported terminal clades. Thus, our analysis suggests
55 rities more than 99% in clades A, B, D and G. The three the occurrence of different fungal guilds as mycobionts 111
liverwort species also shared sequences with similarities of ericads and orchids, respectively.
Please cite this article as: Kottke, I., et al. Guilds of mycorrhizal fungi and their relation to trees, ericads, orchids and liverworts in a neotropical
mountain rain forest. Basic and Applied Ecology (2007), doi:10.1016/j.baae.2007.03.007

5. BAAE : 50160
ARTICLE IN PRESS
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1 57
3 59
5 61
7 63
9 65
11 67
13 69
15 71
17 73
F
19 75
O
21 77
O
23 79
25
PR 81
27 83
D
29 85
TE
31 87
33 89
EC
35 91
37 93
R
39 95
R
41 97
O
43 99
C
45 101
N
47 103
U
49 105
51 107
Fig. 2. Phylogenetic relationships of the detected Tulasnella mycobionts of pleurothallid orchids (clades A–H) and Aneuraceae
53 (clades 1–9) in the tropical mountain rain forest area of South Ecuador. BIONJ analysis of an alignment of nuclear DNA sequences 109
coding for the D1/D2 region of the large ribosomal subunit (nucLSU). The tree was rooted with Auricularia auricula-judae.
55 Numbers on branches designate BIONJ bootstrap values (only values exceeding 50% are shown). Note that genetic distances cannot 111
be directly correlated to branch lengths in the tree, since highly diverse alignment regions were excluded for phylogenetic
reconstruction. The Tulasnella sequence from Aneura pinguis AY298949, marked with a diamond, is not from the study site.

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85
from Cavendishia nobilis AY825047 ECU
from Cavendishia nobilis AY825075 ECU
57
66 from Cavendishia nobilis AY825054 ECU
from Cavendishia nobilis AY825042 ECU
3 59 from Ceratostema reginaldii DQ352049 ECU
from Cavendishia nobilis AY825048 ECU
59
from Gaultheria erecta DQ352045 ECU
from Cavendishia nobilis AY825072 ECU
from Disterigma alaternoides DQ352070 ECU
5 from Disterigma microphyllum DQ352066 ECU 61
from Cavendishia nobilis AY825066 ECU
from Cavendishia nobilis AY825044 ECU
from Cavendishia nobilis AY825052 ECU
7 from Gaultheria erecta DQ352068 ECU 63
from Sphyrospermum cordifolium DQ352061 ECU
from Sphyrospermum cordifolium DQ352063 ECU
82 from Psammisia guianensis DQ352046 ECU
9 from Disterigma microphyllum DQ352052 ECU 65
100 from Stelis superbiens DQ358067 ECU
from Stelis superbiens DQ358068 ECU 1
from Diogenesia cf. floribunda DQ352058 ECU
11 from Cavendishia bracteata DQ352051 ECU
55 from Cavendishia nobilis AY825053 ECU
67
from Cavendishia nobilis AY825057 ECU
from Cavendishia nobilis AY825041 ECU
63
13 from Cavendishia nobilis AY825055 ECU
from Cavendishia nobilis AY825056 ECU
69
72 from Cavendishia nobilis AY825071 ECU
69 89 from Semiramisia speciosa DQ352053 ECU
15 from Cavendishia nobilis AY825043 ECU
from Cavendishia nobilis AY825045 ECU 71
56
from Psammisia guianensis DQ352069 ECU
64 from Cavendishia nobilis AY825049 ECU
from Cavendishia nobilis AY825051 ECU
17 50 86 from Stelis superbiens DQ358058 ECU 73
from Stelis superbiens DQ358066 ECU
from Pleurothallis lilijae DQ358070 ECU
F
from Pleurothallis lilijae DQ358069 ECU 2
19 from Pleurothallis lilijae DQ358071 ECU 75
from Pleurothallis sp DQ358064 ECU
O
88 Sebacina vermifera from Eriochilus scaber AY505548 AUS
Sebacina vermifera from Microtis uniflora AY505554 AUS
21 84 from Stelis superbiens DQ358060 ECU
98 from Stelis superbiens DQ358061 ECU
77
95
3
O
from Stelis superbiensDQ358059 ECU
from Stelis superbiens DQ358062 ECU
23 71 from Pleurothallis lilijae DQ358072 ECU
82 from Pleurothallis lilijae DQ358074 ECU
79
96 85 from Pleurothallis lilijae DQ358063 ECU
PR
100
from Stelis hallii DQ358055 ECU
4
25 from Pleurothallis lilijae DQ358073 ECU
100 from Stelis hallii DQ358057 ECU
81
5
from Stelis superbiens DQ358065 ECU
53 from Gaultheria shallon AF300777 CAN
from Gaultheria shallon AY112930 CAN
27 55
from Gaultheria shallon AF300775 CAN 83
from Gaultheria shallon AF300783 CAN
81 from Gaultheria shallon AF300774 CAN
D
69 from Gaultheria shallon AF284135 CAN
29 89 from Gaultheria shallon AF300785 CAN 85
67 from Gaultheria shallon AF284137 CAN
70 54 from Gaultheria shallon AF300786 CAN
TE
from Gaultheria shallon AF300793 CAN
31 from Gaultheria shallon AF300789 CAN 87
from Gaultheria shallon AF300790 CAN
from Gaultheria shallon AF300792 CAN
from Disterigma alaternoides DQ352055 ECU
33 75
61 from Cavendishia bracteata DQ352048 ECU
from Sphyrospermum cordifolium DQ352050 ECU
89
EC
50 from Cavendishia nobilis AY825061 ECU
69
from Cavendishia nobilis AY825074 ECU
35 76
from Cavendishia nobilis AY825060 ECU
from Cavendishia nobilisAY825059 ECU
91
from Disterigma alaternoides DQ352065 ECU
from Stelis hallii DQ358056 ECU 6
37 from Sphyrospermum cordifolium DQ352062 ECU
from Ceratostema oellgaardii DQ352064 ECU
93
R
from Cavendishia nobilis AY825064 ECU
64 from Macleania benthamiana DQ352047 ECU
from Cavendishia nobilis AY825063 ECU
39 67
from Cavendishia nobilis AY825067 ECU 95
R
84 from Cavendishia bracteata DQ352054 ECU
51 90 from Semiramisia speciosa DQ352056 ECU
from Sphyrospermum cordifolium DQ352059 ECU
41 from Semiramisia speciosa DQ352057 ECU 97
O
Sebacina vermifera from Cyrtostylis reniformis AF291366 AUS
100 Sebacina vermifera from Phyllanthus calycinus AY505552 AUS
94 Sebacina vermifera from Eriochilus scaber AY505549 AUS
43 Sebacina vermifera from Microtis uniflora AY505555 AUS 99
C
64 from Lophozia incisa AY298847 SPA
72 from Calypogeia muelleriana AY298948 FRA
89
from Lophozia sudetica AY298946 SPA
45 from Cavendishia nobilis AY825073 ECU 101
N
from Disterigma microphyllum DQ352067 ECU
100 Multinucleate Rhizoctonia AY505556 AUS
Piriformospora indica AY505557 IND
47 Sebacina vermifera from Caladenia catenata AY505553 AUS 103
U
54 Sebacina vermifera from Caladenia catenata AY505550 AUS
Sebacina vermifera from Glossodia minor AY505551 AUS
from Sebacina vermifera DQ352060 ECU
49 61
59 Sebacina epigaea AF291267 GER
Sebacina incrustans AF291365 GER 105
51 Tremellodendron pallidum AF384862 CAN
88
Efibulobasidium rolleyi AF291317 CAN
Craterocolla cerasi AY505542 GER
51 Sebacina allantoidea AF291367 GER 107
0.005 substitutions/site
53 109
Fig. 3. Phylogenetic relationships of the detected Sebacinales mycobionts of ericads (dotted line) and pleurothallid orchids (clades
1–6) in the tropical mountain rain forest area of South Ecuador. BIONJ analysis of an alignment of nuclear DNA sequences coding
55 111
for the D1/D2 region of the large ribosomal subunit (nucLSU). The tree was rooted with Sebacina allantoidea, Sebacina epigaea,
Sebacina incrustans, Tremellodendron pallidum, Efibulobasidium rolleyi and Craterocolla cerasi. Numbers on branches designate
BIONJ bootstrap values (only values exceeding 50% are shown). Asterisks, dots and triangles mark identical sequences. AUS,
Australia; CAN, Canada; ECU, Ecuador; FRA, France; GER, Germany; IND, India.

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1 57
3 59
5 61
7 63
9 65
11 67
13 69
15 71
17 73
F
19 75
O
21 77
O
23 79
25
PR 81
27 83
D
29 85
TE
31 87
33 89
EC
35 91
37 93
R
39 95
R
41 97
O
43 99
C
45 101
N
47 103
U
49 105
51 107
53 109
Fig. 4. Phylogenetic relationships of the glomeralean mycobionts of 38 arbuscular mycorrhizal trees in the tropical mountain rain
55 forest: BIONJ analysis of an alignment of nuclear DNA sequences coding for the small ribosomal subunit (nucSSU; 1108 111
characters). The tree was rooted with seven sequences of the Gigasporaceae. Glomeralean sequences which clustered together and
showed sequence similarities higher than 99% were regarded as one sequence type. Numbers on branches designate BIONJ
bootstrap values (only values exceeding 50% are shown). Nineteen sequence types which were found at different trees were
numbered continuously, sequence types found at only one tree are indicated by black circles.

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1 Table 1. Occurrence of Glomus sequence types in the arbuscular mycorrhizas of 34 trees from 21 species 57
a b
3 Sequence type 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 59
Tree species
5 Tabebuia chrysantha 1 A x 1 61
Tabebuia chrysantha 2 A 1 1
7 Clethra revoluta F x x 2 63
Clusia elliptica F x 1 1
9 Vismia tomentosa F x x x 3 65
Hyeronima asperifolia F x 1 2
11 Hyeronima moritziana F x x 1 3 67
Hyeronima oblonga F x 1
Hyeronima sp. F x 1 2
13 69
Juglans neotropica 1 A x x 2
Juglans neotropica 2 A x x 1 3
15 Nectandra laevis 1 F x x 1 3 71
Nectandra laevis 2 F x x 1 3
17 Graffenrieda emarginata 1 F x x 1 3 73
Graffenrieda emarginata 2 F x x 2
F
19 Graffenrieda emarginata 3 F x x 2 75
Cedrela montana 1 N x 1
O
21 Cedrela montana 2 A x x 1 3 77
Cedrela montana 3 A x x 2
O
Cedrela sp. F x x 2
23 79
Guarea kunthiana F x x 2
25
Guarea cf. kunthiana F
Guarea pterorhachis F
PR x x x
2
3
2 81
Guarea sp. F x x 1 3
27 Inga acreana 1 F x 1 83
Inga acreana 2 F x 1
D
29 Inga acreana 3 F x 1 85
Inga acreana 4 F x 1
TE
31 Podocarpus oleifolius F x 1 2 87
Cinchona officinalis N x 1
33 Heliocarpus americanus 1 N x 1 89
Heliocarpus americanus 2 A x 1
EC
Heliocarpus americanus 3 A x 1 2
35 91
Heliocarpus americanus 4 F x x 2
Occurrence of sequence types N A F F A A N F F F F F A F F F F F N
37 A F A F 93
R
F
39 95
R
N, nursery; A, afforestation; F, pristine forest. For numbers of sequence types, see Fig. 4.
a
Number of sequence types found only with this tree individual.
41 b 97
O
Total number of sequence types per tree individual.
43 99
C
The Sebacinales sequences from pleurothallid orchids by dots) and C. nobilis one with Semiramisia speciosa
45 were separated into six clades (Fig. 3, clades 1–6; clade 6 (Fig. 3, marked by triangles), all collected in the pristine 101
N
consisting of a single sequence). No identical Sebaci- forest.
47 nales sequences were found in different orchid species 103
U
from the study site. Sequence similarity, for example in
49 the well-supported clade 4, was only 89% among Glomeraceae of tree mycorrhizas 105
mycobionts of Stelis hallii and Pleurothallis lilijae.
51 Identical Sebacinales sequences were, however, found The molecular investigation using primer 107
in different ericad species. Cavendishia nobilis collected GLOM1310rc revealed 33 sequence types of arbuscular
53 from the pristine forest shared a sequence with mycorrhizal fungi in the 34 trees that belonged to 21 109
Gaultheria erecta collected from the road side (Fig. 3, species out of 12 families (see Appendix A, Supplemen-
55 marked by asterisks), Psammisia guianensis shared a tary Table 1). Thirty-two sequence types belonged to 111
sequence with Disterigma microphyllum (Fig. 3, marked Glomus group A, one sequence type belonged to Glomus
Please cite this article as: Kottke, I., et al. Guilds of mycorrhizal fungi and their relation to trees, ericads, orchids and liverworts in a neotropical
mountain rain forest. Basic and Applied Ecology (2007), doi:10.1016/j.baae.2007.03.007

9. BAAE : 50160
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I. Kottke et al. / Basic and Applied Ecology ] (]]]]) ]]]–]]] 9
1 group B as defined by Schußler, Schwarzott, and Walker
¨ genus Tulasnella, because these were either the only 57
(2001) (Fig. 4, numbered lines and points). Eighteen associated fungal guilds (Aneuraceae; M. Preußing
3 sequence types occurred at least with two and at most unpublished) or the dominating fungi (trees, Ericaceae, 59
with four tree species. Fifteen sequence types were found ´
Orchidaceae; Setaro et al., 2006a, b; Suarez et al., 2006,
5 in one tree individual only (Fig. 4, points). One–three I. Haug unpublished). Secondly, problems resulted from 61
sequence types were found with one tree individual. the observation that the ribosomal genes can show
7 Thirty-two tree individuals shared fungal sequence types intraspecific variation especially in case of the Glomer- 63
with other trees, two tree individuals (Tabebuia chry- omycota (Lloyd-Macgilp et al., 1996; Sanders, 2004b;
9 santha 2 A., Guarea pterorhachis F.) had no common Sanders, Alt, Groppe, Boller, & Wiemken, 1995). These 65
fungal sequence type with another tree. Different facts pose general, unresolved challenges to a species
11 individuals of the same tree species showed different concept based on meaningful levels of genetic diversity 67
fungal sequence types in most cases (Table 1). In cases (Husband et al., 2002).
13 were common fungal sequence types occurred in two We decided to use the nucSSU in the case of 69
tree individuals of the same species, e.g. type 15 and 16 Glomeromycota because primers are available, this
15 in Graffenrieda emarginata, this sequence type was also DNA region could be well aligned, and many sequences 71
found in other tree species (Table 1). Thus, no tree- have already been deposited in GenBank. The nucSSU
17 specific fungal sequence types were found. Four has been widely used in ecological studies (Russel & 73
sequence types could be linked with sequences of Bulman, 2004; Saito, Suyama, Sato, & Sugawara, 2004;
F
19 identified morphospecies: sequence type 1 with Glomus Scheublin, Ridgway, Young, & van der Heijden, 2004; 75
intraradices, type 2 with Glomus vesiculiferum, type 5 Wubet, Weiß, Kottke, Teketay, & Oberwinkler, 2006),
O
21 with Glomus proliferum and type 19 with Glomus and was the basis of a new classification system of 77
mosseae (Fig. 4). Glomeromycota based on sequences assigned to species
O
23 by spore morphology (Schußler et al., 2001). Our
¨ 79
phylogenetic analysis including these spore-based se-
PR
25 Discussion quences from GenBank-detected groups where inter- 81
and intraspecific differences were overlapping
27 The investigated mycorrhizal associations occurred at (AY635833 Glomus mosseae/U96139 Glomus mosseae 83
the study site frequently in close vicinity. We therefore proportion of differing sites 2/1025 ¼ 0.2%; Y17653 G.
D
29 expected that few identical or closely related fungi might caledonium/AJ276085 G. fragilistratum 2/1079 ¼ 0.2%). 85
be the main mycorrhizal associates. The results did not Nearly every sequenced clonal insert showed at least
TE
31 support this simple hypothesis but gave a fairly complex 0.2% differences to other clonal inserts of the same PCR 87
picture of the associations. An unexpectedly high product (data not shown). We decided to unite
33 number of sequence types were detected in Glomeraceae sequences with differences o1% into one sequence type 89
EC
from Glomus group A and in Basidiomycota from and evaluated diversity and host preferences of the
35 Tulasnellales and Sebacinales. Furthermore, overlap of Glomus mycobionts at this level. Several authors 91
the sequence types belonging to Sebacinales and regarded clustering with a high bootstrap value and
37 Tulasnella was restricted to the family level in the sequence differences o2.5% to define sequence types 93
R
Ericaceae, Orchidaceae and Aneuraceae, respectively, (Helgason et al., 2002; Husband et al., 2002; Vanden-
39 but was detected in about 55% of the AM fungi koornhuyse et al., 2002). We chose a lower sequence 95
R
colonizing trees of distinct families. These findings are difference level in order not to overestimate the number
41 based on a rather narrow concept of sequence types that of fungal species shared by different plants. It cannot be 97
O
might intrinsically relate to a concept of species. excluded that even within these sequence types several
43 Traditionally, studies on biodiversity and host specificity species were joined together, which would mean that we 99
C
were based on morphologically defined species. No such still overestimated the number of shared fungi. Many
45 approach was feasible in the case of the mycobionts in AM fungi that are clearly separated by morphology and 101
N
our study as the fungi did not display sufficient e.g. ITS sequences cannot be separated on the SSU base
47 structural differences in the mycorrhizas for delimitation (Schußler pers. communication). We found that 55% of
¨ 103
U
of morphospecies. The analysis of host range of the the sequence types were shared by different tree species
49 mycobionts from field samples using DNA sequences, of distinct families. Nearly all the investigated trees 105
however, also poses problems. Firstly, results are limited shared a sequence type with another tree. Seven tree
51 by the available primers (Husband et al., 2002) and the species were represented by more than one individual, 107
amount of material that can be analyzed in appropriate but no species-specific sequence type was recognized.
53 time. The number of fungi that were detected during this Many sequence types were only isolated once, but 109
study is, therefore, far from being complete for the further investigations may show a wider distribution of
55 exceptional species-rich area. We restricted the investi- these sequence types resulting in a broader range of 111
gation to the Glomeraceae, the Sebacinales and the shared species. Our conclusion that a functional net-
Please cite this article as: Kottke, I., et al. Guilds of mycorrhizal fungi and their relation to trees, ericads, orchids and liverworts in a neotropical
mountain rain forest. Basic and Applied Ecology (2007), doi:10.1016/j.baae.2007.03.007