Biodiversity and Conservation

, Volume 21, Issue 9, pp 2269–2285 | Cite as

A clustering optimization strategy to estimate species richness of Sebacinales in the tropical Andes based on molecular sequences from distinct DNA regions

  • Sabrina D. Setaro
  • Sigisfredo Garnica
  • Paulo I. Herrera
  • Juan Pablo Suárez
  • Markus Göker
Original Paper


Fungi are believed to be diverse in the tropics, but because many groups are only known from their DNA sequences this hampers comparative diversity studies. We investigated mycorrhizal Sebacinales (Basidiomycota) of 67 individuals of Ericaceae and Orchidaceae in a tropical mountain ecosystem in Southern Ecuador to provide a first estimate of whether these fungi are particularly diverse in the Northern Andes. We partially sequenced the internal transcribed spacer (ITS) and large subunit (LSU) regions of the nuclear ribosomal DNA and analyzed them together with all Sebacinales sequences available from GenBank. The clustering optimization technique was used to determine clustering parameters that maximize the comparability between molecular operational taxonomic units (MOTUs) obtained from the distinct loci. Sampling effort and species richness were estimated with rarefaction-accumulation curves and non-parametric estimation using Chao2 and compared between Southern Ecuador and France. Clustering optimization indicated that a 1% LSU distance threshold corresponds to the commonly used 3% dissimilarity threshold for ITS, and that a clustering algorithm close to single-linkage clustering is optimal. The resulting clusters show that about 8–9% of observed Sebacinales MOTUs occur in the study area and that most of these MOTUs are endemic (74%). The widespread MOTUs from Southern Ecuador were also found in Panama, North America and Europe. The estimation of species richness revealed unsaturated sampling of Sebacinales in general and also in our study area. Our results suggest a high diversity of Sebacinales associated with Ericaceae and Orchidaceae at the study site in Southern Ecuador, but no hotspot of Sebacinales in comparison with other areas.


Biodiversity Ericaceae ITS LSU Molecular diversity Mycorrhizal fungi Orchids Ribosomal DNA Species richness Tropical mountain rain forest 



Basic local alignment search tool


Linkage fraction


Internal transcribed spacer


Large subunit (beginning of LSU to primer region LR3)


Nuclear ribosomal DNA


Molecular operational taxonomic unit


Modified Rand index


National Center for Biotechnology Information


Polymerase chain reaction


Reserva Biológica San Francisco and surroundings


Small subunit


Threshold (distance)



We thank Jim Luteyn and Paola Pedraza for help with species identification of Neotropical Ericaceae and Michael Weiß for kindly providing Sebacinales-specific primers. We also express our gratitude to Ingrid Kottke for valuable comments and Tanja Schuster for critically revising earlier versions of this manuscript. We thank Jörg Bendix, Rüttger Rollenbeck and Christoph Reudenbach for providing a map of Ecuador. In addition, the support and assistance of the UTPL in Loja, Ecuador and members of the research group FOR 816 are highly appreciated. This project was funded by the DFG as part of the research unit FOR 816.

Supplementary material

10531_2011_205_MOESM1_ESM.tif (404 kb)
Fig. S1: Plot of modified Rand indices (MRI) observed during cluster optimization with a linkage fraction of 0.1 (TIFF 404 kb)
10531_2011_205_MOESM2_ESM.xls (340 kb)
Table S1: General information about sequences from all three data sets (XLS 341 kb)
10531_2011_205_MOESM3_ESM.xls (24 kb)
Table S2: Numbers of molecular operational taxonomic units (MOTUs) of Sebacinales obtained with distinct clustering approaches (XLS 24 kb)


  1. Allen T, Millar T, Berch S et al (2003) Culturing and direct DNA extraction find different fungi from the same ericoid mycorrhizal roots. New Phytol 160:255–272CrossRefGoogle Scholar
  2. Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410PubMedGoogle Scholar
  3. Arnold A, Maynard Z, Gilbert G (2000) Are tropical fungal endophytes hyperdiverse? Ecology 3:267–274Google Scholar
  4. Beck E, Makeschin F, Haubrich F et al (2008) The ecosystem (Reserva Biológica San Francisco). In: Beck E, Bendix J, Kottke I et al (eds) Gradients in a tropical mountain ecosystem of Ecuador. Ecological studies, vol 198. Springer, BerlinCrossRefGoogle Scholar
  5. Bendix J, Rollenbeck R, Reudenbach C (2006) Diurnal patterns of rainfall in a tropical Andean valley of southern Ecuador as seen by a vertically pointing K-band Doppler radar. Int J Climatol 26:829–846CrossRefGoogle Scholar
  6. Bickford D, Lohman D, Sodhi N et al (2007) Cryptic species as a window on diversity and conservation. Trends Ecol Evol 22:148–155PubMedCrossRefGoogle Scholar
  7. Bidartondo MI, Duckett JG (2009) Conservative ecological and evolutionary patterns in liverwort-fungal symbioses. Proc R Soc B 277:485–492PubMedCrossRefGoogle Scholar
  8. Bonfante-Fasolo P (1980) Occurrence of a basidiomycete in living cells of mycorrhizal hair roots of Calluna vulgaris. Trans Br Mycol Soc 72:320–325CrossRefGoogle Scholar
  9. Brummitt N, Lughadha EN (2003) Biodiversity: where’s hot and where’s not. Conserv Biol 17:1442–1448CrossRefGoogle Scholar
  10. Can F (2009) DNA barcoding confirms species rank for a cryptic geometrid species from Turkey and Bulgaria (Lepidoptera: Geometridae: Sterrhinae). Zootaxa 2314:63–68Google Scholar
  11. Chao A (1987) Estimating the population size for capture-recapture data with unequal catchability. Biometrics 43:783–791PubMedCrossRefGoogle Scholar
  12. Colwell R (2011) EstimateS: statistical estimation of species richness and shared species from samples. Version 8.2. Accessed 9 June 2011
  13. Cullings K (1994) Molecular phylogeny of the Monotropoideae (Ericaceae) with a note on the placement of the Pyroloideae. J Evol Biol 7:501–516CrossRefGoogle Scholar
  14. Dayrat B (2005) Towards integrative taxonomy. Biol J Linn Soc 85:407–415CrossRefGoogle Scholar
  15. Douhan GW, Rizzo DM (2005) Phylogenetic divergence in a local population of the ectomycorrhizal fungus Cenococcum geophilum. New Phytol 166:263–271PubMedCrossRefGoogle Scholar
  16. Douhan GW, Vincenot L, Gryta H et al (2011) Population genetics of ectomycorrhizal fungi: from current knowledge to emerging directions. Fungal Biol 115:569–597Google Scholar
  17. Drummond A, Ashton B, Cheung M et al (2009) Geneious v4.7. Accessed 9 June 2011
  18. Eberhardt U (2010) A constructive step towards selecting a DNA barcode for fungi. New Phytol 187:265–268PubMedCrossRefGoogle Scholar
  19. Feuerer T, Hawksworth D (2007) Biodiversity of lichens, including a world-wide analysis of checklist data based on Takhtajan’s floristic regions. Biodivers Conserv 16:85–98CrossRefGoogle Scholar
  20. Gardes M, Bruns T (1993) ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118PubMedCrossRefGoogle Scholar
  21. Gargas A, Taylor J (1992) Polymerase chain reaction (PCR) primers for amplifying and sequencing nuclear 18S rDNA from lichenized fungi. Mycologia 84:589–592CrossRefGoogle Scholar
  22. Glen M, Tommerup I, Bougher N et al (2002) Are Sebacinaceae common and widespread ectomycorrhizal associates of Eucalyptus species in Australian forests? Mycorrhiza 12:243–247PubMedCrossRefGoogle Scholar
  23. Göker M, García-Blázquez G, Voglmayr H et al (2009) Molecular taxonomy of phytopathogenic fungi: a case study in Peronospora. PLoS ONE. doi: 10.1371/journal.pone.0006319
  24. Göker M, Grimm G, Auch A et al (2010) A clustering optimization strategy for molecular taxonomy applied to planktonic Foraminifera SSU rDNA. Evol Bioinform Online 6:97PubMedCrossRefGoogle Scholar
  25. Gotelli N, Colwell R (2001) Quantifying biodiversity: procedures and pitfalls in the measurement and comparison of species richness. Ecol Lett 4:379–391CrossRefGoogle Scholar
  26. Haug I, Weiß M, Homeier J et al (2005) Russulaceae and Telephoraceae form ectomycorrhizas with members of the Nyctaginaceae (Caryophyllales) in the tropical mountain rain forest of southern Ecuador. New Phytol 165:923–936PubMedCrossRefGoogle Scholar
  27. Hawksworth D (1993) The tropical fungal biota: census, pertinence, prophylaxis, and prognosis. In: Isaac S, Frankland JC, Whalley JS (eds) Aspects of tropical mycology. University of Cambridge, MelbourneGoogle Scholar
  28. Hawksworth D (2001) The magnitude of fungal diversity: the 1.5 million species estimate revisited. Mycol Res 105:1422–1432CrossRefGoogle Scholar
  29. Hebert P, Penton E, Burns J et al (2004) Ten species in one: DNA barcoding reveals cryptic species in the Neotropical skipper butterfly Astraptes fulgerator. Proc Natl Acad Sci USA 101:14812–14817PubMedCrossRefGoogle Scholar
  30. Homeier J, Werner F (2007) Spermatophyta—Checklist Reserva Biológica San Francisco (Prov. Zamora-Chinchipe, S-Ecuador). In: Liede-Schumann L, Breckle S (eds) Provisional checklist of flora and fauna of the San Francisco valley and its surroundings [Reserva Biológica San Francisco/Prov. Zamora - Chinchipe, Southern Ecuador]. Ecotropical Monographs No. 4. The German Society for Tropical Ecology, HamburgGoogle Scholar
  31. Hopple J Jr, Vilgalys R (1994) Phylogenetic relationships among coprinoid taxa and allies based on data from restriction site mapping of nuclear rDNA. Mycologia 86:96–107CrossRefGoogle Scholar
  32. Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2319PubMedCrossRefGoogle Scholar
  33. Hubert L, Arabie P (1985) Comparing partitions. J Classif 2:193–218CrossRefGoogle Scholar
  34. Hughes KW, Petersen RH, Lickey EB (2009) Using heterozygosity to estimate a percentage DNA sequence similarity for environmental species’ delimitation across basidiomycete fungi. New Phytol 182:795–798PubMedCrossRefGoogle Scholar
  35. Kottke I, Beiter A, Weiß M et al (2003) Heterobasidiomycetes form symbiotic associations with hepatics: Jungermanniales have sebacinoid mycobionts while Aneura pinguis (Metzgeriales) is associated with a Tulasnella species. Mycol Res 107:957–968PubMedCrossRefGoogle Scholar
  36. Kottke I, Haug I, Setaro S et al (2008) Guilds of mycorrhizal fungi and their relation to trees, ericads, orchids and liverworts in a Neotropical mountain rain forest. Basic Appl Ecol 9:13–23CrossRefGoogle Scholar
  37. Kottke I, Suárez J, Herrera P et al (2010) Atractiellomycetes belonging to the “rust” lineage (Pucciniomycotina) form mycorrhizae with terrestrial and epiphytic Neotropical orchids. Proc R Soc B 277:1289–1298PubMedCrossRefGoogle Scholar
  38. Koufopanou V, Burt A, Taylor JW (1997) Concordance of gene genealogies reveals reproductive isolation in the pathogenic fungus Coccidioides immitis. Proc Natl Acad Sci USA 94:5478–5482PubMedCrossRefGoogle Scholar
  39. Krüger M, Stockinger H, Krüger C et al (2009) DNA-based species level detection of Glomeromycota: one PCR primer set for all arbuscular mycorrhizal fungi. New Phytol 183:212–223PubMedCrossRefGoogle Scholar
  40. Kurtzman CP, Robnett CJ (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Leeuwenhoek 73:331–371PubMedCrossRefGoogle Scholar
  41. Lee C, Grasso C, Sharlow MF (2002) Multiple sequence alignment using partial order graphs. Bioinformatics 18:452–464PubMedCrossRefGoogle Scholar
  42. Legendre P, Legendre L (1998) Numerical ecology. Elsevier, AmsterdamGoogle Scholar
  43. Lynch M, Thorn R (2006) Diversity of Basidiomycetes in Michigan agricultural soils. Appl Environ Microbiol 72:7050–7056PubMedCrossRefGoogle Scholar
  44. Morris M, Smith M, Rizzo D et al (2008) Contrasting ectomycorrhizal fungal communities on the roots of co-occurring oaks (Quercus spp.) in a California woodland. New Phytol 178:167–176PubMedCrossRefGoogle Scholar
  45. Moyersoen B (2006) Pakaraimaea dipterocarpacea is ectomycorrhizal, indicating an ancient Gondwanaland origin for the ectomycorrhizal habit in Dipterocarpaceae. New Phytol 172:753–762PubMedCrossRefGoogle Scholar
  46. Myers N, Mittermeier RA, Mittermeier CG, da Fonseca GAB, Kent J (2000) Biodiversity hotspots for conservation priorities. Nature 403:853–858PubMedCrossRefGoogle Scholar
  47. Newsham K, Bridge P (2010) Sebacinales are associates of the leafy liverwort Lophozia excisa in the southern maritime Antarctic. Mycorrhiza 20:307–313PubMedCrossRefGoogle Scholar
  48. Nilsson R, Kristiansson E, Ryberg M et al (2008) Intraspecific ITS variability in the Kingdom Fungi as expressed in the international sequence databases and its implications for molecular species identification. Evol Bioinform Online 4:193–201PubMedGoogle Scholar
  49. O’Donnell K (1993) Fusarium and its near relatives. In: Reynolds D, Taylor J (eds) The fungal holomorph: mitotic, meiotic and pleomorphic speciation in fungal systematics. CAB International, Wallingford, pp 225–233Google Scholar
  50. Öpik M, Moora M, Liira J et al (2006) Composition of root-colonizing arbuscular mycorrhizal fungal communities in different ecosystems around the globe. Ecology 94:778–790CrossRefGoogle Scholar
  51. Öpik M, Moora M, Zobel M et al (2008) High diversity of arbuscular mycorrhizal fungi in a boreal herb-rich coniferous forest. New Phytol 179:867–876PubMedCrossRefGoogle Scholar
  52. Peay KG, Kennedy PG, Davies SJ et al (2010) Potential link between plant and fungal distributions in a dipterocarp rainforest: community and phylogenetic structure of tropical ectomycorrhizal fungi across a plant and soil ecotone. New Phytol 185:529–542PubMedCrossRefGoogle Scholar
  53. Porter T, Skillman J, Moncalvo J (2008) Fruiting body and soil rDNA sampling detects complementary assemblage of Agaricomycotina (Basidiomycota, Fungi) in a hemlock-dominated forest plot in southern Ontario. Mol Ecol 17:3037–3050PubMedCrossRefGoogle Scholar
  54. Pringle A, Baker D, Platt J et al (2005) Cryptic speciation in the cosmopolitan and clonal human pathogenic fungus Aspergillus fumigatus. Evolution 59:1886–1899PubMedGoogle Scholar
  55. Ryberg M, Kristiansson E, Sjökvist E et al (2009) An outlook on the fungal internal transcribed spacer sequences in GenBank and the introduction of a web-based tool for the exploration of fungal diversity. New Phytol 181:471–477PubMedCrossRefGoogle Scholar
  56. Selosse M-A, Bauer R, Moyersoen B (2002a) Basal hymenomycetes belonging to the Sebacinaceae are ectomycorrhizal on temperate deciduous trees. New Phytol 155:183–195CrossRefGoogle Scholar
  57. Selosse M, Weiß M, Jany J et al (2002b) Communities and populations of sebacinoid basidiomycetes associated with the achlorophyllous orchid Neottia nidus-avis (L.) LCM Rich. and neighbouring tree ectomycorrhizae. Mol Ecol 11:1831–1844PubMedCrossRefGoogle Scholar
  58. Selosse M, Setaro S, Glatard F et al (2007) Sebacinales are common mycorrhizal associates of Ericaceae. New Phytol 174:864–878PubMedCrossRefGoogle Scholar
  59. Selosse M, Dubois M, Alvarez N (2009) Do Sebacinales commonly associate with plant roots as endophytes? Mycol Res 113:1062–1069PubMedCrossRefGoogle Scholar
  60. Setaro SD, Kron KA (2011) Neotropical and North American Vaccinioideae (Ericaceae) share their mycorrhizal Sebacinales—an indication for concerted migration? PLoS Curr 3:RRN1227Google Scholar
  61. Setaro S, Weiß M, Oberwinkler F et al (2006a) Sebacinales form ectendomycorrhizas with Cavendishia nobilis, a member of the Andean clade of Ericaceae, in the mountain rain forest of southern Ecuador. New Phytol 169:355–365PubMedCrossRefGoogle Scholar
  62. Setaro S, Kottke I, Oberwinkler F (2006b) Anatomy and ultrastructure of mycorrhizal associations of Neotropical Ericaceae. Mycol Prog 5:243–254CrossRefGoogle Scholar
  63. Shearer C, Descals E, Kohlmeyer B et al (2007) Fungal biodiversity in aquatic habitats. Biodivers Conserv 16:49–67CrossRefGoogle Scholar
  64. Stielow B, Bubner B, Hensel G et al (2010) The neglected hypogeous fungus Hydnotrya bailii Soehner (1959) is a widespread sister taxon of Hydnotrya tulasnei (Berk.) Berk. & Broome (1846). Mycol Prog 9:195–203CrossRefGoogle Scholar
  65. Stielow B, Bratek Z, Orczán A et al (2011) Species delimitation in taxonomically difficult fungi: the case of Hymenogaster. PLoS ONE 6:e15614PubMedCrossRefGoogle Scholar
  66. Suárez JP, Weiß M, Abele A et al (2008) Members of Sebacinales subgroup B form mycorrhizae with epiphytic orchids in a Neotropical mountain rain forest. Mycol Prog 7:75–85CrossRefGoogle Scholar
  67. Swofford D (2002) Paup* phylogenetic analysis using parsimony (* and other methods). Version 4. Sinauer Associates, SunderlandGoogle Scholar
  68. Tedersoo L, Gates G, Dunk C et al (2009) Establishment of ectomycorrhizal fungal community on isolated Nothofagus cunninghamii seedlings regenerating on dead wood in Australian wet temperate forests: does fruit-body type matter? Mycorrhiza 19:403–416PubMedCrossRefGoogle Scholar
  69. Urban A, Weiß M, Bauer R (2003) Ectomycorrhizas involving sebacinoid mycobionts. Mycol Res 107:3–14PubMedCrossRefGoogle Scholar
  70. Warcup J (1988) Mycorrhizal associations of isolates of Sebacina vermifera. New Phytol 110:227–231CrossRefGoogle Scholar
  71. Weiß M, Selosse M, Rexer K et al (2004) Sebacinales: a hitherto overlooked cosm of heterobasidiomycetes with a broad mycorrhizal potential. Mycol Res 108:1003–1010PubMedCrossRefGoogle Scholar
  72. Weiß M, Sýkorová Z, Garnica S et al (2011) Sebacinales everywhere: previously overlooked ubiquitous fungal endophytes. PLoS ONE 6:e16793PubMedCrossRefGoogle Scholar
  73. White T, Bruns T, Lee S et al (1990) Analysis of phylogenetic relationships by amplification and direct sequencing of ribosomal RNA genes. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to method and applications. Academic Press, New York, pp 315–322Google Scholar
  74. Will KW, Mishler BD, Wheeler QD (2005) The perils of DNA barcoding and the need for integrative taxonomy. Syst Biol 54:844–851PubMedCrossRefGoogle Scholar
  75. Yilmaz P, Kottmann R, Field D et al (2010) The “minimum information about an environmental sequence” (MIENS) specification. Nat Preced. doi: 10.1038/npre.2010.5252.2

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Sabrina D. Setaro
    • 1
  • Sigisfredo Garnica
    • 2
  • Paulo I. Herrera
    • 3
  • Juan Pablo Suárez
    • 3
  • Markus Göker
    • 4
  1. 1.Department of BiologyWake Forest UniversityWinston-SalemUSA
  2. 2.Institut für Evolution und Ökologie, Organismische BotanikUniversität TübingenTübingenGermany
  3. 3.Centro de Biología Celular y MolecularUniversidad Técnica Particular de LojaLojaEcuador
  4. 4.DSMZ—German Collection of Microorganisms and Cell CulturesBraunschweigGermany

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