, Volume 18, Issue 1, pp 15–22 | Cite as

Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a Quercus woodland

  • Matthew E. Smith
  • Greg W. Douhan
  • David M. Rizzo
Original Paper


The Internal Transcribed Spacer (ITS) regions of ribosomal DNA are widely used as markers for phylogenetic analyses and environmental sampling from a variety of organisms including fungi, plants, and animals. In theory, concerted evolution homogenizes multicopy genes so that little or no variation exists within populations or individuals. However, contrary to theory, ITS variation has been confirmed in populations and individuals from a diverse range of eukaryotes. The presence of intraspecific and intra-individual variation in multicopy genes has important implications for ecological and phylogenetic studies, yet relatively little is known about natural variation of these genes, particularly at the community level. In this study, we examined intraspecific and intra-sporocarp ITS variation by DNA sequencing from sporocarps and pooled roots from 68 species of ectomycorrhizal fungi collected at a single site in a Quercus woodland. We detected ITS variation in 27 species, roughly 40% of the taxa examined. Although intraspecific ITS variation was generally low (0.16–2.85%, mean = 0.74%), it was widespread within this fungal community. We detected ITS variation in both sporocarps and ectomycorrhizal roots, and variation was present within species of Ascomycota and Basidiomycota, two distantly related lineages within the Fungi. We discuss the implications of such widespread ITS variability with special reference to DNA-based environmental sampling from diverse fungal communities.


Cloning Ectomycorrhiza Environmental sampling Fungi  ITS variation Ribosomal DNA 

Supplementary material


  1. Aanen DK, Kuyper TW, Hoekstra RF (2001) A widely distributed ITS polymorphism within a biological species of the ectomycorrhizal fungus Hebeloma velutipes. Mycol Res 105:284–290CrossRefGoogle Scholar
  2. Allmer J, Vasiliauskas R, Ihrmark K, Stenlid J, Dahlberg A (2006) Wood-inhabiting fungal communities in woody debris of Norway spruce (Picea abies(L.) Karst.), as reflected by sporocarps, mycelial isolations and T-RFLP identification. FEMS Micro Ecol 43:329–340Google Scholar
  3. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nuc Acids Res 25:3389–3402CrossRefGoogle Scholar
  4. Avis PG, Dickie IA, Mueller GM (2006) A ‘dirty’ business: testing the limitations of terminal restriction length polymorphism (TRFLP) analysis of soil fungi. Mol Ecol 15:873–882PubMedCrossRefGoogle Scholar
  5. Bensoussan E, Nasereddin A, Jonas F, Schnur LF, Jaffe CL (2006) Comparison of PCR assays for diagnosis of cutaneous Leishmaniasis. J Clinic Microbiol 44:1435–1439CrossRefGoogle Scholar
  6. Bidartondo MI, Gardes M (2005) Fungal diversity in molecular terms: profiling, identification, and quantification in the environment. In: Dighton J, White JF, Oudemans P (eds) The fungal community—its organization and role in the ecosystem, third edition. Taylor and Francis, New York, pp 215–239Google Scholar
  7. Bruns TB, Shefferson RP (2004) Evolutionary studies of ectomycorrhizal fungi: recent advances and future directions. Can J Bot 82:1122–1132CrossRefGoogle Scholar
  8. Buchan A, Newell SY, Moreta JIL, Moran MA (2002) Analysis of internal transcribed spacer (ITS) regions of rRNA genes in fungal communities in a southeastern US salt marsh. Micro Ecol 43:329–340CrossRefGoogle Scholar
  9. Buckler ES, Ippolito A, Holtsford TP (1997) The evolution of ribosomal DNA: divergent paralogues and phylogenetic implications. Genetics 145:821–832PubMedGoogle Scholar
  10. Campbell BC, Steffen-Campbell JD, Werren JH (1993) Phylogeny of the Nasonia species complex (Hymenoptera: Pteromalidae) inferred from an internal transcribed spacer (ITS2) and 28S rDNA sequences. Ins Mol Biol 2:225–237Google Scholar
  11. Chenna R, Sugawara H, Koide T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nuc Acids Res 31:3497–3500CrossRefGoogle Scholar
  12. Cruse M, Telerant R, Gallagher T, Lee T, Taylor J (2002) Cryptic species in Stachybotrys chartarum. Mycologia 94:814–822CrossRefGoogle Scholar
  13. Dickie IA, FitzJohn RG (2007) Using terminal restriction fragment length polymorphism (T-RFLP) to identify mycorrhizal fungi: a methods review. Mycorrhiza 17:259–270PubMedCrossRefGoogle Scholar
  14. Dickie IA, Xu B, Koide RT (2002) Vertical niche differentiation of ectomycorrhizal hyphae in soil as shown by T-RFLP analysis. New Phytol 156:527–535CrossRefGoogle 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. Downie SR, Katz-Downie DS, Spalik K (2000) A phylogeny of Apiaceae tribe Scandiceae: evidence from nuclear ribosomal DNA internal transcribed spacer sequences. Am J Bot 87:76–95PubMedCrossRefGoogle Scholar
  17. Elder JF, Turner BJ (1995) Concerted evolution of repetitive DNA-sequences in eukaryotes. Quart Rev Biol 70:297–320PubMedCrossRefGoogle Scholar
  18. Fairley TL, Kilpatrick CW, Conn JE (2005) Intragenomic hetereogeneity of internal transcribed spacer rDNA in neotropical malaria vector Anopheles aquasalis (Diptera: Culicidae). J Med Ent 42:795–800CrossRefGoogle Scholar
  19. Feliner G, Larena BG, Aguilar JF (2004) Fine-scale geographical structure, intra-individual polymorphism and recombination in nucreal ribosomal internal transcribed spacers in Armeria (Plumbaginaceae). Ann Bot 93:189–200CrossRefGoogle Scholar
  20. Fischer M, Binder M (2004) Species recognition, geographic distribution, and host–pathogen relationships: a case study in a group of lignicolous basidiomycetes, Phellinus s.l. Mycologia 96:799–811Google Scholar
  21. Frøslev TG, Matheny PB, Hibbett DS (2005) Lower level relationships in the mushroom genus Cortinarius (Basidiomycota, Agaricales): a comparison of RPB1, RPB2, and ITS phylogenies. Mol Phylo Evo 37:602–618CrossRefGoogle Scholar
  22. Gandolfi A, Bonilauri P, Rossi V, Menozzi P (2001) Intraindividual and intraspecies variability of ITS1 sequences in the ancient asexual Darwinula stevensoni (Crustaceae: Ostracoda). Heredity 87:449–455PubMedCrossRefGoogle Scholar
  23. Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118PubMedGoogle Scholar
  24. Gardes M, Bruns TD (1996) Community structure of ectomycorrhizal fungi in a Pinus muricata forest: above- and below-ground views. Can J Bot 74:1572–1583Google Scholar
  25. Geml J, Laursen GA, O’Neill KO, Nusbaum C, Taylor DL (2006) Beringian origins and cryptic speciation events in the fly agaric (Amanita muscaria). Mol Ecol 15:225–239PubMedCrossRefGoogle Scholar
  26. Glen M, Tommercup IC, Bougher NL, O’Brien PA (2001) Interspecific and intra-specific variation of ectomycorrhizal fungi associated with eucalyptus ecosystems as revealed by ribosomal DNA PCR-RFLP. Mycol Res 105:843–858CrossRefGoogle Scholar
  27. Harris DJ, Crandall KA (2000) Intragenomic variation within ITS1 and ITS2 of freshwater crayfishes (Decapoda: Cambaridae); implications for phylogenetic and microsatelite studies. Mol Biol Evol 17:284–291PubMedGoogle Scholar
  28. Hopple J, Vilgayls R (1994) Phylogenetic relationships among coprinoid taxa and allies based on data from restriction site mapping of nuclear rDNA. Mycologia 86:96–107CrossRefGoogle Scholar
  29. Horton TR (2002) Molecular approaches to ectomycorrhizal diversity studies: variation in ITS at a local scale. Plant Soil 244:29–39CrossRefGoogle Scholar
  30. Hugall A, Stanton J, Moritz C (1999) Reticulate evolution and the origins of ribosomal internal transcribed spacer diversity in apomictic Meloidogyne. Mol Biol Evol 16:157–164PubMedGoogle Scholar
  31. Izzo AD, Agbowo J, Bruns TD (2005) Detection of plot-level changes in ectomycorrhizal communities across years in an old-growth mixed-conifer forest. New Phytol 166:619–630PubMedCrossRefGoogle Scholar
  32. Jasalavich CA, Ostrofsky A, Jellison J (2000) Detection and identification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA. App Environ Microbiol 66:4725–4734CrossRefGoogle Scholar
  33. Kåren O, Hõgberg N, Dahlberg A, Jonsson L, Nylund J-E (1997) Inter- and intra-specific variation in the ITS region of ectomyocrrhizal fungi in Fennoscandia as detected by endonuclease analysis. New Phytol 136:313–325CrossRefGoogle Scholar
  34. Kauserud H, Schumacher T (2003) Ribosomal DNA variation, recombination and inheritance in the basidiomycete Trichaptum abietinum: implications for reticulate evolution. Heredity 91:163–172PubMedCrossRefGoogle Scholar
  35. Kauserud H, Stensrud Ø, Decock C, Shalchian-Tarbriz K, Schumacher T (2006) Multiple gene genealogies and AFLP’s suggest cryptic speciation and long-distance dispersal in the basidiomycete Serpula himantioides (Boletales). Mol Ecol 15:421–431PubMedCrossRefGoogle Scholar
  36. Kõljalg U, Dahlber A, Taylor AFS, Larsson E, Hallenberg N, Stenlid J, Larsson K-H, Fransson PM, Kåren O, Jonsson L (2000) Diversity and abundance of resupinate thelephoroid fungi as ectomycorrhizal symbionts in Swedish boreal forests. Mol Ecol 9:1985–1996PubMedCrossRefGoogle Scholar
  37. Liao D (1999) Concerted evolution: molecular mechanism and biological implications. Am J Hum Gen 64:24–30CrossRefGoogle Scholar
  38. Madani M, Subbotin SA, Moen M (2005) Quantitative detection of the potato cyst nematode, Globodera pallida, and the beet cyst nematode, Heterodera schachtii, using real-time PCR with SYBR green I dye. Mol Cell Probes 19:81–86PubMedCrossRefGoogle Scholar
  39. Maddison DR, Maddison WP (2000) MacClade: interactive analysis of phylogeny and character evolution, version 4.05. Sinauer, Sunderland, MassachusettsGoogle Scholar
  40. Martin FN, Tooley PW, Blomquist C (2004) Molecular detection of Phytophthora ramorum, the causal agent of sudden oak death in California, and two additional species commonly recovered from diseased plant material. Phytopathology 94:621–631CrossRefGoogle Scholar
  41. Mello A, Canitsani A, Vizzini A, Bonfante P (2002) Genetic variability of Tuber unicatum and its relatedness to other black truffles. Environ Microbiol 4:584–594PubMedCrossRefGoogle Scholar
  42. Mello A, Murat C, Vizzinin, Gavazza V, Bonfante P (2005) Tuber magnatum Pico, a species of limited geographical distribution: its genetic diversity inside and outside a truffle ground. Environ Microbiol 7:55–56PubMedCrossRefGoogle Scholar
  43. Murat C, Diez J, Delaruelle C, Dupre C, Chevalier G, Bonfante P, Martin F (2004) Polymorphism at the ribosomal DNA ITS and its relation to postglacial re-colonization routes of the Perigord truffle Tuber melanosporum. New Phytol 164:401–411CrossRefGoogle Scholar
  44. O’Brien HE, Parrent JL, Jackson JA, Moncalvo J-M, Vilgalys R (2005) Fungal community analysis by large-scale sequencing of environmental samples. App Environ Microbiol 71:5544–5550CrossRefGoogle Scholar
  45. O’Donnell K, Cigelnik E, Nirenberg HI (1998) Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycologia 90:465–493CrossRefGoogle Scholar
  46. Petti CA (2007) Detection and identification of microorganisms by gene amplification and sequencing. Clin Infect Dis 44:1108–1114PubMedCrossRefGoogle Scholar
  47. Redecker D (2002) Molecular identification and phylogeny of arbuscular mycorrhizal fungi. Plant Soil 244:67–73CrossRefGoogle Scholar
  48. Reiss E, Tanaka K, Bruker G, Chazalet V, Coleman D, Debeaupuis JP, Hanazawa R, Latge JP, Lortholary J, Makimura K, Morrison CJ, Murayama SY, Naoe S, Paris S, Sarfati J, Shibuya K, Sullivan D, Uchida K, Yamaguchi H (1998) Molecular diagnosis and epidemiology of fungal infections. Med Mycol 36:249–257PubMedCrossRefGoogle Scholar
  49. Rooney AP, Ward TJ (2005) Evolution of a large ribosomal RNA multigene family in filamentous fungi: birth and death of a concerted evolution paradigm. Proc Natl Acad Sci 102:5084–5089PubMedCrossRefGoogle Scholar
  50. Scupham AJ, Presley LL, Wei B, Bent E, Griffith N, McPherson M, Zhu F, Oluwadara O, Rao N, Braun J, Borneman J (2006) Abundant and diverse fungal microbiota in the murine intestine. App Environ Microbiol 72:793–801CrossRefGoogle Scholar
  51. Smith ME, Trappe JM, Rizzo DM (2006) Genea, Genabea, and Gilkeya gen. nov.: ascomata and ectomycorrhiza formation in a Quercus woodland. Mycologia 98:699–716PubMedCrossRefGoogle Scholar
  52. Smith ME, Douhan GW, Rizzo DM (2007) Ectomycorrhizal community structure in a xeric Quercus woodland based on rDNA sequence analysis of sporocarps and pooled roots. New Phytol 170:143–151Google Scholar
  53. Tedersoo L, Köljalg, U, Hallenberg N, Larsson K (2003) Fine scale distribution of ectomycorrhizal fungi and roots across substrate layers including course woody debris in a mixed forest. New Phytol 159:153–165CrossRefGoogle Scholar
  54. Tedersoo L, Hansen K, Perry BA, Kjøller R (2006) Molecular and morphological diversity of pezizalean ectomycorrhiza. New Phytol 170:581–596PubMedCrossRefGoogle Scholar
  55. Villa NO, Kageyama K, Asano T, Suga H (2006) Phylogenetic relationships of Pythium and Phytophthora species based on ITS rDNA, cytochrome oxidase II and β-tubulin gene sequences. Mycologia 98:410–422PubMedGoogle Scholar
  56. White TJ, Bruns TD, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR Protocols: a guide to methods and applications. Academic, San Diego, USA, pp 315–322Google Scholar
  57. Worheide G, Nichols SA, Goldberg J (2004) Intragenomic variation of the rDNA internal transcribed spacers in sponges (Phylum Porifera): implications for phylogenetic studies. Mol Phylo Evol 33:816–830CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Matthew E. Smith
    • 1
  • Greg W. Douhan
    • 3
  • David M. Rizzo
    • 2
  1. 1.Department of Organismic and Evolutionary Biology and the Farlow HerbariumHarvard UniversityCambridgeUSA
  2. 2.Department of Plant PathologyUniversity of California at DavisDavisUSA
  3. 3.Department of Plant Pathology and MicrobiologyUniversity of California at RiversideRiversideUSA

Personalised recommendations