Microbial Ecology

, Volume 56, Issue 4, pp 604–614 | Cite as

Microfungal “Weeds” in the Leafcutter Ant Symbiosis

  • A. Rodrigues
  • M. Bacci Jr
  • U. G. Mueller
  • A. Ortiz
  • F. C. Pagnocca
Original Article


Leafcutter ants (Formicidae: tribe Attini) are well-known insects that cultivate basidiomycete fungi (Agaricales: Lepiotaceae) as their principal food. Fungus gardens are monocultures of a single cultivar strain, but they also harbor a diverse assemblage of additional microbes with largely unknown roles in the symbiosis. Cultivar-attacking microfungi in the genus Escovopsis are specialized parasites found only in association with attine gardens. Evolutionary theory predicts that the low genetic diversity in monocultures should render ant gardens susceptible to a wide range of diseases, and additional parasites with roles similar to that of Escovopsis are expected to exist. We profiled the diversity of cultivable microfungi found in 37 nests from ten Acromyrmex species from Southern Brazil and compared this diversity to published surveys. Our study revealed a total of 85 microfungal strains. Fusarium oxysporum and Escovopsis were the predominant species in the surveyed gardens, infecting 40.5% and 27% of the nests, respectively. No specific relationship existed regarding microfungal species and ant-host species, ant substrate preference (dicot versus grass) or nesting habit. Molecular data indicated high genetic diversity among Escovopsis isolates. In contrast to the garden parasite, F. oxysporum strains are not specific parasites of the cultivated fungus because strains isolated from attine gardens have similar counterparts found in the environment. Overall, the survey indicates that saprophytic microfungi are prevalent in South American leafcutter ants. We discuss the antagonistic potential of these microorganisms as “weeds” in the ant–fungus symbiosis.


Internal Transcribe Spacer Fungus Garden Cunninghamella Oxysporum Strain Syncephalastrum Racemosum 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We would like to thank the “Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)” for a scholarship supporting A Rodrigues, “Fundação de Amparo a Pesquisa Científica do Estado de São Paulo” (FAPESP) for funding the fieldwork; and DC Marini and J Martins Jr for laboratory support. We are also grateful to A Silva for comments on earlier versions of this manuscript as well as NM Gerardo and one anonymous referee for kindly reviewing this manuscript. We also thank SE Solomon for helping with the phylogenetic analyses.


  1. 1.
    Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman D (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1:3389–3402CrossRefGoogle Scholar
  2. 2.
    Bacci M Jr, Ribeiro SB, Casarotto MEF, Pagnocca FC (1995) Biopolymer-degrading bacteria from nests of the leaf-cutting ant Atta sexdens rubropilosa. Braz J Med Biol Res 28:79–82Google Scholar
  3. 3.
    Bandelt H-J, Foster P, Röhl A (1999) Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol 16:37–48PubMedGoogle Scholar
  4. 4.
    Barron GL (1968) The genera of hyphomycetes from soil. Robert E. Krieger, New YorkGoogle Scholar
  5. 5.
    Batra LR (1979) Insect–fungus symbiosis: nutrition, mutualism and commensalisms. Allanheld, Osmun, MontclairGoogle Scholar
  6. 6.
    Bourtzis K, Miller TA (2006) Insect symbiosis, vol 2. CRC Press, FloridaGoogle Scholar
  7. 7.
    Brown JM, Lemmon AR (2007) The importance of data partitioning and the utility of Bayes factors in Bayesian phylogenetics. Syst Biol 56:643–655PubMedCrossRefGoogle Scholar
  8. 8.
    Cafaro MJ, Currie CR (2005) Phylogenetic analysis of mutualistic filamentous bacteria associated with fungus-growing ants. Can J Microbiol 51:441–446PubMedCrossRefGoogle Scholar
  9. 9.
    Carreiro SC, Pagnocca FC, Bueno OC, Bacci M Jr, Hebling MJA, Silva OA (1997) Yeasts associated with nests of the leaf-cutting ant Atta sexdens rubropilosa Forel, 1908. Antonie van Leeuwenhoek 71:243–248PubMedCrossRefGoogle Scholar
  10. 10.
    Craven SE, Dix MW, Michaels GE (1970) Attine fungus gardens contain yeasts. Science 169:184–186PubMedCrossRefGoogle Scholar
  11. 11.
    Currie CR, Mueller UG, Malloch D (1999) The agricultural pathology of ant fungus gardens. Proc Natl Acad Sci U S A 96:7998–8002PubMedCrossRefGoogle Scholar
  12. 12.
    Currie CR (2001) Prevalence and impact of a virulent parasite on a tripartite mutualism. Oecologia 128:99–106CrossRefGoogle Scholar
  13. 13.
    Currie CR, Stuart AE (2001) Weeding and grooming of pathogens in agriculture by ants. Proc R Soc Lond B 268:1033–1039CrossRefGoogle Scholar
  14. 14.
    Currie CR, Wong B, Stuart AE, Schultz TR, Rehner SA, Mueller UG, Sung GH, Spatafora JW, Straus NA (2003) Ancient tripartite coevolution in the attine ant–microbe symbiosis. Science 299:386–388PubMedCrossRefGoogle Scholar
  15. 15.
    Domsch KH, Gams W, Anderson T (1980) Compendium of soil fungi, vol. 1 and 2. Academic, LondonGoogle Scholar
  16. 16.
    Druzhinina IS, Kopchinskiy AG, Komon M, Bissett J, Szakacs G, Kubicek CP (2005) An oligonucleotide barcode for species identification in Trichoderma and Hypocrea. Fungal Genet Biol 42:813–828PubMedCrossRefGoogle Scholar
  17. 17.
    Felsenstein J (1985) Confidence limits on phylogenies: an approach using de bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  18. 18.
    Fernández-Marín H, Zimmerman JK, Rehner SA, Wcislo WT (2006) Active use of the metapleural glands by ants in controlling fungal infection. Proc R Soc Lond B 273:1689–1695CrossRefGoogle Scholar
  19. 19.
    Fisher PJ, Stradling DJ, Sutton BC, Petrini LE (1996) Microfungi in the fungus gardens of the leaf-cutting ant Atta cephalotes: a preliminary study. Mycol Res 100:541–546Google Scholar
  20. 20.
    Gerardo NM, Currie CR, Price SL, Mueller UG (2004) Exploiting a mutualism: parasite specialization on cultivars within the fungus-growing ants symbiosis. Proc R Soc Lond B 271:1791–1798CrossRefGoogle Scholar
  21. 21.
    Gerardo NM, Mueller UG, Currie CR (2006) Complex host–pathogen coevolution in the Apterostigma fungus-growing ant–microbe symbiosis. Evol Biol 6:88–97CrossRefGoogle Scholar
  22. 22.
    Gonçalves CR (1961) O Gênero Acromyrmex no Brasil (Hym. Formicidae). Stud Entomol 4:113–180Google Scholar
  23. 23.
    Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98Google Scholar
  24. 24.
    Hamilton WD, Axelrod R, Tanese R (1990) Sexual reproduction as an adaptation to resist parasites (a review). Proc Natl Acad Sci U S A 87:3566–3573PubMedCrossRefGoogle Scholar
  25. 25.
    Hölldobler B, Wilson EO (1990) The ants. Harvard University Press, CambridgeGoogle Scholar
  26. 26.
    Klich MA (2002) Identification of common Aspergillus species. Centraalbureau voor Schimmelcultures, BaarnGoogle Scholar
  27. 27.
    Lee JS, Ko KS, Jung HS (2000) Phylogenetic analysis of Xylaria based on nuclear ribosomal ITS1–5.8S-ITS2 sequences. FEMS Micro Lett 187:89–93CrossRefGoogle Scholar
  28. 28.
    Liu XY, Huang H, Zheng RY (2001) Relationships within Cunninghamella based on sequence analysis of ITR rDNA. Mycotaxon 80:77–95Google Scholar
  29. 29.
    Möller A (1893) Die Pilzgärten einiger südamerikanischer Ameisen. Bot Mittl Trop 6:1–127Google Scholar
  30. 30.
    Mueller UG, Schultz TR, Currie CR, Adams RMM, Malloch D (2001) The origin of the attine ant–fungus mutualism. Q Rev Biol 76:169–197PubMedCrossRefGoogle Scholar
  31. 31.
    Mueller UG (2002) Ant versus fungus versus mutualism: Ant-cultivar conflict and the deconstruction of the attine ant–fungus symbiosis. Am Nat 160(Suppl.):S67–98PubMedCrossRefGoogle Scholar
  32. 32.
    Mueller UG, Gerardo NM, Aanen DK, Six DL, Schultz TR (2005) The evolution of agriculture in insects. Annu Rev Ecol Evol Syst 36:563–595CrossRefGoogle Scholar
  33. 33.
    Nelson PE, Toussoun TA, Marasas WFO (1983) Fusarium species: an illustrated manual for identification. The Pennsylvania State University Press, PennsylvaniaGoogle Scholar
  34. 34.
    O’Donnell K, Cigelnik E (1998) Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol Phylogenet Evol 7:103–116CrossRefGoogle Scholar
  35. 35.
    Poulsen M, Currie CR (2006) Complexity of insect–fungal associations: exploring the influence of microorganisms on attine ant–fungus symbiosis. In: Bourtzis K, Miller TA (eds) Insect symbiosis, vol. 2. CRC, Boca Raton, FL, USA, pp 57–77Google Scholar
  36. 36.
    Poulsen M, Boomsma J (2005) Mutualistic fungi control crop diversity in fungus-growing ants. Science 307:741–744PubMedCrossRefGoogle Scholar
  37. 37.
    Reynolds HT, Currie CR (2004) Pathogenicity of Escovopsis weberi: The parasite of the attine–microbe symbiosis directly consumes the ant-cultivated fungus. Mycologia 96:955–959CrossRefGoogle Scholar
  38. 38.
    Rodrigues A, Pagnocca FC, Bacci M Jr, Hebling MJA, Bueno OC, Pfenning LH (2005) Variability of non-mutualistic filamentous fungi associated with Atta sexdens rubropilosa nests. Folia Microbiol 50:421–425CrossRefGoogle Scholar
  39. 39.
    Rodrigues A, Pagnocca FC, Bueno OC, Pfenning LH, Bacci M Jr (2005) Assessment of microfungi in fungus gardens free of the leaf-cutting ant Atta sexdens rubropilosa (Hymenoptera: Formicidae). Sociobiology 46:329–334Google Scholar
  40. 40.
    Ronquist F, Huelsenbeck JP (2003) MrBayes 3: bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574PubMedCrossRefGoogle Scholar
  41. 41.
    Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  42. 42.
    Samson RA, Hoekstra ES, Frisvad JC (2000) Introduction to food-airborne fungi, 6th ed. Centraalbureau voor Schimmelcultures, BaarnGoogle Scholar
  43. 43.
    Schultz TR, Meier R (1995) A phylogenetic analysis of the fungus-growing ants (Formicidae: Attini) based on morphological characters of the larvae. Syst Entomol 20:337–370CrossRefGoogle Scholar
  44. 44.
    Silva A, Bacci M Jr, Siqueira CG, Bueno OC, Pagnocca FC, Hebling MJA (2003) Survival of Atta sexdens workers on different food sources. J Insect Physiol 49:307–313PubMedCrossRefGoogle Scholar
  45. 45.
    Silva A, Rodrigues A, Bacci M Jr, Pagnocca FC, Bueno OC (2006) Susceptibility of ant-cultivated fungus Leucoagaricus gongylophorus (Agaricales: Basidiomycota) towards microfungi. Mycopathologia 162:115–119PubMedCrossRefGoogle Scholar
  46. 46.
    Skovgaard K, Nirenberg HI, O’Donnell K, Rosendahl S (2001) Evolution of Fusarium oxysporum f. sp. vasinfectum races inferred from multigene genealogies. Phytopathology 91:1231–1237CrossRefPubMedGoogle Scholar
  47. 47.
    Swofford DL (2002) PAUP*: Phylogenetic analysis using parsimony (*: and other methods), ver. 4. Sinauer Associates, MassachusettsGoogle Scholar
  48. 48.
    Taerum SJ, Cafaro MJ, Little AEF, Schultz TR, Currie CR (2007) Low host–pathogen specificity in the leaf-cutting ant–microbe symbiosis. Proc R Soc Lond B 274:1971–1978CrossRefGoogle Scholar
  49. 49.
    Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedCrossRefGoogle Scholar
  50. 50.
    Weber NA (1972) Gardening ants: the attines, vol. 92 Memoirs of the American Philosophical Society, PhiladelphiaGoogle Scholar
  51. 51.
    White TJ, Bruns T, 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, California, pp 315–321Google Scholar
  52. 52.
    Zwickl DJ (2006) Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion. Ph.D. dissertation, The University of Texas at AustinGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • A. Rodrigues
    • 1
    • 2
  • M. Bacci Jr
    • 1
    • 2
  • U. G. Mueller
    • 3
  • A. Ortiz
    • 4
  • F. C. Pagnocca
    • 1
    • 2
  1. 1.Center for the Study of Social InsectsUNESP-São Paulo State UniversityRio ClaroBrazil
  2. 2.Department of Biochemistry and MicrobiologyUNESP-São Paulo State UniversityRio ClaroBrazil
  3. 3.Section of Integrative BiologyUniversity of Texas at AustinAustinUSA
  4. 4.Conservación, Usos y Biodiversidad, Facultad de CienciasUniversidad Nacional de ColombiaMedellínColombia

Personalised recommendations