Internal transcribed spacer 1 secondary structure analysis reveals a common core throughout the anaerobic fungi (Neocallimastigomycota)

PLoS One. 2014 Mar 24;9(3):e91928. doi: 10.1371/journal.pone.0091928. eCollection 2014.


The internal transcribed spacer (ITS) is a popular barcode marker for fungi and in particular the ITS1 has been widely used for the anaerobic fungi (phylum Neocallimastigomycota). A good number of validated reference sequences of isolates as well as a large number of environmental sequences are available in public databases. Its highly variable nature predisposes the ITS1 for low level phylogenetics; however, it complicates the establishment of reproducible alignments and the reconstruction of stable phylogenetic trees at higher taxonomic levels (genus and above). Here, we overcame these problems by proposing a common core secondary structure of the ITS1 of the anaerobic fungi employing a Hidden Markov Model-based ITS1 sequence annotation and a helix-wise folding approach. We integrated the additional structural information into phylogenetic analyses and present for the first time an automated sequence-structure-based taxonomy of the ITS1 of the anaerobic fungi. The methodology developed is transferable to the ITS1 of other fungal groups, and the robust taxonomy will facilitate and improve high-throughput anaerobic fungal community structure analysis of samples from various environments.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anaerobiosis
  • Base Sequence
  • DNA, Ribosomal Spacer / chemistry*
  • DNA, Ribosomal Spacer / genetics*
  • Genetic Markers / genetics
  • Markov Chains
  • Neocallimastigomycota / classification
  • Neocallimastigomycota / genetics*
  • Neocallimastigomycota / metabolism
  • Nucleic Acid Conformation
  • Phylogeny


  • DNA, Ribosomal Spacer
  • Genetic Markers

Grant support

This work was supported by and research was carried out under contract to the Pastoral Greenhouse Gas Research Consortium (PGgRc;, and the New Zealand Ministry for Primary Industries (MPI; as part of its support for the Global Research Alliance on Agricultural Greenhouse Gases (GRA; The funders had no role in study design, data collection and analysis, or preparation of the manuscript. The authors required the funders' approval to publish.