Intraspecific ITS Variability in the Kingdom Fungi as Expressed in the International Sequence Databases and Its Implications for Molecular Species Identification

The internal transcribed spacer (ITS) region of the nuclear ribosomal repeat unit is the most popular locus for species identification and subgeneric phylogenetic inference in sequence-based mycological research. The region is known to show certain variability even within species, although its intraspecific variability is often held to be limited and clearly separated from interspecific variability. The existence of such a divide between intra- and interspecific variability is implicitly assumed by automated approaches to species identification, but whether intraspecific variability indeed is negligible within the fungal kingdom remains contentious. The present study estimates the intraspecific ITS variability in all fungi presently available to the mycological community through the international sequence databases. Substantial differences were found within the kingdom, and the results are not easily correlated to the taxonomic affiliation or nutritional mode of the taxa considered. No single unifying yet stringent upper limit for intraspecific variability, such as the canonical 3% threshold, appears to be applicable with the desired outcome throughout the fungi. Our results caution against simplified approaches to automated ITS-based species delimitation and reiterate the need for taxonomic expertise in the translation of sequence data into species names

A software pipeline for processing and identification of fungal ITS sequences

<p>Abstract</p> <p>Background</p> <p>Fungi from environmental samples are typically identified to species level through DNA sequencing of the nuclear ribosomal internal transcribed spacer (<it>ITS</it>) region for use in BLAST-based similarity searches in the International Nucleotide Sequence Databases. These searches are time-consuming and regularly require a significant amount of manual intervention and complementary analyses. We here present software – in the form of an identification pipeline for large sets of fungal <it>ITS </it>sequences – developed to automate the BLAST process and several additional analysis steps. The performance of the pipeline was evaluated on a dataset of 350 <it>ITS </it>sequences from fungi growing as epiphytes on building material.</p> <p>Results</p> <p>The pipeline was written in Perl and uses a local installation of NCBI-BLAST for the similarity searches of the query sequences. The variable subregion <it>ITS2 </it>of the <it>ITS </it>region is extracted from the sequences and used for additional searches of higher sensitivity. Multiple alignments of each query sequence and its closest matches are computed, and query sequences sharing at least 50% of their best matches are clustered to facilitate the evaluation of hypothetically conspecific groups. The pipeline proved to speed up the processing, as well as enhance the resolution, of the evaluation dataset considerably, and the fungi were found to belong chiefly to the <it>Ascomycota</it>, with <it>Penicillium </it>and <it>Aspergillus </it>as the two most common genera. The <it>ITS2 </it>was found to indicate a different taxonomic affiliation than did the complete <it>ITS </it>region for 10% of the query sequences, though this figure is likely to vary with the taxonomic scope of the query sequences.</p> <p>Conclusion</p> <p>The present software readily assigns large sets of fungal query sequences to their respective best matches in the international sequence databases and places them in a larger biological context. The output is highly structured to be easy to process, although it still needs to be inspected and possibly corrected for the impact of the incomplete and sometimes erroneously annotated fungal entries in these databases. The open source pipeline is available for UNIX-type platforms, and updated releases of the target database are made available biweekly. The pipeline is easily modified to operate on other molecular regions and organism groups.</p