Limits of nuclear ribosomal DNA internal transcribed spacer (ITS) sequences as species barcodes for <i>Fungi</i>

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.

Nuclear ribosomal genes in most eukaryotes are present in multiple copies and often used for taxonomic and phylogenetic analyses. We comprehensively examined intragenomic polymorphism levels of three nuclear ribosomal loci for four important plant pathogenic fungi by polymerase chain reaction amplification and cloning. Here, we show that single nucleotide polymorphisms are present in an unexpectedly high amount. This might have implications for studies of fungal evolution, phylogenetics, and population genetics. Furthermore, our work demonstrates that the majority of all ribosomal sequences obtained from one individual and gene is identical to the majority rule consensus sequence of all detected sequence variants. Due to the large number of polymorphisms found and the fact that the polymorphism level differed markedly even between ribosomal genes of one and the same individual, we assume that nuclear ribosomal genes might not always evolve in a strictly concerted manner.

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.

The validity of reclassifying filamentous, ascomycetous anamorphs solely on the basis of ribosomal DNA sequences is examined. We suggest that emotional reactions to the debate are a consequence of often unacknowledged philosophical biases. From the perspective of the scientific method, neither morphological nor sequence-based taxonomic studies are inherently superior. A review of published information on the internal transcribed spacer of filamentous Ascomycetes and ascomycetous anamorphs demonstrates that uniform species concepts based on DNA sequences alone are presently infeasible. Because a phylogenetic scheme should classify species, the concept that fungi can be typified or classified solely by DNA sequences is challenged. Similarly, because no adequate nonmorphological species concept exists for anamorphic fungi that lack a sexual state, integration of the Deuteromycetes into the holomorphic classification on the basis of DNA sequences alone is also presently impractical. Key words: DNA sequencing, fungal taxonomy, internal transcribed spacer, species concepts.