ITS1 versus ITS2 as DNA metabarcodes for fungi.

The nuclear ribosomal Internal Transcribed Spacer ITS region is widely used as a DNA metabarcoding marker to characterize the diversity and composition of fungal communities. In amplicon pyrosequencing studies of fungal diversity, one of the spacers ITS1 or ITS2 of the ITS region is normally used. In this methodological study we evaluate the usability of ITS1 vs. ITS2 as a DNA metabarcoding marker for fungi. We analyse three data sets: two comprising ITS1 and ITS2 sequences of known taxonomic affiliations and a third comprising ITS1 and ITS2 environmental amplicon pyrosequencing data. Clustering analyses of sequences with known taxonomy using the bioinformatics pipeline ClustEx revealed that a 97% similarity cut-off represent a reasonable threshold for estimating the number of known species in the data sets for both ITS1 and ITS2. However, no single threshold value worked well for all fungi at the same time within the curated UNITE database, and we found that the Operational Taxonomic Unit (OTU) concept is not easily translated into the level of species because many species are distributed over several clusters. Clustering analyses of the 134 692 ITS1 and ITS2 pyrosequences using a 97% similarity cut-off revealed a high similarity between the two data sets when it comes to taxonomic coverage. Although some groups are under- or unrepresented in the two data sets due to, e.g. primer mismatches, our results indicate that ITS1 and ITS2 to a large extent yield similar results when used as DNA metabarcodes for fungi.

An open source chimera checker for the fungal ITS region.

The internal transcribed spacer (ITS) region of the nuclear ribosomal repeat unit holds a central position in the pursuit of the taxonomic affiliation of fungi recovered through environmental sampling. Newly generated fungal ITS sequences are typically compared against the International Nucleotide Sequence Databases for a species or genus name using the sequence similarity software suite blast. Such searches are not without complications however, and one of them is the presence of chimeric entries among the query or reference sequences. Chimeras are artificial sequences, generated unintentionally during the polymerase chain reaction step, that feature sequence data from two (or possibly more) distinct species. Available software solutions for chimera control do not readily target the fungal ITS region, but the present study introduces a blast-based open source software package (available at http://www.emerencia.org/chimerachecker.html) to examine newly generated fungal ITS sequences for the presence of potentially chimeric elements in batch mode. We used the software package on a random set of 12 300 environmental fungal ITS sequences in the public sequence databases and found 1.5% of the entries to be chimeric at the ordinal level after manual verification of the results. The proportion of chimeras in the sequence databases can be hypothesized to increase as emerging sequencing technologies drawing from pooled DNA samples are becoming important tools in molecular ecology research.

454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity.

* Soil fungi play a major role in ecological and biogeochemical processes in forests. Little is known, however, about the structure and richness of different fungal communities and the distribution of functional ecological groups (pathogens, saprobes and symbionts). * Here, we assessed the fungal diversity in six different forest soils using tag-encoded 454 pyrosequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS-1). No less than 166 350 ITS reads were obtained from all samples. In each forest soil sample (4 g), approximately 30 000 reads were recovered, corresponding to around 1000 molecular operational taxonomic units. * Most operational taxonomic units (81%) belonged to the Dikarya subkingdom (Ascomycota and Basidiomycota). Richness, abundance and taxonomic analyses identified the Agaricomycetes as the dominant fungal class. The ITS-1 sequences (73%) analysed corresponded to only 26 taxa. The most abundant operational taxonomic units showed the highest sequence similarity to Ceratobasidium sp., Cryptococcus podzolicus, Lactarius sp. and Scleroderma sp. * This study validates the effectiveness of high-throughput 454 sequencing technology for the survey of soil fungal diversity. The large proportion of unidentified sequences, however, calls for curated sequence databases. The use of pyrosequencing on soil samples will accelerate the study of the spatiotemporal dynamics of fungal communities in forest ecosystems.

A phylogenetic approach to detect selection on the target site of the antifouling compound irgarol in tolerant periphyton communities.

Using DNA sequence data for phylogenetic assessment of toxicant targets is a new and promising approach to study toxicant-induced selection in communities. Irgarol 1051 is a photosystem (PS) II inhibitor used in antifouling paint. It inhibits photosynthesis through binding to the D1 protein in PS II, which is encoded by the psbA gene found in genomes of chloroplasts, cyanobacteria and cyanophages. psbA mutations that alter the target protein can confer tolerance to PS II inhibitors. We have previously shown that irgarol induces community tolerance in natural marine periphyton communities and suggested a novel tolerance mechanism, involving the amino acid sequence of a turnover-regulating domain of D1, as contributive to this tolerance. Here we use a large number of psbA sequences of known identity to assess the taxonomic affinities of psbA sequences from these differentially tolerant communities, by performing phylogenetic analysis. We show that periphyton communities have high psbA diversity and that this diversity is adversely affected by irgarol. Moreover, we suggest that within tolerant periphyton the novel tolerance mechanism is present among diatoms only, whereas some groups of irgarol-tolerant cyanobacteria seem to have other tolerance mechanisms. However, it proved difficult to identify periphyton psbA haplotypes to the species or genus level, which indicates that the genomic pool of the attached, periphytic life forms is poorly studied and inadequately represented in international sequence databases.