title: Barcoding lichen-forming fungi using 454 pyrosequencing is challenged by artifactual and biological sequence variation.
abstract: Although lichens (lichen-forming fungi) play an important role in the ecological integrity of many vulnerable landscapes, only a minority of lichen-forming fungi have been barcoded out of the currently accepted ∼18 000 species. Regular Sanger sequencing can be problematic when analyzing lichens since saprophytic, endophytic, and parasitic fungi live intimately admixed, resulting in low-quality sequencing reads. Here, high-throughput, long-read 454 pyrosequencing in a GS FLX+ System was tested to barcode the fungal partner of 100 epiphytic lichen species from Switzerland using fungal-specific primers when amplifying the full internal transcribed spacer region (ITS). The present study shows the potential of DNA barcoding using pyrosequencing, in that the expected lichen fungus was successfully sequenced for all samples except one. Alignment solutions such as BLAST were found to be largely adequate for the generated long reads. In addition, the NCBI nucleotide database-currently the most complete database for lichen-forming fungi-can be used as a reference database when identifying common species, since the majority of analyzed lichens were identified correctly to the species or at least to the genus level. However, several issues were encountered, including a high sequencing error rate, multiple ITS versions in a genome (incomplete concerted evolution), and in some samples the presence of mixed lichen-forming fungi (possible lichen chimeras).
authors.affiliation: a Biodiversity and Conservation Biology, Swiss Federal Research Institute WSL, Switzerland.|b Institute of Botany and Ecology, University of Tartu, Estonia.
authors.forename: Kristiina
authors.initials: K
authors.lastname: Mark
authors.affiliation: a Biodiversity and Conservation Biology, Swiss Federal Research Institute WSL, Switzerland.
authors.forename: Carolina
authors.initials: C
authors.lastname: Cornejo
authors.affiliation: a Biodiversity and Conservation Biology, Swiss Federal Research Institute WSL, Switzerland.
authors.forename: Christine
authors.initials: C
authors.lastname: Keller
authors.affiliation: a Biodiversity and Conservation Biology, Swiss Federal Research Institute WSL, Switzerland.
authors.forename: Daniela
authors.initials: D
authors.lastname: Flück
authors.affiliation: a Biodiversity and Conservation Biology, Swiss Federal Research Institute WSL, Switzerland.
authors.forename: Christoph
authors.initials: C
authors.lastname: Scheidegger
mesh_terms: D044822:Biodiversity; D005075:Biological Evolution; D019295:Computational Biology; D016384:Consensus Sequence; D058893:DNA Barcoding, Taxonomic; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation / Q000379:methods; D008011:Lichens / Q000145:classification* / Q000235:genetics*; D058889:Molecular Typing / Q000295:instrumentation / Q000379:methods; D010802:Phylogeny; D017422:Sequence Analysis, DNA
keywords: 454 pyrosequencing; DNA barcoding; champignons lichénisés; codage à barres de l’ADN; espaceur interne transcrit; internal transcribed spacer; intragenomic variation; lichenized fungi; pyroséquençage 454; variation intragénomique