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).
Highlights
Lichens are intimate and long-term symbiotic associations consisting of a heterotrophic fungal partner— called the mycobiont—and photosynthetic algae or cyanobacteria— called the photobiont (Nash 2008)
The internal transcribed spacer region (ITS) of the nuclear ribosomal RNA cistron was proposed as the primary fungal barcode marker (Schoch et al 2012)
The aim of our study was to test whether lichen mycobiont can be successfully identified to a single species using DNA barcoding through pyrosequencing the ITS marker of 100 species of both foliose–fruticose and crustose lichens, using fungal-specific markers in the highthroughput 454 sequencing in the GS FLX+ system, while elucidating and quantifying artifactual and biological sequence variation
Summary
Lichens are intimate and long-term symbiotic associations consisting of a heterotrophic fungal partner— called the mycobiont—and photosynthetic algae or cyanobacteria— called the photobiont (Nash 2008). Lichens are named after the fungal partner, and according to most recent estimates, about 18% of all fungal species are lichen-forming (Feuerer and Hawksworth 2007). Only a minority of lichenforming fungi have been barcoded from the estimated 17 500 species (Feuerer and Hawksworth 2007). The largest and most typical order of lichen-forming fungi (Lecanorales) consists of about 5700 species, but only about 29% of them have publicly available ITS sequences in sequence databases such as the National Institute of Health’s (NIH) genetic sequence database, GenBank (http://www.ncbi.nlm.nih.gov/genbank/; accessed 28 September 2015), the Barcode of Life Data System (BOLD; www.boldsystems.org; Ratnasingham and Hebert 2007), and UNITE (https://unite.ut.ee/; accessed 24 November 2015; Abarenkov et al 2010)
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