Abstract
DNA metabarcoding has become a powerful approach for analysing complex communities from environmental samples, but there are still methodological challenges limiting its full potential. While conserved DNA markers, like 16S and 18S, often are not able to discriminate among closely related species, other more variable markers - like the fungal ITS region, may include considerable intraspecific variation, which can lead to oversplitting of species during DNA metabarcoding analyses. Here we assessed the effects of intraspecific sequence variation in DNA metabarcoding by analysing local populations of eleven fungal species. We investigated the allelic diversity of ITS2 haplotypes using both Sanger sequencing and high throughput sequencing (HTS) coupled with error correction with the software dada2. All the eleven species, except one, included some level of intraspecific variation in the ITS2 region. Overall, we observed a high correspondence between haplotypes generated by Sanger sequencing and HTS, with the exception of a few additional haplotypes detected using either approach. These extra haplotypes, typically occurring in low frequencies, were probably due to PCR and sequencing errors or intragenomic variation in the rDNA region. The presence of intraspecific (and possibly intragenomic) variation in ITS2 suggest that haplotypes (or ASVs) should not be used as basic units in ITS-based fungal community analyses, but an extra clustering step is needed to approach species-level resolution.
Highlights
The fungal kingdomThe Fungal kingdom is believed to have originated between 760 million and 1 billion years ago (Lücking et al 2009)
By denoising the sequence data using DADA2, we tested whether the same ITS2 haplotypes were identified by DNA metabarcoding and Sanger sequencing, and analyzed the allelic diversity of ITS2
There was a high correspondence between haplotypes generated by Sanger sequencing and High throughput sequencing (HTS), but a few additional haplotypes were detected in low frequencies using either approach
Summary
The fungal kingdomThe Fungal kingdom is believed to have originated between 760 million and 1 billion years ago (Lücking et al 2009). The uncertainty of the number of fungal species is mainly based on their microscopic size and that they generally live hidden within their substrate, except during the fruiting stage. Filamentous fungi may form large macroscopic structures, such as fruit bodies commonly found in nature (Figure 1). Some fungal species can be dimorphic and shift between growth forms during different life stages (producing either yeasts or hyphae). Both yeasts and filamentous growth are found in species of both Basidiomycota and Ascomycota
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