The influence of intraspecific sequence variation during DNA
metabarcoding: A case study of eleven fungal species
DNA metabarcoding has become a powerful approach for analyzing 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 over-splitting of species during DNA metabarcoding analyses.
Here we assess the effects of intraspecific sequence variation in DNA
metabarcoding, by analyzing 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 focal 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, often
occurring in low frequencies, were likely 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.