The influence of intraspecific sequence variation during DNA metabarcoding: A case study of eleven fungal species

Abstract

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.

Authors 6

1. Eva Lena F. Estensmo corresponding

   University of Oslo

   Affiliation as printed

   University of Oslo Faculty of Mathematics and Natural Sciences

   Section for Genetics and Evolutionary Biology (Evogene), Department of Biosciences, University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway

2. Sundy Maurice

   University of Oslo

   Affiliation as printed

   University of Oslo Faculty of Mathematics and Natural Sciences

   Section for Genetics and Evolutionary Biology (Evogene), Department of Biosciences, University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway

3. Luís Neves Morgado Aachen Naturalis Biodiversity Center

   University of Oslo · Naturalis Biodiversity Center

   Affiliation as printed

   Naturalis Biodiversity Center

   Naturalis Biodiversity Center, Vondellaan 55, P.O. Box 9517, 2300, RA Leiden, the Netherlands

   Section for Genetics and Evolutionary Biology (Evogene), Department of Biosciences, University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway

   University of Oslo Faculty of Mathematics and Natural Sciences

4. Pedro Maria Martin-Sanchez

   University of Oslo

   Affiliation as printed

   University of Oslo

   Section for Genetics and Evolutionary Biology (Evogene), Department of Biosciences, University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway

   University of Oslo Faculty of Mathematics and Natural Sciences

5. Inger Skrede

   University of Oslo

   Affiliation as printed

   University of Oslo Faculty of Mathematics and Natural Sciences

   Section for Genetics and Evolutionary Biology (Evogene), Department of Biosciences, University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway

6. Håvard Kauserud

   University of Oslo

   Affiliation as printed

   University in Oslo

   Section for Genetics and Evolutionary Biology (Evogene), Department of Biosciences, University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway

   University of Oslo Faculty of Mathematics and Natural Sciences