Abstract
Rapid development of high-throughput (HTS) molecular identification methods has revolutionized our knowledge about taxonomic diversity and ecology of fungi. However, PCR-based methods exhibit multiple technical shortcomings that may bias our understanding of the fungal kingdom. This study was initiated to quantify potential biases in fungal community ecology by comparing the relative performance of amplicon-free shotgun metagenomics and amplicons of nine primer pairs over seven nuclear ribosomal DNA (rDNA) regions often used in metabarcoding analyses. The internal transcribed spacer (ITS) barcodes ITS1 and ITS2 provided greater taxonomic and functional resolution and richness of operational taxonomic units (OTUs) at the 97% similarity threshold compared to barcodes located within the ribosomal small subunit (SSU) and large subunit (LSU) genes. All barcode-primer pair combinations provided consistent results in ranking taxonomic richness and recovering the importance of floristic variables in driving fungal community composition in soils of Papua New Guinea. The choice of forward primer explained up to 2.0% of the variation in OTU-level analysis of the ITS1 and ITS2 barcode data sets. Across the whole data set, barcode-primer pair combination explained 37.6–38.1% of the variation, which surpassed any environmental signal. Overall, the metagenomics data set recovered a similar taxonomic overview, but resulted in much lower fungal rDNA sequencing depth, inability to infer OTUs, and high uncertainty in identification. We recommend the use of ITS2 or the whole ITS region for metabarcoding and we advocate careful choice of primer pairs in consideration of the relative proportion of fungal DNA and expected dominant groups.
Highlights
Fungi are one of the most diverse kingdoms of life on Earth (Blackwell 2011)
This study demonstrates that PCR-free metagenomics and amplicon-based approaches perform in a comparable fashion in recovering major fungal classes in spite of certain statistical differences
We found no evidence for reduced statistical performance in barcodes with relatively conserved sequences, but the use of conserved barcodes seriously hampers biological relevance of the data due to the inability to approximate species level and to assign functional categories such as trophic status
Summary
Fungi are one of the most diverse kingdoms of life on Earth (Blackwell 2011). Traditionally, fungi have been identified based on morphological characters of fruit-bodies or pure cultures in agar medium. High-throughput sequencing (HTS) technologies enable documentation and characterization of hundreds to thousands of species simultaneously from biological samples. These methods have been used successfully to uncover diversity and community composition of fungi in various substrates and localities, including little-studied habitats such as mangroves, aquatic ecosystems, and arctic soils (Arfi et al 2012; Blaalid et al 2013; Livermore and Mattes 2013). The genomes of eukaryotes are typically several orders of magnitude larger compared to bacterial genomes such that the relative proportion of ribosomal DNA in the metagenome will be lower This may complicate their detection without enrichment
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