Whole Genome Shotgun Sequencing Detects Greater Lichen Fungal Diversity Than Amplicon-Based Methods in Environmental Samples

Abstract

In this study we demonstrate the utility of whole genome shotgun (WGS) metagenomics in study organisms with small genomes to improve upon amplicon-based estimates of biodiversity and microbial diversity in environmental samples for the purpose of understanding ecological and evolutionary processes. We generated a database of full-length and near-full-length ribosomal DNA sequence complexes from 273 lichenized fungal species and used this database to facilitate fungal species identification in the southern Appalachian Mountains using low coverage WGS at higher resolution and without the biases of amplicon-based approaches. Using this new database and methods herein developed, we detected between 2.8 and 11 times as many species from lichen fungal propagules by aligning reads from WGS-sequenced environmental samples compared to a traditional amplicon-based approach. We then conducted complete taxonomic diversity inventories of the lichens in each one-hectare plot to assess overlap between standing taxonomic diversity and diversity detected based on propagules present in environmental samples (i.e., the "potential" of diversity). From the environmental samples, we detected 94 species not observed in organism-level sampling in these ecosystems with high confidence using both WGS and amplicon-based methods. This study highlights the utility of WGS sequence-based approaches in detecting hidden species diversity and demonstrates that amplicon-based methods likely miss important components of fungal diversity. We suggest that the adoption of this method will not only improve understanding of biotic constraints on the distributions of biodiversity but will also help to inform important environmental policy.

Comparative Microbiome Study of Mummified Peach Fruits by Metagenomics and Metatranscriptomics

“…A previous study has successfully identified fungi in the animal metagenomes containing 71 fungal species from 39 datasets using newly developed FindFungi program [ 33 ]. Other study demonstrated that DNA shotgun sequencing was superior to detect lichen fungal diversity in environmental samples as compared to amplicon based methods [ 34 ]. RNA-sequencing with poly(A) selection cannot be used for the majority of bacteria, and many viruses without poly(A) tails cannot be selected by oligo dT for mRNA preparation.…”

Extinction risk and threats to plants and fungi

“…Sampling substrata (soil, wood, leaves, and roots) has great potential for documenting the presence of fungi even in the absence of visible spore bodies (e.g., mushrooms). Mycologists use eDNA to assess the potential conservation value of sites (Griffith, Cavalli, & Detheridge, 2019) and monitor, document, and model the distribution of taxa (Hao, Guillera-Arroita, May, Lahoz-Monfort, & Elith, 2020;Keepers et al, 2019;Suz et al, 2015).…”

Metagenomic data reveal diverse fungal and algal communities associated with the lichen symbiosis

“…All reads were filtered using TRIMMOMATIC v0.33 (Bolger et al 2014) before mapping to remove low quality reads and/or included contamination from Illumina adaptors using the following parameters: ILLUMINACLIP; LEADING:3; TRAILING:3; SLIDINGWINDOW:4:15; and MINLEN:36. Previous studies have used assembled metagenomic contigs (Keepers et al 2019) or mapped fungal reads to a fungal protein database (LaBonte et al 2018) to provide crucial insight into fungal diversity in lichens and deciduous trees. Given the expected low coverage for fungi potentially co-occurring with a lichen thallus in short reads generated for this study, we chose to focus on the well-known repeat region which includes the standard fungal DNA barcode, the ITS region of the nuclear ribosomal DNA (nrDNA) (Schoch et al 2012).…”