Whole Genome Shotgun Sequencing Detects Greater Lichen Fungal Diversity Than Amplicon-Based Methods in Environmental Samples
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ORIGINAL RESEARCH published: 13 December 2019 doi: 10.3389/fevo.2019.00484 Whole Genome Shotgun Sequencing Detects Greater Lichen Fungal Diversity Than Amplicon-Based Methods in Environmental Samples Kyle Garrett Keepers 1*, Cloe S. Pogoda 1, Kristin H. White 1, Carly R. Anderson Stewart 1, Jordan R. Hoffman 2, Ana Maria Ruiz 2, Christy M. McCain 1, James C. Lendemer 2, Nolan Coburn Kane 1 and Erin A. Tripp 1,3 1 Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO, United States, 2 Institute of Edited by: Systematic Botany, The New York Botanical Garden, New York, NY, United States, 3 Museum of Natural History, University of Maik Veste, Colorado, Boulder, CO, United States Brandenburg University of Technology Cottbus-Senftenberg, Germany Reviewed by: In this study we demonstrate the utility of whole genome shotgun (WGS) metagenomics Martin Grube, in study organisms with small genomes to improve upon amplicon-based estimates University of Graz, Austria of biodiversity and microbial diversity in environmental samples for the purpose of Robert Friedman, University of South Carolina, understanding ecological and evolutionary processes. We generated a database of United States full-length and near-full-length ribosomal DNA sequence complexes from 273 lichenized *Correspondence: fungal species and used this database to facilitate fungal species identification in Kyle Garrett Keepers [email protected] the southern Appalachian Mountains using low coverage WGS at higher resolution and without the biases of amplicon-based approaches. Using this new database and Specialty section: methods herein developed, we detected between 2.8 and 11 times as many species This article was submitted to Phylogenetics, Phylogenomics, and from lichen fungal propagules by aligning reads from WGS-sequenced environmental Systematics, samples compared to a traditional amplicon-based approach. We then conducted a section of the journal complete taxonomic diversity inventories of the lichens in each one-hectare plot to Frontiers in Ecology and Evolution assess overlap between standing taxonomic diversity and diversity detected based on Received: 10 July 2019 Accepted: 28 November 2019 propagules present in environmental samples (i.e., the “potential” of diversity). From the Published: 13 December 2019 environmental samples, we detected 94 species not observed in organism-level sampling Citation: in these ecosystems with high confidence using both WGS and amplicon-based Keepers KG, Pogoda CS, White KH, methods. This study highlights the utility of WGS sequence-based approaches in Anderson Stewart CR, Hoffman JR, Ruiz AM, McCain CM, Lendemer JC, detecting hidden species diversity and demonstrates that amplicon-based methods likely Kane NC and Tripp EA (2019) Whole miss important components of fungal diversity. We suggest that the adoption of this Genome Shotgun Sequencing Detects Greater Lichen Fungal method will not only improve understanding of biotic constraints on the distributions of Diversity Than Amplicon-Based biodiversity but will also help to inform important environmental policy. Methods in Environmental Samples. Front. Ecol. Evol. 7:484. Keywords: metagenomics, ribosomal RNA, sequence database, biological soil crusts, lichen, potential of diversity, doi: 10.3389/fevo.2019.00484 taxonomic diversity Frontiers in Ecology and Evolution | www.frontiersin.org 1 December 2019 | Volume 7 | Article 484 Keepers et al. WGS Method of Envr. Metagenomics INTRODUCTION whole, received less attention relative to bacterial metagenomics. For example, only 360 NCBI Bioprojects were archived for fungi Microbial diversity present in the environment is recognized using amplicon-based molecular barcodes compared to 5,121 increasingly for its important and varied roles in the health Bioprojects available for studies of microbes using amplicon- of ecosystems (Chen et al., 2018; Nottingham et al., 2018; based molecular barcodes (search conducted on June 21, 2019, Pike et al., 2018), particularly in the face of a changing in NCBI Bioproject Archive). climate (Cavicchioli et al., 2019). Unsurprisingly, a great deal Lichens are a species-rich and evolutionarily heterogeneous of focus has been on quantifying biodiversity—the number, assemblage of fungi that form obligate symbioses with a identity, and functions of species (Gotelli and Colwell, 2001; minimum of one primary photosynthetic partner, often in Faith, 2002; Barlow et al., 2007). It has also helped researchers addition to other endolichenic fungi, algae, and bacteria more fully document the ranges of rare or endangered species (Ahmadjian and Jacobs, 1981; Seaward, 1997; Brodo et al., through environmental DNA detection strategies (Olson et al., 2001; Papazi et al., 2015). Lichens are highly successful and 2012; Thomsen et al., 2012; Spear et al., 2015). Further ecologically important, as is evidenced by their abundance expanding the impacts of this relatively new field, microbial and diversity in terrestrial ecosystems around the world metagenomics has proven exceptionally useful toward informing (Hawksworth, 1991). Along with bryophytes, cyanobacteria, remediation strategies of disturbed habitats such as ecologically and non-lichenized fungi, lichens are a crucial component of sensitive biological soil crusts (BSC; Bowker, 2007; Steven biological soil crust communities and function prominently in et al., 2012). Failure to fully understand the microbial (biotic) ecological restoration processes (Belnap, 2001; Belnap and Lange, community can thus dramatically limit understanding of 2001; Thompson et al., 2006; Bowker, 2007). In addition to their what structures ecological interactions, species distributions, pivotal ecological contributions to such communities, lichenized and environmental sustainability, all of which can, in turn, fungi have relatively small metagenome sizes (Armaleo and May, negatively impact informed conservation decision making 2009; Tripp et al., 2017), making them ideal targets for cost- (Guisan et al., 2013). effective genomics projects (Allen et al., 2018; Brigham et al., Increased accessibility and affordability of high throughput 2018; Funk et al., 2018; Pogoda et al., 2018, 2019). Given the sequencing, has facilitated broad scale exploration of microbial above, lichens serve as an excellent system in which to explore community structure (Logares et al., 2012, 2014; Chen et al., the factors that constrain the establishment and development 2017; Zhang et al., 2018). At present, the majority of broad- of obligate symbioses in nature, including those prevalent scale biotic diversity assessments employ primarily culture- among soil crust communities. Such factors span the dynamics independent, amplicon-based sequencing (Petrosino et al., 2009; of propagule dispersal, the distribution and establishment of Mande et al., 2012; Uyaguari-Diaz et al., 2016). This method individual symbionts in the environment, and biotic interactions relies on sufficiently variable, universally present regions of the between extant symbionts in a given environment. To date, genome, or “barcoding loci” (Hebert and Gregory, 2005; Kress however, few studies have explored such avenues of research, but and Erickson, 2008). Such loci must, first and foremost, be unique these have relied entirely on amplicon-based sequencing methods enough to yield distinctions between species present in a sample (Banchi et al., 2018; Eaton et al., 2018; but see, e.g., Tripp et al., (Kolbert et al., 2004). A proliferation of bioinformatic pipelines 2017; Pizarro et al., 2019). developed for barcode sequencing has resulted in widespread This shortage of fungal genomic resources broadly, and capacity to analyze microbial diversity and community structure lichen genomic resources more specifically, makes it challenging present in a variety of different environments, ranging from to investigate key questions about lichen ecology, evolution, soil to the human body (e.g., QIIME; Caporaso et al., 2010; genetics, and physiology. Moreover, existing studies that have Kuczynski et al., 2012; Navas-Molina et al., 2013). These pipelines investigated fungal metagenomic communities, like bacteria, rely primarily on the 16S ribosomal DNA (rDNA) gene as primarily relied on amplicon-based approaches. Given known a target for PCR amplification and subsequent sequencing complications arising from amplification bias (Acinas et al., to distinguish species (Winker and Woese, 1991; Kolbert 2005; Wang and Qian, 2009), one potential solution is to forego et al., 2004; Petrosino et al., 2009). However, shortcomings of amplification of barcoding loci and instead utilize data from such amplicon-based approaches include moderate to extreme whole-genome shotgun sequencing (WGS). This method avoids amplification bias (Acinas et al., 2005; Wang and Qian, classical PCR amplification biases but has been little employed to 2009), thus effectively investigating only a fraction of total date, likely as a function of one to several challenges. In addition standing diversity. to increased costs of WGS relative to amplicon-based methods, Fungi decompose organic litter (Chapin et al., 2002; the lack of developed, publicly available reference databases (e.g., Osono, 2007), produce secondary compounds of tremendous complete or nearly complete rDNA complexes) as well as a importance to humans such as antibiotics (Keller et al., 2005), paucity of bioinformatics pipelines have limited the utility of are used extensively in food production (e.g., bread, wine, beer;