Use of High Throughput Sequencing and Light Microscopy Show Contrasting Results in a Study of Phytoplankton Occurrence in a Freshwater Environment

Abstract

Assessing phytoplankton diversity is of primary importance for both basic and applied ecological studies. Following the advances in molecular methods, phytoplankton studies are switching from using classical microscopy to high throughput sequencing approaches. However, methodological comparisons of these approaches have rarely been reported. In this study, we compared the two methods, using a unique dataset of multiple water samples taken from a natural freshwater environment. Environmental DNA was extracted from 300 water samples collected weekly during 20 years, followed by high throughput sequencing of amplicons from the 16S and 18S rRNA hypervariable regions. For each water sample, phytoplankton diversity was also estimated using light microscopy. Our study indicates that species compositions detected by light microscopy and 454 high throughput sequencing do not always match. High throughput sequencing detected more rare species and picoplankton than light microscopy, and thus gave a better assessment of phytoplankton diversity. However, when compared to light microscopy, high throughput sequencing of 16S and 18S rRNA amplicons did not adequately identify phytoplankton at the species level. In summary, our study recommends a combined strategy using both morphological and molecular techniques.

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

“…Ten studies focused on a broad range of eukaryotic biodiversity (e.g. Pawlowski et al 2011;Baldwin et al 2013), whereas only three of the studies targeted both prokaryotic and eukaryotic organisms (Xiao et al 2014;Young et al 2014;Kowallik et al 2015). Just over 10% of the studies investigated fungi (e.g.…”

Do current European lake monitoring programmes reliably estimate phytoplankton community changes?

“…In recent years, novel molecular tools have replaced classical DNA Sanger sequencing in aquatic microbiology; quantitative PCR, high throughput sequencing, metatranscriptome and proteome analyses are increasingly being used to characterise taxonomic and functional diversity of (marine) microorganisms including phytoplankton (reviewed by Johnson & Martiny, 2015). However, quantification of the entire phytoplankton species composition and biomass is still difficult with the molecular protocols currently available (Johnson & Martiny, 2015;Xiao et al, 2014), and the application of the molecular tools by monitoring agencies has been limited (e.g. Wollschläger et al, 2014;Wood et al, 2013).…”

Bioassessment of a Drinking Water Reservoir Using Plankton: High Throughput Sequencing vs. Traditional Morphological Method

“…Compared to TOM, HTS can detect a wide variety of plankton, including nanoplankton and rare taxa. Other studies also found similar results in freshwater ecosystems [32,34,50,52]. Xiao et al (2014) [32] found that the species compositions detected by TOM and 454 HTS did not always match at the taxa level after analyzing 300 weekly water samples over 20 years in Lake Gjersjøen, Norway, a drinking water source.…”

“…Other studies also found similar results in freshwater ecosystems [32,34,50,52]. Xiao et al (2014) [32] found that the species compositions detected by TOM and 454 HTS did not always match at the taxa level after analyzing 300 weekly water samples over 20 years in Lake Gjersjøen, Norway, a drinking water source. Studies in Lake Tegel, Germany, showed that because the 480 used diatom sequences of the 18S region were generated from world-wide occurrences, only a small number of individuals precisely matched on the species level [52].…”

“…The method is faster in terms of sampling processing, and may become cheaper as the technology improves, and more importantly with the ability to provide more reliable and richer biological information than the traditional morphology-based method [22][23][24]. Different plankton assemblages as bioindicators were characterized using DNA sequencing including bacterioplankton [18,[24][25][26], and phytoplankton [27][28][29][30][31][32][33]. Baird and Hajibabaei (2012) [17] envisioned a new paradigm (i.e., Biomonitoring 2.0) in ecosystem assessment based on a HTS platform, though a complete paradigm shift may require more research.…”