Use of high throughput sequencing and light microscopy show contrasting results in a study of phytoplankton occurrence in a freshwater environment

PLoS One. 2014 Aug 29;9(8):e106510. doi: 10.1371/journal.pone.0106510. eCollection 2014.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Ribosomal / genetics*
  • Fresh Water*
  • High-Throughput Nucleotide Sequencing / methods*
  • Microscopy
  • Phylogeny
  • Phytoplankton / classification*
  • Phytoplankton / cytology
  • Phytoplankton / genetics
  • RNA, Ribosomal, 16S / genetics
  • RNA, Ribosomal, 18S / genetics
  • Sequence Analysis, DNA / methods


  • DNA, Ribosomal
  • RNA, Ribosomal, 16S
  • RNA, Ribosomal, 18S

Associated data

  • SRA/SRP044824

Grant support

This work was supported by Norwegian Research Council (grant 183360/S30 to TR), and Government-Exchange Scholarship from the Research Council of Norway and China Scholarship Council regarding KSJ and XX. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.