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

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

From Green to Red: Horizontal Gene Transfer of the Phycoerythrin Gene Cluster between Planktothrix Strains

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Gene flow, recombination, and selection in cyanobacteria: population structure of geographically related Planktothrix Freshwater Strains

Several Planktothrix strains, each producing a distinct oligopeptide profile, have been shown to coexist within Lake Steinsfjorden (Norway). Using nonribosomal peptide synthetase (NRPS) genes as markers, it has been shown that the Planktothrix community comprises distinct genetic variants displaying differences in bloom dynamics, suggesting a Planktothrix subpopulation structure. Here, we investigate the Planktothrix variants inhabiting four lakes in southeast of Norway utilizing both NRPS and non-NRPS genes. Phylogenetic analyses showed similar topologies for both NRPS and non-NRPS genes, and the lakes appear to have similar structuring of Planktothrix genetic variants. The structure of distinct variants was also supported by very low genetic diversity within variants compared to the between-variant diversity. Incongruent topologies and split decomposition revealed recombination events between Planktothrix variants. In several strains the gene variants seem to be a result of recombination. Both NRPS and non-NRPS genes are dominated by purifying selection; however, sites subjected to positive selection were also detected. The presence of similar and well-separated Planktothrix variants with low internal genetic diversity indicates gene flow within Planktothrix populations. Further, the low genetic diversity found between lakes (similar range as within lakes) indicates gene flow also between Planktothrix populations and suggests recent, or recurrent, dispersals. Our data also indicate that recombination has resulted in new genetic variants. Stability within variants and the development of new variants are likely to be influenced by selection patterns and within-variant homologous recombination

Phytoplankton in Lake Gjersjøen from 1969 to 1989 detected by high throughput sequencing of 16S rRNA (a) and 18S rRNA (b) hypervariable regions.

<p>Phytoplankton in Lake Gjersjøen from 1969 to 1989 detected by high throughput sequencing of 16S rRNA (a) and 18S rRNA (b) hypervariable regions.</p

Phytoplankton occurrences in Lake Gjersjøen from 1969 to 1989, and comparison of genera/species numbers using high throughput sequencing of 16S rRNA gene and 18S rRNA gene and microscopy (a. at the species level; b. at the genus level).

<p>The M:P stand for the ratio of total species/genus numbers detected by microscopy and HTS.</p

Phytoplankton detected by high throughput sequencing of 16S rRNA gene and 18S rRNA gene and microscopy at the species level in Lake Gjersjøen.

<p>The species which formed frequent blooms of toxigenic cyanobacteria in this lake were marked with “↑”.</p

Rarefaction curves of high throughput sequencing of 16S rRNA (V2) and 18S rRNA (V9) hypervariable regions.

<p>Rarefaction curves of high throughput sequencing of 16S rRNA (V2) and 18S rRNA (V9) hypervariable regions.</p

Shared species and genera detected by both high throughput sequencing and light microscopy based on over 300 water samples from 1969 to 1989 in Lake Gjersjøen.

<p>Shared species and genera detected by both high throughput sequencing and light microscopy based on over 300 water samples from 1969 to 1989 in Lake Gjersjøen.</p

From green to red: horizontal gene transfer of phycoerythrin gene cluster between Planktothrix strains.

<p>This data is part of a published article: From green to red: horizontal gene transfer of phycoerythrin gene cluster between Planktothrix strains.</p> <p>.fasta file of 217.5 kb long alignment of 200 randomly selected core genes from eight Planktothrix genomes (Cyanobacteria).</p