High-resolution metagenomic reconstruction of the freshwater spring bloom

Abstract

Background: The phytoplankton spring bloom in freshwater habitats is a complex, recurring, and dynamic ecological spectacle that unfolds at multiple biological scales. Although enormous taxonomic shifts in microbial assemblages during and after the bloom have been reported, genomic information on the microbial community of the spring bloom remains scarce.

Results: We performed a high-resolution spatio-temporal sampling of the spring bloom in a freshwater reservoir and describe a multitude of previously unknown taxa using metagenome-assembled genomes of eukaryotes, prokaryotes, and viruses in combination with a broad array of methodologies. The recovered genomes reveal multiple distributional dynamics for several bacterial groups with progressively increasing stratification. Analyses of abundances of metagenome-assembled genomes in concert with CARD-FISH revealed remarkably similar in situ doubling time estimates for dominant genome-streamlined microbial lineages. Discordance between quantitations of cryptophytes arising from sequence data and microscopic identification suggested the presence of hidden, yet extremely abundant aplastidic cryptophytes that were confirmed by CARD-FISH analyses. Aplastidic cryptophytes are prevalent throughout the water column but have never been considered in prior models of plankton dynamics. We also recovered the first metagenomic-assembled genomes of freshwater protists (a diatom and a haptophyte) along with thousands of giant viral genomic contigs, some of which appeared similar to viruses infecting haptophytes but owing to lack of known representatives, most remained without any indication of their hosts. The contrasting distribution of giant viruses that are present in the entire water column to that of parasitic perkinsids residing largely in deeper waters allows us to propose giant viruses as the biological agents of top-down control and bloom collapse, likely in combination with bottom-up factors like a nutrient limitation.

Conclusion: We reconstructed thousands of genomes of microbes and viruses from a freshwater spring bloom and show that such large-scale genome recovery allows tracking of planktonic succession in great detail. However, integration of metagenomic information with other methodologies (e.g., microscopy, CARD-FISH) remains critical to reveal diverse phenomena (e.g., distributional patterns, in situ doubling times) and novel participants (e.g., aplastidic cryptophytes) and to further refine existing ecological models (e.g., factors affecting bloom collapse). This work provides a genomic foundation for future approaches towards a fine-scale characterization of the organisms in relation to the rapidly changing environment during the course of the freshwater spring bloom. Video Abstract.

Fig. 1

Time course of different features of the spring bloom in the epilimnion. A Phytoplankton biovolume, picocyanobacterial abundances, and viral-like particle counts (VLP). B The concentration of chlorophyll-a, temperature, counts of total heterotrophic bacteria, heterotrophic nanoflagellates (HNF), ciliates, rotifers, and crustaceans. C Total phosphorus (TP), dissolved nitrogen (DN), nitrate and ammonium concentrations, and D dissolved reactive phosphorus (DRP), dissolved organic carbon (DOC), and silica concentrations. The gray rectangle in the background shows the duration of the spring bloom

Fig. 2

Relative abundances of different taxonomic groups (expressed as a percentage) in the spring bloom as assessed by 16S rRNA (top, prokaryotes) and 18S rRNA (below, eukaryotes) gene read sequences. The results from different filters are shown (left, 0.22 μm; middle, 5 μm and right, 0.8 μm). Epilimnion and hypolimnion samples are also indicated

Fig. 3

A Relative abundance of selected dereplicated MAGs during the spring bloom in the small filter (0.22 µm). Each row was normalized by Z score and clustered by average linkage (with Spearman’s rank correlation method). Different letters represent different patterns of abundance common within groups. The numbers of genomes used for each group are mentioned in parentheses. For more details about other groups, check Additional file 1: Figure S4, S5, and S6. B–G Abundances of selected microbial genomes/groups during the spring bloom: The X-axes show the progression of the bloom (in days), and Y-axes show the abundance of the MAGs. All Y-axes range from 0 to 200 except for Alphaproteobacteria (0–50). The numbers of dereplicated genomes used for each group are given in parentheses. Methylomonadaceae, Methylophilaceae, and Burkholderiaceae are abbreviated as ‘Methylom.’, ‘Methylop.’, and ‘Burkhold.’ respectively, in the figure. H–M Metagenomic abundance vs CARD-FISH relative abundance. H, J, and I Coverage per Gb on the left Y-axis and CARD-FISH relative abundance % on right Y-axis. I, K, and M Correlation between metagenomic abundance vs CARD-FISH relative abundance

Fig. 4

A Phylogenomic trees of Thalassiosira.BChrysochromulina metagenome-assembled genomes. Ultrafast bootstrap values (UFB) are defined by full (95–100) and empty (70–85) circles. Collapsed clades (triangles) followed by square brackets containing the total number of members within the clade. C Metagenomic abundance of MAGs of Chrysochromulina and Thalassiosira along the entire spring bloom timeline (epilimnion and hypolimnion). D Heatmap of metagenomic abundances of MAGs of Chrysochromulina and Thalassiosira across several freshwater metagenomic datasets. A scale is shown to the right. Chrysochromulina is abbreviated as “Chryso.” in C and D

Fig. 5

A Dynamics of cryptophytes and closely related group Katablepharids during the spring bloom assessed by 18S rRNA gene read sequences. Results are shown as the percentage of total cryptophyte reads recovered. B Genomic comparison (nucleotide-nucleotide) of three complete uncultured cryptophytes mitochondrial genomes recovered here with the only available reference mitochondrial genome for Teleaulax amphioxeia. A scale is shown at the top right. C Average nucleotide identity (ANI) of uncultured cryptophyte mitochondrial genomes in comparison to all known cryptophyte mitochondrial genomes. D CARD-FISH abundance estimates for plastidic and aplastidic cryptophytes are shown as % of total eukaryotes. Sampling days are indicated along the X-axis

Fig. 6

Schematic overview of the spring bloom in Římov reservoir. Both Epilimnion and Hypolimnion are shown. Abundances (minimum to maximum, scaled from 0 to 1) from multiple methods are shown (microscopy, CARD-FISH, 18S rRNA abundances, and cumulative MAG abundances from all three filter sizes). Microscopy: HNF and Ciliates, CARD-FISH: Plastidic, Aplastidic, and uncultured CRY1 cryptophytes; 18S rRNA abundances: Perkinsidae; MAG abundances: Chrysochromulina and Thalassiosira; all prokaryotes and viruses. All prokaryotic MAGs and viral/NCLDV contigs were dereplicated. A temperature scale is shown at the bottom left, and the spring bloom period is highlighted in a gray box