Reconstruction of cyanobacterial phylogenetic diversity from sediments.
Our work shows that it is possible to study cyanobacterial communities by sequencing DNA from lake sediment cores. We successfully sequenced amplicons recovered from DNA archived in the sediments of two lakes over the last two centuries, and we were able to validate the data with two independent time series, consisting of 40 years of phytoplankton microscopic identification from the same lakes. We also reconstructed the history of potentially microcystin-producing cyanobacteria over the last century. Our results are consistent with the historical information describing the cyanobacterial community composition in the two lakes.
One of the main limitations in sedimentary DNA studies is the degradation of DNA over time (50
). However, the cold and anoxic/hypoxic conditions at the bottom of the two deep and stratified lakes studied here are ideal for DNA preservation (51
). We first verified the quality of the DNA preserved over the past 200 years in the sediments of Greifensee and Lake Zurich by amplifying an 800-nt-long fragment of the 16S rRNA gene. This test confirmed the possibility of sequencing a shorter DNA fragment of ∼400 bp. Another important limitation of cyanobacterial investigations using sequencing technologies is the lack of exhaustive and well-curated reference databases, which limits the taxonomic assignment of OTUs. While the existing reference databases (40
) are well developed for microbial 16S rRNA analysis, the coverage of cyanobacteria, especially the freshwater taxa, needs to be improved. In this study, however, sequencing a relatively large DNA fragment (400 nt) allowed us to use the OTU reference sequences in a more detailed phylogeny analysis based on Bayesian inferences (Fig. 3
).This opens up the possibility of investigating cyanobacterial phylogenetic diversity and community structure.
Because the diversity of whole natural cyanobacterial communities had never been assessed using HTS technologies on sedimentary records, we had no clear expectations regarding the recovery efficiency of our approach. However, based on the time series of pelagic observations, we had some prior knowledge of the cyanobacterial community composition over the past 5 decades in the two lakes. The sequencing of circa 40 sedDNA samples per lake spanning 200 years yielded a similarly high diversity, covering all major clades of cyanobacteria in the two lakes (Fig. 3
). Even though the PCR primers were thought to be cyanobacterium-specific, they coamplified chloroplasts and heterotrophic bacteria. This coamplification did not have an impact on community reconstruction because the Illumina sequencing run produced millions of amplicons, which were sufficient for an optimal coverage of the cyanobacterial diversity in most samples (see Fig. S3 in the supplemental material). In general, older samples contained less DNA, which led to a lower number of amplicons sequenced. This problem can be solved by pooling DNA extracts and increasing the template DNA for PCR amplifications or by pooling PCRs. In this study, we were mainly interested in the diversity of recent samples (i.e., between 1975 and 2010) for comparison with pelagic samples; therefore, we did not attempt to optimize the coverage in the older sample.
Interestingly, sequencing revealed the presence of unexpected deep-branching groups of cyanobacteria termed Melainabacteria
and ML635J-21 in the sediments of the two lakes (Fig. 3
). Recent evidence from whole-genome sequencing confirmed that Melainabacteria
constitute a class within the Cyanobacteria
phylum because the two groups share common ancestral traits, such as the cell envelope structure and the presence of putative circadian rhythms (54
). Taxa within this group have been detected in various environments, including groundwater, drinking water and wastewater treatment plants (54
), terrestrial plants, and animal guts (56
). To our knowledge, their presence has not been previously reported in lakes. The sequencing of these clades from the sediments of our two lakes shows that the cyanobacterium-specific primers used can target taxa over the whole phylum. From our results, we cannot conclude whether these nonphotosynthetic cyanobacteria live in the water column or colonize the sediments, but the contrasting diversity in the two lakes (Fig. 3
) may reflect the adaptation of these taxa to specific local conditions. More eDNA studies using HTS technologies may help to elucidate the ecological roles and the sensitivity of these clades to environmental changes.
Comparison of sediment and pelagic samples.
The strong and significant relationship observed between the annual cyanobacterial richness estimated at 12 time points from sedDNA and from pelagic samples between the mid-1970s and 2010 in the two lakes (Fig. 6
) reinforces the validity of our reconstruction approach. The greater cyanobacterial richness observed in the sediments of the two lakes compared to the microscopic estimates in the pelagic samples (Fig. 6
) can be partially explained by differences in the detection limit of the two methods. Other studies have shown that diversity estimation based on morphology generally underestimates the true diversity of cyanobacteria, which emerges from genetic methods (57
). Our results suggest that the richness of Chroococcales
was widely underestimated in the microscopy data compared to the genetic data from sedDNA (Fig. 7
). This is probably because several taxa within the two groups are unicellular picocyanobacteria (<2 μm in diameter), which are difficult to classify based on morphology (59
With the sequencing approach, we were able to verify the presence of potentially toxic cyanobacterial taxa that have been observed in the lakes, like Microcystis aeruginosa
and Planktothrix rubescens
. Regular blooms of M. aeruginosa
have been reported in Greifensee over the past 15 years (Eawag, unpublished), and the phytoplankton community of Lake Zurich has been largely dominated by P. rubescens
over the last 3 decades (28
). Our data confirmed the presence of two OTUs that are assigned to Microcystis
species in Greifensee and a single OTU sequence that is related to P. rubescens
in Lake Zurich. The detection of the mcyA
genes started at the same time that there was an increase in the abundance of one of the OTUs assigned to Microcystis
sp. in Greifensee. Although our results show that the number of sequencing reads does not reflect the number of cells counted in pelagic samples (Fig. 5
), it is likely that an increase in the relative OTU abundance reflects a change in the pelagic community, in this particular case, the dominance of a microcystin-producing M. aeruginosa
In Lake Zurich, the detection of an OTU assimilated to Planktothrix rubescens
was supported by the amplification of the mcyA
gene related to the same taxa. The latter finding is consistent with early reports of the presence of P. rubescens
(formerly Oscillatoria rubescens
) forming large reddish blooms known as the Burgundy blood phenomenon at the surface of Lake Zurich in 1897 (61
) and with a recent study showing that a single genotype of P. rubescens
constituted almost 100% of the lake's population over almost 30 years (1980 to 2008) (63
The relative abundances of sequencing reads did not match the relative annual species densities estimated in the water by microscopy (Fig. 5
). Several methodological and biological explanations for this result can be hypothesized. First, PCR and high-throughput sequencing of bacterial 16S rRNA genes introduce biases that can lead to inaccurate population data (64
). Also, the number of 16S rRNA gene copies per cell is known to vary among cyanobacterial taxa (65
). Traditional microscopy methods are also not free of biases, as plankton identification and cell estimations can vary greatly from one person to another. Rare phytoplankton taxa have been shown to be severely underestimated by traditional sampling methods (66
), and it is known that many cyanobacterial taxa are impossible to distinguish on the basis of their morphology only (60
). Furthermore, local phenomena in the lake, such as the presence of grazers, buoyant cells, and water currents, affect the sedimentation rate of plankton and may influence the proportion of phytoplankton cells that can be found in the sediments. For the above-mentioned reasons and others, abundance data should be interpreted with extreme caution in sedDNA studies. Nonetheless, the high richness recovered in this study and the strong relationship observed between independent data sets of cyanobacterial community composition (sediments versus water) confirm that the approach for reconstructing past diversity was successful in both Greifensee and Lake Zurich.
This study presents a validated approach to characterize the past composition of cyanobacterial communities archived in lake sediments. Our results demonstrate that amplicon sequencing of a relatively large DNA fragment is useful for investigating the richness and phylogenetic relatedness of cyanobacteria in lakes where the sediments are undisturbed. Our approach, applied to varved sediments, will allow us to explore phylogenetic diversity and community assembly of cyanobacteria over centuries with high temporal resolution. In a forthcoming paper, we investigate the impact of human-induced environmental changes on cyanobacterial phylogenetic diversity and community structure. The ability to recover and sequence important functional genes, like those underpinning the production of secondary metabolites, will assist us in studying the factors that favored toxic cyanobacterial taxa. This approach can, in principle, be extended to other planktonic organisms to help address ecological questions, such as those related to eutrophication, climate change, colonization processes, and invasive species, which are all relevant to the assessment and management of ecosystem processes and services.