Comparing Quantitative Methods for Analyzing Sediment DNA Records of Cyanobacteria in Experimental and Reference Lakes

Sediment DNA (sedDNA) analyses are rapidly emerging as powerful tools for the reconstruction of environmental and evolutionary change. While there are an increasing number of studies using molecular genetic approaches to track changes over time, few studies have compared the coherence between quantitative polymerase chain reaction (PCR) methods and metabarcoding techniques. Primer specificity, bioinformatic analyses, and PCR inhibitors in sediments could affect the quantitative data obtained from these approaches. We compared the performance of droplet digital polymerase chain reaction (ddPCR) and high-throughput sequencing (HTS) for the quantification of target genes of cyanobacteria in lake sediments and tested whether the two techniques similarly reveal expected patterns through time. Absolute concentrations of cyanobacterial 16S rRNA genes were compared between ddPCR and HTS using dated sediment cores collected from two experimental (Lake 227, fertilized since 1969 and Lake 223, acidified from 1976 to 1983) and two reference lakes (Lakes 224 and 442) in the Experimental Lakes Area (ELA), Canada. Relative abundances of Microcystis 16S rRNA (MICR) genes were also compared between the two methods. Moderate to strong positive correlations were found between the molecular approaches among all four cores but results from ddPCR were more consistent with the known history of lake manipulations. A 100-fold increase in ddPCR estimates of cyanobacterial gene abundance beginning in ~1968 occurred in Lake 227, in keeping with experimental addition of nutrients and increase in planktonic cyanobacteria. In contrast, no significant rise in cyanobacterial abundance associated with lake fertilization was observed with HTS. Relative abundances of Microcystis between the two techniques showed moderate to strong levels of coherence in top intervals of the sediment cores. Both ddPCR and HTS approaches are suitable for sedDNA analysis, but studies aiming to quantify absolute abundances from complex environments should consider using ddPCR due to its high tolerance to PCR inhibitors.

Phototrophic pigment diversity and picophytoplankton in permafrost thaw lakes

Permafrost thaw lakes (thermokarst lakes) are widely distributed across the northern landscape, and are known to be biogeochemically active sites that emit large amounts of carbon to the atmosphere as CH4 and CO2. However, the abundance and composition of the photosynthetic communities that fix CO2 have been little explored in this ecosystem type. In order to identify the major groups of phototrophic organisms and their controlling variables, we sampled 12 permafrost thaw lakes along a permafrost degradation gradient in northern Québec, Canada. Additional samples were taken from five rock-basin reference lakes in the region to determine if the thaw lakes differed in limnological properties and phototrophs. Phytoplankton community structure was determined by high-performance liquid chromatography analysis of their photoprotective and photosynthetic pigments, and autotrophic picoplankton concentrations were assessed by flow cytometry. One of the black-colored lakes located in a landscape of rapidly degrading palsas (permafrost mounds) was selected for high-throughput 18S rRNA sequencing to complement conclusions based on the pigment and cytometry analyses. The results showed that the limnological properties of the thaw lakes differed significantly from the reference lakes, and were more highly stratified. However, both waterbody types contained similarly diverse phytoplankton groups, with dominance of the pigment assemblages by fucoxanthin-containing taxa, as well as chlorophytes, cryptophytes and cyanobacteria. Chlorophyll a concentrations (Chl a) were correlated with total phosphorus (TP), and both were significantly higher in the thaw lakes (overall means of 3.3 µg Chl a L−1 and 34 µg TP L−1) relative to the reference lakes (2.0 µg Chl a L−1 and 8.2 µg TP L−1). Stepwise multiple regression of Chl a against the other algal pigments showed that it was largely a function of alloxanthin, fucoxanthin and Chl b (R2 = 0.85). The bottom waters of two of the thaw lakes also contained high concentrations of bacteriochlorophyll d, showing the presence of green photosynthetic sulphur bacteria. The molecular analyses indicated a relatively minor contribution of diatoms, while chrysophytes, dinoflagellates and chlorophytes were well represented; the heterotrophic eukaryote fraction was dominated by numerous ciliate taxa, and also included Heliozoa, Rhizaria, chytrids and flagellates. Autotrophic picoplankton occurred in biovolume concentrations up to 3.1 × 105 µm3 picocyanobacteria mL−1 and 1.9 × 106 µm3 picoeukaryotes mL−1, with large variations among lakes. Both groups of picophytoplankton were positively correlated with total phytoplankton abundance, as measured by Chl a; picocyanobacteria were inversely correlated with dissolved organic carbon, while picoeukaryotes were inversely correlated with conductivity. Despite their net heterotrophic character, subarctic thaw lakes are rich habitats for diverse phototrophic communities.

Phototrophic pigment diversity and picophytoplankton abundance in permafrost thaw lakes

Permafrost thaw lakes (thermokarst lakes) are widely distributed across the northern landscape, and are known to be biogeochemically active sites that emit large amounts of carbon to the atmosphere as CH4 and CO2. However, the abundance and composition of the photosynthetic communities that consume CO2 have been little explored in this ecosystem type. In order to identify the major groups of phototrophic organisms and their controlling variables, we sampled 12 permafrost thaw lakes along a permafrost degradation gradient in northern Québec, Canada. Additional samples were taken from 5 rock-basin reference lakes in the region to determine if the thaw waters differed in limnological properties and phototrophs. Phytoplankton community structure was determined by high performance liquid chromatography analysis of their photoprotective and photosynthetic pigments, and autotrophic picoplankton concentrations were assessed by flow cytometry. One of the black colored lakes located in a andscape of rapidly degrading palsas (permafrost mounds) was selected for high-throughput 18S rRNA sequencing to help interpret the pigment and cytometry data. The results showed that the limnological properties of the thaw lakes differed significantly from the reference lakes, and were more highly stratified. However, both waterbody types contained similarly diverse phytoplankton groups, with dominance of the pigment assemblages by fucoxanthin-containing taxa, as well as chlorophytes, cryptophytes and cyanobacteria. Chlorophyll a concentrations (Chl a) were correlated with total phosphorus (TP), and both were significantly higher in the thaw lakes (overall means of 3.3 μg Chl a L−1 and 34 μg TP L−1) relative to the reference lakes (2.0 μg Chl a L−1 and 8.2 μg TP L−1). Stepwise multiple regression of Chl a against the other algal pigments showed that it was largely a function of lutein, fucoxanthin and peridinin (R2 = 0.78). The bottom waters of two of the thaw lakes also contained high concentrations of bacteriochlorophyll d, showing the presence of green photosynthetic sulphur bacteria. The molecular analyses indicated a relatively minor contribution of diatoms, while chrysophytes, dinoflagellates and chlorophytes were well represented; the heterotrophic eukaryote fraction was dominated by numerous ciliate taxa, and also included Heliozoa, Rhizaria, chytrids and flagellates. Autotrophic picoplankton occurred in cell concentrations up to 8.8 × 105 mL−1 (picocyanobacteria) and 4.6 × 105 mL−1 (picoeukaryotes). Both groups of picophytoplankton were positively correlated with total phytoplankton abundance, as measured by Chl a; picocyanobacteria were inversely correlated with dissolved organic carbon, while picoeukaryotes were correlated with conductivity. Despite their net heterotrophic character, subarctic thaw lakes are rich habitats for diverse phototrophic communities.