Contrasting histories of microcystin-producing cyanobacteria in two temperate lakes as inferred from quantitative sediment DNA analyses

Figures & data

Table 1. Primer sequences (for qPCR and high-throughput sequencing), amplicon size, and annealing temperatures.

Target	Primers	Sequence (5'–3')	Size (bp)	Ta C	Reference
mcy gene (1)	mcyDksF	TGG GGA TGG ACT CTC TCA CTT C	107	55	Fortin et al. 2010
	mcyDksR	GGC TTC AAC ATT CGG AAA ACG
Microcystis	MICR F	GCC GCR AGG TGA AAM CTA A	247	54.6	Rinta-Kanto et al. 2005
16S rRNA (2)	MICR R	AAT CCA AAR ACC TTC CTC CC
Cyanobacteria	CYAN 108F	ACG GGT GAG TAA CRC GTR A	269	60	Rinta-Kanto et al. 2005
16S rRNA (qPCR)	CYAN 377R	CCA TGG CGG AAA ATT CCC C
(1–4)
Cyanobacteria	CYAN 359F	GGG GAA TYT TCC GCA ATG GG	450	53	Nübel et al. 1997; Jungblut et al. 2005
16S rRNA (HTS) (1–4)	CYAN 809R	GCT TCG GCA CGG CTC GGG TCG ATA
glnA (1)	GS1β	GAT GCC GCC GAT GTA GTA	153-156	60	Hurt et al. 2001
	GS2γ	AAG ACC GCG ACC TTY ATG CC

Note. Numbers in bracket following the target gene refer to the number(s) of copies present per cell reported in the literature (Kaebernick et al. 2000; Kaneko et al. 2007; Engene and Gerwick 2011; Schirrmeister et al. 2012; Stoeva et al. 2014).

Table 2. Kendall’s nonparametric correlations (τ) of molecular data (DNA concentrations and gene copy number per gram of wet sediment) and sediment microcystin content in the Lake of the Woods core.

	MC (ng/g)	mcyD	16S MICR	16S CYAN	glnA	DNA
MC (ng/g)	1.00
mcyD	0.63** (n = 20)	1.00
16S MICR	0.53** (n = 14)	0.53** (n = 15)	1.00
16S CYAN	0.61** (n = 9)	0.69*** (n = 13)	−0.6 (n = 6)	1.00
glnA	0.30 (n = 13)	0.62*** (n = 17)	0.02 (n = 12)	0.64** (n = 12)	1.00
DNA	0.30 (n = 20)	0.26 (n = 24)	−0.10 (n = 15)	0.46** (n= 13)	0.35** (n = 17)	1.00

***p ≤ 0.001; **p ≤ 0.05.

Note. Sample sizes (n) are shown in parentheses for each correlation. MC and mcyD displayed significant temporal autocorrelation; thus, the p values of their bivariate correlation have been corrected using a temporally restricted permutation test. No corrections were applied to the other bivariate correlations as the variables were not temporally autocorrelated.

Figure 1. Lake of the Woods sediment core depth profiles with dating on the right axis of concentrations, on a logarithmic scale. (A) Total microcystin concentrations (ng/g dry weight, from Zastepa et al. 2017a) and microcystin gene (mcyD) copy numbers per gram fresh weight (f.w.) with detection limits (DL) reached approximately midway down. The dashed vertical lines indicate mean values for both through the core. Solid lines in each case represent LOESS trends. (B) Total DNA (ng/g f.w.), cyanobacterial 16S rRNA gene copy numbers/g f.w., bacterial housekeeping gln A gene copy numbers/g f.w., with dashed lines indicating mean values for each. Solid lines in each case represent LOESS trends.

Figure 2. Lake of the Woods through the sediment time series. Gene copy numbers per gram fresh weight as presented in Fig. 1 plotted on a logarithmic scale, including Microcystis specific 16S rRNA gene copy numbers. Solid lines in each case represent LOESS trends for each gene. Gray arrow shown post 1980 CE corresponds to the rise in the diatom Cyclotella relative to Aulacoseira, indicative of climate warming (from Rühland et al. 2010).

Figure 3. Baptiste Lake South (Alberta) sediment core depth profiles of total microcystins concentrations (ng/g d.w., from Zastepa et al. 2017b) and microcystin gene (mcyD) copy numbers/g f.w. on a logarithmic scale with corresponding dating on the right axis. Dashed vertical lines indicate mean values for both through the core. See Fig. 1A for detection limits. Solid lines in each case represent LOESS trends.

Figure 4. Baptiste Lake South (Alberta) across the sediment core time series. mcyD gene copy numbers as presented in Fig. 3 are plotted on a logarithmic scale, along with the bacterial housekeeping gln A, cyanobacterial 16S rRNA, and Microcystis specific 16S rRNA gene copy numbers/g f.w. Solid lines in each case represent LOESS trends. Gray arrow shown in early post 2000 CE corresponds to the rise in the diatom Stephanodiscus hantzschii, indicative of cultural eutrophication (from Adams et al., 2014).

Table 3. Kendall’s nonparametric correlations (τ) of molecular data (DNA concentrations and gene copy number per gram of wet sediment) and microcystin content in the Baptiste Lake core.

	MC (ng/g)	mcyD	16S MICR	16S CYA	glnA	DNA
MC (ng/g)	1.00
mcyD	0.05 (n= 41)	1.00
16S MICR	0.00 (n = 24)	0.30** (n = 25)	1.00
16S CYAN	0.18 (n = 16)	0.58 (n = 16)	0.49** (n = 13)	1.00
glnA	0.18 (n = 16)	0.04 (n = 16)	−0.02 (n = 15)	−0.12 (n =12)	1.00
DNA	0.10 (n = 41)	0.02 (n = 43)	0.06 (n = 25)	0.43 (n = 16)	0.44** (n = 17)	1.00

**p ≤ 0.05.

Note. Sample sizes (n) shown in parentheses for each correlation. All p values have been corrected for temporal autocorrelation by a temporally restricted permutation test (with the exception of correlations with 16S MICR and glnA, which were variables that did not display significant temporal autocorrelation).

Figure 5. Taxa in Baptiste Lake South at selected intervals of the sediment core as determined from high-throughput sequencing of the 16S rRNA gene using primers specific to cyanobacteria (Table 1). Proportions of the main taxa are presented at the level of order (A) and separated further into genera within the Nostocales (B) and Chroococcales (C) orders. Genera containing potentially toxigenic species dominate in both cases.

Table 4. Operational taxonomic units (OTUs based on 97% sequence similarity) and Shannon diversity index through the Baptiste Lake core.

Depth (cm)	Numberoof sequences			Species diversity
	Total original sequences	Post-trimming total sequences	Only cyanobacterial sequences	Number of OTUs	Shannon Index
0	14,154	7788	6100	301	5.60
2	6689	5846	1383	115	4.54
5.5	6797	5772	1634	139	4.93
7.5	5219	4374	1137	94	4.23
11	25,457	13,983	11,168	299	4.75
21	14,733	5994	5518	297	5.06
31	13,751	5543	4689	276	4.88
41	9005	4136	3192	251	5.51
45.5	11,433	5039	1192	200	5.81

Note. Sequences were trimmed using MOTHUR and OTUs were determined using QIIME.

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