Population turnover in a Microcystis bloom results in predominantly nontoxigenic variants late in the season

Abstract

Surface samples of the 2007 Microcystis bloom occurring in Copco Reservoir on the Klamath River in Northern California were analyzed genetically by sequencing clone libraries made with amplicons at three loci: the internal transcribed spacer of the rRNA operon (ITS), cpcBA, and mcyA. Samples were taken between June and October, during which time two cell count peaks occurred, in mid-July and early September. The ITS and cpcBA loci could be classified into four or five allele groups, which provided a convenient means for describing the Microcystis population and its changes over time. Each group was numerically dominated by a single, highly represented sequence. Other members of each group varied by changes at 1 to 3 nucleotide positions, while groups were separated by up to 30 nucleotide differences. As deduced by a partial sampling of the clone libraries, there were marked population turnovers during the season, indicated by changes in allele composition at both the ITS and cpcBA loci. Different ITS and cpcBA genotypes appeared to be dominant at the two population peaks. Toxicity (amount of microcystin per cell) and toxigenic potential (mcyB copy number) were lower during the second peak, and the mcyB copy number fell further as the bloom declined.

FIG. 1.

Progress of the Microcystis bloom in Copco Reservoir in 2007. Microcystis cell counts (⧫) and microcystin content (□) in surface samples taken at site CR01 at the indicated dates were assessed by microscopic phycological analysis and enumeration. Microcystin (extracellular plus intracellular) was determined by ELISA. Cell counts and toxin determinations were conducted with splits of the same samples, some of which were the same samples genetically analyzed in this study. Data were derived from reference .

FIG. 2.

Loss of the mcyB gene during the 2007 bloom season in Copco Reservoir. Quantitative PCR was used to determine the relative copy numbers of the Microcystis-specific mcyB and cpcBA loci (average from three experiments). Samples were taken from Copco Reservoir at the indicated dates and from the MDT8 site in Upper Klamath Lake (UKL) on 21 August 2007.

FIG. 3.

Relationships between ITS genotypes collected from a 2007 Microcystis bloom in Copco Reservoir. The maximum parsimony network created with the TCS version 1.21 program (4) from all sequences generated from clone libraries made from the June through October samples separates the ITS sequences into four allele groups. The larger circles represent the MAS genotypes of each group, which are identified by name. The areas of circles are roughly proportional to the number of times a sequence was found, which is also indicated by the numbers entered within the larger circles. The number of nucleotide differences between two genotypes is the sum of steps on the shortest connecting path, summing crosshatches, intervening genotypes, and junction nodes (small circles). Sampling times are color coded as indicated in the legend. MAS GenBank accession numbers are as follows: ITS-1, GU249289; ITS-2, GU249253; ITS-3, GU249225; ITS-4, GU249216.

FIG. 4.

Changes in ITS and cpcBA allele populations during the 2007 bloom season in Copco Reservoir. (A) The relative abundances of the members of ITS sequence groups 1 to 4 are plotted across the bloom season; poorly clustered sequences GU249218 and GU249219 are represented as “other.” The June sample was from site CRCC, and all others were from CR01. The dashed lines report the relative abundances of the MAS genotypes belonging to groups 1 and 2 (ITS-1 and ITS-2, respectively). (B) The relative abundances of the members of cpcBA sequence groups A to D are plotted across the bloom season. The dashed lines report the relative abundances of the MAS genotypes belonging to groups A, B1, and C (CPC-A, CPC-B1, and CPC-C, respectively).

FIG. 5.

Relationships between cpcBA genotypes collected from a 2007 Microcystis bloom in Copco Reservoir. The maximum parsimony network created from all sequences generated from clone libraries made from June through October separates the cpcBA genotypes into five allele groups. Details are as in Fig. 3. MAS GenBank accession numbers are as follows: CPC-A, GU249203; CPC-B1, GU249162; CPC-B2, GU249171; CPC-C GU249154; CPC-D, GU249176.

FIG. 6.

Microcystis genotypes in Copco Reservoir. The predicted genotypes at the three analyzed loci for most of the population at the 2007 sampling times are indicated at left. Mixed populations were present on 8 August and 23 August. The determined genotypes of three isolated colonies taken from Copco Reservoir on 15 July 2008 (colonies 8 and 15) or 5 August 2008 (colony 24) are given at right. The amplicon sequences derived from these colonies matched exactly the MAS of the indicated sequence groups.