Clonal culture isolations

Three individual cells representing the four dominant genera were isolated from each incubation bottle at the end of the two-week incubation of the natural community, and maintained in long-term culture (52 weeks) at the pCO2 from which they were obtained under conditions of temperature, light, nutrients, CO2 bubbling, etc., identical to the 2-week natural community experiment. Cultures were maintained in exponential phase using autoclave-sterilized enriched seawater growth medium with semi-continuous weekly dilutions based on specific growth rates within each bottle, calculated as in Tatters et al. (2012). The approximate number of generations during this time period was: L. polyedrum (48-62), P. micans (58-71), Alexandrium sp. (34-38) and Gonyaulax sp. (75-126).

Artificial community competition experiments

The conditioned clonal cultures were recombined into artificial communities after 4, 8 and 12 months in the same relative proportions and cell densities as the original natural bloom assemblage. The 8- and 12-month experiments used triplicate communities of all four species, but due to logistical limitations, the 4-month experiment used only L. polyedrum, P. micans, and Alexandrium sp. in duplicate communities. Because Gonyaulax sp. was not included in this preliminary 4-month experiment and replication was different, it is not fully comparable to the other experiments. The dinoflagellates in the artificial community trials were allowed to compete under identical experimental conditions of light, temperature, nutrient availability, and pCO2 for the same time period and diluted exactly as in the original natural bloom incubation. Samples were collected for cell counts and carbonate system parameters (Table 1) in all experiments.

Switch experiment

A triplicated CO2 switch experiment at the 12-month time point consisted of ‘switching’ low CO2-conditioned cell lines to medium and high CO2 (low --> medium and low --> high), medium CO2-conditioned cell lines to low and high CO2 (medium --> low and medium --> high), and high CO2-conditioned clones to low and medium CO2 (high --> low and high --> medium). The same experimental bottles employed in the 12-month artificial community experiments described above were used to provide appropriate controls of low --> low, medium --> medium, and high --> high. Other than the switched pCO2 treatments, protocols for these trials were exactly the same as in the other artificial community experiments.

switch_competition.csv