Experimental design
An overview of the experimental design is depicted in figure 1, and followed the general protocols for recently published dinoflagellate community experiments [21]. Sequentially, the study included a short-term two-week temperature/pCO2 factorial matrix incubation experiment using a natural, mixed diatom assemblage, the isolation of clonal cultures from each treatment and conditioning of the clones to the pCO2 and temperature combinations from which they were isolated for 1 year. Finally, the conditioned clones were recombined into artificial communities and allowed to compete, followed by a comparison of final community structure with that observed in the original two-week natural community experiment.

Short-term natural community incubation experiment
A mixed diatom assemblage that consisted primarily of Cylindrotheca fusiformis Reimann and Lewin 1964, Coscinodiscus spp., Thalassiosira spp., Pseudo-nitzschia delicatissima (Cleve) Heiden 1928, Navicula sp. and Chaetoceros criophilus (Castracene) sensu Hust 1886 was collected off the city of Dunedin on the South Island of New Zealand in January of 2011. The water was collected approximately 3 km offshore from Tairoa Head at the mouth of Otago Harbour halfway to Munida (45, 45.098 S 170, 48.68 E). The ambient sea surface temperature was 14.88C.

Seawater was collected for both the initial incubations and the short-term experimental dilution water. All water was combined into an approximately 500 l container and subsampled after filtering through 80 mm mesh to remove large zooplankton. Volumes (800 ml) were added to triplicate polycarbonate bottles and spiked with an f/50 nutrient derivative (10 mm NaNO3 2, 0.8 mm NaH2PO4 32, 10 mm Na2SiO3 and f/50 vitamin and trace metal concentrations [27,28]) to promote diatom growth. The bottles were incubated on a 12 L : 12 D cycle under 140 mE of cool white fluorescent illumination in free-standing laboratory incubators at 14 or 198C. The temperatures (ambient and þ58C) were selected based on predicted sea surface warming from the IPCC [29]. Triplicate sterilized 1 l polycarbonate bottles were gently bubbled at each temperature using commercially prepared air/ CO2 mixtures (Alphagaz, Air Liquide) at three concentrations also based on IPCC scenarios (approx. 210 matm ¼ pre-industrial pCO2; approx. 370 matm ¼ current pCO2; and approx. 560 matm ¼ future, year 2050 projected pCO2) [29]. Cellular abundances in an unbubbled control treatment did not significantly deviate from results of the current pCO2-bubbled treatment (data not shown). This methodology has been used for other CO2 experiments [21,30,31], including previous diatom studies [10,16,20].

The six pCO2/temperature treatments were maintained in active growth using semicontinuous culture methods [21]. Each bottle was diluted to the original time-zero in vivo chlorophyll a fluorescence value every 2 days with nutrient-amended 0.2 mmfiltered seawater. Aliquots were removed initially and after one and two weeks for examination of carbonate system parameters and community structure using microscopic cell counts.

Establishment of clonal cultures
Two to four individual cells from the six dominant diatom species were isolated from each of the short-term incubation bottles at the termination of the experiment. Inverted light microscopy was used to make taxonomic determinations based on morphological characteristics to make sure the isolates for each cell line were from the same species [32]. These monospecific clones were propagated in 24-well plates prior to being transferred to tissue culture flasks for long-term maintenance under pCO2 and temperature conditions identical to those from which they were isolated. A set of the culture isolates were transported under controlled temperature conditions to the University of Southern California in Los Angeles, CA, USA, where conditioning of the isolates and the 12-month community recombination experiments presented here were carried out. The culture isolates were maintained unreplicated for the first few weeks until they were verified to be established and growing well, at which time they were transferred into triplicate cultures for long-term maintenance; initial growth rates were obtained from these original unreplicated cell lines. These cultures were then maintained for a period of 1 year in exponential growth phase using the same recipe of autoclave-sterilized enriched seawater growth medium, and with other environmental variables such as light, pCO2 bubbling, temperature etc., maintained as in the two-week natural community experiment. Semicontinuous weekly dilutions were performed based on specific growth rates within each bottle, calculated as in [21]. The approximate number of generations during this time period was: C. fusiformis (185-212), Coscinodiscus sp. (169-229), Thalassiosira sp. (179-200), P. delicatissima (178-221), Navicula sp. (188-212) and C. criophilus (194-236).

Artificial community competition experiments
After the 12-month pCO2/temperature conditioning period, the cultures were recombined into artificial communities in the same relative proportions and abundance as in the original natural assemblage collected from Otago Harbour. The incubations of these artificial communities were performed under experimental conditions, duration and dilution frequencies identical to those of the original short-term natural community experiment.

Cell counts and growth rates
Samples for cell counts were obtained at the time of collection, before and after dilution and upon termination of the natural and artificial community incubations to determine abundances of each species. Cell-specific growth rates for each clonal culture were determined in individual culture flasks at the beginning of the 1 year conditioning period and in triplicate replicates after approximately 10 months of conditioning. These were calculated from samples taken 3 days apart using the growth rate equation m ¼ ln(Nt/No)/t12t0 (where N is the number of cells at time t1 and t0 (in days)) and represent a long-term steady-exponential state of growth. Algal cells were collected in 30 ml borosilicate glass scintillation vials, preserved with acidified Lugol’s solution and enumerated using an Accu-Scope v. 3032 inverted microscope using the Utermo¨hl method [33].

Carbonate system characterization
Samples for carbonate system parameter analysis were taken at the time of the natural sample collection and at the termination of the short- and long-term experiments. Spectrophotometric pH for the initial community incubations was measured after [34] as described in [35] using a UV-vis spectrophotometer (Ocean Optics USB4000). For samples from the 12-month community incubations, spectrophotometric pH was determined using a Shimadzu 1800UV spectrophotometer according to a similar method [36]. Temperature was monitored using standard laboratory incubator thermometers and salinity by conductivity with an interchangeable probe using an Orion 5-star plus pH meter. For pH measurements, temperature and salinity values for the initial experiment were 23.68C and 35, respectively. For the conditioned experiment, the temperature was 258C and salinity 35. Dissolved inorganic carbon was analysed using a CM5230 CO2 coulometer (UIC) [37]. Experimental pCO2 was calculated using CO2SYS software [22] with dissociation constants from Dickson & Millero [38] using the combined data of [39,40] and KSO4 from [41] (table 1).