Species isolation and culture conditions

Four diatom species, Chaetoceros sp., Thalassiosira sp., Chaetoceros tenuissimus and Synedra sp., were isolated from coastal Red Sea waters near the Al Fahal Reef (22.2528°N, 38.9612°E). Surface water samples were collected and then were passed through a 45-μm filter. Approximately ten clonal cultures were established by single-cell isolation under a microscope. All cultures were maintained as batch cultures in filtered seawater that was taken from the same location and enriched with f/4 medium and silicate. The cultures incubated at 24 °C in a precise temperature-controlled incubator (Percival, United States). The cultures grew with a light: dark cycle of 12 h: 12 h under 50 μmol photons m−2 s−1.

After pure cultures of each species were established, mono-specific cultures of Chaetoceros sp., Thalassiosira sp., Chaetoceros tenuissimus and Synedra sp. were grown in 200 mL Erlenmeyer flasks at 26 ± 0.1 (experimental ambient, termed as ambient hereafter) and 30 ± 0.1 °C (experimental warming, termed as warming hereafter). The design of the warming temperature that ignoring the regional temperature variation (both annual and seasonal) in the Red Sea rather serves as a temperature increase by average as projected by high-emission scenario (RCP 8.5, IPCC 2014) for the turn of next century, to explore the extent the adaptive capacity. The cultures grew under 400 μmol photons m−2 s−1 with a light: dark cycle of 12 h: 12 h. Four independent replicated cultures (n = 4) were run semi-continuously for about 6 months under ambient and warming conditions by renewing the medium every 3 days for Chaetoceros sp., Thalassiosira sp. and C. tenuissimus and every 7 days for Synedra sp. due to their lower growth rate. This long-term nature of the experiments allowed the diatoms to adapt to the experimental temperature environment. The initial cell concentration was set at 1000 cells mL−1, and the medium was partially renewed every 3 or 7 days to restore the cell density to the initial level (i.e., growth batch cycle). Nutrients were not limiting as the cell abundances achieved at the end of the batch cycles were far from those expected at the stationary phase. Cell abundance was quantified every 3 days for Chaetoceros sp., Thalassiosira sp. and C. tenuissimus and every 7 days for Synedra sp., by examining the samples under an optical microscope (LEICA DMI 3000B-Germany) by hemocytometer. The specific growth rate, μ (d−1), during each batch growth cycle was calculated as

$${\rm{\mu }}=\frac{ln({C}_{1}-{C}_{0})}{({t}_{1}-{t}_{0})},$$ (1)

where C 1 and C 0 are the cell densities at times t 1 and t 0 (t 1 − t 0 = 3 or 7 days), respectively. The number of generations per transfer (g) is equivalent to the number of doubles and was calculated as follows:

$$g=\frac{{t}_{1}-{t}_{0}}{ln(2)/{\rm{\mu }}},$$ (2)

where t 1 − t 0 is the time interval of the transfer (d), ln (2)/μ is the doubling time (d) and μ is the specific growth rate (d−1).

Thermal growth curves

The thermal growth responses for all four diatoms adapted to ambient and warming conditions for about 6 months were determined at ten (Chaetoceros sp., Thalassiosira sp. and Synedra sp.) or twelve (C. tenuissimus, depending on its high thermal capacity) assay temperatures. At the end of the long-term selection period, cultures of ambient and warming conditions were inoculated into 200 mL flasks at an initial cell density of 1000 cells mL−1, and then incubated at 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36 °C (Chaetoceros sp., Thalassiosira sp. and Synedra sp.) and 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40 °C for species C. tenuissimus. To accommodate the different growth rates, batch cycles lasted 3 days for Chaetoceros sp. (~6–8 generations), Thalassiosira sp. (~5–7 generations) and C. tenuissimus (~9–11 generations) and 7 days for Synedra sp. (~7–8 generations) of the different temperatures. At the end of each growth cycle, the cell densities were determined by counting cell abundances under an optical microscope, and specific growth rates (μ) for each assay temperature were calculated from cell abundances versus time.

Photosynthetic responses to temperature

Photosynthetic responses were measured at the end of each growth cycle using Pulse-Amplitude-Modulation (PAM) fluorometry (Phyto-PAM, Walz, Germany) at the ambient and warming temperatures in a reciprocal assay in which warming-adapted and ambient-adapted populations were compared against the respective non-adapted (i.e., 30 °C and 26 °C, for warming and ambient, respectively) and vice versa (Fig. S2). All the measurements were made in the middle of the light phase.

The maximum quantum yield (Fv/Fm) of photosystem II (PSII) was measured on samples that were adapted to the dark for 15 min and then determined by a saturated pulse (5000 μmol photos m−2 s−1). For the rapid light curve (RLC) measurements, we determined the relative electron transport rate (rETR) at 12 different light levels (1, 16, 32, 64, 164, 264, 364, 564, 764, 1064, 1364 and 1664 μmol photons m−2 s−1), each lasting for 20 s. The rETR (an arbitrary unit) was calculated as

$$rETR={\rm{\Phi }}\mathrm{PSII}\times 0.5\times \mathrm{PAR},$$ (3)

where ΦPSII is the photochemical quantum yield of PSII in light, PAR is the actinic light intensity (μmol photons m−2 s−1), and the factor 0.5 accounts for approximately 50% of all the absorbed energy allocated to PSII. RLC was fitted with the model proposed in ref.34. The photosynthetic parameters maximum electron transport rate (ETR max ), light usage efficiency (α) and saturated light intensity (Ik) were derived from the fitted curves (Supplementary Methods).

Statistical analyses

We used the linear mixed-effects model to quantify trajectories of specific growth rates under experimental ambient and warming temperatures. For the analysis, μ was considered the dependent variable, time (in days) and treatment temperature were the fixed effects, while slopes and intercepts were treated as random effects at the level of replicates nested within the selection temperature35. The significance of the parameters was assessed using likelihood ratio tests, comparing models with common slopes and intercepts for each selection temperature (Table 1). Comparisons of models were done using Akaike’s information criterion (AIC) and likelihood ratio tests on models fitted with restricted maximum likelihood (REML) for comparison of random effects, and maximum likelihood (ML) for comparison of fixed effects36.

The thermal reaction norms of the four diatoms adapted to the two treatment temperatures were assessed by applying the equation described in refs14,37:

$$f(T)=a{e}^{bT}[1-{(\frac{T-z}{w/2})}^{2}],$$ (4)