Water mass properties varied moderately during the experiment, with initial surface temperatures of 19°C increasing to 22°C during day 1 (Fig. ). Day 2 surface waters increased in temperature from 22 to 24°C by the end of the experiment. Deeper water also experienced slight warming from 16 to 17°C, with upper layer stratification being apparent. Practical salinity during the sample period ranged from 31.6 (surface) to 32 (deep), with an increase of less than 0.2 in surface water at the 19:00 and 22:00 time points of day 2 (data not shown). An additional intrusion of warmer water at the sample site was also evident between 6 and 10 a.m. during day 1 at approximately 12:00. However, no samples for primary production, rbcL mRNA, or 18S rRNA were collected at these intermediate depths during the experiments.
Temperature versus depth data for day 1 (A) and day 2 (B).
Surface chlorophyll a, particulate organic C, and particulate organic N concentrations all increased from approximately 18:00 on day 1 to 19:00 on day 2, suggesting that a small phytoplankton bloom was under way (Fig. ). The chlorophyll a concentration ranged between 3 and 16 mg m−3 during day 1 and between 2.6 and 34.7 mg m−3 during day 2 with deep water (14 m) chlorophyll a variation substantially lower than surface variation. Particulate organic C concentrations varied between 0.48 and 3.2 mg liter−1 and particulate organic N concentrations ranged between 0.08 and 0.49 mg liter−1 (Fig. ). Both particulate organic C (r2 = 0.86) and particulate organic N (r2 = 0.90) were significantly correlated to chlorophyll a and for the most part followed a temporal pattern similar to that observed for chlorophyll a. Chlorophyll a, particulate organic C, and particulate organic N content were consistently greater near the surface than at depth during the course of the experiment.
Time course for chlorophyll a (Chl a), particulate organic C (POC), and particulate organic N (PON) at the LEO 15 node A site.
Relative to the Redfield C:N ratio of 7.1, surface particulate organic matter (8.8) was significantly depleted in N, while particulate organic matter at depth (5.9) was N rich. These differences may reflect enhanced pigment and protein synthesis in light-limited deep waters relative to greater carbohydrate and lipid synthesis in surface waters. Chlorophyll a values, as determined by HPLC, were about 65% of those reported by bulk fluorometry, indicating positive interference by other chlorophylls in the fluorometric method. Chlorophyll a was the dominant photosynthetic pigment. Fucoxanthin was the dominant accessory pigment representing 28% of the total chlorophyll a, followed by chlorophyll b (15% chlorophyll a). Fucoxanthin was significantly correlated with chlorophyll a (r2 = 0.52, P < 0.01). Other pigments present in minor concentrations (<10% chlorophyll a) were, in order of abundance, chlorophyll c2, zeaxanthin, peridinin, and chlorophyll c1 (Table ).
Concentration of photosynthetic pigments at LEO 15 during the GRIST experiment as determined by HPLC
Surface photosynthetic rates during the first day were only measured during the initial hours of the experiment when chlorophyll a variability was low. Light-saturated photosynthetic rates (Pmax), the maximum achievable C fixation rates, varied little in these samples, as was the case for deep-water samples despite a greater range in phytoplankton abundance, as reflected by chlorophyll a, particulate organic C, and particulate organic N content. During the second diel experiment, Pmax varied substantially (Fig. , Table ).
Photosynthesis-irradiance curves for surface and bottom phytoplankton populations.
Photosynthetic parameters of phytoplankton assemblages during the GRIST experiment
Photosynthetic capacity (PBmax
), the maximum achievable photosynthetic rate normalized to chlorophyll a
concentration, ranged between 1.1 and 11.6 mg of C (mg of chlorophyll a
, well within the range of values reported previously for phytoplankton assemblages of temperate coastal marine waters (2
). Average PBmax
was moderately higher at depth (6.9 m) than near the surface (4.8 m). The response to irradiance at low light levels (α) ranged between 0.03 and 0.25 mg of C (mg of chlorophyll a
and was higher on average at depth (0.13) than near the surface (0.09). Photoinhibition was apparent only in deep-water samples.
In the surface waters, rbcL mRNA levels were highest either at the first-light sampling (dawn; day 1 surface waters) or at 10:30 a.m. (Fig. ). In the subsurface, maximum transcription occurred at the 10:30 or 1 p.m. sampling. The minimum transcriptional activity occurred at about 7:00 p.m. in both surface and bottom samples. During the first diel in the surface samples, form ID rbcL mRNA was the most abundant form of rbcL mRNA during the morning peak in transcriptional activity (Fig. ). During the other three diel samples (day 1 bottom water and both surface and bottom for day 2), diatom rbcL mRNA displayed the greatest dynamic range among the rbcL forms that we quantified.
Time course for rbcL mRNA abundance and light-saturated photosynthetic rate (Pmax). IA, form IA rbcL mRNA; IB, form IB rbcL mRNA; ID, form ID rbcL mRNA; DRT, real-time diatom rbcL mRNA. Open squares, PBmax. Right panels, day 1; left panels, day 2.
Taking the average rbcL mRNA levels for the daylight hours for this group of samples indicated that diatom rbcL was significantly greater than form IA or form IB, but not different from form ID (P = 0.046). Figure shows the daylight average rbcL levels for all four time series. Bottom samples both showed a distinct increase in transcript abundance at the last sampling, 10:30 p.m. to 11:00 p.m., which was also reflected in particulate organic C and PBmax. All three forms of rbcL mRNA quantified by hybridization were significantly correlated to each other at the 1% confidence interval (A to B: P = 0.001, A to D: P = 0.01, B to D: P = 0.001). Form IB and form ID rbcL mRNAs were also significantly correlated with diatom rbcL mRNA quantified by real-time PCR. Form IA was not (P = 0.12). Pmax was significantly correlated with all forms of rbcL that we quantified (A: P = 0.01, B: P = 0.005, D: P = 0.001, diatom: P = 0.007; see Fig. ). Pmax was least well correlated to form IA rbcL (R2 = 0.41). Pmax exhibited progressively tighter correlation to form IB (R2 = 0.47) and to form ID rbcL mRNA (R2 = 0.56). The best predictor of Pmax was the cumulative rbcL mRNA signal obtained by adding form IA, IB, and ID hybridization numbers (R2 = 0.58).
Relative abundance of the different forms of rbcL mRNA observed during the experiment (averages of the daylight hours).
Relationship between PCR rbcL mRNA abundance and light-saturated photosynthetic rate. The linear relationship is given by y = −6 + 95.5x (r2 = 0.58; n = 16).
Cloning and sequencing of rbcL
mRNA from transcriptionally active phytoplankton yielded 22 unique rbcL
clones (Fig. ). Table gives a list of all unique sequences detected and their closest relatives in GenBank as determined by BlastP analysis (1
). Phylogenetic analysis suggested that 14 of these clones were of the form ID type and likely included four diatoms, four prymnesiophytes, four chrysophytes, a rhodophyte, and a deeply rooted sequence most closely related to Olithodiscus luteus
. The remaining eight clones were of the form IB type and included two flagellated chlorophytes related to Pyramimonas
, one sequence closely related to Bathycoccus
, and a small clade of deeply rooted forms. No picocyanobacterium-like sequences (i.e., Synechococcus
) were detected.
Neighbor-joining tree of transcriptionally active rbcL clones obtained from the site (labeled GRIST and clone-specific suffix) and related sequences from GenBank. The clone labeled P994DY5 is from the Gulf of Mexico (25).
Closest relatives of rbcL clones in GenBank
To test whether diatoms, prymnesiophytes, or chrysophytes were actively growing in the water samples during the experiment, we created clonal libraries from intact rRNA purified from the samples with reverse transcriptase. The relationship between RNA content and growth rate has been well characterized for phytoplankton and bacteria (3
). A reconstructed phylogenetic tree for the 18S rRNA subunit is shown in Fig. . Comparison of gene phylogenies obtained for cloned rbcL
and 18S sequences revealed several important similarities and discrepancies. The GRIST CH25 rbcL
clone was found to be most similar (>92% at the amino acid level, Table ) to Detonula confervacea
, as demonstrated by phylogenetic analysis (Fig. ). Similarly, the 18S clones G2D2T4 and C12D2T4 (Fig. ) were found to be most closely related to Detonula
. Two rbcL
sequences were observed to be most similar to the diatom Skeletonema costatum
(GRIST CH19 and CH27), but no corresponding 18S clones were observed. The GRIST CH29 clone was related to Pedinella
, matching the B12D2T4 and G1D2T3 18S clones from both surface and deep samples. Sequences similar to a red alga, green algae, and one almost identical to Bathycoccus prasinos
were not observed among the sequence of the corresponding 18S library. Finally, the GRIST CH36 rbcL
related to E. huxleyi
was similar to the D1D2T3 18S clone. These results support the observation that diatoms and prymnesiophytes were actively photosynthesizing and growing during the experiment.
Phylogenetic tree based on 18S rRNA genes obtained via reverse transcription of intact ribosomes during GRIST with maximum-likelihood methods.