Phytoplankton N utilization

Phytoplankton diversity and N use in upwelling systems:

The ocean’s most productive regions are associated with eastern boundary currents where the upwelling of cold, nutrient-rich waters from depth stimulates phytoplankton blooms. The phytoplankton community in these regions tends to be dominated by diatoms. We hypothesized that the success of diatoms in episodic upwelling systems is attributable to their early, rapid response to newly upwelled nitrate. This strategy allows diatoms to grow quickly and consume a disproportionate fraction of the available nutrients. To test this hypothesis, we simulated an upwelling event – and induced a phytoplankton bloom - in a mesocosm experiment (Figure 1) using Monterey Bay seawater to investigate differential growth of phytoplankton during the early stages of upwelling (Fawcett and Ward 2011). The particulate nitrogen and carbon biomass was initially dominated by the smallest phytoplankton, but the largest fraction increased most rapidly, so that it dominated the biomass and N and C assimilation after a few days. All size fractions achieved similar maximum specific nitrate uptake rates (VNO3), but this occurred most rapidly and was maintained longest by the largest fraction. This initial acceleration of VNO3 appears to be the mechanism by which diatoms exploit upwelling conditions. Pigment measurements and light microscopy independently documented changing phytoplankton abundance, with diatoms demonstrating a characteristic pattern of succession. Changes in diatom diversity (Figure 2) and assemblage composition occurred early, sooner than observable changes in NO3- or biomass implied.

In our current and future research in this area, we are pursuing the following questions:

■ Can we use the environmental transcriptome to learn the molecular basis of rapid diatom response from the patterns of gene expression in experimental manipulations?

■ Can we identify differences in gene expression among members of the phytoplankton assemblage that provide insight into their ecological success under different environmental conditions?

Figure 1

Figure 1. Mesocosm (plastic barrels shaded to approximate light conditions at 10 m) experiments underway at Moss Landing Marine Laboratories. Seawater (200 L) was obtained from 70 m depth in Monterey Bay, inoculated with 2 L of surface seawater, and sampled daily for uptake rates and biomass characteristics.

Figure 2

 

Figure 2. Composition of the diatom assemblages in one of the mesocosms, derived from microscope counts. The community was initially dominated by Pseudonitzschia and Eucampia, but by the end, several Chaetoceros species comprised 80 % of the cells. The initial assemblage was very diverse and low abundance, while the bloom assemblage was much less diverse at higher biomass.

N sources to phytoplankton in the subtropical Sargasso Sea

In the vast subtropical ocean, regenerated N is assumed to fuel most phytoplankton growth, although it is notoriously difficult to measure uptake in such N-poor systems. Even in the open ocean of the Sargasso Sea, one of the most oligotrophic environments on earth, we find nitrate is an important N source for phytoplankton, even though it is present at vanishingly small concentrations. Evidence of nitrate utilization was obtained by using flow cytometry to separate small prokaryotic cyanobacteria (Synchococcus and Prochlorococcus) from slightly larger eukaryotic phytoplankton (Figure 3), and determining the stable nitrogen isotope of the sorted populations (Fawcett et al 2011). We find that prokaryotic cyanobacteria and eukaryotic phytoplankton have distinct isotope signatures, indicating that they rely on different N sources. The cyanobacteria uniformly use regenerated N, consistent with expectations for subtropical ecosystems. Surprisingly however, the eukaryotes appear to rely on subsurface nitrate for more than half of their N. Our findings also suggest that eukaryotic phytoplankton contribute a disproportionately large fraction of the organic N sinking out of the surface. Despite their being about two orders of magnitude less abundant than the cyanobacteria, the eukaryotes appear to be driving the Sargasso Sea’s biological pump.

 

In our current and future research in this area, we are pursuing the following questions:

■ How do the sources of N supporting phytoplankton growth change seasonally?

■ How do stochastic events affect phytoplankton community composition and N use?

Figure 3

 

Figure 3. Flow cytogram of Sargasso Sea seawater assemblage, identifying the particles that can be identified and sorted based on size and fluorescence characteristics.

Functional Diversity of phytoplankton

The structure of phytoplankton communities, in terms of cell size and species composition, is a major determinant in the rate of primary production and its fate in the food web and in biogeochemical cycles. We have developed functional gene microarrays to allow us to investigate community composition in a high throughput manner [Ward, 2008; Ward and Bouskill, 2011]. The Phytoarray is a functional gene microarray that contains probes for genes involved in C and N assimilation representing all phytoplankton types for which sequence data are available (both cultivated and environmental sequences). It is capable of providing relative magnitude data on DNA (abundance) and mRNA (gene expression) for several hundred phylotypes for several N- and C-uptake related genes. By hybridizing targets, made from DNA or RNA extracted from natural samples, to the probes on the array (Figure 4), we obtain a survey of the community composition of the samples, both in terms of presence and gene expression. The current generation phytoarray contains probes for three functional genes in phytoplankton: nitrate reductase (NR/nar), the gene that encodes the first enzyme in the assimilation of NO3- in both eukaryotic and prokaryotic phytoplankton; the gene encoding RuBisCO (rbcL), the primary CO2 assimilation enzyme in photosynthetic autotrophs, and the genes encoding high affinity NO3- transporters (Nrt2/ hnat). Each gene is represented by three probe sets that represent the major phytoplankton groups: Chromophyte algae (diatoms, pelagophytes, dinoflagellates, haptophytes, etc); Chlorophyte algae (prasinophytes and chlorophytes); and Cyanobacteria.

Because of its high throughput capability, the phytoarray is a powerful tool for characterizing phytoplankton assemblages across the surface ocean and in response to experimental manipulations. For example, we find evidence that the phytoplankton assemblage in the surface Sargasso Sea contains diverse eukaryotes, including diatoms and coccolithophorids. These groups have been identified as important components of the Sargasso Sea assemblage previously based on pigment data, but with the array, we can identify individual phylotypes and follow their ecological responses and distributions. We found that Phaeodactylum tricornutum, a diatom often associated with eutrophic coastal areas a common laboratory model organism, was an important component of the phytoplankton assemblage in the English Channel (Ward et al 2011). The P. tricornutum hybridization signal on the array was correlated with f-ratio in a seasonal study of nitrogen uptake. Probes representing sequences unrelated to any cultivated phytoplankton often produce significant contributions to the total signal, implying that, even for relatively well known groups such as diatoms, the diversity of natural phytoplankton assemblages is much greater than previously suspected. In Monterey Bay, we are comparing the community composition with that evaluated by microscopy (Figure 5A) and that identified on the microarray (Figure 5B). Much higher resolution (greater diversity) can be detected on the microarrays, but because many of the important sequences we detect in the samples are not related to known cultivated organisms, we cannot yet say much about their ecology or physiology.

Figure 4

 

Figure 4. Fluorescence image of the phytoarray. Green signal indicates specific hybridization between the environmental sample and gene probes represented in individual features of the array.

Figure 5A

Figure 5B

 

Figure 5. Phytoarray data from Monterey Bay mesocosm incubations. The names of the archetypes probes are listed on the X-axis and the relative fluorescence ratio (RFR) (Y-axis) is the fraction of the total hybridization signal contributed by each archetype. A) Chromophyte rbcL archetypes; B) Chromophyte NR archetypes. The rbcL database does not contain very many diatom sequences, while the NR database is predominantly diatom sequences. Thus the two sets of probes are detecting different components of the assemblage.