Organic Phosphorus Scavenging Supports Efficient Growth of Diazotrophic Cyanobacteria Under Phosphate Depletion.

Considering the reported significant diazotrophic activities in open-ocean regions where primary production is strongly limited by phosphate, we explored the ability of diazotrophs to use other sources of phosphorus to alleviate the phosphate depletion. We tested the actual efficiency of the open-ocean, N2-fixer Crocosphaera watsonii to grow on organic phosphorus as the sole P source, and observed how the P source affects the cellular C, N, and P composition. We obtained equivalent growth efficiencies on AMP and DL-α-glycerophosphate as compared with identical cultures grown on phosphate, and survival of the population on phytic acid. Our results show that Crocosphaera cannot use all phosphomonoesters with the same efficiency, but it can grow without phosphate, provided that usable DOP and sufficient light energy are available. Also, results point out that organic phosphorus uptake is not proportional to alkaline phosphatase activity, demonstrating that the latter is not a suitable proxy to estimate DOP-based growth yields of organisms, whether in culture experiments or in the natural environment. The growth parameters obtained, as a function of the P source, will be critical to improve and calibrate mathematical models of diazotrophic growth and the distribution of nitrogen fixation in the global ocean.

Erratum to "Quantifying Cyanothece growth under DIC limitation" [Comput. Struct. Biotechnol. J. 19 (2021) 6456-6464].

[This corrects the article DOI: 10.1016/j.csbj.2021.11.036.].

Calculation and Interpretation of Substrate Assimilation Rates in Microbial Cells Based on Isotopic Composition Data Obtained by nanoSIMS.

Stable isotope probing (SIP) combined with nano-scale secondary ion mass spectrometry (nanoSIMS) is a powerful approach to quantify assimilation rates of elements such as C and N into individual microbial cells. Here, we use mathematical modeling to investigate how the derived rate estimates depend on the model used to describe substrate assimilation by a cell during a SIP incubation. We show that the most commonly used model, which is based on the simplifying assumptions of linearly increasing biomass of individual cells over time and no cell division, can yield underestimated assimilation rates when compared to rates derived from a model that accounts for cell division. This difference occurs because the isotopic labeling of a dividing cell increases more rapidly over time compared to a non-dividing cell and becomes more pronounced as the labeling increases above a threshold value that depends on the cell cycle stage of the measured cell. Based on the modeling results, we present formulae for estimating assimilation rates in cells and discuss their underlying assumptions, conditions of applicability, and implications for the interpretation of intercellular variability in assimilation rates derived from nanoSIMS data, including the impacts of storage inclusion metabolism. We offer the formulae as a Matlab script to facilitate rapid data evaluation by nanoSIMS users.

Quantifying Cyanothece growth under DIC limitation.

The photoautotrophic, unicellular N2-fixer, Cyanothece, is a model organism that has been widely used to study photosynthesis regulation, the structure of photosystems, and the temporal segregation of carbon (C) and nitrogen (N) fixation in light and dark phases of the diel cycle. Here, we present a simple quantitative model and experimental data that together, suggest external dissolved inorganic carbon (DIC) concentration as a major limiting factor for Cyanothece growth, due to its high C-storage requirement. Using experimental data from a parallel laboratory study as a basis, we show that after the onset of the light period, DIC was rapidly consumed by photosynthesis, leading to a sharp drop in the rate of photosynthesis and C accumulation. In N2-fixing cultures, high rates of photosynthesis in the morning enabled rapid conversion of DIC to intracellular C storage, hastening DIC consumption to levels that limited further uptake. The N2-fixing condition allows only a small fraction of fixed C for cellular growth since a large fraction was reserved in storage to fuel night-time N2 fixation. Our model provides a framework for resolving DIC limitation in aquatic ecosystem simulations, where DIC as a growth-limiting factor has rarely been considered, and importantly emphasizes the effect of intracellular C allocation on growth rate that varies depending on the growth environment.

Independence of a Marine Unicellular Diazotroph to the Presence of NO3.

Marine nitrogen (N2) fixation was historically considered to be absent or reduced in nitrate (NO3-) rich environments. This is commonly attributed to the lower energetic cost of NO3- uptake compared to diazotrophy in oxic environments. This paradigm often contributes to making inferences about diazotroph distribution and activity in the ocean, and is also often used in biogeochemical ocean models. To assess the general validity of this paradigm beyond the traditionally used model organism Trichodesmium spp., we grew cultures of the unicellular cyanobacterium Crocosphaera watsonii WH8501 long term in medium containing replete concentrations of NO3-. NO3- uptake was measured in comparison to N2 fixation to assess the cultures' nitrogen source preferences. We further measured culture growth rate, cell stoichiometry, and carbon fixation rate to determine if the presence of NO3- had any effect on cell metabolism. We found that uptake of NO3- by this strain of Crocosphaera was minimal in comparison to other N sources (~2-4% of total uptake). Furthermore, availability of NO3- did not statistically alter N2 fixation rate nor any aspect of cell physiology or metabolism measured (cellular growth rate, cell stoichiometry, cell size, nitrogen fixation rate, nitrogenase activity) in comparison to a NO3- free control culture. These results demonstrate the capability of a marine diazotroph to fix nitrogen and grow independently of NO3-. This lack of sensitivity of diazotrophy to NO3- suggests that assumptions often made about, and model formulations of, N2 fixation should be reconsidered.

Electron & Biomass Dynamics of Cyanothece Under Interacting Nitrogen & Carbon Limitations.

Marine diazotrophs are a diverse group with key roles in biogeochemical fluxes linked to primary productivity. The unicellular, diazotrophic cyanobacterium Cyanothece is widely found in coastal, subtropical oceans. We analyze the consequences of diazotrophy on growth efficiency, compared to NO3 --supported growth in Cyanothece, to understand how cells cope with N2-fixation when they also have to face carbon limitation, which may transiently affect populations in coastal environments or during blooms of phytoplankton communities. When grown in obligate diazotrophy, cells face the double burden of a more ATP-demanding N-acquisition mode and additional metabolic losses imposed by the transient storage of reducing potential as carbohydrate, compared to a hypothetical N2 assimilation directly driven by photosynthetic electron transport. Further, this energetic burden imposed by N2-fixation could not be alleviated, despite the high irradiance level within the cultures, because photosynthesis was limited by the availability of dissolved inorganic carbon (DIC), and possibly by a constrained capacity for carbon storage. DIC limitation exacerbates the costs on growth imposed by nitrogen fixation. Therefore, the competitive efficiency of diazotrophs could be hindered in areas with insufficient renewal of dissolved gases and/or with intense phytoplankton biomass that both decrease available light energy and draw the DIC level down.

A thermal trade-off between viral production and degradation drives virus-phytoplankton population dynamics.

Marine viruses interact with microbial hosts in dynamic environments shaped by variation in abiotic factors, including temperature. However, the impacts of temperature on viral infection of phytoplankton are not well understood. Here we coupled mathematical modelling with experiments to explore the effect of temperature on virus-phytoplankton interactions. Our model shows the negative consequences of high temperatures on infection and suggests a temperature-dependent threshold between viral production and degradation. Modelling long-term dynamics in environments with different average temperatures revealed the potential for long-term host-virus coexistence, epidemic free or habitat loss states. We generalised our model to variation in global sea surface temperatures corresponding to present and future seas and show that climate change may differentially influence virus-host dynamics depending on the virus-host pair. Temperature-dependent changes in the infectivity of virus particles may lead to shifts in virus-host habitats in warmer oceans, analogous to projected changes in the habitats of macro-, microorganisms and pathogens.

Temporal Patterns and Intra- and Inter-Cellular Variability in Carbon and Nitrogen Assimilation by the Unicellular Cyanobacterium Cyanothece sp. ATCC 51142.

Unicellular nitrogen fixing cyanobacteria (UCYN) are abundant members of phytoplankton communities in a wide range of marine environments, including those with rapidly changing nitrogen (N) concentrations. We hypothesized that differences in N availability (N2 vs. combined N) would cause UCYN to shift strategies of intracellular N and C allocation. We used transmission electron microscopy and nanoscale secondary ion mass spectrometry imaging to track assimilation and intracellular allocation of 13C-labeled CO2 and 15N-labeled N2 or NO3 at different periods across a diel cycle in Cyanothece sp. ATCC 51142. We present new ideas on interpreting these imaging data, including the influences of pre-incubation cellular C and N contents and turnover rates of inclusion bodies. Within cultures growing diazotrophically, distinct subpopulations were detected that fixed N2 at night or in the morning. Additional significant within-population heterogeneity was likely caused by differences in the relative amounts of N assimilated into cyanophycin from sources external and internal to the cells. Whether growing on N2 or NO3, cells prioritized cyanophycin synthesis when N assimilation rates were highest. N assimilation in cells growing on NO3 switched from cyanophycin synthesis to protein synthesis, suggesting that once a cyanophycin quota is met, it is bypassed in favor of protein synthesis. Growth on NO3 also revealed that at night, there is a very low level of CO2 assimilation into polysaccharides simultaneous with their catabolism for protein synthesis. This study revealed multiple, detailed mechanisms underlying C and N management in Cyanothece that facilitate its success in dynamic aquatic environments.

Overproduction of the Flv3B flavodiiron, enhances the photobiological hydrogen production by the nitrogen-fixing cyanobacterium Nostoc PCC 7120.

BACKGROUND: The ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen (H2) is a promising alternative for renewable, clean-energy production. However, the most recent, related studies point out that much improvement is needed for sustainable cyanobacterial-based H2 production to become economically viable. In this study, we investigated the impact of induced O2-consumption on H2 photoproduction yields in the heterocyte-forming, N2-fixing cyanobacterium Nostoc PCC7120. RESULTS: The flv3B gene, encoding a flavodiiron protein naturally expressed in Nostoc heterocytes, was overexpressed. Under aerobic and phototrophic growth conditions, the recombinant strain displayed a significantly higher H2 production than the wild type. Nitrogenase activity assays indicated that flv3B overexpression did not enhance the nitrogen fixation rates. Interestingly, the transcription of the hox genes, encoding the NiFe Hox hydrogenase, was significantly elevated, as shown by the quantitative RT-PCR analyses. CONCLUSION: We conclude that the overproduced Flv3B protein might have enhanced O2-consumption, thus creating conditions inducing hox genes and facilitating H2 production. The present study clearly demonstrates the potential to use metabolic engineered cyanobacteria for photosynthesis driven H2 production.

Picoeukaryotes of the Micromonas genus: sentinels of a warming ocean.

Photosynthetic picoeukaryotesx in the genus Micromonas show among the widest latitudinal distributions on Earth, experiencing large thermal gradients from poles to tropics. Micromonas comprises at least four different species often found in sympatry. While such ubiquity might suggest a wide thermal niche, the temperature response of the different strains is still unexplored, leaving many questions as for their ecological success over such diverse ecosystems. Using combined experiments and theory, we characterize the thermal response of eleven Micromonas strains belonging to four species. We demonstrate that the variety of specific responses to temperature in the Micromonas genus makes this environmental factor an ideal marker to describe its global distribution and diversity. We then propose a diversity model for the genus Micromonas, which proves to be representative of the whole phytoplankton diversity. This prominent primary producer is therefore a sentinel organism of phytoplankton diversity at the global scale. We use the diversity within Micromonas to anticipate the potential impact of global warming on oceanic phytoplankton. We develop a dynamic, adaptive model and run forecast simulations, exploring a range of adaptation time scales, to probe the likely responses to climate change. Results stress how biodiversity erosion depends on the ability of organisms to adapt rapidly to temperature increase.

Diazotrophic community and associated dinitrogen fixation within the temperate coral Oculina patagonica.

Dinitrogen (N2 ) fixing bacteria (diazotrophs) are an important source of new nitrogen in oligotrophic environments and represent stable members of the microbiome in tropical corals, while information on corals from temperate oligotrophic regions is lacking. Therefore, this study provides new insights into the diversity and activity of diazotrophs associated with the temperate coral Oculina patagonica from the Mediterranean Sea by combining metabarcoding sequencing of amplicons of both the 16S rRNA and nifH genes and 15 N2 stable isotope tracer analysis to assess diazotroph-derived nitrogen (DDN) assimilation by the coral. Results show that the diazotrophic community of O. patagonica is dominated by autotrophic bacteria (i.e. Cyanobacteria and Chlorobia). The majority of DDN was assimilated into the tissue and skeletal matrix, and DDN assimilation significantly increased in bleached corals. Thus, diazotrophs may constitute an additional nitrogen source for the coral host, when nutrient exchange with Symbiodinium is disrupted (e.g. bleaching) and external food supply is limited (e.g. oligotrophic summer season). Furthermore, we hypothesize that DDN can facilitate the fast proliferation of endolithic algae, which provide an alternative carbon source for bleached O. patagonica. Overall, O. patagonica could serve as a good model for investigating the importance of diazotrophs in coral recovery from bleaching.

Colonization of Non-biodegradable and Biodegradable Plastics by Marine Microorganisms.

Plastics are ubiquitous in the oceans and constitute suitable matrices for bacterial attachment and growth. Understanding biofouling mechanisms is a key issue to assessing the ecological impacts and fate of plastics in marine environment. In this study, we investigated the different steps of plastic colonization of polyolefin-based plastics, on the first one hand, including conventional low-density polyethylene (PE), additivated PE with pro-oxidant (OXO), and artificially aged OXO (AA-OXO); and of a polyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), on the other hand. We combined measurements of physical surface properties of polymers (hydrophobicity and roughness) with microbiological characterization of the biofilm (cell counts, taxonomic composition, and heterotrophic activity) using a wide range of techniques, with some of them used for the first time on plastics. Our experimental setup using aquariums with natural circulating seawater during 6 weeks allowed us to characterize the successive phases of primo-colonization, growing, and maturation of the biofilms. We highlighted different trends between polymer types with distinct surface properties and composition, the biodegradable AA-OXO and PHBV presenting higher colonization by active and specific bacteria compared to non-biodegradable polymers (PE and OXO). Succession of bacterial population occurred during the three colonization phases, with hydrocarbonoclastic bacteria being highly abundant on all plastic types. This study brings original data that provide new insights on the colonization of non-biodegradable and biodegradable polymers by marine microorganisms.

How do microalgae perceive light in a high-rate pond? Towards more realistic Lagrangian experiments.

Hydrodynamics in a high-rate production reactor for microalgae cultivation affects the light history perceived by cells. The interplay between cell movement and medium turbidity leads to a complex light pattern, whose forcing effects on photosynthesis and photoacclimation dynamics are non-trivial. Hydrodynamics of high density algal ponds mixed by a paddle wheel has been studied recently, although the focus has never been on describing its impact on photosynthetic growth efficiency. In this multidisciplinary downscaling study, we first reconstructed single cell trajectories in an open raceway using an original hydrodynamical model offering a powerful discretization of the Navier-Stokes equations tailored to systems with free surfaces. The trajectory of a particular cell was selected and the associated high-frequency light pattern was computed. This light pattern was then experimentally reproduced in an Arduino-driven computer controlled cultivation system with a low density Dunaliella salina culture. The effect on growth and pigment content was recorded for various frequencies of the light pattern, by setting different paddle wheel velocities. Results show that the frequency of this realistic signal plays a decisive role in the dynamics of photosynthesis, thus revealing an unexpected photosynthetic response compared to that recorded under the on/off signals usually used in the literature. Indeed, the light received by a single cell contains signals from low to high frequencies that nonlinearly interact with the photosynthesis process and differentially stimulate the various time scales associated with photoacclimation and energy dissipation. This study highlights the need for experiments with more realistic light stimuli to better understand microalgal growth at high cell densities. An experimental protocol is also proposed, with simple, yet more realistic, step functions for light fluctuations.

Temperature is a key factor in Micromonas-virus interactions.

The genus Micromonas comprises phytoplankton that show among the widest latitudinal distributions on Earth, and members of this genus are recurrently infected by prasinoviruses in contrasted thermal ecosystems. In this study, we assessed how temperature influences the interplay between the main genetic clades of this prominent microalga and their viruses. The growth of three Micromonas strains (Mic-A, Mic-B, Mic-C) and the stability of their respective lytic viruses (MicV-A, MicV-B, MicV-C) were measured over a thermal range of 4-32.5 °C. Similar growth temperature optima (Topt) were predicted for all three hosts but Mic-B exhibited a broader thermal tolerance than Mic-A and Mic-C, suggesting distinct thermoacclimation strategies. Similarly, the MicV-C virus displayed a remarkable thermal stability compared with MicV-A and MicV-B. Despite these divergences, infection dynamics showed that temperatures below Topt lengthened lytic cycle kinetics and reduced viral yield and, notably, that infection at temperatures above Topt did not usually result in cell lysis. Two mechanisms operated depending on the temperature and the biological system. Hosts either prevented the production of viral progeny or maintained their ability to produce virions with no apparent cell lysis, pointing to a possible switch in the viral life strategy. Hence, temperature changes critically affect the outcome of Micromonas infection and have implications for ocean biogeochemistry and evolution.

Photosystem-II shutdown evolved with Nitrogen fixation in the unicellular diazotroph Crocosphaera watsonii.

Protection of nitrogenase from oxygen in unicellular Cyanobacteria is obtained by temporal separation of photosynthesis and diazotrophy through transcriptional and translational regulations of nitrogenase. But diazotrophs can face environmental situations in which N2 fixation occurs significantly in the light, and we believe that another control operates to make it possible. The night-time shutdown of PSII activity is a peculiar behaviour that discriminates Crocosphaera watsonii WH8501 from any other phototroph, whether prokaryote or eukaryote. This phenomenon is not only due to the plastoquinone pool redox status, and suggests that the sentinel D1 protein, expressed in periods of nitrogen fixation, is inactive. Results demonstrate a tight constraint of oxygen evolution in C. watsonii as additional protection of nitrogenase activity and suggest a possible recycling of cellular components.

Long-term adaptive response to high-frequency light signals in the unicellular photosynthetic eukaryote Dunaliella salina.

Productivity of microalgal cultivation processes is tightly related to photosynthetic efficiency, and therefore to light availability at the cell scale. In an agitated, highly turbid suspension,the light signal received by a single phytoplankton cell moving in a dense culture is a succession of flashes. The growth characteristics of microalgae under such dynamic light conditions are thus fundamental information to understand nonlinear properties of the photosynthetic process and to improve cultivation process design and operation. Studies of the long term consequences of dynamic illumination regime on photosynthesis require a very specific experimental set-up where fast varying signals are applied on the long term. In order to investigate the growth response of the unicellular photosynthetic eukaryote Dunaliella salina (Chlorophyceae) to intermittent light exposure, different light regimes using LEDs with the same average total light dose were applied in continuous cultures. Flashing light with different durations of light flashes (△t of 30 s, 15 s, 2 s and 0.1 s) followed by dark periods of variable length (0.67 ≤ L:D ≤ 2) yielding flash frequencies in the range 0.017-5 Hz, were compared to continuous illumination. Specific growth rate, photosynthetic pigments, lipid productivity and elemental composition were measured on two duplicates for each irradiance condition. The different treatments of intermittent light led to specific growth rates ranging from 0.25 to 0.93 day(-1) . While photosynthetic efficiency was enhanced with increased flash frequency, no significant differences were observed in the particular carbon and chlorophyll content. Pigment analysis showed that within this range of flash frequency, cells progressively photoacclimated to the average light intensity.

Nitrogen fixation and respiratory electron transport in the cyanobacterium Cyanothece under different light/dark cycles.

Incompatibility of nitrogen fixation and oxygen production compels unicellular diazotrophic cyanobacteria to perform photosynthesis during daytime and restrict nitrogen fixation to nighttime. The marine diazotroph Cyanothece BG 043511 was grown in continuous culture under three light/dark regimes (16L : 8D, 12L : 12D, and 8L : 16D h); we monitored nitrogen fixation and potential photosynthetic efficiency simultaneously online to reveal how their temporal separation is affected by different LD regimes. An increase in nitrogen fixation rate at night coincided with a rise in pulse-amplitude modulated fluorescence, indicating that the enhanced respiratory electron transport to fuel diazotrophy affects the oxidation state of the plastoquinone pool. This may offer an alternative approach to assess instantaneous nitrogen fixation activity. Regardless of photoperiod, the maximum rate of nitrogen fixation was conserved at about 20 h after the onset of the light. Consequently, nitrogen fixation rates peaked at different moments in the dark: relatively early in the 16L : 8D cycle, at midnight in 12L : 12D, and relatively late in 8L : 16D. Under 16L : 8D, nitrogen fixation extended into the light, demonstrating the functional plasticity of nitrogen fixation in Cyanothece. Highest daily amounts of nitrogen fixed were obtained in 12L : 12D, which is consistent with the natural LD cycle of subtropical latitudes in which Cyanothece thrives.

Photoperiod length paces the temporal orchestration of cell cycle and carbon-nitrogen metabolism in Crocosphaera watsonii.

We analysed the effect of photoperiod length (PPL) (16:8 and 8:16 h of light-dark regime, named long and short PPL, respectively) on the temporal orchestration of the two antagonistic, carbon and nitrogen acquisitions in the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii strain WH8501 growing diazotrophically. Carbon and nitrogen metabolism were monitored at high frequency, and their patterns were compared with the cell cycle progression. The oxygen-sensitive N2 fixation process occurred mainly during the dark period, where photosynthesis cannot take place, inducing a light-dark cycle of cellular C : N ratio. Examination of circadian patterns in the cell cycle revealed that cell division occurred during the midlight period, (8 h and 4 h into the light in the long and short PPL conditions, respectively), thus timely separated from the energy-intensive diazotrophic process. Results consistently show a nearly 5 h time lag between the end of cell division and the onset of N2 fixation. Shorter PPLs affected DNA compaction of C. watsonii cells and also led to a decrease in the cell division rate. Therefore, PPL paces the growth of C. watsonii: a long PPL enhances cell division while a short PPL favours somatic growth (biomass production) with higher carbon and nitrogen cell contents.

Light-dark (12:12) cycle of carbon and nitrogen metabolism in Crocosphaera watsonii WH8501: relation to the cell cycle.

This study provides with original data sets on the physiology of the unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501, maintained in continuous culture in conditions of obligate diazotrophy. Cultures were exposed to a 12:12 light-dark regime, representative of what they experience in nature and where growth is expected to be balanced. Nitrogen and carbon metabolism were monitored at high frequency and their dynamics was compared with the cell cycle. Results reveal a daily cycle in the physiological and biochemical parameters, tightly constrained by the timely decoupled processes of N(2) fixation and carbon acquisition. The cell division rate increased concomitantly to carbon accumulation and peaked 6 h into the light. The carbon content reached a maximum at the end of the light phase. N(2) fixation occurred mostly during the dark period and peaked between 9 and 10 h into the night, while DNA synthesis, reflected by DNA fluorescence, increased until the end of the night. Consequently, cells in G1- and S-phases present a marked decrease in their C:N ratio. Nitrogen acquisition through N(2) fixation exceeded 1.3- to 3-fold the nitrogen requirements for growth, suggesting that important amounts of nitrogen are excreted even under conditions supposed to favour balanced, carbon and nitrogen acquisitions.

Genome-wide analysis of diel gene expression in the unicellular N(2)-fixing cyanobacterium Crocosphaera watsonii WH 8501.

The unicellular cyanobacterium Crocosphaera watsonii is an important nitrogen fixer in oligotrophic tropical and subtropical oceans. Metabolic, energy and cellular processes in cyanobacteria are regulated by the circadian mechanism, and/or follow the rhythmicity of light-dark cycles. The temporal separation of metabolic processes is especially essential for nitrogen fixation because of inactivation of the nitrogenase by oxygen. Using a microarray approach, we analyzed gene expression in cultures of Crocosphaera watsonii WH 8501 (C. watsonii) over a 24-h period and compared the whole-genome transcription with that in Cyanothece sp. ATCC 51142 (Cyanothece), a unicellular diazotroph that inhabits coastal marine waters. Similar to Cyanothece, regulation at the transcriptional level in C. watsonii was observed for all major metabolic and energy processes including photosynthesis, carbohydrate and amino acid metabolisms, respiration, and nitrogen fixation. Increased transcript abundance for iron acquisition genes by the end of the day appeared to be a general pattern in the unicellular diazotrophs. In contrast, genes for some ABC transporters (for example, phosphorus acquisition), DNA replication, and some genes encoding hypothetical proteins were differentially expressed in C. watsonii only. Overall, C. watsonii showed a higher percentage of genes with light-dark cycling patterns than Cyanothece, which may reflect the habitats preferences of the two cyanobacteria. This study represents the first whole-genome expression profiling in cultivated Crocosphaera, and the results will be useful in determining the basal physiology and ecology of the endemic Crocosphaera populations.