Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • Problem Description
• Parameters
• Instruments
• Project Information
• Funding

All G. huxleyi isolates were obtained from either the National Center for Marine Algae and Microbiota (East Boothbay, ME) or the Roscoff Culture Collection (Roscoff, France) and maintained in incubators with ~100 µE m-2 s-1 light on a 14:10 light:dark cycle in autoclaved L1 media with a 0.2-µm filtered Vineyard Sound (MA) seawater base. Isolates were grown at different temperatures, as determined by their collection location (reported in strain metadata from NCMA and/or Roscoff; e.g., CCMP371 was collected in June 1987 in the Sargasso Sea at 32°N, 62°W with a culture collection maintenance temperature of 20 °C). To generate enough biomass needed for downstream genome sequencing (details reported elsewhere), large (1 L) batch cultures of each isolate were grown. For each separate isolate, a 25 mL nutrient-replete (L1) culture was used to inoculate 1 L nutrient-replete (L1) media in a Fernbach flask. No replicate cultures were grown for this experiment, as replicates were not necessary given the goal of obtaining DNA for genome sequencing.

Growth was monitored daily at the same time of day by in vivo chlorophyll fluorescence (relative fluorescence units, RFUs) on a Turner Designs Aquafluor handheld fluorometer (in vivo chlorophyll a channel). Briefly, each culture was mixed by swirling 3 times clockwise and then 3 times counterclockwise before aseptically removing a 2 mL subsample with a serological pipette. The subsample was transferred to a 10 mm × 10 mm methacrylate cuvette and placed in the Aquafluor for RFU measurement. A blank measurement was also taken daily using deionized water in place of a culture sample in the same type of cuvette. The RFU value obtained from the blank was subtracted from the RFU value of the culture for a “blank-corrected” RFU. Negative values for the blank were a result of instrument drift and were subtracted as described above. No RFUs in the dataset were missing or anomalous.

The exponential population growth rate for each G. huxleyi strain was calculated by measuring changes in blank-corrected RFUs (in vivo chlorophyll a) as a proxy for biomass over time during the exponential growth phase (minimally 4 days), using the formula µ = (ln(N2) - ln(N1))/t2-t1, where N1 is the in vivo chlorophyll a (RFU) at the start, N2 is the in vivo chlorophyll a (RFU) at the end, t1 is the first timepoint (day), and t2 is the final timepoint (day). RFUs were ln-transformed and fitted with a linear regression of ln(RFU) versus time (days). The regression slope over the most linear portion (where R2 > 98%) of the growth curve was taken as the best estimate of mean daily population growth rate.

The resulting datasets from applying this method included G. huxleyi strain growth conditions, G. huxleyi growth (biomass proxied by relative fluorescence units from in vivo chlorophyll a), and G. huxleyi exponential growth rates.

Relative fluorescence unit (RFU) values for each day and for each G. huxleyi strain were calculated by subtracting the blank RFU for that day from the RFU of the strain for that day. This yielded a “blank-corrected” RFU. Negative values for the blank were a result of instrument drift. The exponential population growth rate for each G. huxleyi strain was calculated from RFUs from the linear portion of the growth curve (minimally 4 days). RFUs were ln-transformed in Prism 11 (GraphPad) and fitted with a linear regression of ln(RFU) versus time (days). The regression slope over the most linear portion (where R2 > 98%) of the growth curve was taken as the best estimate of mean daily population growth rate.

NSF Award Abstract:
Emiliania huxleyi is a numerically and ecologically important phytoplankton species in the ocean known for its cosmopolitan distribution and ability to form large blooms in coastal and open ocean regions. Studies of E. huxleyi variants in culture have found differences in growth, function and activity potential among them. The E. huxleyi variants also differ in some of the genes they carry. It has been hypothesized that this genomic variability may underlie the global success of this phytoplankton species by allowing adaptation of variants to diverse environments. Yet, the direct connection between genomic content and ecological success remains unclear. This project investigates how the conserved and variable portions of E. huxleyi genome may be connected to its success and its dynamics under varied environmental conditions. This work is critical to our understanding of how this important phytoplankton species may shift and respond to future changes. This project also supports the development of a series of hands-on activities designed to teach middle school students advance computational data analysis in ocean science. These activities are in collaboration with the Girls Who Code Club at the Our Sisters School, a tuition-free, non-sectarian, independent school for girls from low-income families, located in New Bedford, MA.

Understanding how phytoplankton diversity and phenotype are driven by changes in the environment is crucial for better predicting carbon cycle dynamics in the future ocean. While much work has investigated competition among phytoplankton species, intraspecific diversity and dynamics remain largely unknown for many eukaryotic phytoplankton. The overarching goal of this project is to define the role of the pan genome (set of variable genes) in E. huxleyi ecology and biogeography through a series of genomic analyses, computational field surveys, and laboratory-based experiments. This project is sequencing the genomes of several E. huxleyi isolates from across the global ocean and combining them with existing genome sequences to constrain the core and variable portions of the pan genome. Using this new pan genome reference database and leveraging global scale metagenomics and metatranscriptomic surveys, this project is estimating ecotype diversity of E. huxleyi across ocean regions to identify patterns of environmental selection. This project additionally focuses on identifying the physiological and transcriptional responses of a selection of sequenced strains and their responses to shifts in their nutrient environments in controlled laboratory studies. As E. huxleyi plays such a significant role in marine ecosystems and the global carbon cycle it is important that its pan genome and its impact on the biogeography and ecology of E. huxleyi is taken into consideration. It is likely that these dynamics are acutely important to predicting how this genus, and perhaps others, respond to changing environmental conditions in the future ocean.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.