Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Sample collection:
Four sites experiencing upwelling (E1: 41° 57.152 latitude, 124° 25.005 longitude. L2: 41° 42.078 latitude, 124° 30.865 longitude. L3: 43° 33.183 latitude, 124° 22.312 longitude. L4: 43° 59.988 latitude, 124° 41.28 longitude) were sampled off the coast of Northern California on May 30, 2019 (E1 and L2) and June 05, 2019 (L3 and L4) aboard the R/V Oceanus. Seawater was collected using a CTD-Rosette sampler, at four depths throughout the euphotic zone at each station corresponding to 50%, 30%, 10% and 1% incident irradiance (E1: 5.7 meters (m), 8.5m, 14.5m, 26.9m. L2: 5.7m, 9m, 16m, 27m. L3 and L4: 5m, 7m, 14m, 27m). At each depth, samples for chlorophyll a (Chl a), dissolved inorganic nutrients, dissolved inorganic carbon and nitrate uptake rates, cell counts and gene expression were obtained.

Physiological Measurements:
For chlorophyll a measurements, 250 milliliters (mL) of seawater was gravity filtered through 5 micrometer (μm) Isopore membrane filters (47 millimeters (mm)) and subsequently vacuum filtered onto GF/F (25 mm) filters under 100 mmHg of vacuum pressure. Filters were then rinsed with 0.45 μm filtered seawater and immediately stored at -20 degrees Celsius (°C) until onshore analysis in the lab. Chlorophyll a extraction was performed using a 90% acetone solution at -20 °C for 24 hours and measured on a 10-AU fluorometer (Turner Designs, San Jose, CA) using the acidification method.

Dissolved inorganic nutrients (nitrate + nitrite, phosphate and silicic acid) were measured by filtering 30 mL of water through a 0.2 µm filter, using acid-washed syringes into an acid-cleaned polypropylene FalconTM tube. Dissolved nutrient concentrations were analyzed using an OI Analytical Flow Solutions IV auto analyzer by Wetland Biogeochemistry Analytical Services at Louisiana State University. Concentrations of nitrate measured from the discrete samples were used for the calculation of absolute nitrate uptake rates.

Samples for dissolved iron (dFe) were collected from each cubitainer within a trace-metal clean, positive pressure plastic bubble by filtering through pre-cleaned 0.2 um pore size polyethersulfone membrane Acropak-200® capsule filters into LDPE bottles that had been rigorously cleaned as described in the GEOTRACES cookbook. Sample bottles were rinsed three times with sample before filling. Samples were acidified at sea to pH ~1.7 with optima HCl (2 mL of 12 M HCl per liter of seawater) and were analyzed after the cruise. Briefly, this method involves pre-concentration onto Nobias-chelate PA1 resin followed by analysis with a High Resolution Inductively Coupled Plasma Mass Spectrometer. For quality control, a few samples were rerun with a flow injection analysis method. This method involves pre-concentration on toyopearl resin followed by in-line spectrophotometric analysis.

Seawater from the rosette was collected into polycarbonate bottles (~618 mL) and immediately spiked with both NaH13CO3 and Na15NO3 at approximately 10% of the predicted ambient DIC concentrations and measured nitrate concentrations under a trace metal clean (TMC) flow hood located on the ship. Samples were then incubated for six hours, and vacuum filtered similarly to Chl a measurements, such that cells greater than or equal to 5 μm were collected onto 5 μm Isopore polycarbonate membrane filters (47 mm), with the flow through containing cells smaller than 5 μm which were collected onto 0.7 μm pre-combusted GF/F filters. The polycarbonate filters containing cells greater than 5 μm were then rinsed onto new 0.7 μm pre-combusted GF/F filters using 0.45 μm filtered-seawater. The filters were then preserved at -20 °C until further processing in the laboratory. Prior to analysis, filters were dried at 60 °C for 24 hours, encapsulated in tin, and pelletized. Particulate organic carbon (POC), particulate organic nitrogen (PON), and atom percentages of 13C and 15N were subsequently quantified using an isotope ratio mass spectrometer (EA-IRMS) at the UC Davis Stable Isotope Facility. For each sample, POC and PON concentrations (micromoles per liter (μmol L-1)) were calculated by dividing the measured POC/PON mass (micrograms (μg)) by the respective atomic mass of carbon and nitrogen over the volume filtered (0.5 liter (L)).

GraphPad Prism was used to plot both the environmental and physiological data. RStudio was used to generate a Principal Component Analysis for the environmental and physiological data in order to determine correlation and variability among the four upwelling stations.

- Imported original file "PUPCYCLE_compiled_results_Upwelling_transects.csv" into the BCO-DMO system.
- Renamed fields to comply with BCO-DMO naming conventions.
- Converted dates to YYYY-MM-DD format
- Saved the final file as "949801_v1_pupcycle_2019_upwelling_transects.csv".

NSF Award Abstract:
Upwelling zones are hotspots of photosynthesis that are very dynamic in space and time. Microsocopic algae, known as phytoplankton, bloom when deep, nutrient-rich waters are upwelled into sunlit surface layers of the ocean, providing nourishment that supports productive food webs and draws down carbon dioxide (CO2) from the atmosphere to the deep ocean. Photosynthetic microbes in these regions must constantly adapt to changes in their chemical and physical environments. For example, subsurface populations respond to changes in light as they approach the surface. When upwelled waters move offshore, cells sink out of the illuminated zone, establishing seed populations that remain inactive until the next upwelling event. This process is called the upwelling conveyor belt cycle (UCBC). How phytoplankton respond to these changes in environmental conditions and how they may influence their nutrient requirements remains unknown. With future ocean changes predicted to alter seawater chemistry, including ocean acidification and decreased iron availability, some phytoplankton groups may be more vulnerable than others. Accompanying educational activities provide learning experiences to enhance understanding and awareness of marine microbes. The development of a research hub at UNC aims to provide infrastructure and support for scientists and students conducting research on environmental genomics. A laboratory component for an upper-level undergraduate course focused on marine phytoplankton is being developed. Educational outreach activities to broader communities include creation of a lesson plan on phytoplankton in upwelling zones and a virtual research cruise experience for middle-school students, as well as a hands-on lab activity for a local museum focused on marine phytoplankton and the important roles they play in shaping our planet.

The project examines how phytoplankton respond at the molecular and physiological level to the different UCBC stages, which seed populations (i.e., surface versus subsurface) contribute most to phytoplankton blooms during upwelling events of varying intensity, how phytoplankton elemental compositions are altered throughout UCBC stages, and how future predicted ocean conditions will affect the phytoplankton responses to UCBC conditions. This project contains both laboratory and fieldwork. In the laboratory, phytoplankton isolates recently obtained from upwelling regions are exposed to simulated UCBC conditions to examine changes in gene expression, growth and photosynthetic characteristics and elemental composition. Cultures are subjected to both current and future ocean conditions, including reduced iron availability and higher CO2. In the field, research cruises within upwelling regions study the dynamics of natural phytoplankton communities (both surface and subsurface) experiencing upwelling and relaxation and within simulated upwelling incubation experiments. Knowledge of how phytoplankton are affected by UCBC conditions at an integrated molecular, physiological and elemental level under both current and future scenarios is imperative for the proper conservation and management of these critically important ecosystems.