Table of Contents

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

Six independent lineages of each Ochromonas strain were grown in batch cultures in three light- and temperature-controlled incubators (18°C, 24°C, and 30°C), and regularly tested for several characteristics including growth rate, cell size, chlorophyll content, and photosynthetic efficiency. To show that characteristic changes are indeed from an evolutionary response as opposed to phenotypic plasticity, reciprocal transplant assays were conducted every three months for two years. This involved placing subsamples of each evolving lineage into all three temperatures, and comparing their performance in characteristic tests (growth rate, photosynthetic efficiency etc.). Evolved lineages performing equally at all "acclimation temperatures" is evidence of plasticity, while differences in performance indicates adaptation. Using growth rate and photosynthetic rate to compute the relative contributions of autotrophy versus heterotrophy for each evolved lineage.

Procedures for Photosynthesis-Irradiance curves:
Dark-acclimate cells to be sampled for at least 15 minutes. This operation requires the actinic light source (ALS). In the DOS prompt Type 'fview'. Press enter. The data acquisition program will open. Adjust the number of samples and PAR steps to 20. Adjust the max PAR to 1001. MTF and MTRP stay set to 0. Adjust the acclimation time between each sample to 15 seconds. Homogenize sample before reading. Insert your test tube with dark-acclimated sample. Press 's' (for 'start/stop'). Adjust the 'Gain:' so that the fluorescence trace is as close to, but not greater than, 100%.

Missing Data:
Occasionally, at high light levels and especially high temperatures, the signal to noise ratio of the fluorescence signal is very low. This causes noisy instrument output, which fails at the analysis (fprope.exe) stage. These data are missing from the dataset, and can be identified when a run (unique combination of week, light level, food level, strain, evolutionary temperature, acclimation temperature, and replicate) has fewer than twenty rows of data.

Data processing procedures:
fprope.exe, EGAVGA.bgi (graphic driver), DOSBox emulator

FIRe Analysis: To run the analysis program on a separate PC, copy the program (fprope.exe), together with the file EGAVGA.bgi (a graphic driver). The program fprope.exe must be stored in the same directory as the raw FIRe data files. Use DOSBox emulator to run fprope.exe.

Calculating ETR data from .RES file: Copy the .RES file into the ETR template excel sheet. This sheet has prepopulated equations for base FvFm, base Sigma, and ETR. The base FvFm equation is =IF(Par=0, Fv/Fm, base FvFm) The equation for base Sigma is =IF(par=0, Sigma, base sigma) The equation for ETR is =(+par * sigma * 2.04 * (FvFm/(FvFm)) * .00602). Export this completed excel sheet as a CSV file to be used in the R script.

BCO-DMO Processing:
- concatenated separate .csv files into one dataset;
- re-named fields to comply with BCO-DMO naming conventions (replaced special characters with underscores);
- converted date format to YYYY-MM-DD.

NSF Award Abstract:
Aquatic ecosystems host a wide variety of single-celled, microscopic organisms. Many of these species live near the surface of the water, where they grow and reproduce using different metabolic strategies that shape their place in the marine food web. For example, biologists have traditionally grouped planktonic microbes into either primary producers (which use photosynthesis to create new organic matter) or heterotrophs (which eat organic matter - such as the bodies - produced by other organisms). However, a large number of species are actually mixotrophic: they "mix" these two forms of metabolism by simultaneously conducting photosynthesis and eating smaller cells, including bacteria. Furthermore, many mixotrophs are metabolically flexible: they may rely more or less on each source of metabolism depending on environmental conditions. Because photosynthesis (which takes carbon out of the atmosphere and locks it into organic matter) and heterotrophy (which respires organic matter back into carbon dioxide) control whether or not oceanic food webs act as carbon sinks (having a net removal of carbon dioxide from the atmosphere), understanding mixotroph metabolism is critical to predicting the effects of marine plankton on atmospheric carbon. This project advances understanding of mixotroph metabolism by quantifying the extent to which mixotrophs can alter their reliance on photosynthesis over short and long timescales. The project tests how quickly mixotrophs can adapt to both warmer and colder water conditions, and how these adaptations alter their role in the carbon cycle. Researchers - including graduate students, a postdoctoral researcher, and undergraduate trainees - will measure the physiological responses of experimentally evolved mixotrophs and use mathematical models to connect these changes to global oceanic carbon cycling. As data are collected, they are shared with the public through outreach seminars, annual open house events, and weekly scientific presentations at the local Santa Barbara Museum of Natural History.

In order to predict biologically mediated feedbacks in the climate system, we must understand how marine plankton will respond to future ocean conditions. While a number of studies have sought to quantify the potential evolutionary response of phytoplankton, much less is known about the impacts of shifting conditions (e.g., increased temperature) on mixotrophs. What data are available suggest that mixotrophs may modulate a positive climate feedback loop: when warmed, mixotrophs become more heterotrophic, thus reducing their contribution to the biological pump and enhancing local respiration of organic carbon. Warming may also result in reductions in cell size, reducing sinking fluxes and carbon export from the upper ocean. Furthermore, because the predicted increase in oceanic stratification is expected to favor mixotrophs, their metabolic responses may be increasingly significant to understanding the global carbon cycle. The PI of this project is experimentally evolving mixotrophs under a range of temperature conditions in a fully factorial design that also manipulates the availability of light (photosynthesis) and prey (heterotrophy). She quantifies the carbon budget, grazing activity, nutrient content, and grazer palatability of evolved lineages in order to estimate the impact of any observed adaptations on carbon cycling. Specifically, the investigator asks how evolved lineages compare to ancestral lineages in their ability to tolerate altered thermal conditions, and connects differences in fitness to shifts in reliance on photosynthesis versus heterotrophy. Simultaneously, she incorporates a mixotrophy module into a global ocean biogeochemistry model, allowing the quantification of the impact of mixotrophs with either contemporary or evolved physiological traits. This work will provide some of the first known data on mixotroph plastic and evolutionary responses, and allow the scaling of these responses to their potential impacts on upper ocean biogeochemistry.

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.