Dataset: The Long Term Phytoplankton Evolution Experiment: Culture Analysis

Name: Data and code from an examination of growth rates of cyanobacteria co-cultured with a heterotrophic bacterium, Alteromonas, under either present-day or predicted future pCO2 conditions
Published: 2024-04-24
Keywords: Experimental Evolution, phytoplankton, ocean acidification, Heterotrophic Bacteria, oceans

Cite This Dataset

DOI:10.26008/1912/bco-dmo.925841.1

Laboratories at the University of Alabama at Birmingham

Temporal Extent: 2013-08-01 - 2024-01-31

Project:

Collaborative Research: Ecology and Evolution of Microbial Interactions in a Changing Ocean (LTPE)

Impacts of Evolution on the Response of Phytoplankton Populations to Rising CO2 (P-ExpEv)

Program:

Science, Engineering and Education for Sustainability NSF-Wide Investment (SEES): Ocean Acidification (formerly CRI-OA) (SEES-OA)

Principal Investigator:

James Jeffrey Morris (University of Alabama at Birmingham, UA/Birmingham)

Scientist:

Elizabeth Entwistle (University of Alabama at Birmingham, UA/Birmingham)

Zhiying Lu (University of Alabama at Birmingham, UA/Birmingham)

Student:

Matthew Kuhl (University of Alabama at Birmingham, UA/Birmingham)

Technician:

Alexander Durrant (University of Alabama at Birmingham, UA/Birmingham)

BCO-DMO Data Manager:

Shannon Rauch (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

Version Date:

2024-04-24

Restricted:

Validated:

Yes

Current State:

Final no updates expected

Data and code from an examination of growth rates of cyanobacteria co-cultured with a heterotrophic bacterium, Alteromonas, under either present-day or predicted future pCO2 conditions

Abstract:

The CO2 content of Earth's atmosphere is rapidly increasing due to human consumption of fossil fuels. Models based on short-term culture experiments predict that major changes will occur in marine phytoplankton communities in the future ocean, but these models rarely consider how the evolutionary potential of phytoplankton or interactions within marine microbial communities may influence these changes. Here we experimentally evolved representatives of four phytoplankton functional types (silicifiers, calcifiers, coastal cyanobacteria, and oligotrophic cyanobacteria) in co-culture with a heterotrophic bacterium, Alteromonas, under either present-day or predicted future pCO2 conditions. The data and analysis code in this dataset show that the growth rates of cyanobacteria generally increased under both conditions, and the growth defects observed in ancestral Prochlorococcus cultures at elevated pCO2 and in axenic culture were diminished after evolution. Evolved Alteromonas were also poorer "helpers" for Prochlorococcus, supporting the assertion that the interaction between Prochlorococcus and heterotrophic bacteria is not a true mutualism but rather a competitive interaction stabilized by Black Queen processes. This work provides new insights on how phytoplankton will respond to anthropogenic change and on the evolutionary mechanisms governing the structure and function of marine microbial communities.

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Archival Copy

Version Date	Archive	Persistent Identifier	Date Assigned
2024-04-24	Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)	10.26008/1912/bco-dmo.925841.1	2024-05-06

Methods & Sampling

Detailed methods can be found in the manuscript "Marine phytoplankton and heterotrophic bacteria rapidly adapt to future pCO2 conditions in experimental co-cultures". A summary of major methods are provided here.

The phytoplankton used in this study as well as the media in which they were grown are Prochlorococcus MIT9312 (PEv medium), Synechococcus CC9311 (SEv medium), Synechocystis PCC6803 (SEv medium), Thalassiosira oceanica CCMP1005 (FEv medium), and Emiliania huxleyi CCMP371 (FEv medium). All media types were derived from media commonly used to cultivate each organism detailed in "Algal Culturing Techniques" edited by Andersen. Prior to use in experiments, phytoplankton cultures were rendered clonal and axenic, then mixed with pure cultures of the heterotrophic bacterium Alteromonas EZ55, obtained by streaking for isolation on YTSS agar.

Cultures were experimentally evolved for approximately 500 generations at 22 degrees Celisius (°C) under approximately 75 micromoles photons per square meter per second (µmol photons m-2 s-1) in acid-washed conical-bottom glass tubes with airtight caps where the carbonate system was manipulated by the addition of acid or base to achieve present-day (400 parts per million (ppm)) or year 2100 (800 ppm) pCO2 conditions. Phytoplankton growth was measured every 48 hours using a Guava HT1 flow cytometer equipped with a 488 nanometer (nm) laser. When phytoplankton cell densities crossed a cutoff value, cultures were diluted 26-fold into fresh media, representing log₂26 or 4.7 generations per transfer. Samples from each lineage were cryopreserved every 25 generations and again at the end of the experiment.

At the end of the evolution period, we subcultured clonal evolved Alteromonas strains by spread-plating evolved cultures on YTSS agar and selecting single, isolated colonies for growth in YTSS broth. Prochlorococcus was rendered axenic by the addition of 100 micrograms per milliliter (µg mL-1) streptomycin. All growth experiments were initiated by mixing axenic Prochlorococcus with a specific Alteromonas clone (or else remaining axenic) and acclimating the co-culture for 3 transfer cycles (approximately 14 generations) at the target pCO2 concentration. Growth was then monitored by flow cytometry as described above for at least 3 subsequent transfers under constant conditions.

Data Processing Description

The impact of experimental treatments on growth parameters was statistically analyzed using linear models in R with post-hoc statistical testing using extended marginal means with the emmeans package. Malthusian and exponential growth rates were calculated as described in our previous work (Hennon et al. 2017, ISME Journal). Because these experiments involved thousands of measurements collected over several years, a variety of clearly erroneous data points were recorded that led to several outlier growth rates that had a disproportionate impact on model output; rather than attempt to manually curate all growth rates, we simply removed either the most extreme 5% high and low Malthusian growth rates or eliminated exponential growth rates with r2 values lower than 0.95 for each strain in each experiment before conducting statistical tests.

Data Files

File
FinalGR.csv (Comma Separated Values (.csv), 86.07 KB) MD5:3722d33f5be206e3ecb1875464a3ffe6 Contains growth rates for experiments done at the end of the experiment comparing the ancestral and evolve phytoplankton organisms. Each row represents a single transfer cycle as in the above description. These data were used to generate main text Figure 2 Lu, et al. (2024). Column headings are: Strain: which phytoplankton strain was measured -- Prochlorococcus MIT9312, Synechococcus CC9311, Thalassiosira oceanica CCMP1005, or Emiliania huxleyi CCMP371. EvolTreat: Whether the culture represents an ancestor (Anc), 400 ppm pCO2 evolved population (CO2-), or 800 ppm pCO2 evolved population (CO2+) AssayTreat: Whether the culture was assessed at 400 ppm (CO2-) or 800 ppm (CO2+) pCO2 t: the number of days between culture inoculation and final reading N0: initial cell density Nt: final cell density EGR: exponential growth rate, calculated as described in the Methods rsq: r-squared value of the regression used to calculate the EGR; cultures with r-squared values less than 0.95 were removed from the analysis MGR: Malthusian growth rate, calculated as described in the METHODS
LTPE_RCode_Cultures.R (R Script, 20.39 KB) MD5:452bf22e737ea02e4bc246607a339ec2 Contains the code necessary to run the statistical analyses described in the text (Lu, et al., 2024).
LTPE_Transfers.csv (Comma Separated Values (.csv), 424.63 KB) MD5:6a1f34b9c3019fedcb8e09a66bef937c Contains the day-by-day flow cytometry data collected during the evolution phase of the Long Term Phytoplankton Evolution experiment that forms the basis of the manuscript. Each row represents the results of a single transfer cycle of a single evolving lineage; i.e., the beginning and ending cell densities, dates, and so forth. These data were used to generate main text Figure 1 Lu, et al. (2024). Column heading explanations are: Strain: which phytoplankton strain was measured -- Prochlorococcus MIT9312, Synechococcus CC9311, Synechocystis PCC6803, Thalassiosira oceanica CCMP1005, or Emiliania huxleyi CCMP371. Replicate: which replicate lineage each measured sample was taken from. Each lineage has a unique designation. Treatment: whether the lineage was grown at 400 ppm (CO2-) or 800 ppm (CO2+) pCO2. Method: how the phytoplankton biomass was measured. Most samples were assessed for cell density using a Guava flow cytometer, but some early samples were assessed by in vivo chlorophyll-a fluorescence with a Turner Designs Trilogy fluorometer. Restart: Rarely, a lineage would crash or become contaminated and would have to be restarted. Measurements applying to the first culture transfer after a restart and indicated here. Cultures that would eventually crash are not depicted in main text Figure 1. Transfer: the sequential number of each transfer, representing the evolutionary distance between the culture and its ancestor. Each transfer represents log(2) 26 = 4.7 generations. T0: date of culture inoculation Tend: date of culture transfer deltaT: days elapsed between T0 and Tend InitDens: initial cell density (or chl-a fluorescence) FinalDens: final cell density (or chl-a fluorescence) MGR: Malthusian growth rate, calculated as described in the Methods Notes: special notes pertaining to certain transfers corresponding to reasons for breaks in transfer number or dates (e.g., crash, contamination, cryopreservation prior to moving labs)
MixnMatch.csv (Comma Separated Values (.csv), 53.83 KB) MD5:70be38910633d2f667f28ed4b2982a0d Contains the data from experiments where ancestral and evolved Prochlorococcus were grown with different strains of Alteromonas EZ55 (or axenically). These data were used to generate main text Figure 4B of Lu, et al. (2024). Column headings are as follows: LTPE: LTPE strain designation of the Prochlorococcus strain in the experiment. Pro: Whether the Prochlorococcus strain was ancestral (Anc), evolved at 400 ppm pCO2 (Evo400), or evolved at 800 ppm pCO2 (Evo800) Het: Whether the culture was axenic (X), grown in co-culture with ancestral Alteromonas EZ55 (A), or with an Alteromonas strain evolved at the pCO2 condition the culture was grown at in co-culture with Prochlorococcus (P), Thalassiosira oceanica (TO), or Emiliania huxleyi (EH) t: Elapsed time between culture inoculation and final measurement IniDens: Initial cell density of the culture FinDens: Final cell density of the culture EGR: Exponential growth rate as calculated in the Methods R^2: r-squared value of the regression used to calculate the EGR; cultures with r-squared values less than 0.95 were removed from the analysis MGR: Malthusian growth rate as calculated in the Methods
Viability.csv (Comma Separated Values (.csv), 40.41 KB) MD5:65590822089f426801c26281aeea48be Contains the data from experiments measuring the impact of different EZ55 strains or axenic culture on mortality of Prochlorococcus strains. These data were used to make Figure 4A Lu, et al. (2024). Column headings are as follows: LTPE: LTPE Strain designation of the Prochlorococcus strain in the experiment. Pro: Whether the Prochlorococcus strain was ancestral (Anc), evolved at 400 ppm pCO2 (400ppm), or evolved at 800 ppm pCO2 (800ppm) Het: Whether the culture was axenic (X), grown in co-culture with ancestral Alteromonas EZ55 (A), or with an Alteromonas strain evolved at the pCO2 condition the culture was grown at (E) CO2: The pCO2 condition under which the culture was grown ViaRes: Whether or not the culture survived (s) or failed (f). Failure was considered to occur if the culture did not attain the cutoff cell density for transfer of 2,600,000 cells per milliliter within 30 days.

Supplemental Files

File
ReadMe.cultures.txt (Plain Text, 5.61 KB) MD5:b9399712fe6524cca81b140a60ced517 Explanation of the files in this dataset, how the data files are organized, and how to run the code to replicate the analyses we used in our manuscript (Lu, et al., 2024).

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-1316101
NSF Division of Ocean Sciences	OCE-1540158
NSF Division of Ocean Sciences	OCE-1851085

Deployments

None available yet

Instruments

Algal Growth Chamber

Supplied Name: Percival algal growth chamber

Supplied Description:

Instrument Type

Generic Name: Algal Growth Chamber

Generic Description:

A chamber specifically designed for the growth of algae in flasks. The chamber typically provides controlled temperature, humidity, and light conditions.

Flow Cytometer

Supplied Name: Guava HT1 flow cytometer with 488nm laser

Supplied Description:

Instrument Type

Generic Name: Flow Cytometer

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB37/

Generic Description:

Flow cytometers (FC or FCM) are automated instruments that quantitate properties of single cells, one cell at a time. They can measure cell size, cell granularity, the amounts of cell components such as total DNA, newly synthesized DNA, gene expression as the amount messenger RNA for a particular gene, amounts of specific surface receptors, amounts of intracellular proteins, or transient signalling events in living cells.
(from: http://www.bio.umass.edu/micro/immunology/facs542/facswhat.htm)

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Dataset: The Long Term Phytoplankton Evolution Experiment: Culture Analysis