DOI: 10.5061/dryad.ns1rn8q1h
title: Data from: Plasticity and the evolution of group-level regulation of cellular differentiation in the volvocine algae

description: During the evolution of multicellularity, the unit of selection
 transitions from single cells to integrated multicellular cell groups,
 necessitating the evolution of group-level traits such as somatic
 differentiation. However, the processes involved in this change in units
 of selection are poorly understood. We propose that the evolution of soma
 in the volvocine algae included an intermediate step involving the plastic
 development of somatic-like cells. We show that Eudorina elegans, a
 multicellular volvocine algae species previously thought to be
 undifferentiated, can develop somatic-like cells following environmental
 stress (i.e., cold shock). These cells resemble obligate soma in closely
 related species. We find that somatic-like cells can differentiate
 directly from cold-shocked cells. This differentiation is a cell-level
 trait, and the differentiated colony phenotype is a cross-level byproduct
 of cell-level processes. The offspring of cold-shocked colonies also
 develop somatic-like cells. Since these cells were not directly exposed to
 the stressor, their differentiation was regulated during group
 development. Consequently, they are a true group-level trait and not a
 byproduct of cell-level traits. We argue that group-level traits such as
 obligate somatic differentiation can originate through plasticity and that
 cross-level byproducts may be an intermediate step in the evolution of
 group-level traits.
description: <em>Induction of somatic-like cells over multiple
 generations</em> In order to determine whether
 somatic-like cells develop in response to cold shock, we inoculated a 250
 mL flask of Standard Volvox Media (SVM) with <em>E. elegans
 </em>UTEX 1201 and grew the cultures on a shaker at 25°C with a 16:8
 hour light:dark cycle. After 5 days, we transferred 0.5 mL of the culture
 to a new 250 mL flask with fresh SVM and allowed the culture to grow for
 10 days. We then placed 1 mL of the culture with young unexpanded colonies
 in six Eppendorf tubes. Three of the tubes were cold-shocked in a covered
 ice bath at 1° C for two hours while the other three control tubes were
 placed in the dark at 25° C for two hours. The contents of each tube were
 then transferred to fresh media in separate wells of a 6 well
 plate.  To assess the development of somatic cells over
 multiple generations following cold shock, we imaged the cold-shocked
 colonies (which we refer to as generation 1; G1), their offspring
 (generation 2; G2), and their grand-offspring (generation 3; G3) (Figure
 1F). We did the same for three generations of controls. Wells were
 examined daily for nine days to determine whether colonies were
 reproducing and to track their offspring. The density of each well was
 measured at the time of imaging by mixing the culture and then counting
 the number of colonies contained within 50 µL. During this experiment, we
 recorded the number of somatic-like cells each colony had. We imaged
 approximately 100 expanded adults (Figure 1E and Figure 2) from the cold
 treatment and 100 expanded adult controls from each generation. Each well
 was imaged for a single generation and treatment. When
 imaging, we mixed the content of each well before randomly withdrawing
 colonies and placing them on a grid slid. We then systematically imaged
 all expanded adults on the slide using a Nikon Eclipse Ti-E inverted
 microscope. We used both brightfield and fluorescence microscopy to image
 colonies. For each expanded adult colony, we characterized each cell by
 size and by the presence/absence of chlorophyll autofluorescence.
 Chlorophyll autofluorescence was used as a proxy to determine whether the
 cell was likely to be alive. The basic experimental setup is visually
 summarized in Supplementary Material S1.
 <em>Assessment of the heritability of somatic-like
 cells </em> In order to specifically determine
 when somatic-like cells develop following cold shock and whether the
 phenotype is heritable, we grew two flasks on a shaker in the conditions
 described in the previous section. From these cultures, we inoculated two
 new flasks at a density of 5 colonies per mL and measured the culture
 density every other day. After 10 days, 48 colonies at the same
 developmental stage (unexpanded juveniles; as represented in Figure 1E and
 Figure 2) were haphazardly chosen from the two flasks and imaged to
 confirm they did not have visible somatic-like cells. Each of the 48
 colonies were placed in separate Eppendorf tubes with 40 µL of SVM. We
 placed 24 of the Eppendorf tubes in an enclosed, dark ice bath at 1°C for
 two hours and the other 24 in a dark container at 25°C. After two hours,
 the contents of each Eppendorf tube were transferred into fresh media in
 separate wells of two 24 well plates. We imaged all G1 colonies before
 they divided and gave rise to G2 offspring colonies. After they hatched,
 we transferred individual G2 colonies to separate wells in 24 well plates
 and imaged them. We imaged G3 colonies from the wells they hatched out in.
 Imaging was conducted with a Nikon Eclipse Ti-E (Nikon, Tokyo, Japan). A
 summary of the three generations is shown in Figure 1F and Figure
 2. Our final sample sizes were affected by colony
 survival, successful transfer to or from wells, and the number of
 reproductive cells (and therefore number of offspring) present in
 generations 1 and 2. We collected data from 12 cold-shocked G1 colonies,
 19 control G1 colonies, 72 cold-shocked G2 colonies, 199 control G2
 colonies, 661 cold-shocked G3 colonies, and 1267 control G3
 colonies. Each time we imaged a colony, we recorded the
 number of large (reproductive) and small (somatic-like) cells. Since we
 tracked the differentiation state of each colony and that of their
 offspring, we were able to characterize the heritability of the
 facultatively differentiated phenotype. Specifically, we determined if
 facultatively differentiated colonies had a higher proportion of
 differentiated offspring than undifferentiated colonies. The data reported
 here are the result of two independent experiments completed several
 months apart. <em>Separation of single
 cells</em> To determine whether somatic-like
 cells can develop in the absence of interactions among cells, we grew
 colonies following the protocol described above. We separated two single
 cells from 20 unexpanded undifferentiated colonies using needles to break
 apart the colonies and placed each cell into a separate Eppendorf tube,
 along with 40 µL of SVM. Twenty tubes, each containing 1 cell from the 20
 colonies, were placed in an ice bath for 2 hours. The other 20 tubes were
 placed in the dark at room temperature for 2 hours and used as controls.
 Each of the 40 cells was placed on a separate 1% agar plate (in SVM) to
 prevent cell-cell communication and ensure that cells could be clearly
 seen and tracked. Four to six days after the treatment, each plate was
 imaged using a Nikon Eclipse Ti-E inverted microscope. The experiment was
 replicated with an additional 40 cells from 20 unexpanded
 colonies.   <em>Cell size
 measurements</em> To determine if somatic-like
 cells are significantly smaller than reproductive cells and the same size
 as somatic cells in other species, we measured reproductive and
 somatic-like cells in mature <em>E. elegans </em>UTEX 1201
 colonies as well as reproductive and somatic cells in <em>Pleodorina
 starrii </em>NIES 1362<em> </em>and the somatic cells in
 <em>Volvox carteri </em>Eve<em>. </em>We
 inoculated two 250 mL flasks with <em>P. starrii</em> and
 <em>V. carteri</em> on a shaker for five days in the
 conditions described above. We repeatedly withdrew 10 µL of media from the
 flask and imaged the expanded adult colonies on a slide using a Nikon
 Eclipse Ti-E inverted microscope (Nikon, Tokyo, Japan).
 To measure the sizes of somatic-like cells and reproductive cells
 from <em>E. elegans </em>UTEX 1201 expanded adult G1 and G2
 colonies, we followed the same protocol described in the sub-section
 “Induction of somatic-like cells over multiple generations.” We then
 measured cell sizes using ImageJ (31). We traced the perimeter of each
 cell in a colony using the circle tool in ImageJ, calibrated our
 measurements using the scale bar from each image, and obtained the minimum
 diameter, maximum diameter, and area of each cell.
 <em>Assessment of reproductive
 potential</em> Somatic cells in obligately
 differentiated species are terminally differentiated and do not give rise
 to the next generation. To determine if somatic-like cells reproduce, we
 set up experimental well plates using the protocol described above. After
 the offspring of cold-shocked G1 colonies hatched, the G1 shells composed
 of extracellular matrix and somatic-like cells were transferred to new
 wells. The wells that contained shells were monitored for five days to
 determine if any of the somatic-like cells went on to reproduce. The same
 procedure was followed for the G2 colonies. To address the possibility
 that cold treatment and culture conditions could inhibit reproduction,
 twelve differentiated cold-shocked colonies were transferred to separate
 wells and after reproduction, their offspring remained in the wells.
 Additionally, 12 control colonies were transferred to separate wells and
 their offspring remained in the wells after reproduction. We examined
 these wells after 5 days and recorded whether viable colonies were still
 present to ensure that the growth conditions didn’t inhibit
 reproduction.
description: # Plasticity and the evolution of group-level regulation of cellular
 differentiation in the volvocine algae
 [https://doi.org/10.5061/dryad.ns1rn8q1h](https://doi.org/10.5061/dryad.ns1rn8q1h) #### Description of the data and file structure The datasets and code associated with the manuscript "Plasticity and the evolution of group-level regulation of cellular differentiation in the volvocine algae" are deposited here. There are five datasets associated with this manuscript: 1. Supplementary_data_S1.csv provides the data used to examine the number of plastic somatic-like cells per colony for replicates of two generations in cold-shocked and control conditions in flasks.  2. Supplementary_data_S2.csv provides the data used to follow three generations of cold-shocked and control colonies and to assess the inheritence of the differentiated phenotype. 3. Supplementary_data_S3.csv provides the data used to examine the number of living plastic somatic-like cells per colony for three generations in cold-shocked and control conditions in well plates. 4. Supplementary_data_S4.csv provides the data used to examine the sizes of obligate somatic cells in *Volvox* and *Pleodorina* as well as the sizes of generation 1 and generation plastic somatic-like cells in *Eudorina*. 5. Supplementary data S5.zip provides images of fixed generation 1 and 2 cold-shocked and control *Eudorina* colonies. These images were taken as the data for Supplementary_data_S1.csv was entered into the spreadsheet. 6. Supplementary_data_S6.csv provides the data used to examine cell fate when cells were removed from the colony. 7. Supplementary_data_S7.csv is a manually subsetted version of Supplementary_data_S2.csv. It contains generation 3 control data and was used to generate Figure 2B. 8. Supplementary_data_S8.csv is a manually subsetted version of Supplementary_data_S2.csv. It contains generation 3 cold data and was used to generate Figure 2B. Seven of the datasets are .csv files and can be most easily viewed in Excel. Five of the datasets are the result of a different experiment and is used in separate analyses. Datasets S7 and S8 are manually subsetted from S2 for ease of analysis. The datasets have been cleaned up in that columns with redundant or extraneous information have been removed and the columns have been renamed for clarity. The column names are descriptive. The .zip file contains 291 image files that correspond to the csv dataset S1. Each image file contains at least one adult colony. #### Sharing/Access information The data described here was used to generate the results of *Proceedings of the Royal Society B *paper, "Plasticity and the evolution of group-level regulation of cellular differentiation in the volvocine algae." The data support the conclusions of the this manuscript. ## Code/Software Python and R were used to generate figures and perform analyses. The packages involved are stated in each file. Python was used to generate Figure 2. Individual python files that were used to create each panel of Figure 2 are named Code_SomaProportions_Figure2A.py, Code_Inheritance_Figure2B.py, Code_SomaNumbers_Figure2C.py, and Code_SingleCells_Figure2D.py. Python was used to analyze the effects of cold shock on the development of somatic-like cells; this code is named Code_G1G2Analysis_Table1.py, Code_FishersExactTest_G1G2.py, and Code_WilcoxonRankSumtest_G1G2.py. R was used to generate panels G and H of Figure 4; this code is named Figure4GH_code_v2.txt. R was used to conduct all other statistical analyses. The file containing the code used to perform those analyses is present in Inheritance and cell sizes R code.txt. ### Files and variables #### File: Supplementary\_data\_S1.csv **Description:** The dataset used to examine the number of somatic-like cells per colony for two generations of cold-shocked and control treatments in flasks. ##### Variables * Species: The species used the experiment. All data in this file was collected from Eudorina elegans UTEX 1201. * Replicate: This denotes which replicate (A, B, or C) each colony belonged to for each generation and treatment. * Treatment: The colony's treatment; either Cold shock or Control. * Generation: The generation of each colony. 1 = Generation 1, 2 = Generation 2, and 3 = Generation 3. * Colony number: The number assigned to a colony for each generation and treatment; used to keep track of when I collected data for 50 adult colonies. * Number of soma: The number of somatic cells per colony. * Colony size: The approximate total number of cells per colony (4, 8, 16, or 32). * HasSomatic: Whether or not the colony has somatic-like cells. 1 = somatic-like cells are present; 0 = somatic-like cells are absent. #### File: Supplementary\_data\_S2.csv **Description:** The data used to track the reproduction of three generations of cold-shocked and control colonies and to assess the inheritance of the differentiated phenotype. ##### Variables * Trial number: Which replicate trial the colony is from; either 1 (the first trial) or 2 (the second trial). * Treatment: Which treatment a given colony received; either Cold or Control. * Colony ID: The unique identifier for that colony. * Parent's ID: The unique identifier for that colony's parent. Cells for all colonies from generation 1 are entered as NA, since their parents weren't tracked. * Grandparent's ID: The unique identifier for that colony's grandparent. Cells for all colonies from generations 1 and 2 are entered as NA, since their grandparents weren't tracked. * Generation: The generation of the colony. * Total number of cells: How many cells the colony has. * Number of reproductive cells: How many reproductive cells the colony has. * Number of somatic-like cells: How many somatic cells the colony has. #### File: Supplementary\_data\_S3.csv **Description:** The data used to examine the number of living somatic-like cells per colony for three generation in cold-shocked and control colonies. This experiment was conducted in a six-well plate. ##### Variables * Generation: The generation of each colony. 1 = Generation 1, 2 = Generation 2, and 3 = Generation 3. * Treatment: The colony's treatment; either Cold or Control. * Total number of cells (living and dead): The total number of cells present in the colony. * Number of living somatic-like cells: The total number of living somatic-like cells present in the colony. * Total number of dead cells: The total number of dead cells present in the colony. * Number of dead somatic-like cells: The total number of dead somatic-like cells present in the colony. #### File: Supplementary\_data\_S4.csv **Description:** The data used to examine the cell sizes in obligately differentiated* Volvox carteri* Eve,* Pleodorina californica* NIES 1362, and* Eudorina elegans* UTEX 1201. ##### Variables * Species: The species each cell belongs to; either *Volvox carteri* Eve,* Pleodorina californica* NIES 1362, and* Eudorina elegans* UTEX 1201. * Cell area: The area of that cell. * Major diameter (um): The largest diameter (in um) * Minor diameter (um): The smallest diameter (in um) * Average diameter: The average diameter (in um) * Cell type: The type of cell; either somatic or reproductive. #### File: Supplementary\_data\_S5.zip **Description:** Images of fixed generation 1 and generation cold-shocked and control colonies. These images were taken at the same time data was entered into the Supplementary_data_S1.csv file. #### File: Supplementary\_data\_S6.csv **Description:** The data used to examine cell fate after cells were removed from colonies, cold-shocked or kept as controls, and transferred to individual plates. ##### Variables * Plate: The identifier of the plate each cell was on. * Treatment: The treatment each cell experienced. * Single cell or multicellular: Whether a single cell (single) or a colony (colony) was observed. * Cell Fate: The fate of each cell. It was either Somatic-like, a Colony, or Reproductive (and most likely dead) #### File: Supplementary\_data\_S7.csv **Description:** A manually subsetted version of Supplementary_Data_S2.csv (subsetted for ease of analysis). It contains generation 3 control data. It is included because it's used to generate Figure 2B. ##### Variables * Treatment: The treatment applied to the grandparents of each colony. All colonies are from the control (con) treatment. * Colony ID: The unique identifier for that colony. * Parent: The unique identifier for that colony's parent. * Grandparent: The unique identifier for that colony's grandparent. * Generation: The generation of the colony. * Total number of cells: How many cells the colony has. * Number of reproductive cells: How many reproductive cells the colony has. * Number of somatic-like cells: How many somatic cells the colony has. * SLC: Whether or not the colony has somatic-like cells. 1 = somatic-like cells are present; 0 = somatic-like cells are absent. * Parent_SLC: Whether or not the colony's parent had somatic-like cells. 1 = somatic-like cells were present; 0 = somatic-like cells were absent. #### File: Supplementary\_data\_S8.csv **Description:** A manually subsetted version of Supplementary_Data_S2.csv (subsetted for ease of analysis). It contains generation 3 cold-shocked data. It is included because it's used to generate Figure 2B. ##### Variables * Treatment: The treatment applied to the grandparents of each colony. All colonies are from the cold treatment. * Colony ID: The unique identifier for that colony. * Parent: The unique identifier for that colony's parent. * Grandparent: The unique identifier for that colony's grandparent. * Generation: The generation of the colony. * Total number of cells: How many cells the colony has. * Number of reproductive cells: How many reproductive cells the colony has. * Number of somatic-like cells: How many somatic cells the colony has. * SLC: Whether or not the colony has somatic-like cells. 1 = somatic-like cells are present; 0 = somatic-like cells are absent. * Parent_SLC: Whether or not the colony's parent had somatic-like cells. 1 = somatic-like cells were present; 0 = somatic-like cells were absent. ## Code/software The R files and python files were used to generate the data for figures and conduct statistical analyses. They all specify which datasets and packages to import.  The following four python files were used to generate figure 2: *Code_SomaProportions_Figure2A.py: Used to generate panel A of Figure 2. *Code_Inheritance_Figure2B.py: Used to generate panel B of Figure 2. *Code_SomaNumbers_Figure2C.py: Used to generate panel C of Figure 2. *Code_SomaNumbers_Figure2D.py: Used to generate panel D of Figure 2. The following three python files were used to analyze data on the development of somatic-like cells across generations. *Code_G1G2Analysis_Table1.py: Used to generate the results in Table 1. *Code_FishersExactTest_G1G2.py: Used to perform Fishers exact test on generations 1 and 2. Code_WilcoxonRankSumtest_G1G2: Used to perform Wilcoxon rank sum test on generations 1 and 2. The following .txt files were used to generate Figure 4 and perform additional analyses in R. *Figure4GH_code_v2.txt: Used to generate panels G and H of Figure 4 in R. *Inheritance and cell sizes R code.txt: Used to conduct additional analyses, including cell size analyses, in R.