Data from: Stochastic phenotypic switching arises in response to directional selection in experimentally evolved multicellular yeast.

This BBC_2025__README.txt file was generated on 2025-11-24 by Beatriz Baselga Cervera

GENERAL INFORMATION

1. Title of Dataset and code: Data from: Stochastic phenotypic switching arises in response to directional selection in experimentally evolved multicellular yeast.

2. Author Information

            Corresponding Investigator

                        Name: Ph.D. Beatriz Baselga-Cervera

                        Institution: University of Minnesota Twin cities, Minnesota, US.

                        Email: bbaselga@umn.edu; beabaselga@gmail.com

            Co-investigator 1

                        Name: Ph.D. Nahui Olin Medina-Chávez

                        Institution: University of Minnesota Twin cities, Minnesota, US.

                        Email: nmedinac@umn.edu

            Co-investigator 2

                        Name: Ph.D. Noah Gettle

                        Institution: Wellcome Sanger Institute, Hinxton, UK.

                        Email: nbgettle@gmail.com

Co-investigator 3

                        Name: Ph.D. Michael Travisano

                        Institution: University of Minnesota Twin cities, Minnesota, US.

                        Email: travisan@umn.edu

3. Data collectors: Ph.D. Beatriz Baselga-Cervera, Ph.D. Nahui Olin Medina-Chávez & Ph.D. Noah Gettle.

4. Date of data collection: 2022-2024

5. Geographic location of data collection: Saint Paul, US

6. Funding sources that supported the collection of the data: Fundación Alfonso Martín Escudero, Madrid, Spain (BBC).

7. Recommended citation for this dataset: Baselga-Cervera et al. (2024), Data from: Stochastic phenotypic switching arises in response to directional selection in experimentally evolved multicellular yeast.

DATA & FILE OVERVIEW

8. Description of dataset

In this study, we address whether stochastic phenotypic switching can shape biological diversity contributing to evolutionary change across the transition from singles cells to multicellular clutters in Saccharomyces cerevisiae multicellular yeast system. Populations characterization was conducted with a Coulter Counter multisize 4, a FlowCam 3, under the optic microscope, via ACE2 gene sequencing and RNA sequencing and mathematical modeling. The populations studied were the genetically uniform diploid wild-type Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockouts, and strains containing the missense mutation (ACE2 ^{c.1934 A>T}).

9. File list:

●      Coulter Counter size distribution data: 

o   File 1 name:  File_1_Coulter_Counter_Counts_20h.csv

o   File 1 description: Size distributions of Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockout, and strains containing the missense mutation (ACE2 ^{c.1934 A>T}) in YPD at 20-hours growth. Data for: Fig. 1A, Fig. 3A and Fig. S2, Table S1 and Table S2.

o   File 2 name:  File_2_Coulter_Counter_Counts_24h.csv

o   File 2 description: Size distributions of Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockout, and strains containing the missense mutation (ACE2 ^{c.1934 A>T}) in YPD at 24-hours growth. Data for: Fig. 1A, Fig. 3A, Fig. S1, Fig. S2, Table S1 and Table S2. 

o   File 3 name:  File_3_Coulter_Counter_Counts_48h.csv

o   File 3 description: Size distributions of Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockout, and strains containing the missense mutation (ACE2 ^{c.1934 A>T}) in YPD at 48-hours growth. Data for: Fig. 1, Fig. 3A, Fig. S2, Table S1 and Table S2.

o   File name:  File_4_Coulter_Counter_Counts_Constructed_strains_diversity.csv

o   File 4 description: Size distributions of the constructed ACE2 knockout and a strain containing the homozygous missense mutation (ACE2 ^{c.1934 A>T}) in YPD at 24h growth. Size distributions were obtained from populations before (initial) and five resuspended colonies obtained from small-size particles by plating the top fraction of the population after gravitational selection from three isolates per strain. Data for: Fig. S8.

o   File 5 name:  File_5_Coulter_Counter_Counts_Selection_Experiment.xlsx

o   File 5 description: Size distributions of C1W8.1 and C1W8.2 multicellular evolved strains in YPD at 24h growth. Size distributions from the selection experiment for small-size particles by plating the top fraction of the population after gravitational selection over three cycles of selection. Data for: Fig. 2B, Fig. S6 and Fig. S7. Table S4 and S5.

o   File 6 name:  File_6_Coulter_Counter_Counts_12h.xlsx

o   File 6 description: Size distributions of C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockout, and strains containing the missense mutation (ACE2 ^{c.1934 A>T}) in YPD at 12-hours growth. Data for: Fig. 3A and Fig. S3.

●      FlowCam data:

o   File 7 name: File_7_Rawdata_FlowCam_all.csv

o   File 7 description: FlowCam data from Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockouts, and strains containing the missense mutation (ACE2 c.1934 A>T) in YPD at 24h growth. Data for: Fig. 1B and Fig. S4. 

●      Data generated statistically:

o   File 8 name: File_8_C1W8.2_overlapPairs_Selection_Experiment.csv

o   File 8 description: overlapping indexes (η) of the KDE distributions were computed using the R-package ‘overlapping’ from the Coulter Counter data of the C1W8.2 derived strain over the selection experiment. Data for: Fig. S7D.

o   File 9 name: File_9_C1W8.1_overlapPairs_Selection_Experiment.csv

o   File 9 description: overlapping indexes (η) of the KDE distributions were computed using the R-package ‘overlapping’ from the Coulter Counter data

o   of the C1W8.1 derived strain over the selection experiment. Data for: Fig. S7C.

o   File 10 name: File_10_ overlapPairs_Constructed_strains_diversity.xlsx

o   File 10 description: overlapping indexes (η) of the KDE distributions were computed using the R-package ‘overlapping’ from the Coulter Counter data

of the constructed ACE2 knockout and a strain containing the homozygous missense mutation (ACE2 ^{c.1934 A>T}) in YPD at 24h growth. Size distributions were obtained from populations before (initial) and after gravitational selection of five resuspended colonies from three isolates per strain. Data for: Fig. S8B.

●      Data from ImageJ:

o   File 11 name: File_11_ImageJ_analyses.xlsx

o   File 11 description: ImageJ analyses of the microphotographs from Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockouts, and strains containing the missense mutation (ACE2 ^{c.1934 A>T}). Cultures were grown in culture tubes with 10 ml of media, 50 mL Erlenmeyer flasks with 10 mL and 30 mL of media, in YPD under non-shaking and shaking at 250 rpm. YPD media was used across all conditions. Cultures were assessed after 24 hours growth at 30°C.  Microphotographs of each condition and strain were obtained with a Nikon TE2000 microscope using 10x objective. Data for: Figure S5 and Table S3.

●      Pictures:

o   File 12 name: File_12_ FlowCam_Pictures.zip

o   File 12 description FlowCam IMAGES from Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockouts, and strains containing the missense mutation (ACE2 ^{c.1934 A>T}) in YPD at 24h growth. Images for: Fig. 1B.

o   File 13 name: File_13_Microphotography_controled_experimental_conditions.zip

o   File 13 description: Microphotographs from Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockouts, and strains containing the missense mutation (ACE2 ^{c.1934 A>T}). Cultures were grown in culture tubes with 10 mL of media, 50 mL Erlenmeyer flasks with 10 mL and 30 mL of media, in YPD under non-shaking and shaking at 250 rpm. YPD media was used across all conditions. Cultures were assessed after 24 hours of growth at 30°C. Pictures were obtained with a Nikon TE2000 microscope using 10x objective. Images for: Fig. 2A

●      Mathematical Model

o   File 14 name: File_14_Mathematical_model.zip

o   File 14 description: Mathematical model R code and generated values. Data for: Appendix A, Fig. 3D, and Fig. 3E.

●      ARN data

o   File 15 name: File_15_rnaseq-final-results-Top_v_Bottom.xlsx

o   File 15 description: RNA analyses final results Top vs Bottom phenotypic subdistributions. Top is used as control. Data for: Fig. 4, Fig. S9, S10, S11, and S12. 

o   File 16 name: File_16_Variant_Call_format_file.vcf

o   File 16 description: Variant Calling analyses of the sample ARN sample Top 1. Adhesion number: SRR32105384.

●      Time-lapse videos

o   File 17 name: Supp. Video 1. C1W8.1 from 17 to 22 hours growth

o   File 17 description: Supplementary Video 1. Experimentally evolved multicellular yeast video between 17 and 22 hours of growth (C1W8.1-derived strain) — time-lapse video of the formation of a single-cell propagule from a multicellular cluster. Images for: Fig. 3B.

o   File 18 name: Supp. Video 2. Ace2x2KO over 26 hours growth.

o   File 18 description: Supplementary Video 2. ace2Δ knockout constructed strain growth — time-lapse video of a single large multicellular cluster over 26 hours.

o   File 19 name: Supp. Video 3. C1W8.1 over 6 hours growth

o   File 19 description: Supplementary Video 3. Experimentally evolved multicellular yeast growth between 6 and 12 hours of growth (C1W8.1-derived strain).

o   File 20 name: Supp. Video 4. C1W8.1 over 24 hours growth

o   File 20 description: Supplementary Video 4. Experimentally evolved multicellular yeast growth over 24 hours (C1W8.1-derived strain) — cell division stops in small ancestral-like phenotypes.

o   File 21 name: Supp. Video 5. Ace2x2KO over 24 hours growth

o   File 21 description: Supplementary Video 5. ace2Δ knockout constructed strain growth — time-lapse video of multiple large multicellular clusters over 24 hours.

o   File 22 name: Supp. Video 6. Ace2x2missense from 0 to 3h45m hours growth

o   File 22 description: Supplementary Video 6. ace2Δ missense constructed strain growth — time-lapse video of multiple large multicellular clusters up to 3 hours 45 min.

METHODOLOGICAL INFORMATION

Strains: ancestral wildtype (Y55 strains), C1W8.1 and C1W8.2 multicellular derived strains isolated after 60 days of selection in YPD media, constructed ACE2 gene knockouts, and strains containing the ACE2 missense mutation (ACE2 ^{c.1934 A>T}).

Media: Growth media used in this study were Yeast Peptone Dextrose media (YPD; 1% (v/w) yeast extract, 2% (v/w) peptone, 2% (v/w) D-glucose, pH 5.8).

Phenotypic characterization of the different strains was conducted in a Coulter Counter Multisizer 4 and FlowCam® 3.0 Fluid Imaging Technologies, optic microscopy and a mathematical model. Replicate populations of different individual isolates per strain were analyzed to obtain the population distributions in YPD media.

RNA was extracted using an Invitrogen® PureLink RNA Mini Kit. Three out of four extracted samples per treatment with the highest RNA integrity score were submitted for TrueSeq Stranded RNA-Seq.

10. Detailed description

●      Coulter Counter size distribution data of all the populations: 

o   File 1 name:  File_1_Coulter_Counter_Counts_20h.csv

o   File 1 description: strains naming convention; strain_Isolate_run.pseudoreplicate. Strains: ace2x2m=strains containing the ACE2 missense mutation (ACE2 c.1934 A>T); ace2x2= ACE2 knockout; C1W8.1= C1W8.1 evolved multicellular strain; C1W8.2= C1W8.2 evolved multicellular strain; Y55= ancestral strain.

§  Page 1:

Column 1: Volume (um3)

Column 2: Diameter (um2)

Columns 3 to the last column: strains counts.

o   File 2 name:  File_2_Coulter_Counter_Counts_24h.csv

o   File 2 description: strains naming convention; strain_Isolate_run.pseudoreplicate. Strains: ace2x2m=strains containing the ACE2 missense mutation (ACE2 c.1934 A>T); ace2x2= ACE2 knockout; C1W8.1= C1W8.1 evolved multicellular strain; C1W8.2= C1W8.2 evolved multicellular strain; Y55= ancestral strain.

§  Page 1:

Column 1: Volume (um3)

Column 2: Diameter (um2)

Columns 3 to the last column: strains counts.

o   File 3 name:  File_3_Coulter_Counter_Counts_48h.csv

o   File 3 description: strains naming convention; strain_Isolate_run.pseudoreplicate. Strains: ace2x2m=strains containing the ACE2 missense mutation (ACE2 c.1934 A>T); ace2x2= ACE2 knockout; C1W8.1= C1W8.1 evolved multicellular strain; C1W8.2= C1W8.2 evolved multicellular strain; Y55= ancestral strain.

§  Page 1:

Column 1: Volume (um3)

Column 2: Diameter (um2)

Column 3 to the last column: strains counts.

o   File_4_Coulter_Counter_Counts_Constructed_strains_diversity.csv

o   File 4 description: strains naming convention; strain_Isolate_colony_run.pseudoreplicate. Strains: ace2x2m=strains containing the ACE2 missense mutation (ACE2 c.1934 A>T); ace2x2= ACE2 knockout.

§  Page 1:

Column 1: Volume (um3)

Column 2: Diameter (um2)

Column 3 to the last column: strains counts.

o   File_5_Coulter_Counter_Counts_Selection_Experiment.xlsx

o   File 5 description: strains naming convention; strain_colony.phenotype_selection.cycle_run.pseudoreplicate. Strains: C1W8.2= C1W8.2 evolved multicellular strain and C1W8.1= C1W8.1 evolved multicellular strain.

§  Page 1:

Column 1: Volume (um3)

Column 2: Diameter (um2)

Column 3 to the last column: strains counts.

o   File 6 name:  File_6_Coulter_Counter_Counts_12h.xlsx

o   File 6 description: Size distributions of C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockout, and strains containing the missense mutation (ACE2 ^{c.1934 A>T}) in YPD at 12-hours growth. Data for: Fig. 3A and Fig. S3.

§  Page 1:

Column 1: Volume (um3)

Column 2: Diameter (um2)

Column 3: Time

Column 4: replicate

Column 5: Strain name (strain_f)

Column 6: Isolate (isolate_f)

o   File 7 name: File_3_Rawdata_Flowcam_all.csv

o   File 7 description: strains naming convention; ace2_isolate= ACE2 knockout;

Ace2m_isolate= strain containing the ACE2 missense mutation (ACE2 c.1934 A>T); c1w82_isolate=C1W8.2 evolved multicellular strain; C1W81_isoalte C1W8.1 evolved multicellular strain; Y55_isolate=ancestral strain.

§  Page 1:

                                                                        Column 1: Particle ID

                                                                        Column 2: Area ABD

                                                                        Column 3: Aspect Ratio (Width/Length)

                                                                        Column 4: Circle Fit

                                                                        Column 5: Area base Diameter (ABD)

                                                                        Column 6: Equivalent Spherical Diameter (ESD)

                                                                        Column 7: Elongation

                                                                        Column 8: Perimeter

                                                                        Column 9: Roughness

                        Column 10: Volume ABD-based

                                                                        Column 11: Volume ESD-based

                                                                        Column 12: Width

                                                                        Column 13: Source. Name of the sample.

o   File 8 name: File_8_C1W8.2_overlapPairs_Selection_Experiment.csv

o   File 8 description: C1W8.2 _lineage_selection.cycle= C1W8.2 evolved multicellular strain, lineage (A=ancestral, M1= lineage 1,  M2= lineage 2 , M3= lineage 3) and selection cycle  (0, 1, 2 and 3).

§  Page 1:

Column 1: Var1= strain 1

Column 2: Var2= strain 2

Column 3: overlap value of both strains compared.

o   File 9 name: File_9_C1W8.1_overlapPairs_Selection_Experiment.csv

o   File 9 description: C1W8.1 _lineage_selection.cycle =C1W8.1 evolved multicellular strain, lineage (A=ancestral, M1= lineage 1,  M2= lineage 2 , M3= lineage 3) and selection cycle  (0, 1, 2 and 3).

§  Page 1:

Column 1: Var1= strain 1

Column 2: Var2= strain 2

Column 3: overlap value of both strains compared.

o   File 10 name: File_10_overlapPairs_Constructed_strains_diversity.xlsx

o   File 10 description: variables naming convention; strain _isolate_colony.number. Strains; ace2x2m=strains containing the ACE2 missense mutation (ACE2 ^{c.1934 A>T}); ace2x2= ACE2 knockout. Isolate; 1,2 and 3. Colony.number; Initial=initial population and colony number (1,2,3,4 and 5).

§  Page 1:

Column 1: Var1= strain 1

Column 2: Var2= strain 2

Column 3: overlap value of both strains compared.

o   File 11 name: File_11_ ImageJ _analyses.xlsx

o   File 11 description: ImageJ analyses of the microphotographs from Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockouts, and strains containing the missense mutation (ACE2 ^{c.1934 A>T}). Cultures were grown in culture tubes with 10 mL of media, 50 mL Erlenmeyer flasks with 10 mL and 30 mL of media, in YPD under non-shaking and shaking at 250 rpm. YPD media was used across all conditions. Cultures were assessed after 24 hours growth at 30°C.  Microphotographs of each condition and strain were obtained with a Nikon TE2000 microscope using 10x objective.

§  Page 1:

Column 1: Var1= strain 1

Column 2: Var2 = strain 2

Column 3: overlap value of both strains compared.

o   File 12 name: File_12_ FlowCam_Pictures.zip

o   File 12 description: FlowCam runs, images, and raw data of Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockouts, and strains containing the missense mutation (ACE2 ^{c.1934 A>T}) in YPD at 24h growth. Data for: Fig. 1B.

o   File 13 name: File_13_Microphotography_controled_experimental_conditions.zip

o   File 13 description: 149 microphotographs.

§ Folder 1:  Images of Erlenmeyer flasks with 30ml of YPD

§ Folder 2:  Images of Erlenmeyer’s and tubes with 10ml of YPD

o   File 14 name: File_14_Mathematical_model.zip

o   File 14 description: Mathematical model, R code, and generated values.

§  Document 1:  R code of the model

§  Document 2:  Resulted data from the mathematical model with different inset values of k, alpha, and beta.

§  Document 2:  Resulted data from the mathematical model with different inset values of k, alpha, gamma, and beta.

o   File 15 name: File_15_rnaseq-final-results-Top_v_Bottom.xlsx

o   File 15 description:

§  Page 1:

Column 1: number

Column 2: ID

Column 3: protID

            Column 4: gene_symbol        

            Column 5: chr

            Column 6: chr_latin

Column 7: location

Column 8: baseMean

            Column 9: log2FoldChange

Column 10: lfcSE

Column 11: stat

            Column 12: pvalue     padj

Column 13: test

Column 14: log10padj

            Column 15: log10baseMean

Column 16: blast_pident

Column 17: transcript_length

            Column 18: blast_evalue

Column 19: blast_bitscore

Column 20: rnaID

            Column 21: feature

Column 22: accession

Column 23: strain

            Column 24: gene_accession

o   File 16 name: File_16_Variant_Call_format_file.vcf

o   File 16 description: Variant Calling analyses of the  ARN sample Top 1. Adhesion number: SRR32105384.

o   File 17 name: Supp. Video 1. C1W8.1 from 17 to 22 hours growth

o   File 17 description: Supplementary Video 1. Experimentally evolved multicellular yeast video between 17 and 22 hours of growth (C1W8.1-derived strain) — time-lapse video of the formation of a single-cell propagule from a multicellular cluster. The time-lapse video captures growth dynamics over this period, highlighting the formation of a single-cell propagule from a multicellular cluster on two occasions (visible in the lower left region of the frame). Images were acquired every 15 minutes using a 10x objective lens. 

o   File 18 name: Supp. Video 2. Ace2x2KO over 26 hours of growth.

o   File 18 description: Supplementary Video 2. ace2Δ knockout constructed strain growth — time-lapse video of a single large multicellular cluster over 26 hours. The video captures large, multicellular clusters that produce both large, multicellular and small, ancestral-like clusters. The video shows a single large multicellular cluster fragmenting into two large multicellular clusters at ~ 13 hours of growth (from 02:09 to 02:10 minutes in the time-lapse) and generating two small ancestral-like propagules at ~19 hours of growth (from 03:07 to 03:09 minutes in the time-lapse). Microphotographs were obtained at 3-minute intervals under a 10x objective over 26 hours.  

o   File 19 name: Supp. Video 3. C1W8.1 over 6 hours growth

o   File 19 description: Supplementary Video 3. Experimentally evolved multicellular yeast growth between 6 and 12 hours of growth (C1W8.1-derived strain). The time-lapse video captures large, multicellular clusters of the C1W8.1 strains, which produce both large, multicellular and small, ancestral-like clusters. Additionally, small ancestral-like clusters are observed undergoing cellular division —no separation is observed— during the first 2 to 3 hours, followed by a cessation of division for the remainder of the time-lapse. Images were acquired every 30 seconds using a 10x objective lens.

o   File 20 name: Supp. Video 4. C1W8.1 over 24 hours growth

o   File 20 description: Supplementary Video 4. Experimentally evolved multicellular yeast growth over 24 hours (C1W8.1-derived strain) — cell division stops in small ancestral-like phenotypes. The footage captures multiple large multicellular clusters undergoing fragmentation into propagules. Additionally, a small ancestral-like cluster is observed undergoing division during the first 2 to 3 hours, followed by a cessation of division for the remainder of the time-lapse (visible in the lower left region of the frame). This early division phase is evident during the first 10 seconds of the video. Images were acquired every 5 minutes using a 10x objective lens. 

o   File 21 name: Supp. Video 5. Ace2x2KO over 24 hours growth

o   File 21 description: Supplementary Video 5. ace2Δ knockout constructed strain growth — time-lapse video of multiple large multicellular clusters over 24 hours. The video shows multiple large multicellular clusters fragmenting into large clusters and several small ancestral-like clusters being dragged by Brownian motion and evaporation of the sample. Microphotographs were obtained at fixed intervals of 3 minutes under the 10x objective over 24 hours. 

o   File 22 name: Supp. Video 6. Ace2x2missense from 0 to 3h45m hours growth

o   File 22 description: Supplementary Video 6. ace2Δ missense constructed strain growth — time-lapse video of multiple large multicellular clusters up to 3 hours 45 min. The video shows multiple large multicellular clusters fragmenting into large clusters, generating two small ancestral-like propagules before being dragged by Brownian motion and evaporation of the sample. Microphotographs were obtained at 3-minute intervals under the 10x objective.