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This BBC_2024__README.txt file was generated on 2024-07-15 by Beatriz Baselga Cervera GENERAL INFORMATION 1. Title of Dataset and code: Data from: Phenotypic variation across the first steps of experimentally evolved multicellularity. 2. Author Information Corresponding Investigator Name: Dr Beatriz Baselga-Cervera Institution: University of Minnesota Twin cities, Minnesota, US. Email: bbaselga@umn.edu; beabaselga@gmail.com Co-investigator 1 Name: Dr Noah Gettle Institution: Wellcome Sanger Institute, Hinxton, UK Email: noah.gettle@sanger.ac.uk Co-investigator 2 Name: Dr Michael Travisano Institution: University of Minnesota Twin cities, Minnesota, US. Email:travisan@umn.edu 4.Data collector: Dr Beatriz Baselga-Cervera 5. Date of data collection: 2021-2022 6. Geographic location of data collection: Saint Paul, US 5. Funding sources that supported the collection of the data: Fundación Alfonso Martín Escudero, Madrid, Spain. 6. Recommended citation for this dataset: Baselga-Cervera et al. (2024), Data from: Phenotypic variation across the first steps of experimentally evolved multicellularity. Zenodo. Data set. DATA & FILE OVERVIEW 1. Description of dataset In this study, we study the genotype to phenotype map that provides the basis for the diverse morphologies and unique adaptations observed in nascent multicellularity carried over from their unicellular ancestors under high and low nutrient availability conditions. We carried out a NetLogo agent-based model simulation of the multicellular snowflake yeast growth. Populations characterization was conducted with a Coulter Counter multisize 4 and FlowCam 3. 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). The population data represent the six independent isolates per strain in two different media, YPD and SD. 2. File list: NetLogo Agent Base model: File 1 name: Netlogo Snowflake 3D.nlogo3d File 1 Description: Netlogo code for the 3D model/ File name: Data_NETLOGO_model.csv File 2 Description: Data obtained from the Netlogo model. Coulter Counter size distribution data of all the populations: File 3 name: Counter_Counter_Raw_data.xls File 3 description: 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 and SD at 24h growth. File 4 name: Coulter_Counter_Raw_data_heterozygous_contructions File 4 description: Size distributions of the heterozygous construct knockout (ACE2/ace2Δ) and homozygous missense (ACE2/ace2Δ) in YPD and SD at 24h growth. FlowCam image data: File 5 name: FlowCamPicturesData.zip File 5 description: Pictures generated by the FlowCam. File 6 name: Flow_Cam_Raw data.xls File 6 desciption: 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 and SD at 24h growth. Data generated statistically: File 7 name: bootstrapped_means.csv File 7 description: bootstrapped means from the Coulter Counter data to calculate the relative contributions to phenotypic variation. File 8 name: bootstrapped_vars.csv File 8 description: bootstrapped variance from the Coulter Counter data to calculate the phenotypic noise. File 9 name: overlapPairs_values.xlsx File 9 description: overlapping indexes (η) of the KDE distributions were computed using the R-package ‘overlapping’ from the Coulter Counter data. R codes for data generated statistically: File 10 name: R_code_bootstrapping.docx File 10 description: R code to obtain bootstrapped means and variance from the Counter_Counter_Raw_data.xls data to calculate the relative contributions to phenotypic variation. File 11 name: R_code_Overlap_KDE_distributions_from _the_Coulter_Counter_data File 11 description: R code to obtain overlapping indexes (η) of the Kernel density estimations (KDE) distributions from Counter_Counter_Raw_data.xls R codes for figures: File 12 name: R_code_Fig2.docx File 12 description: R code for Figure 2 panels B and C. Panels are created from Data_NETLOGO_model.csv. File 13 name: R_code_Fig3.docx File 13 description: R code for Figure 3 panels A to E. Panels are created from Counter_Counter_Raw_data.xls File 13 name: R_code_Fig4.docx File 13 description: R code for Figure 4, data from Counter_Counter_Raw_data.xls File 14 name: R_code_FigS1.docx File 14 description: R code for Figure S1. Raw data from Data_NETLOGO_model.csv File 15 name: R_code_FigS2.docx File 15 description: R code for Figure S2. Raw data from Counter_Counter_Raw_data.xls File 16 name: R_code_FigS3.docx File 16 description: R code for Figure S3. Raw data from Coulter_Counter_Raw_data_heterozygous_contructions.xlsx File 16 name: R_code_FigS4.docx File 16 description: R code for Figure S4. Raw data from Counter_Counter_Raw_data.xls 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) and Standard minimal (SD; Yeast nitrogen base with amino acids (YNB w/AA) 6.7 g L-1, 0.5% (v/w) D-glucose). Phenotypic characterization of the different strains was conducted in a Coulter Counter Multisizer 4 and FlowCam® 3.0 Fluid Imaging Technologies. Replicate populations of different individual isolates per strain were analyzed to obtain the population distributions in both YPD and SD media. Agent Base Model data was generated in Netlogo (https://ccl.northwestern.edu/netlogo/). 3. Detailed description NetLogo Agent Base model: File 1 name: Netlogo Snowflake 3D.nlogo3d o File 1 Description: Netlogo code for the 3D model. o File name: Data_NETLOGO_model.csv o File 2 Description: Data obtained from the Netlogo model. § Page 1: Agent Base Model data generated with Netlogo. Column 1: run number Column 2: size-of-turtle(cells) Column 3: [step] Column 4: N Column 5: ticks(generations) Column 6:distance-turtle(cells) Column 7: diameter cells Column 8: radius cells Column 9: diameter cluster Column 10: radius cluster Column 11: volume Column 12: SAVr (surface area/volume ratio) Column 13: packing Column 14: Ratio Coulter Counter size distribution data of all the populations: File 3 name: Counter_Counter_Raw_data.xls File 3 description: 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 and SD at 24h growth. § Page 1: Coulter Coulter data runs at 24h Column 1: Volumen (um3) Column 2: Diameter (um2) Column 3: ace2Δ _k_1_SD. Homozygous ACE2 knockout construct (ace2Δ /ace2Δ) isolate one is SD media. Column 4: ace2Δ _k_1_YPD. Homozygous ACE2 knockout construct (ace2Δ /ace2Δ) isolate one is YPD media. Column 5: ace2Δ _k_2_SD. Homozygous ACE2 knockout construct (ace2Δ /ace2Δ) isolate two is SD media. Column 6: ace2Δ _k_2_YPD. Homozygous ACE2 knockout construct (ace2Δ /ace2Δ) isolate two is YPD media. Column 7: ace2Δ _k_3_SD. Homozygous ACE2 knockout construct (ace2Δ /ace2Δ) isolate three is SD media. Column 8: ace2Δ _k_3_YPD. Homozygous ACE2 knockout construct (ace2Δ /ace2Δ) isolate three is YPD media. Column 9: ace2Δ _k_4_SD. Homozygous ACE2 knockout construct (ace2Δ /ace2Δ) isolate four is SD media. Column 10: ace2Δ _k_4_YPD. Homozygous ACE2 knockout construct (ace2Δ /ace2Δ) isolate four is YPD media. Column 11: ace2Δ _k_5_SD. Homozygous ACE2 knockout construct (ace2Δ /ace2Δ) isolate five is SD media. Column 12: ace2Δ _k_5_YPD. Homozygous ACE2 knockout construct (ace2Δ /ace2Δ) isolate one is YPD media. Column 13: ace2Δ _k_6_SD. Homozygous ACE2 knockout construct (ace2Δ /ace2Δ) isolate five is SD media. Column 14: ace2Δ _k_6_YPD. Homozygous ACE2 knockout construct (ace2Δ /ace2Δ) isolate five is YPD media. Column 15: ace2Δ _m_1_SD. Homozygous ACE2 missense construct (ace2Δ /ace2Δ) isolate one is SD media. Column 16: ace2Δ _m_1_YPD. Homozygous ACE2 knockout construct (ace2Δ /ace2Δ) isolate one is YPD media. Column 17: ace2Δ _m_2_SD. Homozygous ACE2 missense construct (ace2Δ /ace2Δ) isolate two is SD media. Column 18: ace2Δ _m_2_YPD. Homozygous ACE2 missense construct (ace2Δ /ace2Δ) isolate two is YPD media. Column 19: ace2Δ _m_3_SD. Homozygous ACE2 missense construct (ace2Δ /ace2Δ) isolate three is SD media. Column 20: ace2Δ _m_3_YPD. Homozygous ACE2 missense construct (ace2Δ /ace2Δ) isolate three is YPD media. Column 21: ace2Δ _m_4_SD. Homozygous ACE2 missense construct (ace2Δ /ace2Δ) isolate four is SD media. Column 22: ace2Δ _m_4_YPD. Homozygous ACE2 missense construct (ace2Δ /ace2Δ) isolate four is YPD media. Column 23: ace2Δ _m_5_SD. Homozygous ACE2 missense construct (ace2Δ /ace2Δ) isolate five is SD media. Column 24: ace2Δ _m_5_YPD. Homozygous ACE2 missense construct (ace2Δ /ace2Δ) isolate one is YPD media. Column 25: ace2Δ _m_6_SD. Homozygous ACE2 missense construct (ace2Δ /ace2Δ) isolate five is SD media. Column 26: ace2Δ _m_6_YPD. Homozygous ACE2 missense construct (ace2Δ /ace2Δ) isolate five is YPD media. Column 27: C1W8.1 _m_1_SD. C1W8.1 evolved strain isolate one is SD media. Column 28: C1W8.1 _m_1_YPD. C1W8.1 evolved strain isolate one is YPD media. Column 29: C1W8.1 _m_2_SD. C1W8.1 evolved strain isolate two is SD media. Column 30: C1W8.1 _m_2_YPD. C1W8.1 evolved strain isolate two is YPD media. Column 31: C1W8.1 _m_3_SD. C1W8.1 evolved strain isolate three is SD media. Column 32: C1W8.1 _m_3_YPD. C1W8.1 evolved strain isolate three is YPD media. Column 33: C1W8.1 _m_4_SD. C1W8.1 evolved strain isolate four is SD media. Column 34: C1W8.1 _m_4_YPD. C1W8.1 evolved strain isolate four is YPD media. Column 35: C1W8.1 _m_5_SD. C1W8.1 evolved strain isolate five is SD media. Column 36: C1W8.1 _m_5_YPD. C1W8.1 evolved strain isolate five is YPD media. Column 37: C1W8.1 _m_6_SD. C1W8.1 evolved strain isolate six is SD media. Column 38: C1W8.1 _m_6_YPD. C1W8.1 evolved strain isolate sic is YPD media. Column 39: C1W8.2 _m_1_SD. C1W8.2 evolved strain isolate one is SD media. Column 40: C1W8.2 _m_1_YPD. C1W8.2 evolved strain isolate one is YPD media. Column 41: C1W8.2 _m_2_SD. C1W8.2 evolved strain isolate two is SD media. Column 42: C1W8.2 _m_2_YPD. C1W8.2 evolved strain isolate two is YPD media. Column 43: C1W8.2 _m_3_SD. C1W8.2 evolved strain isolate three is SD media. Column 44: C1W8.2 _m_3_YPD. C1W8.2 evolved strain isolate three is YPD media. Column 45: C1W8.2 _m_4_SD. C1W8.2 evolved strain isolate four is SD media. Column 46: C1W8.2 _m_4_YPD. C1W8.2 evolved strain isolate four is YPD media. Column 47: C1W8.2 _m_5_SD. C1W8.2 evolved strain isolate five is SD media. Column 48: C1W8.2 _m_5_YPD. C1W8.2 evolved strain isolate five is YPD media. Column 49: C1W8.2 _m_6_SD. C1W8.2 evolved strain isolate six is SD media. Column 50: C1W8.2 _m_6_YPD. C1W8.2 evolved strain isolate sic is YPD media. Column 51: Y55_1_SD. Y55 ancestral strain isolate one is SD media. Column 52: Y55_1_SD. Y55 ancestral strain isolate one is YPD media. Column 53: Y55_2_SD. Y55 ancestral strain isolate two is SD media. Column 54 Y55_2_SD. Y55 ancestral strain isolate two is YPD media. Column 55: Y55_3_SD. Y55 ancestral strain isolate three is SD media. Column 56: Y55_3_SD. Y55 ancestral strain isolate three is YPD media. Column 57: Y55_4_SD. Y55 ancestral strain isolate four is SD media. Column 58: Y55_4_SD. Y55 ancestral strain isolate four is YPD media. Column 59: Y55_5_SD. Y55 ancestral strain isolate five is SD media. Column 60: Y55_5_SD. Y55 ancestral strain isolate five is YPD media. Column 61: Y55_6_SD. Y55 ancestral strain isolate six is SD media. Column 62: Y55_6_SD. Y55 ancestral strain isolate sic is YPD media. o File 4 name: Coulter_Counter_Raw_data_heterozygous_contructions File 4 description: Size distributions of the heterozygote construct knockout (ACE2/ace2Δ) and heterozygous missense construct (ACE2/ace2Δ) in YPD and SD at 24h growth. § Page 1: Coulter Coulter data runs at 24h Column 1: Volumen (um3) Column 2: Diameter (um2) Column 3: ACE2/ace2Δ missense_1_SD. Heterozygous missense construct (ACE2/ace2Δ) isolate one is SD media. Column 4: ACE2/ace2Δ missense_1_YPD. Heterozygous missense construct (ACE2/ace2Δ) isolate one is YPD media. Column 5: ACE2/ace2Δ missense_2_SD. Heterozygous missense construct (ACE2/ace2Δ) isolate two is SD media. Column 6: ACE2/ace2Δ missense_2_YPD. Heterozygous missense construct (ACE2/ace2Δ) isolate two is YPD media. Column 7: ACE2/ace2Δ missense_3_SD. Heterozygous missense construct (ACE2/ace2Δ) isolate three is SD media. Column 8: ACE2/ace2Δ missense_3_YPD. Heterozygous missense construct (ACE2/ace2Δ) isolate three is YPD media. Column 9: ACE2/ace2Δ knockout_1_SD. Heterozygous knockout construct (ACE2/ace2Δ) isolate one is SD media. Column 10: ACE2/ace2Δ knockout _1_YPD. Heterozygous knockout construct (ACE2/ace2Δ) isolate one is YPD media. Column 11: ACE2/ace2Δ knockout _2_SD. Heterozygous knockout construct (ACE2/ace2Δ) isolate two is SD media. Column 12: ACE2/ace2Δ knockout _2_YPD. Heterozygous knockout construct (ACE2/ace2Δ) isolate two is YPD media. Column 13: ACE2/ace2Δ knockout _3_SD. Heterozygous knockout construct (ACE2/ace2Δ) isolate three is SD media. Column 14: ACE2/ace2Δ knockout _3_YPD. Heterozygous knockout construct (ACE2/ace2Δ) isolate three is YPD media. FlowCam image data: File 5 name: FlowCamPicturesData.zip File 5 description: Pictures generated by the FlowCam. File 6 name: Flow_Cam_Raw data.xls File 6 desciption: 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 and SD at 24h growth. § Page 1: FlowCam data runs at 24 hours of Y55 strain. Column 1:Particle ID Column 2: Class. Classification as; single cells and mother-daughter(s). Column 3: Area ABD Column 4: Aspect Ratio (Width/Length) Column 5: Circle Fit Column 6: Area base Diameter (ABD) Column 7: Equivalent Spherical Diameter (ESD) Column 8: Elongation Column 9: Perimeter Column 10: Roughness Column 11: Volume ESD-based Column 12: Width Column 13: Source. Name of the sample. Column 14: Strain Y55. Column 15: media. Values: YPD and SD Column 16: clone. Isolate. § Page 2: FlowCam data runs at 24 hours of C1W8.1 multicellular strain. 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 ESD-based Column 11: Width Column 12: Source. Name of the sample. Column 13: Strain C1W8.1. Column 14: media. Values: YPD and SD Column 15: clone. Isolate. § Page 3: FlowCam data runs at 24hours of C1W8.2 multicellular strain. 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 ESD-based Column 11: Width Column 12: Source. Name of the sample. Column 13: Strain C1W8.2. Column 14: media. Values: YPD and SD Column 15: clone. Isolate. Data generated statistically: File 7 name: bootstrapped_means.csv File 7 description: bootstrapped means from the Coulter Counter data to calculate the relative contributions to phenotypic variation. § Page 1: Bootstrap mean values. Column 1: row number Column 2: sample. ID of the sample from whom the value was generated. Column 3: diameter_um. Mean diameter (um) values. File 8 name: bootstrapped_vars.csv File 8 description: bootstrapped variance from the Coulter Counter data to calculate the phenotypic noise. § Page 1: Bootstrap variance values. Column 1: row number Column 2: sample. ID of the sample from whom the value was generated. Column 3: diameter_um. Variance in diameter (um) values. File 9 name: overlapPairs_values.xlsx File 9 description: overlapping indexes (η) of the KDE distributions were computed using the R-package ‘overlapping’ from the Coulter Counter data. § Page 1: Overlapping indexes (η) of the KDE distributions by strain, media and isolate. Column 1: var1. Population 1. Column 2: var2. Population 2. Column 3: value. Overlap value. § Page 2: Overlapping indexes (η) of the KDE distributions by strain and media. Column 1: var1. Strain 1. Column 2: var2. Strain 2. Column 3: value. Overlap value. Column 4: media.

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