DOI: 10.5061/dryad.w9ghx3g2c
title: Constraints in temperature adaptation reinforce differences in thermal niche between mesophilic and psychrotolerant Bacillus cereus group species

description: Experimental evolution has demonstrated that mesophilic microbes readily
 adapt to increases in temperature. However, many microbes are
 psychrotolerant and resistant to cold, which is associated with
 physiological specializations, suggesting constraints in thermal
 adaptation. We hypothesized that constraints would limit adaption
 differently in a mesophilic species (B. thuringiensis) compared to its
 psychrotolerant relative B. mycoides – with adaptation at cooler
 temperatures and adaptation at higher temperatures being constrained in
 each species respectively. To test this hypothesis, we imposed 140
 generations of selection at temperatures at and below the optimum for
 productivity for both species, conducting competition fitness assays,
 thermal profiling via thermal performance curves and SNP variant analysis
 to determine the changes that occurred during experimental
 evolution.  The data and code enclosed is that used to create
 each figure in the paper and the raw data generated in this study; the
 only exception is the genetic sequencing data, which is available in NCBI
 via the Bioproject PRJNA826440. The fitness and thermal performance of
 evolved bacteria showed ancestral thermal niche plays a role in thermal
 adaptation over this timescale, in support of our hypothesis of adaptive
 constraints. Temperature-dependent trade-offs appeared common
 in B. mycoides, with fitness gains associated with decreases in
 operational niche width; fitness gains at one temperature caused a
 decrease in the range of temperatures that the bacterium showed
 appreciable growth. Genome resequencing showed that variation in mutation
 supply and selection strength could not explain temperature-dependent
 responses to selection. Importantly, metabolic theory only held true for
 mesophilic B. thuringiensis, showing abundant but less studied
 psychrotolerant species could follow different adaptive
 trajectories. 
description: # Constraints in temperature adaptation reinforce differences in thermal
 niche between mesophilic and psychrotolerant Bacillus cereus group species
 Access this dataset on Dryad DOI:10.5061/dryad.w9ghx3g2c ## Introduction
 Experimental evolution has demonstrated that mesophilic microbes readily
 adapt to increases in temperature. However, many microbes are
 psychrotolerant and resistant to cold, which is associated with
 physiological specializations, suggesting constraints in thermal
 adaptation. We hypothesized that constraints would limit adaption
 differently in a mesophilic species (B. thuringiensis) compared to its
 psychrotolerant relative B. mycoides – with adaptation at cooler
 temperatures and adaptation at higher temperatures being constrained in
 each species respectively. To test this hypothesis, we imposed 140
 generations of selection at temperatures at and below the optimum for
 productivity for both species. The fitness and thermal performance of
 evolved bacteria showed ancestral thermal niche plays a role in thermal
 adaptation over this timescale, in support of our hypothesis of adaptive
 constraints. Temperature-dependent trade-offs appeared common in B.
 mycoides, with fitness gains associated with decreases in operational
 niche width; fitness gains at one temperature caused a decrease in the
 range of temperatures that the bacterium showed appreciable growth. Genome
 resequencing showed that variation in mutation supply and selection
 strength could not explain temperature-dependent responses to selection.
 Importantly, metabolic theory only held true for mesophilic B.
 thuringiensis, showing abundant but less studied psychrotolerant species
 could follow different adaptive trajectories. The dataset contains data
 and methods to reproduce the results of White et al., as detailed in their
 manuscript "Constraints in temperature adaptation reinforce
 differences in thermal niche between mesophilic and psychrotolerant
 Bacillus cereus group species". We hypothesized that constraints
 would limit adaption differently in a mesophilic species (B.
 thuringiensis) compared to its psychrotolerant relative B. mycoides – with
 adaptation at cooler temperatures and adaptation at higher temperatures
 being constrained in each species respectively. We conducted experimental
 evolution using a mesophilic Bacillus thuringiensis strain and a
 psychrotolerant Bacillus mycoides which both belong to the Bacillus cereus
 group. After ~140 generations of adaptation to temperatures close to and
 far below thermal optimum, we used a three-step analytical approach.
 Competition assays were used to test for fitness changes via direct
 competition with the strain’s ancestor. SNP variant calling was used to
 identify genetic changes that emerged during experimental evolution. ##
 Description of the data and file structure Cell_size_checks.zip; Contains
 the raw image files collected to determine the cell size of each
 strain/lineage. Cell size of strains was determined by suspending cells in
 saline prior to staining (incubation at 4°C in dark) with BacLight
 bacterial viability and counting staining kit. Images were acquired using
 a confocal fluorescence microscope (Leica AF6000) set up as follows:
 objective ×63 plan apochromatic; SYTO9 excitation wavelength 488 nm,
 emission wavelength 493-547 nm; propidium iodide excitation wavelength
 552 nm, emission wavelength 567-725 nm. Analysis of images used Leica
 software (LAS X version 3.5.1.18803) to measure cells over five
 independent fields of view per strain. Dimensions were recorded as
 pole-equator lengths in μm for a total of 30 cells/strain in focus and
 planarity. Cell volume was calculated assuming an ellipsoid form (cell vol
 = 4⁄3 π × ρ/2× ϵ ) where ρ is the polar long axis and ϵ is the equatorial
 short axis. Image files are arranged in series, each containing fifteen
 files with the numbering system Series001-060. Identities for lineage to
 number is as follows; * In folder brhwcellsize_Series 1.zip
 brhwcellsize_Series001.tif to brhwcellsize_Series005.tif; Bacillus
 mycoides ancestor brhwcellsize_Series006.tif to
 brhwcellsize_Series010.tif; Bacillus mycoides, lineage 1 in 30°C selection
 regime brhwcellsize_Series011.tif to brhwcellsize_Series015.tif; Bacillus
 mycoides, lineage 2 in 30°C selection regime * In folder
 brhwcellsize_Series 2.zip brhwcellsize_Series016.tif to
 brhwcellsize_Series020.tif; Bacillus mycoides, lineage 3 in 30°C selection
 regime brhwcellsize_Series021.tif to brhwcellsize_Series025.tif; Bacillus
 mycoides, lineage 4 in 30°C selection regime brhwcellsize_Series026.tif to
 brhwcellsize_Series030.tif; Bacillus mycoides, lineage 5 in 30°C selection
 regime * In folder brhwcellsize_Series 3.zip brhwcellsize_Series031.tif to
 brhwcellsize_Series035.tif; Bacillus thuringiensiss ancestor
 brhwcellsize_Series036.tif to brhwcellsize_Series040.tif; Bacillus
 thuringiensis, lineage 1 in 30°C selection regime
 brhwcellsize_Series041.tif to brhwcellsize_Series045.tif; Bacillus
 thuringiensis, lineage 2 in 30°C selection regime * In folder
 brhwcellsize_Series 4.zip brhwcellsize_Series046.tif to
 brhwcellsize_Series050.tif; Bacillus thuringiensis, lineage 3 in 30°C
 selection regime brhwcellsize_Series051.tif to brhwcellsize_Series055.tif;
 Bacillus thuringiensis, lineage 4 in 30°C selection regime
 brhwcellsize_Series056.tif to brhwcellsize_Series060.tif; Bacillus
 thuringiensis, lineage 5 in 30°C selection regime Alongside these image
 files, there are two files. #### "cellsize.txt": this file
 contains the measurements of cell size obtained from the image files
 listed above. Variables are: * *Species:* The species examined (either Bm
 for B. mycoides or Bt for B. thuringiensis). * *Lineage*: Lineage of the
 strain/lineage tested (either "ancestor" for ancestral strain,
 or "30X", showing lineage was result of experimental evolution
 at 30°C (X being a number between 1-5 indicating a different replicate
 lineage). * *polar*: polar long axis length in μm * *equator:* equatorial
 short axis length in μm * *vol_cyl*: Cell volume, calculated assuming an
 ellipsoid form (cell vol = 4⁄3 π × ρ/2× ϵ ) where ρ is the polar long axis
 and ϵ is the equatorial short axis. This data was analysed using the R
 script "hugh_cell_size.R". ###
 Raw\_thermal\_profiling\_data.zip; This file contains the raw OD data used
 to generate thermal performance curves in this study. Raw data is arranged
 by the temperature each strain was grown at, each with its own folder
 (15°C, 17°C, 20°C, 23°C, 27°C, 29°C, 33°C). In each X folder; ##### 26
 files beginning "Hw start od . . ."; each containing the output
 of the plate reader at a given timepoint during the experiment. There are
 five essential sheets for these files; * *Absorbance results*; the optical
 density reading (at wavelength of 600nm) for each well at the timepoint *
 *Raw data 600nm*; the raw data formatted and presented in *Absorbance
 results*. Headers are; Well (cell row and number of 96-well plate),
 Wavelength(s) [nm] (wavelength using for reading, in nanometres), Raw
 absorbance (measure of growth in data, unitless data) and measurement time
 (seconds). * *General information*; Information regarding the session
 information of the Multiskan Sky OD reader (including execution time,
 device name, serial number, firmware version, software version, set
 temperature and whether cuvette and incubator are on) and protocol
 parameters (including assay type, plate template, wavelength, measurement
 type, shake (to homogenise the samples) and shake duration. * *Plate
 layout*; Layout of plate. None specified here, can be ignored. * *Run
 log*; the optical density reading (at wavelength of 600nm) for each well
 at the timepoint. Can be ignored. #### "Data formatting template
 X.xlsx"; Taking information from the sheet \*Absorbance results in
 the above files, and entering it into cells A1-M9 (for replicate 1-2) and
 A11-M19. Colours are used to show layout (cells containing water in blue,
 cells containing blank controls in yellow, and strains containing samples
 in red). The table underneath details * *Strain*: Strain examined. Either
 BtA (B. thuringiensis, ancestor), BmA (B. mycoides, ancestor), Bt30/Bt15
 (B. thuringiensis selected at 30°C or 15°C) and Bm30/Bm15 (B. mycoides
 selected at 30°C or 15°C) * *Temp*: Experimental temperature (°C) * *Rep*:
 Biological replicate (1, 2, 3 or 4) * *ID*; concatenated Strain and Rep
 information. * *Time*: Experimental timepoint (in hours, changed manually)
 * *Raw absorbance*: Absorbance at wavelength 600nm of blanks * *Average OD
 of blanks*: Average absorbance at wavelength 600nm of blanks Data from
 each "Hw start OD ..." file is copied and pasted into cells
 A1-M9 or A11-M19. Time can be used to discriminate plates, as the plate
 occupying A1-M9 was always read first at each timepoint. The collated
 results are stored in the sheet "collated data" within this
 file, and in "X output.csv" These "X output.csv"
 results are processed to produce the file "X well values" which
 contains parameters from the growth curves extracted using R package
 GrowthCurver * *IDnames*: concatenated Strain and Rep information e.g.
 Bm15-1 means replicate 1 of B. mycoides selected at 15°C. * *Rcurve*:
 maximum growth rate of sample at experimental temperature * *Kcurve*:
 maximum carrying capacity of sample at experimental temperature *
 *auc1curve*: area under the logistic curve obtained by taking the integral
 of the logistic equation * *aucEcurve*: empirical area under the curve
 which is obtained by summing up the area under the experimental curve from
 the measurements in the input data * *temp*: experimental temperature We
 recommend using the "X well values STATIONARY.csv" for analysis,
 as these record only to stationary phase of bacterial growth and so avoid
 die-off which could skew results. "All well R and auc1 STATIONARY
 PHASE REDUCED.csv" contains concatenated results of "X well
 values STATIONARY.csv". #### Checking\_activation\_energy.zip ####
 "Activation energy check rTPC 11 5 2024.csv" Output from growth
 curve data to obtain the activation energy (e) from thermal performance
 curves for each strain/lineage. * *Strain*: Species of bacteria (either B.
 thuringiensis or B. mycoides) * *Type*: Identifies strain as the ancestral
 strain ("Anc") or a lineage derived from experimental evolution
 ("evo"). * *Temp*: The selection regime for experimental
 evolution the strain is from (either Anc for ancestor strains that did not
 undergo any experimental evolution, 15°C or 30°C). * *e*: activation
 energy, defined as the minimum energy required for growth to occur. Blanks
 indicate missing data. The data was analysed using the script
 "Activation energy check script 11 5 2024". ####
 Fig\_1\_and\_supp\_fig\_1.zip #### All well values output ALL STRAINS BOTH
 24 08 23.csv * *IDnames*: concatenated Strain and Rep information e.g.
 Bm15-1 means replicate 1 of B. mycoides selected at 15°C. * *Rcurve*:
 maximum growth rate of sample at experimental temperature * *Kcurve*:
 maximum carrying capacity of sample at experimental temperature *
 *auc1curve*: area under the logistic curve obtained by taking the integral
 of the logistic equation * *aucEcurve*: empirical area under the curve
 which is obtained by summing up the area under the experimental curve from
 the measurements in the input data * *Strain*: Species of bacteria (either
 B. thuringiensis or B. mycoides) with lineage (either A for ancestral or
 XYY to reflect lineage number (X; 1-5) and experimental evolution
 treatment (YY; 30°C or 15°C). * *Selection temperature*; The selection
 regime for experimental evolution the strain is from (either Anc for
 ancestor strains that did not undergo any experimental evolution, 15°C or
 30°C). * *Rep*: Replicate number * *Growth temperature*: Growth
 temperature (°C) at which Rcurve/Kcurve/auc1curve/aucEcurve were
 calculated. This file was fed into "plotting rTPCs 4 9 25 FIGURE
 1.R" and "plotting Supplementary figure 1.R" to produce
 figure 1 and Supplementary figure 1 respectively. #### Fig\_2.zip Scripts
 and data for plotting figure 2 from the article "Constraints in
 temperature adaptation reinforce differences in thermal niche between
 mesophilic and psychrotolerant Bacillus cereus group species".
 Analysis focuses on determining responses to selection at 15°C and 30°C in
 Bacillus thuringiensis and Bacillus mycoides by competition assays.
 Contains raw data and processed data for analysis.  #### WT results for
 analysis LINEAGES 27 2 24.csv * *Genotype*: Species (Bt or Bm). *
 *SelectionTemp*: Selection regime (either "A" if ancestor, or
 30°C or 15°C). * *Lineage*: Number 1-5 indicating the experimental
 lineage. Note that only one lineage exists for ancestral strain, marked as
 3. * *Speclineage*: Genotype, SelectionTemp and Lineage concatenated into
 a single variable. * *Line*: Binary variable defining sample as ancestral
 (A) or selected (S). * *Facet*: When plotting, indicates where data should
 be presented (either in the Bt30 facet, or in the Bm30 facet). *
 *CompTemperature*; Temperature in which competition experiment took place
 (30°C or 15°C) * *Replicate*: Biological replicate for competition
 experiment. * *Selectionrateconstant*: Selection rate constant of wildtype
 strain. Average across three replicates. Defined as the difference between
 the Malthusian parameters of the wildtype strain of interest and the
 antibiotic resistant standard competitor. * *Relativefitness*: Relative
 fitness of wildtype strain. Average across three replicates. Defined
 as the ratio between the Malthusian parameters of the wildtype strain of
 interest and the antibiotic resistant standard competitor. NA = missing
 data, where there was a general die-off in the media during competition.
 *WT results for analysis LINEAGES 27 2 24.csv* is fed into "Analyses
 27 2 24 STATISTICS.R" for analysis. #### Analysis data for 15c with
 means 10 1 23.csv/Analysis data for 30c with means 1 23.csv Files
 containing the individual fitness parameters for each wildtype strain
 replicate, and the means used in "WT results for analysis LINEAGES 27
 2 24.csv" * *Strain*: Species (Bt or Bm) and lineage information (A
 for ancestor, 1-5 for lineages from experimental evolution and 30 or 15
 for experimental evolution temperature). - *Temperature*: Competition
 temperature for these strains (15 or 30°C) * *Replicate*: Biological
 replicate (1, 2 or 3) - *Selectionrateconstant*: Selection rate constant
 of wildtype strain. Average across three replicates. Defined as the
 difference between the Malthusian parameters of the wildtype strain of
 interest and the antibiotic resistant standard competitor. *
 *Relativefitness*: Relative fitness of wildtype strain. Average across
 three replicates. Defined as the ratio between the Malthusian parameters
 of the wildtype strain of interest and the antibiotic resistant standard
 competitor. Further columns detail the mean values - *Strain*: Species (Bt
 or Bm) and lineage information (A for ancestor, 1-5 for lineages from
 experimental evolution and 30 or 15 for experimental evolution
 temperature). * Mean Selection Rate constant: Average selection rate
 constant for Strain across replicates. - Mean relative fitness: Average
 selection rate constant for Strain across replicates. #### Fig\_3.zip
 Scripts and data for plotting figure 3 from the article "Constraints
 in temperature adaptation reinforce differences in thermal niche between
 mesophilic and psychrotolerant Bacillus cereus group species". ###
 Productivity rTPC parameter correlations by lineage WOP 7 12 24 *
 *Species*: Species (Bt or Bm). * *Regime*: Selection regime (either
 "Anc" if ancestor, or 30°C or 15°C). * *Topt*: Temperature at
 which productivity is highest, derived from thermal performance curve of
 each lineage. * *Bpk*: Maximum productivity derived from thermal
 performance curve of each lineage. * *Wop*: Operational thermal niche
 width, over which productivity is at least half Bpk, derived from thermal
 performance curve of each lineage. ### rTPC parameter correlations by
 lineage WOP 7 12 24 * *Species*: Species (Bt or Bm). * *Regime*: Selection
 regime (either "Anc" if ancestor, or 30°C or 15°C). * *Topt*:
 Temperature at which growth rate is highest, derived from thermal
 performance curve of each lineage. * *Bpk*: Maximum growth rate derived
 from thermal performance curve of each lineage. * *Wop*: Operational
 thermal niche width, over which growth rate is at least half Bpk, derived
 from thermal performance curve of each lineage. Figure 3 in the manuscript
 is produced using "rTPC correlations script FIGURE 3 4 9 25.R".
 #### Fig\_4\_and\_supp\_fig\_2.zip Scripts and data for plotting figure 4
 and supplementary figure 2 from the article "Constraints in
 temperature adaptation reinforce differences in thermal niche between
 mesophilic and psychrotolerant Bacillus cereus group species". ###
 Productivity rTPC parameter correlations by lineage WOP 7 12 24 *
 *Species*: Species (Bt or Bm). * *Regime*: Selection regime (either
 "Anc" if ancestor, or 30°C or 15°C). * *Topt*: Temperature at
 which productivity is highest, derived from thermal performance curve of
 each lineage. * *Bpk*: Maximum productivity derived from thermal
 performance curve of each lineage. * *Wop*: Operational thermal niche
 width, over which productivity is at least half Bpk, derived from thermal
 performance curve of each lineage. ### rTPC parameter correlations by
 lineage WOP 7 12 24 * *Species*: Species (Bt or Bm). * *Regime*: Selection
 regime (either "Anc" if ancestor, or 30°C or 15°C). * *Topt*:
 Temperature at which growth rate is highest, derived from thermal
 performance curve of each lineage. * *Bpk*: Maximum growth rate derived
 from thermal performance curve of each lineage. * *Wop*: Operational
 thermal niche width, over which growth rate is at least half Bpk, derived
 from thermal performance curve of each lineage. Figure 4 and supplementary
 figure 2 in the manuscript are produced using "rTPC correlations
 script GROWTH AND PRODUCTIVITY PLOTS" and data is analysed using
 "rTPC correlations ANALYSIS.R" #### Fig\_5.zip Scripts and data
 for plotting figure 5 from the article "Constraints in temperature
 adaptation reinforce differences in thermal niche between mesophilic and
 psychrotolerant Bacillus cereus group species". ### dn ds ratios 8 9
 22.csv * *Strain*: Species, either B. thuringiensis (Bt) or B. mycoides
 (Bm) * *Phenotype*: Rifampicin resistance phenotype, either susceptible
 (wildtype, WT) or resistant. * *Lineage*: Experimental lineage, numbered
 1-5. * *Selection Temp*: Temperature (°C) at which selection took place. *
 *dN*: Number of non-synonymous single nucleotide polymorphisms (SNPs)
 differentiating the strain from its ancestor. * *dS*: Number of synonymous
 single nucleotide polymorphisms (SNPs) differentiating the strain from its
 ancestor. * *dN/dS*: Ratio of non-synonymous SNPS to synonymous SNPS in
 each strain, indicator of strength and direction of selection. Data is
 analysed using "dn ds analysis PLOTS 7 18 24.R" and used to
 produce the plots. #### Fig\_6.zip Scripts and data for plotting figure 6
 from the article "Constraints in temperature adaptation reinforce
 differences in thermal niche between mesophilic and psychrotolerant
 Bacillus cereus group species". ### Venn for r Bm WT(in).csv * *Bm15:
 Product of genes showing convergent single-nucleotide polymorphisms within
 B. mycoides lineages selected at 15°C. Convergence is defined as the same
 gene possessing a non-synonymous SNP in at least 3 lineages within the
 given selection regime. * *Bm30: Product of genes showing convergent
 single-nucleotide polymorphisms within B. mycoides lineages selected at
 30°C. Convergence is defined as the same gene possessing a non-synonymous
 SNP in at least 3 lineages within the given selection regime. ### Venn for
 r Bt only WT(in).csv * *Bt15: Protein ID of genes showing convergent
 single-nucleotide polymorphisms within B. mycoides lineages selected at
 15°C. Convergence is defined as the same gene possessing a non-synonymous
 SNP in at least 3 lineages within the given selection regime. * *Bt30:
 Protein ID of genes showing convergent single-nucleotide polymorphisms
 within B. mycoides lineages selected at 30°C. Convergence is defined as
 the same gene possessing a non-synonymous SNP in at least 3 lineages
 within the given selection regime. ## Sharing/Access information Link to
 Bioproject containing sequences used to derive data for Figure 5 and
 Figure 6: *
 [https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA826440](https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA826440) R is required to run all scripts. Annotations are provided throughout the script through 1) library loading, 2) dataset loading and cleaning, 3) analyses, and 4) figure creation.