DOI: 10.25349/d9fk6q
title: Testing the adaptive value of sporulation in budding yeast using experimental evolution

description: Saccharomyces yeast grow through mitotic cell division, converting
 resources into biomass. When cells experience starvation, sporulation is
 initiated and meiosis produces haploid cells inside a protective ascus.
 The protected spore state does not acquire resources and is partially
 protected from desiccation, heat, and caustic chemicals. Because cells
 cannot both be protected and acquire resources simultaneously, committing
 to sporulation represents a trade-off between current and future
 reproduction. Recent work has suggested that passaging through insect guts
 selects for spore formation, as surviving insect ingestion represents a
 major way that yeasts are vectored to new food sources. We subjected
 replicate populations from five yeast strains to passaging through
 insects, and evolved control populations by pipette passaging. We assayed
 populations for their propensity to sporulate after resource depletion. We
 found that ancestral domesticated strains produced fewer spores, and all
 strains evolved increased spore production in response to passaging
 through flies, but domesticated strains responded less. Exposure to flies
 led to a more rapid shift to sporulation that was more extreme in
 wild-derived strains. Our results indicate that insect passaging selects
 for spore production and suggest that domestication led to genetic
 canalization of the response to cues in the environment and initiation of
 sporulation.
description: Data was collected by freezing samples from the start (baseline)
 middle (midpoint) and end of an experimental evolution study. We performed
 evolution experiments which repeatedly exposed yeast cells to ingestion
 and digestion by the fruit fly,  Drosophila melanogaster, over the course
 of 31 one-week cycles and measured the phenotypic response in terms of
 sporulation timing. We used a set of regionally diverse and genetically
 distinct strains of Saccharomyces cerevisiae with different historical
 selection pressures cultured and adapted by Liti and Louvel (Cubillos,
 Liti and Louvel et al., 2014). We compared phenotypic characteristics of
 yeast multiple replicates of each of these regional strain lineages
 repeatedly exposed to an insect vector (treatment) relative to those same
 characteristics of the same strains repeatedly transferred by pipette
 (control).<br> <br> We used a set of five genetically distinct
 strains of  diploid <i>S. cerevisiae</i> that had received
 genetic barcodes and antibiotic resistance to G418. All strains were wild
 isolates or wild-derived (commercial) isolates transformed with resistance
 to Geneticin (G418) a yeast orthologue to Kanamycin (Louvel et al.,
 2014). For each ancestral strain, we created four replicates by
 inoculating into YPD (Yeast-Peptone-Dextrose)  broth culture  and growing
 for five days with shaking (230 rpm) at 30 degrees Celsius. Cultures were
 always grown in  YPD media with G418,  tetracycline, and ampicillin added
 (YPDA).    Strains were grown in 2
 milliliters of antibiotic media over a 120-hour (5 day) period. Samples of
 these initial strains (baseline samples) were then frozen in 15% glycerol
 solution at -80 degrees Celsuis.  Four initial
 concentration-adjusted samples of the five regional strains were then
 proportioned into 4 replicates each (Replicates 1-20). Each replicate was
 then split into two Eppendorf tubes: a control sample and a treatment
 sample, and two cryotubes: a control sample and a treatment sample, for
 freezing. Each control sample remained in the Eppendorf tube and was
 placed in proximity to the treatment vials (same ambient conditions) for
 the duration of the treatment time. After 48 hours, each control tube was
 vortexed and 10uL  of each control was moved to 1.49 mL  of YPDA in a new
 culture tube (1.5 mL total volume). Each new culture tube was incubated as
 the sample for the next round of control trials.   
   Each concentration-adjusted treatment was
 offered to 3-4 clean, sexed flies using a CaFe apparatus. Fly Capillary
 Feeder (CaFe) apparatus tops were assembled per design adapted from Ja and
 colleagues (2007).  Flies were allowed to feed for 48 hours and then
 removed from the vials.  The vials containing fly fecal material (frass)
 and body transfer yeast were then rinsed with $1.6 mL$ YPDA media and the
 supernatant ($1.5 mL$ total volume because some volume is reabsorbed into
 the agar in the vial) was collected. Each new culture tube was incubated
 as the sample for the next round of experimental trials.
 <br> Each new generation of control and treatment tubes
 were incubated at 30 degree Celsius for 120 hours without shaking to allow
 yeast to form diploids and grow. After incubation,  optical density
 adjustments to a consistent OD across all strains were repeated using
 established lineages (1-20) of all strains, both control and treatment.
 This process was repeated for 31 generations (treatment cycles) freezing
 every odd sample (G1, G3 etc…) of both the treatment and control lineages
 after the initial (G0) samples in 15% glycerol at $-80 degrees
 Celsius.<br> <br> Single colony isolates of the Ancestral (
 frozen sample before the first interval of the experimental evolution
 trials and evolved ( frozen sample at the 31st interval of the
 experimental evolution trials, both control and treatment) strains were
 grown for 6 hours without shaking and then assessed for their optical
 densities. Equal concentrations (2 mL of media at an Optical Density of
 1.5) of each ancestral, control, and treatment replicate were then washed
 and sporulated in 2mL of Potassium Acetate ( 2% KAc at pH 6.7), and
 incubated at  30 degrees Celsius with shaking (230 rpm). Sporulation
 percentage was checked and recorded at 2.5 days (time 1) and 5 days (time
 2).  <br> <br> The sporulation assay was performed in two
 experimental blocks with each block containing samples of all treatment
 and control replicates at 31 generations. Two single colony isolates for
 each of 20 experimental replicates (evolutionary lineages) were taken for
 both the control and the treatment evolved strains. Two technical
 replicates were taken of each colony isolate for a total of 160 samples.
 For the ancestral strain, three single colony isolates were taken for each
 of the 5 represented lineage backgrounds, which were previously frozen at
 the start of the experimental evolution procedure to ensure comparison of
 a true ancestral strain. Two technical replicates were taken for each of
 the single colony isolates, these samples were also repeated across two
 dates for a total of 60 samples.  At two time points in the sporulation
 process, 2.5 days and 5 days, each sample was diluted to 10^-2
 concentration (5uL in 95 uL), photographed at 40X magnification and
 assessed for its sporulation rate by counting the number of spores and
 vegetative cells in each similarly dense objective frame. The sporulation
 rate of the evolved treatment and control strains were quantified by
 counting both the spores and vegetative cells in a similarly diluted
 concentration of cells, which was photographed at  40X  magnification.
 These counts were then compared to samples of the ancestral strain which
 had been frozen at the start of the experimental procedure, and was
 quantified in the same manner as the evolved strains. Count data was
 recorded for cells determined to be spores, cells determined to be
 vegetative and the summed total cell value.     
  
description: YeastSporulationData.csv: Data is in numerical format (count
 data) and separated by category of strain region. Each strain region has
 four experimental replicates. Each experimental replicate was evaluated in
 two assays. In each assay, each experimental replicate has 3
 colony replicates, and each colony replicate has two technical replicates.
 For each replicate, there are two data points of time point 1 (2.5 days)
 and time point 2 (5 days) across three treatments: baseline, control and
 experimental. Data has been minimally processed but the percent spore
 composition has been calculated for each photograph (taken at 2.5 or 5
 days).    RawDataFigures.Rmd is
 provided to produce figure S.1. It plots the raw proportion of cells that
 are spores for each of the yeast strains.
 LTEE_Main_Stats.Rmd is the main analysis file. This includes all
 the statistical procedures in the main text as well as some supplementary
 analyses. FittedModels.RData : An RData file containing
 the brms fitted models for the full dataset.
 AncModels.RData : An RData file containing the brms fitted models
 for the ancestral yeast strains.