Electronic Supplementary Material
Strains
Our goal was to arrange antibiotic and auxotrophic markers such that a unique
combination of each was expressed in each cell type (diploid, MATa, and MATα cells).
This allowed selection of a specific cell type from a culture containing one or more
undesirable types of cells. We therefore constructed a MATa strain that alone could grow
in the presence of the antibiotic hygromycin and without the amino acid histidine, and a
MATα strain that could alone grow without the addition of the amino acid leucine. This
was accomplished using genetic constructs linked to MATa or MATα-specific promoters
[1] that either restored an auxotrophy or conferred antibiotic resistance. Additionally, we
placed two different antibiotic markers, kanMX4 [2] and natMX4 [3], at the HO locus
(one in each haploid strain) to ensure that heterozygous diploids would be able to grow in
the presence of both G418 and cloNAT. Specifically, we created (see the next section for
details on their construction and Table S1 for genotypes of each strain):
(1) A MATa strain (YLR12) containing a gene conferring resistance to hygromycin [3]
coupled to the STE2 promoter (which codes for the receptor of α-pheromone and is only
expressed in MATa cells). This strain also contains the promoter of the MFA1 gene
(which encodes a-pheromone and is only expressed in MATa cells) coupled to HIS3,
restoring the ability to synthesize histidine. A pure culture of MATa cells could thus be
achieved by growth in media containing hygromycin and leucine, but not histidine. (All
media recipes are given below.)
(2) A MATα strain (YLR13) containing the construct lyp1Δ::STE3-LEU2 [1], a deletion
of the lyp1 gene replaced by the LEU2 gene coupled to the promoter of STE3 (which
codes for the receptor for a-pheromone and is only expressed in MATα cells). Selection
for MATα cells could therefore be accomplished by placing a culture of cells in media
containing histidine but lacking leucine.
(3) A diploid strain (YLR11) was the product of the mating of the MATa and MATα
constructs and was therefore deficient in producing either histidine or leucine. However,
it was resistant to both cloNat and G418, as it contained a gene conferring resistance to
each at the HO locus. Heterozygous diploids could therefore be selected through growth
in the presence of both antibiotics supplemented by both amino acids; however,
homozygous diploids at the HO locus would be selected out.
(4) A “marked” ancestor (YLR14) was also created that appeared pink when plated (by
addition of the ade2-101ochre mutation) in order to make estimations of fitness in each
population during the experiment.
Construction Details
The main genetic background of the experimental strain was YPS670, a woodland isolate
from Pennsylvania. First, we crossed a previously described version of the strain [4]
(YPS3340- MATα, hoΔ::natMX4) with YJN27 (a YPS670 derivative transformed to be
hoΔ::natMX4, 191647-191819::MFA1pr-HIS3 and chrIII 205554-206553::STE2pr-
hphMX4, generously provided by JN Jasmin, Wake Forest University). Both of these
regions on chromosome III are intergenic; the MFA1 promoter contains 394bp upstream
from the start codon and the STE2 promoter contains 557bp. From this cross, we isolated
a strain that was MATa, chrIII 191647-181819::MFA1pr-HIS3, chrIII 205554206553::STE2pr-hphMX4, hoΔ::natMX4 and contained the rest of the YPS670
background.
Next, we crossed this strain with BY8205 (MATα, can1Δ::STE2pr-Sp_his5,
lyp1Δ::STE3pr-LEU2, leu2Δ, his3Δ, ura3Δ, derived from S288c, gift of Charles Boone,
University of Toronto), to incorporate leu2Δ, his3Δ, and lyp1Δ::STE3pr-LEU2. Through
numerous backcrosses, all other auxotrophies associated with the lab strain were selected
out. Finally, we crossed the MATα strain with YPS3062 (MATa, hoΔ::kanMX4, YPS670
derivative) to switch the antibiotic marker at the HO locus. We estimate that the final
strain was 85% YPS670 and 15% S288c.
Finally, a marked ancestor was created by crossing the ancestral strain to
YMZ336, a YPS670 derivative with an adenine auxotrophy (ade-101ochre).
Media
Evolution Medium-Diploids
0.1% dextrose, 0.17% yeast nitrogen base, 0.1% glutamic acid, 0.2M NaCl, 250 mg/L
histidine, 250 mg/L leucine, 375mg/L G418, and 250 mg/L cloNat.
To improve the effectiveness of the antibiotics, glutamic acid was substituted for
ammonium sulfate (http://mgm.duke.edu/faculty/mccusker/lab/resources/). Histidine and
leucine were added, as diploids cannot endogenously produce either of these amino acids.
The presence of G418 and cloNat was necessary to ensure populations of pure diploid
cells, but also removed all diploids homozygous at the HO locus for either kanMX4 or
natMX4 (see ‘Diploid selection’ in ‘Experimental Cycle’ below). This should not bias
our results, as these alleles segregate at random during meiosis.
For the fitness assays, this medium was supplemented with 100 mg/L adenine, more than
5 times the amount found in typical medium.
Evolution Medium- MATa selection
0.1% dextrose, 0.17% yeast nitrogen base, 0.1% glutamic acid, 0.2M NaCl, 250 mg/L
leucine, and 400 mg/L Hygromycin.
This concentration of leucine is roughly 10 times the concentration in typical media and
used in our experiment in order to minimize selection for autotrophic cells at this stage of
the experimental cycle. Only MATa cells express resistance to Hygromycin and produce
histidine endogenously.
Evolution Medium- MATα selection
0.1% dextrose, 0.17% yeast nitrogen base, 0.1% glutamic acid, 0.2M NaCl, and 250
mg/L histidine.
Only MATα cells produce leucine endogenously. Unlike MATa cells, histidine must be
added to media to allow growth.
The selective environment for both types of haploids therefore closely paralleled that of
the diploids, with small changes in media composition to allow selection for the two
types of haploids.
Sporulation Medium
1% potassium acetate, 250 mg/L histidine, 250 mg/L leucine, and 1.5% agar.
Addition of histidine and leucine were necessary because diploids cannot produce
histidine and leucine endogenously. These concentrations of amino acids are 5-10 times
that in typical media to ensure minimal selection during this phase of the experimental
cycle.
Mating Medium
0.1% dextrose, 0.17% yeast nitrogen base, 0.1% glutamic acid, 0.2M NaCl, 250 mg/L
histidine, 250 mg/L leucine, 1.5% agar.
The medium was sterilized and poured into the wells of a 24-well plate and allowed to
cool to create a solid surface for mating. After mating occurred, liquid evolution medium
supplemented with antibiotics (see above) was added to the wells and pipetted up and
down to suspend cells.
Alternate Medium- Sucrose-based
0.1% sucrose, 0.17% yeast nitrogen base, 0.1% glutamic acid, 0.2M NaCl, 250 mg/L
histidine, 250 mg/L leucine, 375mg/L G418, and 250 mg/L cloNat.
Experimental Cycle
A single diploid colony was used to found all populations to begin the experiment. The
colony was grown in evolution medium supplemented with antibiotics (see “Evolution
Medium-Diploids” above) for 24h at 30°C in a standing incubator.
Serial transfers. To begin the experiment, 12 wells of a 24-well plate each containing
2mL of EM supplemented with antibiotics were inoculated with 20µL of culture (from
the single diploid colony). After 24h of asexual growth in a 30°C standing incubator,
20μL of each population were transferred to a new 24-well plate containing fresh medium
and incubated at 30°C. This was done a total of 3 times during each cycle, representing 4
24-hour periods of diploid growth in a standing incubator.
Meiosis. 24h after the final transfer, each population (with the exception of the four
asexual populations, which were placed at 4°C until the serial transfer phase) was
induced to undergo meiosis (i.e. sporulation). Populations were washed, resuspended in
50μL water, transferred to a small sporulation plate (which allows for >90% sporulation
rate), and incubated at 30°C.
Roughly 36h later, sporulation was verified for a subsample of all populations via
microscopy. The sporulated cells were resuspended in 1mL water and incubated at 55°C
for 45 min to kill all diploid cells [5]. Elimination of diploids was necessary to ensure
only cells undergoing meiosis and subsequent sex were passed through to the next cycle.
Isolation of pure haploid cultures. Populations were treated with 100μL zymolyase
solution (18% sorbitol, 0.01% zymolyase 100T, 5% 2-mercaptoethanol) and rotated for 4
- 4.5h at 30°C. 2μL Triton X was then added to each tube and vortexed on high for at
least 5 min to separate spore clumps. Each population was then centrifuged and
resuspended in 500μL distilled water. Half of each population was transferred to MATa or
MATα selective medium.
Mating and sexual selection. After 24h, pure MATa and MATα cultures were obtained.
MATa and MATα cells from the same original population were combined in the
prescribed ratio by volume. The number of MATa cells was constant across treatment
groups; the MATα cells were varied, leading to a larger effective population size in the
strong sexual selection treatment than in the weak at this part of the cycle (see ‘Effective
Population Size’ below). The mixed culture was centrifuged and resuspended in a small
amount (~20μL) of water and placed on a solid mating plate. The populations were then
left overnight to mate at 30°C.
Diploid selection. The following morning, ~12h after mating, the mated cultures were
suspended in the evolution medium supplemented with antibiotics to select for diploids.
The serial transfer process began again after 24h.
Effective Population Size
Serial transfer: The average effective population size for each population during the
serial transfer phase was approximately 4 x 106 individuals [6]. The final number of cells
after 24h was 1.5 x 107.
Sporulation and Haploid Selection: The entire population was induced to sporulate; each
diploid cell produces four haploid spores (2 MATa, 2 MATα) on average. Half of each
sporulated population, containing haploids of both mating types, were placed in Evolving
Medium-MATa selection, the other half in Evolving Medium-MATα selection (see Media
above). Half of all haploids (MATa cells in MATα selection medium and MATα cells in
MATa selection medium) were eliminated in this process.
On average, each diploid cell should be represented by one spore of each mating type in
the inoculum of the haploid cultures. This assumes a 100% sporulation efficiency and no
loss of spores during the digestion process. We observed a rate of closer to 90%
sporulation efficiency and a recovery rate ~80%. The pure haploid cultures reached the
same density as the original diploid culture. (As long as sporulation and spore recovery
did not decrease the population to 0.01 of its original size, it is not qualitatively different
than a round of serial transfer.) Given the sporulation and recovery rate, that each diploid
produces on average two haploids of each mating type, and that half the haploids were
eliminated, the pure MATa or MATα cultures used in the mating phase contained ~107
individuals of each mating type.
Mating: 106 MATa cells were combined with an equal number of MATα cells in the weak
selection treatment and 107 MATα cells in the strong selection treatment; in both
treatments, on average, 106 diploids should form.
Diploid Selection: Only heterozygous diploids were selected for; therefore, the first day
of diploid growth started with approximately 5 x 105 diploids and ended with 1.5 x 107
diploids, giving an effective population size of about 2 x 106.
Overall, we conservatively estimate the effective population size each complete cycle to
be approximately 3.7 x 106 (the harmonic mean of the effective population size of all the
days of growth). Note that because the number of MATa cells was held constant while the
number of MATα cells was increased to effect our 1:10 mating type ratio, the effective
population size of the strong sexual selection populations is roughly 1.8x higher than that
of the weak sexual selection populations for the mating component of the cycle.
Relative Fitness Assays
Cultures of the marked ancestor (YLR14) and evolved strain to be competed were
allowed to grow for 48h (24h followed by a 1:100 serial transfer and additional 24h of
growth) in glucose media + adenine, as YLR14 carries an adenine auxotrophy. The
strains were combined in equal volumes and 10 replicates of this master mix were plated
to YPD in a 1:5000 dilution to determine the initial relative abundance of each strain.
20µL of this mix was then added to 10 wells of a 24-well plate containing 2mL glucose
media + adenine. After 24h at 30°C, the cultures were transferred in a 1:100 dilution and
allowed to grow for an additional 24h. 2 samples from each of the 10 wells were then
plated in a 1:5000 dilution to YPD. The number of white (evolved) and pink (marked
ancestral) cells were counted on each plate. Raw colony counts are available in the
supplementary Excel file.
Calculations of relative fitness were based on ref [7]. A Malshusian parameter (m) was
determined for each of the 10 samples for each population. This was calculated as:
m(A) = ln((# A colonies after 48h competition period x 10,000) / # of initial A colonies)
The number of colonies after the 48h competition period was taken as the average of the
two samples for a given well, while the initial number of colonies was taken as the
average number of colonies from the ten samples of the original master mix. Relative
fitness (W) was calculated as the ratio of Malthusian parameters, i.e.,
W = m(evolved) / m(marked ancestral)
Biologically, this term represents that the evolved strain increased at a rate W times that
of the ancestral strain.
Population Mating Assays
Diploids strains to be mated were grown in YPD for 24h at 30°C and sporulated as above.
Half of each culture of digested asci from each strain were added to 30mL MATa or
MATα selective media and grown for 24h at 30°C.
All cultures were then centrifuged at 3000 rpm for 3 min, resuspended in 10mL distilled
water, and sonicated for 30s to break up cell clumps. Each culture was centrifuged once
again and resuspended in 200μL water. 100μL each of both mating types was mixed
thoroughly, plated on a small (35mm diameter) glucose media plate, and allowed to dry
in a sterile hood for 30 min. Samples were scraped from plates immediately after drying
(hour 0), then 3h thereafter (hour 3). 3 replicates were taken for each sample, diluted
1:1000 and plated to YPD.
Although equal volumes of both mating types were mixed together, we controlled for
possible deviations from this optimal 50:50 MATa:MATα. Deviations from this ratio
result in fewer diploid (mated) yeast, as haploids of one mating type are more plentiful
than the other and remain unmated. To determine the exact proportion of MATa:MATα,
and thus the theoretically maximum proportion of mated yeast, all hour 0 plates were
replica-plated to glucose media plates containing hygromycin, which only allows the
growth of MATa haploids.
To determine the proportion of mated cells after 3 hours, the hour 3 plates were replicaplated to glucose media plates containing G418 and cloNAT, which only allows for the
growth of diploid colonies stemming from cells heterozygous at the HO locus. The
proportion of cells mated after 3 hours was calculated as the proportion of heterozygous
diploids after 3 hours divided by the maximum proportion of cells that could theoretically
mate. This number was then multiplied by two, as on average half of the diploids formed
during the mating period were homozygous at the HO locus and thus would not have
survived plating on double antibiotic media.
By 3 hours, many of the cells have mated. We ended our assay at this time point because
observations under the microscope showed mated diploids beginning to bud. Data after
this time point would inflate the estimate of mated cells, as it would be biased by growth
of diploids (i.e., not indicative of the mating dynamics).
Supplementary Tables
Table S1: Strains used in this experiment.
Strain Name
Genotype
YLR11
MATa/α, chrIII 191647-181819::MFA1pr-HIS3/chrIII+, chrIII
205554-206553::STE2pr-hphMX4/chrIII+, his3Δ/his3Δ,
leu2Δ/leu2Δ, lyp1Δ::STE3pr-LEU2/lyp1Δ::STE3pr-LEU2,
hoΔ::natMX/hoΔ::kanMX
YLR12
MATa, chrIII 191647-181819::MFA1pr-HIS3, chrIII 205554206553::STE2pr-hphMX4, his3Δ, leu2Δ, lyp1Δ::STE3prLEU2, hoΔ::natMX4
YLR13
MATα, his3Δ, leu2Δ, lyp1Δ::STE3pr-LEU2, hoΔ::kanMX4
YLR14
MATa/α, chrIII 191647-181819::MFA1pr-HIS3/chrIII+, chrIII
205554-206553::STE2pr-hphMX4/chrIII+, his3Δ/his3Δ,
leu2Δ/leu2Δ, lyp1Δ::STE3pr-LEU2/lyp1Δ::STE3pr-LEU2,
hoΔ::natMX/hoΔ::kanMX, ade-101ochre/ade-101ochre
*Naming note: The ‘pr’ here refers to the promoter, so ‘STE3pr-hphMX4’ means that the
gene conferring resistance to hygromycin (hphMX4) is linked to the promoter of the
STE3 gene (Tong et al 2001).
Table S2: Fitness in Glucose Medium*
model effect
test statistic
p-value
% variance
component
cycle
F1,413 = 72.25
<0.0001
treatment
F2,~9 = 15.747
0.0012
cycle*treatment
F2,~9 = 5.91
0.0232
population[treatment]
9.3
cycle*population[treatment]
2.6
residual
88.1
Table S3: Fitness in Sucrose Medium*
model effect
test statistic
p-value
cycle
F1,336 = 191.878
< 0.0001
treatment
F2,9 = 15.535
0.0012
cycle*treatment
F2,9 = 10.259
0.0048
% variance
component
population[treatment]
33.8
cycle*population[treatment]
1.3
residual
64.9
*F-ratios were constructed based on the recommendations of ref. 8 (see section 12.7.3).
Table S4: Mating Affinity After 6 Experimental Cycles
model effect
test statistic
p-value
treatment
F2,14 = 1.65259
0.227
References
1 Tong, A. H. Y., Evangelista, M., Parsons, A. B., Xu, H., Bader, G. D., Pagé, N.,
Robinson, M., Raghibizadeh, S., Hogue, C. W. V., Bussey, H. et al. 2001 Systematic
genetic analysis with ordered arrays of yeast deletion mutants. Science 294, 2364-2368.
(doi:10.1126/science.1065810)
2 Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. 1994 New heterologous modules
for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10,
1793–1808. (doi:10.1002/yea.320101310)
3 Goldstein, A. L. & McCusker, J. H. 1999 Three new dominant drug resistance cassettes
for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541-1553.
(doi:10.1002/(SICI)1097-0061(199910)15:14<1541::AID-YEA476>3.3.CO;2-B)
4 Murphy, H.A. & Zeyl, C.W. 2012 Prezygotic isolation between Saccharomyces
cerevisiae and Saccharomyces paradoxus through differences in mating speed and
germination. Evolution 66, 1196-1209.
5 Rogers, D. W. & Greig, D. 2009 Experimental evolution of a sexually selected display
in yeast. P. Roy. Soc. Lond. B Bio. 276, 543-549. (doi:10.1098/rspb.2008.1146)
6 Wahl LM & Gerrish PJ. 2001. The probability that beneficial mutations are lost in
populations with periodic bottlenecks. Evolution 55, 2606–2610. (doi:10.1111/j.00143820.2001.tb00772.x)
7 Lenski, R. E., Rose, M. R., Simpson, S. C. & Tadler, S. C. 1991 Long-term
experimental evolution in Escherichia coli. I. Adaptation and divergence during 2,000
generations. Am. Nat. 138, 1315-1341. (doi:10.1086/285289)
8 Quinn, G. & Keough, M. 2002. Experimental Design and Data Analysis for Biologists.
Cambridge University Press, Cambridge, UK.