Supporting online materials for “An experimental demonstration of ecological
character displacement using diversified Escherichia coli” by Tyerman et al.
Methods & Methods
Strain evolution and description
Ten replicate populations of E. coli B initiated from two isogenic lines (alternately)
kindly provided by R. Lenski, were evolved for 1000 generations in batch culture.
One isogenic line was ara- while the other was ara+, allowing us to plate on
Tetrazolium arabinose agar plates with an indicator dye to check for cross
contamination between lines(1). Test tubes were filled with 10 mL fresh media
that consisted of Davis Minimal Salts media (DM) supplemented with 250 g/mL
glucose and 575 g/mL acetate as the sole carbon sources. These resources
were selected as diversification in the presence of these resources has
previously occurred (2, 3). We selected these concentrations of glucose and
acetate to balance yields obtained by the ancestor.
Cultures were incubated at 37˚ and vigorously shaken (250 rpm) for 24 h.
At the end of each 24 h “season,” 100 L of culture was transferred to 10 mL of
fresh media (~1/100 dilution) and thus the seasonal cycle was reset. Each
season yielded log2100 = 6.7 generations. By generation 1000, two discernable
E. coli ecotypes, Fast switching (FS) and Slow-switching (SS) were identified in
all ten replicate populations.
1
Growth parameter extraction
Growth curves were obtained by inoculating 1.5 L of cell culture into 150 L of
fresh media in individual wells of a 96-well microplate. Microplates were grown in
a Biotek 808UI Optical Density reader, under similar conditions to the original
evolutionary environment (37˚, well shaken). Measurements consisted of optical
densities (OD, 600 nm) obtained every 10 min over the course of 24 h. Data files
were converted to a usable format using Microsoft Excel, and growth curve
parameters were extracted with a program written in object oriented C++.
Table S1 summarizes the parameters extracted from growth curves (in
addition to the parameters mentioned in the main text). rmaxace was found by
calculating the slope of the growth curve, for all points between the Switching
Point, SP, and the final measurement, using a sliding window algorithm, that
calculated the linear slope over 9 data points (modified from 2). The switching
lag, lagace was calculated as the total time from the SP to the time point that
corresponded to rmaxace.
The ODmax was simply the highest OD obtained in a run.
Character Displacement experiment
i. Competitive Release
Fifteen samples were inoculated into 3 mL fresh media for 24 h (37˚, 250
rpm) from frozen stock of population 18, isolated at the 1000-generation mark in
our evolution experiment. From each sample, we isolated a large and small
2
colony by plating the sample on Tetrazolium agar plates. We treated each
sample as an independent replicate of Population 18. We felt this was
appropriate because, in the past, initiating cultures from frozen stock has resulted
in variable ratios of SS and FS ecotypes. Further, this allows us to conduct
paired statistical analyses (see below). Each large and small colony selected
above, was individually used to inoculate 3 mL of Davis Minimal media enriched
with glucose and acetate (DMGA) as the only carbon sources. After 24 hours,
~1.5 L of each culture was inoculated into a single well containing 150 L
evolutionary media of a 96-well microplate (~1/100 dilution), and was grown up in
a microplate optical density reader (Biotek 808UI) for 24 h at 37˚ and high
shaking. The growth profiles from all cultures confirmed that thirteen of fifteen
pairs of colony-size variants (i.e., large and small) reflected the correct SS/FS
ecotype (the remaining two pairs had small colony variants that corresponded to
the SS ecotype, and were thus discarded). Even though growth curves were
measured on ecotypes grown in isolation (i.e., no competition), we took the initial
growth parameter values as those reflecting the sympatric condition of the
ecotype. Because the comparison will be made to the allopatric values (see
below) this is a conservative estimate). All cultures were propagated in isolation
(allopatry) for about 50 generations by transferring 1/100 of the culture to fresh
media every 24 hours for 7 days, using a 96-well microplate and the microplate
reader. After 7 days, the values obtained from growth curves were taken to
reflect the allopatric condition (for ~50 generations) facing the ecotypes. Figure
S2a illustrates this protocol.
3
We plated all cultures on Tetrazolium agar plates every other day to
ascertain if contamination (indicated by the presence of the opposite colony size
variant) between wells occurred (none was detected).
ii.) Character divergence
From the final time-point in (i), which is recently allopatric, a single genotype (FS
or SS) was isolated and selected from Tetrazolium agar plates. These were
inoculated into 3 mL of fresh media, and grown for 24 h (37˚, 250 rpm), to give
the growth parameter values for that genotype. In addition to initiating pure
cultures of each ecotype, pairs of ecotypes were mixed to create a sympatric
treatment. The FS and SS were selected from cultures that were initially paired at
the outset of the first phase of the experiment. The mixture was initiated by
mixing 50% of each ecotype (by volume) for each replicate pair into a single
source culture. This source culture was used to inoculate a single well of a 96well microplate. The mixed cultures were serially propagated for 7 days (as
above). At the final time period, mixed cultures were plated out, so individual FS
and SS ecotypes could be isolated and their growth parameters determined.
Figure S2b illustrates this protocol.
Statistical analysis
Paired t-tests were conducted for all three traits, for both ecotypes (FS and SS) for both
phases of the experiment. The paired difference for each trait was calculated, and the
distributions were tested for normality. If normal, then a parametric t-test was conducted
to test for departures from zero (i.e., no difference). If the distribution of paired
4
differences was not normal, then signed rank tests were employed. Because character
displacement usually makes the prediction of less extreme values in allopatry (i.e., less
trait exaggeration) and more extreme values in sympatry (but see 4), we’ve employed one
tailed tests throughout. All statistics were done with SAS JMP 4.0.
Results
During phase (i), competitive release, the distributions of the paired trait
differences were found to differ from normality for the lagace difference (ShapiroWilk test, W=0.503237, p<0.0001), but not for rmaxace (Shapiro-Wilk test,
W=0.877849, p<0.0650) and ODmax (Shapiro Wilk test, W=0.900246, p<0.1314).
Accordingly, we conducted a one-tailed non-parametric signed-rank test on lagace
(p<0.005), and one-tailed parametric t-tests on rmaxace (t=-2.8010, df=12,
p=0.0160) and maxOD (t=-1.6585, df=12, p=0.1231).
Figure S3 is raw data for lagace and Figure S4 is the raw data for ODmax.
5
Table S1 Parameters extracted from growth curves.
Parameter
lagglu
rmaxglu
SP
Explanation
Initial lag, until until rmaxglu
Maximum growth rate during “glucose phase” of
diauxie.
Switching Point (time) from glucose to acetate
phase.
ODSP
Optical Density of switching point.
Lagace
Switching lag from glucose to acetate growth
rmaxace
Maximum growth rate during “acetate phase” of
diauxie.
ODmax
Yield, or maximum OD
6
Supplemental Online Material - Figure captions
Figure S1. Illustration of batch culture evolution experiment, consisting of serial
transfers for 150 days.
Figure S2. Protocols for Competitive release. See text for further explanation.
Figure S3. Protocols for Character divergence. See text for further explanation.
Figure S4. Character displacement for lagace (a) when competitors were
removed (sympatry to allopatry) and (b) when competitors were added (allopatry
to sympatry). Filled triangles are SS ecotype, open triangles are FS ecotypes.
Figure S5. Character displacement for ODmax (a) when competitors were
removed (sympatry to allopatry) and (b) when competitors were added (allopatry
to sympatry). Filled triangles are SS ecotype, open triangles are FS ecotypes.
Figure S6. The proportion of Slow-switching (SS) during competition phase of
character displacement.
7
References and notes
1.
2.
3.
4.
R. Lenski, M. Rose, S. Simpson, S. Tadler, Am Nat 138, 1315-41 (1991).
M. L. Friesen, G. Saxer, M. Travisano, M. Doebeli, Evolution 58, 245-60. (2004).
J. G. Tyerman, N. Havard, G. Saxer, M. Travisano, M. Doebeli, Proc R Soc Lond.
B 272, 1393-8 (2005).
P. A. Abrams, Theor Pop Biol 29, 107-60 (1986).
8
Figure S1
9
Figure S2
10
Figure S3
11
a
ALLO
SYM
0
20
40
60
80
100
120
80
100
120
lagace
b
ALLO
SYM
0
20
40
60
lagace
Figure S4
12
a
ALLO
SYM
0.15
0.20
0.25
0.30
0.35
0.40
0.30
0.35
0.40
ODmax
b
ALLO
SYM
0.15
0.20
0.25
ODmax
Figure S5
13
0
1
2
3
Time (days)
4
5
6
7
Figure S6
14
0.0
0.2
0.4
0.6
Proportion SS
0.8
1.0