Supporting information
Title: Rapid adaptation of herbivore consumers to nutrient limitation: eco-evolutionary feedbacks to
population demography and resource control
Authors: Steven A.J. Declerck, Andrea R. Malo, Sebastian Diehl, Dennis Waasdorp, Kimberley
Lemmen, Konstantinos Proios and Spiros Papakostas
Appendix S1 Additional methodological information on the second stage chemostat set-up and the
analysis of rotifer cultures
Set-up of phytoplankton chemostats in the selection experiment
We used phytoplankton chemostats (volume: 1.2 L) with Chlamydomonas reinhardtii as food source
for the rotifer populations (Fig. S1). We manipulated phytoplankton stoichiometry by varying the
nutrient concentrations in the medium. The medium for the P-replete cultures (HP) consisted of WCmedium with 0.1 mmol L-1 K2HPO4 and 0.48 mmol L-1 NaNO3. The P-limited cultures (LP) received
medium composed of WC-medium with 0.02 mmol L-1 K2HPO4 and 2 mmol L-1 NaNO3. Both cultures
received 300 mL day-1 of fresh medium and equal light.
Figure S1 Photograph of the second stage chemostat system with a phytoplankton chemostat at the
top left and four rotifer reactors at the bottom.
Collection and culturing of rotifer genotypes prior to selection experiment
We incorporated two different pond origins in the design of our experiment to increase the
generality of our results. We wanted to increase the robustness of our conclusions by avoiding that
our results would be contingent on the genetic architecture of one single population. The distance
between the ponds spans approximately 20 km. ‘Pond 7’ (Lat.: 51.854065°, Long.: 5.893175°) is
situated in the flood plain of a branch of the lower River Rhine near the city of Nijmegen and has a
surface area of about 10 ha. ‘Pond 22’ (Lat.: 51.985263°, Long.: 5.665691°) is located at the campus
of Wageningen University, Wageningen and has a surface of approximately 0.25 ha. These two ponds
are not hydrologically connected and their B. calyciflorus rotifer populations were found to have
moderate levels of genetic differentiation at assumedly neutral microsatellite markers (FST: 0.158; P =
0.001, 10000 permutations; see Tables S2, S3 and S5 for microsatellite data). We refer to Appendix
S3 for more details on the development of the microsatellite markers.
Resting eggs were separated from pond sediments using a sugar flotation technique (Gómez and
Carvalho, 2000) and hatched under light in Petri dishes in distilled water. Upon hatching, B.
calyciflorus females were individually transferred into cell culture plates with 2 ml culture medium.
Culture medium consisted of C. reinhardtii. The volume of the cultures was progressively increased
over the span of one to two weeks to reach 40 ml. Meanwhile, cultures were also divided in two
duplicates. Clonal cultures were maintained by transferring each week about half of the culture to a
clean tube with 20 ml of fresh culture medium.
Set-up of rotifer chemostats in the selection experiment
A convenient property of the rotifer reactors in our second stage chemostat system is that they had
two ports for medium input, one for food and one for additional medium. Despite the fact that
Chlamydomonas concentrations in the phytoplankton chemostats differed among treatments (see
above), the two input ports allowed us to provide equal supply rates of food to the rotifer reactors of
the different food quality treatments. This was accomplished by blending Chlamydomonas from the
first stage chemostats with nutrient-free medium to an incoming food concentration of 1550 µmol C
L-1 while keeping dilution rates equal (i.e. 0.19 day-1).
We inoculated the rotifer chemostats twice with the same sets of clones, once on the 14/15th of
August (day 0 and day 1 of the experiment) and once on the 26/27th of August 2013 (day 12 and day
13). We performed a second inoculation in an effort to reduce initial loss of genetic diversity at low
population densities. Clones were represented each by 10 individuals per chemostat during the first
inoculation and by 25 individuals during the second inoculation. The dilution medium consisted of
WC-medium without N and P. The dilution regime was started on 28 August 2013 (day 14). Soon
after, we noticed a systematically lower population density in the wash-out than within the
chemostats, indicating avoidance of the chemostat outlets by the rotifers. For this reason, we
decided to dilute the rotifer chemostats manually by removing 20% of the medium on a daily basis
using a syringe.
Rotifer sample analysis
Instead of counting rotifer cultures microscopically, we used a FlowCam. A FlowCam is an automatic
imaging device (Sieracki et al. 1998) that captures and stores individual images of particles. This
allows processing larger volumes of sample per unit of time and effort, higher numbers of particles
counted and therefore more reliable density estimates, especially for the rarer objects of interest
(e.g. resting eggs and males). We operated our FlowCam with a syringe pump to allow maximum
control over sample processing speed and volume. We used a ‘field of view’ (FOV) flow chamber of
300µm depth. Pictures were taken at sample flow rates of 45 ml hour-1 with a frequency of 17 s-1 and
an optical magnification of 4. Pictures of particles with a radius above 50µm were all visually
inspected, identified and counted by a trained person.
Estimates obtained with the FlowCam correlated well with conventional microscopic counts (r=0.83;
P<0.0001; n = 71) although we discovered that the FlowCam method resulted in a systematic
underestimation of rotifer densities compared to microscopic counts. We accounted for this bias
using the regression equation: manual counts = 1.68058 * FlowCam counts.
References
Gómez, A. & Carvalho, G.R. (2000). Sex, parthenogenesis and genetic structure of rotifers:
microsatellite analysis of contemporary and resting egg bank populations. Mol. Ecol., 9, 203–214.
Sieracki, C.K., Sieracki, M.E. & Yentsch, C.S. (1998). An imaging in-flow system for automated analysis
of marine micro-plankton. Mar. Ecol. Prog. Ser.,168, 285−296.
Appendix S2 Stoichiometric seston quality in the phytoplankton and rotifer chemostats of the
selection experiment
Phytoplankton biomass in the phytoplankton chemostats ranged between 5 and 15 mmol C L-1 in the
LP treatment and between 5.6 and 8.5 mmol C L-1 in the HP treatment (Fig. S2). The stoichiometric
quality of the phytoplankton food source differed consistently between treatments throughout the
course of the selection experiment. Average molar C:P equaled 414 and 61 in the LP and HP
chemostats (Fig. S3a), whereas average molar N:P ratios equaled 40 and 4 in the HP chemostat,
respectively (Fig. S3c). Dissolved P remained below detection level in both the LP and HP treatments.
20
HP selection
LP selection
-1
Phytoplankton biomass (mmol L C)
The elemental ratios of the seston in the rotifer chemostats differed consistently between
treatments and reflected strongly the differences in the phytoplankton chemostats throughout the
course of the selection experiment (Fig. S3). Average molar C:P equaled 320 and 72 in the LP and HP
chemostats (Fig. S3b), whereas average molar N:P ratios equaled 59 and 11 in the HP chemostats,
respectively (Fig. S3d).
15
10
5
0
0
50
100
150
200
250
300
Time (days)
Figure S2 Temporal dynamics of the total carbon biomass of C. reinhardtii in the phytoplankton
reactors of the second stage chemostat system during the selection experiment. Circles refer to the
P-replete chemostat whereas triangles refer to the P-limited chemostat.
(a)
800
600
C:P of algae
600
C:P of algae
(b)
800
400
400
200
200
0
0
0
50
100
150
200
250
0
300
50
(c)
200
250
300
250
300
(d)
80
60
60
N:P of algae
N:P of algae
150
Time (days)
Time (days)
80
100
40
20
40
20
0
0
0
50
100
150
200
Time (days)
250
300
0
50
100
150
200
Time (days)
Figure S3 (a-d) Temporal dynamics of the elemental composition of C. reinhardtii in the
phytoplankton and of seston in the rotifer reactors of the second stage chemostat system during the
selection experiment. C:P ratio of C. reinhardtii in the phytoplankton reactor (a), seston C:P ratio in
the rotifer reactor (b); N:P ratio of C. reinhardtii in the phytoplankton reactor (c), seston C:P ratio in
the rotifer reactor (d). Circles refer to the P-replete chemostat whereas triangles refer to the Plimited chemostat. Due to methodological problems we were unable to obtain reliable data on N
during the first 90 days of the experiments.
Appendix S3 Microsatellite primer development and application
We developed two microsatellite multiplex panels for B. calyciflorus. For this, we used a total of
approximately 300 rotifers from a randomly selected clonal line. DNA from these rotifers was
extracted with the NucleoSpin® Tissue kit (Macherey-Nagel). Upon testing for DNA quality,
construction of the 454 library and 454 GS FLX genome sequencing (Roche Applied Science,
Penzburg, Germany) were performed according to the manufacturer’s protocol at the Cytogenetics
and Genome Research laboratory of the University of Leuven (Belgium). The 454 run resulted in
473744 DNA sequence reads with an average length of about 393 nucleotides. The raw reads were
imported into the QDD v.2.1 pipeline (Meglecz et al. 2010), a tool for microsatellite detection and
primer design. We used the software with the default settings, omitting mononucleotide repeats.
We selected 24 candidate loci that would give the highest chance of polymorphism (e.g. bi- and trinucleotide repeats) and that would have product sizes and primer sequences that would help
generate multiplex panels. Upon testing these candidate loci for successful PCR amplification and
polymorphism using rotifers from multiple clonal lines, we developed two multiplex panels of six
microsatellite loci each, named SSR1 and SSR2.
Microsatellite genotyping
We applied the newly developed primers to genotype the clonal lines used to inoculate the rotifer
chemostats of the selection experiment. We also applied the same procedure to monitor the
chemostat populations during the course of the selection experiment. Rotifer genotyping was
performed on DNA extracts from single rotifers using the HotSHOT method (Montero-Pau et al.
2008). In each panel we used, for a 5 μl PCR reaction, 0.5 μl template DNA, 0.1 μl from each primer
(from 10 pmol/μl concentration), 2.5 μl Master Mix of the QIAGEN Multiplex PCR kit (2× stock
concentration), and 0.8 μl Milli-Q water. PCR thermocycler conditions involved an initial denaturation
of 95 oC for 15 min required to activate the HotStarTaq DNA polymerase of the QIAGEN Multiplex
PCR kit. The next step was 30 cycles of 30 sec at 95 oC, 90 sec at 56 oC, and 60 sec at 72 oC. A final
elongation step of 30 min at 60 oC completed the amplification. The PCR amplicon was then diluted
1:20 with Milli-Q water and 1 μl from this dilution was then mixed with 8.8 μl of formamide and 0.2
μl of GeneScanTM 500 LIZTM size standard (Applied Biosystems, CA) prior loading 1 μl to an ABI Prism
3130 DNA Analyzer (Applied Biosystems, CA). Samples were run for 30 min at 15000 voltage using 36
cm capillaries. Allele calling was performed with the software GeneMapper® v. 4.0 using GS500 (250) LIZ as an option for size standard. Peak threshold was set to 50 rfu for each dye but two
different users also did manual inspection and correction, when necessary, of the allele calls.
Multilocus genotype (MLG) assignment and analyses of genetic relationship were conducted with
GenoDive v.2.b27 (Meirmans & Van Tienderen 2004), a program designed for the analysis of genetic
diversity of clonal organisms. To determine the MLG of clonal lines to be inoculated in the
chemostats of the selection experiment, three to five rotifers per culture were genotyped.
Results for clonal lines originally inoculated in the selection experiment
Table S1 shows the nucleotide sequences of the microsatellite primers, the approximate size of the
expected PCR products, and the number of alleles observed for the total of clonal lines analyzed. The
MLGs of the clonal lines are shown in Tables S2 and S3. Some clonal lines exhibited two different
MLGs. For example, in the clonal line BE from ‘Pond 7’, five rotifers were genotyped and two
different MLG’s were observed, differing at the genotype of the microsatellite loci A14, 96/96 vs.
96/99, and A15, 87/87 vs. 87/90 (Table S2). Most likely this is the result of selfing as these MLG’s
always had the lowest clonal distance and the highest kinship coefficient with each other than with
any of the other MLG’s from the same pond (for example, see Table S4). In these cases, both MLGs
were kept as representatives of the particular clonal line and they were denoted with different
numbers (Tables S2 and S3).
During MLG assignment, we omitted information from three loci: (1) locus A3, because of the
frequent presence of stutter bands that made genotyping difficult, (2) locus A10, because we found
evidence for the presence of null alleles, and, (3) in the case of ‘Pond 22’, from the locus A4, because
of too many missing data (Tables S2 and S3). Regardless, the polymorphism present in the rest of the
loci was adequate to distinguish each of the clonal lines introduced in the selection experiment.
Table S1 Primer sequences, motifs, approximate estimated size (in base pairs, bp) of the PCR
product, and number of observed alleles in the clonal lines for the developed microsatellite loci.
Locus
name
Panel
Primer sequence (5’  3’)
Motif
PCR product
(bp)
Alleles
found
(CAA)9
149
5
(AG)7
100
6
(AG)7
161
3
(AC)7
109
3
(TGT)6
96
4
(TAT)6
90
6
(TC)8
125
3
(TGA)8
105
5
(AT)7
181
5
F: AAATGGGCATAAAACGCAAG
A1
R: TTCGCGACGAGTTTGTATTG
F: AAGCTGTCAACATTGATTTGTTAAA
A5
R: CCAAATGTATCCTTCAGGTCA
F: AGTTTTGCCTTCAAATTGTATCA
A9
R: CTTGTACCCTTGCAATTAACAAC
SSR1
F: TTATCTCAACGCCGTTTTGT
A11
R: TCAAAACCATTGAATCTAGAAGAA
F: TGATATTGAGTTGGCATTGGC
A14
R: GCACACCTCAAGCTCAAACA
F: AATCGGTACCATTCGTGTCC
A15
R: TTGGTTTATTTAAAATCGAGTGATGA
F: TGTCTCGTATAGATTAGTTCGACTCC
A3
R: CGCTTTAGAAATTGCATTTAGGA
F: CGATGTCATTGATGAAGAATCATTT
A4
SSR2
R:
GAAAATACAGATCAATCAGAATCACC
F: CATTGGTAATTCCCGAATTTTG
A7
R: TCTTCTTTCCTTAAAGATCAAGTGT
F: CGGATTACACAACCATCGAG
A8
(AAT)7
238
5
(TTG)7
262
5
(AT)7
145
3
R: TTGGCCTCATCGTCTAAATTG
F: TTGTTGGGTTGAATTGACCA
A10
R: TGGATGCCATGTTACAAATGA
F: TCACGGTCTATTTGGAAACCA
A12
R: GGAATTTCGCAAGGCACTTA
Table S2 Multilocus genotypes (MLG) for the clonal lines established from the ‘Pond 7’. The size in base pairs (bp) of each allele is separated with “/”.
Grey-shaded cells indicate loci that have not been used for clonal assignment of the rotifer samples from the chemostats due to the presence of many
stutter bands (=locus A3) or of null alleles (=locus A8).
SSR1
SSR2
Culture
Name
A1
A5
A9
A11
A14
A15
A3
A10
A4
A12
A8
A7
A
143/146
100/100
161/163
109/111
96/99
87/90
121/121
262/268
99/105
147/147
232/235
183/183
H
143/146
96/100
161/163
111/111
96/99
87/90
121/121
262/268
93/105
147/149
229/238
181/181
K
146/146
96/96
163/163
109/109
96/96
87/90
121/121
268/268
99/105
147/147
214/229
181/181
P
146/146
98/98
163/163
109/111
96/99
87/87
121/121
268/268
96/96
147/147
232/232
181/181
S
146/146
98/100
163/165
111/111
96/96
84/90
121/121
262/268
96/105
147/147
235/235
181/181
AD
143/146
98/98
163/163
111/111
99/99
90/90
123/123
262/268
96/96
147/147
238/238
181/181
AE
146/146
96/98
161/163
111/111
96/99
87/87
123/123
262/268
96/105
149/149
238/238
181/183
AG
146/146
96/100
161/163
111/111
96/99
87/90
123/123
268/268
96/102
147/151
238/238
181/187
BD
146/146
96/100
163/163
109/111
96/96
90/93
123/123
262/268
96/105
147/149
235/235
181/183
BE1
143/143
96/96
161/161
109/113
96/99
87/90
123/123
262/268
105/105
149/151
238/238
181/183
BE2
143/143
96/96
161/161
109/113
96/96
87/87
123/123
262/268
105/105
149/151
238/238
181/183
BM
146/146
98/100
161/163
111/111
96/99
87/87
123/123
265/268
105/105
147/149
229/238
181/181
BZ
143/143
96/96
163/163
109/111
96/96
87/96
125/125
262/268
93/105
147/149
229/235
181/187
CW
140/146
96/96
163/163
109/111
96/99
87/90
123/123
Note: For clonal line BE we detected two different MLG’s, possibly as the result of selfing.
262/268
93/93
147/149
235/235
181/183
Table S3 Multilocus genotypes for the clonal lines established from the ‘Pond 22’. The size in base pairs (bp) of each allele is separated with “/”. Grey-shaded
cells indicate loci that have not been used for clonal assignment of the rotifer samples from the chemostats due to the presence of many stutter bands (=locus
A3) or of null alleles (=locus A8), or of many missing data (= locus A10).
SSR1
SSR2
Culture
Name
A1
A5
A9
A11
A14
A15
A3
A10
A4
A12
A8
A7
E
134/143
96/98
163/163
109/109
96/96
87/93
123/125
259/268
93/93
147/147
232/238
179/185
M
134/143
96/100
161/163
109/111
96/99
90/90
121/125
268/268
96/105
147/149
229/238
183/185
P
143/143
96/96
163/165
109/111
99/99
90/90
125/125
262/271
96/105
147/147
232/238
185/185
Q
143/149
96/100
161/163
109/111
99/99
90/90
123/125
262/268
93/93
147/147
232/238
179/185
U
134/143
96/96
165/165
109/111
99/99
87/90
121/125
262/268
93/93
147/147
232/238
185/187
AD1
134/143
96/98
163/163
109/111
96/99
90/90
123/123
259/268
96/105
147/147
238/238
187/187
AD2
134/143
98/98
163/163
109/111
96/99
90/90
123/123
259/268
96/105
147/147
229/238
185/187
AE
134/143
96/96
161/163
109/111
96/99
81/96
123/125
259/268
96/96
147/149
232/232
181/185
AO
134/143
96/100
163/163
109/111
99/99
90/90
125/125
262/268
93/102
147/147
238/238
185/185
AQ
134/134
96/96
163/163
111/111
99/99
90/90
125/125
259/268
96/96
147/147
232/232
185/185
AZ
143/143
96/98
161/161
109/111
96/99
87/87
121/125
262/262
93/102
147/147
238/238
181/187
BA1
134/143
96/100
161/163
111/111
96/99
87/93
121/125
262/262
96/102
147/149
232/238
181/185
BA2
143/143
96/100
161/163
111/111
96/99
87/93
121/125
262/271
96/102
147/149
232/238
181/185
BC1
134/143
96/96
161/165
109/109
99/99
87/90
125/125
259/268
96/96
147/147
232/238
185/185
BC2
134/143
96/96
161/165
109/109
99/99
87/90
125/125
259/259
96/96
147/147
232/238
185/185
BE
134/143
94/96
161/163
109/109
99/99
90/90
121/125
262/268
96/102
147/147
238/238
185/187
BF
134/143
96/96
161/163
109/109
96/99
90/90
125/125
262/268
93/96
147/147
232/238
185/187
BG1
143/146
98/98
165/165
109/109
96/99
93/93
125/125
262/268
93/93
147/147
232/238
185/185
BG2
143/146
98/100
165/165
109/109
96/99
93/93
123/125
262/268
93/93
147/147
238/238
185/185
BH
146/146
96/96
163/163
109/111
96/96
93/93
123/123
259/262
93/96
147/147
232/238
187/187
BJ1
134/143
96/96
163/165
109/109
96/99
87/90
125/125
262/262
102/105
147/147
232/238
185/185
BJ2
143/143
96/96
163/165
109/109
96/99
87/90
125/125
262/268
102/105
147/147
232/238
185/185
BL
143/143
96/96
161/161
109/109
99/99
81/81
125/125
259/262
93/93
147/147
232/238
185/185
BM1
143/143
96/96
163/163
109/109
96/99
81/81
123/125
262/268
96/102
147/147
232/235
181/181
BM2
143/143
96/96
163/163
109/109
96/99
81/81
125/125
262/268
96/96
147/147
235/235
181/185
BO
134/134
96/96
163/163
109/111
96/99
81/90
125/125
268/268
93/102
147/147
238/238
183/185
BP
143/143
94/98
163/163
109/109
99/99
90/93
125/125
262/262
96/99
147/147
238/238
181/185
BQ
134/143
96/98
163/163
111/111
99/99
90/90
123/123
262/262
93/96
147/147
238/238
179/181
BZ1
146/146
100/100
163/163
109/109
93/99
93/93
125/125
262/271
93/99
147/147
238/238
185/187
BZ2
134/146
100/100
163/163
109/109
93/99
93/93
125/125
262/271
93/93
147/147
238/238
185/187
CA1
143/143
96/100
163/165
109/109
96/96
81/81
121/121
262/268
93/96
147/147
232/238
181/185
CA2
134/143
96/100
163/165
109/109
96/96
81/81
121/121
262/268
93/96
147/147
232/238
181/185
CE
134/146
96/98
165/165
109/109
96/99
87/90
125/125
262/268
93/93
147/147
235/238
181/185
CH
143/143
98/100
163/163
109/109
96/96
81/93
123/125
259/259
93/93
149/149
232/238
179/179
CJ
143/143
96/96
161/161
109/111
96/96
90/90
123/125
268/268
96/96
147/147
238/238
181/181
CK
134/143
96/96
163/163
111/111
90/96
90/90
123/125
259/268
96/102
147/147
238/238
181/185
CM
143/143
96/108
163/165
109/111
96/96
87/90
123/125
262/268
93/102
147/147
235/235
179/181
CN
143/143
96/96
163/163
109/111
96/96
81/90
125/125
262/268
102/102
147/147
238/238
181/181
Note: In each of the clonal lines AD, BA, BC, BG, BJ, BM, BZ and CA we detected two different MLG’s, possibly as the result of selfing.
Table S4 Clonal distances and kinship coefficients among the MLG from clonal line BE1 and all other
MLG’s from Pond 7. Clonal distance was lowest with the MLG of BE2. Similarly, the kinship coefficient
with that MLG was much higher, suggesting that BE1 and BE2 resulted from selfing.
BE1
Culture
name
Clonal distance
Kinship coefficient
A
42
0.106
H
44
0.243
K
74
0.041
P
67
-0.232
S
55
-0.184
AD
47
0.064
AE
28
0.339
AG
42
0.224
BD
44
-0.043
BE1
-
-
BE2
6
1.006
BM
39
0.163
BZ
51
0.185
CW
46
-0.088
Table S5 Multilocus genotypes of clones that were additionally genotyped for the assessment of the degree of population genetic differentiation between
Pond 7 and Pond 22. Clones were obtained from resting eggs of ‘Pond 7’ and ‘Pond 22’. Fst-values were calculated from these genotypes combined with
those of the established clonal lines (see Tables S2 and S3). The size in base pairs (bp) of each allele is separated with “/”.
Sample
Name
SSR1
A1
A5
A9
SSR2
A11
A14
A15
A3
A10
A4
A12
A8
A7
Pond 7
7R
146/146
98/100
163/165
111/111
96/96
84/90
123/123
262/268
96/105
147/147
235/235
181/181
7V
146/146
98/100
163/165
111/111
96/96
84/90
121/121
262/268
96/105
147/147
235/235
181/181
7X
146/146
98/100
163/165
111/111
96/96
84/90
121/121
262/268
96/105
147/147
235/235
181/181
7AA
146/146
98/100
163/165
111/111
96/96
84/90
121/121
262/268
96/105
147/147
235/235
181/181
7AB
146/146
98/100
163/165
111/111
96/96
84/90
123/123
262/268
96/105
147/147
235/235
181/181
7AC
146/146
98/100
163/165
111/111
96/96
84/90
121/121
262/268
96/105
147/147
235/235
181/181
7AH
143/146
96/98
163/165
109/111
96/99
87/87
123/123
268/268
96/105
147/149
232/238
181/185
7AI
143/146
96/98
163/165
109/111
96/99
87/87
121/121
268/268
96/105
147/149
232/238
181/185
7AJ
143/146
96/98
163/165
109/111
96/99
87/87
123/123
268/268
96/105
147/149
232/238
181/185
7AK
143/146
98/98
163/163
109/111
99/99
87/87
123/123
268/268
93/96
147/147
232/238
181/181
7AL
134/146
100/100
163/163
109/109
96/99
90/93
121/121
262/268
93/96
147/149
235/235
181/181
7AM
146/146
100/100
163/163
109/111
99/99
87/90
123/123
268/268
102/105
149/149
229/235
181/185
7AN
134/146
98/98
163/163
109/111
96/96
87/90
123/123
262/262
102/105
147/147
null
179/179
7AP
134/143
96/98
163/165
109/111
93/99
81/87
121/121
262/268
93/99
147/147
238/238
181/181
7AS
146/146
96/96
163/163
111/111
99/99
87/87
123/123
262/268
99/105
147/149
235/235
181/181
7BK
143/146
98/100
163/163
109/109
96/99
81/96
123/123
262/268
93/99
147/147
229/229
181/181
7BN
146/146
98/100
161/163
111/111
96/99
87/87
123/123
265/268
105/105
147/149
229/238
181/181
7BS
134/146
96/96
163/163
111/111
96/99
87/90
123/123
268/268
96/105
147/147
235/235
181/181
7CB
143/146
96/96
161/161
109/111
99/99
87/90
123/123
268/268
96/105
147/149
235/235
181/181
7CQ
134/143
98/98
163/163
109/109
99/99
81/87
125/125
262/268
93/102
147/149
229/229
181/181
Pond 22
22A
134/143
96/96
161/163
109/111
96/96
81/93
121/121
259/268
93/96
147/147
238/238
181/185
22D
143/143
96/96
161/163
111/111
99/99
87/90
125/125
259/268
93/96
147/149
232/232
181/185
22F
134/143
96/96
163/165
109/109
96/99
90/90
123/123
259/268
93/99
147/147
232/232
181/185
22H
134/143
96/98
161/163
109/109
96/99
90/90
123/125
262/268
96/105
147/149
235/235
181/185
22I
134/143
96/98
163/165
109/109
93/96
87/93
123/125
259/268
93/96
147/149
238/238
185/185
22K
134/143
96/100
161/163
111/111
99/99
90/90
123/125
259/268
93/96
147/149
238/238
185/185
22L
134/143
96/102
163/165
109/111
96/96
90/90
123/123
262/268
93/99
147/149
232/232
179/181
22O
134/143
96/98
163/163
109/109
96/99
87/90
125/125
259/268
93/96
147/147
238/238
185/187
22T
134/146
96/98
163/165
109/109
99/99
81/93
121/123
262/268
93/102
147/147
238/238
181/181
22W
134/143
96/96
163/165
109/111
99/99
90/93
123/123
259/268
93/93
147/147
238/238
185/185
22AC
134/134
96/96
161/163
109/109
96/96
90/90
125/125
259/268
93/105
147/149
null
185/187
22AG
134/146
96/96
163/163
109/111
96/96
87/90
123/123
262/268
96/96
149/149
232/232
185/185
22AH
134/146
98/98
165/165
109/109
96/96
87/90
125/125
262/271
105/105
147/147
235/238
179/185
22AJ
143/143
96/96
163/163
109/109
96/96
90/90
121/125
262/262
93/93
147/147
238/238
185/185
22AL
134/134
96/100
161/163
109/109
93/99
90/93
125/125
262/268
93/93
147/147
238/238
185/187
22AP
134/143
100/100
161/163
109/111
96/99
90/90
123/123
259/268
96/102
147/149
232/232
181/185
22AW
134/143
96/96
163/163
109/111
96/99
90/90
121/121
262/268
93/96
147/149
238/238
185/185
22AY
134/143
96/96
163/163
109/111
93/93
90/90
121/125
259/262
93/93
147/147
238/238
181/185
22BD
134/143
96/96
161/161
109/109
96/96
90/90
125/125
262/268
99/105
151/151
null
185/185
22BK
134/143
100/100
161/161
109/109
93/93
90/90
121/121
259/268
96/96
147/147
232/232
181/181
22BS
134/143
96/100
161/163
109/109
96/96
93/93
121/125
262/271
93/93
147/149
238/238
181/183
22BT
134/143
96/96
165/165
109/109
99/99
90/90
121/121
262/268
102/102
147/147
232/238
187/187
22BW
143/143
96/96
163/165
109/111
99/99
90/93
121/121
259/268
93/93
147/147
238/238
185/185
22BX
134/143
98/98
163/165
109/109
96/96
90/90
125/125
262/268
93/102
147/149
232/232
181/187
22BY
134/143
96/100
161/163
109/109
96/99
90/93
123/123
262/268
93/96
147/147
null
181/185
22CB
143/143
94/96
161/163
109/109
99/99
90/90
121/125
262/268
93/96
147/149
null
185/185
22CC
143/146
96/96
163/163
109/109
93/96
93/93
121/121
262/268
93/99
147/147
238/238
185/187
22CD
143/146
96/98
163/163
111/111
96/96
87/90
121/125
262/268
93/105
147/147
238/238
185/187
22CG
143/146
98/100
161/163
109/111
99/99
90/90
121/125
262/268
93/96
147/149
232/238
185/187
22CI
134/143
96/96
163/165
109/111
96/99
90/93
123/125
262/271
99/102
147/147
238/238
185/187
22CL
134/143
96/96
161/163
109/109
96/99
90/90
123/125
262/268
96/96
147/147
232/238
181/185
22CO
143/143
96/100
163/163
109/111
96/99
90/90
125/125
259/268
93/96
147/149
238/238
185/187
References
1.Meglécz, E., Costedoat, C., Dubut, V., Gilles, A., Malausa, T., Pech, N., et al. (2010). QDD: a userfriendly program to select microsatellite markers and design primers from large sequencing projects.
Bioinformatics, 26, 403–404.
2.Meirmans, P.G. & Van Tienderen, P.H. (2004). GENOTYPE and GENODIVE: two programs for the
analysis of genetic diversity of asexual organisms. Molecular Ecology Notes, 4, 792–794.
3.Montero-Pau, J., Gómez, A. & Muñoz, J. (2008). Application of an inexpensive and high-throughput
genomic DNA extraction method for the molecular ecology of zooplanktonic diapausing eggs.
Limnology and Oceanography-Methods, 6, 218–222.
Appendix S4 Summary of linear mixed effects analyses for the selection experiment (see also Fig. 1
in manuscript). Effects of time and the food treatment are presented as the fixed components of the
models.
Fixed effect
SS†
MS†
d.f. †
F†
P†
Rotifer population density
Food
4.69
4.69
15
71.5
<0.001
Time
0.21
0.21
397
7.1
0.008
Food x Time
0.19
0.19
397
6.3
0.013
Food
5.61
5.61
49
63.4
<0.001
Time
0.27
0.27
99
6.2
0.014
Food x Time
0.20
0.20
99
4.5
0.037
Food
3.93
3.93
13
11.7
0.004
Time
67.28
67.28
397
100.9
<0.001
4.21
4.21
397
6.3
0.012
Residual food concentration
Per capita number of sexual eggs
Food x Time
†SS, sum of squares; MS, mean squares; d.f., degrees of freedom; F, F-ratio; P, P-level.
Appendix S5 Summary of linear mixed effects analyses for common garden transplant Experiment 2
(see also Figs. 2-4 in manuscript). Effects of the treatments Food, Selection history and Population
origin are presented as the fixed components of the models.
Fixed effect
SS†
MS†
d.f.†
F†
P†
Rotifer population performance
Steady state population biomass
Food
1.446
1.446
6
164.0
<0.001
Selection history
0.116
0.116
5
13.2
0.015
Food x Selection history
0.111
0.111
6
12.6
0.012
Population origin
0.067
0.067
5
7.6
0.040
Food
0.689
0.689
6
74.2
<0.001
Selection history
0.068
0.068
5
7.3
0.043
Food x Selection history
0.061
0.061
6
6.6
0.042
Population origin
0.055
0.055
5
5.9
0.060
Food
0.589
0.589
6
326.4
<0.001
Selection history
0.006
0.006
6
3.5
0.109
Food x Selection history
0.055
0.055
6
30.4
0.001
Food
0.235
0.235
38
34.9
<0.001
Selection history
0.028
0.028
6
4.1
0.088
Food x Selection history
0.041
0.041
38
6.1
0.018
Yield
Residual seston concentration
Rotifer demographic variables
Birth (b) and death (d) rates*
Per capita number of fecund asexual females
Food
3.634
3.634
38
29.1
<0.001
Selection history
0.406
0.406
6
3.3
0.121
Food x Selection history
0.472
0.472
38
3.8
0.059
3.509
3.509
38
9.2
0.004
22.492
22.492
6
58.7
<0.001
6.942
6.942
38
18.1
<0.001
11.142
11.142
38
41.0
<0.001
Selection history
2.594
2.594
6
9.6
0.021
Food x Selection history
2.291
2.291
38
8.4
0.006
0.424
0.424
38
229.5
<0.001
Food
0.087
0.087
38
10.4
0.003
Food
0.896
0.896
38
90.9
<0.001
Food
2.532
2.532
7
1646.2
<0.001
Population origin
0.020
0.020
6
12.8
0.012
Food
2.532
2.532
7
1646.2
<0.001
Population origin
0.020
0.020
6
12.8
0.012
Per capita number of sexual eggs
Food
Selection history
Food x Selection history
Per capita number of males
Food
Rotifer stoichiometry
C-content
Food
P-content
C:P
Seston stoichiometry
Seston C:P
Seston N:P
*Birth rate b and death rate d yielded identical analysis results
†SS, sum of squares; MS, mean squares; d.f., degrees of freedom; F, F-ratio; P, P-level. Degrees of
freedom for the food quality effect and its interactions with other factors vary among response
variables because replicates for seston stoichiometry and nutrient analyses were pooled before
analysis.
Appendix S6 Results of common garden transplant experiment 1
Experiment 1 only included treatments with a Pond 22 origin. The results from Experiment 2 that are
shown for comparison are therefore also from the treatments with a Pond 22 origin (treatment
means across both pond origins are reported in Figs. 2-4).
Figure S4 (a-c) Responses of performance related population variables to ambient food quality and
selection history in the two common garden transplant experiments. Only treatments with a Pond 22
origin are shown. Steady state rotifer population biomass (a) and yield (b), and residual seston
concentration (c). Circles are treatments with a P-replete selection history, triangles are treatments
with a P-limitation history. Ambient food quality treatments are: HP: food with high P-content; LP:
food with low P-content. Shown are means across chemostat origins ± 2 standard errors (n = 2). Note
the log-scale in (a).
300
(a)
0,14
Experiment 1
Experiment 2
Experiment 1
Experiment 2
0,12
0,10
100
90
80
70
60
0,08
Yield
Population biomass (µmol C L -1 )
200
50
0,06
40
30
0,04
HP-selected
20
LP-selected
10
1400
0,02
0,00
HP
LP
HP
LP
(c)
Experiment 2
1200
1000
800
600
400
200
0
HP
HP
LP
HP
Food quality
Experiment 1
Residual seston (µmol C L -1 )
(b)
LP
HP
Food quality
LP
LP
Figure S5 (a-d) Responses of demographic rotifer variables to ambient food quality and selection
history in the two common garden transplant experiments. Only treatments with a Pond 22 origin
are shown. Death rate (a), per capita number of fecund asexual females (b), per capita number of
sexual eggs (c) and males (d). Circles are treatments with a P-replete selection history, triangles are
treatments with a P-limitation history. Ambient food quality treatments are: HP: food with high Pcontent; LP: food with low P-content. Shown are means across chemostat origins ± 2 standard errors
(n = 2).
(a)
0,6
Experiment 1
Experiment 2
death rate d (day-1 )
0,4
0,3
0,2
0,1
HP-selected
LP-selected
Per capita number of fecund asexual females
0,5
0,0
LP
HP
Experiment 2
0,5
0,4
0,3
0,2
0,1
HP
LP
(c)
0,4
Experiment 1
Experiment 2
0,3
0,2
0,1
LP
HP
LP
(d)
Experiment 1
Per capita number of males
Per capita number of sexual eggs
Experiment 1
0,0
HP
0,4
(b)
Experiment 2
0,3
0,2
0,1
0,0
0,0
HP
LP
HP
Food quality
LP
HP
LP
HP
Food quality
LP
Figure S6 (a-c) Response of rotifer elemental content and elemental ratios to ambient food quality
and selection history in the two common garden transplant experiments. Only populations with a
Pond 22 origin are shown. Rotifer C-content (a), rotifer P-content (b), molar rotifer C:P ratio (c).
Circles are treatments with a P-replete selection history, triangles are treatments with a P-limitation
history. Ambient food quality treatments are: HP: food with high P-content; LP: food with low Pcontent. Shown are means across chemostat origins ± 2 standard errors (n = 2).
20
(a) Experiment 1
0,18
Experiment 2
(b) Experiment 1
Experiment 2
0,16
HP-selected
Rotifer P content (nmol ind. -1 )
Rotifer C content (nmol ind. -1 )
LP-selected
15
10
5
0,14
0,12
0,10
0,08
0,06
0,04
0,02
0
0,00
HP
200
LP
(c) Experiment 1
HP
LP
Experiment 2
HP-selected
LP-selected
Rotifer C:P
150
100
50
0
HP
LP
HP
LP
HP
LP
HP
LP