Rapid evolution of highly variable competitive abilities in a key phytoplankton species

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1 SUPPLEMENTARY Brief Communication INFORMATION In the format provided by the authors and unedited. Rapid evolution of highly variable competitive abilities in a key phytoplankton species Lennart T. Bach 1 *, Kai T. Lohbeck 1,2, Thorsten B. H. Reusch 1 and Ulf Riebesell 1 1 GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel, Germany. 2 Present address: Department of Marine Sciences, University of Gothenburg, Göteborg, Sweden. * lbach@geomar.de Nature Ecology & Evolution Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

2 1 Title: Rapid evolution of highly variable competitive abilities in a key phytoplankton species 2 3 Supplementary Tables Table S1. Summary of the statistical analysis. The statistical analysis was conducted as 2x2 factorial design with the fixed factors selection and complex environment, in combination with a sub-replication (n=3) at the level of independent long-term lineages, permitting to compare lineages (=replicates) within treatment combinations (the final abundance was log-transformed to meet the prerequisites of parametric statistical testing (i.e. variance homogeneity, normal distribution of residuals)). Neither the main factors of the complex environment (complex) nor the selection environment (select) or the interaction of both (complex*select) had a significant influence on E. huxleyi final abundance after 26 days in the complex environments. In contrast, the variation among replicates within treatment combinations, assessed as random factor nested in complex*select had a significant effect on the final abundance (P=0.001). The affiliation to a particular lineage explained 43.9 % of the variance between lineages after 26 days in the complex environments. 17 Nested analysis traditional least square variance component by nested factor MS df F P Var component % of total complex nested[complex,select]&random select residual complex*select total nested[complex,select]&random residual

3 Table S2. Microzooplankton abundance. Microzooplankton populations (mostly ciliates) were distinguished from other particles based on FSC-A and FL3-A/FL2-A (red/orange) fluorescence signal strength using the Cytosense imaging flow cytometer (Cytobuoy) which is specifically designed for the quantification of plankton in natural samples. The microzooplankton population in the cytogram had a slightly elevated red/orange fluorescence ratio and could therefore be distinguished from other particles. The population was photographed during the measurement to reassure that the particles in the gated population were ciliates. It should be kept in mind, however, that the gated population may only represent part of the microzooplankton present in the microcosms. Organisms with other scatter and fluorescence properties may have co-existed but not been included in the designated microzooplankton gate. Nevertheless, these measurements support our statement that microzooplankton grazers were present in all microcosms (see main text). Please note that the measurement is time consuming (10 minutes per sample) so that we could not determine the abundances in all 60 replicates on every sampling day. All abundances are given in cells ml -1. Day of experiment Environment Selection line Replicate Control Control Control Control Control Control Control Control Control Control Control Control Control Control

4 Control Control Control Control Control Control Control Control Control Control Control Control Control Control Control Control OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA

5 34 OA OA OA OA Table S3. Copepod abundance at the end of the experiment. Copepods were sampled on the last day of the experiment (day 26). Therefore, 550 ml of microcosm volume were filtered with a 55 µm (mesh size) net while emptying the bottles. The individuals collected on the net were transferred into 30 ml beakers and conserved with Lugol solution (1%) until quantification with a Leica stereomicroscope in a Bogorov chamber. Copepodites and smaller copepods (<1 mm) were identified mostly as Pseudocalanus acuspes or Oithona similis. Larger copepods (>1 mm) were mostly Pseudocalanus acuspes or Temora longicornis. All abundances are shown in individuals L -1. Environment Selection line Replicate Nauplii Copepodites Smaller copepods Larger copepods control control control control control control control control control control control control control control control control control control control control control

6 43 control control control control control control control control control OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA OA Supplementary Figures

7 Figure S1. Development of abundance and relative fluorescence of the 10 lineages during the 26 days challenge experiment. (A, B) Abundance. (C, D) Relative fluorescence. Each line represents the mean development of one lineage (average of triplicate incubations). Visualization of standard deviations were omitted to maintain clarity. Please note that standard deviations on days 12 and 26 are shown in the bar charts of Fig. 1C, D. Cool and warm colours show control and OA lineages, respectively. Subplots on the left (A, C) and on the right (B, D) show the developments in the control and the OA environment, respectively.

8 Figure S2. Initial growth rate vs. final abundance relationship. E. huxleyi growth rate at the beginning (average of days 0 4) of the experiment in the complex environment vs. E. huxleyi cell abundance at the last day (day 26) in the complex environment. µ was calculated as: (ln(t 4 )-ln(t 0 ))/4, where t 0 and t 4 are E. huxleyi cell abundances on day 0 and day 4, respectively. The figure legend shows the selection environment (first term) and the test environment (second term). For example, control->oa refers to lineages that were cultured for 1700 generations in the simplified control environment and then transferred into the complex OA environment. The positive correlation underlines that high growth rates at the onset of a bloom largely determined peak abundance 1.

9 Figure S3. Fitness of E. huxleyi lineages in the simplified environment in relation to final abundance or growth in the complex environment. Growth rates of the 10 E. huxleyi lineages in the simplified laboratory experiment after 1600 generations 2,3 (x-axis) vs. (A) cell abundance of the lineages after 26 days in the complex environments or vs. (B) growth rates of the lineages in the complex environments at the beginning of the experiment (days 0 4) where all lineages were growing exponentially (see Fig. S1). The figure legend shows the simplified selection environment (first term) and the test environment (second term). For example, control->oa refers to lineages that were cultured in the simplified control environment but growth rates were tested in the OA environment. The test environment was a simplified one in case of the growth rate data taken from the Lohbeck and Schlüter et al studies 2,3 (x-axis) or a complex environment for the experiments

10 73 presented here (y-axis). Neither of the two correlations is significant (p>>0.05), demonstrating that 74 growth rates determined in the simplified environment are poor indicators for growth rate and 75 bloom intensity in a complex environment. (Please note that Schlüter et al.8 determined growth rates 76 in the simplified test environment after 1600 generations of selection in the simplified environment 77 whereas the experiment in the complex test environment (this study) was performed after generations of selection in the simplified environment. Thus, it is important to keep in mind that the 79 correlations shown here include a 100 generations mismatch.) Figure S4. Experimental design. Step A: E. huxleyi cells were collected in Norway (Raunefjord) 82 and transported to Kiel, Germany. Step B: A single genotype was isolated from the E. huxleyi 83 assemblage. Five control and five OA lineages were established from this genotype and grown 84 exponentially for 3 years in semi-continuous batch cultures under simplified laboratory conditions2 85 (the selection environment). Step C: Natural plankton assemblages were collected after 3 months of 86 growth in a control and an OA mesocosm and split in 30 microcosms, respectively. (Step D) The 87 OA-adapted and control E. huxleyi lineages were incubated in the OA-acclimated and control

11 88 microcosms and attached to a plankton wheel for 26 days (i.e. the experiment in the complex 89 environments). The objective of the experiment in the complex experiment was to assess whether 90 adaptive evolution to a common stressor (in this case OA) in a simple laboratory environment is 91 associated with equivalent fitness gains in a complex natural environment Figure S5. Gating strategy in the flow cytometer analysis. (A) Population I could be distinguished 94 best from electronic noise on the FL3-A vs. FL4-A plot. (B) Several phytoplankton populations and 95 E. huxleyi were gated on the SSC-A vs. FL3-A plot. Plots (C) and (D) illustrate the differences in 96 forward scatter (FSC-A) and side scatter (SSC-A) characteristics of the E. huxleyi population. E. 97 huxleyi was close to, and in some samples indistinguishable from, population III on the FSC-A 98 channel but there was never overlap with any other populations when using the SSC-A channel for 99 gating.

12 Figure S6. Temporal development of ph. Blue and red lines show the average development of 5 control and 5 OA lineages, respectively. Shaded areas represent ± 1 standard deviations. ph was calibrated to the free scale with certified seawater standard as described by Bach et al 4. The slight ph increase over time was most likely due to autotrophic biomass build-up in the course of the experiment. Please note that the 10 lineages were accustomed to ph drift within this range as they experienced the same degree of ph drift in the semi-continuous batch culture during the laboratory adaptation phase in the simplified environments 2, Supplementary references 1. Riebesell, U. et al. Nat. Geosci. 10, (2017). 2. Lohbeck, K. T., Riebesell, U. & Reusch, T. B. H. Nat. Geosci. 5, (2012). 3. Schlüter, L., Lohbeck, K. T., Gröger, J. P., Riebesell, U. & Reusch, T. B. H. Sci. Adv. 2, e (2016). 4. Bach, L. T., Bauke, C., Meier, K. J. S., Riebesell, U. & Schulz, K. G. Biogeosciences 9, (2012).