Evolution in temperature-dependent phytoplankton traits revealed from a sediment archive: do reaction norms tell the whole story?

Abstract

The high evolutionary potential of phytoplankton species allows them to rapidly adapt to global warming. Adaptations may occur in temperature-dependent traits, such as growth rate, cell size and life cycle processes. Using resurrection experiments with resting stages from living sediment archives, it is possible to investigate whether adaptation occurred. For this study, we revived resting cysts of the spring bloom dinoflagellate Apocalathium malmogiense from recent and 100-year-old sediment layers from the Gulf of Finland, and compared temperature-dependent traits of recent and historic strains along a temperature gradient. We detected no changes in growth rates and cell sizes but a significant difference between recent and historic strains regarding resting cyst formation. The encystment rate of recent strains was significantly lower compared with historic strains which we interpret as an indication of adaptation to higher and more rapidly increasing spring temperatures. Low encystment rates may allow for bloom formation even if the threshold temperature inducing a loss of actively growing cells through resting cyst formation is exceeded. Our findings reveal that phenotypic responses of phytoplankton to changing temperature conditions may include hidden traits such as life cycle processes and their regulation mechanisms. This study emphasizes the potential of living sediment archives to investigate plankton responses and adaptation to global warming.

1. Introduction

Owing to their large census population sizes and short generation times, phytoplankton are expected to rapidly adapt to changing environmental conditions [1]. In the light of global warming, we investigate whether and how phytoplankton, here represented by the cyst-forming cold-water dinoflagellate Apocalathium malmogiense, made use of this potential within the past 100 years and responded to increasing sea surface temperatures (SST) in the Baltic Sea.

Temperature represents one of the important environmental variables essentially influencing phytoplankton productivity, phenology, range expansion and community composition [2–5]. Global warming may cause drastic changes in temperature-dependent phytoplankton traits including thermal reaction norms, cell size and life cycle transitions. Thermal reaction norms describe the performance or growth rate of organisms for a range of different temperatures; and due to global warming their shape may vary by shifting vertically, horizontally or by changing in width [6]. Laboratory studies using experimental evolution approaches for phytoplankton organisms confirmed that such adaptive changes in reaction norms can take place after 100–500 generations [7–9]. Elevated temperatures also cause a linear decrease in cell size in most phytoplankton species for the range of preferred growth conditions, and diverging responses in cell size for high sublethal temperature conditions [10]. Cell size is generally regarded as a trait with high adaptation potential; for example, in response to ocean acidification and nutrient limitation [1,11,12]. Thus, natural selection via increasing temperatures may lead to smaller cell sizes, in general, and changes in the cell-size response at high sublethal temperatures. Temperature is moreover known to affect phytoplankton life cycle processes such as resting stage formation and germination [13–16], and may thereby control the success of bloom formation [17]. It can be expected that such temperature-regulated life cycle processes are particularly sensitive to long-term temperature changes over times scales of global climate change.

Previous research on thermal adaptation of phytoplankton has concentrated on experimental evolution in laboratory and mesocosm studies and on field studies comparing organisms from different temperature environments [7–9,18,19]. However, sediment archives of living resting stages provide a so far unused opportunity to examine long-term responses of phytoplankton to temperature changes. Living sediment archives are formed by resting stages conserved in undisturbed, stratified sediments. Many phytoplankton species form such resting stages to outlast short term or seasonally unfavourable environmental conditions, and in some species resting stages are capable to stay alive for as long as 100 years [20–22]. Previous resurrection studies using sediment archives have focused on genetic patterns and phenotypic responses to salinity and pH changes of phytoplankton from different sediment layers [20,22–24]. Temperature-dependent traits of phytoplankton trapped in sediment archives have so far remained unexplored though.

For this study, we chose a site with a significant temperature trend over the past 100 years. In the cold-temperate Gulf of Finland (north Baltic Sea), global warming is particularly noticeable by an earlier break-up of sea ice. The onset of melting is now 10–15 days earlier compared with 1923 [25]; and consistent with global trends [26], SST re-analyses for the central Gulf of Finland from the BALTIC dataset [27] reveal an increase in spring SST by 0.8°C over the past 100 years. One of the most notable consequences of this development is an earlier onset of Baltic spring blooms [28].

The warming trend should particularly affect cold-adapted spring bloom phytoplankton species with a stenotherm temperature tolerance spanning approx. 10°C [29,30]. Stenotherm organisms may adapt faster than eurytherm ones, as their narrow temperature window usually implicates a steeper temperature tolerance curve and thus a higher phenotypic plasticity, which in turn favours evolutionary adaptation [31]. One of these stenotherm species is the dinoflagellate Apocalathium malmogiense, formerly known as Scrippsiella hangoei. This species belongs to a group of dinoflagellates that dominates the spring bloom in the Baltic Sea and whose seasonal dynamics is regulated by temperature-dependent life cycle processes [17]. Apocalathium malmogiense produces resistant asexual resting cysts when water temperatures increase above a threshold temperature [32]. The transition of a large fraction of the population to a benthic resting phase secures the survival of the species through periods of adverse warm conditions when growth cannot be sustained and the species disappears from the water column [15]. After a mandatory dormancy period of several months the cysts germinate at low temperatures [32], leading to the return of a large part of the encysted population to the water column in late winter/early spring. Apocalathium malmogiense cysts have a high long-term survival capacity and remain viable in century-old layers of Gulf of Finland bottom sediments [33]. These features make the species an ideal model organism to examine potential long-term responses of phenotypic traits to increasing temperatures in a living sediment archive.

Here, we use revived individuals of the spring-blooming dinoflagellate A. malmogiense from recent and 100-year-old sediment layers, and compare the temperature-dependent traits growth rate, cell size and cyst formation. This approach will allow us to detect trait changes that indicate adaptation of the organism to increasing temperatures. In addition, these data are useful for ecosystem modelling, specifically for long-term simulations that need to take into account changes in the functional relationship between temperature and key traits.

2. Material and methods

(a). Sediment processing

Sediment cores were collected onboard RV Aranda at HELCOM monitoring station LL7 in the Gulf of Finland (lat 59° 50.79, lon 24° 50.27, water depth 100 m) during a HELCOM monitoring cruise in February 2015. A gravity corer (Gemax) was used to retrieve three replicate cores from anoxic sediments. The cores were sliced into 1 cm slices and individual slices were stored cool and dark in plastic bags under oxygen-free conditions until further processing. The material of one of the three replicate cores was freeze-dried and used for gamma-spectrometric dating. Radioactive Pb- and Cs-isotope analyses were performed and the age of the sediment slices was estimated using the CSR model. The procedures have been described in detail in [33]. Model estimates showed that the surface layer of the sediment corresponds to an age of approximately 2 years, while 16 cm core depth was estimated to be roughly 100–110 years old. For the isolation of A. malmogiense strains, subsamples from both, the surface sediment layer and the 16 cm deep layer were processed as described in [33] to extract the dinoflagellate cyst fraction. In short, 2.5 ml of well-mixed sample material were suspended in 20 ml filtered sea water (FSW, 0.2 μm filter, 6 psu), sonicated for 30 s at 30% intensity using a Bandelin Sonoplus Ultrasonicator and sieved through a 70 μm sieve onto a 20 μm sieve using FSW. The resulting 20–70 μm fraction, potentially containing dinoflagellate cysts, was collected into a 15 ml centrifuge tube and stored dark and cool until germination experiments were set up.

(b). Culture establishment

The processed samples were microscopically examined for the presence of intact A. malmogiense resting cysts before sediment slurries were distributed into wells of 24-well tissue culture plates. Each well was filled with 500 μl of the sediment slurry and 1000 μl f/8-Si medium. The cyst slurries were placed in a temperature- and light-controlled culturing cabinet, and incubated at 4°C, 50–100 μmol photons m⁻² s⁻¹ for 16 L : 8 D cycle to allow for germination. After six weeks of incubation, well plates were checked for emergence of motile A. malmogiense cells and several cells were isolated from each well. To grow clonal cultures single cells were transferred through several washes into separate new wells containing f/8-Si culture medium using a micropipette. The isolated cells were grown for two months at 4°C, 50–100 μmol photons m⁻² s⁻¹ and 14 L : 10 D cycle. Four well-growing strains were established from the recent sediment surface layer and five strains from the 16 cm deep, 100-year-old sediment layer.

(c). Determination of temperature tolerance ranges

For the temperature tolerance experiment three recent and three historic strains were randomly chosen. Stock cultures of the strains were maintained in 6 psu f/2-Si medium (made from North Sea water that was diluted using MilliQ water) at 3.5°C, 50–100 μmol photons m⁻² s⁻¹, and 16 L : 8 D cycle. To examine temperature tolerances of recent and historic A. malmogiense strains temperature reaction norms were generated using a temperature gradient table [34,35]. The table contained 10 horizontal rows and six vertical slots for culturing containers. Along the horizontal axis a temperature gradient from 0°C to 10°C was built up in steps of 0.9–1.4°C; along the vertical axis the temperatures differed slightly by approx. 0.4°C. In each of the 10 horizontal rows, the samples of three recent and three historic strains were randomly distributed among the six vertically oriented slots. In this way, each of the three recent and three historic strains was investigated at 10 temperature steps between 0°C and 10°C, at 50 μmol photons m⁻² s⁻¹ and 16 L : 8 D cycle. The experiment was repeated for the overlapping temperature range from 6.5°C to 16.5°C.

Inoculum cultures for the temperature gradient experiment were set up from exponentially growing stock cultures by transferring 5 ml of stock culture into experimental vessels filled with 200 ml medium. Vessels were distributed across the table as described above to allow inoculum cultures to acclimate to the respective experimental temperatures. After two weeks, the acclimated subsamples were inoculated into new culture vessels containing fresh medium and placed in the same temperature slots. Starting cell concentrations were set to 700 cells ml⁻¹. The 0–10°C experiment was run for 18 days, the 6.5–16.5°C experiment was run for 14 days to capture the exponential growth phase. Cell concentrations were monitored every 2–3 days from 1 ml subsamples fixed with a drop of Lugol's solution. Cells were counted microscopically in a gridded Sedgewick Rafter chamber. At least 400 cells were counted per sample, except when cell concentrations were very low. Here, at least 200 squares of the chamber were examined. The cell diameter was determined several times during the experiment to examine the effect of temperature on cell size. For this purpose, 7 ml sample material were fixed with Lugol's solution and analysed with a FlowCam VS-IV. The temperature and pH of all samples were measured twice a week using a WTW 340i pH metre. Nitrate and phosphate concentrations were measured at the start, in the middle and at the end of the respective experimental run. Therefore, each 7 ml sample volume was filtered through a 0.2 μmol filter and frozen until later analysis with a Seal-Analytik AA3 Autoanalyser. Particulate organic carbon and nitrogen (POC/PON) analyses were performed at the end of the acclimation period, in the middle and at the end of both experimental runs. For the first measurement 50 ml were extracted from each acclimation culture vessel; for the latter two measurements during the experiment, 14 ml were extracted from each experimental sample. The subsamples were filtered onto precombusted, acid-washed GF/C filters. The filters were dried in a compartment drier and analysed with an Eurovector EA-3000 elemental analyser.

(d). Estimation of reaction norms

The carbon-based growth rate was calculated as follows: the POC data were interpolated to obtain POC values for all time points for which the cell concentration (c) was monitored. For each time point, the cell concentration was multiplied with the POC concentration and divided by the molecular mass of carbon:

2.1

The exponential growth rate based on POC was then calculated for all time steps using

2.2

The time interval of the maximum growth rate was identified graphically using Matlab. The mean growth rate for this time interval was calculated including at least three data points in the analysis, corresponding to at least 5–7 days in the experiment. A modified Gauss function was fitted to the obtained maximum growth rates of the three recent and three historic strains in the temperature range 0–16.5°C:

2.3

This modified Gauss function has an advantage compared with other functions used for fitting temperature reaction norms: the parameters have a biological meaning. represents the maximum growth rate at the optimum temperature T_opt, (T_l1 − T_l2) determines the slope on the left side of the curve, and (T_l1 + T_l2) determines the slope on the right side of the curve. We fitted function (2.3) to the growth rate data of both recent and historic strains using maximum-likelihood to obtain one reaction norm for the recent strains and one for the historic strains.

(e). Examination of life cycle responses to temperature

To study the sensitivity of life cycle processes to temperature changes in recent and historic A. malmogiense strains, a separate experiment was performed where the temperature signal which triggers life cycle transitions in this species was modified in a temperature gradient. Based on previous observations, we assumed that cyst formation is triggered by a temperature shift from preferred growth conditions at low temperatures to the higher end of the temperature window [15,32]. To analyse the flexibility of this temperature trigger and to test whether strains isolated from different temperature regimes distinguish, two recent and two historic strains were acclimated to 3°C and 6°C. After six weeks of acclimation, subsamples were inoculated in experimental containers filled with fresh f/2-Si medium which were then spread in the temperature table set for a temperature range of 0°C to 10°C. All experimental cultures were kept in the table at their respective incubation temperatures for 53 days. Numbers of motile cells, distinct small cells (10–16 μm) and resting cysts were determined every 2–3 days in a Sedgewick Rafter chamber. Samples for cell diameter, POC/PON, and nutrient analysis were taken on days 0, 11, 25 and 53 of the experiment, and analysed as described for the temperature tolerance experiment. The POC content of distinct small cells was extrapolated from their diameter using a linear correlation of cell size and POC from experimental data. Diameter estimates were generated from FlowCam data compiled from thousands of small cells measured from samples containing significant amounts of them. Based on the mean abundance of small cells and their POC content, a POC budget was calculated for this cell fraction. The cyst POC budget was calculated using maximum cyst concentrations and the previously measured mean POC content of 2321 pg C per cyst for A. malmogiense [15]. The encystment rate was calculated for the time interval from the first detection of cysts in a sample until the end of the experiment as the average percentage of the POC concentration of vegetative cells which transform into cysts per day.

(f). Statistical analysis

To investigate the influence of temperature on the growth rate and to validate if there are differences in the growth rate between ages (recent/historic) and between single strains (strain no.), we performed a statistical analysis in R. As the POC-based growth rate data were not normally distributed (analysed using Shapiro test and visual diagnostic methods), we used a generalized additive model (GAM) for significance tests. We tested for significant influence of temperature, age and strain no. on the growth rate and identified the model which explains the data best using the Akaike information criterion (AIC). A similar analysis was performed for the cell diameter. The relative plasticity was calculated as the mean slope of the reaction norm between the coldest tested temperature and the optimum temperature, and a Wilcox test was performed to compare the plasticity of recent and historic strains. The differences in cyst and gamete concentrations between recent and historic strains were investigated in R using a linear model; a similar analysis was performed for the encystment rate. The effect of prior acclimation temperatures (3°C and 6°C) on the growth rate was analysed using a GAM, and the effect of acclimation temperatures on gamete and cyst production was tested with a linear model.

3. Results

(a). Reaction norm

Growth experiments in an experimental temperature gradient from 0°C to 16.5°C revealed typical, left-skewed reaction norms for recent and historic strains (figure 1). Generally, the growth rates increased from 0°C to 7°C, reached a plateau between 7°C and 11°C and decreased drastically thereafter; no growth was observed above 14°C. The variance explained by the fitted Gauss functions (R²) was 0.844 for historic and 0.836 for recent strains. Growth rates of both recent and historic strains were highly variable within each layer; the historic strain hist-3 had notably lower growth rates compared to all other strains at low temperatures and contained a high concentration of small, lightly pigmented cells, presumably sexual life cycle stages (gametes). The performed GAM did not reveal significant differences in growth rates between single strains though (electronic supplementary material, table S1).

Figure 1.

Reaction norms of recent and historic strains of A. malmogiense based on carbon concentrations. Parameter estimates for both fits are summarized in electronic supplementary material, table S2. (Online version in colour.)

Recent strains had a slightly higher growth rate compared with historic strains at higher temperatures. But overall, the reaction norms of recent and historic strains were surprisingly similar; the applied GAM did not indicate a statistically significant difference in growth rates between recent and historic strains (electronic supplementary material, table S1).

Similar to the maximum growth rate, the relative plasticity, expressed by the slope of the reaction norms, was slightly, but not significantly higher in recent compared to historic strains (electronic supplementary material, table S1).

(b). Cell size

As expected, we observed a linear decrease in cell size with higher temperatures for all strains (figure 2). However, in contrast to what may be assumed under global warming, the linear trend was similar for both recent and historic strains, except for strain hist-3. Excluding this strain, again there was no significant difference in the cell diameter between recent and historic strains (electronic supplementary material, table S1; adjusted R² = 0.549).

Figure 2.

Cell size of recent and historic strains of A. malmogiense in relation to temperature including a linear regression. Visualized are the data from all experimental runs combined. Parameter estimates for the regression are summarized in electronic supplementary material, table S3. (Online version in colour.)

(c). life cycle transitions

In a second experiment, we compared the life cycle transitions of recent and historic strains in a temperature range from 0°C to 10°C (figures 3 and 4). Prior acclimation temperatures (3°C and 6°C) before the start of the experiment did not influence life cycle transitions or growth rates significantly (electronic supplementary material, table S1). Noticeable amounts of distinct small cells, possibly gametes, occurred in experimental cultures incubated at low temperatures; their POC concentrations were elevated at approximately 0.5–5.5°C. POC concentrations of resting cysts increased at the higher end of the temperature gradient (approx. 8–10°C). Distinct small cells appeared on day 23 and their concentration remained more or less constant from day 28 onwards until the end of the experiment on day 53, whereas the cysts developed later, from day 30 onwards and increased in concentration until the end of the experiment or until the remaining motile cells died.

Figure 3.

Life cycle processes in relation to temperature: mean small cell and maximum cyst POC concentration in the stationary phase for recent and historic strains. (Online version in colour.)

Figure 4.

Encystment rate per day at the end of the stationary phase for two historic and two recent strains; for each temperature step, three historic and three recent samples are averaged. The boxes indicate the interquartile range; the inner line of each box represents the median; small circles represent the mean; and the whiskers represent the total range of data. (Online version in colour.)

In contrast with reaction norm and cell size, the differences in life cycle responses between historic and recent strains were pronounced: significantly less cysts were produced by recent strains compared to historic strains (LM, F = 7.03, p = 0.020, adjusted R² = 0.499). Likewise, the encystment rate (the POC of vegetative cells which is transformed into POC of cysts per day) was significantly lower in recent compared with historic strains (LM, F = 6.16, p = 0.025, adjusted R² = 0.466).

4. Discussion and conclusion

Here, we revived dinoflagellate cysts from recent and 100-year-old sediment layers in the Baltic Sea, and compared their temperature-dependent traits to assess potential long-term responses to global warming.

Germination of cysts from different sediment layers has been successfully induced previously [20–22,36]. However, our study is the first looking at potential effects of global warming in revived phytoplankton. The comparison of cultures established from living sediment archives covering periods of significant environmental changes represents an attractive possibility to detect signs of adaptation to such changes. Here, we focus on traits known to be sensitive towards temperature: reaction norm, cell size and life cycle transitions.

We detected a slightly higher growth rate at high temperatures in recent compared to historic strains, but against our expectations the differences were not significant. Apparently, our study organism, A. malmogiense has not altered its reaction norm over the past 100 years despite the significant SST increase that has happened in the Baltic since industrialization. Instead, we noted a high variability in the growth rate among the examined strains from each sediment layer. Such high phenotypic variation probably reflects the reservoir function of the dormant seed pool of cyst-forming phytoplankton species, which is thought to integrate different cohorts of a population and thereby preserve diversity [21]. The lack of a clear change in the thermal reaction norm might be furthermore explained by the presence of simultaneous environmental changes. It has been suggested that when selection via multiple environmental changes acts simultaneously on phytoplankton traits, genetic correlations among those traits may limit or constrain adaptation [1]. Apart from temperature, other environmental parameters such as pH, light intensity and nutrient supply have changed due to global change and probably influence phytoplankton traits [37]. In case of the Baltic Sea, the nutrient supply has increased particularly due to a severe eutrophication since the 1950s [38,39].

Our results showed a linear decrease in cell size with increasing temperature, which is typical for protists [10]. Over a century of global warming the decrease in cell size could become stronger due to selection for earlier cell division; offspring formed earlier can establish a larger fraction of the population than those formed later [10] and so in the long run, mean cell sizes could decrease. Additionally, temperature–size correlations diverge at high sublethal temperatures [10], and may therefore be more adaptive than the temperature–size correlation at preferred growth conditions. Nevertheless, in this study recent and historic strains did not differ in their cell size–temperature relationship over the whole investigated thermal reaction norm.

Apart from altering their reaction norm and cell size phytoplankton can respond to higher temperatures in other ways, for example by altering life cycle processes. Typically, resting stages are formed to endure unfavourable environmental conditions. In contrast with the majority of dinoflagellate species which form cysts when nutrients are exhausted, Baltic spring bloom dinoflagellates and specifically A. malmogiense transform into resting cysts when temperatures shift from preferred cold growth conditions to higher temperatures [15,32]. In a cold water system such as the Baltic Sea, the spring transition from cold water conditions to higher surface temperatures is rapidly taking place. Induction of encystment at favourable growth conditions is necessary to ensure successful production of enough resting stages before the spring bloom ceases. The amount of produced resting stages then determines the extent of the next year's bloom [17]. Our data show that the temperature threshold for cyst formation remained constant in both historic and recent strains. Instead we detect an intrinsic change in the amount of cysts produced: cyst concentrations and encystment rates in recent strains were on average 70% lower compared with historic strains. But what could be the benefit of reduced cyst production in recent A. malmogiense populations?

Cyst formation reduces the pool of actively growing cells and can lead to the termination of the spring bloom [40]. Under present climatic conditions with an early and rapidly increasing SST in spring, a high encystment rate would be unfavourable, as it would cause high losses to the vegetative population at an early stage of growth, which would hinder the development of a bloom [41]. A low intrinsic encystment rate, on the contrary, would have a less drastic effect on the growing cell population. Even if the threshold temperature for encystment is exceeded, there remain still enough cells in the water column which can form a bloom. By allowing for bloom formation at rapidly increasing spring temperatures, the strategy of a reduced encystment rate would support population survival and long-term persistence more effectively than a high encystment rate.

As the temperature window of A. malmogiense has remained unchanged over the past 100 years, global warming, including earlier ice melting and earlier increase in spring SST, can be expected to cause a shift in bloom timing. Indeed, the 10–15-day earlier onset of melting over the past 100 years [25] matches the 10-day phenological shift of phytoplankton spring bloom timing observed over the past three decades in the study area [28]. In this context, it is plausible to consider the changed cyst formation behaviour of A. malmogiense as an adaptation to the changed temperature regime.

The life cycle experiment conducted here revealed a notable increase in small and lightly pigmented cells at the lower end of the temperature gradient. We consider them as sexual life cycle stages as they have the typical appearance of dinoflagellate gametes. We did not find significant differences in small cell POC budgets of historic and recent strains, and conclude that sexual reproduction has remained unaffected by changes in the temperature regime. Unlike in most other dinoflagellates, the sexual reproduction of A. malmogiense is largely decoupled from resting cyst formation [32]. Low temperature preference for sexual reproduction implies that this process happens at optimal growth conditions, which should increase the chance for the success of immediate recombination and support preservation of genetic diversity [42]. Being uncoupled from temperature-sensitive encystment, sexual reproduction may not be equally subjected to global warming.

Here, resurrection experiments are used for the first time to investigate responses of phytoplankton temperature traits to global warming. Our results show that trait changes indicative of temperature adaptation have occurred over the past century in the investigated A. malmogiense population, and that these trait changes do not manifest themselves in reaction norms but involve complex life cycle processes. These findings emphasize that the resurrection approach used here represents a promising tool for studies on phytoplankton adaptation to global warming.

This first attempt to use a phytoplankton sediment archive in the context of temperature adaptation, however, also revealed some challenges that need to be addressed by future studies. First, it is difficult to establish comparable samples with a representative number of revived strains from different sediment layers with traditional experimental approaches. Variation among strains is very common in phytoplankton populations [43,44] and ideally samples should consist of a high number of strains to get representative responses for the respective temporal subpopulations. This study comprises a fairly small amount of strains that were tested for each sediment layer. This small sample size expresses itself in low R² values for the analyses of cell sizes and encystment rates, and implies an uncertainty regarding the interpretation of our results for the whole A. malmogiense population. New high-throughput phenomic approaches that begin to emerge in phytoplankton experimentation [45–48] will help to overcome these limitations and make phenotypic trait comparisons more powerful. Second, it is challenging to assess the randomity in sample representation when working with living sediment archives: we cannot exclude the possibility of selective cyst germination even though according to a previous study [33] the recent and the deep sediment layer had equally good germination success under the same conditions. Similarly, it is impossible to assess potential selection in the regulation of encystment a 100 years ago and selective processes affecting cyst survival. The rapidly advancing field of single cell genomics may provide solutions to these limitations of using living sediment archives. When genomic DNA is quantitatively analysed including estimates of effective population sizes and potential storage effects, conclusions on evolutionary adaptation will be possible. Still, genetic analyses cannot replace the more traditional experimental set-up applied here. Only if the functional relationships between traits and environmental factors are quantitatively described and mathematically formulated, they are of use for ecosystem modelling.

Data accessibility

All data used in this manuscript are available in table format in the electronic supplementary material.

Author's contributions

J.H., A.K. and I.H. designed research; J.H. and A.K. performed laboratory experiments; J.H. analysed data; J.H., A.K. and I.H. discussed the results; and J.H., A.K. and I.H. wrote the paper.

Competing interests

We have no competing interests.

Funding

J.H. acknowledges the support of Landesforschungsförderung Hamburg LFF-OS 20-2014. Funding for A.K. was provided by the Academy of Finland grants 282061 and 251564 and the Walter and Andre de Nottbeck Foundation. I.H. was supported through the Cluster of Excellence CliSAP (EXC177), University of Hamburg, funded through the German Science Foundation (DFG).