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General shape of a thermal performance curve. Relationship between environmental temperature and a physiological rate of an ectotherm expressed as a thermal performance curve (grey line). The optimum temperature ( T opt ) specifies the temperature at
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Ectothermic organisms are thought to be severely affected by global warming since their physiological performance is directly dependent on temperature. Latitudinal and temporal variations in mean temperatures force ectotherms to adapt to these complex environmental conditions. Studies investigating current patterns of thermal adaptation among popul...
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... is one of the most important environmental factors determining a variety of ecosystem elements, e.g. species ecophysiology, abundance and distribution, as well as species diversity and population dynamics [1–4]. Due to the current climate change, scientists started to re-evaluate the impact of elevated temperatures on the ecology of species. Here, ectothermic organisms are of special interest as their physiological performance is highly dependent on environmental temperature. To make predictions of organisms’ and population responses to global warming, studies on genetic and phenotypic diversity over a species’ geographic range are important. Such investigations can unveil patterns of evolutionary temperature adaptation to the current thermal heterogeneity on Earth by determining which ectotherms have a high acclimatisation capacity and which only occur at specific temperatures. Adaptive phenotypic plasticity, for instance, may cause a higher tolerance to changing thermal conditions [5,6], while local temperature adaptation might be detrimental. Several studies could show a co-variation of latitude and thermal tolerance (e.g. [7,8]) suggesting that organisms are adapted to the mean temperatures of their environment, but others failed (e.g. [9,10]). Climate change is supposed to affect both climate averages and variability [11] and it has been shown that the thermal tolerance of many organisms is proportional to the magnitude of variation they are exposed to [12]. Organisms are also expected to be adapted to the thermal heterogeneity of their particular environment. This thermal heterogeneity increases with latitude. Therefore, organisms from variable climates, such as the temperate zone, should evolve a broad thermal tolerance resulting in thermal generalists . In contrast, tropical ectotherms, experiencing less variation in temperature, should be selected for narrow thermal niches resulting in thermal specialists [13,14]. Consequently, analysing thermal niches of different populations along a latitudinal transect is necessary to understand the process of adaptation to novel thermal environments. It has been shown that populations and individuals at the edge of the species range may suffer the most from increasing temperatures, because they often live close to the limit of their species’ physiological thermal tolerance [15]. Therefore, it is not only important to investigate the intraspecific variation in species’ thermal tolerance, but also to consider populations from the margins of their current distribution range. Especially if one would expect a thermal generalist pattern for ubiquitous species, populations at the ‘warm edge’ (such as the tropics or subtropics) might be most at risk due to global warming (cf. [16,17]). Because of the anticipated increasing risk of more intense, more frequent and longer-lasting heat waves during summer [18], species heat resistances are of particular significance [15,19,20,21]. Here, thermal safety margins as well as the maximum warming tolerance are suitable characters to qualitatively elucidate the impact of climate change effects across latitude on different populations. These indicators are based on an organism’s thermal tolerance and its relation to the local temperature regime [17]. Studies investigating species’ current thermal adaptation patterns with respect to present-day and future environmental temperatures therefore allow predictions of species’ and population responses to elevated temperatures. Beside these patterns of evolutionary temperature adaptation obviously related to climate change, many other patterns are important in thermal adaptation with respect to species evolution and ecology. For example, the warmer is better hypothesis [22,23,24], which predicts a positive correlation between an organism’s optimal temperature and its maximum performance; or the Jack-of-all-temperatures is a master of none hypothesis [25], which assumes an evolutionary trade-off between the performance breadth and the maximal performance of an organism, are controversially discussed. These patterns are relevant in a climate change context, too, but only few investigators have experimen- tally tested these basic ideas of evolutionary temperature adaptation [26–29]. For the investigation of such elementary hypotheses, the determination of thermal performance curves (Figure 1) provides a suitable framework to evaluate an organism’s thermal tolerance [30]. Thermal performance curves (TPCs) allow estimations on how basic physiological functions are influenced by environmental temperature [17]. Furthermore, TPCs permit the calculation of ecophysiological key characteristics like the lower and upper critical thermal limits ( CT min and CT max ) as well as the optimum temperature ( T opt ) and the maximum performance ([31]; cf. Figure 1]). Such key parameters are useful indicators for the thermal tolerance or thermal niche as well as for a potential environmental adaptation of different genotypes. As before mentioned, these ecophysiological characteristics can show a co- variation with latitude in metazoan species (e.g. [12,32]), although other studies unveiled that the upper thermal limits of ectotherms vary little with latitude (e.g. [33,34]). However, this has never been critically evaluated for microbial eukaryotes, which are not only important for aquatic ecosystems [35,36], but also constitute well suited organisms for experimental evolution [37,38]. While some recent studies have investigated the response of protozoan species to increasing temperatures (e.g. [39–42]), little is known about thermal adaptation patterns of globally distributed eukaryotic microbes and how temperature might affect the genetic diversity of natural populations. Furthermore, investigations on the intraspecific variation in species’ thermal tolerance by considering populations from the margins of their current distribution range are rare as well. In the present study, the microbial eukaryote Paramecium caudatum was used to investigate the intraspecific variation in temperature reaction norms of different genotypes. These were isolated from natural habitats along a latitudinal transect in Europe, while three genotypes from tropical habitats (Indonesia) served as a genetic and phenotypic outgroup. This globally distributed ciliate species inhabits the mud-water interface of littoral freshwater environments, which are considerably affected by atmospheric temperature changes [43,44]. Consequently, P. caudatum has to cope with large temporal and spatial variations in temperature. It therefore constitutes a suitable ectotherm to test hypotheses in thermal adaptation as well as the consequences of climate change on such ubiquitous protists. Here, we performed temperature dependent growth experiments to ( i ) test for a hypothesized local temperature adaptation of different P. caudatum genotypes; ( ii ) investigate thermal constraints resulting from evolutionary temperature adaptation; and ( iii ) understand the sensitivity of P. caudatum to predicted future temperatures. Paramecium caudatum cells were isolated from freshwater samples of 12 different natural habitats along a north-south transect in Europe as well as from three tropical habitats in Indonesia, Sulawesi (see Figure 2 and Table 1 for specifications). No specific permits were required for the described field studies. In Europe and Indonesia, work with Paramecium does not require specific permission and samples were not taken from water bodies where private property was indicated or from nature reserves where sampling is prohibited. The field studies did not involve endangered or protected species. The food bacteria Enterobacter aerogenes were obtained from the American Type Culture Collection (ATCC 35028) and the kanamycin-resistant strain Pseudomonas fluorescens SBW25 EeZY- 6KX [45] was acquired from the University of Oxford. The investigated P. caudatum stock cultures were maintained in a 0.25% C EROPHYL infusion, prepared according to the methods of Sonneborn [46] with minor modifications [31]. Isolated cells were separated in 1 ml of filtrated habitat water in 24-well tissue culture plates (TPP H AG) to establish clonal cultures. Afterwards, cells were washed and maintained at 22 u C in a C EROPHYL infusion inoculated with Enterobacter aerogenes to establish mass cultures. Later, cultures were kept at 10 u C, lowering the growth and ageing of P. caudatum . Previous to the start of the experiments, monoxenic P. caudatum cultures were established at 22 u C in a C EROPHYL infusion with Pseudomonas fluorescens serving as the only food bacteria (for details see [31]). Cells from exponentially growing, monoxenic P. caudatum cultures were transferred to tissue culture flat tubes and acclimatised to experimental temperatures between 7 u C and 35.5 u C in steps of 6 1.5 K d 2 1 . Cultures were kept in exponential growth phase (500–1000 cells ml 2 1 ) during the acclimation period by doubling the culture volume with Pseudomonas fluorescens inoculated C EROPHYL infusion (CMP; pH 7.0) as appropriate (1– 5 ml per day). Due to the different acclimation phases from 22 C up to 35.5 u C or down to 7 u C, respectively, temperature- dependent experiments were conducted time-delayed. All experiments were performed in microprocessor-controlled, cooled incubators obtained from BINDER GmbH (Type KB 53). Before the experimental start, acclimatised P. caudatum pre- cultures were adjusted to , 250 cells ml 2 1 with CMP. Two millilitres of these starting cultures were added to each microcosm containing 18 ml CMP and resulting in an initial abundance of , 25 cells ml 2 1 . Growth experiments were run in triplicate in 60- ml tissue culture flasks with filter lids (TPP H AG) over two to eight days depending on the ...
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... the kanamycin-resistant strain Pseudomonas fluorescens SBW25 EeZY- 6KX [45] was acquired from the University of Oxford. The investigated P. caudatum stock cultures were maintained in a 0.25% C EROPHYL infusion, prepared according to the methods of Sonneborn [46] with minor modifications [31]. Isolated cells were separated in 1 ml of filtrated habitat water in 24-well tissue culture plates (TPP H AG) to establish clonal cultures. Afterwards, cells were washed and maintained at 22 u C in a C EROPHYL infusion inoculated with Enterobacter aerogenes to establish mass cultures. Later, cultures were kept at 10 u C, lowering the growth and ageing of P. caudatum . Previous to the start of the experiments, monoxenic P. caudatum cultures were established at 22 u C in a C EROPHYL infusion with Pseudomonas fluorescens serving as the only food bacteria (for details see [31]). Cells from exponentially growing, monoxenic P. caudatum cultures were transferred to tissue culture flat tubes and acclimatised to experimental temperatures between 7 u C and 35.5 u C in steps of 6 1.5 K d 2 1 . Cultures were kept in exponential growth phase (500–1000 cells ml 2 1 ) during the acclimation period by doubling the culture volume with Pseudomonas fluorescens inoculated C EROPHYL infusion (CMP; pH 7.0) as appropriate (1– 5 ml per day). Due to the different acclimation phases from 22 C up to 35.5 u C or down to 7 u C, respectively, temperature- dependent experiments were conducted time-delayed. All experiments were performed in microprocessor-controlled, cooled incubators obtained from BINDER GmbH (Type KB 53). Before the experimental start, acclimatised P. caudatum pre- cultures were adjusted to , 250 cells ml 2 1 with CMP. Two millilitres of these starting cultures were added to each microcosm containing 18 ml CMP and resulting in an initial abundance of , 25 cells ml 2 1 . Growth experiments were run in triplicate in 60- ml tissue culture flasks with filter lids (TPP H AG) over two to eight days depending on the experimental growth temperature. The bacterial start density was regulated to a saturating prey level of about 2 N 10 8 cells ml 2 1 . If the P. caudatum pre-culture densities were below 250 cells ml 2 1 because of growth-limiting temperatures (e.g. 7 u C or $ 34 u C), initial cell abundance was adjusted to the highest possible cell number ( $ 10 cells ml 2 1 ). Paramecium cell abundance was estimated by sampling 1 ml every nine to 41 hours depending on the experimental growth temperature. This resulted in five to eight samples per replicate. For precise counting, cells were fixed by the addition of Bouin’s solution [47] to a final concentration of 1%. Cell numbers were enumerated microscopically by threefold counting 100 m l to 300 m l subsamples using a dark field stereoscopic microscope (Olympus GmbH). The population growth rate ( m , d 2 1 ) for each replicate and at each experimental temperature was calculated over the period of exponential increase using the slope of the linear regression of log -transformed cell densities versus time ( t ). To identify and distinguish the individual, clonal cultures of natural P. caudatum genotypes from different geographic regions, the mitochondrial cytochrome c oxidase subunit I (COI) gene was sequenced following the protocol of Barth et al. [48]. Five cells from each stock culture were washed four times in sterile Eau de Volvic H and then incubated overnight with 100 m l of 10% Chelex H solution and 10 m l Proteinase K (10 mg ml 2 1 ) at 56 u C. Afterwards, the mixture was boiled for 20 min and frozen at 2 20 u C; the supernatant was used for subsequent PCR reactions. Each PCR reaction mix contained 10 m l of Chelex H extracted genomic DNA, 10 pmol of each primer, 1 U Taq-polymerase (SIGMA, Taufkirchen, Germany), 1 6 PCR buffer with 2 mM MgCl 2 and 200 m M dNTPs in a total volume of 50 m l. PCR conditions were as follows: 5 min initial denaturation (95 u C); 35 cycles of 1 min at 95 u C, 1 min at 50 u C and 45 s at 72 u C; and a final extension step of 5 min (72 u C). Using the primers CoxL11058 and CoxH10176 (see [48]), an 880-bp fragment of the mitochondrial COI gene was amplified. After purification with the Rapid PCR Purification System (Marligen Bioscience, Ijamsville, USA), PCR products were directly sequenced. Sequencing reactions were performed in both directions and analysed on an ABI 3100 Genetic Analyzer (Applied Biosystems). The calculation of thermal performance curves (TPCs) was performed to describe the temperature dependent growth rate data of the individual P. caudatum clones and to determine clone specific key ecophysiological characteristics. TPCs have a common general shape with a gradual increase from a lower critical temperature ( CT min ) to a thermal optimum ( T opt ) where the investigated biological function reaches its maximum. With a further increase in temperature above T opt the TPCs show a rapid decline towards a critical temperature maximum ( CT max ; Figure 1). It was shown that the nonlinear Lactin-2 optimum function [49] can adequately describe the temperature – growth rate relationship of Paramecium caudatum resulting in typically skewed TPCs with a right-shift towards warmer temperatures [31]. The TPC estimation was done by fitting nonlinear mixed-effects models [50] simultaneously to the whole data set. The mixed- effects models were compared with AIC based model selection at three hierarchical levels; the whole data set with common fixed effects for all 18 clones (null models nm0a and nm0b , cf. Table 2), with separate fixed effects for the two regions, Europe and Indonesia (model nm2 , cf. Table 2) and with separate fixed effects for the four regions northern, central, southern Europe and Indonesia (model nm4 , cf. Table 2). In all cases, all four original parameters of the Lactin-2 function ( r , T max , D and l ) were used as fixed effects. In model nm0a , all four parameters were also used as random effects while for models nm0b , nm2 , and nm4 only T max , D and l were used because of the high correlation between the parameters r and l resulting in a low model convergence. The decision which of the two parameters had to be omitted for nm2 and nm4 was made by comparing the respective AIC values (not shown). Model nm0b is shown for comparison only (cf. Table 2). Then, the ecophysiological characteristics CT min and CT max were derived numerically as the intersection points of the resulting thermal performance curve with the temperature axis ( m = 0). The maximum growth rate ( m max, cal ) was calculated analytically as the growth rate ( m ) at T opt using the Lactin-2 function (Eq.1), while T opt was calculated using its first derivative (Eq.2) as follows: where, the parameter r is a constant influencing m max and the slope of the low-temperature branch, T max is the maximum temperature, and D defines the temperature range of the thermal inhibition above T opt . Parameter l is an intercept parameter that forces the curve to intersect the abscissa at low temperatures and allows the estimation of CT . Finally, standard errors for both, the original Lactin-2 function parameters (see Table S1) and the derived ecophysiological key parameters (Table 3) were estimated by nonparametric residual bootstrapping [51] with 1000 bootstrap replicates. For all further analyses based on these key ecophysiological characteristics, estimated data derived from the nm0a mixed-effects model fitting and bootstrapping procedure were used if not otherwise stated. Investigating an organism’s local temperature adaptation or its extinction risk due to climate change requires specific knowledge about the thermal conditions within its natural habitats. Here, we used site-specific temperature data to compare the clonal specific ecophysiological characteristics T and CT with the current climate conditions of the specific habitats. Climate data were obtained from nearby meteorological stations (Table S2) or derived from the WorldClim database [52] using the program DIVA-GIS. In case of the meteorological station data, daily mean and maximum air temperature data of the years 2000–2011 (if available) were used to calculate the mean surface air temperature ( T hab, mean ) as well as the mean maximal surface air temperature ( T hab, max ), both for the warmest three months of the specific habitat. The WorldClim database is a set of interpolated global climate layers considering monthly precipitation as well as mean, minimum and maximum temperatures of the years , 1950–2000. The database also provides 19 derived bioclimatic variables. The 2.5 arc-minutes resolution database was used to obtain the habitat mean temperature of the warmest quarter (bioclimatic variable 10 o T hab, mean ) and to calculate the habitat mean maximum temperature of the warmest three months ( T hab, max ). Furthermore, we used climate change data (2.5 arc-minutes) provided by DIVA-GIS to calculate future conditions for the respective habitats of the investigated P. caudatum clones. These data were derived from high-resolution simulations of global warming [53] using the CCM3 model and assuming a CO 2 doubling until 2100 (see Table S2). The use of such temperature data has proven controversial and it is well known that local and microhabitat temperature extremes and fluctuations can differ significantly from the regional average [54,55]. However, it could be demonstrated that the summer lake surface water temperature of shallow lakes clearly correlates with the local air temperature [56]. Therefore, the use of local air temperature data seems to be a valid approach to estimate an aquatic ectotherm’s performance temperature such as for P. caudatum that inhabits the littoral zone of freshwater environments. Correlation analyses between each key ecophysiological characteristic ( CT min , T opt , CT max ) and the latitude of the respective natural habitats were performed to compare these thermal adaptation ...
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... is one of the most important environmental factors determining a variety of ecosystem elements, e.g. species ecophysiology, abundance and distribution, as well as species diversity and population dynamics [1–4]. Due to the current climate change, scientists started to re-evaluate the impact of elevated temperatures on the ecology of species. Here, ectothermic organisms are of special interest as their physiological performance is highly dependent on environmental temperature. To make predictions of organisms’ and population responses to global warming, studies on genetic and phenotypic diversity over a species’ geographic range are important. Such investigations can unveil patterns of evolutionary temperature adaptation to the current thermal heterogeneity on Earth by determining which ectotherms have a high acclimatisation capacity and which only occur at specific temperatures. Adaptive phenotypic plasticity, for instance, may cause a higher tolerance to changing thermal conditions [5,6], while local temperature adaptation might be detrimental. Several studies could show a co-variation of latitude and thermal tolerance (e.g. [7,8]) suggesting that organisms are adapted to the mean temperatures of their environment, but others failed (e.g. [9,10]). Climate change is supposed to affect both climate averages and variability [11] and it has been shown that the thermal tolerance of many organisms is proportional to the magnitude of variation they are exposed to [12]. Organisms are also expected to be adapted to the thermal heterogeneity of their particular environment. This thermal heterogeneity increases with latitude. Therefore, organisms from variable climates, such as the temperate zone, should evolve a broad thermal tolerance resulting in thermal generalists . In contrast, tropical ectotherms, experiencing less variation in temperature, should be selected for narrow thermal niches resulting in thermal specialists [13,14]. Consequently, analysing thermal niches of different populations along a latitudinal transect is necessary to understand the process of adaptation to novel thermal environments. It has been shown that populations and individuals at the edge of the species range may suffer the most from increasing temperatures, because they often live close to the limit of their species’ physiological thermal tolerance [15]. Therefore, it is not only important to investigate the intraspecific variation in species’ thermal tolerance, but also to consider populations from the margins of their current distribution range. Especially if one would expect a thermal generalist pattern for ubiquitous species, populations at the ‘warm edge’ (such as the tropics or subtropics) might be most at risk due to global warming (cf. [16,17]). Because of the anticipated increasing risk of more intense, more frequent and longer-lasting heat waves during summer [18], species heat resistances are of particular significance [15,19,20,21]. Here, thermal safety margins as well as the maximum warming tolerance are suitable characters to qualitatively elucidate the impact of climate change effects across latitude on different populations. These indicators are based on an organism’s thermal tolerance and its relation to the local temperature regime [17]. Studies investigating species’ current thermal adaptation patterns with respect to present-day and future environmental temperatures therefore allow predictions of species’ and population responses to elevated temperatures. Beside these patterns of evolutionary temperature adaptation obviously related to climate change, many other patterns are important in thermal adaptation with respect to species evolution and ecology. For example, the warmer is better hypothesis [22,23,24], which predicts a positive correlation between an organism’s optimal temperature and its maximum performance; or the Jack-of-all-temperatures is a master of none hypothesis [25], which assumes an evolutionary trade-off between the performance breadth and the maximal performance of an organism, are controversially discussed. These patterns are relevant in a climate change context, too, but only few investigators have experimen- tally tested these basic ideas of evolutionary temperature adaptation [26–29]. For the investigation of such elementary hypotheses, the determination of thermal performance curves (Figure 1) provides a suitable framework to evaluate an organism’s thermal tolerance [30]. Thermal performance curves (TPCs) allow estimations on how basic physiological functions are influenced by environmental temperature [17]. Furthermore, TPCs permit the calculation of ecophysiological key characteristics like the lower and upper critical thermal limits ( CT min and CT max ) as well as the optimum temperature ( T opt ) and the maximum performance ([31]; cf. Figure 1]). Such key parameters are useful indicators for the thermal tolerance or thermal niche as well as for a potential environmental adaptation of different genotypes. As before mentioned, these ecophysiological characteristics can show a co- variation with latitude in metazoan species (e.g. [12,32]), although other studies unveiled that the upper thermal limits of ectotherms vary little with latitude (e.g. [33,34]). However, this has never been critically evaluated for microbial eukaryotes, which are not only important for aquatic ecosystems [35,36], but also constitute well suited organisms for experimental evolution [37,38]. While some recent studies have investigated the response of protozoan species to increasing temperatures (e.g. [39–42]), little is known about thermal adaptation patterns of globally distributed eukaryotic microbes and how temperature might affect the genetic diversity of natural populations. Furthermore, investigations on the intraspecific variation in species’ thermal tolerance by considering populations from the margins of their current distribution range are rare as well. In the present study, the microbial eukaryote Paramecium caudatum was used to investigate the intraspecific variation in temperature reaction norms of different genotypes. These were isolated from natural habitats along a latitudinal transect in Europe, while three genotypes from tropical habitats (Indonesia) served as a genetic and phenotypic outgroup. This globally distributed ciliate species inhabits the mud-water interface of littoral freshwater environments, which are considerably affected by atmospheric temperature changes [43,44]. Consequently, P. caudatum has to cope with large temporal and spatial variations in temperature. It therefore constitutes a suitable ectotherm to test hypotheses in thermal adaptation as well as the consequences of climate change on such ubiquitous protists. Here, we performed temperature dependent growth experiments to ( i ) test for a hypothesized local temperature adaptation of different P. caudatum genotypes; ( ii ) investigate thermal constraints resulting from evolutionary temperature adaptation; and ( iii ) understand the sensitivity of P. caudatum to predicted future temperatures. Paramecium caudatum cells were isolated from freshwater samples of 12 different natural habitats along a north-south transect in Europe as well as from three tropical habitats in Indonesia, Sulawesi (see Figure 2 and Table 1 for specifications). No specific permits were required for the described field studies. In Europe and Indonesia, work with Paramecium does not require specific permission and samples were not taken from water bodies where private property was indicated or from nature reserves where sampling is prohibited. The field studies did not involve endangered or protected species. The food bacteria Enterobacter aerogenes were obtained from the American Type Culture Collection (ATCC 35028) and the kanamycin-resistant strain Pseudomonas fluorescens SBW25 EeZY- 6KX [45] was acquired from the University of Oxford. The investigated P. caudatum stock cultures were maintained in a 0.25% C EROPHYL infusion, prepared according to the methods of Sonneborn [46] with minor modifications [31]. Isolated cells were separated in 1 ml of filtrated habitat water in 24-well tissue culture plates (TPP H AG) to establish clonal cultures. Afterwards, cells were washed and maintained at 22 u C in a C EROPHYL infusion inoculated with Enterobacter aerogenes to establish mass cultures. Later, cultures were kept at 10 u C, lowering the growth and ageing of P. caudatum . Previous to the start of the experiments, monoxenic P. caudatum cultures were established at 22 u C in a C EROPHYL infusion with Pseudomonas fluorescens serving as the only food bacteria (for details see [31]). Cells from exponentially growing, monoxenic P. caudatum cultures were transferred to tissue culture flat tubes and acclimatised to experimental temperatures between 7 u C and 35.5 u C in steps of 6 1.5 K d 2 1 . Cultures were kept in exponential growth phase (500–1000 cells ml 2 1 ) during the acclimation period by doubling the culture volume with Pseudomonas fluorescens inoculated C EROPHYL infusion (CMP; pH 7.0) as appropriate (1– 5 ml per day). Due to the different acclimation phases from 22 C up to 35.5 u C or down to 7 u C, respectively, temperature- dependent experiments were conducted time-delayed. All experiments were performed in microprocessor-controlled, cooled ...
Citations
... The list of the sequences in the collapsed clades is provided in Figure S1. separate clade within P. caudatum (Krenek et al., 2012(Krenek et al., , 2015. Five Thai strains in our study matched the general characteristics of this morphospecies, but their micronuclei lack the achromatin cap; interestingly, they cluster with the Indonesian isolates with strong support (Figure 4; Figure S1), corroborating the genetic divergence of this cluster. ...
... Although the other paramecia discovered in the present study do not drastically change general diversity picture of the genus, they provide a useful set of morphological and molecular information for further Paramecium revisions. Paramecium hiwatashii n. sp., a new cryptic sister species of P. caudatum Paramecium caudatum-like strains collected by us in Thailand and those from Indonesia (Krenek et al., 2012(Krenek et al., , 2015 formed a separate group on both phylogenetic trees with high or maximum supports (Figures 4 and 5; Figure S1). Paramecium caudatum was originally considered to consist of several syngens (Sonneborn, 1957;Wichterman, 1986), although the molecular identification of these syngens was not possible (Stoeck et al., 2000;Tsukii, 1994). ...
... Paramecium caudatum was originally considered to consist of several syngens (Sonneborn, 1957;Wichterman, 1986), although the molecular identification of these syngens was not possible (Stoeck et al., 2000;Tsukii, 1994). However, the strains from Indonesia that form the cluster together with the strains collected by us have not been used either for syngen identification, or for thorough morphological and molecular analyses (Krenek et al., 2012(Krenek et al., , 2015. The COI gene sequences between strains of two subclades (subclade 1. BKK28-4, ChMa2-1, and KohMak3-1; subclade 2. BKK11-4, BKK11-6, and three Indonesian strains) of this new Asian cluster deviated by 4.2%-4.3%, ...
Paramecium (Ciliophora, Oligohymenophorea) is a good model to study ciliate biogeography. Extensive sampling mainly in northern hemisphere has led to 16 valid morphological species description thus far. However, a majority of hard‐to‐reach regions, including South East Asia, are underinvestigated. Our study combined traditional morphological and molecular approaches to reveal the biodiversity of Paramecium in Thailand from more than 110 samples collected in 10 provinces. Representatives of seven morphological species were identified from our collection, including the rare species, such as P. gigas and P. jenningsi . Additionally, we detected five different sibling species of the P. aurelia complex, described a new cryptic species P. hiwatashii n. sp. phylogenetically related to P. caudatum, and discovered a potentially new genetic species of the P. bursaria species complex. We also documented a variety of bacterial cytoplasmic symbionts from at least nine monoclonal cultures of Paramecium .
... Despite the promise of predicting evolutionary and plastic potential with climatic variation, we still need to understand the scales at which these relationships apply. Most studies testing for relationships between climatic variation and climate change vulnerability compare species or populations across broad latitudinal gradients (e.g., temperate versus tropical) (Deutsch et al., 2008;Diamond, 2017;Hof et al., 2012;Krenek et al., 2012;Simon et al., 2015;Vasseur et al., 2014), where gene flow is likely low and therefore selection can act more effectively (Lenormand, 2002). Yet, climatic variation often differs at fine-spatial scales where gene flow can be higher. ...
Rapid evolutionary adaptation could reduce the negative impacts of climate change if sufficient heritability of key traits exists under future climate conditions. Plastic responses to climate change could also reduce negative impacts. Understanding which populations are likely to respond via evolution or plasticity could therefore improve estimates of extinction risk. A large body of research suggests that the evolutionary and plastic potential of a population can be predicted by the degree of spatial and temporal climatic variation it experiences. However, we know little about the scale at which these relationships apply. Here, we test if spatial and temporal variation in temperature affects genetic variation and plasticity of fitness and a key thermal tolerance trait (critical thermal maximum; CTmax) at microgeographic scales using a metapopulation of Daphnia magna in freshwater rock pools. Specifically, we ask if (a) there is a microgeographic adaptation of CTmax and fitness to differences in temperature among the pools, (b) pools with greater temporal temperature variation have more genetic variation or plasticity in CTmax or fitness, and (c) increases in temperature affect the heritability of CTmax and fitness. Although we observed genetic variation and plasticity in CTmax and fitness, and differences in fitness among pools, we did not find support for the predicted relationships between temperature variation and genetic variation or plasticity. Furthermore, the genetic variation and plasticity we observed in CTmax are unlikely sufficient to reduce the impacts of climate change. CTmax plasticity was minimal and heritability was 72% lower when D. magna developed at the higher temperatures predicted under climate change. In contrast, the heritability of fitness increased by 53% under warmer temperatures, suggesting an increase in overall evolutionary potential unrelated to CTmax under climate change. More research is needed to understand the evolutionary and plastic potential under climate change and how that potential will be altered in future climates.
... Organisms can survive temperatures beyond their pejus temperature thanks to physiological mechanisms such as the heat shock response, metabolic depression and antioxidative defense but they can only do so for a limited time (Pörtner et al., 2017). In general, the pejus temperatures of tropical marine ectotherms are closer to their critical thermal maximum (CT max ) than their temperate counterparts, making them particularly vulnerable to warming temperatures (Sunday et al., 2014;Huey et al., 2009;Krenek et al., 2012). Thus, it is crucial that we understand the factors that can affect the pejus temperature of tropical marine species to understand their thermal limits. ...
Understanding factors affecting ectothermic fishes' capacity to cope with warming temperature is critical given predicted climate change scenarios. We know that a fish's social environment introduces plasticity in how it responds to high temperature. However, the magnitude of this plasticity and the mechanisms underlying socially-modulated thermal responses are unknown. Using the amphibious, selfing hermaphroditic mangrove rivulus fish (Kryptolebias marmoratus) as a model, we tested three hypotheses: 1) social stimulation affects physiological and behavioural thermal responses of isogenic lineages of fish, 2) social experience and acute social stimulation result in distinct physiological and behavioural responses, and 3) a desensitization of thermal receptors is responsible for socially modulated thermal responses. To test the first two hypotheses, we measured the temperature at which fish emerged (i.e., pejus temperature) with acute warming with socially naïve, isolated fish and with fish that were raised alone and then given a short social experience prior to exposure to increasing temperature (i.e., socially experienced fish). Our results did not support our first hypothesis as fish socially-stimulated by mirrors during warming (i.e., acute social stimulation) emerged at similar temperatures as isolated fish. However, in support of our second hypothesis, a short period of prior social experience resulted in fish emerging at a higher temperature than socially naïve fish suggesting an increase in pejus temperature with social experience. To test our third hypothesis, we exposed fish having had a brief social interaction and naïve fish to capsaicin, an agonist of TRPV1 thermal receptors. Socially experienced fish emerged at significantly higher capsaicin concentrations than socially naïve fish suggesting a desensitization of their TRPV1 thermal receptors. Collectively, our data indicate that past and present social experiences impact the behavioural response of fish to high temperature. We also provide novel data suggesting that brief periods of social experience affects the capacity of fish to perceive warm temperature.
... 101 We used isogenic lineages of the tropical mangrove rivulus in different social situations to 102 determine the possible effects of sociality on thermal biology. In general, the pejus temperatures 103 of tropical marine ectotherms' are closer to their critical thermal maximum (CTmax) than their 104 temperate counterparts, making them particularly vulnerable to warming temperatures (Sunday et Krenek et al., 2012). The mangrove rivulus is a simultaneous 106 hermaphrodite fish and is capable of producing fertilized eggs (Taylor et al., 2001). ...
Understanding factors affecting ectothermic fishes' capacity to cope with warming temperature is critical given predicted climate change scenarios. We know that a fish's social environment introduces plasticity in how it responds to high temperature. However, the magnitude of this plasticity and the mechanisms underlying socially-modulated thermal responses are unknown. Using the amphibious, selfing hermaphroditic mangrove rivulus fish (Kryptolebias marmoratus) as a model, we tested three hypotheses: 1) social stimulation affects physiological and behavioural thermal responses of isogenic lineages of fish, 2) social experience and acute social stimulation result in distinct physiological and behavioural responses, and 3) a desensitization of thermal receptors is responsible for socially-modulated thermal responses. To test the first two hypotheses, we measured the temperature at which fish emerged (i.e., pejus temperature) with acute warming with socially naive, isolated fish and socially experienced fish. Our results did not support our first hypothesis as fish socially-stimulated by mirrors during warming (i.e., acute social stimulation) emerged at similar temperatures as isolated fish. However, in support of our second hypothesis, prior social experience resulted in fish emerging at a higher temperature than socially naive fish suggesting an increase in pejus temperature with social experience. We measured whole-body cortisol concentrations of socially naive and socially experienced fish and determined that socially experienced fish had significantly higher cortisol concentrations than socially naive fish. To test our third hypothesis, we exposed socially experienced and naive fish to capsaicin, an agonist of TRPV1 thermal receptors. Socially experienced fish emerged at significantly higher capsaicin concentrations than socially naive fish suggesting a desensitization of their TRPV1 thermal receptors. Collectively, our data indicate that past and present social experiences impact the behavioural response of fish to high temperature. We also provide novel data suggesting that social experience affects the capacity of fish to perceive warm temperature.
... In the course of global warming, adaptation to elevated temperatures is key for the survival of ciliates and other organisms. If most freshwater ciliates have developed a "thermal safety margin" known from Paramecium (Krenek et al. 2012), then they should be able to tolerate and grow at temperatures above their current temperature environment. Due to the paucity of evidence discussed above, it is an open question if this also applies to marine ciliates. ...
On the global average, the temperature increase in the ocean is lower than in lakes. Moreover, most freshwater organisms must cope with wider temperature fluctuations than marine organisms. Knowing if the organisms' thermal sensitivity differs in the two realms is crucial for predicting the respective climate-related changes at community and ecosystem levels. We investigated the thermal sensitivity of planktonic ciliates, which are of tremendous significance for biogeochemical cycling in all aquatic ecosystems. Marine and freshwater ciliates differ in their thermal performance; therefore, system-specific activation energies should be applied in models predicting ciliate responses to altered temperatures. This work may serve as a model study for other taxa and be of interest to many marine and freshwater ecologists. Abstract Predicting the performance of aquatic organisms in a future warmer climate depends critically on understanding how current temperature regimes affect the organisms' growth rates. Using a meta-analysis for the published experimental data, we calculated the activation energy (E a) to parameterize the thermal sensitivity of marine and freshwater ciliates, major players in marine and freshwater food webs. We hypothesized that their growth rates increase with temperature but that ciliates dwelling in the immense, thermally stable ocean are closely adapted to their ambient temperature and have lower E a than ciliates living in smaller, thermally more variable freshwater environments. The E a was in the range known from other taxa but significantly lower for marine ciliates (0.390 AE 0.105 eV) than for freshwater ciliates (0.633 AE 0.060 eV), supporting our hypothesis. Accordingly, models aiming to predict the ciliate response to increasing water temperature should apply the environment-specific activation energies provided in this study.
... A low responsiveness of protozoans to external forcing was also observed in marine systems (Dolan and Gallegos, 2001) and in experiments with epibenthic ciliates . Ciliate species may comprise a high diversity of functionally different clones, which may buffer their response to environmental changes (Weisse et al., 2001;Krenek et al., 2012). ...
Ciliates represent a crucial link between phytoplankton and bacteria and mesozooplankton in pelagic food webs, but little is known about the processes influencing the dynamics of individual species. Using long-term, high-frequency observations, we compared the diversity and the temporal variability in biomass and species composition of the ciliate community in large, deep, mesotrophic Lake Constance to that of the phytoplankton and rotifer communities in the same lake. Furthermore, we used boosted regression trees to evaluate possible environmental predictors (temperature, three prey groups, four predator/competitor groups) influencing ciliate net growth. The biomass of all ciliate species showed a common, recurrent seasonal pattern, often with peaks in spring and summer. The ciliate community was more diverse than the rotifer community, exhibited highly synchronous dynamics and its species were regularly encountered during the season. The top-down control by copepods likely contributes to the ciliates’ synchronized decline prior to the clear-water phase when food concentration is still high. The high temporal autocorrelation of the ciliate biomasses together with the inter-annual recurrent seasonal patterns and the low explanatory power of the environmental predictors suggest that the dynamics of individual ciliate species are strictly controlled, yet it remains difficult to determine the responsible factors.
... Thermal performance curves have been applied, frequently through meta-analyses, to reveal trends across widely divergent taxa (Sinclair et al., 2016). In contrast, comparisons of closely related taxa are relatively rare (e.g., Hoffmann et al., 2013;Krenek et al., 2012), yet trends arising from these should offer insights into mechanisms of adaptation, as closely related taxa diverge. Here, we use experimental data to test if theory-based hypotheses associated with cross-phyletic thermal performance analyses apply equally to closely related taxa. ...
... Further mutations may result in allozymes that confer greater stability at high and low temperatures, shifting T min or T max (Figure 1c). There is good evidence that when environmental temperature extremes differ, there is a commensurate adaptation of species to these (Hoffmann et al., 2013;Krenek et al., 2012;Payne et al., 2016). When both the above adaptations occur, this will shift the entire curve ( Figure 1d). ...
... Furthermore, the matrix distancing analysis on our review (n = 18) of the maximum temperature at which growth occurs (T max ) suggests a significant correlation between this one aspect of thermal performance and phylogeny ( Figure 6). These analyses, therefore, seem to support the heritability of thermal responses, as has been seen for metazoa such as Drosophila; and other protists (Krenek et al., 2012). Coupled with this correlation, the intriguing steady increase in T max , across species (Figure 7)-albeit with occasional poor replication within species-suggests that this adaptation may be polygenic and incremental rather than saltatory. ...
Temperature drives performance and therefore adaptation; to interpret and understand these, thermal performance curves (TPC) are used, often through meta‐analyses, revealing trends across divergent taxa. Four discrete hypotheses—thermodynamic‐constraint; biochemical‐adaptation (hotter is not better); specialist‐generalist; thermal‐trade‐off—have arisen to explain cross‐phyletic trends. In contrast, detailed comparisons of closely related taxa are rare, yet trends arising from these should reveal mechanisms of adaptation, as taxa diverge. Here, we combine experimental work with TPC theory to assess if the current hypotheses apply equally to closely related taxa. We established TPC for six species (and two strains of one species) of the animal model Tetrahymena (Ciliophora)—characterized by SSU rDNA/COX1 sequences—by examining specific growth rate (r), size (V), production (P = rV), and metabolic rate (rV−0.25) across 15–20 temperatures. Using parameters derived from the mechanistic “Sharpe and DeMichele” function, we established a framework to test which hypothesis best represented the data. We conclude that superficially the “hotter is not better” hypothesis is best but argue that the mechanistic theory underlying it cannot apply at the genus level: trends are likely to arise from little rather than substantial adaptation. Our further analysis suggests: (1) upward shift in the maximum‐functioning temperature (Tmax) is more constrained than the optimal temperature (Topt), leading to a decreased safety margin (Topt−Tmax) and suggesting that species initially succeed in warmer environments through an increase in Topt, followed by increasing Tmax; and (2) thermal performance traits are correlated with phylogeny for closely related species, suggesting that species gradually adapt to new thermal environments.
... A low responsiveness of protozoans to external forcing was also observed in marine systems (Dolan and Gallegos, 2001) and in experiments with epibenthic ciliates . Ciliate species may comprise a high diversity of functionally different clones which may buffer their response to environmental changes (Weisse et al., 2001;Krenek et al., 2012). ...
Plankton food webs are the basis of marine and limnetic ecosystems. Especially aquatic ecosystems of high biodiversity provide important ecosystem services for humankind as providers of food, coastal protection, climate regulation, and tourism. Understanding the dynamics of biomass and coexistence in these food webs is a first step to understanding the ecosystems. It also lays the foundation for the development of management strategies for the maintenance of the marine and freshwater biodiversity despite anthropogenic influences. Natural food webs are highly complex, and thus often equally complex methods are needed to analyse and understand them well. Models can help to do so as they depict simplified parts of reality. In the attempt to get a broader understanding of the complex food webs, diverse methods are used to investigate different questions. In my first project, we compared the energetics of a food chain in two versions of an allometric trophic network model. In particular, we solved the problem of unrealistically high trophic transfer efficiencies (up to 70%) by accounting for both basal respiration and activity respiration, which decreased the trophic transfer efficiency to realistic values of ≤30%. Next in my second project I turned to plankton food webs and especially phytoplankton traits. Investigating a long-term data set from Lake Constance we found evidence for a trade-off between defence and growth rate in this natural phytoplankton community. I continued working with this data set in my third project focusing on ciliates, the main grazer of phytoplankton in spring. Boosted regression trees revealed that temperature and predators have the highest influence on net growth rates of ciliates. We finally investigated in my fourth project a food web model inspired by ciliates to explore the coexistence of plastic competitors and to study the new concept of maladaptive switching, which revealed some drawbacks of plasticity: faster adaptation led to higher maladaptive switching towards undefended phenotypes which reduced autotroph biomass and coexistence and increased consumer biomass. It became obvious that even well-established models should be critically questioned as it is important not to forget reality on the way to a simplistic model. The results showed furthermore that long-term data sets are necessary as they can help to disentangle complex natural processes. Last, one should keep in mind that the interplay between models and experiments/ field data can deliver fruitful insights about our complex world.
... .3390/d13110589/s1, Table S1: Paramecium bursaria complex strains used in the current study [25,29,36,75,95,96], Table S2: Paramecium strains (except P. bursaria) used in the current study [16,36,[59][60][61]83,95,[97][98][99][100][101][102][103]. Two Tetrahymena strains were used as outgroup, Table S3: p-distance matrix of the studied COI mtDNA fragments. ...
Ciliates are a diverse protistan group and many consist of cryptic species complexes whose members may be restricted to particular biogeographic locations. Mitochondrial genes, characterized by a high resolution for closely related species, were applied to identify new species and to distinguish closely related morphospecies. In the current study, we analyzed 132 sequences of COI mtDNA fragments obtained from P. bursaria species collected worldwide. The results allowed, for the first time, to generate a network of COI haplotypes and demonstrate the relationships between P. bursaria strains, as well as to confirm the existence of five reproductively isolated haplogroups. The P. bursaria haplogroups identified in the present study correspond to previously reported syngens (R1, R2, R3, R4, and R5), thus we decided to propose the following binominal names for each of them: P. primabursaria, P. bibursaria, P. tribursaria, P. tetrabursaria, and P. pentabursaria, respectively. The phylogeographic distribution of P. bursaria species showed that P. primabursaria and P. bibursaria were strictly Eurasian, except for two South Australian P. bibursaria strains. P. tribursaria was found mainly in Eastern Asia, in two stands in Europe and in North America. In turn, P. tetrabursaria was restricted to the USA territory, whereas P. pentabursaria was found in two European localities.
... To estimate TPCs for each population, we fit the r max estimates to the Lactin-2 function (Krenek et al. 2011(Krenek et al. , 2012Lactin et al. 1995;Salsbery and DeLong, 2018) using ordinary nonlinear least-squares regression: ...
How and if organisms can adapt to changing temperatures has drastic consequences for the natural world. Thermal adaptation involves finding a match between temperatures permitting growth and the expected temperature distribution of the environment. However, if and how this match is achieved, and how tightly linked species change together, are poorly understood. Paramecium bursaria is a ciliate that has a tight physiological interaction with endosymbiotic green algae (zoochlorellae). We subjected a wild population of P. bursaria to a cold and warm climate (20℃ and 32℃) for ∼300 generations. We then measured the thermal performance curve (TPC) for intrinsic rate of growth (rmax ) for these evolved lines across temperatures. We also evaluated number and size of the zoochlorellae populations within paramecia cells. TPCs for warm-adapted populations were shallower and broader than TPCs of cold-adapted populations, indicating that the warm populations adapted by moving along a thermal generalist/specialist trade off rather than right-shifting the TPC. Zoochlorellae populations within cold-adapted paramecia had fewer and larger zoochlorellae than hot-adapted paramecia, indicating phenotypic shifts in the endosymbiont accompany thermal adaptation in the host. Our results provide new and novel insight into how species involved in complex interactions will be affected by continuing increasing global temperatures. This article is protected by copyright. All rights reserved.
