Temporal environmental change, clonal physiology and the genetic structure of a Daphnia assemblage (D. galeata?hyalina hybrid species complex) (original) (raw)

Temporal environmental change, clonal physiology and the genetic structure of a Daphnia assemblage (D. galeata-hyalina hybrid species complex)

OLAF PINKHAUS*, SUSANNE SCHWERIN*, RALPH PIROW*, BETTINA ZEIS*, INA BUCHEN*, ULRIKE GIGENGACK*, MARITA KOCH*, WOLFGANG HORN †{ }^{\dagger} AND RÜDIGER J. PAUL*
*Institut für Zoophysiologie, Westfälische Wilhelms-Universität, Hindenburgplatz, Münster, Germany
†{ }^{\dagger} Saxon Academy of Sciences at Leipzig, Ecological Station Neunzehnhain, Neunzehnhainer Str., Lengefeld, Germany

SUMMARY

1. In a combined field and laboratory study, seasonal relationships between water temperature and oxygen content, genetic structure (composition of MultiLocus Genotypes, MLGs) of a Daphnia assemblage (D. galeata-hyalina hybrid species complex), and the physiological properties of clones of frequent MLGs were studied. In accordance with the oxygen-limited thermal tolerance hypothesis, essential physiological variables of oxygen transport and supply were measured within the tolerable temperature range.
2. A few MLGs (types T1-T4) were frequent during early spring and late autumn at surface temperatures below 10∘C10^{\circ} \mathrm{C}. Clones of T1-T4 showed a low tolerance towards higher temperatures (above 20∘C20^{\circ} \mathrm{C} ) and a high phenotypic plasticity under thermal acclimation in comparison to clones derived from frequent MLGs from later seasons, and stored highmedium quantities of carbohydrates at 12 and 18∘C18^{\circ} \mathrm{C}.
3. Another MLG (T6) succeeded the MLGs T1-T4. T6 was frequent over most of the year at temperatures above 10∘C10^{\circ} \mathrm{C} and below 20∘C20^{\circ} \mathrm{C}. A clone derived from T6 exhibited a high tolerance towards warm temperatures and a more restricted phenotypic plasticity. It stored high-medium quantities of carbohydrates at 12,18 and 24∘C24^{\circ} \mathrm{C} and showed a high capacity for acclimatory adjustments based on haemoglobin expression.
4. During the summer period at temperatures ≥20∘C\geq 20^{\circ} \mathrm{C}, the MLG T6 was found mainly near to the thermocline, where temperature and oxygen content were distinctly lower, and to a lesser extent in surface water. At the surface, another MLG (T19) was predominant during this period. A clone of this MLG showed a very high tolerance towards warm temperatures, minimal phenotypic plasticity, low carbohydrate stores and a high capacity for circulatory adjustments to improve oxygen transport at higher temperatures.
5. This study provides evidence for connections between the spatio-temporal genetic heterogeneity of a Daphnia assemblage and the seasonal changes of water temperature and oxygen content. The data also suggest that not only the actual temperature but also the dynamics of temperature change may influence the genetic structure of Daphnia populations and assemblages.

Keywords: genetic heterogeneity, oxygen, plasticity, temperature, thermal tolerance

[1]

Introduction

Most physiological processes are affected by temperature. However, various adaptations allow organisms to reduce these effects and to diminish or avoid the

1. Correspondence: Prof. Dr Rüdiger J. Paul, Institut für Zoophysiologie, Westfälische Wilhelms-Universität, Hindenburgplatz 55, D-48143 Münster, Germany. E-mail: paulr@uni-muenster.de ↩︎

impact of temperature extremes. The specific nature of these adaptations defines the particular thermal niche of a species. Given these differences in thermal adaptation, it is not surprising that the distribution patterns of organisms usually reflect gradients or discontinuities in temperature (Hochachka & Somero, 2002). Although the thermal range of a species is essentially fixed by its specific repertoire of adaptive mechanisms, a varying degree of phenotypic plasticity allows the individual to optimise its performance at the prevailing ambient temperature.

Daphnia, a small zooplanktonic organism with low capacities to maintain its internal milieu (extracellular fluid) constant (e.g. with regard to oxygen partial pressure (Po2)\left(\mathrm{Po}_{2}\right) or pH ; e.g. Pirow, Bäumer & Paul, 2004), is challenged by severe temporal and spatial changes of temperature and ambient oxygen concentration. In Daphnia magna, a major regulatory strategy to cope with variations in oxygen concentration involves adjustments in the quantity and quality of the respiratory protein haemoglobin (Hb)(\mathrm{Hb}) via gene expression (e.g. Kobayashi & Hoshi, 1982; Kobayashi, Fujiki & Suzuki, 1988; Paul et al., 2004b; Lamkemeyer et al., 2005). This strategy is advantageous to an animal of low homeostatic capacities (see above), because an alternative allosteric control of Hb function (e.g. Bohr effect) might lead to uncontrolled effects during environmental change (e.g. by variations in haemolymph pH ). Regulation via gene expression, however, is a slow process, which takes several days to become effective. Accordingly, D. magna also exhibits rapid regulatory responses, which mitigate the impact of changing oxygen conditions on the animal’s internal milieu. These regulatory responses comprise adjustments in the ventilatory (e.g. Pirow & Buchen, 2003) and circulatory systems (e.g. Paul et al., 1997) as well as in the standard metabolic rate (metabolic depression; e.g. Paul et al., 1998; Seidl, Paul & Pirow, 2005a). To complete acclimation (i.e. acclimatory processes in the laboratory) in all its sub-processes requires up to 1-2 weeks (Zeis et al., 2004).

The present study focuses on the thermal tolerance of clones derived from MultiLocus Genotypes (MLG) (cf. Weider, 1984) of a Daphnia galeata-hyalina hybrid species complex (D. galeata Sars, D. hyalina Leydig). An MLG was characterized by cellulose acetate electrophoresis (Hebert & Beaton, 1989) at four enzyme loci. An MLG may comprise only one clone
or many clonal lineages that share the same four-loci pattern. The physiological mechanisms underlying thermal tolerance include oxygen transport processes (oxygen-limited thermal tolerance hypothesis; e.g. Pörtner, 2001, 2002) as well as subordinated processes. These mechanisms may be plastic because of gene expression-mediated alterations that improve the animals’ ability to perform in a specific temperature range. Environmentally induced (acclimatory) changes in clone animals (e.g. in oxygen transport parameters) are one prominent example of phenotypic plasticity.

The oxygen-limited thermal tolerance hypothesis relates the thermal tolerance of an animal directly to its temperature-dependent aerobic scope. The aerobic scope is the ratio between maximal and minimal (standard) metabolic rate and defines the amount of oxygen available for the different life processes (maintenance, activity and production). If the aerobic scope reaches a maximal value, all cellular energy demands are matched with oxygen supply, and sufficient energy is available for all life processes. In ectotherms, the aerobic scope changes with temperature, and the temperature range of a maximal or near-to-maximal aerobic scope can be defined as an animal’s thermal optimum. At temperatures deviating from this thermal optimum, the aerobic scope of ectotherms decreases because of mismatches between oxygen supply and energy demand. Increasing temperatures are associated with a higher oxygen and energy demand in ectotherms. As a standard response, the rates of ventilation and circulation also increase with temperature. If energy demand surpasses the maximal ventilation or circulation rate of an animal, the aerobic scope starts to decrease. At even higher temperatures, the increasing gap between oxygen supply and energy demand may result in anaerobic metabolism altogether. A decrease of ventilation/circulation rate with increasing temperature is indicative of such a severely reduced aerobic scope. Long-term acclimation to higher temperatures may result in a horizontal shift of the response curve (ventilation/heart rate versus ambient temperature) towards higher temperatures occasionally combined with changes in its slope and/or shape. With decreasing temperature, oxygen and energy demand decrease in ectotherms. However, the lowered energy demand may not be matched with oxygen supply when e.g. minimal rates of ventilation and circulation

are approached or reached. At even lower temperatures, anaerobiosis has to provide energy for the remaining life processes. In this way, the aerobic scope changes between low and high temperatures (minimum, rise, optimum, decline, minimum) defining the thermal tolerance window of an ectotherm animal. Due to adaptation to habitats of different mean temperature or thermal acclimatisation (i.e. acclimatory processes in the field), the position and shape of the thermal tolerance window may change. It has been suggested (see Pörtner, 2002) that alterations of mitochondrial density and/or capacity, with the latter depending on the quantity and quality of mitochondrial enzymes (e.g. Guderley, 1998; St.-Pierre, Charest & Guderley, 1998), can be a key mechanism for these shifts of thermal tolerance. For the daphnid D. magna, behavioural, physiological and biochemical studies have proven the validity of this hypothesis, and changes in Hb expression have been added as a further key mechanism for the unidirectional shifts of the thermal tolerance window (Lamkemeyer, Zeis & Paul, 2003; Paul et al., 2004a; Zeis et al., 2004; Seidl, Pirow & Paul, 2005b).

As cyclic parthenogens, daphnids exhibit a clonal population structure with genetic diversity varying in time and space (e.g. Hebert & Crease, 1980; Hebert & Moran, 1980; Korpelainen, 1986). An impact of spatiotemporal changes in ambient temperature and oxygen conditions on shifts in the genetic structure of Daphnia populations has been reported (Weider, 1985; Weider & Lampert, 1985; Carvalho, 1987; LaBerge & Hann, 1990; Geedey, Tessier & Machledt, 1996; Mitchell & Lampert, 2000).

The main aim of this study was to relate environmental temperature and oxygen concentration to the genetic structure and clone-specific physiological properties of a Daphnia assemblage to assess the role of these abiotic factors as possible driving forces for changes in genetic structure. We studied, simultaneously with ambient temperature and oxygen concentration, the seasonal changes in genetic structure (MLG composition) of a Daphnia assemblage, and the physiological properties of clones of frequent or prominent MLGs isolated at different times of the year. Physiological experiments were carried out within the tolerable temperature range avoiding excessive cold and heat, and focused on oxygen transport processes including the essential variables of ventilation and circulation as well as the Hb concentration. In
addition, the NADH fluorescence of leg muscles was determined to assess (via the redox state) the oxygen supply to tissues. Carbohydrate stores (glycogen and glucose) were also measured in the different clones. Before experiments, the clones were long-term acclimated at three different temperatures (12, 18 and 24∘C24^{\circ} \mathrm{C} ) within the natural range to assess the clonespecific phenotypic plasticity of physiological function.

Methods

Study species

The daphnids (D. galeata-hyalina hybrid species complex) were regularly sampled throughout 2005 from the Saidenbach Reservoir, Saxony (Germany), at a site in the middle of the lake approximately 200 m from the dam. The Saidenbach Reservoir has a mean depth of 15 m and a maximal depth of 45 m . To sample many hundreds of Daphnia from surface water (0−5 m)(0-5 \mathrm{~m}) while excluding copepods, a plankton net of 780−μm780-\mu \mathrm{m} mesh size and 74−cm74-\mathrm{cm} mouth diameter was used. To sample a hundred or more Daphnia around the thermocline (varying depth: 7−22 m7-22 \mathrm{~m}; sampling depth: thermocline ±2 m\pm 2 \mathrm{~m} ) during the period of stratification, a closing net ( 175−μm175-\mu \mathrm{m} mesh size, 9−cm9-\mathrm{cm} mouth diameter) was utilised. In this case, the separation of Daphnia from copepods was more time consuming. Starting immediately after ice off in mid-April and finishing in December 2005, animals were collected 12 times at a sampling interval of once every 2 weeks in spring and early summer and every 4 weeks later in the year. The animals were always sampled in the morning ( 9.30−11AM9.30-11 \mathrm{AM} ). Due to the identical sampling time, any possible effects of vertical migration on the determined genetic structures (see below) were probably similar at each sampling date. Water temperature and oxygen content of the Saidenbach Reservoir were recorded at different depths using an automatic hydro-meteorological station and data loggers.

To analyse the respective genetic structure of the Daphnia assemblage at each sampling date, approximately 100 individuals from each sampling site (surface, thermocline) were isolated in individual glass vessels containing 40 mL filtered water from the Saidenbach Reservoir. Clone breeding was carried out at 18∘C18^{\circ} \mathrm{C} and took approximately 1 week. Then the isolated individuals (mothers) were used for allozyme characterisation of the genotype, and the clonal

offspring for further experimentation (see below). Cellulose acetate electrophoresis (Hebert & Beaton, 1989) was conducted at four enzyme loci: aspartate amino transferase ( AATA A T, EC 2.6.1.1), aldehyde oxidase (AO, EC 1.2.3.1), phosphoglucoisomerase (PGI, EC 5.3.1.9) and phosphoglucomutase (PGM, EC 5.4.2.2). A clone of the MLG designated as T1 was used as standard on all gels.

Individuals of frequent or prominent MLGs with respect to the seasonally changing genetic structure of the Daphnia assemblage (i.e. MLGs of high genotype frequency; see Fig. 1b,c) were chosen to establish clonal lineages, and clone breeding was carried out for at least 3 weeks at three different acclimation temperatures within the environmental range (12, 18 and 24∘C24^{\circ} \mathrm{C} ) for further physiological experimentation. The clones were kept in 1.5 L culture medium (M4; Elendt & Bias, 1990) within 2.5 L glass beakers at airsaturated conditions under a 16:8 h16: 8 \mathrm{~h} light : dark cycle. To avoid density stress, the population density was kept below 65 individuals L−1\mathrm{L}^{-1} by the regular removal of animals either for experimentation or for a next culture. This way, several cultures were kept for each clone at the different acclimation temperatures. Maintenance of clonal identity was regularly checked by allozyme characterisation. The clones were fed with algae (Chlamydomonas reinhardtii) once daily with the food supply increasing with acclimation temperature (2−4mgCL−1)\left(2-4 \mathrm{mg} \mathrm{C} \mathrm{L}^{-1}\right) to avoid food stress. To guarantee sufficient food supply under all conditions (12, 18 and 24∘C24^{\circ} \mathrm{C} acclimation; 65 individuals L−1\mathrm{L}^{-1} ), several control experiments were carried out to check that the food concentrations (even after a 24−h24-\mathrm{h} period of nonfeeding) were always above 1mgCL−11 \mathrm{mg} \mathrm{C} \mathrm{L}^{-1}. One-half of the culture medium was renewed weekly.

Measurement of ventilation rate, heart rate and NADH fluorescence

Physiological variables representative of oxygen transport processes or indicative of the oxygen supply of mitochondria were simultaneously measured by optophysiological methods (Colmorgen & Paul, 1995) in the differently acclimated clones at varying ambient temperature. These variables included (i) the ventilation rate, the movement frequency of the thoracic limbs, which serve for filter feeding as well as ventilatory oxygen transport (e.g. Pirow, Wollinger & Paul, 1999a; Pirow & Buchen, 2003); (ii) the heart
rate, which is representative of the rate of haemolymph oxygen transport (e.g. Bäumer, Pirow & Paul, 2002; Seidl et al., 2005a,b) and (iii) the NADH fluorescence intensity of the leg muscles, which is indicative of the mitochondrial reduction-oxidation state of these tissues (e.g. Pirow, Bäumer & Paul, 2001).

The only difference to the previously described experimental setup for this type of measurements (cf. Pirow et al., 2001; Seidl et al., 2005a,b) was the technique for recording the NADH fluorescence. In this study, a photomultiplier (H5784; Hamamatsu, Herrsching, Germany) was utilised. In 20-s intervals, the specimen was illuminated by UV light ( 365 nm ) for 1 s , and the fluorescence at 450 nm was recorded by the photomultiplier. The photomultiplier signal was digitised and recorded by a computer equipped with an A/D converter (DAS1602; Keithley Metrabyte, Taunton, MA, U.S.A.).

For the different clonal lineages and acclimation temperatures, replicated experiments were carried out on single females (at least n=3n=3 different individuals) with carapace lengths (see Seidl et al., 2005a) of 1.251.32 mm and carrying three to five parthenogenetic embryos of the developmental stages 1 to 2 (Green, 1956). Before experimentation, animals from the different (acclimated) cultures were incubated for 30 min in nutrient free, air-saturated M4 medium. After this incubation period, the animals were immobilised by glueing their dorsal carapace with adhesive to a thin insect needle attached to a small PVC cube (see Pirow et al., 2001). The immobilised individual was then transferred into a thermostat-controlled ( ±0.1∘C\pm 0.1^{\circ} \mathrm{C} ) animal chamber (cf. Paul et al., 1997). The chamber was perfused with 50%50 \% air saturated, normocapnic (i.e. atmospheric CO2\mathrm{CO}_{2} content) M4 medium at a flow rate of 5 mL min−15 \mathrm{~mL} \mathrm{~min}^{-1} using a peristaltic pump. The moderate air saturation of 50%50 \% was chosen to specifically emphasise differences in the tissue NADH fluorescence signal. Temperature and air saturation of the perfusion medium were set using a thermostatcontrolled ( ±0.1∘C\pm 0.1^{\circ} \mathrm{C} ) glass mixing vessel in front of the animal chamber. The vessel was supplied (via a peristaltic pump) with M4 medium, which was equilibrated with gas provided by a gas-mixing pump (Wösthoff, Bochum, Germany). Temperature and oxygen content of the medium was measured by a temperature sensor (dm. 0.3 mm , Type MT-29/1; Science Products, Hofheim, Germany) within the chamber and by a combined oxygen and temperature

Fig. 1 Seasonal changes of temperature (circles and lines), oxygen concentration (squares and lines) and Daphnia abundance (dashed and dotted lines) after ice off (day 90) in surface water (0−5 m;Ts,Os,as)\left(0-5 \mathrm{~m} ; T_{\mathrm{s}}, O_{\mathrm{s}}, a_{\mathrm{s}}\right) and at the thermocline (7−22 m;Tb,Ob,ab)(a)\left(7-22 \mathrm{~m} ; T_{\mathrm{b}}, O_{\mathrm{b}}, a_{\mathrm{b}}\right)(\mathrm{a}) in conjunction with the genetic structure (b-f) of the Daphnia assemblage (D. galeata-hyalina hybrid species complex). [b, c show only MultiLocus Genotypes (MLGs) with a frequency higher than 2%2 \% at a sampling date]. A few MLGs (T1-T4) frequent or prominent in mid-April almost vanished during summer, but increased their genotype frequency again at the end of the year (b, d). The MLG T6 succeeded T1 in dominance (b, c, e). In summer, another group of MLGs (particularly T19) contributed substantially to the genetic structure of the assemblage (b, f).

sensor (Oxi 3000; WTW, Weilheim, Germany) within the outflow from the animal chamber. After habituation to the experimental conditions for 30 min , the physiological variables were monitored for 10 min and then the ambient temperature was gradually increased from 6∘C6^{\circ} \mathrm{C} to 30∘C30^{\circ} \mathrm{C} in steps of 6∘C6^{\circ} \mathrm{C}. Each temperature level except the first one was kept for 20 min . The first 10 min of each 20−min20-\mathrm{min} period served to obtain a constant temperature within the chamber and to allow the animal to respond physiologically to the new condition. The second-half of each period was used for data acquisition (ventilation and heart rate, NADH fluorescence, temperature). Control experiments included a testing of animals for 2 h at a constant temperature of 12,18 or 24∘C24^{\circ} \mathrm{C} to prove the constancy of all measured physiological variables over longer periods of time.

Measurement of glycogen and glucose concentration

Glycogen and glucose concentrations were determined according to the method of Kunst, Draeger & Ziegenhorn (1984) via the NADPH fluorescence intensity, which was measured in a luminescence spectrometer (LS 50 B; Perkin Elmer, Boston, MA, U.S.A.). The metabolites were determined in the crude extracts of animals taken from different clonal lineages and acclimation temperatures. For each measurement, 10 animals ( 1.5−2.0 mm1.5-2.0 \mathrm{~mm} in body length) were collected by sieving and adhering water was removed with a tissue. Animals were then shock frozen in liquid nitrogen and weighed (BP 211D; Sartorius, Göttingen, Germany), and if necessary the sample was kept frozen at −70∘C-70^{\circ} \mathrm{C} until the start of the analysis. After adding a defined quantity of water, extracts were prepared using a Teflon ®{ }^{\circledR} pistil (Kontes, Vineland, NJ, U.S.A.) in a 1.5−mL1.5-\mathrm{mL} reaction tube, and proteins were then denatured at 70∘C70^{\circ} \mathrm{C} for 30 min . The metabolite concentrations were determined in two aliquots. In the first aliquot, only glucose was measured. In the second aliquot, glucose was measured after hydrolysing glycogen to glucose using amyloglucosidase (enzymes from Sigma, Taufkirchen, Roche, Mannheim, Germany). The glycogen concentration was calculated from the difference in both aliquots. After adding 200μ L200 \mu \mathrm{~L} of acetate buffer to both aliquots and amyloglucosidase only to the second one, the samples were incubated for 5 h at 37∘C37^{\circ} \mathrm{C}, centrifuged ( 14000 gat4∘C14000 \mathrm{~g} \mathrm{at} 4^{\circ} \mathrm{C} for 20 min ), and the supern-
atants were immediately used for enzymatic analysis or were kept frozen at −70∘C-70^{\circ} \mathrm{C} until the start of the analysis. Based on a calibration curve of known glucose concentrations, the glucose concentration in the samples could be quantified. To express carbohydrate concentration in general terms, the measured concentrations of glycogen and glucose were combined as (carbohydrates) =(=( glycogen )+2/3)+2 / 3 (glucose), thereby taking into account the different quantities of ATP available from the glucosyl units of glycogen (3 ATP) or glucose (2 ATP).

Measurement of haemoglobin concentration

Haemoglobin concentration was determined in crude extracts. Twenty animals (1.5-2.0 mm in body length) were collected by sieving and adhering water was removed with a tissue. Animals were then shock frozen in liquid nitrogen and weighed, and if necessary the sample was kept frozen at −70∘C-70^{\circ} \mathrm{C} until the start of the analysis. After adding 600μ L600 \mu \mathrm{~L} of M4 medium, extracts were prepared using a Teflon ®{ }^{\circledR} pistil in a 1.5 mL reaction tube. To prevent proteolysis, phenylmethylsulfonylfluoride (PMSF) and EDTA were added to a final concentration of 1 mm each. After centrifugation ( 12000 gat4∘C12000 \mathrm{~g} \mathrm{at} 4^{\circ} \mathrm{C} for 15 min ), the supernatants were used for photometric measurement (UV/Visible spectrophotometer Ultrospec 3000; Pharmacia Biotech, Uppsala, Sweden). Absorption spectra of oxygenated Hb were recorded in the range of 250−800 nm250-800 \mathrm{~nm} using M4 medium containing PMSF and EDTA as reference. After adding some crystals of sodium dithionite, a second spectrum of the deoxygenated Hb was recorded. The Hb concentration was calculated from the difference of both spectra by comparing the area of the difference spectrum between 422 and 452 nm with that of a purified Hb solution of known concentration from Hb-rich D. magna (cf. Zeis et al., 2003).

Calculations based on physiological data

Calculation of perfusion rates and maximum haemolymph oxygen concentrations were carried out according to Bäumer et al. (2002). Perfusion rate (i.e. the amount of haemolymph pumped by the heart per unit time into the body) is the product of heart rate and stroke volume. The stroke volume ( 1.7 nL ) was derived from the body size - stroke volume relationship given in Seidl et al. (2005a). The maximum

haemolymph oxygen concentration is the sum of (i) the amount of oxygen maximally bound to the available haem groups (i.e. Hb concentration) and (ii) the physically dissolved oxygen concentration. There is a 1:11: 1 ratio between the concentrations (in nmolμL−1\mathrm{nmol} \mu \mathrm{L}^{-1} ) of oxygen and haem groups. However, Hb concentrations were measured on the basis of fresh body mass (nmol haem per unity mass). To calculate Hb concentrations on the basis of haemolymph volume (i.e. nmol haem per unity volume), haemolymph volume has to be estimated from fresh body mass. Kobayashi (1983) reported that the haemolymph volume comes to approximately 60%60 \% of fresh body mass. Accordingly, the volume-based Hb concentrations are higher by approximately 70%70 \% than the mass-based Hb concentrations. The physically dissolved oxygen was calculated from the ambient Po2\mathrm{Po}_{2} neglecting the small Po2\mathrm{Po}_{2} gradient between ambient water and haemolymph (cf. Pirow et al., 2004), and the physical solubility for oxygen in the haemolymph (cf. Bäumer et al., 2002) taking into account the tempera-ture-dependence of this value.

Data analysis

Data are given as mean ±SD\pm \mathrm{SD} with nn indicating the number of animals tested if not stated otherwise. The influence of acclimation temperature and clone on ventilation, circulation, NADH fluorescence, carbohydrate and Hb concentration were assessed by a twoway analysis of variance (two-way ANOVA). Statistical differences were considered to be significant at P<0.05P<0.05. In the case of a statistically significant difference, multiple comparisons (Holm-Sidak test) among pairs of mean using an experimentwise (overall) significance level of 0.05 were carried out to determine the differing pairs of mean. Comparisons of the carbohydrate and Hb concentration between different groups of identically acclimated clones were performed by a non-parametric two-sample rank test (Mann-Whitney test). SIGMAPlot 8.0 and SIGMAstat 3.01 (Systat Software, ErKrath, Germany) were used for graphs, non-linear regression analyses and statistical analyses.

Results

From April until December, the surface temperature of the Saidenbach Reservoir (Saxony, Germany) rose
from approximately 3∘C3^{\circ} \mathrm{C} to maximal values near to 23∘C23^{\circ} \mathrm{C} and decreased then to 6∘C6^{\circ} \mathrm{C} at the end of the year (Fig. 1a). At the thermocline, which persisted during the period of stratification from early May until early November, the temperature varied between 8 and 15∘C15^{\circ} \mathrm{C} during the seasons. The oxygen concentration of surface water was between 0.22 and 0.44mmolO2 L−10.44 \mathrm{mmol} \mathrm{O}_{2} \mathrm{~L}^{-1} (Fig. 1a). At the thermocline, it decreased between June and November from 0.34 to 0.09mmolO2 L−10.09 \mathrm{mmol} \mathrm{O}_{2} \mathrm{~L}^{-1}.

The Daphnia assemblage (D. galeata-hyalina hybrid species complex) from the Saidenbach Reservoir showed two seasonal maxima in abundance ( 7−107-10 individuals L−1\mathrm{L}^{-1} ) in surface water and at the thermocline, one at the beginning of July and one at the beginning of October, with a reduced abundance in between (Fig. 1a). In respect to the abundance in surface water, the abundance at the thermocline increased from June until the end of October.

The genetic structure (composition of MLGs) of the Daphnia assemblage from Saidenbach Reservoir was investigated from mid-April (directly after ice off) until December 2005. In total, 47 MLGs (types T1-T47) were identified during the year by allozyme electrophoresis for the enzymes AAT, A0, PGI and PGM. Per sampling date, 7-21 different MLGs were found. The most frequent MLGs in 2005 were T1, T6, T8, T9 and T19 (Fig. 1b,c).

MultiLocus Genotypes present in mid-April (mainly T1, but also T2-T4) almost disappeared during the next weeks and months except T1, which was still found although at low frequency (Fig. 1d). At the end of the year, the frequency of T1, T2 increased again. T6 succeeded T1 in dominance (Fig. 1e). In the summer months, however, a few MLGs (particularly T19) reached or even surpassed the frequency of T6 (Fig. 1f). They reached a maximal frequency in surface water just at the time when T6 showed an intermediate minimum there. At that time, T6 reached a maximal frequency at the thermocline (Fig. 1e). Other MLGs such as T1-T4 or T19 were found only in minor quantities at the thermocline with the exception of T8 and T9 (Fig. 1c).

A set of clones derived from frequent or prominent MLGs (T1, T2, T3, T4, T6, T8, T9, T19) was selected for thermal acclimation ( 12,18 and 24∘C24^{\circ} \mathrm{C} ) and subsequent physiological experimentation (Fig. 2). The rate of thoracic limb movements (ventilation rate) and the heart rate usually increased with ambient tempera-
© 2007 The Authors, Journal compilation © 2007 Blackwell Publishing Ltd, Freshwater Biology, 52, 1537-1554

ture. Acclimation to higher temperatures caused the ventilatory response curves (ventilation rate versus ambient temperature) to shift towards higher temperatures markedly in the T1-derived clone, designated as T1c (the ’ cc ’ is added to differentiate between MLG and clone), less in T6c and scarcely in T19c. In T1c, these shifts were combined with changes in shape of the response curves. Similar acclimation effects were found for the circulatory response curves of this clone. Irrespective of shape, the temperatures, at which ventilation rates were maximal, were lower in T1c than in T6c or T19c. Accordingly, the ability to transport oxygen and maintain a high aerobic scope
at higher temperatures (20−30∘C)\left(20-30^{\circ} \mathrm{C}\right) was much lower in T1c in comparison to T6c or T19c. The NADH fluorescence decreased with increasing ambient temperature in all clones except for T1c, where the NADH fluorescence increased at higher temperatures almost in synchrony with the decrease of ventilatory movements.

The ventilatory response proved to be particularly suitable for a more detailed analysis (Fig. 3). The ventilation rate at identical ambient and acclimation temperatures (12, 18 and 24∘C24^{\circ} \mathrm{C}; see grey-filled circles in Fig. 2), which corresponds more to the natural condition, increased in most clones (T1c, T2c, T4c,

Fig. 2 Oxygen transport variables (ventilatory movements, heart rate, and NADH fluorescence) (mean ±SD;n=3\pm \mathrm{SD} ; n=3 animals each) simultaneously measured at different ambient temperatures in clones derived from MultiLocus Genotypes (MLGs) most frequent during mid-April (T1 clone: T1c), spring-autumn (T6c), and summer (T19c). The dotted, dashed and solid regression curves (Lorentzian peak functions) indicate the mean response of 12,18 and 24∘C24^{\circ} \mathrm{C} acclimated clones. Grey-filled circles mark measurements at identical ambient and acclimation temperatures. The slope of the grey dashed lines (top, left) corresponds to the increase in ventilation rate per unit change in temperature. The NADH fluorescence intensity was set to 100%100 \% at 6∘C6^{\circ} \mathrm{C} ambient temperature.

T8c) between 12 and 18∘C18^{\circ} \mathrm{C} (Fig. 3a). Between 18 and 24∘C24^{\circ} \mathrm{C}, however, this specific ventilation rate decreased in mid-April clones (T1c, T2c, T4c), but increased in summer clones (T9c, T19c). The heart rate at identical ambient and acclimation temperatures (data not shown) increased statistically significantly between 12 and 18∘C18^{\circ} \mathrm{C} in T1c, T2c, T8c, T19c and between 18 and 24∘C24^{\circ} \mathrm{C} in T2c, T6c, T8c, T9c, T19c. The NADH fluorescence at identical ambient and acclimation temperatures (data not shown) decreased more or less linearly between 12 and 24∘C24^{\circ} \mathrm{C} in all clones.

The increase of ventilation rate per unit change in temperature, ΔV/ΔT\Delta \mathrm{V} / \Delta \mathrm{T} (see grey broken lines in Fig. 2),

Fig. 3 Ventilatory response (mean + SD; n=3n=3 animals each) of 12,18 and 24∘C24^{\circ} \mathrm{C} acclimated clones derived from frequent or prominent MultiLocus Genotypes (MLGs) from mid-April (T1c, T2c, T4c), spring-autumn (T6c) and summer (T8c, T9c, T19c) ( 12∘C12^{\circ} \mathrm{C} acclimation was not tested in T9c\mathrm{T9c} ). (a) Ventilation rate (= thoracic limb movement rate) of the clones at identical ambient and acclimation temperatures (see grey-filled circles in Fig. 2). Statistically significant differences (P<0.05)(P<0.05) between 12 and 18∘C18{ }^{\circ} \mathrm{C} are marked by the letter aa and between 18 and 24∘C24^{\circ} \mathrm{C} by the letter bb. (b) Increase of ventilation rate per unit change in temperature (see grey dashed lines in Fig. 2) of the 12,18 and 24∘C24^{\circ} \mathrm{C} acclimated clones. Statistically significant differences (P<0.05)(P<0.05) between 18 and 24∘C24^{\circ} \mathrm{C} are marked by the letter bb. (Vertical dashed lines separate clones derived from MLGs from midApril, spring-autumn and summer).
which indicates the intensity of the ventilatory response to changing ambient temperatures, did not change significantly between 12 and 18∘C18^{\circ} \mathrm{C} acclimation (Fig. 3b). Between 18 and 24∘C24^{\circ} \mathrm{C} acclimation, however, there was a pronounced decrease of the ΔV/ΔT\Delta \mathrm{V} / \Delta \mathrm{T} values in mid-April clones (T1c, T2c, T4c) in contrast to clones of other MLGs. For the increase of heart rate per unit change in temperature (data not shown), clear tendencies were mostly not detected. Depending on clone, this quantity either increased or decreased between the different acclimation temperatures. However, the increase of heart rate per unit change in temperature was the (statistically significantly) highest in T19c at all acclimation temperatures. The decrease of NADH fluorescence per unit change in temperature (data not shown) frequently became stronger between 18 and 24∘C24^{\circ} \mathrm{C} acclimation with the (statistically significantly) highest change towards a stronger decrease in T19c.

Acclimation to higher temperatures frequently caused shifts of response curves (ventilation/heart rate and/or NADH fluorescence versus ambient temperature) towards higher temperatures (see Fig. 2). The ventilatory response curves most distinctly exhibited these shifts, thereby reflecting a changed temperature range at which the aerobic scope is maximal. This phenotypic plasticity because of thermal acclimation was particularly high in T1c, whereas T19c exhibited almost none (Fig. 2). To test for phenotypic plasticity in all clones, and to get a coarse measure of its degree, a simple numeric procedure was carried out (see Fig. 4). Minor differences in phenotypic plasticity were found at lower ambient temperatures and major differences at higher ambient temperatures (Fig. 4). Mid-April clones, however, showed for all tested physiological variables a much higher degree of phenotypic plasticity, particularly at higher temperatures, than the other ones. The phenotypic plasticity because of thermal acclimation was the highest in T1c, T2c and T4c; it was reduced in T6c and T8c and the lowest in T19c.

In addition to variables related to oxygen transport, carbohydrate concentration as a measure of stored and rapidly available energy, and Hb concentration as a potentially essential variable of circulatory oxygen transport, were determined in the differently acclimated clones. At 12∘C12^{\circ} \mathrm{C} acclimation, T6c showed higher carbohydrate stores than T1c-T4c (Fig. 5a). At 18∘C18^{\circ} \mathrm{C} acclimation, T1c had the highest carbohydrate

Fig. 4 Degree of phenotypic plasticity of ventilatory movements (a), heart rate (b) and NADH fluorescence © in clones derived from frequent or prominent MLGs as a function of ambient temperature. To quantify the degree of phenotypic plasticity due to thermal acclimation separately for each physiological variable, each ambient temperature and each clone, mean values of the oxygen transport variables were pooled irrespective of acclimation temperature, and the standard deviation was taken as a measure of the degree of phenotypic plasticity. The phenotypic plasticity (particularly at higher temperatures) was much higher in mid-April clones (T1c,T2c, T4c; grey symbols and lines) than in clones derived from other MultiLocus Genotypes (MLGs) (T6c, T8c, T19c; black symbols and lines).
concentration of all clones and moreover, all other mid-April clones (T2c-T4c) as well as T6c exhibited a higher carbohydrate concentration than clones from summer (T9c, T19c). Between 18 and 24∘C24^{\circ} \mathrm{C} acclimation, there was a general decrease in carbohydrate concentration, which was statistically significant in T1c, T2c and T4c. The Hb concentration increased to a varying extent between 12 and 18∘C18^{\circ} \mathrm{C} acclimation in almost all clones (Fig. 5b). Between 18 and 24∘C24^{\circ} \mathrm{C} acclimation, however, T6c was the only clone, which showed a pronounced further increase in Hb concentration starting already from the highest Hb concentration of all clones at 18∘C18^{\circ} \mathrm{C} acclimation.

Fig. 5 Carbohydrate (Ch) (a) and haemoglobin (Hb) (b) concentration (mean +SD ; for Ch:n=3\mathrm{Ch}: n=3 pools of 10 animals each; for Hb:n=3\mathrm{Hb}: n=3 pools of 20 animals each) measured in 12, 18 and 24∘C24^{\circ} \mathrm{C} acclimated clones derived from MultiLocus Genotypes (MLGs) from mid-April (T1c-T4c), spring-autumn (T6c) and summer (T8c, T9c, T19c). (Some acclimation conditions were not tested in T8c, T9c and T19c). Statistically significant differences ( P<0.05P<0.05 ) between 18 and 24∘C24^{\circ} \mathrm{C} are marked by the letter bb. Statistically significant differences (P<0.05)(P<0.05) between different clones/ groups of clones are indicated by the letters cc and dd (in a: T6c versus T1c-T4c; in b: T6c versus T1c-T4c, T8c, T9c, T19c), the letters ee and ff (in a: T1c versus T2c-T4c, T6c, T9c, T19c) and the letters gg and hh (in a: T2c-T4c, T6c versus T9c, T19c). (Vertical dashed lines separate clones derived from MLGs from midApril, spring-autumn and summer).

Discussion

Daphnids exhibit a clonal population structure with genetic diversity varying over the year (e.g. Hebert & Crease, 1980; Hebert & Moran, 1980; Korpelainen, 1986). Accordingly, variation in thermal adaptation may be present not only at the species level, but also at the clonal level. For that reason, we have studied, together with ambient temperature and oxygen content, the seasonal changes in genetic structure of a Daphnia assemblage in conjunction with the physiological characteristics of clones derived from frequent or prominent MLGs. Before physiological experimentation, the clones have been acclimated at three different temperatures to be able to distinguish between genetic determination and phenotypic plasticity of physiological properties. The selected acclimation temperatures were within the natural temperature range.

A determination of taxon affinity by cellulose acetate electrophoresis is particularly important in a hybridising and backcrossing assemblage, such as the D. galeata-hyalina hybrid species complex. The enzyme AATA A T has been reported to be a reliable taxonspecific marker (Wolf & Mort, 1986), which has been used to identify daphnids from many lakes. Daphnia galeata was found to be homozygous for the fast allele (F), D. hyalina was homozygous for the slow allele (S) and D. galeata ×\times D. hyalina was heterozygous (SF). A study by Gießler (1997) reported that the FF and M alleles of the enzyme AOA O are specific for DD. galeata (FF, MF, MM), and various S alleles are specific for D. hyalina (SS). In our study on daphnids from the Saidenbach Reservoir, we have found three banding
patterns for the AATA A T and AOA O : SS (one slow band), SF (three bands: slow, medium and fast), and FF (one fast band) (Table 1). Based on genotype, T1, T4, T12 and T19 are related to D. galeata, and T6 and T9 to D. hyalina. If individuals show for one species-specific marker a homozygote pattern and for the other a heterozygote pattern, a cross within a hybrid or a backcross of a hybrid with one of the parental species is indicated (Spaak et al., 2004). In T2, the parental species was presumably D. galeata (BPgal )\left(\mathrm{BP}_{\text {gal }}\right). Based on their genotype, T3 and T8 were secondary (F2) hybrids (cf. Jankowski & Straile, 2004). In addition to genotypic classification, frequent or prominent MLGs of this study were also repeatedly examined by an eminent expert of Daphnia morphology (D. Flößner, Jena). The investigated MLGs were morphologically classified (Table 1) as either D. galeata (T1-T4, T12, T19) or D. galeata ×\times D. hyalina (T6, T8, T9). The difference between genotypic and morphological classification may result from the aforementioned problem of classification in a hybridising and backcrossing assemblage.

With changing surface temperature of the Saidenbach Reservoir (Fig. 1a), a succession of MLGs was found with a dominance or prominence of T1 in midApril and December at 10∘C10^{\circ} \mathrm{C} and below (Fig. 1b,d), of T6 at temperatures above 10∘C10^{\circ} \mathrm{C} (Fig. 1b,e), and of T19 in summer at a more-or-less constant temperature of 20∘C20^{\circ} \mathrm{C} (Fig. 1b,f). During the maximum of T19 abundance, T6 was dominant at the oxygen-depleted thermocline with a temperature of approximately 14∘C14^{\circ} \mathrm{C} (Fig. 1c,e). However, this MLG may perform diel vertical migration and inhabit the thermocline mainly during daytime.

Table 1 Genotypes as well as genotypic and morphological classification of the most frequent MultiLocus Genotypes (MLG) of the Saidenbach Reservoir ( AATA A T, aspartate amino transferase; AOA O, aldehyde oxidase; PGIP G I, phosphoglucoisomerase; PGMP G M, phosphoglucomutase)

[1]

1. a{ }^{a} Deduced from AATA A T and AOA O banding patterns.
   †{ }^{\dagger} Classification by D. Flößner (Jena). ↩︎

Clones derived from frequent or prominent MLGs were physiologically investigated in the laboratory. A MLG, which was characterised by cellulose acetate electrophoresis at four enzyme loci, may comprise either one clone or many clonal lineages that share the same four-loci pattern. The remarkably coherent relationships between the environmental conditions and the outdoor frequency and physiological properties of MLGs and MLG-derived clones suggest that either one clone per MLG has dominated or the investigated physiological properties of the clones belonging to one MLG have been similar. Independent of any specific interpretation, however, the fourlocus allozyme electrophoresis technique turned out to be a sufficiently valid genetic platform to relate the physiological properties of a MLG-derived clone to the outdoor frequency of this MLG and to the prevailing environmental conditions.

In accordance with the oxygen-limited thermal tolerance hypothesis (e.g. Pörtner, 2001, 2002), the thermal tolerance of thermally acclimated clones of frequent or prominent MLGs was studied by measuring physiological variables related to oxygen transport and supply at different ambient temperatures. Generally, the increase of energy demand with rising ambient temperature caused an increase of ventilation and circulation rate (Fig. 2). The NADH fluorescence signal, which mainly originates from mitochondrial protein-bound NADH species (e.g. Wakita, Nishimura & Tamura, 1995; Paul et al., 2000), allows assessment (via the redox state) of the oxygen supply to tissues. The redox balance left(mathrmNADH/mathrmNAD+right)\left(\mathrm{NADH} / \mathrm{NAD}^{+}\right)left(mathrmNADH/mathrmNAD+right)of the tissues shifted to the oxidation state (decrease of NADH fluorescence) with rising temperature, probably indicating a higher rate of mitochondrial electron transport and oxidative phosphorylation. The ability for transporting oxygen at 20−30∘C20-30^{\circ} \mathrm{C} was lower in the T1-derived clone (T1c) than in T6c and T19c. In T1c, the ventilation rate decreased at ambient temperatures above 18∘C(12∘C18^{\circ} \mathrm{C}\left(12^{\circ} \mathrm{C}\right. acclimation), 22∘C(18∘C22^{\circ} \mathrm{C}\left(18^{\circ} \mathrm{C}\right. acclimation) and 24∘C(24∘C24^{\circ} \mathrm{C}\left(24^{\circ} \mathrm{C}\right. acclimation). In 12 and 18∘C18^{\circ} \mathrm{C} acclimated T 1 c , the heart rate also decreased at higher temperatures and the NADH fluorescence increased. The shifts of redox balance to the reduction state (NADH)(\mathrm{NADH}) indicate the onset of a temperatureinduced shortage of oxygen (hypoxia) in thoracic limb muscle tissues.

In comparison to T6c and T19c, T1c exhibited a high phenotypic plasticity because of thermal acclimation.

Generally, mid-April clones (T1c, T2c, T4c) showed a much higher phenotypic plasticity of physiological properties because of thermal acclimation (see Fig. 4) than clones derived from MLGs from later periods of the year (T6c, T8c, T19c). Thermal acclimation may take up to 2 weeks in Daphnia (Zeis et al., 2004). In this study, therefore, the clones have been acclimated to 12,18 and 24∘C24^{\circ} \mathrm{C} for several weeks, before physiological experimentation has been carried out. In natural environments, however, ambient temperature may change rapidly and strongly. At the Saidenbach Reservoir, the temperature of surface water rose in early spring from 2.5 to 13.8∘C13.8^{\circ} \mathrm{C} within a few weeks (Fig. 1a). This rapid temperature increase may have been one of the factors which explained the replacement of T1 by T6 (Fig. 1b,d,e) despite the high phenotypic plasticity of T1c. T1 (T1c) may be competitive with T6 (T6c) in terms of oxygen transport at temperatures ≤18−20∘C\leq 18-20^{\circ} \mathrm{C} only if it had enough time to acclimatise. Nevertheless, T1 and a few other MLGs from mid-April (T2, T4) remained present over the year and increased their frequency again (Fig. 1b,d) when surface temperature dropped at the end of the year.

The MLG T6 was already present at a temperature below 10∘C10^{\circ} \mathrm{C} (mid-April). When the temperature of surface water exceeded 10∘C,T610^{\circ} \mathrm{C}, \mathrm{T6} rapidly reached a maximal frequency of more than 70%70 \% (Fig. 1b,e). Physiological experimentation showed that T6c is able to transport oxygen over a wide range of ambient temperatures (Fig. 2). The phenotypic plasticity of T6c because of thermal acclimation was reduced in comparison to T1c, but still pronounced. This inherent potential for thermal acclimatisation may be one of the factors responsible for the particularly high genotype frequency of T6 (Fig. 1c,e) during periods of changing water temperature (spring, autumn) and at the variable temperature and oxygen conditions in deeper water layers.

A clone of the MLG T19, which was frequent during the summer period (Fig. 1b,f) when water temperature was nearly constant (approximately 20∘C20^{\circ} \mathrm{C} ), also had the ability to transport oxygen over a wide range of ambient temperatures (Fig. 2). However, in accordance with a thermally almost constant environment, T19c had almost no ability to vary physiological functions after thermal acclimation.

Analysing the ventilatory response in more detail confirmed that mid-April clones (T1c, T2c, T4c)

reduce oxygen transport at higher temperatures (20$30^{\circ} \mathrm{C}$ ). In these clones, the ventilation rate at identical ambient and acclimation temperatures (Fig. 3a) was much higher after 18∘C18^{\circ} \mathrm{C} than 24∘C24^{\circ} \mathrm{C} acclimation. The same was valid for the increase of ventilation rate per unit change in temperature (Fig. 3b). In T6c and T8c, the ventilatory response after 18 and 24∘C24^{\circ} \mathrm{C} acclimation remained more or less constant. However, T9c and T19c exhibited, between 18 and 24∘C24^{\circ} \mathrm{C}, significant increases of the ventilation rate at identical ambient/ acclimation temperatures (Fig. 3a). T19c also showed, at each acclimation temperature, the highest increase of heart rate per unit change in temperature, and between 18 and 24∘C24^{\circ} \mathrm{C} acclimation, the highest change of the NADH fluorescence per unit change in temperature towards a stronger decrease of NADH fluorescence with temperature.

From all these data, it can be concluded that T1c and other mid-April clones have a low, T6c a high, and T19c and other summer clones a very high ability to transport oxygen and maintain a high aerobic scope between 18 and 24∘C24^{\circ} \mathrm{C}.

Clone-specific differences were also found in biochemical variables. At 12∘C12^{\circ} \mathrm{C} acclimation, the carbohydrate stores (Fig. 5a) proved to be high in T6c and medium in mid-April clones (T1c-T4c). At 18∘C18^{\circ} \mathrm{C} acclimation, they were high (T1c) or medium (T2cT4c) in mid-April clones, medium also in T6c, but low in summer clones (T9c, T19c). At 24∘C24^{\circ} \mathrm{C} acclimation, these stores strongly dropped in mid-April clones (T1c, T2c, T4c) but not in T6c. The high-medium quantities of stored carbohydrates in T1c-T4c and T6c at 12−18∘C12-18^{\circ} \mathrm{C} acclimation prevent a permanent dependency on food supply and allow a rapid mobilisation of energy provision mechanisms. At least a partial compensation for the restriction of food resources (phytoplankton) is possible via a reduction of the zooplankton’s energy demands. However, clones (MLGs) with a lower susceptibility to periods of starvation because of higher carbohydrate stores will have fitness advantages during food restriction.

The highest concentration of the respiratory protein Hb was found in T6c already after 18∘C18^{\circ} \mathrm{C} acclimation, and a further increase to approximately 100 nmol haem g−1\mathrm{g}^{-1} fresh weight occurred in this clone after 24∘C24^{\circ} \mathrm{C} acclimation (Fig. 5b). The high capacity for Hb synthesis improves the oxygen transport capacity and consequently, the tolerance of T6c towards higher temperatures and/or lower oxygen concentrations.

This unique quality of T6c may have been another factor responsible for the particularly high genotype frequency of T6 during periods of changing water temperature (spring, autumn) and at the oxygendepleted thermocline. Similarly, Weider & Lampert (1985) and Weider (1985) showed for a Daphnia pulex population, clone-specific variations in hypoxia tolerance and Hb production as well as an enrichment of those MLGs during summer, whose clones exhibited a high capacity for Hb synthesis.

Combining the data on Hb concentration (Fig. 5b) and heart rate (Fig. 2; data at identical ambient and acclimation temperatures, grey-filled circles) gives deeper insights into the mechanisms of thermal tolerance in Daphnia. From Hb concentration, the maximum amount of oxygen in the haemolymph (Hbbound and physically dissolved oxygen) was computed (see Methods). From heart rate, the perfusion rate (i.e. the amount of haemolymph pumped by the heart per unit time into the body) was calculated. The product of perfusion rate (Fig. 6d-f) and the maximum amount of oxygen in the haemolymph (Fig. 6a-c) yields the maximum convective oxygen supply of an animal (cf. Bäumer et al., 2002), which is the maximum amount of oxygen pumped by the heart per unit time into the body. The comparison of the maximum convective oxygen supply with the oxygen consumption rate of an animal (Fig. 6g-i) allows assessment of the relative importance of circulatory (convective) and diffusive oxygen supply in Daphnia (Bäumer et al., 2002). As a complete removal of oxygen from the circulating haemolymph is very unlikely, a distinct surplus of the maximum convective oxygen supply over the oxygen consumption rate is necessary for a higher share of convective oxygen supply. Conversely, a higher share of diffusive oxygen supply can be inferred from a low or an even not existing surplus. In T1c (Fig. 6g), a surplus of the maximum convective oxygen supply over the oxygen consumption rate was found at 12 and 18∘C18^{\circ} \mathrm{C}. At 24∘C24^{\circ} \mathrm{C}, however, there was a deficit of convective oxygen supply, which mainly resulted from the decrease of the maximum amount of oxygen in the haemolymph ( Hb concentration). Consequently, T1c has to rely on a higher share of diffusive oxygen supply at 24∘C24^{\circ} \mathrm{C}. In T6c (Fig. 6h) and T19c (Fig. 6i), a surplus was found at each temperature. The more or less constant increase of the maximum amount of oxygen in the haemolymph ( Hb concentration) with

Fig. 6 Maximum oxygen concentration in the haemolymph (a-c; mean + SD; based on the data in Fig. 5b), perfusion rate (d-f; mean + SD; based on the data on heart rates shown in Fig. 2: grey-filled circles), and maximum convective oxygen supply ( g−i\mathrm{g}-\mathrm{i} ) in clones derived from the MultiLocus Genotypes (MLGs) T1, T6, and T19 at identical ambient and acclimation temperatures (12, 18 and 24∘C24^{\circ} \mathrm{C} ). Maximum convective oxygen supplies are shown in comparison with oxygen consumption rates (open squares; unpublished data, mean ±SD,n=3−4\pm \mathrm{SD}, n=3-4 experiments on four animals each). See text for details.
temperature in T6c (Fig. 6b) or the steady increase of perfusion rate (heart rate) with temperature in T19c (Fig. 6f) allows a higher share of convective oxygen supply in these clones. However, a mechanism based on Hb induction (T6c) is less sensitive to changing oxygen availabilities than an adjustment of the oxy-gen-dependent heart function (T19c).

Convective oxygen supply can be regarded as a very reliable source of oxygen for the tissues’ oxygen demands. Particularly, tissues and organs near to the animal’s core (e.g. intestine, ovaries) require convective oxygen supply even in small-sized Daphnia (cf. Bäumer et al., 2002). Depending on the share of diffusive oxygen transport, which is functionally related to the mass-specific oxygen consumption rate, body size and ambient Po2\mathrm{Po}_{2}, the oxygen offered by the circulating haemolymph will be differently used, and the oxygen concentration in the haemolymph returning to the respiratory exchanges areas (cf. Pirow, Wollinger & Paul, 1999b) will be reduced to a greater or lesser extent. Diffusive oxygen transport, however, is not always a reliable source of oxygen. Increases in mass-specific oxygen consumption rate brought about
by rising temperatures or intensified energy-consuming processes (e.g. swimming activity, feeding, growth and reproduction) as well as an increasing body size during growth may exceed the capacity of diffusive oxygen supply and require a higher utilisation of the convective oxygen supply. Seasonal changes of Po2\mathrm{Po}_{2} in ambient water because of e.g. phytoplankton blooms or diurnal vertical migration between normoxic (oxygen-rich) water at the surface and hypoxic (oxygen-depleted) water near the thermocline may impose problems in oxygen supply, which cannot be solved by diffusive oxygen transport alone. A well-developed capacity for convective oxygen supply allows to rapidly adjust oxygen supply to varying oxygen conditions in the environment and variable oxygen demands of the animals. Accordingly, combined adjustments of convective processes (ventilation, perfusion) and/or Hb expression are necessary to stabilise the oxygen supply of Daphnia during critical periods of life. The alterations in ventilation and heart rate either induced by acclimatisation or determined by evolutionary adaptation may be related to adjustments at the mitochondrial

level (increase or decrease of mitochondrial density/ capacity; cf. St.-Pierre et al., 1998; Guderley, 1998; Pörtner, 2002).

This study has provided evidence for connections between the temporal and spatial genetic heterogeneity of a Daphnia assemblage and seasonal variations of water temperature and oxygen content. Apart from other factors, such as seasonal changes of food quantity and quality, predation or parasitism (e.g. Cousyn et al., 2001; Decaestecker, De Meester & Ebert, 2002; Mitchell, Read & Little, 2004; Weider et al., 2005), environmental temperature and oxygen content seem to affect the genetic structure in addition. This linkage seems to be caused by differences in the physiological properties of the Daphnia clones, which included (i) oxygen transport, and accordingly thermal tolerance and tolerance towards a shortage of oxygen; (ii) the degree of phenotypic plasticity and (iii) metabolism. In mid-April and December at low water temperatures (10∘C\left(10^{\circ} \mathrm{C}\right. and below), MLGs (particularly T 1 ) were frequent, the clones of which exhibited a low tolerance towards higher temperatures (20−30∘C)\left(20-30^{\circ} \mathrm{C}\right) but a high phenotypic plasticity and highmedium levels of carbohydrate stores (at 12 and 18∘C18^{\circ} \mathrm{C} ). At moderate temperatures ( 10∘C10^{\circ} \mathrm{C} and above), the MLG T6 was predominant. The T6-derived clone showed a high tolerance towards higher temperatures, a more restricted phenotypic plasticity, highmedium levels of carbohydrate stores (at 12, 18 and 24∘C24^{\circ} \mathrm{C} ), and a high capacity for Hb synthesis, which also helps to invade oxygen-depleted water zones. In summer at water temperatures around 20∘C20^{\circ} \mathrm{C}, MLGs (particularly T19) became frequent, the clones of which showed a very high tolerance towards higher temperatures, a very limited phenotypic plasticity, low levels of carbohydrate stores and a high capacity for convective oxygen transport.

The D. galeata-hyalina hybrid species complex is a hybridising and backcrossing assemblage. Hybridisation in animals was previously regarded as disadvantageous caused by genetic incompatibilities and reductions in fitness (cf. Arnold, 1997). However, hybrids may dominate sympatric populations (Spaak, Eggenschwiler & Bürgie, 2000) and exhibit a higher fitness than the parental species (e.g. Spaak & Boersma, 2001). Accordingly, the ‘temporary hybrid superiority model’ suggests that hybrids may have a higher fitness under particular environmental conditions (Spaak & Hoekstra, 1995). Hybridising species
might be better in quickly adapting to changing situations as they can pick up genes of both species (Jankowski & Straile, 2004). Daphnia hyalina has been reported to grow and reproduce better than D. galeata at low food concentrations (Stich & Lampert, 1984), and to perform diurnal vertical migration during summer with the consequence that DD. hyalina has to cope with a low food and temperature environment in its day-time refuge (e.g. Stich & Lampert, 1981; Weider & Stich, 1992). Recent studies (Jankowski & Straile, 2004) on the D. hyalina-galeata hybrid complex of Lake Constance showed D. galeata to be present only from spring to autumn, to reproduce sexually in early summer and not to overwinter in the plankton, and DD. hyalina to reproduce sexually in autumn and to overwinter. Concerning these variables, D. hyalina ×D\times D. galeata hybrids showed intermediate patterns, whereas proposed backcross hybrids were more similar to their respective parentals. In the Saidenbach Reservoir, MLGs of D. galeata were frequent at the low temperatures immediately after ice off and in December (T1) and at the higher temperatures during summer (T19). Clones derived from these MLGs were either intolerant (T1c) or very tolerant (T19c) towards higher temperatures and stored either high-medium (T1c) or low (T19c) quantities of carbohydrates. Accordingly, large variation exists among the MLGs (clones) of one taxon (D. galeata). The MLG T6, which was genotypically classified as D. hyalina, was dominant at intermediate temperatures. A striking feature of a clone of this MLG was, apart from its welldeveloped phenotypic plasticity, its high capacity for Hb synthesis, which improves the oxygen supply of tissues, and accordingly the thermal tolerance, and favours the dominance of this MLG at the oxygendepleted thermocline. Whether the MLG T6 from Saidenbach Reservoir performs diurnal vertical migration, as was already reported for D. hyalina, cannot be answered yet. From the results, however, it may be inferred that T6 is fitter than other MLGs in thermally changing and/or oxygen-depleted environments, at least partly because of the adaptive character of an improved Hb synthesis.

Concerning thermal tolerance, the Daphnia clones exhibited a different degree of phenotypic plasticity. However, climate changes caused by global warming may alter, apart from water temperature, e.g. the timing of ice off, the period of spring full circulation and, consequently, the development of phytoplank-

ton biomass. Combined changes in temperature and food resources may create seasonal windows with new abiotic and biotic conditions, which may prevent Daphnia clones just to shift with their clone-specific thermal tolerance in warm years. Accordingly, studies on the coupling or non-coupling of thermal tolerance and food uptake/utilisation or energy allocation have to be carried out to assess the potential of present clones to adjust to future scenarios of global warming. In addition, the impact of further factors, such as predation or parasitism has also to be considered.

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (priority program SPP 1162 ‘Aquashift’; Pa 308/10). We particularly thank Dr Dietrich Flößner, Jena, for the morphological classification of the Daphnia genotypes, Dr Lothar Paul and his team at the Ecological Station Neunzehnhain near Saidenbach Reservoir for all their support and Dr Hans-Ulrich Steeger, Münster, for technical assistance. We also thank Prof. Dr Thorsten Reusch, Münster, for carefully reading the manuscript.

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(Manuscript accepted 30 March 2007)