Annals of Botany 89: 283-292, 2002
© 2002 Annals of Botany Company
Variation Amongst Survivor Populations of White Clover Collected from Sites Across Europe: Growth Attributes and Physiological Responses to Low Temperature
1Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, SY23 3EB, UK and 2The Agricultural Research Institute, Keldnaholt, 112 Reykjavík, Iceland
* For correspondence, e-mail rosemary.collins{at}bbsrc.ac.uk
Received: 21 August 2001; Returned for revision: 9 October 2001; Accepted: 19 November 2001.
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ABSTRACT |
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Experiments were carried out at IGER, Aberystwyth, UK to investigate traits of direct relevance to the processes of overwintering and spring growth in white clover (Trifolium repens L.). The plant material used was derived from baseline populations of the cultivar AberHerald and survivor populations generated after 2–3 years’ growth in Germany (Kiel), Sweden (Uppsala) and Switzerland (Zürich). The aims of the experiments were to measure the level of genetic shift that had occurred in certain traits due to selection in the survivor populations by comparing these with the baseline population. The adaptive significance of traits was assessed by determining the extent to which stabilizing selection had operated to reduce levels of intra-population variation. Significant differences were found in the responses of leaf production to two temperature treatments in the survivor populations from Germany and Sweden compared with the Swiss and baseline material. Plants of the former two populations produced much more leaf than the others at the higher temperature, but leaf production rates at the lower temperature did not differ. As this experiment used cloned genotypes in the two treatments, the result suggests that a higher degree of phenotypic plasticity for this trait had been selected for in the German and Swedish populations. These populations also showed greater rates of regrowth of leaves from terminal buds exposed to sub-zero temperatures, but there were no differences between populations in levels of freezing tolerance, or in stolon carbohydrate content. Genetic shift occurred in the degree of unsaturation of stolon lipids, with all three survivor populations possessing higher proportions of unsaturated fatty acids than the baseline. Stabilizing selection also operated on this trait in the survivor populations, suggesting that it is of adaptive significance in cool climates.
Key words: Trifolium repens L., white clover, morphology, freezing tolerance, carbohydrates, unsaturated fatty acids, adaptation.
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INTRODUCTION |
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In a previous paper, Collins et al. (2001) described a single-site experiment investigating patterns of variation in morphological traits amongst survivor and baseline populations of the white clover cultivars Grasslands Huia and AberHerald. These survivor populations had been generated from vegetative samples collected after 2–3 years’ growth at a range of sites across Europe. Since the seed provided to these sites was from the same original seed stocks of the two cultivars, the baseline populations were similar in composition at each site. The results obtained (Collins et al., 2001) showed that directional selection had operated in survivor populations within both cultivars, resulting in genetic shift in certain morphological and reproductive traits at various sites.
The present study sought to extend this analysis by investigating traits of direct relevance to the processes of overwintering and spring growth in white clover, using four populations within cultivar AberHerald. In the previous study (Collins et al., 2001), analysis of morphological traits using multivariate techniques showed that the AberHerald populations clustered into three distinct groups, with the population from Finland remaining separate. Populations for the present study were chosen on the basis of this clustering, and the baseline population was included for comparative purposes. In the previous experiment, this population had clustered in a group along with the survivors from Wales. The most contrasting group to this was that containing populations from the two German sites, and for the current study the population from Kiel (KI) was chosen. The two other populations chosen for the present study—Sweden (UP) and Switzerland (ZU)—were from the ‘intermediate’ morphological cluster.
Morphological traits that might discriminate between white clover cultivars in terms of overwintering ability and subsequent spring growth have been identified in several studies carried out at low temperatures in controlled environments (e.g. Ollerenshaw and Baker, 1981; Eagles and Othman, 1988; Corbel et al., 1999) and in field plots (Collins et al., 1991; Stäheli et al., 1996). An extension of this approach is the analysis at low temperature of the growth attributes of survivor populations in comparison with their baseline cultivar (Frankow-Lindberg, 1999; Helgadóttir et al., 2001). In the current study, clones of survivors from the three chosen sites, plus the baseline population, were grown at two simulated spring temperature regimes in controlled environments.
The ability of stolon terminal buds to survive and regrow after exposure to sub-zero temperatures is crucial for successful overwintering and subsequent spring growth in this species (Collins et al., 1991). Artificial freezing tests have been used to predict winter hardiness in white clover by measuring the survival rates of pieces of stolon bearing a terminal bud after exposure to sub-zero temperatures (e.g. Junttila et al., 1990; Collins and Rhodes, 1995; Svenning et al., 1997; Annicchiarico et al., 2001). Similar tests have also been used to assess cold tolerance using the percentage of leaves damaged as an index (e.g. Caradus et al., 1989; Caradus and Christie, 1998). In general, freezing tests using pieces of stolon with terminal buds as the assay material have shown some degree of correlation with field survival. For example, Annicchiarico et al. (2001) found that terminal bud survival rates in a wide range of white clover populations measured in a freezing test were highly correlated with the survival rates of tagged terminal buds of the same populations grown under field conditions in Lodi, northern Italy—a location that experiences predictably cold winters. In addition to bud survival, the extent of leaf regrowth from terminal buds exposed to freezing is of interest as a component of cold tolerance in white clover (Caradus, 2000).
Alteration in the physical properties of cell membranes is considered to be a primary cause of freezing injury in plants (Steponkus, 1984). Decreases in ambient temperature result in a reduction in the fluidity of cell membranes (Murata and Los, 1997), which impairs their function (Cossins, 1994). Consequently, a fundamental acclimatory response of plants to low temperature is to increase their production of unsaturated fatty acids (Lynch and Steponkus, 1987), since the degree of unsaturation of membrane lipids is the major factor influencing the fluidity of cell membranes. There is some evidence from higher plants that differences in the degree of unsaturation of lipids are correlated with inter-population variation in cold tolerance, and a study comparing three white clover populations growing in Iceland indicated that genetic shift in favour of increased lipid unsaturation had occurred in the cold-adapted material (Dalmannsdóttir et al., 2001).
Carbohydrates accumulate in plants during the autumn as rates of photosynthesis begin to exceed growth (Pollock, 1990). Many authors have proposed a positive link between carbohydrate accumulation and increased freezing tolerance in various plant species. For example, cold-induced accumulation of cryoprotective sugars appears to be associated with increased freezing tolerance in alfalfa (Castonguay et al., 2000). In white clover, research has also suggested a positive effect of increases in levels of various water-soluble sugars, sugar alcohols and proline on freezing tolerance, possibly via their influence on the osmotic potential of the cell sap (Røsnes et al., 1993; Svenning et al., 1997). Conversely, Annicchiarico et al. (2001) reported no significant variation in the stolon contents of either water-soluble or total non-structural carbohydrates in a sample of 11 white clover populations that differed widely in their levels of freezing tolerance. Thomas and James (1993) also failed to establish a direct link between solute accumulation and freezing tolerance in Lolium perenne. It has proved difficult to establish causal rather than merely correlative relationships between carbohydrate accumulation and cold tolerance. However, in terms of plant breeding, the existence of correlations between physiological/biochemical traits, such as the degree of unsaturation of tissue lipids and carbohydrate accumulation, and cold tolerance, is of potential interest for the development of indirect selection criteria for complex and expensive-to-evaluate traits such as winter survival in field plots (Annicchiarico et al., 2001).
The present study investigated variation between the chosen survivor populations and the baseline population in: (1) growth attributes at two simulated spring temperatures in controlled environments; (2) rates of survival and regrowth of terminal buds after exposure to sub-zero temperatures in an artificial freezing test; (3) the extent of unsaturation of lipids present in stolon tissue; and (4) levels of stolon carbohydrates. Such an approach assumes that different selective forces have operated at different sites, potentially resulting in specific adaptational changes in the survivor populations (Collins et al., 2001). Levels of intra-population variation in stolon fatty acid profiles and carbohydrate contents were also analysed to provide more information on the nature of the selective forces acting on these traits at different sites. In addition, relationships among all the traits were investigated for patterns of correlation.
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MATERIALS AND METHODS |
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Plant material
A full description of the development of the survivor populations within each cultivar is given in Collins et al. (2001). Briefly, after 2–3 years’ growth, random samples of survivor plants were collected from each site participating in the pan-European COST 814 Programme (described fully in Wachendorf et al., 2001). These were sent to IGER, Aberystwyth, UK, where they were propagated so that polycross seed could be produced for each site x cultivar combination. Material for all the experiments described in this paper was derived from a spaced plant nursery at IGER, Aberystwyth, containing 40 genotypes from the baseline population and from each of the survivor populations of cultivar AberHerald. The populations were arranged as randomized blocks with four replicates, and genotypes within populations were labelled 1–40. The survivor populations chosen for assessment in the following experiments were from Germany (KI), Sweden (UP) and Switzerland (ZU). The letters in brackets correspond to the site codes used in Wachendorf et al. (2001). The baseline population (BASE) was included in all experiments as a reference against which traits in the survivor populations were measured.
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Measurements |
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(A) Growth attributes at low temperatures
On 7 Jan. 2000, two stolon cuttings, each approx. 5 cm long and possessing a terminal bud, were collected from genotypes 1–20 in each of the chosen populations. The cuttings were individually marked with their population and genotype number using a plastic label and were planted into deep trays (60 x 38 x 11 cm) of Levington’s peat-based potting compost (Levington Ltd, Ipswich, UK) at a density of ten cuttings per tray. All cuttings in a single tray were from genotypes within one population and two trays were assigned to each population in each temperature treatment (i.e. genotypes 1–10 and 11–20 were in separate trays within each treatment). The cuttings were arranged so that there was enough space around each one to allow unimpeded stolon extension, and to permit individual genotypes to be readily identified. Trays were kept in a cool glasshouse (temperature maintained at a minimum of 5 °C) for 2 weeks to allow the cuttings to establish. The two sets of cuttings were then allocated to either of two temperature treatments: temperature 1 (11/8 °C day/night), or temperature 2 (8/4 °C day/night), with two trays per population in each. These treatments were chosen as typical of spring temperatures in a temperate, maritime climate, such as that prevailing in Aberystwyth, UK. The temperature treatments were imposed in growth cabinets under a 12 h daylength and a mean photosynthetic photon flux density of 500 µM m –2 s–1 at the height of the plant canopy. On day 1, a dot of red acrylic paint was placed on a standard internode on each cutting (third internode back from the stolon apex). They were then left to grow for 5 weeks, with water supplied as required, and their morphology was analysed. First, non-destructive measurements were carried out: the length of stolon between the marked node and the youngest identifiable node was measured, and all the leaves and branches produced after the paint mark were counted. Each of these leaves was given a score using the Carlson visual scale of leaf development, where 1·0 = fully expanded and 0·1 = leaf just visible as it emerges from its stipule (Carlson, 1966). The sum of these leaf development scores was calculated for each cutting, providing a value for ‘total Carlson score’. To quantify the allocation of dry matter to different organs in a rapid and standardized way, the concept of the ‘metamer’ (a basic plant modular unit comprising a fully expanded leaf, internode, axillary bud and root initial) was used (White, 1979). On each cutting, a metamer was identified and dissected into its components. The chosen metamer on each plant was that which possessed the youngest fully expanded leaf. Roots, where present, were discarded as they were invariably too small to process. The diameter of the dissected internode was measured using calipers; the area of the leaf was measured using a leaf area meter (Delta-T Devices, Cambridge, UK), and the length of its associated petiole was also obtained. Each component was oven-dried and weighed.
(B) Physiological traits
All measurements of physiological traits were made on stolon cuttings removed from genotypes 1–20 in each of the four chosen populations in the spaced plant nursery. The field measurements were carried out in Aberystwyth, UK, in mid-February, a period which is usually relatively cold at this location. It was anticipated that inter-population differences, if present, in traits related to winter survival would be expressed at this time of year.
Rates of survival of terminal buds and regrowth after freezing.
An artificial freezing test was carried out on material collected from the field on 15 February. Seven stolon cuttings, each approx. 4 cm long and possessing a terminal bud, were taken from each of the genotypes to be sampled. All leaf laminae were removed from these cuttings so that leaf regrowth after freezing would be measured from standardized samples. The cold tolerance of the populations was evaluated using the ‘glycol tank’ method of Fuller and Eagles (1978), in which cuttings are placed in Perspex tubes and suspended in a temperature-controlled tank of ethylene glycol/water (50/50, v/v). The tubes were labelled so that population and genotype number were readily identifiable. The stolon cuttings were placed in their tubes and the coolant temperature in the tank was lowered from ambient, then maintained at 2 °C overnight. The surface of the coolant was insulated from the ambient temperature by a thick polystyrene cover placed over the tank. During the following day, the temperature of the coolant was gradually lowered to –12 °C at a constant rate of 1·5 °C h –1. From each population, 20 cuttings were removed from the tank when the coolant reached the following seven temperatures: –2·5, –5, –7, –8, –9, –10 and –12 °C. The experiment was designed so that, in each population, genotypes 1–20 were replicated at each pre-set temperature. In order to assess the possible impact of the overnight pre-treatment on the survival of terminal buds, a control treatment was imposed. In this treatment, stolon cuttings taken from each of 20 genotypes of the baseline population were kept overnight in the tank at 2 °C and their survival and regrowth subsequently monitored.
After artificial freezing, the cuttings were kept in their temperature x population groups in shallow plastic boxes on a bed of absorbent cotton wool covered in strong paper tissue. The boxes were placed in a glasshouse heated to a minimum temperature of 15 °C and were regularly supplied with water via a fine spray. After 12 d recovery each cutting was assessed visually for the survival of its terminal bud and classified as ‘live’ or ‘dead’. Dead cuttings were obvious because their bud tissue had become flaccid. In this way a ‘% mortality’ value was obtained for each population at each pre-set temperature. Stolon cuttings with a live terminal bud were given a regrowth score which was the sum of the Carlson leaf development stages (Carlson, 1966) of any leaves produced during the recovery period after the freezing test.
Fatty acid profile of stolon lipids.
On 18 February, five stolon cuttings were removed from each of the chosen genotypes in the spaced plant nursery. To standardize the samples, only meristematic tissue was used, i.e. stolon apices, and all leaves and roots were removed. The tissue was immediately frozen in liquid nitrogen, then freeze dried for 24 h. Samples were then ground in a mill through a 1 mm mesh, keeping tissue from each genotype separate. Individual fatty acids were quantified after conversion to their corresponding fatty acid methyl esters (FAMES) using the one-step extraction–transesterification method of Sukhija and Palmquist (1988). The procedure used a solvent system consisting of chloroform–methanol–acetyl chloride in the ratio 20 : 27 : 3 (v/v/v) which was added to each sample along with an internal standard (19 : 0 nonadecanoic acid). After being tightly capped, the sample tubes were vortexed, then heated to 70 °C for 2 h. The tubes were cooled and 6 % K2 CO3 was added to adjust the pH to neutrality. The samples were centrifuged and the esterified fatty acids were taken from the chloroform phase. Samples were analysed using an HP 5890 gas chromatograph fitted with a CP Sil 88 50 m x 0·25 mm (i.d.) column with a film thickness of 0·2 µm (‘Chrompak’, Varian Ltd, Warrington, UK). The temperature programme for the analysis was 80 °C for 1 min, increasing at a rate of 5 °C min–1 to 240 °C, which was held for 7 min. Fatty acids were identified by comparison of their retention times with that of the internal standard. The fatty acids measured were: 16 : 0 (palmitic), 16 : 1 (palmitoleic), 16 : 3 (cis,cis,cis -7,10,13 hexadecatrienoic), 18 : 0 (stearic), 18 : 1 (oleic), 18 : 2 (linoleic) and 18 : 3 (linolenic), where the number preceding the colon represents the number of carbon atoms in the fatty acid and the number following the colon indicates the number of double bonds present (Lynch and Steponkus, 1987).
Stolon carbohydrate content.
Samples for carbohydrate measurement (sufficient to provide approx. 0·5 g d.wt from each genotype) were removed from genotypes in the spaced plant nursery on 23 February by cutting lengths of approx. 10 cm from live stolons. The stolon pieces were trimmed so that neither terminal buds nor leaves were included in the samples. To minimize changes in the tissue, the collected samples were taken to a nearby laboratory at regular intervals during the sampling and put into a pre-heated oven at 70 °C. They were dried overnight, then ground in a mill through a 1-mm mesh sieve, keeping the samples from individual genotypes separate. The milled samples were analysed for their contents of: (1) water-soluble carbohydrates (WSC), i.e. chiefly sucrose, glucose and fructose, using an automated anthrone procedure (Thomas, 1977); and (2) total non-stuctural carbohydrates (TNC), i.e. all carbohydrates except cellulose, estimated by the anthrone reaction after extraction in a weak acid solution as described by Smith (1973). In white clover TNC mainly comprises starch.
Statistical analyses
Statistical analyses were carried out using the appropriate programs in Genstat 5 release 4·1 (Lawes Agricultural Trust, 1987).
Growth attributes at low temperatures.
Analysis of variance (ANOVA) was carried out on data collected from the controlled environment experiment. The temperature treatments were analysed together using ANOVA (without blocking). Mean values for each population were based on 20 replicate genotypes. The model selected allowed the effects of temperature treatment, population and their interaction to be evaluated. Where significant effects were detected, comparisons between factors were made using LSD (least significant difference) values calculated for P < 0·05.
Physiological traits.
Rates of terminal bud survival after freezing were calculated using a probit transformation procedure for each population. This transformation rendered linear the relationship between percentage mortality and pre-set temperature. A regression was fitted to these data and the resulting linear equation allowed LT50 the temperature at which 50 % mortality occurred) to be estimated by calculating a/b, where a is the intercept on the log mortality axis and b is the slope of the mortality change (log mortality °C–1 ).
Rates of leaf regrowth at each temperature were calculated for each population using the genotype values as replicates. Because regrowth could only occur from live terminal buds, dead buds were counted as missing values, resulting in reduced replication at lower temperatures. To overcome this statistical problem, regrowth scores were calculated from regression using a model incorporating the factors pre-set temperature, population and their interaction, taking into account genotype differences within populations, using the Generalised Linear Mixed Model procedure in Genstat. ANOVA partitioned the variances associated with the factors in the model and tested the significance of the appropriate variance ratios.
Levels of fatty acids, WSC and TNC measured in stolon tissue in each population were analysed using one-way ANOVA (without blocking). Population means were based on values for 20 replicate genotypes. The model selected allowed the effect of population to be evaluated. Where a significant effect was detected, comparisons between populations were made using LSD values calculated for P < 0·05.
The amount of intra-population variation present in these traits was estimated by calculating the coefficient of variation [% CV = (
variance/mean) x 102 ). As discussed in Collins et al. (2001), it may be assumed that differences in % CV are due primarily to genetic variation, indicating the action of different selection pressures at different sites. The significance of differences in intra-population % CV was tested using Bartlett’s test of homogeneity of variances (Snedecor and Cochran, 1967). For traits in which non-homogeneity was identified, a 95 % confidence interval, based on the 
2 distribution, was calculated for the baseline population (Snedecor and Cochran, 1967). This allowed differences in % CV between the baseline and other populations to be analysed.
The pattern of relationships between growth attributes and physiological traits was investigated using simple correlation analysis.
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RESULTS |
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Growth attributes at low temperatures
Growth temperature had a significant effect on all traits, whether these were measured directly or derived mathematically from the primary data. Exposure to the lower temperature (8/4 °C; temperature 2) resulted in a reduction in the growth rates of plant parts, the formation of stolon branches and the allocation of dry matter to stolons. For a number of traits there were statistically significant interactions between temperature treatment and population. As such interactions were found in several variables related to the production of leaf area, these were combined by calculating values of ‘total Carlson score x metamer leaf area’. This gave a measure of the leaf area produced by the plant during the experiment (‘total leaf area’). At the higher temperature (11/8 °C; temperature 1) the Swedish population produced much more total leaf area than the baseline population (Table 1). However, at the lower temperature, total leaf area values for the three survivor populations did not differ from the baseline population. For the number of leaves produced during the experiment (i.e. the rate of leaf appearance), higher values were observed in the German and Swedish populations than in the baseline population at the higher temperature, but lower rates of leaf appearance were found in the Swedish material compared with the baseline population at the lower temperature. In the case of internode diameter there was greater variation among populations at the lower temperature. All populations produced thinner stolons at this temperature than at the higher temperature.
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Physiological traits
The control treatment showed that keeping stolon cuttings at 2 °C overnight had no adverse effect on their survival. Table 2 shows that there were no statistical differences in freezing tolerance (LT50) among the populations used in this study, although the baseline had the lowest LT50 value. As expected, the rate of regrowth of leaves from terminal buds was significantly affected by the pre-set temperature during the freezing test, and no buds survived at –12 °C. Regrowth differed significantly between populations (P = 0·031), and there was a significant interaction between pre-set temperature and population (P = 0·057). Consequently, comparisons between populations could be made only within a given pre-set temperature. Comparisons made by using the standard error attached to the mean regrowth score per plant at each temperature to calculate a confidence interval showed that there was no difference in regrowth between the baseline and any other population after freezing at –2·5 °C. However, the Swedish material maintained its regrowth potential better than the baseline after freezing at –5 °C and –7 °C.
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Results showing levels of individual fatty acids, ‘UFA’ (the sum of the unsaturated fatty acids 16 : 1, 16 : 3, 18 : 1, 18 : 2 and 18 : 3), ‘SFA’ (the sum of the saturated fatty acids 16 : 0 and 18 : 0) and ‘% unsaturated’ (UFA expressed as a percentage of the total fatty acid content—a measure of the degree of unsaturation of tissue lipids) in each population are presented in Table 3. Significant variation between populations was found in all these traits, except for 16 : 1 and 18 : 0. Averaged over populations, the most abundant fatty acid in stolon tissue was 18 : 3, closely followed by 18 : 2, but only small amounts of 16 : 1 were detected. The population from Germany had the highest values of 18 : 1, 18 : 2 and 18 : 3, and the baseline population had the highest levels of 16 : 0 and 16 : 3. Of particular interest in the present study were the UFA content and the degree of unsaturation of tissue lipids. The German population had the highest values for both of these traits. The populations from Sweden and Switzerland also showed significantly greater degrees of unsaturation of tissue lipids than the baseline.
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Analysis of variance showed that concentrations of WSC and TNC did not differ significantly among populations. Values of WSC ranged from 11·16 to 13·75 g 100 g–1 d. wt and those of TNC from 22·53 to 25·67 g 100 g–1 d. wt.
Levels of intra-population variation were found to differ significantly between populations for a number of traits (Table 4). In the case of all fatty acids except 16 : 3, there was a significant decrease in variation within the survivor populations relative to the baseline. For WSC and TNC a reduction in variation was evident only in the Swiss material.
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There was a general lack of correlation between regrowth of terminal buds after freezing and various physiological traits, with the exception of a tendency for regrowth to be negatively associated with levels of the fatty acid 16 : 3 (r = –0·23; P
0·05 at 78 d.f.). Growth attributes measured on plants in controlled environments showed considerably more evidence of correlation with physiological traits (Table 5), and at 8/4 °C, stolon carbohydrate content was significantly negatively correlated with total plant leaf area and dry weight. However, this relationship was not observed at 11/8 °C. There was no evidence of relationships between fatty acid levels and growth at the lower temperature, except for a positive correlation between leaf area per plant and levels of 16 : 3, but significant correlations were found at the higher temperature between leaf area per plant and 16 : 1 (negative), and 18 : 1, 18 : 2 and 18 : 3 (positive). The degree of unsaturation of tissue lipids was also positively correlated with this trait at the higher temperature.
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DISCUSSION |
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The analysis of growth of white clover plants at two temperatures in this experiment has provided clear evidence for the occurrence of genetic shift in survivor populations in certain characters. For these traits, the populations that differed most from the baseline were those from Germany and Sweden. The observation that the baseline and Swiss material tended to respond to temperature in a similar way may reflect the fact that the cultivar AberHerald was developed from plant collections made in the Zürich uplands, close to the site in which the Swiss survivor population was collected. The similarity of their morphological responses to temperature is therefore not unexpected.
Analysis of traits in cloned material at different temperatures allows separation of the effects of genetic shift (i.e. differences expressed within temperature treatments between survivor populations and the baseline population) from those of phenotypic plasticity (i.e. differences in the response of populations to the temperature treatments). Genetic shift reflects changes in the population gene pool as a response to selection, whereas phenotypic plasticity is an individual response to environmental fluctuations.
In this experiment, differences among populations at the lower temperature in total leaf area per plant were not evident, but, at the higher temperature, the increase in this trait was substantial in the German and Swedish material. In another controlled environment experiment, Helgadóttir et al. (2001) also found that in AberHerald the survivor population from Iceland produced more leaf area per plant than the baseline population at a higher temperature, but maintained a similar level at a low temperature. They suggested that in the Icelandic environment, selection had occurred for genotypes that could increase their rate of leaf production during the cool summer months, i.e. exhibited a greater level of phenotypic plasticity than the baseline material. A similar interpretation could be made of the results obtained in the present study. Corroborative evidence is provided by the spaced plant data presented in Collins et al. (2001), which showed a significant trend towards larger leaves in the Kiel (Germany) and Swedish populations compared with the baseline population. Selection at these sites thus appears to have favoured competitive ability, and this potential is expressed as growing conditions become more favourable. It should be emphasized that this increase in plasticity would itself be under genetic control, since the ability to respond to environmental changes is genetically based. Plasticity and genetic shift are not opposing concepts (Pérez de la Vega, 1997), and it is quite feasible that adaptation to a changing environment would involve both processes.
The level of freezing tolerance expressed by plants at any given time represents the end result of many interacting processes and factors (Dale and Heiberg, 1984). In a cool maritime climate such as that prevailing in the UK, levels of freezing tolerance during the winter are likely to be strongly influenced by the frequent occurrence of dehardening temperatures (Eagles et al., 1997). Rapid dehardening at temperatures above 6 °C has been observed in white clover (Svenning et al., 1997). Consequently, artificial freezing tests carried out on samples collected from the field are useful in providing a comparison that is relevant under the specific conditions pertaining at that time, but may not be of predictive value if considered in isolation. The wide variation reported in the literature for values of LT50 obtained from AberHerald (equivalent to the baseline population in the current study) and its progenitor population, Ac3789, illustrates this point. When comparing the freezing tolerance of populations, high (i.e. less negative) values of LT50 indicate inferior levels of tolerance. Levels of freezing tolerance in all populations in the present experiment were inferior to those obtained for AberHerald in many other studies. For example, Dalmannsdóttir et al. (2001) reported an LT50 of –14·6 °C for a baseline AberHerald population hardened outdoors in Iceland, and in tests carried out by Svenning et al. (1997) on AberHerald plants hardened in a controlled environment the maximum level of cold hardiness attained was an LT50 of –13 °C. These results demonstrate that a reasonable level of freezing tolerance can be induced in this cultivar, given sufficiently uninterrupted cold conditions for acclimation to occur. Winter conditions in the UK are often sufficiently cold to produce significant cold hardening but this may be counteracted by frequent intervals of mild weather. For example, an artificial freezing test carried out by Annicchiarico et al. (2001) on populations grown in an unheated polythene tunnel in Aberystwyth in January 1998 gave a relatively low LT50 of –9·2 °C for AberHerald. This level of cold tolerance was markedly superior to that of the baseline material in the current experiment (–6·7 °C) and to the value (–6·2 °C) obtained by Collins and Rhodes (1995) for Ac3789 collected from field plots in Aberystwyth in January 1990. On the basis of such wide intra-cultivar variation in freezing tolerance from year to year in the UK, it can be concluded that artificial freezing tests are of predictive value only when they are carried out on fully cold-hardened material. In a temperate, maritime climate the use of controlled environment facilities appears to be essential for this purpose, as field-based evaluations are influenced by fluctuating weather conditions to an unacceptable extent.
In contrast to the assessment of survival rates and estimation of LT50 values, it was evident that measurement of the rates of regrowth of terminal buds exposed to sub-zero temperatures provided a useful method of discriminating among populations in this study. The use of the Carlson leaf growth scale for this purpose has not previously been described. It clearly provided a rapid, objective and non-destructive method of assessing recovery after freezing. The technique may have predictive value, in that the superior regrowth of the German and Swedish material after exposure to –5 and –7 °C was reflected in their greater production of leaf area in the controlled environment experiment at 11/8 °C.
Results obtained for white clover in an earlier study (Dalmannsdóttir et al., 2001) provided evidence for the occurrence of genetic shift in levels of UFA in stolon tissue. The populations compared in that study were AberHerald (baseline population) and an AberHerald survivor population that had undergone selection under Icelandic conditions. Their results showed that higher levels of UFA were present in the AberHerald survivors than in the baseline population, at least during the autumn. The three survivor populations assessed in the current study also had significantly higher proportions of UFA than the baseline population, confirming that selection in climates colder than the site of origin of the baseline cultivar acts to increase the degree of unsaturation of tissue lipids. The present study also found a substantial and significant reduction in intra-population variation in levels of UFA and in the degree of unsaturation of tissue lipids, which strongly suggests that genetic shift in these traits has been followed by stabilizing selection operating to maintain the best-adapted phenotypes in the population (Harper, 1977). Traits of high adaptive value in a specific environment tend to be forced to uniformity (Tigerstedt, 1994) and it may, therefore, be concluded from these results that tissue UFA content is a trait of adaptive significance in cool climates.
The results of the correlation analyses between fatty acid levels and (1) regrowth of terminal buds after freezing, and (2) growth of larger plant structures at low temperatures, identified a number of points of interest. However, the biological, as opposed to statistical, significance of these relationships evidently requires further analysis. The chief limitation of correlative studies of changes in lipid composition and increases in cold tolerance, or growth at low temperatures, is that they are often based on analyses of whole tissues or crude membrane preparations, rather than the plasma membrane per se (Lynch and Steponkus, 1987). This was the case in the current experiment. However, estimation of levels of the major fatty acids in whole tissues by the FAMES method is relatively simple to carry out, and in this case provided a good separation of the populations. Thus, from the perspective of using simple traits as selection criteria in breeding programmes, the existence of correlations such as those reported in this paper may be of potential use in the evaluation of complex traits such as winter survival and spring growth in field plots. However, before adopting increased UFA content as a breeding criterion, it would be necessary to confirm that the changes observed in stolon tissue also occur in leaves. In addition, seasonal fluctuations in fatty acid profiles have been observed in Lolium spp. in the UK (Dewhurst et al., 2001) and these should be also be assessed in white clover under UK conditions.
Stolon carbohydrate content, whether in the form of TNC or WSC, did not differ among populations in this study and neither was there any evidence of a reduction in intra-population variation, except in the survivors from Switzerland. This result casts some doubt on the adaptive significance of stolon carbohydrate content as a trait in relation to cold tolerance. However, the material sampled was from non-cold hardy plants and a different result might have been obtained under colder conditions. Carbohydrate content did not correlate positively with any of the other physiological traits measured in this study. Indeed, TNC and WSC were both found to be negatively correlated with growth rates in the lowest temperature treatment (8/4 °C) of the controlled environment experiment. These results suggest that re-evaluation is necessary, either of the often-stated role of carbohydrates as causal agents in increasing cold tolerance in plants, or of the analytical techniques employed to measure carbohydrate levels in studies such as this one. For example, in contrast to the results obtained here, Dalmannsdóttir et al. (2001) found that inter-population variation in winter levels of sucrose, the chief water-soluble carbohydrate present in white clover, was positively reflected in levels of frost and ice-encasement tolerance measured at the same time. However, levels of other water-soluble carbohydrates, e.g. fructose, glucose, stachyose and raffinose, were not found to be good predictors of cold tolerance in that study. Therefore it appears that the type of carbohydrate selected for measurement is critical in experiments that seek to correlate this with levels of cold tolerance. The use of simple analytical techniques to assess levels of WSC and TNC in this experiment may not have provided a sufficiently detailed discrimination between the various types of carbohydrate present in each sample.
The results of this study exemplify the importance of the choice of traits examined in analyses of plant adaptation to environments outside that in which a cultivar was developed. The previous paper in this series (Collins et al., 2001) reported an absence of evidence for the occurrence of stabilizing selection on any of the traits measured. These traits were general indicators of plant size and competitive ability, and were morphological, rather than physiological/biochemical in nature. Collins et al. (2001) proposed that, although these morphological traits themselves appeared to be of little adaptive significance, it was possible that they might be correlated with other traits which were of more direct relevance to winter survival in cold climates. The results of the current study confirm this hypothesis, demonstrating correlations between levels of fatty acids and various morphological traits measured at moderately low temperatures. Expansion of the range of environments in which white clover cultivars can be grown may therefore be achieved by using traits such as these as selection criteria.
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ACKNOWLEDGEMENTS |
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We thank Ms S. Venerus (SAASD, Italy) and Dr J. Leto (University of Zagreb, Croatia) for technical assistance. S.V. was funded as part of a COST short-term scientific mission. Analysis of fatty acids was carried out by Analytical Services at the Macaulay Land Use Research Institute, Scotland. Carbohydrates were analysed by the Analytical Chemistry Group, IGER, Aberystwyth. Statistical advice was kindly provided by Dr H. Björnsson (RALA, Reykjavík, Iceland) and Drs. R. Sackville-Hamilton and A. Cresswell (IGER, Aberystwyth). R.P.C., M.F. and I.R. were funded by the UK Ministry of Agriculture, Fisheries and Food.
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LITERATURE CITED |
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Annicchiarico P, Collins RP, Fornasier F, Rhodes I. 2001. Variation in cold tolerance and spring growth among Italian white clover populations. Euphytica 122: 407–416.[CrossRef]
Caradus JR. 2000. Breeding for cold tolerance in white clover. In: Parente G, Frame J. eds. Crop development for the cool and wet regions of Europe. Proceedings of the final conference of COST Action 814. Luxembourg: OOPEC, 173–180.
Caradus JR, Christie BR. 1998. Winter hardiness and artificial frost tolerance of white clover ecotypes and selected breeding lines. Canadian Journal of Plant Science 78: 251–255.
Caradus JR, MacKay AC, van den Bosch J, Greer DH, Wewala GS. 1989. Intraspecific variation for frost hardiness in white clover (Trifolium repens). Journal of Agricultural Science 114: 151–155.
Carlson GE. 1966. Growth of clover leaves—developmental morphology and parameters at ten stages. Crop Science 6: 293–294.
Castonguay Y, Nadeau P, Laberge S, Michaud R. 2000. Physiological and molecular bases of alfalfa adaptation to harsh winter conditions. In: Parente G, Frame J. eds. Crop development for the cool and wet regions of Europe. Proceedings of the final conference of COST Action 814. Luxembourg: OOPEC, 161–172.
Collins RP, Rhodes I. 1995. Stolon characteristics related to winter survival in white clover. Journal of Agricultural Science 124: 11–16.
Collins RP, Glendining MJ, Rhodes I. 1991. The relationships between stolon characteristics, winter survival and annual yields in white clover (Trifolium repens L.). Grass and Forage Science 46: 51–61.[CrossRef][Web of Science]
Collins RP, Helgadóttir Á, Fothergill M, Rhodes I. 2001. Variation amongst survivor populations of two white clover cultivars collected from sites across Europe: Morphological and reproductive traits. Annals of Botany 88: 761–770.
Corbel G, Robin C, Frankow-Lindberg BE, Ourry A, Guckert A. 1999. Regrowth of white clover after chilling: Assimilate partitioning and vegetative storage proteins. Crop Science 39: 1756–1761.
Cossins AR. 1994. Homeoviscous adaptation of biological membranes and its functional significance. In: Cossins AR, ed. Temperature adaptation of biological membranes. London: Portland Press, 63–76.
Dale A, Heiberg N. 1984. Studies in black currant (Ribes nigrum L.) on the relationship between frost tolerance in winter and spring and on the relationships of dehardening and rehardening in spring. Crop Research (Horticulture Research) 24: 73–83.
Dalmannsdóttir S, Helgadóttir Á, Guðleifsson B. 2001. Fatty acid and sugar content in white clover in relation to frost tolerance and ice-encasement tolerance. Annals of Botany 88: 753–759.
Dewhurst RJ, Scollan ND, Youell SJ, Tweed JKS, Humphreys MO. 2001. Influence of species, cutting date and cutting interval on the fatty acid composition of grasses. Grass and Forage Science 56: 68–74.[CrossRef]
Eagles CF, Othman OB. 1988. Variation in growth of overwintered stolons of contrasting white clover populations in response to temperature, photoperiod and spring environment. Annals of Applied Biology 112: 563–574.
Eagles CF, Thomas H, Volaire F, Howarth CJ. 1997. Stress physiology and crop improvement. In: Christie BR, ed. Proceedings of the XVIII International Grassland Congress, Canada 3: 141–150.
Frankow-Lindberg BE. 1999. Effects of adaptation to winter stress on biomass production, growth and morphology of three contrasting white clover cultivars. Physiologia Plantarum 106: 196–202.[CrossRef]
Fuller MP, Eagles CF. 1978. A seedling test for cold hardiness in Lolium perenne L. Journal of Agricultural Science 91: 217–222.
Harper JL. 1977. Population biology of plants. London: Academic Press.
Helgadóttir Á, Dalmannsdóttir S, Collins RP. 2001. Adaptational changes in populations of contrasting white clover cultivars selected under Icelandic conditions. Annals of Botany 88: 771–780.
Junttila O, Svenning MM, Solheim B. 1990. Effects of temperature and photoperiod on frost resistance of white clover (Trifolium repens) ecotypes. Physiologia Plantarum 79: 435–438.[CrossRef]
Lawes Agricultural Trust. 1987. Genstat V reference manual. Oxford: Clarendon Press.
Lynch DV, Steponkus PL. 1987. Plasma membrane lipid alterations associated with cold acclimation of winter rye seedlings (Secale cereale L. cv Puma). Plant Physiology 83: 761–767.
Murata N, Los DA. 1997. Membrane fluidity and temperature perception. Plant Physiology 115: 875–879.[Web of Science][Medline]
Ollerenshaw JH, Baker RH. 1981. Low temperature growth in a controlled environment of Trifolium repens plants from northern latitudes. Journal of Applied Ecology 18: 229–239.[CrossRef]
Pérez delaVega M. 1997. Plant genetic adaptedness to climatic and edaphic environment. In: Tigerstedt PMA, ed. Adaptation in plant breeding. Proceedings of the XIV EUCARPIA Congress, Jyväskylä, Sweden, 27–38.
Pollock CJ. 1990. The response of plants to temperature change. Journal of Agricultural Science 115: 1–5.
Røsnes K, Junttila O, Ernsten A, Sandli N. 1993. Development of cold tolerance in white clover (Trifolium repens L.) in relation to carbohydrate and free amino acid content. Acta Agriculturae Scandinavica 43: 151–155.
Smith D. 1973. The nonstructural carbohydrates. In: Butler GW, Bailey RW, eds. Chemistry and biochemistry of herbage. Vol. 1. London: Academic Press, 106–155.
Snedecor GW, Cochran WG. 1967. Statistical methods. 6th edn. Ames, Iowa: Iowa University Press.
Stäheli B, Lüscher A, Nösberger J. 1996. Growth of white clover plants in relation to temperature in winter and spring. In: Parente G, Frame J, Orsi S, eds. Grassland and land use systems. Proceedings of 16th meeting of the European Grassland Federation. Italy: ERSA, 309–312.
Steponkus PL. 1984. Role of the plasma membrane in freezing injury and cold acclimation. Annual Review of Plant Physiology 35: 543–584.[CrossRef][Web of Science]
Sukhija PS, Palmquist DL. 1988. Rapid method for determination of total fatty acid content and composition of feedstuffs and feces. Journal of Agriculture and Food Chemistry 36: 1202–1206.[CrossRef]
Svenning MM, Røsnes K, Junttila O. 1997. Frost tolerance and biochemical changes during hardening and dehardening in contrasting white clover populations. Physiologia Plantarum 101: 31–37.[CrossRef]
Thomas H, James AR. 1993. Freezing tolerance and solute changes in contrasting genotypes of Lolium perenne L. acclimated to cold and drought. Annals of Botany 72: 249–254.
Thomas TA. 1977. An automated procedure for the determination of soluble carbohydrates in herbage. Journal of the Science of Food and Agriculture 28: 639–642.[CrossRef]
Tigerstedt PMA. 1994. Adaptation, variation and selection in marginal areas. Euphytica 77: 171–174.[CrossRef]
Wachendorf M, Collins RP, Connolly J, Elgersma A, Fothergill M, Frankow-Lindberg BE, Ghesquiere A, Guckert A, Guinchard MP, Helgadóttir Á, Lüscher A, Nolan T, Nykänen-Kurki P, Nösberger J, Parente G, Puzio S, Rhodes I, Robin C, Ryan A, Stäheli B, Stoffel S, Taube F. 2001. Overwintering of Trifolium repens and succeeding spring growth: results from a common protocol carried out at twelve European sites. Annals of Botany 88: 669–682.
White J. 1979. The plant as a metapopulation. Annual Review of Ecology and Systematics 10: 109–145.[CrossRef][Web of Science]
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