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Genetics and Molecular Biology |
2Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences (SLU), P.O. Box 7027, S-750 07, Uppsala, Sweden; 3Systematic Botany, Department of Ecology, University of Lund, Sölvegatan 37, S-223 62, Lund, Sweden
Received for publication October 17, 2002. Accepted for publication March 25, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Acer platanoides • Aceraceae • Betula pendula • Betulaceae • fluctuating asymmetry • Norway maple • plant growth • silver birch
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Freeman et al., 1994
; Møller and Swaddle, 1997
). Given the common occurrence of bilateral or radially symmetric structures in both plants and animals, this parameter offers a unique tool for comparative studies of developmental stability in a wide variety of traits and organisms (Møller and Swaddle, 1997
). For fluctuating asymmetry to be a useful measure of developmental instability, it is necessary to account for size dependence, measurement error, and possible departures from ideal fluctuating asymmetry (normally distributed deviations from symmetry with a mean of zero) such as directional symmetry (deviations from symmetry with a mean different from zero) and antisymmetry (deviations from symmetry with a bimodal distribution) (Palmer and Strobeck, 1986
).
Although numerous studies have examined how the mean level of fluctuating asymmetry responds to environmental or genetic perturbations such as pollution, herbivory, competition, inbreeding, and hybridization (Palmer and Strobeck, 1986
; Leary and Allendorf, 1989
; Kozlov et al., 1996
; Møller and Swaddle, 1997
; Wilsey et al., 1998
; Martel et al., 1999
; Møller and Shykoff, 1999
; Roy and Stanton, 1999
; Andalo et al., 2000
; Lappalainen et al., 2000
), only a few attempts have been made to include more than one species or population in experimental studies of developmental stability (e.g., Waldmann, 1999
; Rao et al., 2002
). Hence, little is known about the extent to which stress-related changes in developmental stability are species- or population-specific or whether they also apply more generally. Furthermore, despite evidence that increased stress can induce significant levels of fluctuating asymmetry (Palmer and Strobeck, 1986
; Leary and Allendorf, 1989
; Møller and Swaddle, 1997
), it remains uncertain whether the disruption of developmental stability occurs before detrimental effects on growth, fecundity, or survival come into play, i.e., whether fluctuating asymmetry is a robust predictor of future performance (Palmer, 1996
).
Plants have a number of suitable attributes for genetic and experimental analyses of developmental stability. For example, many plants can be cloned or raised in large numbers under uniform growth conditions, making it possible to distinguish between genetic and environmental sources of variation. In addition, plants are modular and therefore possess a number of repeated structures (leaves, petals, flowers), providing an opportunity to obtain more than one measure of fluctuating asymmetry from each individual (Freeman et al., 1994
). As yet, there have been relatively few experiments in which investigators have related measures of performance to the level of asymmetry in "early" plant structures such as cotyledons or early leaves (Anne et al., 1998
; Rao et al., 2002
).
Nutrient availability is one of the most important factors besides temperature and photoperiod for the performance of plants in natural and agricultural environments (Tamm, 1991
). Characteristic symptoms of nutrient deficiency include reduced growth, pale leaves, and a disproportionately high allocation of biomass to the root system (Ericsson, 1995
). Despite the great importance of nutrients for plant vigor, there is little consensus regarding the effects of nutrient availability on measures of developmental stability. Andalo et al. (2000)
found fluctuating asymmetry to be a poor indicator of performance when plants of Lotus corniculatus were raised under nitrogen-poor conditions, whereas a recent study of Betula pubescens documented increased asymmetry after nitrogen enrichment (Lappalainen et al., 2000
).
In the present investigation, we have obtained family-structured data from large cultivation experiments with young plants of Acer platanoides (Norway maple) and Betula pendula (silver birch) (Black-Samuelsson and Eriksson, 2002
) in order to compare the expression of developmental instability in two contrasting environments: one with free access to nutrients and the other with a limited supply of nutrients. Using the difference between the right and left side of each leaf as a measure of fluctuating asymmetry and plant biomass (dry mass) as a performance variable, we addressed the following questions: (1) Does nutrient stress increase leaf asymmetry, and is this response of the same magnitude as that recorded for more conventional characters, e.g., leaf size and plant biomass? (2) Is early leaf asymmetry a sensitive indicator of plant growth within each nutrient treatment? Our data also provided an opportunity to evaluate patterns of leaf asymmetry for plants derived from two geographically distant populations of both species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; De Jong, 1976
). Plants used in the present study represent seed-derived progeny from each of 10 open-pollinated trees of both species in two geographically isolated localities, one on the Baltic island of Öland, Southeastern Sweden (latitude 56°43' N, longitude 16°40' E), and another in Lillehammer, Southern Norway (latitude 61°10' N, longitude 10°25' E). Each seed family was represented by 20 seedlings in each nutrient environment, resulting in a total of 800 experimental plants per species (2 environments x 2 populations x 10 families x 20 seedlings).
Experimental procedures
Following stratification and/or germination, the seedlings were planted in separate pots with mineral wool and cultivated in a phytotron for 3 (maple) or 4 (birch) wk before the assignment of plants to the two nutrient treatments (for a summary of experimental procedures and growth conditions, see Table 1). Plants in the "high-nutrient treatment" had free access to a 50% nutrient solution, 2L-6513, as described by Ingestad (1967)
. Plants in the "low-nutrient treatment" were watered with nutrient-free water, followed by the addition of nutrient solution 2L-6513 pipetted in quantities calculated for a growth rate of 3%. This method accounts for the exponential increase in plant biomass and allows high accuracy so that the amounts of nutrients added correspond to the amounts of nutrients taken up (Ingestad and Lund, 1979
). The irradiance was 300 µmol·m–2·s–1 in the 400–650 nm spectrum, and relative air humidity was 75%. Photoperiod and temperature regimes were chosen to simulate natural conditions in Southern Sweden: the night length increased from 8 h (week 1) to 16 h (week 17), whereas the night temperature decreased from 15°C to 2°C over the same period. The day temperature was usually 5°C higher than the night temperature (Table 1).
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Leaf sampling and measurements
Leaves for the analysis of developmental stability were sampled when all plants had spent 13 or 14 wk in the nutrient environments (Table 1) and ceased growing because of the progressively decreasing day length in the grown chambers (Black-Samuelsson and Eriksson, 2002
). In the case of birch, the low- and high-nutrient plants differed in height and node number, the mean height being 18 and 62 cm, respectively. To standardize the sampling procedure, we used two adjacent leaves at the midpoint of each plant. Leaves were sampled from 381 and 373 plants in the low- and high-nutrient treatment, respectively. As for maple, the low-nutrient plants had 3–5 nodes and a mean height of 12 cm, whereas the high-nutrient plants had 5–7 nodes and a mean height of 18 cm. In this species, we used the two opposite leaves at the fourth node (counted from the bottom up), which turned out to procedure the largest leaves in both treatment groups. Leaf samples were obtained from a total of 375 and 368 maple plants in the low- and high-nutrient treatment, respectively. All leaves were pressed and photocopied before the measurements.
Three weeks after the sampling of leaves (about 17 wk after the initiation of the nutrient treatments), all plants were harvested and scored for biomass after drying for 40 h at 70°C.
To quantify fluctuating asymmetry in birch leaves, we used a digital caliper to determine the distance from the midrib to the right and left margin at a point half-way between the tip of the leaf and the base of the petiole (Fig. 1; see also Wilsey et al., 1998
). The absolute value of the right-minus-left value was then averaged across the two leaves for each plant to obtain an estimate of individual asymmetry, whereas the mean of the measurements provided a measure of leaf size. The absolute values of the asymmetries were subjected to a logarithmic transformation to meet the normality assumption in parametric analyses. To account for scale dependency, we also calculated "relative asymmetries," obtain by dividing each asymmetry value by leaf size before the log transformation (Palmer and Strobeck, 1986
). Similar procedures were used to assess fluctuating asymmetry in maple leaves, except that the asymmetry scores were based on the distance from the base of the petiole to the tip of the largest side lobe on each side of the leaf (Fig. 1).
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), we employed a bootstrap analysis with 2000 random samples from the original data sets to calculate the 95% confidence interval (CI) of the moment statistics g1 and g2 (Sokal and Rohlf, 1995
). The null hypothesis of no skewness (g1 = 0) or no kurtosis (g2 = 0), was rejected if any of these CIs excluded 0. To estimate measurement error, we remeasured a sample of leaves from each species and then compared the mean of the absolute differences between the repeat measurements with the mean of the absolute asymmetry values for each subsample. A mixed model, two-way analysis of variance (ANOVA) was performed to determine whether the between-sides variance was significantly greater than the variance arising from measurement error (Palmer and Strobeck, 1986
).
Individual data on log (leaf asymmetry), leaf size, and plant dry mass were subjected to nested factorial ANOVA (PROC GLM, SAS, 1989
), using type III sum of squares, to determine the effects of nutrient treatment, population, maternal family (nested within population), the treatment-by-population interaction and the treatment-by-family interaction, on each variable. Initial analyses revealed a significant effect of block (nested within treatment), so this factor was included in all analyses. Environment and population were considered as fixed, while the other factors were regarded as random (for other details, see Black-Samuelsson and Eriksson, 2002
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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According to the two-way ANOVAs (Table 2), there was no directional asymmetry (no significant effect of "side"), whereas the leaf-by-side interaction was declared significant in both species, indicating significant levels of asymmetry relative to measurement error. Based on the mean of the absolute differences between repeat measurements and the mean absolute asymmetry (data not shown), the ratio of measurement error to leaf asymmetry was 7.5% for maple and 16.2% for birch.
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Leaf size showed moderate to strong positive correlations with plant biomass in both species (Table 3), not only when individuals were pooled over the two nutrient treatments (r = 0.68–0.76), but also when the analyses were restricted to low-nutrient plants (r = 0.59–0.63) or high-nutrient plants (r = 0.31–0.58). Leaf asymmetry varied independently of plant biomass, once the effect of leaf size had been accounted for (|r| < 0.12).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), the low-nutrient treatment induced significant levels of stress in juvenile plants of both maple and birch. However, despite relatively large sample sizes (370–380 plants per treatment) and evidence for nutrient stress in plant biomass, we found no support for the idea that adverse growth conditions disrupt developmental stability in foliar characters. Although the mean asymmetry was significantly affected by nutrient stress, there was no significant difference in mean asymmetry between high- and low-nutrient plants once the effect of overall leaf size had been accounted for. These patterns were consistent over populations, as shown by the nonsignificant treatment-by-population interaction term for all variables. Based on these observations, there is no evidence to suggest that developmental stability in early maple and birch leaves is related to the amount of nutrient stress experienced by each individual.
Results of this study contrast with Wilsey et al. (1998)
, who found genetic and environmental stress to induce significant levels of leaf asymmetry in treeline birches: interspecific hybrids between Betula pendula, B. pubescens, and B. nana had more asymmetric leaves than the parental species, and there was a positive association between mean expression of leaf asymmetry and elevation. In a study of B. pubescens seedlings, nitrogen fertilization increased aboveground biomass and had a negative effect on developmental stability in leaf morphology (Lappalainen et al., 2000
). In another study (Kozlov et al., 1996
), the mean asymmetry of B. pendula and B. pubescens leaves increased with foliar nickel concentration and decreased with increasing distance from four different pollution sources. In contrast, Martel et al. (1999)
provided no evidence to suggest a relationship between leaf asymmetry and moisture stress: B. pubescens trees growing on the wettest (and most stressful) parts of a mire had relatively small leaves but showed no increase in asymmetry compared to trees experiencing less stressful conditions. The only published report on developmental instability in Acer platanoides (Freeman et al., 1994
) documented significantly higher levels of leaf asymmetry in trees growing around a chemical production facility in Russia than in trees occupying a control site 20 km away. When combined with the results of the present study, data for deciduous trees indicate considerable variation in the extent to which genetic or environmental perturbations influence patterns of variation in developmental instability.
Both maple and birch showed moderate or strong positive correlations between leaf size and plant biomass, regardless of whether or not the data were pooled over nutrient treatments, whereas "size-corrected" estimates of asymmetry turned out to be poor predictors of plant performance, both in the low- and high-nutrient group. Similarly, data available for Sinapis arvensis (Roy and Stanton, 1999
) and Brassica cretica (Rao et al., 2002
) provide no evidence that measures of leaf asymmetry are reliable predictors of performance or that different measures of asymmetry are positively correlated at the within- or between-population levels. In view of these findings, there is no reason to consider measures of leaf asymmetry as more sensitive indicators of individual performance than other variables. Hence, we urge caution in the use of fluctuating asymmetry as a tool for identifying stressed individuals or populations, unless a consistent association between asymmetry and plant performance has been demonstrated.
In this context, it is necessary to ask whether data on leaf asymmetry conform to "ideal" fluctuating asymmetry (normally distributed deviations from symmetry with a mean of zero) and whether our estimates of leaf asymmetry are inflated over their true values as a result of measurement error (Palmer and Strobeck, 1986
). The distribution tests revealed a higher frequency of right-minus-left scores around the mean than expected for a normal distribution (leptokurtosis), but there was little evidence for serious departures from ideal fluctuating asymmetry such as directional asymmetry or antisymmetry. In view of these observations, the level of leaf asymmetry can be considered as a suitable descriptor of developmental instability in young plants of maple and birch. Regarding measurement error, we found differences between repeat measurements to account for a minor, though non-negligible, proportion of the between-sides variance, the ratio of mean measurement error to mean asymmetry being 8% for maple and 16% for birch. These estimates are not consistently higher than those reported for foliar structures in other plant species (e.g., Rao et al., 2002
), but raise the possibility that our analyses lacked the statistical power to detect small differences in foliar asymmetry between stressed and nonstressed individuals, particularly in birch.
Previous studies have found cotyledons or true leaves to be less developmentally stable than reproductive organs (Møller and Shykoff, 1999
; Roy and Stanton, 1999
; Waldmann, 1999
; Andalo et al., 2000
; for an exception, see Rao et al., 2002
). This observation, combined with the high levels of phenotypic plasticity characterizing leaf characters (Bradshaw, 1965
), leads to the suggestion that plastic responses represent a major determinant of the between-sides variance observed in this and other studies of leaf asymmetry. To the extent that such within-leaf plasticity represents a functional response to small-scale variation in light intensity, there is no reason to consider perfect symmetry as the ideal developmental program for foliar structures (cf. Palmer, 1996
). These interpretations do not exclude developmental noise (random accidents) as a major cause of leaf asymmetry, nor do they rule out leaf asymmetry as a potentially useful tool for experimental studies of developmental stability. Indeed, data from wild plant species provide clear evidence that genetic or environmental stress sometimes increases levels of asymmetry in foliar characters (Anne et al., 1998
; Wilsey et al., 1998
; Møller and Shykoff, 1999
; Andalo et al., 2000
).
Conclusion
Based on the present study of maple and birch, two distantly related tree species with widely different leaf morphologies, the degree of leaf asymmetry was found to be a poor indicator of nutrient deficiency and the performance of each individual within each nutrient treatment (as determined by plant biomass), contrasting with overall leaf size, which showed strong positive relationships with both mean and individual performance. As a consequence, leaf asymmetry will be difficult to use as a tool for monitoring the negative effects of nutrient stress in these species. Our data also illustrate the advantage of performing large manipulative studies before any broad generalizations are made regarding the relationship between environmental stress and developmental stability.
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FOOTNOTES |
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4 Author for reprint requests (e-mail: Sanna.Black{at}vbsg.slu.se
)
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Anne P. F. Mawri S. Gladstone D. C. Freeman 1998 Is fluctuating asymmetry a reliable biomonitor of stress? A test using life history parameters in soybean. International Journal of Plant Sciences 159: 559-565[CrossRef]
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Freeman D. J. J. H. Graham J. M. Emlen 1994 Developmental stability in plants: symmetries, stress and epigenesis. In T. A. Markow [ed.], Developmental instability: its origins and evolutionary implications, 99–121. Kluwer Academic Publishers, Dordrecht, Netherlands
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SAS. 1989 SAS/STAT user's guide, version 6, 4th ed., vol. 2. SAS Institute, Cary, North Carolina, USA
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Tamm C. O. 1991 Nitrogen in terrestial ecosystems—questions of productivity, vegetational changes and ecosystem stability. Ecological studies 81. Springer-Verlag, Berlin, Germany
Waldmann P. 1999 The effect of inbreeding and population hybridization on developmental instability in petals and leaves of the rare plant Silene diclinis (Caryophyllaceae). Heredity 83: 138-144
Wilsey B. J. E. Haukioja J. Koricheva M. Sulkinoja 1998 Leaf fluctuating asymmetry increases with hybridization and elevation in tree-line birches. Ecology 79: 2092-2099[ISI]
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