SEARCH

SEARCH BY CITATION

Keywords:

  • fluctuating asymmetry;
  • developmental stability;
  • leaf expansion;
  • Dalechampia scandens;
  • Euphorbiaceae

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    We studied patterns of fluctuating asymmetry (FA) in leaves of four populations of the neotropical vine Dalechampia scandens to obtain insight into the origin of leaf FA and the level at which it is controlled. We analysed correlations in signed and unsigned asymmetry at different organizational levels. We also analysed the ontogeny of FA during leaf expansion to test whether asymmetry is regulated during cell expansion, and whether fast-expanding leaves are more or less asymmetrical.
  • • 
    Signed asymmetry was negatively correlated between successive leaves, that is, when the right side of a leaf was larger than the left side, the next leaf on the shoot tended to show the opposite pattern. The magnitude of FA, however, was very weakly correlated among successive leaves or among leaves measured on different shoots.
  • • 
    The direction of asymmetry did not change during leaf expansion, but the relative asymmetry, that is, asymmetry corrected for difference in trait size, decreased during expansion. We found a weak negative relationship between leaf expansion rate and relative asymmetry on the fully expanded leaves.
  • • 
    These results suggest that leaf asymmetry in Dalechampia originates from perturbations in cell proliferation in the stem, creating asymmetries in opposite directions in successive leaves. These asymmetries persist during leaf expansion, but tend to be reduced by unknown mechanisms.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Developmental stability is the ability of an organism to buffer nonadaptive phenotypic variation resulting from stochastic perturbations during development (developmental noise) (Waddington, 1957; Palmer & Strobeck, 1986; Debat & David, 2001; Nijhout & Davidowitz, 2003). Variation in developmental stability will affect the ability of organisms to reach their target phenotype (the phenotype that would be reached from a given genetic and environmental background without noise of any kind: Nijhout & Davidowitz, 2003). Therefore developmental stability is an important variational property related to fitness (Tracy et al., 2003; see Clarke, 2003 for discussion). However, measuring developmental stability remains difficult because it consists of estimating within-individual variance around an optimal value, which is unknown in most cases. Because both sides of bilateral characters depend on the same genes and share the same environment, the measure of small, directionally random differences between the two sides, referred to as fluctuating asymmetry, has been suggested to reflect the composite effect of developmental noise and developmental stability (Van valen, 1962; Palmer & Strobeck, 1986).

Although fluctuating asymmetry, as a measure of developmental stability, has been studied intensively over the past two decades, both theoretically and empirically (see Polak, 2003 for review), the relationship between developmental stability and fluctuating asymmetry remains poorly understood. Part of the problem resides in a lack of understanding of the processes that control the development of both sides of bilateral characters, and whether these processes have organism-wide or more localized effects.

Analysis of the correlation pattern between fluctuating asymmetry measurements on different characters or repeated modules, such as leaves or flowers, provides valuable information on the level at which fluctuating asymmetry, and possibly developmental stability, are regulated. Indeed, correlation in unsigned fluctuating asymmetry (independent of the direction of asymmetry) among traits should reflect the existence of an organism-wide regulation of developmental stability (Leamy, 1993; Polak et al., 2003). However, correlation in signed asymmetry among traits might also result from the effects of developmental noise shared among structurally or developmentally related traits (Van Dongen et al., 1999; Klingenberg et al., 2001; Polak et al., 2003).

Another series of questions underlying the relationship between fluctuating asymmetry and developmental stability concerns the origin and possible control of fluctuating asymmetry during ontogeny. Several models have been put forward to describe the ontogeny of fluctuating asymmetry (Emlen et al., 1993; Swaddle & Witter, 1997; Aparicio, 1998; see Kellner & Alford, 2003; Klingenberg, 2003 for reviews). Distinction between these different models depends on whether asymmetry is regulated during ontogeny, and whether this regulation involves feedback mechanisms between the two sides (Kellner & Alford, 2003). Data concerning the ontogeny of fluctuating asymmetry, such as the level of changes in magnitude and direction of asymmetry that occur during trait development, are therefore crucial for a better understanding of the mechanisms that control/affect developmental stability.

Plants are particularly useful for studying the ontogeny of fluctuating asymmetry and correlation patterns in the level of fluctuating asymmetry across traits. Their modular structure provides additional organizational levels at which fluctuating asymmetry can be measured and compared. Additionally, measurements on leaves and flowers provide the opportunity to test for correlation in fluctuating asymmetry among homologous traits within individuals.

Using observations in a twining vine, Dalechampia scandens (Euphorbiaceae), we address questions related to the developmental origin of fluctuating asymmetry. We first tested whether the observed asymmetry resulted from the twining behaviour of the vine, or from the phenotypic expression of developmental noise. We further tested whether fluctuating asymmetry reveals perturbations in developmental processes at the level of the leaf, shoot or plant. To answer this question, we analysed the pattern of correlation in signed and unsigned asymmetry at these different levels of organization. We then analysed the asymmetry pattern during leaf expansion to test whether cell expansion amplified already existing asymmetries produced during cell division, or whether regulatory processes might decrease the level of asymmetry. Finally, we analysed the relationship between fluctuating asymmetry and the rate of leaf expansion, to test whether fast-expanding leaves are less asymmetrical, as predicted if fluctuating asymmetry is negatively related to fitness (fitness-indicator model: Teather, 1996; Valkama & Kozlov, 2001); or more asymmetrical, as predicted if a trade-off occurs between growth rate and the ability to correct developmental errors (developmental-homeostasis model, Martel et al., 1999; Lempa et al., 2000).

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study organism and breeding conditions

Dalechampia scandens L. is an alternate-leaved, neotropical vine producing unisexual flowers aggregated into bisexual pseudanthial inflorescences or blossoms (Webster & Webster, 1972; Webster & Armbruster, 1991). Both blossoms and leaves are bilaterally symmetrical (Fig. 1; Pélabon et al., 2004a, 2004b, 2005). Individual plants measured in this study came from seeds collected at two locations in Venezuela and two in Mexico (see Hansen et al., 2000 for location of populations). Blossoms from the two Mexican populations (Tulum and Chetumal) have large, resin-producing glands; those from the two Venezuelan populations (Tovar and Caracas) have small glands (Hansen et al., 2003a). Leaves from all populations are very similar. The only difference observed in the glasshouse was the more frequent occurrence of leaves with five lobes in the Venezuelan populations (we did not measure the five-lobed leaves in this study).

image

Figure 1. (a) Sketch of a shoot of Dalechampia scandens. (b) Diagram of the traits measured on leaves of D. scandens. The illustration shows a normal, three-lobed leaf. Measurements: LCL, length of central lobe; LRL and LLL, length of right and left lobe, respectively; wl and wr, left and right width of central lobe, respectively. Widths of central lobe left and right of the mid-vein were measured perpendicular to the central vein where the lobe was the widest.

Download figure to PowerPoint

Infructescences (comprising three pistillate flowers and containing up to nine seeds) were collected from separate individuals in each population in early 1998. Several seeds from each infructescence were germinated in March–May 1998 in the glasshouse of the Biology Department, NTNU (Trondheim, Norway). Leaf measurements were taken on individuals from this first glasshouse generation for all populations except Tulum. For the Tulum population, individuals from the first glasshouse generation were crossed in a diallel design to estimate the genetic variance in floral traits (Hansen et al., 2003a, 2003b; Pélabon et al., 2004a). For this population, measurements were therefore taken from the second glasshouse generation. Measurements on growing leaves were made on individuals originating from the Chetumal population in 1999, and from the Tovar population in 2003.

Conditions in the glasshouse were maintained as constant as possible during the whole study with an average temperature of 28°C by day and 22°C by night, 60–80% humidity and 13 : 11 light : dark light regime. Plants were fertilized weekly.

Measurements of asymmetry and fluctuating asymmetry

Leaf asymmetry was defined as the difference in length between the left and right lobes (Fig. 1). We also measured asymmetry in the width of the central lobe as the difference in distance between the left and right margins and the central vein (Fig. 1). We defined (signed) asymmetry as the signed difference L − R. Fluctuating asymmetry (FA) corresponds to the absolute value | L − R |. Relative asymmetry and relative FA were calculated as: relative asymmetry = 100 × [ln(L) − ln(R)]; and relative FA = 100 × | ln(L) − ln(R) |, respectively, where ln is the natural log-transformed character value (Clarke, 1998). The relative asymmetry and relative FA can be read as measures of asymmetry in percentage of the trait size. The length of the central lobe was used as a measure of overall leaf size. All measurements were performed with a digital calliper to the nearest 0.01 mm using an optical binocular magnifier (×5 magnification) by a single observer (C.P.).

Asymmetry pattern on successive leaves

We measured asymmetry on two successive leaves on the same shoot, one shoot per plant, on 29–309 plants per population, across four populations (Tulum n = 309; Chetumal n = 31; Caracas n = 30; Tovar n = 29). Measurements were made on leaves borne by long shoots projecting out from the crown of the plant. The two successive leaves shared similar light conditions, as did leaves measured on different plants. In these measurements we did not include the first leaf after the shoot branched, which was studied separately, because it usually exhibited a different pattern of development. We collected the complete shoot and measured two successive leaves flattened under transparent sheet within few minutes of collection. The mean length of these leaves was comparable with that of fully expanded leaves measured at the same time, in the study investigating leaf expansion.

We also measured the first leaf on the branch (most proximal leaf) for 73 individual plants from the Tulum population. These data were used to test the hypothesis that leaf asymmetry results from the twining pattern of the shoot. We predicted that, if leaf asymmetry results from twining, then asymmetries measured at a constant position (here the first leaf of the shoot) should display a pattern of directional asymmetry. We also measured one additional leaf on the same shoot for comparison of the size and fluctuating asymmetry level.

We measured two successive leaves on two different shoots (four leaves per plant) in the same 73 individuals from the Tulum population, to test the correlation in developmental stability across shoots from the same plant.

Fluctuating asymmetry and rate of leaf expansion

Ontogeny of leaf asymmetry during leaf expansion was studied first on a set of 22 leaves on 11 plants (two leaves per plant on different shoots) from the Chetumal population (Mexico). We measured the length of each of the three lobes on each leaf, as described in Fig. 1, every 5 d from when leaves were approximately 20 mm long until the final size was reached (approx. 30 d later). Measurements were stopped when no increase in leaf length was observed between two consecutive measurements. Fully grown leaves were collected and measured immediately after being flattened under a transparent sheet. The relationship between expansion rate and final asymmetry was further analysed on individuals from the distinct population of Tovar (Venezuela). From early September to October 2003, we measured the expansion rate and the final asymmetry on one leaf from each of 31 individuals.

Expansion rate was measured on actively expanding leaves that were 44% (SE = 2.6%) of their final size in the Chetumal population, and from 17.2% (SE = 0.6%) of their final size in the Tovar population. The difference in percentage of the final size at which initial measurements were made has two different causes. First, the initial measurements on the leaves of the Tovar population were made on younger (smaller) leaves because we did not record the length of both lateral lobes, which would have implied measurements on unfolded leaves. Additionally, Tovar leaves achieved a larger size when fully expanded than the leaves from the Chetumal population, presumably because of unknown changes in the glasshouse environment between the first series of measurements in 1999 and the second series in 2003.

The expansion rate of each leaf was estimated by fitting the data with an asymptotic exponential model:

  • Y = A − BeCt

where Y is the length of the central lobe and t the age of the leaf in days after the first measurement; and A, B and C are the parameters of the model. Models were fitted for each leaf independently using nonlinear least-squares analysis (nls in s-plus; Crawley, 2002). We considered C as an index of the leaf expansion rate and analysed the relationship between C and the final asymmetry. Because two leaves were measured per individual plant in the Chetumal data set, we analysed the relationship between the expansion rate and the final fluctuating asymmetry using a linear mixed model, where individual identity was entered as a random factor nested into population, also entered as random factor.

Statistics and measurement errors

Measurement error biases the estimation of fluctuating asymmetry (Palmer, 1994). Measurement error on unexpanded leaves was estimated by repeated measurements on 17 young leaves of average size (SD) of 23.15 (3.73) mm (range 14–36 mm). Measurement error on fully expanded leaves was estimated from the last two measurements on each leaf (n = 22) when expansion had ceased. Repeatability of FA measurement on both young and adult leaves was high (young leaves: R = 0.98, F16,17 = 80.49; adult leaves: R = 0.91, F21,22 = 21.39). Additionally, the means and variances of FA were corrected for measurement error (see Table 1 and Pélabon et al., 2004a for further explanation).

Table 1.  Descriptive statistics for trait size and asymmetry in adult leaves in four populations of Dalechampia scandens. Two successive leaves were measured on a single shoot per individual. The size of both leaves is reported, but only the leaf in proximal position is taken into account to estimate descriptive statistics in FA. Data are in mm except for relative FA which is in percentage of trait size. The 95% confidence intervals were estimated using bootstrapping methods. Mean and variance in FA and relative FA are corrected for measurement error as follows: FAcorr = inline image where FAobs is the observed FA, and inline image is the measurement variance calculated as Var(m1 − m2), where m1 and m2 are the signed asymmetry calculated from the first and second measurements. Variance in FA corrected for measurement error was obtained by removing inline image(1 − 2/π) from the observed variance in FA (see Pélabon et al. (2004a) for further details)
 ChetumalTulumTovarCaracass
  • a

    Test for difference in leaf size among populations: F3,398 = 0.995, P = 0.395.

  • b

    Test for differences in fluctuating asymmetry (FA) among populations: anova on square-root transformed data: FA, F3,395 = 0.84, P = 0.47; relative-FA, F3,395 = 0.73, P = 0.54.

N 31 309 29 30
Leaf size (SD)a
 Proximal leaf 57.04 (11.31) 57.60 (12.67) 54.95 (8.24) 60.33 (9.58)
 Distal leaf 55.35 (8.47) 56.18 (11.29) 57.50 (11.32) 57.32 (10.37)
Signed asymmetry
 Mean  (95% CI) 0.37  (−0.55; 2.26) −0.06  (−0.25; 0.08) 0.57  (−0.03; 2.43) −0.27  (1.53; 0.89)
 Kurtosis 1.62 2.37 0.29 1.92
 Skew −0.001 0.55 −0.16 0.56
FAb
 Mean 2.35 2.56 1.76 2.80
 Variance 5.58 5.72 1.98 7.33
 CV 1.01 0.93 0.80 0.97
Correlation FA, size
 r (95% CI) 0.23  (0.08; 0.55) 0.14  (0.09; 0.25) 0.15  (−0.05; 0.57) −0.08  (−0.44; 0.21)
Unsigned relative FAb
 Lobe length 4.95 5.44 3.61 5.53
 Lobe width 15.59 14.08 12.69 14.50
Correlation between relative asymmetries in lobe length and width
 r (95% CI) 0.25  (−0.22; 0.62) 0.19  (0.10; 0.23) 0.32  (−0.06; 0.68) 0.39  (−0.07; 0.73)

The size of the samples used to test the different hypotheses varies substantially in this study, as does the power of the tests. We therefore focused on the effect size of the patterns, especially when similar effect sizes (r2) were observed in tests with different sample sizes. All statistical analyses were done in s-plus (Venables & Ripley, 2002).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Asymmetry: general pattern

The twining pattern of D. scandens was identical in the four populations observed, the stem showing a similar right-handed rotation (clockwise; see Hashimoto, 2002 for definition of right-handed sense of rotation). Descriptive statistics for leaf size and asymmetries in the four populations are reported in Table 1. We found no evidence of directional asymmetry (zero included in the 95% CI of the mean signed asymmetry in all populations; Table 1) or antisymmetry (no negative kurtosis of the distribution of signed asymmetries in all populations; Table 1). Therefore the asymmetry pattern is consistent with fluctuating asymmetry (Palmer & Strobeck, 1986).

There were no significant differences between the four populations in leaf size or in the level of leaf FA or relative FA (Table 1). Fluctuating asymmetry increased weakly with leaf size (FA in lobe length vs leaf size, r2 = 0.03, P = 0.005; no interaction effect with population, Table 1). Signed asymmetry in the width of the central lobe was positively, although weakly, correlated with the signed asymmetry in the length of lateral lobes in all populations (average r2 = 0.08; Table 1), indicating that leaf fluctuating asymmetry results partly from developmental disturbance between the two halves on each side of the central vein. However, the low r2 of this relationship, and the larger relative asymmetry in the width of the central lobe compared with the asymmetry in lobe length (mean relative FA, width central lobe = 14.21%, lobe length = 5.76%), suggest that these different measurements of leaf asymmetry reflect partly independent sources of developmental disturbance.

As reported earlier (Webster & Webster, 1972), the most proximal leaf on a D. scandens shoot (first leaf after the shoot branches off) displays a different pattern of development from the other leaves. Although we did not notice a systematic change in the number of lobes, as reported by Webster & Webster (1972), the most proximal leaves measured in the Tulum population were, on average, smaller than more distal leaves on the same shoot [mean leaf length (SD): most proximal leaf = 61.33 (15.10) mm; distal leaf = 77.36 (13.65) mm; paired t-test = −8.55, n = 73, P < 0.001]. Furthermore, these leaves displayed a greater level of asymmetry than more distal leaves [mean relative FA (SD) in lobe length: proximal leaf = 6.74 (6.04); distal leaf = 4.05 (3.37); paired t-test = 3.74, n = 73, P < 0.001]. However, we did not observe directional asymmetry on these leaves (mean signed asymmetry = 0.47; 95% CI = −0.50; 1.44). This suggests a random origin of the side of the asymmetry.

Correlations in signed and unsigned FA

The signed asymmetries of two successive leaves on a shoot were negatively correlated; this pattern was similar in the four populations. Although correlations between the two successive leaves ranged from −0.15 to −0.50, the slopes of the regression analyses were very similar (Fig. 2). Thus, when the right side of a leaf was larger than the left side, the next leaf on the shoot tended to show the opposite pattern. Analysis of asymmetry in the width of the central lobe gave similar results (average r = −0.16).

image

Figure 2. Regression of the signed asymmetry of the distal leaf on the signed asymmetry of the proximal leaf for different populations of Dalechampia scandens (•, Chetumal, r = −0.53, n = 31; ○, Tulum, r = −0.149, n = 309; ▾, Tovar, r = −0.295, n = 29; ▿, Caracas, r = −0.43, n = 30). Results of ancova: interaction, F3,391 = 1.56, P = 0.20; population, F3,391 = 0.50, P = 0.68; asymmetry distal leaf, F1,391 = 21.04, P < 0.001.

Download figure to PowerPoint

Fluctuating asymmetry was, at most, weakly correlated among successive leaves (r ranging from −0.20 to 0.36 in the four populations, average r = 0.09), and there was no significant correlation in FA among leaves measured on two different shoots on the same plant (mean r2 = 0.005, n = 73). We conducted a variance component analysis to estimate the proportion of variance in FA expressed at the different levels: individuals; shoots within individuals; and leaf within shoot. This analysis revealed that the vast majority of the variance in FA (98%) was caused by differences among leaves, the remaining 2% being caused by differences among individuals.

Ontogeny of fluctuating asymmetry and leaf expansion rate

Asymmetry increased during leaf expansion, as indicated by the slope > 1 of the linear regression between the initial and the final asymmetry [slope (95% CI) = 1.36 (1.00; 1.63), Fig. 3a]. Relative asymmetry, however, decreased significantly during leaf expansion, as indicated by the slope < 1 of the regression between initial and final relative asymmetry [slope (95% CI) = 0.54 (0.41; 0.67) Fig. 3b]. Forcing the regression through the origin does not affect the slope in either case (not shown). Representation of the asymmetry and relative asymmetry observed during leaf expansion (Fig. 4) also shows that individual asymmetries do not fluctuate in direction during this period.

image

Figure 3. Regression of final asymmetry on initial asymmetry measured during leaf expansion in Dalechampia scandens: asymmetry, estimates (95% CI), intercept = −0.05 (−0.63; 0.57), slope = 1.36 (1.00; 1.63); (b) relative asymmetry (log-transformed value), intercept = −0.008 (−0.02; 0.005), slope = 0.54 (0.41; 0.67). Black lines, regression; dashed lines, line with a slope of 1 forced through the origin.

Download figure to PowerPoint

image

Figure 4. Asymmetry (a) and relative asymmetry (b) in 22 leaves of Dalechampia scandens measured during leaf expansion.

Download figure to PowerPoint

The Chetumal and Tovar populations differed significantly in both leaf expansion rate and final leaf size (Table 2). The Chetumal population had smaller leaves and a similar level of FA, leading to a higher level of relative FA compared with the Tovar population. Therefore analysis of the relationship between leaf expansion rate and fluctuating asymmetry was done using the relative FA. Despite this difference, we found a weak negative relationship between relative FA and the expansion rate, similar in both populations (r2 = 0.08; Fig. 5). This indicates that fast-expanding leaves were less asymmetrical than leaves with a lower expansion rate.

Table 2.  Descriptive statistics for size, signed asymmetry and fluctuating asymmetry (FA) of Dalechampia scandens leaves measured during their expansion
 Chetumal (n= 24)Tovar (n= 31)
InitialFinalInitialFinal
  1. Leaf size is length of central lobe; asymmetry is measured as difference in length between left and right lobes. Asymmetry was not recorded on initial leaves from the Tovar population. Means and variances of FA and relative FA are corrected for measurement error as described in Table 1.

Trait size (mm)
 Mean  (SD) 23.74  (5.73) 54.23  (8.13)16.18    (3.34) 96.11  (13.70)
L − R (mm)
 Mean−0.25−0.40−0.33
 (95% CI)(−1.13; 0.61)(−1.74; 0.95)(−2.25; 1.58)
 Kurtosis 0.99 0.04 0.21
FA (mm)
 Mean 1.56 2.30 4.06
 Var 1.46 3.90 9.63
 CV 0.78 0.86 0.76
Relative FA
 Mean 8.52 4.99 4.40
 Var 52.69 19.88 12.39
 CV 0.85 0.89 0.80
image

Figure 5. Relationship between relative fluctuating asymmetry (FA) and leaf expansion rate in Dalechampia scandens. Omitting the most extreme point on the left of the graph does not affect the results. The line represents the regression slope for pooled data (•, Tovar population; ○, Chetumal population). We performed a mixed-model analysis where population and plant identity, nested in population, were entered as random factors (lme in s-plus, Crawley, 2002). Based on the Akaike criteria of information (AIC), the model with common slope and different intercept was the best. Estimates (± SE) of the model on square-root transformed data: intercept = 0.318 (± 0.048), slope =−0.695 (0.287), P = 0.032.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Correlation pattern in fluctuating asymmetry and the origin and control of leaf asymmetry

In this study, we used a hierarchical analysis of the correlation patterns in signed and unsigned leaf asymmetry in D. scandens to obtain insight into the origin and developmental control of leaf fluctuating asymmetry. We observed a negative correlation between signed asymmetry of successive leaves. Correlation in signed asymmetry is expected to result from common developmental noise shared among structurally integrated characters (Van Dongen et al., 1999; Klingenberg et al., 2001). Therefore a possible interpretation of this result is that fluctuating asymmetry originates from developmental perturbations in the apical meristem that simultaneously affect the development of successive leaves. Successive alternate leaves are formed on both sides of the shoot apical meristem, with their upper side facing each other (making them mirror images of each other). Consequently, any disturbance in the shoot apical meristem that can produce a difference between the left and the right side in one leaf, for example in the number of cells, could produce a difference in the opposite direction in the next leaf. This should result in the negative correlation in signed asymmetry observed among successive leaves. These observations suggest that part of the asymmetry observed in the fully grown leaf originates in the apical meristem during cell proliferation.

Considering the physiological processes involved in leaf initiation in the shoot apical meristem, we can speculate on possible mechanisms creating such a pattern. Leaf initiation depends on the relative local concentration of morphogen and inhibitory morphogen that flows form the apex of the apical meristem to prevent leaf initiation on the tip of the apical meristem (Fleming, 2005). Because the initiation of a leaf will create a gradient of inhibitory morphogen on the periphery of the apical meristem, the next leaf initiation will take place at the opposite side of the apical meristem (Fleming, 2005), creating the alternate pattern in leaf formation. An asymmetry in the relative concentration of morphogen and/or inhibitory morphogen, provoking left–right variations in cell proliferation in a first leaf, could easily provoke symmetrical disturbances in the next leaf. Further testing of this hypothesis could be conducted by applying morphogen or inhibitory morphogen on different sides of the apical meristem. Alternatively, differences in the partitioning of shoot apical meristem cells into leaf primordia could also create a pattern of asymmetry that repeats itself across successive leaves. Indeed, if leaf primordia are formed from a constant proportion of the surface area of the shoot apical meristem, then a meristem that was larger on one side could produce successive leaves with correlated asymmetries.

Surprisingly, we observe only a weak correlation in signed asymmetry between traits measured on the same leaf (asymmetry in central lobe width and asymmetry in the length of the lateral lobes), and this is not larger than the correlation in signed asymmetry observed between successive leaves. This indicates a relatively weak integration of the leaf, where growth of the lateral lobes is somewhat independent of growth of the central lobe. Such a lack of integration may occur if the growth in lobe width depends on the activity of lateral primordia, while the growth in lobe length depends primarily on initial cell distribution in the leaf primordium.

Despite the correlation in signed asymmetry observed among successive leaves, we found only a weak correlation in fluctuating asymmetry among these leaves, and no correlation at all among leaves measured on different shoots on the same plant. Correlation in unsigned asymmetry among traits indicates organism-wide control of developmental stability (Leamy, 1993). Considering that the correlation in fluctuating asymmetry among successive leaves is probably caused by an effect of common developmental noise, as suggested by the negative correlation in signed asymmetries, our results suggest that no overall control of developmental stability, or at most a weak, undetectable control, occurs in D. scandens at either the individual or the shoot level. This is confirmed by the variance component analysis, which indicates that most variability in FA is confined at the leaf level.

Ontogeny of asymmetry

Asymmetry measurement on small leaves is difficult and imprecise without damaging the leaf. Therefore the first asymmetry measurements on leaves were made when they were an average of 40% of final size. Although lateral primordia may contribute to leaf growth via cell division during later stages of leaf ontogeny, the increase in leaf size observed in this study essentially corresponds to cell expansion (Maksymowych, 1973).

We observed an increase in fluctuating asymmetry from the initial measurement on young leaves to the stage of full leaf expansion, without a change in the direction of asymmetry. This pattern is consistent with the pattern observed by Møller & Van Dongen (2003) on expanding leaves of Ulmus glabra, and fits the persistent asymmetry model, where asymmetries determined during early ontogeny persist during growth (Kellner & Alford, 2003). The origin of asymmetry during cell division in the apical meristem, suggested by the negative correlation in signed asymmetry between successive leaves, also fits this hypothesis. However, a simple magnification of early determined asymmetries by cell expansion later during leaf ontogeny should result in the maintenance of the relative (size-corrected) asymmetry during leaf expansion. We observed that relative asymmetry decreased during leaf expansion, suggesting a regulation of symmetry during this part of the leaf ontogeny.

Fluctuating asymmetry in the leaves therefore appears to result primarily from developmental disturbances during cell division, and while regulatory mechanisms can partly reduce the level of relative fluctuating asymmetry, the absolute asymmetry is magnified by later cell expansion. Because of the difficulties in analysing fluctuating asymmetry in leaf primordium, occurrence of similar regulatory mechanisms during the stage of cell division remains unknown.

Fluctuating asymmetry and leaf expansion rate

Both positive and negative relationships between growth rate and fluctuating asymmetry have been suggested. The developmental-homeostasis hypothesis suggests that rapid growth beyond the selected optimum may negatively affect developmental stability because of a trade-off between growth rate and the ability to correct developmental errors (Calow, 1982; Arendt, 1997). In this case rapid growth, such as compensatory growth, should be positively correlated with fluctuating asymmetry (Martel et al., 1999). Note that the simultaneous effects of some stressful events, such as defoliation or plant damage, on growth and developmental stability could produce the same positive correlation between growth rate and fluctuating asymmetry without any direct relationship between growth rate and developmental stability (Kozlov, 2003). Alternatively, rapid growth may be the consequence of a good individual condition (genetic or phenotypic), and if there is a positive relationship between condition and developmental stability, a negative relationship is expected between growth rate and developmental instability (Valkama & Kozlov, 2001). These two hypotheses are not mutually exclusive. Indeed, the negative relationship between growth and developmental stability is expected only when the growth rate is beyond the selected optimum. Below the optimal level, a positive relationship can occur. If we take rate of leaf expansion as a proxy for leaf growth, the observed negative correlation between leaf expansion rate and fluctuating asymmetry in Dalechampia supports the second hypothesis. Interestingly, the effect size observed in this relationship is similar to the effect size observed by Valkama & Kozlov (2001) on leaves from mountain birch (Betula pubescens). Note, however, that Wilsey & Saloniemi (1999) did not find any relationship between leaf fluctuating asymmetry and shoot growth in mountain birches.

Leaf development is highly dependent on environmental factors such as light and water availability (Van Volkenburgh, 1999). Furthermore, Montalvo (1994) provided evidence that leaf expansion rate could reflect the fitness of the plant. Therefore, in principle, a negative relationship between fluctuating asymmetry and leaf expansion rate suggests that fluctuating asymmetry reflects individual fitness. But the small effect sizes observed in this study show that a relationship between fluctuating asymmetry and fitness must be very weak. Furthermore, in a study of the genetic basis of developmental stability in blossoms and leaves of D. scandens, we found no evidence of additive genetic variance for developmental stability, and we found also no consistent effect of inbreeding or outbreeding on the level of developmental stability (Pélabon et al., 2004a, 2004b). Therefore the positive correlation between leaf expansion rate and developmental stability is not expected to reflect individual genetic quality, but appears more likely to result from covariation in the effects of the environment on both growth rate and developmental stability. In conclusion, a component of fluctuating asymmetry appears to originate during early ontogeny via disturbance of cell proliferation in the leaf primordium. Although partially corrected by some regulating mechanisms, these asymmetries are not completely eliminated in expanding leaves. Whether regulation is achieved by compensatory growth or targeted growth remains unresolved, and requires additional study. The absence of a correlation in FA among leaves from the same plant suggests that there is no detectable variation in overall control of developmental stability at the plant level. Furthermore, the weak correlation in fluctuating asymmetry observed in successive leaves seems to result essentially from the sharing of common developmental noise, not from a common developmental stability at the shoot level. Finally, although a negative correlation was found between fluctuating asymmetry and expansion rate, the weakness of this correlation, and the apparent absence of genetic variation in fluctuating asymmetry observed in this species (Pélabon et al., 2004a), suggest that such correlation is unimportant for fitness and is purely phenotypic.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank L. Antonsen, L. Dalen and T. Berge for seed collection in the field, and G. Fyhn-Hanssen for glasshouse assistance. We also thank M. Kozlov and two anonymous referees who provided very valuable comments on an earlier draft of this manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Aparicio JM. 1998. Patterns of fluctuating asymmetry in developing primary feathers: a test of the compensational growth hypothesis. Proceedings of the Royal Society of London, B 265: 23532357.
  • Arendt JD. 1997. Adaptive intrinsic growth rates: an integration across taxa. Quarterly Review of Biology 72: 149177.
  • Calow P. 1982. Homeostasis and fitness. American Naturalist 120: 416419.
  • Clarke GM. 1998. The genetic basis of developmental stability. IV. Individual and population asymmetry parameters. Heredity 80: 553561.
  • Clarke GM. 2003. Developmental stability – fitness relationships in animals: some theoretical considerations. In: PolakM, ed. Developmental Instability: Causes and Consequences. Oxford, UK: Oxford University Press, 187195.
  • Crawley MJ. 2002. Statistical Computing: An Introduction to Data Analysis Using s-plus. New York, USA: John Wiley and Sons.
  • Debat V, David P. 2001. Mapping phenotypes: canalization, plasticity and developmental stability. Trends in Ecology and Evolution 16: 555561.
  • Emlen JM, Freeman DC, Graham JH. 1993. Nonlinear growth dynamics and the origin of fluctuating asymmetry. Genetica 89: 7796.
  • Fleming AJ. 2005. The control of leaf development. New Phytologist 166: 920.
  • Hansen TF, Armbruster WS, Antonsen L. 2000. Comparative analysis of character displacement and spatial adaptations as illustrated by the evolution of Dalechampia blossoms. American Naturalist 156: S17S34.
  • Hansen TF, Pélabon C, Armbruster WS, Carlson ML. 2003a. Evolvability and genetic constraint in Dalechampia blossoms: components of variance and measures of evolvability. Journal of Evolutionary Biology 16: 754766.
  • Hansen TF, Armbruster WS, Carlson ML, Pélabon C. 2003b. Evolvability and constraint in Dalechampia blossoms: genetic correlation and conditional evolvability. Journal of Experimental Zoology 296B: 2339.
  • Hashimoto T. 2002. Molecular genetic analysis of left–right handedness in plants. Philosophical Transactions of the Royal Society of London, B 357: 799808.
  • Kellner JR, Alford RA. 2003. The ontogeny of fluctuating asymmetry. American Naturalist 161: 931947.
  • Klingenberg CP. 2003. A developmental perspective on developmental instability: therory, models, and mechanisms. In: PolakM, ed. Developmental Instability: Causes and Consequences. Oxford, UK: Oxford University Press, 1434.
  • Klingenberg CP, Badyaev AV, Sowry SM, Beckwith NJ. 2001. Inferring developmental modularity from morphological integration: analysis of individual variation and asymmetry in bumblebee wings. American Naturalist 157: 1123.
  • Kozlov MV. 2003. Are fast growing birch leaves more asymmetrical? Oikos 101: 654658.
  • Leamy L. 1993. Morphological integration of fluctuating asymmetry in the mouse mandible. Genetica 89: 139153.
  • Lempa K, Martel J, Koricheva J, Haukioja E, Ossipov V, Ossipova S, Pihlaja K. 2000. Covariation of fluctuating asymmetry, herbivory and chemistry during birch leaf expansion. Oecologia 122: 354360.
  • Maksymowych R. 1973. Analysis of Leaf Development. Cambridge, UK: Cambridge University Press.
  • Martel J, Lempa K, Haukioja E. 1999. Effects of stress and rapid growth on fluctuating asymmetry and insect damage in birch leaves. Oikos 86: 208216.
  • Møller AP, Van Dongen S. 2003. Ontogeny of asymmetry and compensational growth in elm Ulmus glabra leaves under different environmental conditions. International Journal of Plant Sciences 164: 519526.
  • Montalvo AM. 1994. Inbreeding depression and maternal effects in Aquilegia caerulea, a partially selfing plant. Ecology 75: 23952409.
  • Nijhout HF, Davidowitz G. 2003. Developmental perspective on phenotypic variation, canalization, and fluctuating asymmetry. In: PolakM, ed. Developmental Instability: Causes and Consequences. Oxford, UK: Oxford University Press, 313.
  • Palmer AR. 1994. Fluctuating asymmetry analyses: a primer. In: MarkowA, ed. Development Instability: Its Origins and Evolutionary Implications. Dordrecht, the Netherlands: Kluwer Academic, 335364.
  • Palmer AR, Strobeck C. 1986. Fluctuating asymmetry: measurement, analysis, patterns. Annual Review of Ecology and Systematic 17: 391421.
  • Pélabon C, Carlson ML, Hansen TF, Armbruster WS. 2005. Effects of crossing distance on offspring fitness and developmental stability in Dalechampia scandens (Euphorbiaceae). American Journal of Botany 92: 842851.
  • Pélabon C, Hansen TF, Carlson ML, Armbruster WS. 2004a. Variational and genetic properties of developmental stability in Dalechampia scandens. Evolution 58: 504514.
  • Pélabon C, Carlson ML, Hansen TF, Yoccoz NG, Armbruster WS. 2004b. Consequences of inter-population crosses on developmental stability and canalization of floral traits in Dalechampia scandens (Euphorbiaceae). Journal of Evolutionary Biology 17: 1932.
  • Polak M, ed . 2003. Developmental Instability: Causes and Consequences. Oxford, UK: Oxford University Press.
  • Polak M, Møller AP, Gangestad SW, Kroeger DE, Manning JT, Thornhill R. 2003. Does an individual asymmetry parameter exist? A meta-analysis. In: PolakM, ed. Developmental Instability: Causes and Consequences. Oxford, UK: Oxford University Press, 8196.
  • Swaddle JP, Witter MS. 1997. On the ontogeny of developmental stability in a stabilized trait. Proceedings of the Royal Society of London, B 264: 329334.
  • Teather K. 1996. Patterns of growth and asymmetry in nestling Tree Swallows. Journal of Avian Biology 27: 302310.
  • Tracy M, Freeman DC, Duda JJ, Miglia KJ, Graham JH, Hough RA. 2003. Developmental instability: an appropriate indicator of plant fitness components?. In: PolakM, ed. Developmental Instability: Causes and Consequences. Oxford, UK: Oxford University Press, 196212.
  • Valkama J, Kozlov MV. 2001. Impact of climatic factors on the developmental stability of mountain birch growing in a contaminated area. Journal of Applied Ecology 38: 665673.
  • Van Dongen S, Sprengers E, Lofstedt C. 1999. Correlated development, organism-wide asymmetry and patterns of asymmetry in two moth species. Genetica 105: 8191.
  • Van Valen L. 1962. A study of fluctuating asymmetry. Evolution 16: 125142.
  • Van Volkenburgh E. 1999. Leaf expansion – an integrating plant behaviour. Plant, Cell & Environment 22: 14631473.
  • Venables WN, Ripley BD. 2002. Modern Applied Statistics with s-plus. Berlin, Germany: Springer-Verlag.
  • Waddington CH. 1957. The Strategy of the Genes. New York, USA: Macmillan.
  • Webster GL, Armbruster WS. 1991. A synopsis of the neotropical species of Dalechampia (Euphorbiaceae). Biological Journal of the Linnean Society 105: 137177.
  • Webster GL, Webster BD. 1972. The morphology and relationships of Dalechampia scandens (Euphorbiaceae). American Journal of Botany 59: 573586.
  • Wilsey BJ, Saloniemi I. 1999. Leaf fluctuating asymmetry in tree-line mountain birches, Betula pubescens ssp. tortuosa: genetic or environmentally influenced? Oikos 87: 341345.