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Plant Ecology 143: 51–66, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
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Tolerance assessment of Cistus ladanifer to serpentine soils by
developmental stability analysis
Concepcion L. Alados1 , Teresa Navarro2 & Baltasar Cabezudo2
1 Instituto Pirenaico de Ecologı́a, CSIC. Avda. Montañana, 177. Aptdo 202, 50080 Zaragoza, Spain; 2 Departmento
de Biologı́a Vegetal, Universidad de Málaga, Aptdo. 59. 29080 Málaga, Spain
Received 14 November 1997; accepted in revised form 28 December 1998
Key words: Cistus ladanifer, Developmental instability, Fluctuating asymmetry, Serpentine, Translational
asymmetry
Abstract
Developmental instability is the result of random environmental perturbations during development. Its absence
(developmental stability) depends on an organism’s ability to buffer environmental disturbances. Both genotype
and environment influence the phenotypic expression of developmental instability and it is susceptible to selection
pressure. We studied developmental instability (as indicated by increased within-individual asymmetry of repeated
traits) in vegetative and reproductive structures of three populations of Cistus ladanifer L. living in different soil
substrates (serpentine, siliceous and contact zone) to detect tolerance to serpentine soils. Serpentine soils, characterized by high concentrations of heavy metals (Ni, Cr, and Co), low levels of Ca/Mg ratio and high water deficit,
can adversely affect plant performance. In this study we demonstrated that asymmetry and within-plant variance
were higher in the contact zone population than either the silica or serpentine populations, proving the adaptation
of C. ladanifer to serpentine soils. Within-population estimates of developmental instability were concordant for
both vegetative and reproductive traits. There was little or no within-individual correlation among estimates of
developmental instability based on different structures, i.e., plants that had highly asymmetric leaves always had
high developmental instability in translational symmetry. Radial asymmetry of petals was negatively correlated
with petal size, especially in silica soil plants, providing evidence of selection for symmetric and large petals.
While leaf size was positively correlated with absolute fluctuating asymmetry, suggesting selection for small or
intermediate size leaves. Serpentine soils presented the largest foliar and floral traits, as well as shoot elongation,
while silica soil plants had the smallest scores. On the contrary, aboveground plant biomass was larger in silica soil
plants, while the contact zone plants had the lowest biomass.
Introduction
The phenotypic variation of a character has genetic,
environmental and random components. Developmental ‘noise’ or instability represents the cumulative
effects of small, random developmental perturbations
of environmental origin (Waddington 1957). It is reflected by exaggerated intra-individual variation in
repeated traits and patterns. Developmental stability, its opposite, is the ability of an organism to
buffer environmental disturbances encountered during
development (Mather 1953; Thoday 1955). Indeed,
developmental stability requires the suppression of
both genetic and environmental variation (Leary et al.
1992). An apparent confusion between developmental
instability and plasticity may appear in plants. Plasticity is the ability of an organism to alter its morphology
in response to changes in environmental conditions,
that is, it is constant in sign (Bradshaw 1965), and
it is controlled by a different genetic system than developmental stability, as demonstrated in Arabidopsis
thaliana (L.) Heynh. by Bagchi & Iyama (1983).
In principle, any phenotypic trait can be used
to measure developmental instability, provided one
knows a priori what phenotype should be produced
in the absence of stress. Bilateral symmetry is one
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such developmentally invariant trait. Both sides of a
bilateral symmetrical character are assumed to be controlled by the same genes. Random deviations from
bilateral symmetry, i.e., fluctuating asymmetry (Sumner & Huestis 1921; Ludwig 1932; Van Valen 1962;
Palmer & Strobeck 1986), reflects the inability of
an organism to control environmental perturbations
during development, and increases under genetic or
environmental stress (Mather 1953; Thoday 1958;
Valentine & Soulé 1973; Parsons 1992). Developmental instability has the advantage that as a nonspecific
measure of developmental disturbances, it can be used
as an early sign of anthropogenic impacts on animals and plants (Zakharov et al. 1987; Parsons 1992;
Graham et al. 1993; Freeman et al. 1993). Moreover, fluctuating asymmetry has an optimum reference
value (zero), while other indices of phenotypic quality
such as body size do not (Watson & Thornhill 1994).
Not all developmental instability characters are
equally sensitive. Stability will depend on the ability
of the organism to buffer development against environmental perturbations (degree of canalization according
to Waddington 1942) and characters not directly related to individual fitness are expected to display low
developmental stability compared with characters important to fitness (Leary et al. 1985; Clarke 1995).
In order for developmental instability to be consistently useful in environmental diagnosis, we must perform an integrated analysis of several phenotypic traits
(Watson & Thornhill 1994; Swaddle 1997). Indeed,
complex traits whose structure depends upon the coordination of several mechanisms have higher potential
for integration, and thus buffering. Therefore, they
may more accurately represent overall instabilities.
Graham et al. (1993) and Freeman et al. (1993)
have argued that bilateral symmetry is not the only
useful within-individual developmentally invariant
trait to detect developmental stability in organisms.
For example, radial, spiral and translational (longitudinal) symmetries are within-plant measures of variation
(Paxman 1956; Graham 1992; Graham et al. 1993;
Freeman et al. 1993; Alados et al. 1994, 1998; Escós et al. 1995, 1997, Sherry & Lord 1996a) and
have proven reliable and often superior alternatives to
bilateral symmetry. Many plants present radial symmetry in flowers, leaves or fruits and, their within-trait
variance may be used as a manifestation of radial
asymmetry (Graham et al. 1993). The modular nature
of plants allows the examination of acropetal growth,
where, for example, internode length varies regularly
with node order, and represents a form of transla-
tional symmetry with scale (Freeman et al. 1993), and
node order represents the scale of measurement. That
is, they are invariant with multiplicative changes of
scale (Schroeder 1991). The phenotypic expression of
developmental instability reflects the inability of the
organism to buffer environmental disturbances during
development. The genetic basis of developmental stability is still under discussion (Clarke 1993; Markow
& Clarke 1997; Møller & Thornhill 1997). Several
authors have demonstrated variation in the phenotypic
expression of developmental instability between lines
and populations (Sakai & Shimamoto 1965, in Nicotiana tabacum L.; Bagchi et al. 1989, in Tectona grandis L; Barrett & Harder, 1992, in genus Eichhornia
Kth.). Maynard Smith et al. (1985) hypothesized that
recently evolving phenotypes display more instability
than well-established ones. Later studies presented evidence to support this theory in the study of hybrid
populations (Graham 1992; Freeman et al. 1995), in
the evolution of pesticide resistance (Clarke 1993),
and in gynodioecious species (Alados et al. 1998).
Serpentine areas are important sources of metals,
especially nickel (Ni), chromium (Cr) and cobalt (Co).
The adverse effect of heavy metal is enhanced by the
low levels of calcium (Ca) in relation to magnesium
(Mg), the lack of organic matter and the poor physical texture of the soil. This soil structure restricts
soil depth, reducing soil penetration and water content and contributing to water stress in plants. As a
result, serpentine areas have several endemic species
adapted to high concentrations of heavy metals and
generally adverse edaphic conditions (Proctor 1971;
Brooks 1987; Proctor & Woodell 1975; Roberts &
Proctor 1992; Arianoutsou et al. 1993, Freitas &
Mooney 1996). Serpentine soil provides a distinct environment, which has persisted for millennia, allowing
a specialized and characteristic flora to evolve. It is
postulated that directional selection reduces developmental homeostasis during the evolutionary response
(Maynard Smith et al. 1985; Watson & Thornhill
1994). That is, recently evolving phenotypes display
more instability (Levin 1970; Maynard Smith et al.
1985) than well established ones, but, when the rate
of evolution stabilizes, readaptation of the regulatory mechanisms maintain the trait at a new state. If
adaptation to serpentine soil takes place, we expect
plants living in serpentine substrate to be developmentally more stable than plants living in the contact
zone. Indeed, silica plants should also display developmental stability. However a narrow contact zone
should be less developmentally stable because col-
53
onizing plants can come from either serpentine or
silica adapted populations, producing a preponderant
number of non-adapted individuals.
In this study we used not only fluctuating asymmetry, but also radial asymmetry, translational symmetry, and statistical noise in allometric relations to
detect random intra-individual variability during development (Freeman et al. 1993; Alados et al 1994,
1998; Escós et al. 1995, 1997). Not only we did look
at homeostasis disruption, but also at trait size and
biomass in Cistus ladanifer (Cistaceae). We wanted
to know whether adaptation to serpentine soils had occurred so that we could select stress tolerant ecotypes
to use in future restoration of contaminated soils.
Methods
Study area and species
C. ladanifer is an important component of Mediterranean matorral scrub, distributed in SW Europe
(France, Spain and Portugal) and N Africa (Morocco
and Algeria). It is a calcifuga species that grows
on various soil and rock types, with preference for
siliceous and ultrabasic soils.
The study area is located in Sierra Parda de Tolox
(Málaga province), in the southwest of the Iberian
Peninsula (latitude 36◦380 N, longitude 4◦ 560 E) with
an area of 30 km 2 . This area has serpentine soils
(ultrabasic rocks of plutonic origin with serpentines
and peridotites), siliceous soils (schists, gneises and
pizarras), and contact zones (around 5 m wide) with
materials from both substrates. The climate is Mediterranean with an average annual rainfall between 800
and 1000 cc. The mean annual temperature ranges between 10 to 15 ◦ C, but in summer mean temperatures
can reach 25 ◦ C. The plant community is Mediterranean degraded matorral scrub dominated by Cistus
ladanifer, an erect evergreen shrub, 275–290 cm high.
In this area C. ladanifer forms extensive and densely
aggregated populations with isolated individuals scattered in the periphery of the clump. The nomenclature
of taxa and syntaxa used follows Muñoz Garmendia &
Navarro (1993).
The pheno-morphological behavior of C. ladanifer
(Cabezudo et al. 1992; Talavera et al. 1993) is characterized by seasonal vegetative growth at the apex of
the last season’s shoot, or from shorter basal axillary
brachyblasts, between April and September. Flowerbuds develop in the axils of the upper leaves of the last
season’s dolicoblasts between the first-half of April to
the beginning of May and flowering occurs from the
end of April to mid May.
Data collection
We selected three locations with similar sclerophilous
Mediterranean vegetation communities, slope exposure, inclination, altitude and general appearance,
differing only in soil characteristics (Table 1). The first
area was located on serpentine soils, the second in
siliceous substrate, and the third on the contact zone.
Soil samples were collected from the three locations,
air dried and sieved with a 2 mm screen. Concentrations of exchangeable ions in soils were determined
with extracts of 1 M ammonium acetate (soil: extractant ratio of 1:10 for 1 h). In order to evaluate the
plant absorption response to the different soil concentrations we analyzed ions (Ca2+ and Mg2+ ) and Ni2+
concentrations in C. ladanifer leaves. For this, leaves
were oven dried at 60 ◦ C for 48 h and wet ashed with
2 mL of concentrated HNO3 in Teflon pressure vessels for 7 h. Concentrations of Ca2+ and Mg2+ were
determined by flame AAS (Perkin-Elmer 380) and
concentrations of Ni2+ were determined by graphite
furnaces AAS (Perkin-Elmer 5100).
C. ladanifer densities were similar in the three areas: 2.1 ind m−2 in serpentine soils and 2.7 ind m−2 in
the silica and contact zones. A total of 120 C. ladanifer
branches were collected, 40 per plot, at the end of
July 1996, when shoots stopped growing and annual
shoot lignification began. In order to separate the soil
types from other non-controlled ecological variables
we performed four transects per soil type, collecting
10 plants per transect. We chose plants of similar size
to reduce variability in the analysis. We performed a
randomized branch sampling, by randomly selecting
one well-developed 3-year-old branch, from a specific
orientation. Later, in the laboratory, we measured internode lengths from one developed annual shoot per
branch. We also selected six leaves located in the same
position (7th or 8th node counting from the base of the
annual shoot, corresponding to L7 and L8 of Figure 1)
from six different shoots per plant.
Because flowers develop on the previous year’s
stem, before the current shoot finishes elongation, we
collected flowers at the beginning of June, from different individuals than those we took leaves and branches
from. Six flowers per plant were selected from 96 individuals, 32 per soil type and 8 plants per transect.
Floral and leaf asymmetries, internode lengths, and
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Table 1. Concentrations of Ni2+ , Mg2+ , Ca2+ and Ca/Mg ratio Cistus ladanifer leaves in serpentine, siliceous and contact soils. Exchangeable concentrations in soils were obtained from 1 M ammonium acetate soil extracts. (Mean
± sd, n), n.d. not detected. Means with the same letters indicate no significant
differences at the 0.05 level, Bonferroni test.
Serpentine
Contact
Siliceous
1.19 ± 0.01 (2)
0.11 ± 0.02 (3)
3.53 ± 0.08 (3)
0.03 ± 0.01 (3)
0.15 ± 0.01 (2)
0.33 ± 0.02 (2)
0.84 ± 0.06 (2)
0.40 ± 0.06 (2)
n.d.(2)
0.39 ± 0.00 (2)
0.20 ± 0.08 (2)
2.05 ± 0.08 (2)
10.10 ± 2.01 (5)a
4.85 ± 1.31 (9)a
3.24 ± 0.78 (9)a
1.63 ± 0.72 (9)a
7.92 ± 0.83 (4)a
2.43 ± 0.82 (8)b
1.72 ± 0.58 (8)b
1.54 ± 0.70 (8)a
3.75 ± 2.00 (5)b
4.08 ± 0.53 (6)a
2.48 ± 0.44 (6)ab
1.70 ± 0.44 (6)a
Soil
Ni2+ µg/g
Ca2+ mg/g
Mg2+ mg/g
Ca/Mg
Leaves
Ni2+ µg/g
Ca2+ mg/g
Mg2+ mg/g
Ca/Mg
floral and leaf allometries were all examined to detect consistency in developmental stability. To reduce
measurement error, measurements were performed in
the laboratory by the same person, with an electronic
caliper recording to the nearest 0.01 mm. Additionally,
dried plant biomass was estimated for one 3-year-old
branch from 36 different plants, 12 from each soil
type. Plants were oven dried at 70 ◦ C for 48 h and
weighed with a precision balance.
Data measurement and analysis
(a) Fluctuating asymmetry
The traditional way to assess developmental instability is via fluctuating asymmetry. The leaves are
40–120×10–21 mm in size and are malacophyllous,
sessile or subsessile with linear-lanceolate shape, and
with 14–16 months old. We examined the asymmetry
of leaves by measuring the width on each side of the
midrib at the mid point of the leaf blade. Absolute
fluctuating asymmetry (AFA) was calculated as the
unsigned left (L) minus right (R) difference. Relative
fluctuating asymmetry (RFA) was calculated as the absolute value of left (L) minus right (R) divided by the
average (L + R)/2, correspond to index 2 of Palmer &
Strobeck (1986). This scaling procedure has been used
by many authors to correct for possible associations
between asymmetry and leaf size. The validity of fluctuating asymmetry interpretation depends, according
to Palmer & Strobeck (1986, 1992), on an absence
of directional asymmetry (skew), anti-symmetry (bimodality or platykurtosis), and a normal distribution
for (L − R) with mean zero.
(b) Translational symmetry
In addition to examining fluctuating asymmetry, we
also examined the allometric relationship between the
internode lengths and the node order (Alados et al.
1994, 1998; Escós et al. 1995, 1997). Its organization patterns and architectural diagrams are shown in
Figure 1. C. ladanifer is a phanerophyte species with
acropetal branch-shedding, and with two branch types:
glabrous long shoots (dolicoblasts) and glabrous and
very viscid short shoots (brachyblasts). The scaling
relationship has previously been applied to plant developmental stability (Paxman 1956; Freeman et al.
1993; Sherry & Lord 1996a) although none of these
earlier studies examined internode lengths per se. The
relationship between internode length and node order
follow a self–similar (or self-affine) sequence, invariant with multiplicative changes of scale (Schroeder
1991), where internode order (counted from the stem
base) is interpreted as a scaling factor (Freeman et al.
1993). The relation between internode length (L) and
node order (N), starting from the shoot base to its apex,
fits the general equation.
L = kN a e−bN ,
(1)
where e is the natural base, and k, a, and b are fitted
constants.
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instability (a decline in the accuracy of the curve fitting). Thus, one minus the coefficient of determination
R 2 , the standard error of the regression (Syx ), and the
standard error of the parameters a and b (Sa and Sb ),
are good estimators of developmental instability or
homeostasis disruption. Regressions were performed
separately for each plant and the resulting R 2 , Syx,
and, Sa or Sb were then analyzed. Because the number of internodes varies among shoots, we used R 2
adjusted to the mean square sum.
Figure 1. (a) Organization pattern and architectural diagram of
C. ladanifer (according to Scarrone and Leeuwenberg models) corresponding to a three year old branched system: current season shoot
(I), former season shoots (II, III, IV), sylleptic dolicoblasts (sd),
single flowers from last season shoot (f). (b) Architectural unit of
C. ladanifer: axillary buds for the next flower formation (d), scale
node from current season shoot (s). L1 to L10 are internode length.
This equation has two components. The first component (kN a ) corresponds to the allometric relationship between internode length and node order (Escós
et al. 1997). The second component (e−bN ) represents
the inhibition mechanism of flower formation (Meinhardt 1984). The parameter values of this equation do
not have intrinsic biological meaning. It is simply a
convenient function to express individual differences.
Taking the logarithm of both sides of the equation
gives:
ln L = ln k + a ln N − b N
(2)
The constants can be obtained from a regression
analysis. Under stress, the impact of random perturbations increases, leading to enhanced developmental
(c) Radial symmetry
The flower is actinomorphic and solitary and among
the largest in the Spanish flora (Talavera et al. 1993).
The single flowers are flat, with radial symmetry
in five planes of reflectional symmetry, and with a
dish-like appearance during pollination (Faegri & Pijl
1971). Flowers are white and shortly pedicellated,
with five petals of 38–46 mm size, and basal bracts.
The calyx bears 3 sepals 13–19 mm long. The variance
in petal or sepal length affects the radial symmetry of
the flower. We used the standard deviation of petal and
sepal length as a measure of developmental instability
instead of the coefficient of variation, since the latter
measure declines with trait size when developmental
instability is independent of trait size (Soulé 1982).
Another way of estimating radial asymmetry in
floral traits is by performing a nested ANOVA with
trait size nested within flowers, flowers nested within
individuals, and individuals nested within transects.
The nested analysis yielded estimates of variance due
to differences between floral traits (petals or sepals
length or width) within flowers, or radial asymmetry.
(d) Allometric error
Leaf or petal length (L) and width (W) is related by a
power law, as also is calix allometry (see details in:
Alados et al. 1998).
La
_ W b.
Thus, ln L and ln W are linearly related, and we can
write
β1 ln L + β2 ln W = 1 + ,
where is an error term. From the same plant structure
(leaf, petal or sepal) we measured n repeated units to
obtain the matrix
x11 x12 . . . x1n
X=
x21 x22 . . . x2n
where row 1 is length and row 2 is width.
56
In matrix form,
+ βX = 1.
Allometry error is calculated as: 0 /n − 1
AL = (n − β X 10 )/n − 1
being β transposed equal to
β 0 = (XX0 )−1 X10 .
Statistical analysis
Measurement error was estimated by re-measuring
the same plant after all the measurements had been
taken once. Repeatability of morphological measures
of the same individuals was estimated by a two way
nested ANOVA where leaf side petals or sepals were
nested within individuals. Individual was the random
effect factor and within error variance, the measurement error (Sokal & Rohlf 1980; Merilä & Björklund,
1995). Measurement error was estimated from 30 individuals measured twice and then calculated as the
proportion of within-measurement MS (mean square)
to the among-individual MS (fraction of trait size variation due to measurement error) and, to the individual
within-trait MS (fraction of developmental instability variation due to measurement error) (Appendix1).
Measurement error in foliar and floral structures was
insignificant; values ranged between 0.002 for half
leaf width and 0.04 for petal length and width (Appendix 1). The proportion of variance in organ length
or width due to measurement error ranged between
0.56% for sepal width to 0.02% of petal width. Finally,
the proportion of within individual variation (developmental instability) due to measurement error was close
to 1% for the less precise measurements of sepal width
and half leaf width.
Fluctuating asymmetry refers to randomly distributed differences between right and left sides in
bilateral characters. Tests for fluctuating asymmetry
depend on an absence of directional asymmetry (systematic biases towards greater development of one
side, i.e., skewness), antisymmetry (systematic nondirectional deviations from bilateral symmetry, i.e.,
bimodality or platykurtosis) and, according to Palmer
& Strobeck (1986) a normal distribution of the unsigned difference. To detect confounding factors, a
series of preliminary tests were performed. We first
did a mixed model ANOVA with sides and soil type
as fixed effect factors and three level nested analysis,
with transect nested in soil, individuals nested in transect, and leaves nested within individuals as an error
term. Side effects represented the directional asymmetry, soil effects represented size and shape variations,
and the interaction term represented the antisymmetry.
Inter-individual variation was represented by the individual within transect term. The error term referred
to the intra-individual variation between leaves in a
plant and represents the overall intra-individual variation in half leaf width including not only AFA but also
interleaf variability. Estimating the variance components (after Sokal & Rohlf 1981) completed the nested
model ANOVA.
Normality tests were then done on the distribution of signed (L − R) differences to test skewness
and kurtosis. Correlations between the magnitude of
AFA (L − R) and character size (L + R)/2 were also
investigated to prevent size scaling.
Variation in fluctuating asymmetry due to soil type
was investigated by three levels nested analysis of
variance with soil type as the fixed effect factor, transect nested in soil and individuals nested in transect.
The error term represented the among-leaf withinindividual variance. In the same way we compared
floral trait size and within flower variance, where
the error term represented the among-flower withinindividual variance. Within trait variance was also calculated from the error term of three nested ANOVAs,
one per soil type, where trait sizes were nested within
flowers, flowers nested within individuals, and individuals nested in transects.
A two-level nested analysis of covariance, with
transects nested within soil types and, the naperian
logarithm of the base stem diameter as covariate, was
performed to measure the effect of soil type on curve
fitting estimators: R 2 , Syx , Sa and, Sb ; and on the
regression parameters: a, b and ln k.
Comparisons between soil types were assessed by
a two-tailed F test with sequential Bonferroni correction on the P values, to protect against group-wide
type errors associated with multiple tests (Rice 1989).
When intermediate level mean squares were not significant we pooled the implicated source of variation
and calculated F values from pooled mean squares.
All variables were tested for normality. When
the variables were not normally distributed we transformed the data as suggested by Sokal & Rholf (1981).
57
Table 2. Partitioning analysis of variance with two fixed effect factors
(sides and soil type) and three level nested analysis, with transect nested within soil (T ⊂ S), individuals nested within transect
(I ⊂ T ), and leaves nested within individuals as the error term. Dependent variable is half leaf width. ∗∗∗ P < 0.001, ∗∗ P < 0.01,
∗ P < 0.05, no asterisks indicates P > 0.05, s 2 is the variance
components.
Source
MS
df
F
s2
0.22
544.24
0.19
3.68
4.53
0.49
(1, 1202)
(2, 1202)
(2, 1200)
(18, 216)
(216, 1200)
1200
0.44
1110.1∗∗∗
0.39
0.81
9.23∗
0.0%
79.2%
0.2%
0.2%
11.8%
8.6%
Half leaf width
Sides
Soil (S)
Sides × Soil
(T ⊂ S)
(I ⊂ T )
Error
Table 3. Partitioning analysis of variance with the three
level nested analysis, with soil as fixed effect, transept
nested in soil (T ⊂ S), individuals nested within transept
(I ⊂ T ) and, leaves nested within individuals as the error term. Dependent variable is AFA. ∗∗∗ P < 0.001,
∗∗ P < 0.01, ∗ P < 0.05, no asterisks indicates P > 0.05,
s 2 is the variance components.
Source
MS
df
F
s2
17.87∗∗∗
1.54
1.24∗
7.4%
1.2%
3.6%
87.8%
Absolute fluctuating asymmetry
Soil (S)
(T ⊂ S)
(I ⊂ T )
Error
1.677
0.178
0.115
0.092
(2, 9)
(9, 708)
(108, 600)
600
Results
(a) Fluctuating asymmetry
Signed (L − R) averaged for all leaf data (n =
720) gave a mean ± se of −0.024 ± 0.02, which
is not significantly different from zero (t-test =1.33,
NS). The skewness of the distribution was −0.029
(t-test=0.32 NS) and the kurtosis 0.404 (t-test=2.21,
P < 0.05). That is, the distribution was leptokurtic.
The mixed model ANOVA which held the side of
the leaf and soil type as fixed effects (Table 2) showed
that leaf side explained an insignificant amount of the
total variance, indicating an absence of directional
asymmetry, i.e. skewness. Thus, one side of the leaf
was not consistently larger than the opposite. Leaf
side with soil interaction, which represents the antisymmetry, was not significant. Soil type significantly
affected the half leaf width, which represents the size
and shape variation in leaves, and explained 79.2%
of the variance. The transect within soil nested factor was also not significant, indicating no transect
effect. The individual within transect term was significant due to the great inter-individual variability.
Finally, intra-individual variation, represented by the
error term, absorbed 8.6% of the explained variance,
and was responsible for the non-directional asymmetry plus the intra-individual variance in half leaf width.
That is, this term included the variation due to fluctuating asymmetry plus the variation between leaves
within individual. Size scaling appeared unnecessary
as the correlation between the magnitude of absolute
fluctuating asymmetry and character size is very low,
although significant (r = 0.157, n = 720, P <
0.05). Multiple analysis of variance with absolute
fluctuating asymmetry and average half leaf width as
dependent variables was significant (Wilks’ lambda =
0.03, F2,707 = 13960.8, P < 0.001), with a significant effect of soil type (Wilks’ lambda = 0.57,
F4,1414 = 117.3, P < 0.001). Posterior analyses were
performed separately for each variable.
In order to determine the effect of soil type on absolute (L − R) fluctuating asymmetry of leaves we
performed a three levels nested analysis of variance
with soil type as a fixed effect factor. The partitioning nested analysis of variance presented in Table 3
revealed that soil type accounts for 7.4% of variance, significantly affecting the absolute fluctuating
asymmetry (AFA) of C. ladanifer leaves. Average absolute fluctuating asymmetry increased significantly
for plants living in contact and in serpentine areas
in comparison with plants living in the silica zone
(Table 4).
Relative fluctuating asymmetry (RFA) was analyzed by the same procedure. Average relative fluctuating asymmetry increased significantly for plants
living in the contact zone (P < 0.05, Bonferroni
adjustment), although the overall difference was not
significant (F2,9 = 1.91, NS). The decline in significance of relative fluctuating asymmetry in comparison
with absolute fluctuating asymmetry was due to the
relationship between soil type and half leaf width. Half
leaf width in silica soil was smaller than in serpentine or in contact areas (Table 4, F2,717 = 252.46,
P < 0.001). Therefore, once asymmetry was adjusted
for leaf size, differences among soil types declined.
58
Table 4. Means ± se (n) of absolute fluctuating asymmetry (AFA), relative fluctuating
asymmetry (RFA), half leaf width (HLW), and leaf allometry error (ALH) for C. ladanifer inhabiting silica, serpentine and contact zones. Means with the same letters indicate no
significant differences at the 0.05 level, Bonferroni test.
Trait
AFA
RFA
HLW
ALH
Silica
Contact
Serpentines
0.29 ± 0.02 (240)a
0.46 ± 0.02 (240)b
0.06 ± 0.03 (240)a
0.07 ± 0.03 (240)b
10.20 ± 0.03 (240)a
0.0044 ± 0.0007 (40)a
13.55 ± 0.03 (240)b
0.0038 ± 0.0007 (40)a
0.40 ± 0.02 (240)b
0.06 ± 0.03 (240)a
14.16 ± 0.03 (240)c
0.0037 ± 0.0007 (40)a
Table 5. Means ± se (n) and F values of the two level nested analysis of covariance, with transect
nested in treatments and naperian logarithm of the base stem diameter as covariate. Dependent
variables are the scale asymmetry parameters of the relation between internode length and node
2 ), and equation parameters a, b and ln k, are from the leaf arrangement
order (Syx , Sa , Sb and Radj
equation L = k · N a · e−bN . ∗∗∗ P < 0.001, ∗∗ P < 0.01, ∗ P < 0.05, no asterisks indicates
P > 0.05. Means with the same letters show no significant differences at the 0.05 level, Bonferroni
test.
Syx
Sa
Sb
2
Radj
a
-b
ln k
Silica
Contact
Serpentine
F(2, 116)
0.678±0.035 (40)ab
0.854±0.043 (40)a
0.183±0.014 (40)a
0.462±0.040 (40)ab
1.613±0.242 (40)a
−0.247±0.085 (40)a
1.388±0.193 (40)a
0.790±0.033 (40)a
1.188±0.040 (40)b
0.296±0.013 (40)b
0.382±0.038 (40)a
2.395±0.226 (40)ab
−0.531±0.079 (40)b
1.763±0.180 (40)a
0.567±0.035 (40)b
1.011±0.042 (40)c
0.288±0.014 (40)b
0.582±0.040 (40) b
2.615±0.238 (40) b
−0.819±0.084 (40)c
3.156±0.190 (40)b
10.64∗∗∗
15.33∗∗∗
19.12∗∗∗
6.39∗∗
4.24∗
10.10∗∗∗
22.20∗∗∗
(b) Translational symmetry
The relation between internode length (L) and node order (N) is given by L = 8.18 N 2,21 e−0.53N , averaged
over all individuals, and represents the translational
symmetry of internode length. The parameter values
of the relationships between internode length and node
order are presented in Table 5, for each soil type separately. The intercept of Equation (1) represents the
starting conditions in the relationship between internode length and node order. A larger intercept indicates
larger internode length at the shoot base. In this study
we observed that the intercept changed significantly
within the soil substrate. Plants living in serpentine
soils presented the larger intercept. Parameter a represents the rate of internode enlargement with node
order, which changes significantly with soil type and
increases in the serpentine zone. Plants living in silica
soil show a lower a, significantly different from plants
living in serpentines. The parameter b, representing
the inhibition mechanism of growth patterns due to
flower formation, describes internode shrinking as we
go up the stem. In consequence the larger the absolute
value of b, the faster internode shortening occurs. The
parameter b varied significantly between soil types,
reaching its lowest value in serpentinophytes, and the
highest in siliceous substrata plants (Table 5). A clear
picture of the morphological aspect that produces the
variations in the parameter values averaged per soil
type is presented in Figure 2. This figure shows that
shoots produced by plants living in silica grow slower
during the first internodes, but as node order increases,
the decline in internode length with order is less steep
than in serpentinophytes, where it drops drastically
after a few internodes. The number of internodes per
annual shoot varied significantly between populations
(F2,117 = 6.16, P < 0.01). The silica population
presented more internodes (11.1 ± 0.4, n = 40) than
serpentinophytes (9.2 ± 0.4, n = 40), while the contact population was intermediate (9.9 ± 0.4, n = 40).
In spite of this, the total length achieved at the end of
the growth period was significantly larger in serpentine plants than in either silica or contact zone plants
59
and Sb , respectively, indicating that variables are normally distributed. The partitioning nested analysis of
variance revealed that soil type explains 9.8% of the
variance in R 2 . Soil differences accounted for 17.1%,
18.6% and 16.7% of variance in Syx , Sa and Sb , respectively. The results, presented in Table 5, show a
lower value for R 2 , and a higher Syx , indicating lower
developmental stability, in the contact zone. The best
fit was observed in the serpentine substrate. Sa and
Sb also were the worst fit for plants from the contact
substrate. Comparisons between soil types were significantly different. There were no differences among
the transects within soil types. Comparisons between
soil types were assessed using a Bonferroni correction. Above ground biomass of 3 year-old branches
varied significantly between soil types (F2,33 = 3.22,
P = 0.05). Contact zone plants had lower biomass
(29.47 ± 2.4 g, n = 12) than serpentine plants (33.50
± 2.4 g, n = 12), while C. ladanifer inhabiting
siliceous soil presented the largest biomass (38.30 ±
2.4 g, n = 12).
Figure 2. Scatterplots of the non-linear, best-fit equation
L=4.01N 1.61 e−0.25N for plants living in silica, L=5.83N 2.39
e−0.53N from transition and, L=23.48N 2.61 e−0.82N from
serpentine soil. L is internode length and N is node order. Equation
parameters were obtained by least square regression analyses of the
linearized equation (ln L = ln k + a ln N − bN ).
(P < 0.001 for Bonferroni test, F2,117 = 31.13,
P < 0.001). Average ± se shoot length in cm. over
all the shoots measured was 26.85 ± 0.57 (40) for
serpentinophytes, 21.21 ± 0.57 (40) for silica substrate plants, and 21.45 ± 0.57 (40) in the contact zone
plants.
To determine how soil type affects developmental
stability of internode length, we performed a nested
analysis of covariance of the adjusted coefficient of determination, R 2 , standard error of the regression, Syx ,
standard error of the parameter a, Sa , and standard
error of the parameter b, Sb , as dependent variables. A
Kolmogorov-Smirnov one sample test for the normal
distribution gave a maximum difference of D = 0.08,
D = 0.03, D = 0.06, D = 0.07, lower than the
critical value 0.094 for P < 0.01, for R 2 , Syx , Sa ,
(c) Radial symmetry
Average petal and sepal length and width were
normally distributed (maximal difference of the
Kolmogorov–Smirnov normality test are: 0.04, 0.03,
0.06, 0.03 respectively; P < 0.01). Table 6 shows
the variation of floral trait size with soil characteristics. Results of the three level nested analysis of
variance show that petal and sepal length (F2,9 =
19.33; F2,489 = 227.98) and petal and sepal width
(F2,9 = 38.50; and F2,489 = 330.84), are significantly
larger in serpentine soils (P < 0.001) than in silica or
contact soils.
Before we performed the analysis of variance, we
tested for normality of floral symmetry variables. Results of the Kolmogorov–Smirnov normality test gave
a maximal difference of D = 0.09, for standard deviation of petal length (SDLP); D = 0.10, for standard
deviation of petal width (SDWP); D = 0.11, for standard deviation of sepal length (SDLS), D = 0.07
for standard deviation of sepal width (SDWS). Because each variable was larger than the critical value
D = 0.043, for P < 0.01, we log transformed the
data to meet the assumptions of analysis of variance.
Correlation analyses between floral asymmetry
and trait size revealed that this relationship changed
depending on whether all the flowers are analyzed together or were separated by soil type. While a low
negative correlation was found between petal length
and the standard deviation of petal length (r = −0.16)
60
Table 6. Means ± sd (n) of average floral trait per flower, and floral asymmetry. MS represents mean squares of the nested analysis of
variance for the within flower variation term. Means with different letters indicate significant difference at the 0.05 level, sequential
Bonferroni test; g1 and g2 are the skewness and kurtosis statistics. ∗ Indicates the statistic is significantly different from 0 at the 0.05
level.
Trait
Silica
Mean ± sd (n)
g1
Av. petal length 33.79±3.71(192)a −0.41∗
Av. petal with
28.95±3.16(192)a −0.30∗
Av. sepal length 10.02±0.96(192)a −0.12
Av. sepal with
8.11±0.81(192)a
0.40∗
0.97∗
Sd. petal length 1.44±0.79(192)a
a
Sd. petal with
1.90±0.99(192)
1.32∗
a
Sd sepal length
0.55±0.35(192)
2.20∗
a
Sd. sepal with
0.84±0.47(192)
1.01∗
a
MS petal length 2.75
MS petal with
4.60a
MS sepal length 0.43a
MS sepal with
0.92a
g2
Contact
Mean ± sd (n)
g1
g2
0.35 38.44±4.64(192)b
0.54∗ −0.02
b
0.35 35.07±4.70(192) −0.15 −0.26
−0.57 11.17±0.85(192)b −0.09 −0.35
−0.36 9.17±1.04(192)b
0.65∗
1.19∗
0.70∗
1.84±0.96(192)b
1.31∗
3.58∗
∗
b
∗
1.75
2.67±1.43(192)
1.51
4.28∗
∗
b
∗
9.88
0.71±0.43(192)
1.08
1.32∗
∗
a
∗
1.05
0.85±0.48(192)
0.74
0.52
4.37b
9.20b
0.68b
0.95a
for all the 576 flowers together. Separated analyses
showed that plants living in silica soil presented a correlation coefficient: r = −0.31, P < 0.001, n = 192;
while, serpentinophytes presented no such correlation
(r = −0.05, NS), and contact soil plants presented
intermediate values (r = −0.18, P < 0.05).
Analyses of floral asymmetry, measured by the
standard deviation of petal or sepal length or width,
are presented in Table 7. Multiple analysis of variance including all the floral asymmetry variables and
average floral traits revealed a significant effect of
soil type (Wilks’ lambda = 0.34, F20,1128 = 78.62,
P < 0.001). Results of the analysis are presented for
average floral traits and floral asymmetry separately.
The percent of the variance explained by soil type
effects ranged from 10% in petal length, to 0% in sepal
width. No significant differences in transects nested
within soil types were observed. In consequence, treatment effects were tested over the error term plus
the variance components due to the interaction term.
Within flower variance was significantly influenced by
soil type for petal and sepal length and petal width.
Variation among flowers within individuals was lower
than variation among individuals within transects (see
MSE vs MSI ⊂T in Table 7). Mean and se values
of floral asymmetry are presented in Table 6. Floral
asymmetry measured by standard deviation of floral
traits or by mean square of the nested ANOVA for
the within flower variation term give the same results.
Plants living in the contact zone had higher develop-
Serpentine
Mean ± sd (n)
41.83±3.93(192)c
40.65±4.88(192)c
11.42±1.11(192)c
10.01±0.91(192)c
1.29±0.78(192)a
2.11±1.08(192)a
0.77±0.54(192)b
0.90±0.48(192)a
2.28a
5.61a
0.87c
1.04a
g1
g2
0.15 −0.23
0.25 −0.02
0.43∗
1.22∗
0.14 −0.25
1.65∗
3.55∗
∗
1.00
0.86∗
∗
2.19
8.31∗
∗
0.68
0.30
mental instability in petal length and width than those
from silica and serpentine zones. Sepals behaved more
like leaves, tending towards increasing instability in
serpentinophytes, even presenting higher sepal length
asymmetry there than in the contact zone. Sepal width
asymmetry was similar in the three populations. No
significant difference between transects nested within
treatments appeared in any case.
(d) Allometric error
Errors in the allometric relationship among length and
width of leaves, petals and sepals were not normally
distributed (Kolmogorov–Smirnov test D = 0.18,
0.14, P < 0.01 for ALP and ALS, respectively).
Consequently, a logarithmic transformation was applied. No significant effect of soil type was observed
on the naperian logarithm of leaf allometric error
(F2,107 = 0.02, NS). The partitioning analysis of
variance revealed that petal allometric error, which
explained 4.7% of the variance, varied significantly
with soil type. That is, plants in the contact zone
presented a significantly higher petal allometric error
(1.6 × 10−4 ± 0.1 × 10−4 ) than those in serpentine
(0.6 × 10−4 ± 0.1 × 10−4 ). Sepal allometric error explained only 0.8% and did not present any significant
variation.
A Pearson correlation matrix of comparisons
among developmental instability measurements (Table 8) is presented for floral structures separately from
61
Table 7. Partitioning analysis of variance of the three level nested analysis, with transect nested in soil (T ⊂ S), individuals
nested with transect and (I ⊂ T ), flowers nested on individuals as error term. Dependent variables are petal and sepal asymmetries measured as the standard deviation of petal length (SDLP), petal width (SDWP), sepal length (SDLS) and sepal width
(SDWS), and petal and sepal allometry error (ALP, and ALS respectively) after their logarithmic transformation. ∗∗∗ P < 0.001,
∗∗ P < 0.01, ∗ P < 0.05, no asterisks indicates P > 0.05, s 2 is the variance components.
Source
SDLP
MS df
3.58
0.38
0.25
0.13
Source
SDLS
MS df
1.38
0.27
0.14
0.10
s2
(2, 489) 25.82∗∗∗ 9.7%
(9, 84)
1.53
1.6%
(84, 480) 1.83∗
10.8%
480
77.9%
Soil (S)
(T ⊂ S)
(I ⊂ T )
Error
Soil
(T ⊂ S)
(I ⊂ T )
Error
F
F
s2
(2, 489) 12.87∗∗∗ 4.8%
(9, 84)
1.90
2.3%
(84, 480) 1.38∗
5.5%
480
87.4%
SDWP
MS df
3.44
0.20
0.23
0.15
(2, 489) 22.56∗∗∗
(9, 84)
0.87
(84, 480) 1.55∗
480
SDWS
MS df
0.11
0.24
0.19
0.10
s2
F
(2, 489)
(9, 84)
(84, 480)
480
foliar structures and stem growth patterns because
floral measurements were performed on different individuals. The results revealed that no correlations
existed between different organs.
Discussion
Serpentine soils, characterized by impoverished substrate with high concentrations of heavy metals, in particular Ni, provide an excellent opportunity to study
the tolerance of plants to metalipherous substrate. Soil
showed a clear increase in Ni2+ and Mg2+ and a decrease in Ca2+ from siliceous soil to serpentine soils
(table 1). The high concentration of Ni2+ in serpentine soil lead to a high concentration of Ni2+ in leaves
of this soil type, reaching critical levels observed in
other moderately tolerant species (Marschner 1986).
Serpentine soils had high amounts of Mg2+ and low
Ca2+ , with a Ca/Mg ratio lower than one. That the
Ca/Mg ratio in leaves is higher than one indicates that
the plants on serpentine soils actively take up Ca2+ .
Amounts of Ni2+ , Ca2+ and Mg2+ were intermediate in soils from the contact zone. While leaves from
this zone maintained a Ca/Mg ratio similar to plants on
silica and serpentine soils. Actual levels of both Ca2+
and Mg2+ in leaves from the contact zone were lower
than in plants from the other two zones. The lower
amounts in the contact zone may due to Ni2+ , a divalent cation that competes with other divalent cations
9.27%
0%
7.7%
83.4%
s2
F
1.09
1.26
1.80∗
0%
0.86%
11.7%
87.9%
ALP
MS
df
2.1 10−6
3.8 10−7
2.8 10−7
1.5 10−7
(2, 489) 13.07∗∗∗ 4.7%
(9, 84)
1.35
1.1%
(84, 480) 1.83∗
11.4%
480
82.7%
ALS
MS
df
1.2 10−6
4.5 10−7
5.2 10−7
4.8 10−7
(2, 489)
(9,564)
(84, 480)
480
F
F
2.6
0.92
1.1
s2
s2
0.8%
0%
1.6%
97.9%
(Ca2+ or Mg2+ ) and induces a deficiency of these elements. Apparently, there was been no adaptation in
the contact zone for selective uptake of these elements
in the presence of Ni2+ . This result is concordant with
the ecology of C. ladanifer, which prefers acid soils
with low Ca2+ concentrations, where can successfully
compete with other species.
Environmental stress has been correlated with abnormal traits in several plant species (Grant 1956,
1975; Huether 1969; Barret & Harder 1992; Kozlov
et al. 1996). However patterns of instability may result from complex interactions between the genotype
and the environment (Ellstrand & Mitchell 1988; Barrett & Harder 1992), providing evidence that several
vegetative and reproductive traits are required to assess developmental stability as a stress detector. In
this study we observed that plants living in the contact
zone presented larger absolute fluctuating asymmetry
of leaves (relative fluctuating asymmetry was not significant), larger radial asymmetry in petal length and
width, and larger translational symmetry of internode
length in the contact area than in silica or serpentine
soils. This is probably because directional selection
has ceased in serpentine areas where regulatory homeostatic mechanisms have restored developmental stability in vegetative and reproductive traits. However, in
the contact zone, destabilization occurs because colonizing plants can come from either serpentine or silica
adapted populations (Talavera et al. 1993). Talavera
et al. (1993) demonstrated that C. ladanifer has a
62
Table 8. Pearson correlation matrix of comparisons between the developmental instability
measurements used in this study: absolute fluctuating asymmetry (AFA), leaf allometry
2 ), standard deviaerror (ALH), translatory asymmetry parameters (Syx , Sa , Sb and Radj
tion of petal length (SDLP), petal width (SDWP), sepal length (SDLS) and sepal width
(SDWS), and petal and sepal allometry error (ALP, and ALS respectively) ∗∗∗ P < 0.001,
∗∗ P < 0.01, ∗P < 0.05, no asterisks indicates P > 0.05.
AFA
ALH
2
Radj
Syx
Sb
Sa
SDLP
SDWP
ALP
SDLS
SDWS
ALS
AFA
ALH
1
−0.043
0.068
1
−0.118
−0.030
0.083
0.056
0.144
0.003
0.076
2
Radj
Syx
Sb
Sa
−0.806∗∗∗
−0.141
−0.518∗∗∗
1
0.273∗∗
0.710∗∗∗
1
0.868∗∗∗
1
1
SDLP
SDWP
ALP
SDLS
SDWS
ALS
1
0.683∗∗∗
0.800∗∗∗
0.067
0.068
−0.086
1
0.488∗∗∗
0.129
0.106
−0.021
1
−0.058
−0.018
0.034
1
0.202∗
0.368∗∗∗
1
0.338∗∗
1
gametophytic mechanism of incompatibility together
with a low pollinator mobility that results in declining
plant fecundity with increased nearest neighbor distance. This self-incompatibility limits gene exchange,
ultimately resulting in the selection of serpentine and
silica ecotypes.
Evidence supporting the restoration of stability after a change in environmental conditions has been
found by McKenzie & O’Farrel (1993). They reported
that, after resistance was widespread, the continued
use of pesticides selected for a modifier allele in Lucilia cuprina blowfly that restored the relative fitness
of resistant phenotypes in the absence of pesticides
(McKenzie et al. 1982). In the absence of modifier
phenotypes, resistant strains are less successful than
susceptible strains when pesticides were absent, while
in the presence of the modifiers success is similar
(McKenzie 1994).
Differences in values of bilateral traits are usually small (often <1–5% of the total variation of a
given trait; Merilä & Björklund, 1995). To prevent
differences in values of bilateral traits due to measurement error, Palmer & Strobeck (1986) and Merilä
& Björklund (1995) pointed out the necessity of controlling measurement error in fluctuating asymmetry
studies. In the latter study, the percentage of variance
explained by floral or foliar trait asymmetry ranged
between 5 and 10%. In our study, measurement error
accounted for only 1% of the developmental instability index in the worst case. This, together with
the fact that measurement error was evenly distributed between the different treatments, led us to discard
the possibility of a reduction in our accuracy due to
measurement error.
Symmetry occurs because organs develop mechanisms to buffer environmental disturbances during
development (Mather 1953; Thoday 1955). As a result, leptokurtosis in bilaterally symmetrical organs
should be more pronounced since selection acts to
minimize asymmetry. Leptokurtic asymmetries are
found in several studies (Harvey & Walsh 1993; Polak
1993). According to Leung & Forbes (1997), leptokurtosis could also arise when developmental noise is
normally distributed and so should not be excluded
from asymmetry studies. We observed leptokurtosis
in floral and foliar asymmetries, proving the existence
of a selection mechanism for symmetrical flowers and
leaves.
Constancy of floral traits within taxa is well documented, but deteriorates near the limit of ecological
tolerance (Stebbins 1951; Bradshaw 1965). In consequence, we may expect floral structures to be under
high homeostatic control (Sherry & Lord 1996b), and
as a result, to be reliable indicators of developmen-
63
tal homeostasis and of phenotypic quality. Petals are
a major visual attractant for animal pollinators, and
petal size and symmetry are means of competing for
pollen vectors (Bell 1985; Willson 1990; Møller &
Eriksson 1994; Møller 1995a; Cronk & Möller 1997).
In concordance with that, we found a negative correlation between asymmetry and petal size, especially
in silica plants, indicating a directional selection towards large and symmetrical flowers. However, leaf
width asymmetry was positively related to mean leaf
width, as also observed by Møller (1995b) in Ulmus
glabra Hudson, and Kozlov et al. (1996) in Betula
pubescens Ehrh. The higher fluctuating asymmetry of
larger leaves may be due to the larger cost of the bigger
traits. As a result, leaf size will be under stabilizing
selection with an optimal leaf size that may depend on
environmental factors such as resource availability.
Traditionally, studies of developmental instability
have been based on fluctuating asymmetry and number of phenodeviantes (Markow 1994). In this study,
we prove that other traits can indicate developmental instability, i.e., variation in translational symmetry
is concordant with other within plant variations (see
also Alados et al. 1994, 1998; Escós et al. 1995,
1997). Growth rhythm varies among individuals and
with developmental stage (Comte 1993). In this study
we also demonstrate that variation in growth patterns
may be an indicator of environmental disturbance.
Thus, serpentinophytes grow faster at the beginning
of the growing season and produce fewer internodes,
while the silica soil population has a sustainable and
slower growing period but with a larger number of
internodes. After internode enlargement reaches its
maximum, the production of sylleptic branches takes
place. In general, foliar and floral traits are larger
in serpentine plants both for vegetative (annual shoot
length and leaf width) and reproductive (petal and
sepal length and width) structures, whereas silica populations show lower floral and foliar trait size, and the
contact population is intermediate.
Phenological delay between individuals of the
same species is also an indicator of low plant vigor
(Borchert 1976; Ng 1979). The phenological phase
of vegetative growth occurs at the same time for the
serpentine and siliceous soil populations, and with a
delay of around 15 days in the contact zone. A similar
trend was observed during flowering, with individuals
growing in the contact zone flowering later than the
others. The higher developmental stability of C. ladanifer inhabiting serpentine substrate together with its
rapid growth at the beginning of the growing season,
just before the sylleptic extension for brachyblast formation, may suggest that serpentine plants show a
competitive strategy based on the highest shoot vigor
related to syllepsis (Champagnat 1950). The competition between the growth of the main shoots and
developement of sylleptic axillary branches (extension
of main shoot), as well as the brachiblast and floral
stalk formation produces an inhibition of elongation
of subsequent internodes (Borchert 1976; Remphrey
& Powel 1985). This inhibition is more drastic in the
serpentine populations than in the contact zone. Plants
living in silica substrate present a more conservative
strategy with a less pronounced growth rhythm, due to
reduced competition between main shoot growth and
syllepsis. Individual branches of single plants respond
to local conditions (Stebbins 1963), allocating limiting
resources to the organs located in better environmental
conditions. As a result, we might expect high levels of
within individual variation in developmental instability measurements. For example, branches in full light
are larger and more branched than the corresponding
branches in relative shade (Hallé et al. 1978; Jones &
Harper 1987). In addition, light favors new branch formation (Thiébaut et al. 1985; Pickett & Kempf 1980),
whereas shade favors branch death (Willson 1990). In
spite of this, we observed that variation among measurements within individuals was lower than variation
between individuals within soil type. Additionally, different traits are under different selection pressures and
development, resulting in different responses to the
changing environment. For example, coastal Teucrium
lusitanicum Schreb populations exhibit a conservative
strategy with high homeostatic vegetative structures
and low stability in reproductive organs. Inland populations, by contrast, show a colonizing strategy, resulting in larger stability of reproductive organs that
enhances the colonization of new territories (Alados
et al. 1998). Indeed, we observed that the asymmetry of different characters varied independently among
structures, providing no support for the existence of
organism-wide developmental stability, as noted previously (Van Valen 1962; Evans & Marshall 1996;
Leung & Forbes 1997). However developmental instability values for different traits are concordant at the
population level, as observed by other authors (Evans
& Marshall 1996; Sherry & Lord 1996a; Leary et al.
1985).
Correlations between developmental instability of
several traits were probably limited by morphological
compensation and functional integration of traits. Floral instabilities were positively correlated with each
64
other at the level of petals and sepals, separately. At
the vegetative structure level, only curve fitting estimators of the stem growth pattern equation were correlated with each other. Other authors also observed
foliar instabilities correlated with each other (Sakai
& Shimamoto 1965; Bagchi et al. 1989), although
foliar instability was not significantly correlated with
floral instabilities (Sakai & Shimamoto 1965; Evans &
Marshall 1996).
When fitness depends upon the phenotypic characteristic of a particular trait, organisms should buffer
the effects of environmental disturbances on the development of those traits (Fowler & Whitlock 1994).
Natural selection should act to minimize phenotypic
variation in traits that are functionally important to an
organism. However, the way this occurs will differ in
animals and plants. Whilst animals may avoid environmental disturbances by evolved physiological and
behavioral mechanisms, plants resist environmental
perturbations by modifying their structures. In consequence, plants present highly variable structures that
change in response to light interception, soil moisture
or wind exposure. The difficulty involved in providing
perfect symmetrical traits may be determined by the
coefficient of variation of each developmental instability measurement. We observed that floral asymmetry
presents a lower coefficient of variation (0.58 for standard deviation of petal length, and 0.55 for standard
deviation of petal width) than leaf asymmetry (0.84),
with curve fitting accuracy of the internode length
equation showing a lower coefficient of variation (0.34
for Syx , or 0.29 for Sa ). Sherry & Lord (1996b)
also demonstrated, in genus Clarkia Pursh. that floral
asymmetry values were about half those of leaves.
In summary we conclude that asymmetry and
within-plant variance were higher in the contact zone
populations than in either silica or serpentine populations, proving the adaptation of C. ladanifer to
ultrabasic serpentine soils. Probably the competitive
abilities of C. ladanifer inhabiting acidified soils with
low Ca2+ concentrations allowed the plant to perform
adequately in serpentine soils in spite of high Ni2+
concentrations.
Acknowledgements
We thank H. Freitas and C. Nabais for performing
chemical analyses in soil and plant samples. We are
grateful to J. Emlen, H. Freitas, S. Talavera and M. A.
Quesada for their valuable suggestions and comments
on earlier drafts of the manuscript. We also thank J.
Toro for measuring plant samples. This study was partially supported by Ministerio de Educación y Cultura
(PSPGC) into the cooperative program between Spain
and Portugal, project HP96-42.
Appendix 1
Appendix 1. Mean square (MS) measurement error
and the proportion of within-measurement MS to the
among-individual MS (variation due to trait size) and,
to the within-trait MS (variation due to developmental
instability, DI).
Trait
MS error
Error as % of
Trait size
DI
Half leaf width
Petal length
Petal width
Sepal length
0.002
0.04
0.04
0.006
0.02%
0.08%
0.02%
0.1%
0.95%
0.5%
0.25%
0.32%
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