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Annals of Botany 88: 387±391, 2001
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Herbivory and Calcium Concentrations A€ect Calcium Oxalate Crystal Formation in
Leaves of Sida (Malvaceae)
B R E N D A M O L A N O - F LO R E S *
Illinois Natural History SurveyÐCenter for Biodiversity, 607 E. Peabody Drive, Champaign, Illinois 61820, USA
Received: 23 January 2001 Returned for revision: 30 March 2001 Accepted: 9 May 2001
Calcium oxalate crystals have potential roles in plants as part of a defence mechanism against herbivores and/or in
accumulating excess calcium. To date, these potential roles have been studied independently. In this experimental
study the e€ects of calcium levels and herbivory on the production of calcium oxalate crystals (i.e. druse, spherical
crystal aggregates) were examined in seedlings of Sida rhombifolia. Seedlings were subjected to three calcium levels
(low, normal or high) and an arti®cial herbivory treatment. Calcium levels and herbivory both a€ected density of
crystals in leaves. Leaves from seedlings grown in low calcium had a greater crystal density than those grown in high
calcium. Leaves from seedlings subjected to herbivory had a greater crystal density than those from seedlings not
subjected to herbivory. This study provides additional evidence that calcium oxalate crystal production depends not
only on calcium levels but can also be in¯uenced by external pressures such as herbivory. In addition to their
physiological role in plants, these results suggest that calcium oxalate crystals can also act as a defence mechanism
# 2001 Annals of Botany Company
against herbivores.
Key words: Calcium concentrations, calcium oxalate crystals, herbivory, Malvaceae, Sida rhombifolia.
I N T RO D U C T I O N
Calcium oxalate crystals (COC) are found in over 215 plant
families (McNair, 1932; Franceschi and Horner, 1980; Ward
et al., 1997), among them Cactaceae, Malvaceae, Orchidaceae and Rubiaceae. They can occur in any part of the plant:
in the ¯owers (Kenda, 1956; Buss and Lersten, 1972; Horner
and Wagner, 1980), leaves (Price, 1970; Zindler-Frank,
1975; Horner and Zindler-Frank, 1982; Franceschi, 1984;
Sunell and Healey, 1985; Doaigey, 1991), stems (Scott, 1941;
Brander, 1987; Doaigey, 1991), roots (Franceschi, 1989;
Horner et al., 2000) and seeds (Buttrose and Lott, 1978;
Sunell and Healey, 1979). The distribution and shapes
(raphides, druses, etc.) of these crystals have been used as
taxonomic characters for a number of plant families. For
example, the presence of raphides has been used to separate
subfamilies of Rubiaceae (Lersten, 1974) and subgenera of
Prunus (Lersten and Horner, 2000) in the dicots, and in
monocots crystals have been used as a synapomorphy for
some families (Prychid and Rudall, 1999).
In plants, calcium oxalate crystals may have roles in
defence against herbivores and/or in accumulating excess
calcium. Although both roles have been investigated, no
resolution has been achieved. Raphide crystals are generally
thought to play an ecological role as a herbivore defence
mechanism (Sakai et al., 1972; Sunell and Healey, 1985;
Perera et al., 1990; Ward et al., 1997). These crystals are
found in large cells that are ®lled with mucilage and, in
most cases, are dead at maturity. Accumulation of mucilage
increases pressure against the cell wall; this facilitates
release of the crystals when the cell is damaged by grazing
(Esau, 1965). However, raphide crystals are not always
* Fax ‡001 217 265 5110, e-mail [email protected]
0305-7364/01/090387+05 $35.00/00
associated with defence. For example, Colocasia esculenta
(Araceae) has two types of raphides: non-defensive raphides
found in elongated cells which are usually embedded in
tissues of the leaf margins; and defensive raphides, also
found in elongated cells, but these cells are usually
suspended between mesophyll cells in leaf airspaces (Sunell
and Healey, 1985). It is these defensive crystals that have the
irritative property. To date, crystals have been thought to
have a physiological role, sequestering excess calcium
within plant cells (Borchert, 1984, 1985, 1986; Franceschi,
1987, 1989; Fink, 1991; Webb, 1999). High concentrations
of calcium can interfere with many cell processes (e.g.
calcium-dependent signalling, microskeletal dynamics) in
plants (Webb, 1999).
In general, most of the studies associated with production
of COC have emphasized the e€ect of calcium concentration
on crystal production, and several studies have found a
positive relationship between calcium concentration in the
growth medium and crystal production (Frank, 1972;
Zindler-Frank, 1975; Borchert, 1985; Franceschi, 1989).
Other factors, such as herbivory, have been studied less
frequently. A few studies have shown that COC crystals can
deter herbivores (Sunell and Healey, 1985; Perera et al.,
1990; Ward et al., 1997). With the exception of the study by
Ward et al. (1997), no direct link between herbivory, calcium
levels and COC production has been found.
In this study, COC production was examined in Sida
rhombifolia L. (Malvaceae) seedlings exposed to di€erent
calcium levels and herbivory treatments. I addressed the
following questions: (1) do di€erent concentrations of
calcium a€ect COC production; (2) does herbivory a€ect
COC production; and (3) do herbivory and calcium levels
interact to a€ect COC production? Sida rhombifolia is an
# 2001 Annals of Botany Company
388
Molano-FloresÐCalcium Oxalate and Herbivory
excellent experimental species because the plant has
crystals, is attacked by herbivores, and is found in a wide
range of soils (i.e. di€ering in calcium levels).
M AT E R I A L S A N D M E T H O D S
Seed collection, germination and experimental setup
Sida rhombifolia L. (Malvaceae) is a perennial shrubby
herb, commonly found in pastures and wasteland, particularly at low and middle elevations in Puerto Rico (Liogier
and Martorell, 1982). In 1990, seeds of S. rhombifolia were
collected from ten randomly selected plants at Barrio Indio,
Carolina, Puerto Rico. Seeds were segregated by parent
plant and germinated in washed sand. After 3±4 weeks, 24
seedlings of approximately the same height and biomass
were selected from each parental plant and were transplanted into 10 cm pots containing sterile sand, giving a
total of 240 seedlings. Four seedlings with similar origin
(i.e. parental plant) were placed in each pot. Pots were
placed randomly in a glasshouse to minimize position
e€ects on treatments (see below). To minimize uncontrolled
herbivory, pots were surrounded by slug and snail bait and
seedlings were occasionally sprayed with an insecticide
(0.02 % pyrethrins: 0.20 % piperonyl butoxide: 99.78 %
inert ingredients; Schultz Instant1) to control aphids, white
¯ies and mealybugs. Each pot was then randomly assigned
to one of the treatments described below.
Plant treatments
Sida rhombifolia seedlings were assigned to two sets of
treatments: calcium level (low, normal or high) and
herbivory (no herbivory or arti®cial herbivory). Seedlings
were randomly assigned to one of three groups of calcium
treatment: low (6.5 10 ÿ4 M), normal (1.3 10 ÿ3 M), and
high (2.6 10 ÿ3 M) (Whitham et al., 1986). Calcium
solutions were applied every 2 weeks with a liquid fertilizer
lacking calcium (20 : 20 : 20/N:P:K, Evergreen Company1).
Evidence of calcium de®ciency, such as twisted leaves at
maturity and light green colouration at the base of the leaf,
indicated that seedlings in the low calcium treatment
received low levels of calcium.
The arti®cial herbivory treatment was applied to half the
seedlings in each of the three calcium treatments. Twelve
percent of the area from the right-hand side of the blade of
each fully developed leaf was removed by removing a
triangular section without severing the central vein. The
area of the leaf removed was similar to that lost under
natural conditions (Molano-Flores, pers. obs.). This treatment was applied to every other leaf, excluding the
cotyledons. Young leaves in the arti®cial herbivory treatment were periodically cut until the leaves reached maturity,
maintaining the area removed at 12 %.
Sida rhombifolia seedlings thus received one of six
treatments (three calcium levels two herbivory treatments). All treatments lasted 5 months (March to August
1990). At the end of the treatment period, terminal (young)
and basal (mature) leaves were collected from plants in the
non-herbivory treatment. In the case of plants in the
herbivory treatment, two sets of young and mature leaves
were collected: the ®rst set included one young and one
mature leaf that had been arti®cially grazed and the second
set included one young and one mature intact leaf that were
adjacent to the grazed leaves. All leaves were placed in 70 %
included ethanol for later determination of crystal density.
To determine crystal densities, leaves were cleared
following the methods of Sunell and Healey (1985). Leaves
were placed in 70 % ethanol at 608C for 2 h, then in 95 %
ethanol at room temperature for 1 h. Leaves were then
washed brie¯y with distilled water, placed in 5 % NaOH for
1 h at room temperature, and washed three times with
distilled water. Crystal density was determined on the lefthand side of the leaf blade at the tip, middle and base. In
each of these regions, all crystals within a 0.25 cm2 area
were counted. Crystal density was de®ned as number of
crystals/area counted.
The crystals found in Sida rhombifolia are of the druse
type (i.e. spherical crystal aggregates). Solubility tests
following Frey (1929) and Pohl (1965) were conducted to
determine the chemical composition of the crystals. A
section of the leaf (0.25 cm2) was immersed in 1, 2 and 5 %
acetic acid, 70 % ethanol, 10 % hydrochloric acid, 3 %
nitric acid, 4 % sodium hydroxide and 4 % sulfuric acid.
Druses were not soluble in 1, 2 or 5 % acetic acid, 70 %
ethanol or 4 % sodium hydroxide. However, they were
soluble in 4 % sulfuric acid, 3 % nitric acid and 10 %
hydrochloric acid. All these tests con®rmed that the druses
were calcium oxalate. Other crystalline deposits such as
carbonate and phosphate salts of calcium are very soluble
in acetic acid (Franceschi and Horner, 1980; Franceschi,
1984). These druses (one per cell) were located in the
palisade parenchyma, spongy mesophyll cells, and bundlesheath cells of the veins. Druses near the veins were
arranged in straight lines. The diameter of druses ranged
from 0.5 to 5 mm.
Statistical analyses
Results were analysed using two-way analysis of variance
(ANOVA) followed by Tukey tests. In the herbivory
treatment, data were collected for non-grazed and grazed
leaves; thus a series of two-way ANOVAs were performed.
In these ANOVAs, the data were separated to determine
whether di€erences existed between the non-grazed and
grazed leaves from the herbivory treatment and leaves from
the non-herbivory treatment, and also within the leaves
from the herbivory treatment. For all the above ANOVAs,
the number of crystals in the tip, middle and base was
pooled to obtain the crystal density for a leaf: (i.e. crystal
density is the total number of crystals counted/total areas
counted). Data sets were checked for normality and
homogeneity of variance. ANOVA tests were conducted if
variances were homogeneous, regardless of whether data
passed the normality test (Green, 1979; Zar, 1999). Crystal
density data were logarithmically transformed to meet
requirements of both normality and homogeneity of
variance. Means and standard errors are reported. All
statistical tests were performed using Systat 7.0.1 (Systat,
1997).
Molano-FloresÐCalcium Oxalate and Herbivory
R E S U LT S
Log 10 crystal density (cm2)
The e€ect of calcium and herbivory on crystal density
Signi®cant di€erences were found for crystal density (CD)
in the calcium treatment (F ˆ 5.179, d.f. ˆ 2, P ˆ 0.006,
Fig. 1). Leaves collected from seedlings grown in the low
calcium treatment had greater CD than leaves collected
from seedlings grown at high calcium levels (Tukey test,
P ˆ 0.015). However, although not signi®cant, a trend
toward lower CD was found for leaves collected from
seedlings grown in the normal calcium treatment when
compared to leaves collected from seedlings grown at low
calcium levels (Tukey test, P ˆ 0.077). No signi®cant
di€erences were found between leaves collected from
seedlings grown in the normal and high calcium treatments
(Tukey test, P ˆ 0.823).
Signi®cant di€erences were found for CD in the
herbivory treatment (F ˆ 14.302, d.f. ˆ 1, P ˆ 0.000).
Overall, the CD in seedlings that were arti®cially grazed
was signi®cantly greater (mean + s.e. ˆ 2.842 + 0.010)
than in intact leaves in the non-herbivory treatment (mean
+ s.e. ˆ 2.777 + 0.014). Crystal density was signi®cantly
greater in both grazed and non-grazed leaves of the
herbivory treatment compared to leaves in the nonherbivory treatment (Tables 1 and 2; Fig. 2A and B).
However, no signi®cant di€erence was found for CD
between grazed and non-grazed leaves within the herbivory
treatment (Tables 1 and 2; Fig. 2C).
Finally, no interaction was found between calcium level
and herbivory treatment (F ˆ 0.6850, d.f. ˆ 2, P ˆ 0.505).
2.88
2.86
389
F = 5.179, P = 0.006
a
2.84
ab
bc
2.82
2.8
2.78
2.76
2.74
2.72
Low
Normal
High
Calcium levels
F I G . 1. Mean crystal density (log 10) in leaves of seedlings grown in
di€erent calcium treatments. Bars represent s.e. Means with di€erent
letters di€er signi®cantly, based on Tukey's multiple comparisons
(P 5 0.05).
DISCUSSION
Calcium oxalate crystals (COC) may have an ecological role
in plants as a defence mechanism, and a physiological role
accumulating excess calcium. This study demonstrated that
both calcium availability and herbivory can increase the
production of COC. Many studies have found a positive
relationship between calcium concentrations and crystal
production (Frank, 1972; Franceschi and Horner, 1979;
T A B L E 1. Summary of ANOVA tables for the e€ects of calcium and herbivory on crystal density
NHNH'
Main e€ect and
interaction
d.f.
Calcium
Herbivory
Calcium Herbivory
2
1
2
F
4.152
11.114
1.018
NHH
P
d.f.
0.016
0.001
0.362
2
1
2
F
4.484
10.254
1.305
NH'H
P
d.f.
F
P
0.012
0.002
0.272
2
1
2
1.427
0.020
2.692
0.241
0.888
0.069
Leaves from the non-herbivory treatment and non-grazed leaves from the herbivory treatment (NHNH'); leaves from the non-herbivory
treatment and grazed leaves from the herbivory treatment (NHH); and non-grazed and grazed leaves from the herbivory treatment (NH'H) are
compared.
T A B L E 2. Summary of means and standard error (s.e.) tables for ANOVA in Table 1
NHNH'
NHH
NH'H
Mean
s.e.
n
Mean
s.e.
n
Mean
s.e.
n
Calcium level
Low
Normal
High
2.852
2.783
2.797
0.015
0.020
0.018
114
121
127
2.846
2.810
2.774
0.018
0.018
0.017
104
119
136
2.869
2.837
2.825
0.019
0.017
0.016
100
120
127
Herbivory treatment
Non-herbivory
Herbivory
2.777
2.845
0.014
0.014
187
175
2.777
2.839
0.014
0.014
187
172
2.844
2.839
0.014
0.014
175
172
n, Sample size.
See Table 1 for abbreviations.
Log 10 crystal density (cm2)
Log 10 crystal density (cm2)
Log 10 crystal density (cm2)
390
Molano-FloresÐCalcium Oxalate and Herbivory
2.88
2.86
2.84
2.82
2.8
2.78
2.76
2.74
2.72
2.7
2.88
2.86
2.84
2.82
2.8
2.78
2.76
2.74
2.72
2.7
2.88
2.86
2.84
2.82
2.8
2.78
2.76
2.74
2.72
2.7
F = 11.114, P = 0.001
Nonherbivory
treatment
A
Non-grazed
leaves in
herbivory
treatment
B
F = 10.254, P = 0.002
Nonherbivory
treatment
Grazed leaves
in herbivory
treatment
Herbivory treatment
C
F = 0.020, P = 0.888
Non-grazed
leaves
Grazed leaves
Treatment
F I G . 2. Mean crystal density (log 10) in leaves of seedlings: (A) in the
non-herbivory treatment compared to non-grazed leaves of seedlings in
the herbivory treatment; (B) in the non-herbivory treatment compared
to grazed leaves of seedlings in the herbivory treatment; and (C) in the
herbivory treatment: non-grazed leaves compared to grazed leaves.
Bars represent s.e.
Borchert, 1985, 1986; Franceschi, 1989; Zindler-Frank,
1991, 1995); this was not the case in this study. Leaves from
seedlings grown in low calcium concentrations had greater
crystal densities than leaves from seedlings grown in high
calcium concentrations. In a study by Ward et al. (1997), a
similar result was found, but no explanation was provided.
One potential explanation may be associated with the
absorption of calcium by the plant. Kirkby and Pilbeam
(1984) suggested that calcium uptake may be regulated by
the solubility of calcium in the soil rather than by plant
demand. In addition, Frank (1972) reported that if calcium
concentrations in nutrient solutions are high, then calcium
will precipitate as Ca-phosphate and the amount of Ca
taken up by the plant will be similar to that absorbed at a
`normal' level of calcium. Therefore, it is possible that fewer
crystals were formed in seedlings grown in normal and high
calcium levels compared to low calcium levels because these
seedlings absorbed less calcium.
Unlike many studies of COC formation, the approach
taken in this study mimicked a natural setting, with the root
system in direct contact with the soil. Most previous studies
have placed leaves in solution, and thus they do not re¯ect
true root-calcium interactions. In a natural setting, even if
soil calcium levels are high, calcium might not be available
to the plant. Absorption of calcium by the root system is
a€ected not only by other nutrients, such as K, NH4 and
Mg (Mengle and Kirkby, 1982), but also by the availability
of calcium to the plant. It is possible that due to potential
interference of calcium absorption, concentrations of
calcium in the cytoplasm of plants in the normal and
high Ca treatments were lower than those of plants in the
low calcium treatment. This could explain why more
crystals were produced in the lower calcium treatment
than in the normal or high calcium treatments. This ®nding
does not contradict results of previous studies and the
physiological role of COC, it merely con®rms that the
higher the concentration of calcium found in the cytoplasm
the more crystals will be produced (Franceschi, 1989). It
should be pointed out that symptoms of calcium de®ciency
were noted in seedlings grown in the low calcium treatment.
This raises the question of whether calcium was allocated to
COC production at the cost of plant growth.
Another signi®cant ®nding of this study is the e€ect of
herbivory on COC production. Herbivory can increase the
number of crystals in seedling leaves independently of
calcium level. Other studies have shown that crystals, in
particular raphides, act as a defence against herbivores
(Sunell and Healey, 1985; Perera et al., 1990; Ward et al.,
1997). In the study by Ward et al. (1997), plants subjected
to herbivory by gazelles had higher numbers of crystals
than non-grazed plants. In this study, arti®cial herbivory
produced similar results. This suggests that an increase in
COC in seedlings and adult plants of Sida rhombifolia may
deter herbivores, as has been shown in other plants (Sunell
and Healey, 1985; Perera et al., 1990; Ward et al., 1997).
This ®nding is important because a cost and e€ect
relationship has been found between COC production and
herbivory. Regardless of its calcium level, a plant subjected
to herbivory will produce more COC.
Sida rhombifolia is found in habitats in which levels of
calcium and herbivory vary. Arti®cial herbivory and low
calcium concentrations were found to independently
increase the number of calcium oxalate crystals (i.e. druses)
in S. rhombifolia seedlings. Under such conditions, my
results suggest that COC may protect S. rhombifolia
seedlings. This study also suggests that in addition to
raphide crystals other COC such as druses may serve as a
defence for plants by increasing amounts of COC and
Molano-FloresÐCalcium Oxalate and Herbivory
potentially changing the palatability of the plant. However,
this must be con®rmed in natural populations of
S. rhombifolia, along the lines of a study by Ward et al.
(1997). Additional research is needed to better understand
the roles of calcium oxalate crystals in plants. This study
and that by Ward et al. (1997) appear to be the only ones to
have shown an increase in COC production as a result of
herbivory, even if calcium availability is limited. My results
also suggest that the traditional studies into the physiological role of crystals are of little help in understanding the
defensive role of COC in plants. The ecological role of
calcium oxalate crystals must be addressed more closely.
AC K N OW L E D GE M E N T S
I thank Mary Ann Feist, Geo€rey Levin, Chris Whelan and
two anonymous reviewers for editorial and useful comments that improved the manuscript, and Je€ Brawn and
Steve Heard for statistical advice.
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