This study examined variation in glucosinolate content between leaves and petals of wild radish (Raphanus sativus) to test predictions of optimal defence theory. The theory predicts that tissues most important for fitness, like reproductive parts, will be constitutively more defended. The study found that petals had higher constitutive glucosinolate levels than leaves, supporting the prediction. However, individual glucosinolates differed in their degree of inducibility between tissues. Petal colour variants also differed in their induced responses but not constitutive levels. The results provide evidence that selection from pollinators and herbivores could maintain variation in defence chemistry and petal colour in wild radish.

1. Journal of
Optimal defence theory and flower petal colour predict
Blackwell Publishing, Ltd.
Ecology 2004
92, 132 –141 variation in the secondary chemistry of wild radish
SHARON Y. STRAUSS, REBECCA E. IRWIN* and
VIRGINIA M. LAMBRIX†‡
Center for Population Biology, One Shields Avenue, UC Davis, Davis, CA 95616, and †Max-Planck-Institute for
Chemical Ecology, Jena, Germany
Summary
1 The presence, concentration and composition of plant secondary compounds, which
confer plant resistance to herbivores and pathogens, vary greatly both within and
among individuals. Optimal defence theory predicts that plant tissues most closely tied
to plant fitness should be most defended at the constitutive level, and that more expend-
able tissues should be inducible with damage.
2 We examined variation in glucosinolate content between leaves and petals, as well as
among four petal colour morphs of wild radish, Raphanus sativus. We predicted greater
levels of constitutive defences in petals, and greater inducibility of glucosinolates in
leaves, based on previous studies that could relate leaves and petals to plant fitness.
3 While, overall, optimal defence predictions were supported, individual glucosinolates
differed in both their degree of inducibility as well as in their distribution between tissue
types.
4 Petal colour variants differed in their induced responses to damage, but not in their
constitutive levels of compounds. Yellow and white morphs, which are preferred by the
dominant bee pollinators as well as by herbivores, were generally less inducible than
anthocynanin-containing pink and bronze petal morphs.
5 Pleiotropic effects between petal colour and defence loci, or tight linkage between
these loci, may allow pollinators to maintain variation in secondary chemistry, as well
as allow herbivores to influence colour morph fitness and prevalence.
Key-words: glucosinolates, optimal defence, petal colour, pollination, Raphanus sativus,
resistance
Journal of Ecology (2004) 92, 132–141
1994; Mitchell-Olds & Bradley 1996; Berenbaum &
Introduction
Zangerl 1998), and among populations (Bryant et al.
The presence, concentration and composition of plant 1994; Mithen et al. 1995). Several adaptive explanations
secondary compounds are well known to confer plant have been proposed to explain variation in defence
resistance to herbivores and pathogens. Variation in within plants. One of these, optimal defence theory,
defensive chemistry can occur at many scales: within a predicts that tissues that are the most valuable to the
leaf on a single plant (Gibberd et al. 1988), among tissues plant are expected to be the most defended, and to
on the same plant (Nitao & Zangerl 1987; Van Dam have chemistry that is the least inducible with damage
et al. 1995, 1996; Zangerl & Rutledge 1996; Ohnmeiss (McKey 1979). Thus, plant tissues most closely linked
& Baldwin 2000; Pavia et al. 2002), among genotypes to fitness, like reproductive parts, are predicted to be
and phenotypes within the same species (Fritz 1990; constitutively defended at high levels (Nitao & Zangerl
Strauss 1990; Fritz & Simms 1992; Han & Lincoln 1987; Van Dam et al. 1996; Zangerl & Rutledge 1996;
Ohnmeiss & Baldwin 2000).
Along with variation in secondary chemistry among
Correspondence: Sharon Y. Strauss tissue types within plants, secondary chemistry may
(e-mail systrauss@ucdavis.edu).
also vary among individual plants. A variety of hypo-
*Present address: University of Georgia, Institute of Ecology,
Athens, GA 30602, USA. theses have been proposed to explain the maintenance
© 2004 British ‡Present address: Department of Vegetable Crops, One Shields of variation in secondary chemistry among plants in the
Ecological Society Avenue, UC Davis, CA 95616, USA. same population. Costs of defence are one mechanism

2. 133 through which variation in defence levels may be the selective forces maintaining both the petal colour
Patterns of plant present in non-equilibrial populations. In one scenario, polymorphism and variation in defence in this species.
variation in defence well-defended plants have higher fitness in years with Variation in defence may be maintained if defence is
high herbivore damage, whereas less-defended plants somehow linked (sensu lato) to petal colour, and if petal
have higher fitness in low-herbivory years, due to colour is under selection from agents other than
reduced costs, as recently reviewed in Bergelson & herbivores (see Irwin et al. 2003). In Raphanus sativus,
Purrington (1996), Koricheva (2002) and Strauss et al. dominant bee pollinators prefer yellow petal morphs
(2002). Variation in defence may be similarly maintained (anthocynanin recessive) to pink and bronze anthocy-
in a non-equilibrial state when there are ecological costs anin dominant morphs (Stanton 1987; Stanton et al.
of defence, e.g. when different defences work against 1989), and therefore pollinators may exert selection
different herbivores, and herbivore composition varies on defensive chemistry, if petal colour morphs differ
from year to year. in defensive chemistry. A first step to understanding
Here, we consider another hypothesis to explain whether conflicting selection could maintain variation
variation in defence among individuals of Raphanus among genotypes of wild radish is to determine whether
sativus, wild radish. Variation in defence may be a result flower colour is associated with consistent differences
of pleiotropic effects of genes at other loci, or of selection in secondary chemical variation. While this study focuses
on genes tightly linked to defence. For example, her- on variation in defensive chemistry within and among
bivores have been shown to discriminate among petal plants of R. sativus, many of the predictions will also apply
colour morphs in some species (Simms & Bucher 1996; to understanding variation in traits that influence import-
Irwin et al. 2003). Beetle larvae performed better on ant mutualists and antagonists simultaneously for any
leaves of pink-flowering morphs (anthocyanin-producing species.
colour morphs) than on leaves of white-flowering morphs
of morning glory, Ipomaea purpurea (Simms & Bucher
    
1996). In addition, some floral and pollen herbivores
      R . S A T I V U S
discriminate among flower-colour morphs, including
thrips (Vernon & Gillespie 1990; Gaum et al. 1994; Chyzik The predictions of optimal defence theory are that
et al. 1995) and pollen-feeding beetles (Giamoustaris tissues most closely tied to plant fitness should be max-
& Mithen 1996). For R. sativus, many herbivores imally defended. Several studies have shown that the
exhibited better performance on leaves of anthocyanin highest levels of plant secondary compounds are
recessive (white and yellow petal colour) compared associated with reproductive tissues and tend not to be
with anthocyanin-dominant (pink and bronze petal inducible (Van Dam et al. 1996; Zangerl & Rutledge
colour) (Irwin et al. 2003). Armbruster (2002) showed 1996). Here, we compare constitutive and induced
that petal and leaf anthocyanin expression were levels of glucosinolates sampled from damaged and
linked in the Acer and Dalechampia clades; however, in undamaged plant siblings in both petal and leaf tissues
a study of R. sativus, there was no relationship between of R. sativus. Glucosinolates have been shown to deter
leaf anthocynanin content and herbivore performance, and reduce the performance of many herbivores (Blau
and only petal anthocyanins, or traits linked closely et al. 1978; Glen et al. 1990; Kliebenstein et al. 2002;
to petal colour, influenced herbivores (S. Y. Strauss, Renwick 2002), though they have also been speculated
unpublished data). to serve other plant functions, such as in sulphate
One explanation for the links between defensive com- storage and/or involvement in IAA production (Bones
pounds and petal colour may be that genes controlling & Rossiter 1996). They are induced after damage in a
flower colour directly influence plant resistance to her- large number of species in the Brassicaceae (Louda &
bivores, if pleiotropic effects exist between the synthesis Rodman 1983; Bennett & Wallsgrove 1994; Birch et al.
of floral pigments and defensive plant compounds 1996; Siemens & Mitchell-Olds 1998; Li et al. 1999;
(Simms & Bucher 1996; Fineblum & Rausher 1997). McCaffrey et al. 1999; O’Callaghan et al. 2000). While
Alternatively, if petal colour genes and defence genes glucosinolates act as deterrents for many herbivores,
are tightly linked, then selection on one trait may cause they can also be attractants to specialists (Chew &
correlated changes in values of the other trait. Both petal Cutler 1988; Moyes et al. 2000).
colour and glucosinolates are known to be heritable traits Petals are often tightly linked to plant fitness, espe-
in R. sativus (Panetsos 1964; Carlson et al. 1985; Ishii cially in obligately outcrossing species like R. sativus,
et al. 1989; Schuetze et al. 1999). In addition, Hemm et al. because of their important role in attracting pollinators
(2003), using Arabidopsis mutants, showed that altering (reviewed in Proctor et al. 1996). The link between
alkylglucosinolate biosynthesis simultaneously affected petals and fitness in R. sativus is substantive. Young &
phenylpropanoid metabolism, from which anthocy- Stanton (1990) and Stanton & Preston (1988) found
nanin pigments are derived. Thus, there is evidence for that increased petal size was associated with greater
© 2004 British
pleiotropic effects of genes affecting both the glucosino- components of male fitness (pollen removal). In addi-
Ecological Society, late and anthocyanin pathways in a related mustard. tion, female fitness was positively associated with petal
Journal of Ecology, Understanding differences in defensive chemistry size in field experiments on a close relative of R. sativus,
92, 132–141 among colour morphs of wild radish may shed light on Raphanus raphanistrum (Conner et al. 1996), whose

3. 134 flowers are virtually indistinguishable from those of
Methods
S. Y. Strauss, R. sativus in morphology. Thus, petal size may be
R. E. Irwin & related to fitness through both male and female fitness
  
V. M. Lambrix components in R. sativus. In R. raphanistrum, plants
with larger petals often receive more pollinator visits Raphanus sativus L. (Brassicaceae) is a naturalized,
(Strauss et al. 1996; Lehtilä & Strauss 1997). In addi- herbaceous annual, which is common along roadsides
tion, damage to petals by florivores has been associ- and disturbed areas in valley and coastal areas of Cali-
ated with decreased pollinator attraction in a variety fornia, USA. Seeds germinate early in the rainy season
of plant species (Karban & Strauss 1993; Krupnick (October/November) with plants blooming in March
& Weis 1998; Krupnick et al. 1999; Mothershead & for approximately 3–4 months. In California, R. sativus
Marquis 2000; Adler et al. 2001). Moreover, symmet- individuals possess one of four different petal colours:
rical flowers are more attractive to pollinators than yellow, white, pink or bronze. Petal colour is determined
asymmetrical flowers (Moller 1996), so damage directly by two independently assorting loci, each with two
to petals could also reduce attractiveness to pollinators alleles controlling the expression of carotenoids and
through asymmetry. R. sativus experiences extensive anthocyanins (Panetsos 1964) Carotenoid pigments
petal herbivory in some locations and years; we produce yellow petals with yellow (presence of carotenoid)
have observed woolly bear caterpillars at Bodega recessive to white (absence of carotenoid). Anthocyanin
Bay feeding extensively on both leaf and petal tissue pigments produce pink petals with white (absence of
in outbreak years (S. Y. Strauss, personal observa- anthocyanin) recessive to pink (presence of anthocyanin).
tions). Thus, small changes in petal area or shape may Bronze-flowered plants express both anthocyanin and
have large impacts on pollinators and plant fitness. We carotenoids and thus have at least one dominant allele
therefore predict that petals should be constitutively at the anthocyanin locus and only recessive alleles at the
well defended, especially for self-incompatible annuals carotenoid locus.
such as R. sativus that rely on pollinators for plant
reproduction.
 
Leaves are also important to plant fitness, and high
levels of damage to leaves can reduce fitness in Rapha- All plants were glasshouse-grown progeny whose grand-
nus spp. through both direct and indirect pathways (e.g. parents were collected as seed from a naturalized R.
Mauricio & Bowers 1990; Strauss et al. 1996; Lehtilä & sativus population at Bodega Bay, California. After
Strauss 1997; Agrawal et al. 1999). However, Raphanus field collection, seeds were grown and all pollen donors
spp. are relatively tolerant to herbivory and suffer little were crossed with yellow (double recessive) mothers.
to no fitness costs with small amounts of leaf damage We crossed all plants into a yellow background to try to
(Mauricio & Bowers 1990; Lehtilä & Strauss 1999; homogenize plants for traits other than flower colour.
Strauss et al. 2001). When 25% leaf area was removed Thus, non-yellow plants were heterozygous (at least one
from each of the first four leaves of R. sativus by Pieris locus) for flower colour because they were the result of
rapae larvae, reproduction and growth of these dam- a mating between a yellow parent and another pigmented
aged plants was indistinguishable from that of controls parent. These heterozygous families produced progeny
(Mauricio & Bowers 1990). Damage levels in the with multiple flower colours (see below).
field vary among years, and range from a mean of 5% to Experimental plants were grown in the glasshouse in
20% overall damage in adult plants (Strauss and Irwin, individual 10 cm square pots using University of Cali-
unpublished data). Thus, we expect leaves to exhibit fornia glasshouse soil mix. Plants were watered using a
inducible defences. Another hypothesis to explain the subirrigation system ad libitum and fertilized at the
greater inducibility of leaves over petals hinges on the two-leaf stage with 2 g of Osmocote Plus 15-11-13 slow
costs of defence and the timing of defence expres- release fertilizer (Scott’s, Marysville, Ohio, USA).
sion. Costs of defence incurred early in the ontogeny of At the four-leaf stage, plants were randomly assigned
the plant, i.e. if leaves are constitutively defended or to one of two treatments: 50% of all leaves, except the
induced early in the plant lifetime, may have long- fifth and eighth true leaves, consumed by caged Pieris
lasting impacts on plant fitness through diminished rapae larvae, or unmanipulated controls. Pieris rapae
resource acquisition; in contrast, for tissues like petals, are naturalized specialist herbivores of R. sativus and
which are created and defended later in the lifetime are a dominant herbivore in many CA populations of
of the plant, costs may have a lesser overall impact on R. sativus. In the leaf removal treatment, we caged third
plant resources (P. Klinkhamer, personal communica- to fifth instar larvae in clip cages. Cages were placed
tion). This argument is also consistent with the pre- along the mid-vein of a leaf, and caterpillars fed on the
diction that leaf defences should be inducible with leaf tissue in the cages. We moved the cages along the
damage. Moreover, we expect that plants should invest mid-vein until one-half of the leaf was consumed. This
© 2004 British
more defences in petal tissue than in leaf tissue because general pattern of damage mimics natural damage
Ecological Society, low levels of damage to petal tissue may have greater by P. rapae larvae in the field (S. Y. Strauss, personal
Journal of Ecology, impacts on plant fitness than low levels of leaf damage observation). On the leaves of unmanipulated control
92, 132–141 in this annual plant. plants, we placed clip cages with no larvae to control

4. 135 for clip-cage effects. As plants initiated flowering, the (1988). Samples were placed into deep-well microtiter
Patterns of plant fifth (i.e. undamaged) true leaf was removed with a tubes. We added four 2.3-mm ball bearings, and the
variation in defence razor blade, weighed, and immediately microwaved for samples were ground into a fine powder in a paint shaker
approx. 30 – 45 s to denature endogenous myrosinases. by high-speed agitation. To extract glucosinolates, we
Samples were then dried for 48 h at 60 °C and stored at added 400 µL of methanol, 10 µL of 0.3  lead acetate,
0 °C until further chemical analysis. Larvae damaged and 120 µL of water. The samples were mixed for 1 min
plants over the course of c. 3 weeks, and the damage and then allowed to incubate for 60 min at 180 g on a
treatment was completed by the time plants started rotary shaker. The tissue and protein were pelleted by
flowering. centrifugation, and the supernatant was used for anion-
To sample petal tissue for glucosinolate analysis, we exchange chromatography.
removed the petals from at least 25 flowers per plant. We loaded 96-well filter plates from Millipore (model
Petals were removed from the base of flowers using MAHVN4550) with 45 µL of DEAE Sephadex A-25.
fine-point forceps and care was taken to ensure that petal We then added 300 µL of water to each column and
samples did not contain calyces or pollen. We only allowed the mixture to equilibrate for 2– 4 h. We removed
sampled petals from flowers that were 1–2 days old, the water with 2–4 s of vacuum and then added 150 µL
and petal samples were collected over several dates. We of the supernatant to the 96-well columns. The liquid
had to combine the petals across multiple flowers on was removed by 2–4 s of vacuum, and this step was
the same plant to obtain enough petal tissue for chem- repeated once to bring the total volume of plant extract
ical analysis. Samples within plants were combined, to 300 µL. The columns were washed four times with
weighed and processed, as described above. Because we 150 µL of 67% methanol, three times with 150 µL of
required large numbers of flowers of an appropriate water, and three times with 150 µL of 1  sodium acetate.
stage from a single individual to accumulate sufficient To desulphate the glucosinolates on the columns, we
biomass for petal analysis, not all plants could be added 10 µL of water and 10 µL of sulphatase solu-
used for petal analyses. We used a total of 21 maternal tion to each column, and the plates were incubated
families in the experiment. Sample sizes for chemical overnight at room temperature (Hogge et al. 1988). To
analysis ranged from one to seven samples per tissue elute the desulphoglucosinolates, the DEAE Sephadex
type per family. Each sample came from a single plant. was washed twice with 100 µL of 60% methanol and
A total of 139 tissue samples were analysed; 68 petal twice with 100 µL of water. We ran 40 µL of the glu-
samples and 71 leaf samples. cosinolate extract on a Hewlett-Packard 1100 series
As parents of families were heterozygous, progeny HPLC with a Hewlett-Packard Lichrocart 250–4 RP18e
from any single family often expressed multiple flower 5-µm column. Glucosinolates were detected at 229 nm
colours. For both flower and leaf samples, we maximized and separated and identified using the following pro-
the use of families with diverse progeny. Blocking on grammes with aqueous acetonitrile: (i) a 6-min gradi-
family allows us to detect differences among flower ent from 1.5 to 5.0% acetonitrile; (ii) a 2-min gradient
colour types while controlling for genetic background. from 5 to 7% acetonitrile; (iii) a 7-min gradient from 7
Of the 68 plants that provided petal samples, 14 were to 25% acetonitrile; (iv) a 2-min gradient from 25 to
bronze, 14 pink, 20 white and 23 were yellow, from a 92% acetonitrile; (v) 6 min at 92% acetonitrile; (vi) a 1-
total of 20 families. Because we needed copious petal min gradient from 92 to 1.5% acetonitrile; and (vii) a
tissue of specified stages, we could not use all the pro- final 5 min at 1.5% acetonitrile.
geny each maternal plant produced. We collected petal
samples of a single colour from four families, of two
 
different coloured progeny from nine families, and of
three different colours from seven families. Families Total glucosinolate concentration was estimated by adding
often had more diverse progeny than the ones we the concentration of all compounds, after conversion
sampled, but sufficient petal tissue may not have been to SI units of µg/mg leaf tissue. Conversion to µg/mg
available for progeny of all colours. from milli-absorption units was not possible for the two
Because we could readily collect leaf tissue, we sam- unknown compounds, but these comprised only 0.7%
pled more plants per family in order to take advantage of the total investment in glucosinolates prior to con-
of the diversity of progeny produced by families, but version to SI units; therefore, these compounds were not
sampled only 13 families in total. These were a subset included in our analysis of total glucosinolates. Con-
of the same families used for petal colour. Of 71 plants, centrations were log-transformed to meet assumptions
16 were bronze, 11 pink, 20 white and 21 yellow. Leaf of normality.
samples were collected from two families represented To investigate the differences between tissue types, the
by two colours in progeny, from four families represented relationship between colour morph and glucosinolate
by three colours, and from seven families represented content, and the effects of induction via herbivore
© 2004 British
by four colours. feeding on glucosinolates, we used maximum likelihood
Ecological Society, To quantify glucosinolate content, we followed the estimation (type III; PROC MIXED; SAS V.8) with
Journal of Ecology, basic sephadex/sulphatase glucosinolate extraction colour morph (bronze, pink, white, yellow), damage treat-
92, 132–141 and purification protocols described in Hogge et al. ment (50% removal on all leaves but 5th and 8th/no

5. 136 damage), and plant tissue (petals/leaves) as main effects, defensive properties against insects and non-ruminant
S. Y. Strauss, and plant family, family × damage and family × flower mammals (e.g. McDanell et al. 1988, 1989), and thus
R. E. Irwin & colour as random effects. Satterthwaite’s approxima- represents a good choice for examination a priori.
V. M. Lambrix tion was used to account for unequal sample sizes of
colour morphs and families. To test the significance of
Results
random effects in PROC MIXED, the best approach is
to run the model both with and without the random The following glucosinolate compounds were identified
factor(s) included in the model and then to use the like- as present in leaves and petals: 4-methylsulphinylbutyl
lihood ratio statistic (Littel et al. 1996). This statistic is (MSO), 4-methylsulphinyl but-3-enyl (MSOBUT), 4-
computed by taking difference between the REML log- methylthiobutyl (MT), 4-methylthio-but-3-enyl
likelihood of the model containing the random effect (MTBUT) and indol-3-ylmethyl (I3MTRP) glucosino-
and the log-likelihood of the model without the ran- late, plus two unknowns. All compounds were found in
dom effect. The critical value for this difference is half both leaves and petals of all colour morphs.
the probability of a greater chi-squared distribution
from a chi-squared distribution with one degree of free-
  
dom (Littell et al. 1996); i.e. the difference in REML
with and without the random factor in the model must Constitutively, colour morphs did not differ in total
exceed 2.71 at alpha = 0.05. For our data on total glu- glucosinolate content, and petal tissue contained about
cosinolate content, the difference between REMLs of 20% higher overall levels of glucosinolates than did leaves
models with the family × flower colour and the family (Table 1, Fig. 1a). When both damaged and undamaged
× damage interactions compared with the model with plants were included in the model, there was a highly
just family as a random factor was less than 1.5 and thus significant three-way interaction among flower colour,
neither interaction was significant. Family main effects damage and tissue type (Table 2a, Fig. 2a). Glucosinolates
were, however, significant (REML difference between were generally not induced in petals, except for in pink
models with and without random family effect = 4.6). morphs, but were highly inducible in leaves (Figs 1a
Although random family effects are included in all and 2a). Damage tended to increase glucosinolate
the models reported, F-statistics in the tables are only content by 28% compared with undamaged plants (back-
reported for fixed effects and random effects will not be transformed LS means, Table 2a), although this trend
included in tables. was only marginally significant (P = 0.07). Overall, pink-
We also wanted to explore tissue-specific differences flowered plants tended to show the greatest post-damage
in the expression of individual glucosinolates; unfortu- induction, and yellow morphs the least, in fact, a 5%
nately, multivariate, maximum likelihood methods to decrease (Fig. 2a). Overall, there was a marginally signi-
explore the overall changes in compounds are not available ficant damage ¥ flower colour interaction (Table 1a).
in PROC MIXED. Instead, we examined the two most
common glucosinolates: indol-3-ylmethyl glucosino-
  
late (I3MTRP) and 4-methylthio-but-3-enyl (MTBUT).
Together, these comprised 71% of the total glucosino- Patterns for MTBUT were generally similar to those
lates produced by plants. In addition, I3MTRP is an of total glucosinolate content (Table 2b, Fig. 1b) be-
indole glucosinolate, a class of compounds with known cause MTBUT was the most abundant glucosinolate.
Table 1 Results from PROC MIXED analysis of effects of tissue type and petal colour morph on the constitutive concentration
of glucosinolates (ln-transformed) in undamaged plants. Family and Family × flower colour were included in the model as
random factors, but those effects are not presented below (see Materials and methods). Convergence criteria were met.
Satterthwaite’s approximation to estimate degrees of freedom was used because of unbalanced representation of flower colour
among families and treatments
Effect DFNUM DFDENOM F-value P
(a) Total glucosinolates
Tissue 1 47.3 7.71 0.0078
Flower colour 3 23.8 1.03 0.3968
Tissue × flower colour 3 53.1 1.52 0.2201
(b) MTBUT
Tissue 1 48.9 7.35 0.0092
Flower colour 3 27.1 1.02 0.3974
Tissue × flower colour 3 54.5 1.07 0.3688
(c) I3MTRP
© 2004 British
Tissue 1 42 39.29 < 0.0001
Ecological Society,
Flower colour 3 24.4 0.11 0.9526
Journal of Ecology,
Tissue × flower colour 3 38.1 2.78 0.0539
92, 132–141

6. 137 Table 2 Results from PROC MIXED of effects of tissue type, petal colour morph and damage on the concentration of
Patterns of plant glucosinolates (ln-transformed). Family, Family × damage and Family × flower colour were included as random factors in the
variation in defence model; only family main effects were significant and are presented in the Materials and methods. Convergence criteria were met.
Satterthwaite’s approximation to estimate degrees of freedom was used because of unbalanced representation of flower colour
among families and treatments
Effect DFNUM DFDENOM F-value P
(a) Total glucosinolates
Tissue 1 103.0 0.08 0.7764
Flower colour 3 35.7 1.34 0.2764
Damage 1 19.1 3.77 0.0670
Damage × flower colour 3 107.0 2.37 0.0743
Tissue × flower colour 3 90.5 0.03 0.9946
Tissue × damage × flower 4 90.2 6.48 0.0001
(b) MTBUT
Tissue 1 84.5 0.00 0.9977
Flower colour 3 30.5 1.48 0.2403
Damage 1 12.9 1.76 0.2076
Damage × flower colour 3 107.0 1.05 0.3732
Tissue × flower colour 3 89.9 0.04 0.9879
Tissue × damage × flower 4 91.2 4.84 0.0014
(c) I3MTRP
Tissue 1 116.0 105.59 < 0.0001
Flower colour 3 114.0 0.95 0.4192
Damage 1 33.1 13.78 0.0008
Damage × flower colour 3 114.0 1.76 0.1597
Tissue × flower colour 3 104.0 0.81 0.4888
Tissue × damage × flower 4 107.0 2.94 0.0238
Fig. 1 Concentrations (in µg mg−1 dry mass) of (a) total Fig. 2 Concentrations (in µg mg−1 dry mass) of (a) total glucosino-
glucosinolates, (b) 4-methylthio-but-3-enyl (MTBUT ), and lates, (b) 4-methylthio-but-3-enyl (MTBUT), and (c) indol-3-
© 2004 British (c) indol-3-ylmethyl glucosinolate (I3MTRP) by tissue type and ylmethyl glucosinolate (I3MTRP) among tissue type, damage
Ecological Society, damage treatment in Raphanus sativus. Error bars represent treatment and colour morphs of Raphanus sativus. Error bars
Journal of Ecology, standard error. Raw least-squares means are presented here, represent standard error. Raw least-squares means are presented
92, 132–141 but analyses were conducted on ln-transformed data. here, but analyses were conducted on ln-transformed data.

7. 138 Constitutively, petals had 40% greater levels of MTBUT (Fig. 3, see also Irwin et al. 2003). Petals induced
S. Y. Strauss, than did leaves, there were no differences in constitutive indole glucosinolates less than leaves, and in bronze
R. E. Irwin & levels among flower colour morphs, nor was the inter- morphs there was no induction in petals (Fig. 3).
V. M. Lambrix action between flower colour and tissue type significant. Divergent behaviours among tissue types and colour
When both damaged and undamaged plants were included morphs in induction resulted in the highly significant
in the model, only the three-way interaction among three-way interaction among damage, tissue type and
tissue, damage and flower colour was significant flower colour (Fig. 1c, Table 2c). There was also a trend
(Table 2b). For all but the pink morphs, damage for damage to increase indole glucosinolate levels over-
generally caused a decrease in the amount of MTBUT all (P = 0.07).
in petals, but an increase in the amount of MTBUT in
leaves; decreases were particularly pronounced in yellow
Discussion
and bronze petal morphs (Fig. 2b).
Optimal defence theory predicts that tissues linked to
reproduction should be more highly defended than
  
leaf tissue because of their closer ties to plant fitness
Indole glucosinolates behaved very differently from (McKey 1979). We found that, constitutively (i.e. in the
MTBUT. Petals, overall, had 48% lower constitutive undamaged state), total glucosinolate concentrations
levels of indole glucosinolates when compared with in petals were indeed generally greater than in leaves;
leaves (undamaged plants; Table 1c, Figs 1c and 3). this effect was due primarily to the concentration of the
There was also a marginally significant interaction single, most abundant glucosinolate, MTBUT; how-
between tissue type and flower colour; constitutively, ever, petals had lower overall constitutive and induced
petals of pink flowers had the lowest indole glucosi- levels of indole glucosinolates. Neither the leaves nor
nolate content; in contrast, leaves of the pink morph the petals from which we took our measurements were
had the greatest concentration of this glucosinolate. damaged, so changes in glucosinolate content reflected
Leaf indole glucosinolates were highly inducible, systemic responses to damage. Our results are in
with pink and bronze morphs inducing more indole general agreement with other investigations of overall
glucosinolates than white and yellow morphs in leaves concentrations of defensive chemicals in leaf and
reproductive tissues. For plants in the diverse families
Solanaceae, Apiaceae, Asteraceae and Boraginaceae,
floral parts all had greater levels of secondary compounds
than did leaves (Nitao & Zangerl 1987; Van Dam et al.
1996; Zangerl & Rutledge 1996; Ohnmeiss & Baldwin
2000). Overall, these results suggest that patterns of total
glucosinolate expression match predictions of optimal
defence theory; however, the behaviour of individual
glucosinolates varies, and the key will be to understand
the function of individual compounds in relation to
plant fitness.
Another prediction of optimal defence theory is
that less valuable tissue, i.e. leaf tissue, should be more
inducible with damage. This prediction was also gen-
erally upheld. Damage to leaf tissue increased both
indole and MTBUT glucosinolates in leaf tissue much
more than it did in petal tissues. However, an alternative
explanation is that leaf tissue samples were collected
much closer in time to the damage event than were petal
samples, which were necessarily collected over a 2–3-
week period in order to obtain sufficient biomass for
chemical analysis.
Another non-adaptive hypothesis to explain responses
of chemicals in floral tissues is that they change as a by-
product of leaf induction (Adler 2000). Induction in R.
sativus petals was both colour morph- and compound-
specific. For MTBUT, in all but the pink morphs, petal
concentrations decreased or remained the same while
© 2004 British
leaf concentrations increased with damage; this result
Ecological Society, Fig. 3 Inducibility of glucosinolates by flower colour and suggests independent control of petal and leaf concen-
Journal of Ecology, tissue type. Inducibility is defined as percentage change from trations of MTBUT. In contrast, both petals and leaves
92, 132–141 undamaged state [(D/U × 100) − 100]. showed induction of the indole glucosinolates, with

8. 139 petals inducing much lower concentrations than leaves. agents on this flower colour polymorphism (see Irwin
Patterns of plant In this case, we cannot rule out non-adaptive induction et al. 2003). As a corollary, strong and consistent pref-
variation in defence in petal tissues of indole glucosinolates. The herbivores erences by pollinators for particular colour morphs,
damaging leaves and petals may differ, and induced as documented by Stanton (1987) and by Irwin and
responses may vary with herbivore. In our study, the P. Strauss for this population (unpublished data), suggest
rapae larvae we used to damage plants typically dam- that pollinators could also act to maintain variation in
age only leaves and fruits; in contrast, woolly bears and defensive chemistry. In this case, pollinators strongly
mollusk herbivores can damage both petals and leaf preferred less-defended, yellow petal morphs. These yellow
tissues of R. sativus (S. Y. Strauss, personal observation). petal variants exhibited the smallest degree of induction
How plant tissues respond to different herbivores may of glucosinolates in our study, and therefore may be
also explain differential tissue-specific induction. In selected against by herbivores. Both petal colour for this
general, very few studies have examined induction in petal species and glucosinolates are known to be heritable traits
tissue. This study, and one showing nicotine induction (Panetsos 1964; Carlson et al. 1985; Ishii et al. 1989;
in Nicotiana attenuata corollas (Euler & Baldwin 1996), Schuetze et al. 1999). Thus, conflicting selection pres-
show that there can be induction in petal tissue in response sures exerted by pollinators and herbivores, coupled
to leaf damage. with pleiotropy or tight linkage between colour and
Predictions of optimal defence theory rely on the defence loci, could maintain variation in both traits.
relationship between tissue type and fitness (Pavia et al.
2002). While we did not evaluate these relationships
Acknowledgements
here, results from previous experiments provide
strong evidence for the greater value of petals (Stanton We thank Dylan Burge for help with damage treat-
& Preston 1988; Young & Stanton 1990; Conner et al. ments and tissue collection. Lynn Adler, Rick Lankau,
1996; see Introduction) than of leaf tissue (Lehtila & Peter Klinkhamer and anonymous reviewers provided
Strauss 1999; Mauricio & Bowers 1990) to fitness. If, helpful comments on the manuscript. The work was
bite for bite, damage to petal tissue is more injurious supported by U.S. National Science Foundation grant
to plant fitness than is damage to leaf tissue, our data DEB 98–07083 to SYS. Partial support was also provided
support overall constitutive patterns of defence predicted by the Department of Genetics and Evolution, Max-
by optimal defence theory. Planck Institute of Chemical Ecology, Max-Planck
Another important aspect of our results is that col- Gesellschaft, as well as by travel funds provided by the
our morphs differed significantly from one another in Bodega Marine Laboratory, UC Davis.
the degree to which indole and MTBUT glucosinolates
were induced after damage, although there were no
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