Heredity 81 (1998) 546–555
Received 27 January 1998, accepted 11 May 1998
Phenotypic plasticity in the expression of
self-incompatibility in Campanula
rapunculoides
DONNA W. VOGLER, CHANDREYEE DAS & ANDREW G. STEPHENSON*
Department of Biology, The Pennsylvania State University, University Park, PA 16802, U.S.A.
Plants with self-incompatibly (SI) frequently exhibit variable expression of this trait. The study
reported here investigates the breakdown of SI in a perennial bellflower (Campanula rapunculoides) using a standard quantitative genetics approach to examine the relative influences of
genotype, prior fruit-set and floral age on seed-set from self-pollinations with standardized
pollen loads. Cross-pollen was used on separate flowers for comparison. The results obtained
indicate that genotype (clone) explained a significant amount of the total variation and plants
with few developing fruits showed stronger expression of SI on young flowers, and weaker
expression of SI on old flowers than plants with many fruits (fruit-by-floral-age interaction,
Ps0.02). A second experiment determined that the stigmatic curling accompanying floral age
does not influence expression of SI. A significant clone-by-floral-age interaction suggests that
continuous variation in self seed-set of putatively SI species may be the result of genotypeby-environment interactions. It is concluded that SI is a phenotypically plastic trait in C. rapunculoides and its breakdown responds to conditions that are indicative of low pollinator activity.
Keywords: breeding systems, gametic self-incompatibility, phenotypic plasticity, resource
allocation, selfing.
Introduction
Yet natural populations often exhibit marked
phenotypic variation in self-incompatibility, some of
which may be the result of environmental conditions
(e.g. Becarra & Lloyd, 1992; Reinartz & Les, 1994)
and some of which may be genetic in origin (e.g.
Rick & Chetelat, 1991). Breeding experiments with
cultivars of almond (Dicenta & Garcia, 1993), Phlox
(Bixby & Levin, 1996) and Brassica (Ruffio-Chable
et al., 1997) all suggest that genetic modifiers of the
S-locus may be unlinked to that locus and thus
explain the occurrence of continuous variation in
self-incompatibility. Consequently, the traditional
view of self-incompatibility as a qualitative trait is
challenged by evidence demonstrating that, for many
species, self-incompatibility can be quantitative and,
in part, moderated by environmental conditions
(Stephenson & Bertin, 1983; Levin, 1996).
The analysis of quantitative traits requires quantitative statistical methods, yet, surprisingly, the established tools of quantitative genetics, including
analysis of variance (ANOVA) and factorial designs,
have not been widely applied to the analysis of
factors that may affect self-incompatibility, although
they are well suited to the task. Basically, the analy-
Self-incompatibility is a widespread phenomenon
among flowering plants. Nearly half of the angiosperm families are reported to include species
exhibiting one of several forms of self-incompatibility (de Nettancourt, 1977). The major forms of
self-incompatibility (including heteromorphic, sporophytic and gametophytic) appear to be independently derived (Matton et al., 1994) and many
genera that contain self-incompatible species also
contain species that are self-compatible (e.g. Lycopersicon, Rick & Chetelat, 1991; Campanula,
Nyman, 1993a; and Brassica, Ruffio-Chable et al.,
1997). Models for the evolution of incompatibility
alleles typically assume simple (qualitative) inheritance (Charlesworth et al., 1990; Uyenoyama et al.,
1993) and recent studies of the molecular biology of
self-incompatibility indicate that both sporophytic
and gametophytic forms of self-incompatibility function as one- or two-locus multiallelic traits (reviewed in Newbigin, 1996; Richman & Kohn, 1996).
*Correspondence. E-mail: [email protected]
546
©1998 The Genetical Society of Great Britain.
PHENOTYPIC PLASTICITY IN CAMPANULA RAPUNCULOIDES 547
sis of the potential sources of variation in the
expression of self-incompatibility is a study of
phenotypic plasticity where the total phenotypic
variation observed in a population with respect to
some trait is attributed to variation among genotypes (genetic determination or heritability), and/or
variation induced by the environment (Schlichting,
1986). Developmental differences among flowers on
an inflorescence (ontogenetic plasticity) may also
contribute to phenotypic variation (Mazer & Delesalle, 1996) and there may be interactions between
sources
of
variation,
such
as
genotypeby-environment interactions, where the response to
changing environmental conditions is not uniform
among genotypes (de Jong, 1990). Phenotypic plasticity has been of continuing interest to evolutionary
biologists for decades as a potential explanation for
how organisms adapt to heterogeneous environments (reviewed in Schlichting, 1986). In the context
of self-incompatibility, phenotypic variation in the
ability to self-fertilize is thought to be advantageous
in small populations (where the number of S-alleles
is limited) and in environments with variable pollinator or resource conditions (see Lloyd & Schoen,
1992; Uyenoyama et al., 1993).
Previous studies have shown that Campanula
rapunculoides has a gametophytic self-incompatibility
system and an abundant stylar protein with a molecular weight, relative abundance, temporal and
spatial distribution and RNase activity similar to that
reported for the S-locus glycoproteins of the Solanaceae (Richardson et al., 1990; Stephenson et al.,
1992). Moreover, previous studies of plants from a
natural population of C. rapunculoides have revealed
that the flowers of most individuals are strongly selfincompatible when the stigmas first reflex, but
become more self-fertile as the flowers age
(Richardson et al., 1990). That is, C. rapunculoides
has an age-dependent breakdown in self-incompatibility. Here, the results of two experiments are
reported, which examine the effects of additional
environmental factors on the expression of selfincompatibility in C. rapunculoides.
Because developing fruits can draw resources
from later flowers in an inflorescence, they have the
potential to alter the expression of self-incompatibility in later-developing flowers on that inflorescence (Becarra & Lloyd, 1992; Reinartz & Les,
1994). In the first experiment, the effects are
examined of floral position within the inflorescence,
floral age, genotype and prior fruit-set on the
strength of the self-incompatibility system in
C. rapunculoides. By treating these factors in a
factorial design, it is possible to test for genetic vari© The Genetical Society of Great Britain, Heredity, 81, 546–555.
ation and phenotypic plasticity in self-incompatibility
(as main effects in the analysis of variance) and as
interactions among factors.
The second experiment tests whether morphological changes in the stigma of C. rapunculoides
contribute to greater success of self-pollen in older
flowers relative to young flowers. In Campanula, the
stigmatic lobes continue to spread and curl downwards as the flowers age (Fægri & van der Pijl, 1979;
Nyman, 1993a). Thus, pollinations on the first day of
stigmatic opening occur on the distal portion of the
stigmatic lobes, whereas pollinations on subsequent
days are more likely to be placed near the junction
of the stigmatic lobes and closer to the stylar canal,
which is the route pollen tubes travel towards the
ovary.
Using the same SDS-PAGE techniques as
Richardson et al. (1990), it was recently determined
that the putative S-protein of C. rapunculoides was
most concentrated in the stigmatic lobes (Vogler
et al., 1994). Thus, it was reasoned that on the first
day of stigmatic receptivity, pollen tubes must grow
through about 3 mm of the S-protein-rich stigma
lobe before reaching the stylar canal. In contrast,
pollinations 4 days later are more likely to be placed
closer to the stylar canal and, consequently, pollen
tubes may encounter less S-protein. By conducting
this study as a factorial, it was possible to separate
the effect of a change in floral morphology from the
separate effect of floral ageing. By using cloned individuals for replicates, interactions between floral age
and location of pollen placement (proximal vs. distal
stigma lobe) could be quantified, while controlling
for possible genotypic differences.
Materials and methods
The experimental species
Campanula rapunculoides L. (Campanulaceae) is a
naturalized perennial herb, locally abundant along
roadsides and open woods across the north-eastern
United States and Canada (Rosatti, 1986). It overwinters as a rosette and in July each rhizomatous
cluster produces one to eight flowering racemes
containing 20–30 blue, bell-shaped flowers which
open acropetally (bottom upwards). Campanula is
dichogamous: when the flower first opens, pollen is
deposited onto specialized stylar hairs (Nyman,
1993a) and the stigmatic lobes remain tightly
appressed. Thus, at anthesis, the flower is phenotypically male and cannot be pollinated. After several
days, during which the pollen is removed by bees
(primarily Bombus and Apis), the stigmatic lobes
548 D. W. VOGLER ET AL.
reflex and the flower becomes phenotypically
female. Richardson & Stephenson (1989) demonstrated that pollen removal shortens the duration of
the male phase and Richardson et al. (1990) and
Stephenson et al. (1992) quantified the agedependent breakdown of SI in the population of
plants used in the present study. They found that
self-pollinations on young flowers yield fewer than
15 seeds per capsule, whereas cross-pollinations
yield 60–80 seeds. But on older flowers, self-pollinations yield 19–45 seeds per capsule (Richardson
et al., 1990; Stephenson et al., 1992).
Floral position and fruiting experiment
During the summer of 1994, multiple clones of eight
genotypes were brought to flower in a greenhouse.
These eight genotypes were originally collected from
a natural population outside State College, PA
(described in Richardson et al., 1990) and propagated by rhizome cuttings. Eight to ten replicate
clones of each genotype were matched for overall
size and vigour and split into two treatment groups:
FRUIT, in which all flowers on the lower third of
the raceme were hand-pollinated with cross-pollen;
and NO FRUIT in which all flowers on the lower
third of the raceme were prevented from being pollinated by removal of the stigmatic lobes. In the
remaining two-thirds of the raceme, most of the
flowers of each plant continued to receive the
appropriate FRUIT/NO FRUIT treatments, but
4–12 flowers were selected and marked to receive
one of four experimental pollinations: (i) Self Day 1,
selfed on the first day of stigmatic opening; (ii) Out
Day 1, outcrossed on the first day of stigmatic
opening; (iii) Self Day 4, selfed on the fourth day of
stigmatic opening; and (iv) Out Day 4, outcrossed
on the fourth day of stigmatic opening.
The order of these four treatments was randomized for each plant, and each pollination was
performed on flowers that were bracketed by other
flowers receiving the appropriate FRUIT/NO
FRUIT treatment for that plant. Even with that
spacing, at least one and often two replicates could
be made for each of the four experimental pollinations/plant. As each of the designated treatment
flowers opened, pollen was removed daily with a
camel-hair brush to prevent self-pollination. Pollen
removal continued until the stigmatic lobes reflexed,
signalling the first day of stigmatic receptivity (Day
1). Flowers were either pollinated immediately or
marked for pollination three days later (Day 4).
Because of the unusual secondary pollen presentation mechanism of Campanula, mature pollen is
available only on the stylar hairs, and has its highest
viability prior to stigmatic opening. Therefore, the
styles of male-phase flowers were used for pollen
sources in the treatments. Pollen loads were standardized according to the method of Richardson et al.
(1990) to deliver 2500 pollen grains — an amount
just sufficient to produce a full complement of seeds
when cross-pollen is used. Self-pollen was obtained
from a flower on the same plant; cross-pollen was
obtained from one of two genotypes known to be
cross-compatible with the eight genotypes used in
this experiment. All flowers that were experimentally
pollinated were tagged to indicate the genotype,
type of pollination treatment, fruiting treatment of
plant and position of the flower on the raceme
(bottom third, middle third, upper third). Capsules
were allowed to ripen over a 1-month period; they
were then collected and the seeds counted.
Stigmatic lobe experiment
During the winter of 1995, clones of the same eight
genotypes used in the first experiment, plus an additional genotype, were brought out of the cold room,
repotted and placed in the greenhouse where they
began to bloom in mid-March. Four types of pollination treatments were performed; the age of flowers
at the time of pollination was varied as in the
previous experiment (i.e. Day 1 of the female phase
vs. Day 4 of the female phase) as well as the area of
the stigmatic lobe on which pollen was placed (i.e. at
the distal or proximal portion). In order to make the
Day 1 pollinations on the proximal portion of the
stigmatic lobe (near the junction of the three lobes),
it was often necessary to pull apart the stigmatic
lobes that were not completely reflexed. These
pollination treatments were conducted using selfpollen (n = 653) as well as cross-pollen from donors
in the same population (n = 677). The order of the
pollination treatments was random, and at least two
unpollinated flowers separated each pollinated
flower.
Statistical analyses
Histograms of seed-set data were used initially to
evaluate the overall shape and spread of the data.
Cross-pollinations were slightly skewed to the right
and a square-root transformation produced a normal
distribution of error variances from an analysis of
variance. In contrast, the self-pollinations were
severely leptokurtic. There were many zero seed and
low seed counts, as would be expected for a selfincompatible plant. Moreover, square-root trans© The Genetical Society of Great Britain, Heredity, 81, 546–555.
PHENOTYPIC PLASTICITY IN CAMPANULA RAPUNCULOIDES 549
formation failed to remove heteroscedasticity among
the genotypes. Interestingly, although other transformations [natural log (x+1) and negative reciprocal] likewise failed to eliminate heteroscedasticity,
they also produced similar interpretations from
ANOVA. This was interpreted to mean that the tests
for significant effects are relatively robust to these
departures from normality, as might be expected
given the large sample sizes. However, because the
data from self- and cross-pollinations differed with
respect to overall means, shape and response to
transformation, it was decided to treat these data
sets as separate populations for analysis. Differences
in the mean square error estimates in the separate
ANOVAs further support the need for separate analysis (Neter et al., 1990).
For the first experiment, a 4-factorial model was
initially used in the ANOVA to test for all main
effects and interactions of floral position (POS),
genotype (GEN), the presence of prior fruits (FNF)
and floral age (DAY) for both self- and cross-pollinations. However, neither the main effect for position nor any of its interactions was significant in this
full model. Because the inclusion of the position
term greatly complicated the model but did not
provide much information, all but the main effect
was eliminated for position in the final model, while
the remaining three main effects were retained with
all possible interactions among them.
In the second experiment, a 3-factorial design was
used to test for all main effects and all possible
interactions of genotype (GEN), location of pollination on the stigma (LOC) and floral age (DAY). As
in the first experiment, seed-set per capsule was
square-root-transformed and data from self-pollinations and cross-pollinations were analysed
separately.
The data from both experiments were analysed
using the SAS restricted mixed-model ANOVA, treating genotype and all interactions with genotype as
random variables (Fry, 1992). Approximate F-tests
were constructed for components in the model for
which there were no exact tests using the PROC
GLM command with the TEST option (SAS, 1990).
Least square means from the ANOVA were used to
quantify the strength of self-incompatibility for the
eight genotypes. The least square means were first
back-transformed and the average seed numbers
from self-pollinations were divided by the average
seed numbers from cross-pollinations as a index of
the self-compatibility (S-index), with low values
(near 0) indicating strong self-incompatibility and
high values (approaching 1) indicating selfcompatibility.
© The Genetical Society of Great Britain, Heredity, 81, 546–555.
Results
Floral position and fruiting experiment
(NOTE: All seed counts reported here are back-transformed least square means from the ANOVA.) Differences in seed-set were not attributable to position of
that flower on the inflorescence (bottom, middle vs.
upper) for either self- (P = 0.326) or cross-pollinations (P = 0.833, Table 1). Prior fruit-set (FNF) was
significant for cross-pollinations (P = 0.009) but not
for self-pollinations (P = 0.068, Table 1). Because
prior fruiting also had a significant interaction with
floral age for both self- (FNFÅDAY, P = 0.019) and
cross-pollinations (FNFÅDAY, P = 0.049), these
effects are best considered together (Fig. 1). This
interaction for self-pollinations (Fig. 1a) was of the
cross-over type, such that the flowers on plants with
no prior fruits produced the fewest seeds on Day 1
(NO FRUIT = 16.9 seeds/capsule vs. FRUIT = 22.7
seeds/capsule) but also the most seeds on Day 4
(NO FRUIT = 30.0 seeds/capsule vs. FRUIT = 24.2
seeds/capsule). Cross-pollinated flowers yielded
nearly the same number of seeds per capsule on
either FRUIT or NO FRUIT plants on the first day
of stigmatic receptivity (FRUIT = 90.2 vs. NO
FRUIT = 102.0 seeds/capsule, Fig. 1b). Yet on the
fourth day, fruiting plants produced far fewer seeds
from cross-pollinations than non-fruit treated plants
(FRUIT = 59.3 vs. NO FRUIT = 86.5 seeds/capsule,
Fig. 1b).
Genotypic variation among the eight clones tested
was significant in the seed-set from both self(Ps0.001) and cross-pollinations (P = 0.002,
Table 1). Moreover, there were significant differences for the effects of DAY (P = 0.009) and the
GENÅDAY interaction (P = 0.041) for the crosspollinations. For the self-pollinations, both DAY
(P = 0.068) and the GENÅDAY interaction
(P = 0.081) were marginally nonsignificant (Table 1).
Because the three-way interaction term was not
significant but the FNFÅDAY interaction was significant, the GENÅDAY interaction is illustrated in
Table 2 using only the data from NO FRUIT plants
for simplicity. This analysis revealed that genotypes
varied widely in seed production/capsule from both
self- (range 1–118 seeds) and cross-pollinations
(range 46–151 seeds, Table 2). Although most genotypes were strongly self-incompatible on the first day
of stigmatic opening, genotypes nos 3 and 14 were
able to produce nearly half as many seeds from selfpollinations (72 and 79 seeds, respectively) as
outcross (151 and 141, respectively). Self-pollinations on older flowers produced more seeds/capsule
than on young flowers for six out of the eight geno-
550 D. W. VOGLER ET AL.
types, and cross-pollinations on older flowers
produced fewer seeds/capsule for five out of the
eight genotypes. The variation in seed-set over the
course of floral ageing among the genotypes was
particularly high for cross-pollinations; for example,
genotypes nos 4 and 8 produced nearly the same
numbers of seeds per capsule when outcrossed on
either Day 1 or Day 4 (114.9 vs. 114.5 seeds/capsule,
and 72.3 vs. 75.7 seeds/capsule for genotypes nos 4
and 8, respectively, Table 2). Yet other genotypes,
including nos 3, 6 and 13, produced nearly one-third
fewer seeds as the flowers aged (Table 2).
Self-incompatibility, as measured by an index of
the ratio of self : outcross seeds for each combination of genotype and day (S-index, Table 2), is
strong in some genotypes and shows little breakdown over time (e.g. genotypes nos 4 and 13). At
the other extreme, two genotypes (nos 3 and 14) are
only weakly self-incompatible on the first day of stigmatic receptivity, and break down to nearly
complete self-compatibility by the fourth day
(S-index on day 4 = 0.95 and 0.84 for genotypes nos
3 and 14, respectively; Table 2). With the exception
of genotype no. 4, the S-index increased with
increased floral age (Table 2).
Stigmatic lobe experiment
As in the first experiment, there were significant
differences in seed-set among genotypes for selfpollinations (P = 0.001) and for cross-pollinations
(P = 0.035, Table 3). Similarly, as in the first experiment, floral ageing resulted in more seeds set per
self-pollination (5.7 vs. 9.9 seeds) and fewer seeds
set per cross-pollination (86.5 vs. 65.2 seeds). These
differences, however, were not statistically significant
for either self- or cross-pollinations (P = 0.174,
P = 0.100, respectively).
The location of the pollen deposition on the stigmatic surface (distal vs. proximal portion of the
stigma lobe) was not a significant main effect for
either self- (P = 0.222) or cross-pollinations
Table 1 Fruiting experiment results of mixed-model analysis of variance (ANOVA) for (a) self- and (b) cross-pollinations of
Campanula rapunculoides. Main effects are: position of the flower on the raceme (POS), genotype of the seed parent
(GEN, a random effect), prior fruit-set of the plant, fruiting or nonfruiting (FNF), and floral age in days since stigmatic
receptivity, 1 vs. 4 (DAY)
Source
d.f.
Type III SS
MS
F-value
P-value
(a) ANOVA for self-pollinations
POS
GEN
FNF
DAY
GENÅFNF
GENÅDAY
FNFÅDAY
GENÅFNFÅDAY
ERROR
TOTAL
2
7
1
1
7
7
1
7
620
653
19.02
6063.41
0.32
74.84
47.63
127.86
47.71
41.72
5249.46
11831.12
9.51
866.20
0.32
74.84
6.80
18.26
47.71
5.96
8.46
1.12
45.39
0.04
4.42
1.14
3.06
7.56
0.70
0.326
0.001
0.834
0.068
0.433
0.081
0.020
0.669
R-squared
0.556
CV
52.94
Root MSE
2.90
Mean
5.49
Back-transformed mean
30.2
(b) ANOVA for cross-pollinations
POS
GEN
FNF
DAY
GENÅFNF
GENÅDAY
FNFÅDAY
GENÅFNFÅDAY
ERROR
TOTAL
2
7
1
1
7
7
1
7
644
677
4.94
1886.31
179.83
245.47
115.19
159.76
30.73
38.74
8690.17
11156.27
2.47
269.47
179.83
245.47
16.45
22.82
30.74
5.54
13.49
0.18
7.98
11.17
11.32
2.97
4.12
4.73
0.89
R-squared
0.22
CV
38.96
Root MSE
3.67
Mean
9.49
Back-transformed mean
89.7
0.833
0.002
0.009
0.009
0.087
0.041
0.049
0.896
© The Genetical Society of Great Britain, Heredity, 81, 546–555.
PHENOTYPIC PLASTICITY IN CAMPANULA RAPUNCULOIDES 551
(P = 0.445). However, there was significant variation
among the genotypes for this response in the selfpollinations (GENÅLOC, P = 0.022, Table 3).
When self-pollen was applied to the proximal
portion of the stigmatic lobe, three out of nine genotypes yielded a greater number of seeds than pollinations on the distal portion, as predicted. Four
genotypes (nos 3, 8, 9 and 14) had greater fertilization success when self-pollen was applied to the
distal portion (Table 4). These four genotypes were
not similar in S-index; genotypes nos 3 and 14 had
moderately high S-indices of 0.60 and 0.51, respectively, whereas the others had relatively low
S-indices (S-indexE0.05 for genotypes nos 8 and 9).
ment (30.2 vs. 7.3 seeds/capsule, Tables 1 and 3,
respectively), but practically the same for the crosspollinations (89.7 vs. 84.6 seeds/capsule, means of
Tables 1 and 3, respectively). Because both experiments included the same eight genotypes, the clonal
repeatability of self seed-set was tested by comparing the data from Table 2 with the back-transformed
least squares means from the second experiment.
The correlation of seed-set by genotypes between
experiments was high (r = 0.89), and the linear
regression of the least squares means from the first
experiment on the least squares means of the second
experiment was highly significant (P = 0.004) and
explained 77% of the variance (data not shown).
Clonal repeatability
Discussion
Average seed-set per self-pollination from the first
experiment was higher than in the second experi-
Plant breeders have discovered a number of ways to
circumvent self-incompatibility in order to produce
inbred lines in crop plants that are normally selfincompatible (reviewed in de Nettancourt, 1977),
some of which are likely to operate in natural
environments. Many of these conditions occur late
in the season, including floral ageing and temperature variations; conditions which, coupled with lack
of fruit-set, may produce a flexible breeding system
where selfing becomes possible only after most
opportunities for outcrossing have passed. This
would produce a breeding system known as delayed
selfing, a strategy which Lloyd & Schoen (1992)
suggested has the most advantages and least
disadvantages for species with mixed mating.
Plants with delayed selfing must have two sequential pollination phases: (i) there must be a period
where cross-pollen can be received but self-pollination and/or self-fertilization is restricted; and (ii) late
in floral life, the earlier barriers to self-fertilization
must be reversed or broken down. That is, the plant
must be phenotypically plastic with respect to the
ability to be self-fertile. In plants with a morphological or temporal separation of stigma and anthers,
delayed selfing requires coordinated development of
styles, stigma and/or filaments to close the gap of
stigma–anther separation, as has been shown for
self-compatible species of Campanula (Fægri & van
der Pijl, 1979; Nyman, 1993b) and, more recently, in
Hibiscus (Klips & Snow, 1997). For SI species,
delayed selfing further requires a reduction or
circumvention of S-proteins in the style. At the
molecular level, self-incompatibility may be
controlled by the breakdown of S-RNases or the
secondary action of modifiers, which have been
implicated in the loss of self-incompatibility in cultivars of Petunia (Ai et al., 1990). Based on the
Fig. 1 Seed production from (a) self- and (b) cross-pollinations performed on plants with prior fruit development
(fruit) and those with no prior fruits (no fruit). Error
bars¹standard error.
© The Genetical Society of Great Britain, Heredity, 81, 546–555.
552 D. W. VOGLER ET AL.
Table 2 Seed production per pollination for self and outcross pollinations on young (Day 1) and old (Day 4) flowers of
Campanula rapunculoides from non-fruit-treated plants of experiment 1. Data are back-transformed least squares means
from ANOVA (Table 1) with minus one and plus one standard error in parentheses. Self-incompatibility index is the ratio of
self : outcross seeds/capsule
Day 1 pollinations
Genotype
3
14
5
6
1
8
4
13
Day 4 pollinations
Self
Outcross
S-index
Self
Outcross
S-index
72.2
(61.6–83.7)
79.2
(70.4–88.5)
9.0
(6.1–12.4)
2.3
(0.2–6.3)
11.5
(7.3–16.8)
4.4
(1.9–7.9)
17.6
(13.1–22.8)
1.2
(0.05–3.9)
151.3
(129.9–174.2)
141.6
(125.4–158.7)
77.4
(67.2–88.4)
106.1
(86.5–127.7)
64.0
(51.8–77.4)
72.3
(59.3–86.5)
114.5
(100.1–129.9)
108.6
(90.3–127.7)
0.48
106.1
(91.2–122.1)
118.8
(108.6–129.5)
36.1
(29.6–43.0)
17.6
(10.2–27.0)
10.9
(6.8–15.8)
14.4
(9.7–20.1)
11.6
(7.1–16.9)
2.3
(0.3–6.1)
110.3
(94.1–127.7)
136.9
(121.0–153.7)
84.6
(73.9–96.0)
72.3
(59.3–94.0)
46.2
(33.6–60.8)
75.7
(62.4–90.3)
114.9
(98.0–132.3)
64.0
(49.0–81.0)
0.95
0.55
0.14
0.05
0.21
0.09
0.17
0.03
0.84
0.45
0.28
0.26
0.22
0.12
0.07
Table 3 Stigma experiment results of mixed-model analysis of variance (ANOVA) for (a) self-, and (b) cross-pollinations of
Campanula rapunculoides. Main effects are: genotype of the seed parent (GEN, a random effect); location of the
pollination, distal vs. proximal, on the stigma (LOC); and floral age in days since stigmatic receptivity (DAY)
Source
d.f.
Type III SS
MS
F-value
P-value
(a) ANOVA for self-pollinations
GEN
LOC
DAY
GENÅLOC
GENÅDAY
LOCÅDAY
GENÅLOCÅDAY
ERROR
TOTAL
8
1
1
8
8
1
8
426
461
1979.52
4.87
47.64
23.13
194.50
0.09
4.97
1243.21
4171.41
247.44
4.87
47.64
2.89
24.31
0.09
0.62
2.92
9.30
1.68
2.21
4.65
39.13
0.09
0.21
0.001
0.222
0.174
0.022
0.001
0.756
0.989
R-squared
0.70
CV
63.36
Root MSE
1.71
Mean
2.69
Back-transformed mean
7.3
(b) ANOVA for cross-pollinations
GEN
LOC
DAY
GENÅLOC
GENÅDAY
LOCÅDAY
GENÅLOCÅDAY
ERROR
TOTAL
8
1
1
8
8
1
8
139
171
782.38
2.57
48.33
28.02
127.19
17.20
57.73
97.79
2.57
48.33
3.50
15.89
17.20
7.21
8.02
0.61
3.29
0.48
2.20
2.34
0.91
R-squared
0.52
CV
30.57
Root MSE
2.82
Mean
9.21
Back-transformed mean
84.6
0.035
0.445
0.101
0.837
0.142
0.152
0.510
© The Genetical Society of Great Britain, Heredity, 81, 546–555.
PHENOTYPIC PLASTICITY IN CAMPANULA RAPUNCULOIDES 553
present study, it is suggested that in C. rapunculoides
the environmental cues signalling the breakdown of
SI include a lack of pollen removal (a trigger
involved in stigmatic development; Richardson &
Stephenson, 1989) and resource allocations within
the inflorescence in response to fruit development.
Interestingly, both conditions would occur during
times of low pollinator activity, and hence would be
associated with fewer opportunities for outcrossing.
The results obtained from this study show that as
flowers age, self-pollination becomes more successful in terms of numbers of seeds set, whereas crosspollinations become less successful, and the
response is greater on plants with few developing
fruits (Fig. 1). Thus, self-incompatibility in C. rapunculoides is most plastic when ovule numbers are the
highest. The greater success of self-pollination on
aged flowers on plants with low fruit development
may be the result of the greater number of ovules
available for fertilization (self or otherwise), or
perhaps the ovules are longer lived and the slower
growing self-pollen tubes can reach them.
Thus the results of this study demonstrate that
C. rapunculoides has some capacity for delayed
selfing, both as a result of floral ageing (also shown
Table 4 Seed production per pollination on distal and
proximal portions of the stigmatic lobes for nine
genotypes of Campanula rapunculoides. Genotypes are
ranked according to their self-incompatibility index
(S-index) using seed-set from self- and cross-pollinations
on older flowers from this experiment. Data are backtransformed least squares means (¹SE) from ANOVA
(Table 3)
Genotype S-index
3
0.60
14
0.51
5
0.16
6
0.06
1
0.06
8
0.05
4
0.05
9
0.03
13
0.02
Proximal
Distal
73.4
96.9
(88.3–105.9) (65.9–81.3)
21.8
26.9
(19.1–24.7) (23.9–30.1)
7.4
4.4
(5.9–9.1)
(3.2–5.7)
4.7
3.4
(3.5–6.3)
(2.4–4.7)
2.2
0.9
(1.1–3.5)
(0.3–1.8)
3.8
4.5
(2.5–5.5)
(3.0–6.3)
2.9
2.9
(1.9–4.0)
(1.8–4.1)
1.2
1.4
(0.7–2.0)
(0.7–2.2)
0.1
0.1
(0.1–0.7)
(0.1–0.7)
Difference
+23.5
µ5.1
+3.0
+1.3
+1.3
µ0.7
0
µ0.2
0
© The Genetical Society of Great Britain, Heredity, 81, 546–555.
by Richardson et al., 1990) and also with respect to
an interaction between floral age and prior fruit
development (Fig. 1). In young (unpollinated)
flowers, the development and provisioning of prior
fruits may be drawing resources away from later
flowers, reducing the production of ovules and relaxing the maintenance of self-incompatibility (Fig. 1).
Inversely, in plants with few/no developing fruits,
more ovules are available for fertilizations in individual, young flowers but those flowers also exhibit the
strongest expression of self-incompatibility. In
C. rapunculoides, as well as in other species with the
Solanaceous type of gametophytic self-incompatibility, the S-proteins controlling the expression of
self-incompatibility are often the most abundant
proteins in the style (Newbigin, 1996). Thus it is
possible that the resource allocations to successful
prior fruits may reduce allocation to S-protein
production in later flowers.
The position of the flower on the raceme did not
significantly alter the expression of self-incompatibility when the effects of prior fruit-set were also
considered (Table 1). This was interpreted to mean
that the development of prior fruits adjacent to a
newly opened flower is more important than where
that flower is located on the raceme. Under conditions of high fruit-set early in the season, laterdeveloping flowers would exhibit a ‘position’ effect,
not by virtue of their position on the raceme, but by
the presence of prior fruits. As Mazer & Delesalle
(1996) point out, positional or developmental effects
must be experimentally separated from effects
caused by seasonal or resource environments if
sources of variation in phenotypically plastic traits
are to be resolved.
No evidence was found to show that self-incompatibility in C. rapunculoides is moderated by
morphological changes in stigmatic curling
(Table 3). Specifically, the present study revealed
that if self-pollen contacts the distal portion of the
stigma by autogamous self-pollination, it is no more
at a disadvantage than pollen placed nearer the
stylar canal (location on stigma, LOC, not significant, Table 3). Curiously, the ANOVA results revealed
a significant stigma-location-by-genotype interaction
for self-pollen, meaning that seed-set was higher
when pollen was placed on the distal portion of the
stigma in some genotypes, but in other genotypes
seed-set was higher when pollen was placed on the
proximal portion of the stigma (Table 3). Because
this variation appears to be unrelated to the S-index
(Table 4), this may be more indicative of genotypic
variation in the location of the ‘sweet spot’ for
pollen receipt generally rather than variation in
554 D. W. VOGLER ET AL.
S-protein concentrations. The results obtained by
the present study, however, concur with Nyman
(1993b) in that stigmatic curling in Campanula can
lead to autogamous delayed selfing.
This present study also reveals significant genotypic variations in the ability to set seeds from selfand cross-pollinations (Tables 1 and 3). Despite the
limited number of individuals examined, the range
of self-incompatibility phenotypes included strongly
self-incompatible and nearly self-compatible individuals, especially on older flowers (S-index range
0.07–0.95, Table 2). The strong correlation among
genotypes between experiments (r = 0.89) suggests
that the magnitude in the strength of self-incompatibility is heritable. Moreover, the presence of a
significant genotype-by-day interaction in the data
from self-pollinations (Table 3) suggests that there is
variation within this population with respect to plasticity in the temporal breakdown of self-incompatibility. If selection favours plasticity in the expression
of self-incompatibility, then this trait has some
potential to evolve in C. rapunculoides.
Acknowledgements
We thank Tony Omeis and his staff at the Buckhout
Greenhouse, Matt Herbison and Christine DiFolco
for greenhouse and laboratory assistance, and James
Rosenberger for statistical advice. This research was
supported by NSF Grants DEB 93–18224 and DEB
95–27739 to A.G.S.
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