Journal of
Ecology 2005
93, 373–383
Effects of seed size, inbreeding and maternal sex on
offspring fitness in gynodioecious Plantago coronopus
Blackwell Publishing, Ltd.
HANS PETER KOELEWIJN and JOS M. M. VAN DAMME*
ALTERRA – Wageningen UR, Centre for Ecosystem Studies, PO Box 47, 6700 AA Wageningen, the Netherlands, and
*Department of Plant Population Biology, Netherlands Institute of Ecology, Heteren, the Netherlands
Summary
1 Male steriles (MS) must have a fitness advantage relative to hermaphrodites (H) if
they are to be maintained in gynodioecious species. We report experiments in which we
disentangle the relative contributions of seed size, inbreeding and maternal sex to the
fitness advantage of male steriles in Plantago coronopus L.
2 Seed size effects were observed throughout growth experiments in the glasshouse and
were reflected in all biomass measurements. In the field, seed size effects resulted in a
fourfold increase in standardized seed production per initial buried seed after 2 years
between small (mean weight = 0.13 mg) and large (0.20 mg) seeds.
3 Inbreeding depression, calculated from seed to seed was δ = 0.37 after one generation
of selfing and δ = 0.93 after the second generation of selfing. Regression of log(1 − δ) on
inbreeding level suggested synergistic epistasis in fitness.
4 Even after taking into account the effects of seed size and inbreeding level, the offspring of a male sterile mother had a 16% advantage over a hermaphrodite, but this
disappeared when the progeny sex ratio (the ratio of MS : H individuals among the offspring) was taken into account.
5 In the field, offspring of large seeds had both a higher overall incidence of flowering,
and a higher probability of flowering in their first year, thus generating an extra cohort
of individuals. The high inbreeding depression in fitness after two generations of selfing
was also due to a very low incidence of flowering among the S2 individuals. Flowering
probability therefore appears to be a critical trait in this system.
6 In the field, the contributions of seed size variation (15%) and inbreeding (9%)
combine with 48% higher seed production to give a total fitness advantage of 70% of
male steriles relative to hermaphrodites. This is probably sufficient for maintenance of
gynodioecy under nuclear-cytoplasmic inheritance of male sterility.
7 Both inbreeding effects (as a consequence of the sexual system) and pleiotropic effects
(of the genes coding for male sterility) play a role in the maintenance of gynodioecy in
this species, with an apparently greater role for the latter.
Key-words: compensation, flowering, germination, growth analysis, inbreeding depression, male sterility, maternal sex, seed size, sex ratio, spatial variation
Journal of Ecology (2005) 93, 373–383
doi: 10.1111/j.1365-2745.2004.00940.x
Introduction
Gynodioecy is a sexual system in which hermaphroditic (H) and male sterile (MS, functionally female)
individuals coexist in an interbreeding population. As
male steriles can only transmit genes to the next generation through ovules, they are at a disadvantage
compared with hermaphrodites that pass on copies
© 2005 British
Ecological Society
Correspondence: Hans Peter Koelewijn (tel. +31 317477924,
fax +31 317419000, e-mail [email protected]).
through both ovules and pollen: to persist in natural
populations they must incur a compensatory advantage for this asymmetry in gene transmission, with the
amount of compensation required depending on the
mode of inheritance (Lewis 1941; Charlesworth 1981).
The necessary compensatory advantage may be
obtained directly via greater lifetime seed production
(i.e. a sex-related difference) or indirectly via higher
quality seeds (i.e. an offspring quality effect). Two
main hypotheses have been formulated to explain the
maintenance of gynodioecy (Lloyd 1975). First, as
male steriles are obligate outcrossers, they might suffer
374
H. P. Koelewijn &
J. M. M. van
Damme
© 2005 British
Ecological Society,
Journal of Ecology,
93, 373–383
less from inbreeding depression compared with (at least
partially) selfing hermaphrodites. Self-fertilization
might reduce the seed set of hermaphrodites or inbred
seeds may exhibit reduced fitness due to increased
homozygosity or expression of recessive alleles with
deleterious effects. Alternatively, the advantage of male
steriles may originate from pleiotropic effects of the
sterility genes if the expression of these genes influences
other fitness-related life-cycle characters. Because male
sterile plants are relieved of the cost of male function, the
additional resources available might compensate their
genetic disadvantage to increase their female function
by producing more and / or larger seeds (‘compensation
hypothesis’ sensu Darwin 1877; Ashman 1994; Poot 1997 ).
Pleiotropic effects may also be expressed as differences in vegetative development, such as more efficient
use of limiting resources, a higher growth rate or more
general effects (e.g. higher competitive ability) that can
lead to a higher future reproductive success (Ashman
1992; Eckhart & Chapin 1997; Delph et al. 1999). Provided sterility genes are expressed during early stages of
growth, effects on fitness-related plant characteristics
could cause a difference in performance between MS
and H individuals. These differences, which will be
reflected in a difference in performance between the
offspring originating from a H × H and MS × H cross
when there is a difference in sex ratio (MS : H) among
the offspring of these crosses, suggest a maternal sex
effect. Reports of maternal sex effects in gynodioecious
species are equivocal: several studies report that offspring of females germinate in higher proportions
or grow larger than offspring of hermaphrodites (e.g.
Assouad et al. 1978; Ashman 1992; Wolfe & Shmida 1997),
while others report no differences (e.g. Kohn 1988;
Eckhart 1992; Sakai et al. 1997; Mutikainen & Delph
1998). Part of these inconsistencies is due to confounding
the effects of progeny sex ratio and differences in seed size,
thus making it difficult to ascribe the observed difference
solely to a maternal sex effect. However, even in studies
where care was taken to control for confounding effects,
contradictory results are reported (Delph et al. 1999).
Seed size is an important component of life history
in plants ( Harper et al. 1970; Michaels et al. 1988), as
even a small variation may influence seedling emergence
( Winn 1988), seedling growth and survival (Wulff 1986a;
Winn 1988; Bell et al. 1991), seedling competition
( Wulff 1986b; Geritz 1995) and fecundity (Wulff 1986a).
Male steriles tend to produce larger seeds than hermaphrodites in some gynodioecious species (van Damme
1984; Agren & Willson 1991; Klinkhamer et al. 1994;
Wolfe & Shmida 1997) but not in others (Ashman
1992; Eckhart 1992; Sakai et al. 1997; Delph et al. 1999;
Delph & Mutikainen 2003), although self-fertilization
(in self-compatible species) might be expected to give rise
to smaller seeds (Sakai et al. 1997). A positive maternal
effect on seed size in male steriles might thus contribute
to their maintenance in gynodioecious populations.
Inbreeding depression, or the reduction in fitness of
self-fertilized progeny relative to outcrossed progeny, is
considered to be an important selective force governing
the evolution and maintenance of reproductive systems
that enhance cross-fertilization (Lande & Schemske
1985; Husband & Schemske 1996). Self-fertilization
causes deleterious mutations to be expressed in homozygotes, and both the number of such mutations and the
interaction between mutations at different loci (epistasis) influence the level of inbreeding depression (Crow
1970; Charlesworth 1990). When deleterious mutations
act synergistically, such that the log fitness declines at a
greater than linear rate with the number of mutations,
the equilibrium level of inbreeding depression is increased
over that expected if mutations acted independently
(Charlesworth et al. 1991). Because it is often impossible
to determine this relationship directly for individuals
in natural populations, epistasis among deleterious
alleles can only be studied indirectly by monitoring the
effect of different levels of inbreeding on fitness components (Willis 1993) and testing for a non-linear negative relationship between log-transformed fitness and
the expected inbreeding coefficient (Crow & Kimura
1970). Results are, however, equivocal, with inbreeding
effects in hermaphrodites demonstrated to play a role
in maintaining the presence of male steriles in a number of
self-compatible gynodioecious species (e.g. Kohn 1988;
Maki 1993; Sakai et al. 1997; Thompson et al. 2004),
but differences being absent, or too small to play an important role, in others (e.g. Ashman 1992; Eckhart 1992).
This study is concerned with the effects of seed size,
inbreeding and maternal sex on offspring fitness in
the gynodioecious and self-compatible plant Plantago
coronopus. All three factors could contribute to the
maintenance of the gynodioecious breeding system,
but have rarely been studied together. Previous studies
have regularly found differences in quality of offspring
from female and hermaphrodite plants (see Shykoff et al.
2003 for a meta-analysis), although many used openpollinated seeds (confounding maternal sex with inbreeding effects) or neglected seed size differences, and
straightforward interpretation of the results was thus
not possible. We therefore controlled for seed size differences and employed controlled matings in experiments
carried out under optimum nutrient conditions in growth
chambers and in the field. The main questions addressed
were: (i) What are the consequences of variation in seed
size for plant performance during the course of the
experiments? (ii) What are the consequences of prolonged inbreeding for offspring fitness? (iii) Is there an
independent maternal sex effect on offspring performance? (iv) To what extent do these different effects
contribute to the fitness advantage of male steriles,
needed for their coexistence with hermaphrodite plants.
Materials and methods
 
Plantago coronopus is an herbaceous, short-lived perennial
rosette plant with indeterminate growth that mainly
375
Size, inbreeding
and sex effects on
offspring
occurs in salt marshes along the Dutch coast. The species
has perfect flowers on top of long flowering stalks (spikes),
which consist of a stem and an ear part. Each leaf axil has
an axillary meristem that can produce either a spike or a
lateral side rosette, or stay dormant. It is a wind pollinated,
gynodioecious and predominantly outcrossing species
(Koelewijn 1998). Male steriles (MS) are frequently
observed in most populations (Koelewijn & Van Damme
1996), though frequencies never exceed those of hermaphrodites ( H). The species flowers from the beginning of May
through to September and overwinters as a rosette. The
flowers are protogynous, with an overlap in sexual phase.
Flowering and subsequent maturation occur from the base
of the ear upwards and each flower produces five ovules.
Mean seed weight in P. coronopus is on average
18% higher in male steriles than in hermaphrodites and
self-fertilization of hermaphrodites produces slightly
smaller seeds compared with cross-fertilization (average
8%). Although small, and variable, among experiments
and treatments (summarized in Table 1), both results
were significant when calculated over all experiments
using either a combined probability test (Sokal & Rohlf
1980) or a meta-analysis (Hedges’ d, the standard metaanalytical metric; Hedges & Olkin 1985).
   
Maternal plants were germinated from seeds of plants
collected at the Westplaat, a salt-meadow in the southwest of the Netherlands (Koelewijn & Van Damme 1995a).
One hermaphrodite and one male-sterile individual were
selected from the offspring of each of five collected plants.
The male-sterile maternal individuals were crossed with
mixed pollen from a random selection of hermaphrodites
(outcross MS × H, expected inbreeding coefficient
F = 0). Hermaphrodites were outcrossed (H × H, F =
0) or self-pollinated (S1, F = 0.5) and five S1 seeds from
each mother were subsequently sown and self-pollinated
(S2, F = 0.75). After ripening, seeds were taken out of
the capsules, pooled per cross type, and divided into
two groups. One group was used as an unsorted control
group, while the other group was separated into seven
seed size classes (class range 0.02 mg, class means from
0.11 to 0.21 mg and > 0.22 mg; Fig. 1) by means of an
Astell Hearson SCB016 Ottawa seed blower (Waterreus
C.V., The Hague, the Netherlands). Seeds were stored
in a cold room at 4 °C until further usage. No difference
in seed set was observed among the cross types.
  (  )
© 2005 British
Ecological Society,
Journal of Ecology,
93, 373–383
The aim of the experiment was to compare the performance of seeds from two size classes (small, 0.12–
0.14 mg; large, 0.18 – 0.22 mg) and three cross types
(MS × H, H × H and S1) during the juvenile stage of
plant development, i.e. a classical growth analysis (Evans
1972). Seeds were germinated in a climate cabinet on
wet (demineralized water) glass beds (day = 16 hours,
20 °C; night = 8 hours, 15 °C). After 14 days 60 seed-
Fig. 1 Size frequency distribution of seeds of Plantago coronopus
originating from four different cross types: male steriles crossed
with hermaphrodites (MS × H); hermaphrodites crossed with
hermaphrodites (H × H); self-fertilization of the hermaphrodites
(S1) and self-fertilization of offspring from these S1 plants
(S2). Also indicated are the three categories of seed size (small,
medium and large) used in the experiment.
lings of each combination of size class and cross type
were transferred to three separate growth units placed
together in a growth room maintained at 14/10 hours
day/night, 23° ± 0.5 C day/night, quantum flux density
at mean plant height 305 µmol m−2 s−1 (photo synthetically active radiation) and 70% relative humidity. Light
was provided by fluorescent lamps and incandescent
bulbs. Each growth unit was connected to a 5-L container of a 0.125 strength Hoagland nutrient solution
(625 µ KNO3, 625 µ Ca(NO3)2, 250 µ MgSO4,
125 µ KH2PO4, 90 µ Fe-EDTA, 45 µ H3BO3,
9 µ MnCl2, 0.75 µ ZnSO4, 0.5 µ Na2MoO4 and
0.3 µ CuSO4; Hewitt 1966) whose pH was automatically adjusted to 6.0 by a Radiometer pH-stat unit
(Radiometer Analytical, Lyon, France) using H2SO4. The
nutrient solution was applied as a mist (see Freijsen
et al . 1990 for further details). To prevent nutrient
depletion, the solution was renewed each week. Seedlings for the growth units were randomly selected and
the remainders were pooled in groups of 10 to estimate
seedling dry weight. To prevent root or shoot competition the plants were gradually moved apart over the
course of the experiment as harvesting reduced the
initial number of 120 plants. To minimize the effect of
spatial variation in growth conditions the units were
regularly rotated within the growth room. At 4, 8, 11,
14, 16 and 18 days after transfer to the growth unit, 27
plants per size class (nine from each cross type), were
selected and, when possible, dissected into roots and
leafs. The experiment was finished 3 days after initiation
of spikes was first observed. Dry weights were determined on oven-dried (48 hours at 70 °C) material.
Because no significant differences in weight or growth
rate were observed among the three growth units, the
data were analysed as a completely randomized design
(procedure GLM, SAS 1988). A full factorial model
including time, cross type, seed size and their interactions was fitted. Data were log transformed prior to
376
H. P. Koelewijn &
J. M. M. van
Damme
Table 1 Summary of experiments with Plantago coronopus where seed weight (mean ± SE, number of plants) was determined as
a function of either sex morph (MS or H) or inbreeding level (outcross (H), one (S1) or two (S2) generations of selfing). Paired t-tests
were used when crosses were made on the same plant or when the relatedness among the maternal parents was known (sibling
comparison, i.e. male sterile and hermaphrodite parent originate from the same maternal plant). Combined analysis over all studies
was done in two ways: (i) a combined probability test (Sokal & Rohlf 1980); (ii) calculate Hedges’ d from a random-effects model
meta-analysis (Hedges & Olkin 1985). When d is positive male steriles have a larger effect size than hermaphrodites or outcrossing
has a larger effect size than selfing. If 95% confidence intervals (CIs) do not include 0 the effect is significant over all studies
Type of experiment
MS
H
S1
S2
MS vs. H
(A) Random selection of plants
Growth (glasshouse, water
129.1 ± 9.3
culture, free access to
(n = 11)
nutrients; µg C)a
122.8 ± 4.9
(n = 29)
Two sample t-test
t38 = 0.64
P = 0.527
Growth (glasshouse,
water culture, limiting
access to nutrients; µg C)a
111.0 ± 8.2
(n = 13)
89.2 ± 2.7
(n = 30)
Two sample t-test
t41 = 2.79
P = 0.008
Hand pollination (field; mg)b
0.130 ± 0.008 0.094 ± 0.006
(n = 9)
(n = 20)
Growth analysis (glasshouse,
water culture; mg)b
H vs. Self
Two sample t-test
t27 = 4.59
P < 0.001
0.175 ± 0.009 0.154 ± 0.01
(n = 12)
(n = 12)
0.16 ± 0.01
(n = 18)
0.134 ± 0.008
(n = 11)
One-way

F2,32 = 4.78
P = 0.015
Crossing studies
(glasshouse; mg)c
0.179 ± 0.012 0.185 ± 0.01
(n = 14)
(n = 18)
Crossing studies
(glasshouse; mg)c
0.160 ± 0.009 0.146 ± 0.005
(n = 19)
(n = 12)
Two sample t-test
t29 = 1.58
P = 0.126
(B) Sibling comparison
Hand pollination
(garden; mg; n = 10)d
0.202 ± 0.013 0.168 ± 0.011 0.152 ± 0.01
Paired t-test
t9 = 2.85
P = 0.019
Transplant experiment
(field; mg; n = 29)d
0.118 ± 0.005 0.101 ± 0.004
Paired t-test
t28 = 4.14
P < 0.001
Crossing studies
(glasshouse; mg; n = 11)b
0.167 ± 0.014 0.128 ± 0.006 0.131 ± 0.009
Paired t-test
t10 = 2.60
P = 0.026
Paired t-test
t10 = 0.22
P = 0.833
(1) Combined probability
test (−2 Σ ln P)
χ216 = 55.7
P < 0.001
χ28 = 28.5
P < 0.001
(2) Hedges’ d
95% CI
0.65
0.31 – 0.98
0.45
0.07–0.84
Two sample t-test Paired t-test
t30 = 0.36
t17 = 4.54
P = 0.721
P < 0.001
Paired t-test
t9 = 1.52
P = 0.168
Meta-analysis
a
Koelewijn & Hundscheid (2000); bH. P. Koelewijn, unpublished data; cKoelewijn & Van Damme (1995a,b); dKoelewijn (1996).
analysis to improve homogeneity of variance. Difference in relative growth rate (RGR) between the seed
size classes and among cross types was tested according
to Poorter & Lewis (1986) as treatment by time interaction of the natural logarithm of total dry weight.
Second-order polynomials were fitted to test for a trend
of RGR with time.
 
© 2005 British
Ecological Society,
Journal of Ecology,
93, 373–383
In March 1997 seeds of four size classes (small, 0.12–
0.14 mg; medium, 0.14 – 0.18 mg; large, 0.18–0.22, see
Fig. 1, plus unsorted) and four cross types (MS × H,
H × H, S1 and S2) were buried in a salt meadow at the
site from which the source parents were collected. Five
plots of 1.25 by 1.00 m were laid out randomly in
an apparently homogeneous area of 25 × 25 m. These
plots were considered to be replicates for the different
treatments. Plots were subdivided into 20 subplots in a
5 by 4 grid. Each combination of size and cross type
was randomly assigned to one subplot in each plot and
seeds (n = 25) were buried 3 mm deep at 5-cm spacing
in a 5 by 5 grid. Germinates were located by laying a 25
by 25 cm square dish containing 25 holes with a 1-cm
diameter over the existing vegetation and scoring the
appearance of seedlings in the holes. No seeds were
377
Size, inbreeding
and sex effects on
offspring
© 2005 British
Ecological Society,
Journal of Ecology,
93, 373–383
buried in the remaining four subplots, which served as
controls for natural germination and to adjust for
possible misidentifications in the treated subplots. Plots
were checked once a month for germination, survival,
growth and flowering (April through September). In
April 1997 rabbits destroyed one plot. In August 1997
the number of leaves, length of the longest leaf and
number of ripe spikes of germinated seedlings were
measured. Monthly checks continued in 1998 but size
measurements were now also made monthly and at
least one ripe spike was sampled from each flowering
plant to estimate seed production. Plant size was estimated from the linear regression of the product of leaf
length and number of leaves (total leaf length, TLL) on
shoot dry weight: SDW (mg) = − 1.509 + 0.191 × TLL
(mm), r 2 = 0.78, F1,66 = 235.6, P < 0.001. This relationship was based on plants dug up in another experiment
in the same area (Koelewijn 1998).
The germination potential of the different size– cross
type combinations was assessed at the start of the field
experiment, except for the S2 cross type, where there
were too few seeds. For each combination 100 seeds
were placed in Petri dishes filled with 0.5% aqueous
agar, and germinated under natural light conditions in
the glasshouse during March 1997.
Analysis of categorical variables was performed by
fitting generalized linear models with GLIM (Baker 1987).
For data involving proportions (germination, survival
and number of plants alive) a log-linear model with a
binomial error distribution was used. Probability of
flowering was analysed by fitting a multinomial logit
model (three flowering states were distinguished: 0 =
non-flowering; 1 = flowering in only one season; 2 =
flowering in both seasons). In all cases the analysis
started with a null model with all main effects and
interactions. Subsequently, a χ2 test was used to determine
whether dropping a term from the model significantly
reduced the explained variance. The difference in unexplained variance between the categorical models
(deviance) is approximately χ2 distributed, with the
number of degrees of freedom equal to the difference
between the model with and without the term to be
tested ( McCullagh & Nelder 1983). Contrasts were used to
test for specific differences within the main factors seed
size (small vs. large) and cross type (MS vs. H, and H vs.
selfing). Quantitative variables were analysed using the
SAS statistical package (procedures GLM and t-test,
SAS 1988).
Field experiments are often prone to difficulties
in statistical analysis and interpretation. Because of
variation in germination and the death of plants during
the experiment, the experimental design can become
unbalanced or, even worse, incomplete. This is especially
true for traits related to reproduction, as only a fraction
of all plants produce seeds. Therefore, only three plots
were used for the statistical analysis of germination and
survival (plots 1–3), and only two for the analysis of
flowering data (plots 1 and 3), because the log-linear
models based on all four plots did not converge. Quan-
titative traits of flowering plants (size measurements,
number of seeds) could only be analysed by one-way
, because of small sample sizes.
Epistasis at the population level can be detected by
calculating the linear ( f ) and quadratic ( f 2 ) coefficients
of the quadratic regression of (log)-fitness on inbreeding level (Crow & Kimura 1970). To examine the effect
of inbreeding on fitness, calculated as the number of
seeds produced per initial buried seed (standardized
seed production), we performed a linear regression
with linear and quadratic components as the independent variables. A significant effect of the quadratic
component indicates a non-linear relationship between
inbreeding level and fitness.
Cumulative fitness for selfed (F > 0) and outcrossed progeny (F = 0) was calculated as the product of
germination, probability of flowering and number of
seeds per flowering plant at the end of the second year
(standardized seed production). Inbreeding depression
(δ) per inbreeding level was estimated as:
δ = 1 − ws /wo
where ws and wo are, respectively, the mean fitness of
selfed and outcrossed progeny.
Results
  
Frequency distributions of seed weights differed signi2
ficantly between cross types ( χ18 = 44.3, P < 0.001, Fig. 1).
This was mainly caused by male steriles producing
2
more seeds in the larger size classes ( χ 6 MS × H vs. others
2
= 36.2, P < 0.001; χ 12 among others = 10.0, NS). Thus,
MS × H crosses produced heavier seeds than H × H
crosses, which in turn produced slightly heavier seeds
compared with self-fertilization (MS × H, 0.165 ±
0.003; H × H, 0.152 ± 0.003; S1, 0.148 ± 0.003; S2,
0.145 ± 0.003 (mean (mg) ± SE)).
  (  )
Seedling dry weight (mean ± SE) at the start of the
growth experiment was directly proportional to seed
size: for small and large seeds, respectively, seedlings
dry weight was 0.49 mg ± 0.02 and 0.73 mg ± 0.06
(t11 = 3.83, P < 0.01). No significant difference among
cross types was detected (small, MS × H 0.50, H × H
0.51 and S1 0.45 mg; large, MS × H 0.85, H × H 0.70
and S1 0.64 mg; F2,6 = 1.3, NS).
No difference in relative growth rate was observed
between the two seed size classes or among the cross types
(Table 2, no significant time by treatment interactions).
RGR (mg g−1 day−1, mean ± SE), based on the whole
experimental period, was 284 ± 6 and 292 ± 5 for the
small and large seed size classes, respectively. As
a consequence, plants originating from small seeds
remained smaller than those from large seeds throughout
378
H. P. Koelewijn &
J. M. M. van
Damme
Fig. 2 Time course of the ln-transformed total dry weight of two seed size classes (small = 0.13 mg, closed symbols, and
large = 0.20 mg, open symbols) and three cross types (MS × H, H × H and S1) of Plantago coronopus.
Table 2  of the time course of ln-transformed total dry
weight in Plantago coronopus during the growth experiment.
Effects of time, seed size, cross type, their interactions and a
trend analysis for time. Differences in RGR among treatments
can be inferred from the significance of the interaction with
time (see also Fig. 2). Indicated are the degrees of freedom
(d.f.), sums of squares (SS) and the resulting value from the
F-test. Level of significance indicated as: ***P < 0.001;
**P < 0.01; NS = not significant, P > 0.05
Source
F
P
Time
Linear
Quadratic
5 686.54 1506.9
1 682.59 7491.4
1
3.12
34.3
***
***
***
Treatment
Seed size
Cross type
MS vs. (H + S1)
H vs. S1
Seed size by cross type
1
2
1
1
2
8.72
0.96
0.74
0.21
0.40
96.2
5.3
8.22
2.35
2.2
***
**
**
NS
NS
5
10
10
0.76
0.55
0.71
1.7
0.6
0.8
NS
NS
NS
295
26.75
Time by treatment
Time by seed size
Time by cross type
Time by seed size by cross type
Error
© 2005 British
Ecological Society,
Journal of Ecology,
93, 373–383
d.f. SS
the experiment (Fig. 2). The trend analysis indicated
a significant quadratic component (Table 2), implying
a change in RGR with time, and second-order polynomials indeed gave a slightly better fit (results not
shown). There was also a significant main effect of
cross type (Table 2) because plants originating from the
larger seeds of the MS × H cross were taller at all harvests (Table 2, Fig. 2) as they maintained the small size
advantage present at the seedling stage. Dry weights at
final harvest (day 18), however, were not significantly
different among cross types (small, MS × H 66.8 ± 6.7,
H × H 72.1 ± 9.4 and S1 58.6 ± 5.2; large, MS × H
121.5 ± 11.5, H × H 90.0 ± 6.0 and S1 81.5 ± 12.0;
mean (mg) ± SE, n = 10; F2,48 = 2.1, NS).
Fig. 3 Time course of the germination of different seed size
classes of Plantago coronopus in the field.

The fraction of germinated seeds at the end of March in
the glasshouse did not differ among cross types (MS ×
2
H 86%, H × H 89%, S1 91%, χ 2 = 4.2, NS), but did differ
among size classes (small 82%, medium 90%, large
2
92%, χ 2 = 14.9, P < 0.01). Total germination in the field
after 2 years was much lower (41% in the field vs. 88%
in the glasshouse), but results were otherwise the same
2
(cross type, MS × H 40%, H × H 43%, S1 40%, χ 2 = 0.7,
NS; seed size, small 34%, medium 40%, large 45%,
2
χ 2 = 7.1, P < 0.05). In the field, the higher germination
in larger size classes was established within 2 months
(Fig. 3). Total germination in the control plots was
only 2%, indicating that the chance of contamination
by natural germination is negligible.
 
Despite being laid out in an apparently homogeneous
part of the salt marsh, considerable variation existed
among the four plots in the probability of germination,
survival and flowering and in shoot dry weight (Table 3).
Few plants flowered in the two plots with the smallest
plant sizes and lowest survival (Table 3).
379
Size, inbreeding
and sex effects on
offspring
Table 3 Spatial heterogeneity in the field: the probability to germinate, survival, presence after 2 years, probability to flower, and
shoot dry weight in August 1998 of Plantago coronopus seeds in four plots
Probability to
germinate
Plot
Plot 1
Plot 2
Plot 3
Plot 4
0.49
0.48
0.43
0.16
Test for heterogeneity
among plots (χ23 )
Alive after
2 years
Survival
0.54
0.22
0.51
0.57
127.6
P < 0.001
Probability to
flower
0.27
0.10
0.23
0.10
65.7
P < 0.001
40.7 ± 2.1
23.8 ± 1.7
37.9 ± 1.9
28.9 ± 2.6
0.18
0.02
0.22
0.01
66.3
P < 0.001
Shoot dry weight
(mg, mean ± SE)

F3,343 = 12.9
P < 0.001
82.6
P < 0.001
Table 4 Analysis of the fraction of germinated Plantago coronopus seeds, survival of seedlings, number present after 2 years,
probability to flower and shoot dry weight in 1998 in relation to environment (plot), seed size and cross type. Characteristics were
analysed by fitting log-linear models to the binary data (1 = yes, 0 = no), a multinomial-logit model in the case of probability to
flower and an  in the case of plant size. See also Table 5
Fraction
germinated
Survival of
seedlings
Plants alive
after 2 years
Probability to flower
Source
d.f.
χ2
P
χ2
P
χ2
P
d.f.
Plot
Size × Cross
Size
Small vs. Large
Cross
MS vs. H
H vs. Self
Error
2
6
2
1
3
1
1
0.9
8.3
10.1
9.8
8.8
1.9
3.9
0.632
0.216
0.006
0.002
0.032
0.157
0.047
41.5
18.7
2.8
2.6
6.2
0.3
4.8
0.000
0.005
0.245
0.102
0.102
0.581
0.028
29.7
12.3
5.7
5.6
11.5
1.5
6.7
0.000
0.058
0.057
0.017
0.009
0.217
0.010
2
12
4
2
6
2
2
χ2
4.6
23.0
15.8
9.3
24.1
0.8
9.6
Plant size
P
d.f.
SS
P
0.100
0.028
0.003
0.009
0.001
0.681
0.008
3
6
2
1
3
1
1
233
119.1
23.0
10.8
8.8
36.3
3.2
16.9
913.6
0.000
0.490
0.286
0.150
0.030
0.379
0.046
Table 5 Fate of Plantago coronopus seeds in relation to size and cross type. Standardized seed production represents a final fitness
measure for comparison of the performance of the different groups, i.e. the number of seeds produced per initial buried seed. The
fitness of the unsorted seed size class and the H × H cross type have been arbitrarily set to 1. See Table 4 for the accompanying
statistical analysis
Survival
Alive after
2 years
Probability
to flower
at least once
# Seeds per
flowering
plant
Standardized
seed production
Relative
fitness
Shoot dry
weight
(mg ± SE)
0.335
0.403
0.448
0.384
0.425
0.447
0.494
0.431
0.145
0.180
0.225
0.166
0.105
0.115
0.184
0.130
47
56
80
62
1.65
2.59
6.59
3.10
0.53
0.83
2.12
1
30.5 ± 2.5
32.8 ± 2.3
34.5 ± 2.0
31.3 ± 1.9
0.400
0.430
0.400
0.350
0.480
0.521
0.427
0.352
0.193
0.230
0.170
0.123
0.217
0.155
0.108
0.029
65
73
71
33
5.64
4.87
3.07
0.34
1.16
1
0.63
0.07
38.2 ± 2.8
34.8 ± 2.6
28.3 ± 2.3
27.8 ± 1.9
Size class
Probability
to germinate
Small
Medium
Large
Unsorted
Cross type
MS × H
H×H
S1
S2
  
© 2005 British
Ecological Society,
Journal of Ecology,
93, 373–383
Seed size had a pronounced effect on plant performance
in the field (Tables 4 and 5). Probability to germinate,
number of plants alive after 2 years and the probability
to flower ( Fig. 4a) were significantly different between
small and large seeds, with the medium sized seeds
mostly in between (Table 5). Unlike the glasshouse
results, seed size had no influence on plant size ( Tables 4
and 5). Standardized seed production, i.e. the number
of seeds produced per initial buried seed, was four
times higher for large seeds compared with small seeds
(Table 5). Large seeds gave rise both to a higher overall
incidence of flowering plants and to a higher percentage
flowering in their first year, and thus producing seeds
during two seasons (Fig. 4a).
380
H. P. Koelewijn &
J. M. M. van
Damme
Fig. 5 Fitness, expressed as standardized seed production
(number of seeds produced per initial buried seed), in relation
to inbreeding level (mean ± SE, n = 2). Due to absence of
flowering plants, only data from plot 1 and 3 could be used
(see Table 3).
Fig. 4 Frequency distribution of the number of flowering
seasons expressed as a fraction of the total number of
germinated seedlings of Plantago coronopus, (a) in relation to
seed size and (b) in relation to cross type.
Discussion
 
 
Self-fertilization had a pronounced effect on plant performance in the field, especially on the probability to
flower (Fig. 4b, Table 5). Based on standardized seed
production the S1 and S2 suffered from moderate to
high inbreeding depression (δ (S1) = 0.37 and δ (S2) =
0.93, Table 5).
Only plots 1 and 3 produced sufficient flowering
plants (Table 3) to allow testing for synergistic epistasis,
but an effect was, nevertheless, apparent (Fig. 5). The
decrease in fitness after two generations of selfing was
much larger than after one generation of selfing. The
quadratic regression of standardized seed production
on inbreeding level (F ) was significant (ln(ssp) = 1.49 +
2.93 × f – 7.61 × f 2, r 2 = 0.82, F2,3 = 11.87, P = 0.038).
Including the plots with zero standardized seed
production produced the same result as Fig. 5, but the
large standard errors meant that the effect was no
longer significant.
Many researchers have investigated the effect of offspring size on fitness. Almost without exception, larger
offspring outperform smaller offspring in plants (see
reviews in McGinley et al. 1987 and Geritz 1995); here
smaller seeds performed worse than larger seeds. This
study is one of the few where the effects of seed size
have been traced for a long enough period to evaluate
differences in seed production and to identify the traits
responsible. In the glasshouse, large seeds gave larger
seedlings and, as no differences in RGR were observed,
this resulted in significant plant size differences (Fig. 2)
as large seeds maintained their head start. In the field,
variation in germination, survival and probability of
flowering were related to seed size, leading to a fourfold
difference in fitness between small and large seeds
(Table 5). Size differences, although still in favour of
large seeds, were much less pronounced under field
conditions than in the glasshouse (Table 5). Thus an
advantage of 53% in terms of seed size was reduced to
13% after 2 years in the field. Several studies have shown
that the transition from vegetative growth to reproduction is accompanied by a sharp decrease in growth rate
of vegetative plant parts (King & Roughgarden 1983;
Freijsen & Otten 1993; Koelewijn 2004), suggesting
that investment in reproductive structures gets priority.
As a higher fraction of plants from the large seed size group
flowered, individuals from small seeds might be able to
catch up (in terms of vegetative growth) when those from
large seeds switch their resources towards reproduction.
  
© 2005 British
Ecological Society,
Journal of Ecology,
93, 373–383
There was a marked difference in sex ratio among the
flowering progeny of the MS × H and H × H cross
types: MS × H, 48% male steriles; H × H, 7% male steriles
2
( χ1 = 8.23, P < 0.01).
No significant differences in offspring plant performance were observed between the MS × H and H × H
cross types in the field (Table 4). Summed over the
research period of 2 years, however, the slight differences
between the offspring from the MS × H and H × H cross
types resulted in a fitness advantage of 16% in favour of
the offspring of the MS × H cross type (Table 5), due
mainly to their greater probability to flower (Fig. 4b).
381
Size, inbreeding
and sex effects on
offspring
Seed size was studied because previous research
indicated a difference in seed size between the two sex
morphs (Koelewijn 1996; Table 1) and to enable a
proper evaluation of possible maternal sex effects by
controlling for seed size. Seeds originating from MS × H
crosses were 9% heavier than seeds from H × H crosses,
which in turn were about 4% heavier than seeds from
self-fertilization, consistent with previous reports
(summarized in Table 1) of slight, but consistent, differences in seed weight among cross types. Male steriles
produced more seeds in the larger size classes, although
there were no differences in seed set, suggesting that
compensation may operate at the flower level. The
advantage gained by male steriles by producing more
seeds, as reflected in their offspring performance, was
estimated to be 15% (multiplying the fitness estimates
(Table 5) with the seed size frequency data from Fig. 1).
Thus, male steriles might have an advantage over hermaphrodites through a maternal effect on seed size.

© 2005 British
Ecological Society,
Journal of Ecology,
93, 373–383
The results of this study demonstrate substantial
inbreeding depression for important fitness components in the field, but not in the glasshouse. Thus, the
expression of genetic load was strongly influenced by
the environment. Inbreeding depression after one generation of selfing (δ = 0.37, F = 0.5) was moderate, but
inbreeding depression after two generations of selfing
(δ = 0.93, F = 0.75) was high, suggesting that hardly
any highly inbred individuals contributed to the next
generation. These results are in line with a previous,
more detailed, transplant study from the same area
(Koelewijn 1998), where, starting from pre-cultured
seedlings, inbreeding depression after one and two generations of selfing was estimated to be 0.39 and 0.70.
The more pronounced inbreeding depression after two
generations of selfing found in the present study might
be related to our starting with seeds, i.e. also including
the seedling establishment stage. The magnitude of
inbreeding depression after one generation of selfing
is in close agreement with those from other partially
self-fertilizing species and between the observed mean
values for selfing (δ = 0.23) or obligately outcrossing
(δ = 0.53) species (Husband & Schemske 1996).
The data suggest evidence for synergistic epistasis in
fitness. The loss in fitness from one to two generations
of selfing was much higher than the loss in fitness from
outcrossing to one generation of selfing (Fig. 5), resulting
in a significant negative curvilinear regression. Epistasis
influences the rate at which a population purges the
load due to inbreeding. Theoretical studies of the effect
of synergism on the evolution of sexual systems have
shown that even very slight epistasis between harmful
mutations can have considerable effects on the maintenance
of sexual reproduction and outcrossing (Charlesworth
1990). The decrease in the probability of flowering for
S2 plants (F = 0.75) was large (Table 5, Fig. 4b) and
inbreeding depression for this trait was 0.80, implying
that the majority of the plants with high inbreeding
coefficients are present in the population in a vegetative
state and do not take part in reproduction.
Estimates of inbreeding depression in self-compatible
gynodioecious species after one generation of selfing
vary from moderate to high: 0.27 in Sidalcea oregana
(Ashman 1992), 0.36 in Chionographis japonica (Maki
1993), 0.54 in Lobelia siphilitica (Johnston 1992), 0.60
in Schiedea salicaria (Sakai et al. 1999), 0.73 in Thymus
vulgaris (Assouad et al. 1978), and 0.75 in Geranium
maculatum (Agren & Willson 1991). Thus, considerable reduction in fitness of hermaphrodites might occur.
However, these values are for complete selfing and the
effectiveness of inbreeding depression will depend, above
all, on the selfing rate. In the case of P. coronopus,
assuming a selfing rate of 0.25 (Wolff et al. 1988;
Koelewijn 1998), male steriles would gain a 9% advantage over hermaphrodites after one generation of selfing.
 
In the current study no significant differences were
detected in the quality of offspring from outcrossed
female (MS) or hermaphrodite (H) plants, either in the
juvenile stages of the life cycle or in reproductive adults,
provided both seed size and level of inbreeding were
equivalent. Thus, no direct maternal-sex effect was
detectable. Results concerning maternal sex effects in
gynodioecious species are equivocal. The main problem is that there are several confounding sources, e.g.
differences in seed size, inbreeding and variation in offspring sex ratio that are sometimes difficult to control
for. Eckhart (1992) found no relation between maternal sex and either germination, survival, growth rate
or vegetative size in Phacelia linearis. Poot (1996) also
reports no influence of maternal sex on early vegetative
traits in Plantago lanceolata. In the present study, no
differences in germination, growth rate, flowering, plant
size and fitness in P. coronopus were detected with
respect to maternal sex. On the other hand, Ashman
(1992) reports differences in germination, growth rate
and fitness in Sidalcea oregana and Shykoff (1988)
mentions differences in survival in Silene acaulis in
favour of the offspring from male steriles. The latter
two results could indicate the presence of maternal-sex
effects. However, as in these two studies nothing was
known about the sex ratio of the surviving offspring,
i.e. the ratio of MS : H, an alternative explanation is
still possible when there are inherent differences related
to sex itself.
Crosses with females as a mother (MS × H) are more
likely to generate a more female biased sex ratio than
H × H crosses, due to females possessing fewer restorer
genes for male fertility (Koelewijn & van Damme 1995b;
Charlesworth & Laporte 1998; Koelewijn 2003). If
differences in vigour exist between the two sex morphs
and crosses differ in sex ratio among the offspring, sex
morph differences will be reflected in the mean offspring performance of the crosses. Differences in
382
H. P. Koelewijn &
J. M. M. van
Damme
performance between sex morphs themselves are often
observed in gynodioecious species (Agren & Willson
1991; Eckhart 1992; Klinkhamer et al. 1994; Koelewijn
1996; Wolfe & Shmida 1997; Shykoff et al. 2003).
Although not significant, the present study detected a
difference in performance of 16% in favour of the offspring from a female mother (Table 5). However, the
sex ratio among the flowering offspring was different
(MS × H cross, 48% male steriles; H × H cross, 7%
male steriles) and MS plants also tended to produce
more seeds (MS 93 ± SE 20; H 64 ± SE 9; F1,65 = 1.61,
NS). When comparing the offspring performance of
females and hermaphrodites, taking this difference in
sex ratio into account removed the advantage in favour
of MS offspring, indicating the absence of a maternal
sex effect. The observed difference of 16% between the
offspring of the two cross types could be explained by
the difference in frequency of the two sex morphs
among the offspring ((0.48 × 93 + 0.52 × 64)/(0.07 ×
93 + 0.93 × 64) = 1.18, thus a 18% difference). However,
one could also argue that this still implies a maternal sex
effect, because the mother causes the sex ratio difference.
The same explanation might apply to the Siadalcea
and Silene results. In Silene acaulis females had higher
fruit set (Delph et al. 1999), for Sidalcea no information
was available. For neither of the species, however, was
information on the offspring sex ratio available.
  
© 2005 British
Ecological Society,
Journal of Ecology,
93, 373–383
Gynodioecy is an exceptional breeding system because
continuous selection is necessary to maintain it. The
unisexual male steriles (females) only reproduce through
ovules, while the cosexual hermaphrodites reproduce
through ovules and pollen. This sexual asymmetry (i.e.
the non-constant pollen : ovule ratio among individuals
in a population) means that, to maintain the gynodioecious state, differences in fitness characters other
than male function must exist between male steriles
and hermaphrodites that prevent male steriles from
becoming extinct. When inheritance is nuclear, male
steriles must produce twice as many viable seeds as
hermaphrodites, although when inheritance is cytoplasmic male steriles only need a small fitness advantage.
With nuclear-cytoplasmic inheritance, as in P. coronopus
(Koelewijn & van Damme 1995ab), the fitness advantage varies between the two extremes. While most studies
on how male steriles compensate for their disadvantage
deal with only one or two of the components responsible (seed size, maternal sex effects and inbreeding
depression effects, see Introduction for references) we
undertook an integrated study of all three components
in relation to each other. Our results indicate that both
inbreeding (9%) and variation in seed size (15%) provide male sterile plants with small fitness advantages. A
previous study has shown that male steriles themselves
already produce 48% more seeds than hermaphrodites
(Koelewijn 1996). Together these results indicate that
male steriles have a 70% fitness advantage over her-
maphrodites. This is sufficient to compensate for the
loss in male function under nuclear-cytoplasmic inheritance. The results also indicate that the compensation
for the loss of male function via increased resource
allocation to seeds is much more important than the
contribution of inbreeding.
Acknowledgements
Thanks to two anonymous referees for comments on
the manuscript and to Lindsay Haddon, whose amazing editorial skills definitely improved our manuscript.
References
Agren, J. & Willson, M.F. (1991) Gender variation and sexual
differences in reproductive characters and seed production
in gynodioecious Geranium maculatum. American Journal
of Botany, 78, 470 – 480.
Ashman, T.L. (1992) The relative importance of inbreeding
and maternal sex in determining progeny fitness in Sidalcea
oregana ssp. spicata, a gynodioecious plant. Evolution, 46,
1862 – 1874.
Ashman, T.L. (1994) Reproductive allocation in hermaphrodite
and female plants of Sidalcea oregana ssp. spicata (Malvaceae)
using four currencies. American Journal of Botany, 81,
433 – 438.
Assouad, M.W., Dommee, B., Lumaret, R. & Valdeyron, G.
(1978) Reproductive capacities in the sexual form of the
gynodioecious species Thymus vulgaris L. Botanical Journal
of the Linnaean Society, 77, 29 – 39.
Baker, R.J. (1987) GLIM 3.77 Reference Manual. Numerical
Algorithms Group, Oxford.
Bell, G., Lechowicz, M.J. & Schoen, D. (1991) The ecology
and genetics of fitness in forest plants. III. Environmental
variance in natural populations of Impatiens pallida.
Journal of Ecology, 79, 697 – 713.
Charlesworth, D. (1981) A further study of the problem of the
maintenance of females in gynodioecious populations.
Heredity, 46, 27 – 39.
Charlesworth, B. (1990) Mutation-selection balance and the
evolutionary advantage of sex and recombination. Genetical
Research, 55, 199 –221.
Charlesworth, D. & Laporte, V. (1998) The male-sterility
polymorphism in Silene vulgaris. Analysis of genetic data
from two populations, and comparison with Thymus
vulgaris. Genetics, 150, 1267 – 1282.
Charlesworth, B., Morgan, M.T. & Charlesworth, D. (1991)
Multilocus models of inbreeding depression with synergistic
selection and partial self-fertilization. Genetical Research,
57, 177 – 194.
Crow, J.F. (1970) Genetic load and the cost of natural selection. Mathematical Models in Population Genetics (ed.
K.I. Kojima), pp. 128 – 177. Springer, Berlin.
Crow, J.F. & Kimura, M. (1970) An Introduction to Population
Genetic Theory. Harper & Row, New York.
van Damme, J.M.M. (1984) Gynodioecy in Plantago lanceolata III. Sexual reproduction and the maintenance of male
steriles. Heredity, 52, 77 – 93.
Darwin, C. (1877) The Different Forms of Flowers on Plants of
the Same Species. Murray, London.
Delph, L., Bailey, M.F. & Marr, D.L. (1999) Seed provisioning
in gynodioecious Silene acaulis (Caryophyllaceae). American
Journal of Botany, 86, 140 – 144.
Delph, L. & Mutikainen, P. (2003) Testing why the sex of the
maternal parent affects seedling survival in a gynodioecious
species. Evolution, 57, 231 – 239.
383
Size, inbreeding
and sex effects on
offspring
© 2005 British
Ecological Society,
Journal of Ecology,
93, 373–383
Eckhart, V.M. (1992) Resource compensation and the evolution of gynodioecy in Phacelia linearis (Hydrophyllaceae).
Evolution, 46, 1313 – 1328.
Eckhart, V.M. & Chapin, F.S. III (1997) Nutrient sensitivity
and the cost of male function in gynodioecious Phacelia
linearis (Hyfrophyllaceae). American Journal of Botany, 84,
1092 – 1098.
Evans, G.C. (1972) The Quantitative Analysis of Plant Growth.
Blackwell, Oxford.
Freijsen, A.H.J., de Zwart, A.J. & Otten, H. (1990) A culture
system for steady state nutrition in optimum and suboptimum
treatments. Plant Nutrition – Physiology and Applications
(ed. M.L. van Beusichem), pp. 389–395. Kluwer Academic
Publishers, Dordrecht.
Freijsen, A.H.J. & Otten, H. (1993) Utilization of the ambient
concentration as a criterion for steady state after exponential
growth. Some culture experiments with optimum or suboptimum N nutrition. Plant and Soil, 151, 256 – 271.
Geritz, S.A.H. (1995) Evolutionary stable seed polymorphism
and small-scale spatial variation in seedling density.
American Naturalist, 146, 685 – 707.
Harper, J.L., Lovell, P.H. & Moore, K.G. (1970) The shapes
and sizes of seeds. Annual Review of Ecology and Systematics,
1, 327 – 356.
Hedges, L.V. & Olkin, I. (1985) Statistical Methods for MetaAnalysis. Academic Press, New York.
Hewitt, E.J. (1966) Sand and Water Culture Methods Used in
the Study of Plant Nutrition. Technical communication 22.
Commonwealth Agricultural Bureaux, England. East
Malling, Maidstone, Kent UK.
Husband, B. & Schemske, D.W. (1996) Evolution of the
magnitude and timing of inbreeding depression in plants.
Evolution, 50, 54 – 70.
Johnston, M.O. (1992) Effects of cross and self-fertilization
on progeny fitness in Lobelia cardinalis and L. siphilitica.
Evolution, 48, 1736 – 1741.
King, D. & Roughgarden, J. (1983) Energy allocation patterns
of the california grasslands annuals Plantago erecta and
Clarkia rubicunda. Ecology, 64, 16 – 24.
Klinkhamer, P., de Jong, T. & Nell, H.W. (1994) Limiting
factors for seed production and phenotypic gender in the
gynodioecious species Echium vulgare (Boraginaceae). Oikos,
71, 469 – 478.
Koelewijn, H.P. (1996) Sexual differences in reproductive
characters in gynodioecious Plantago coronopus. Oikos, 75,
443 – 452.
Koelewijn, H.P. (1998) Effects of different levels of inbreeding on
progeny fitness in Plantago coronopus. Evolution, 52, 692–702.
Koelewijn, H.P. (2003) Variation in restorer genes and primary sexual investment in Plantago coronopus: the tradeoff between male and female function. Proceedings of the
Royal Society Series B, 270, 1939 – 1945.
Koelewijn, H.P. (2004) Rapid change in relative growth rate
between the vegetative and reproductive stage of the life
cycle in Plantago coronopus. New Phytologist, 163, 67 – 76.
Koelewijn, H.P. & Hundscheid, M.P.H. (2000) Intraspecific
variation in sex allocation in hermaphroditic Plantago
coronopus. Journal of Evolutionary Biology, 13, 302 – 315.
Koelewijn, H.P. & van Damme, J.M.M. (1995a) Genetics of
male sterility in Plantago coronopus. I. Cytoplasmic variation.
Genetics, 139, 1749 – 1758.
Koelewijn, H.P. & van Damme, J.M.M. (1995b) Genetics of
male sterility in Plantago coronopus. II. Nuclear genetic
variation. Genetics, 139, 1759 – 1776.
Koelewijn, H.P. & van Damme, J.M.M. (1996) Gender variation,
partial male sterility and labile sex expression in gynodioecious Plantago coronopus. New Phytologist, 132, 67–76.
Kohn, J.R. (1988) Why be female? Nature, 335, 431 – 433.
Lande, R. & Schemske, D.W. (1985) The evolution of selffertilization and inbreeding depression in plants. I. Genetic
models. Evolution, 39, 24 – 40.
Lewis, D. (1941) Male sterility in natural populations of
hermaphrodite plants. New Phytologist, 40, 56–63.
Lloyd, D.G. (1975) The maintenance of gynodiecy and
androdieocy in angiosperms. Genetica, 45, 325–339.
Maki, M. (1993) Outcrossing and fecundity advantage of
females in gynodioecious Chionogrpahis japonica var.
kurohimensis (Liliaceae). American Journal of Botany, 80,
629 – 634.
McCullagh & Nelder, J.A. (1983) Generalized Linear Models.
Chapman & Hall, London.
McGinley, M.A., Temme, D.H. & Geber, M.A. (1987) Parental
investment in offspring in variable environments, theoretical
and empirical considerations. American Naturalist, 130,
370 – 398.
Michaels, H.J., Benner, B., Hartgerink, A.P., Lee, T.D. &
Rice, S. (1988) Seed size variation, magnitude, distribution
and ecological correlates. Evolutionary Ecology, 2, 157–166.
Mutikainen, P. & Delph, L.F. (1998) Inbreeding depression in
gynodioecious Lobelia siphilitica: among-family differences
override between-morph differences. Evolution, 52, 1572–
1582.
Poorter, H. & Lewis, C.W. (1986) Testing differences in relative growth rate, a method avoiding curve fitting and pairing.
Physiologia Plantarum, 67, 223 – 226.
Poot, P. (1996) Ecophysiological aspects of maintenance of
male sterility in Plantago lanceolata. PhD thesis, Utrecht
University, The Netherlands.
Poot, P. (1997) Reproductive allocation and resource compensation in male-sterile and hermaphroditic plants of Plantago
lanceolata (Plantaginaceae). American Journal of Botany,
84, 1256 – 1265.
Sakai, A.K., Weller, S.G., Chen, M.-L., Chou, S.-Y.
& Tasanont, C. (1997) Evolution of gynodioecy and
maintenace of females, the role of inbreeding depression,
outcrossing rates, and resource allocation in Schiedea
adamantis (Caryophyllaceae). Evolution, 51, 724–736.
SAS. (1988) SAS/STAT Users Guide. SAS Institute, Cary,
North Carolina.
Shykoff, J.A. (1988) Maintenance of gynodioecy in Silene
acaulis (Caryophyllaceae), stage specific fecundity and
viability selection. American Journal of Botany, 75, 844–850.
Shykoff, J.A., Klokotronis, S.O., Collin, C.L. & Villavicencio, M.L.
(2003) Effects of male sterility on reproductive traits in
gynodioecious plants, a meta-analysis. Oecologia, 135, 1–9.
Sokal, R. & Rohlf, F.J. (1980) Biometry. Freeman, San Francisco.
Thompson, J.D., Tarayre, M., Gauthier, P., Litrico, I. &
Linhart, Y.B. (2004) Multiple genetic contributions to plant
performance in Thymus vulgaris. Journal of Ecology, 92,
45 – 56.
Willis, J.H. (1993) Effects of different levels of inbreeding
on fitness componentsin Mimulus guttatus. Evolution, 47,
864 – 876.
Winn, A.A. (1988) Ecological and evolutionary consequences
of seed size in Prunella vulgaris. Ecology, 68, 1527–1544.
Wolfe, L. & Shmida, A. (1997) The ecology of sex expression
in a gynodioecious Israeli shrub (Ochradenus baccatus).
Ecology, 78, 101 – 110.
Wolff, K., Friso, B. & van Damme, J.M.M. (1988) Outcrossing rates and male sterility in natural populations
of Plantago coronopus. Theoretical and Applied Genetics, 76,
190 – 196.
Wulff, R.A. (1986a) Seed size variation in Desmodium paniculatum. II. Effects on seedling growth and physiological
performance. Journal of Ecology, 74, 99 – 114.
Wulff, R.A. (1986b) Seed size variation in Desmodium paniculatum. III. Effects on reproductive yield and competitive
ability. Journal of Ecology, 74, 115 – 121.
Received 1 March 2004
revision accepted 16 August 2004
Handling Editor: Spencer Barrett