Journal of
Ecology 2004
92, 15 – 23
Temporal variation in sex allocation in hermaphrodites
of gynodioecious Thymus vulgaris L.
Blackwell Publishing, Ltd.
BODIL K. EHLERS and JOHN D. THOMPSON
Centre d’Ecologie Fonctionelle et Evolutive, CNRS, 1919 Route de Mende, 34293 Montpellier cedex 5, France
Summary
1 Theory predicts that hermaphrodites adjust sex allocation in relation to the relative
fitness contributions of male and female functions. Few studies have, however, focused
on temporal variation in sex allocation within individuals.
2 We quantified temporal variation in allocation to male and female function of
hermaphrodites of gynodioecious Thymus vulgaris across a single flowering season, in
families from four natural populations grown in an experimental garden.
3 Allocation to pollen production and to seed set and germination varied significantly
among sample dates, with male function highest when female function was lowest, and
a significant negative correlation between pollen production and seed set among families indicated a trade-off between male and female function.
4 Females and hermaphrodites showed gender-specific flowering phenology: female
plants achieved peak flowering later and flowered for a significantly shorter period than
hermaphrodites.
5 The number of open female flowers was significantly positively correlated with pollen
production by hermaphrodites. Hermaphrodite male function was maximized at the
time of peak flowering by females in all four populations.
6 These results fit theoretical predictions that across-season variation in the mating
environment could select for a temporal variation in hermaphrodite sex allocation, but
other factors (e.g. variation in the selfing rate or resource levels) may also contribute to
the observed pattern.
Key-words: gender, mating environment, phenology, sex allocation
Journal of Ecology (2004) 92, 15–23
Introduction
Sex allocation theory predicts that allocation of resources
to male and female function reflects the relative fitness
contribution obtained by hermaphrodite plants via
pollen and seed production (Charnov 1982). Indeed,
variation in resource allocation to male and female
functions among outcrossing hermaphrodite plants
is well documented (e.g. Horovitz 1978; Devlin &
Stephenson 1987; Brunet 1992). A range of factors,
such as plant size and age, the selfing rate and the mating
environment, have been shown, both theoretically and
empirically, to be important in shaping such variation
(e.g. Lloyd 1976; Brunet 1992; Klinkhammer et al. 1997).
The focus of the present study is on temporal variation in sex allocation within individual plants across a
single flowering season. This may depend on competition for resources among flowers. Position-dependent
effects such as first-produced and/or basal-positioned
© 2004 British
Ecological Society
Correspondence: Bodil K. Ehlers (tel. +33 4 67 61 32 14;
e-mail [email protected]).
flowers closer to the resource pool receiving more
resources than later or more distal ones have been demonstrated in Fragaria virginia and were also shown to
interact with the size of individual plants (Ashman
& Hitchens 2000; Ashman et al. 2001). Ashman &
Baker (1992) showed an overall decline in allocation to
secondary sexual structures (e.g. sepals and petals)
in Sidalcea oregana, but no changes in allocation to
primary sexual structures among early- and lateproduced flowers, although there was a trend towards
increased maleness in late-season flowers.
Theoretical studies predict that temporal variation in sex allocation of hermaphrodites may also arise
due to a variation in the mating environment. Brunet
& Charlesworth (1995) demonstrated that, for protandrous hermaphrodites with sequential blooming,
variation in the availability of ovules could select
for a variation in sex allocation among hermaphrodite
flowers across a single flowering season (even in the
absence of among-flower differences in resource allocation). Flowers with a high probability of pollen transfer
should allocate proportionally more of their resources
16
B. K. Ehlers &
J. D. Thompson
© 2004 British
Ecological Society,
Journal of Ecology,
92, 15–23
to male function than flowers with a lower probability
of pollen transfer. When the possibility of pollinating
flowers which specialize in female function is high, sex
allocation to male function will be further enhanced.
The seasonal variation in sex allocation predicted
by their model is a female-biased sex allocation in
early-produced flowers (where pollen is abundant
and competition for ovules is high) and a shift towards
male-biased sex allocation in later produced flowers
(where the number of available ovules has increased).
In gynodioecious populations, where hermaphrodites coexist with females, gender-specific flowering
phenology may additionally influence temporal variation in the mating environment across the flowering
season. Gender-specific flowering phenology in which
females flower less often or over shorter periods than
males in order to reduce their cost of reproduction has
been reported in several species (Delph 1999). As in
many gynodioecious species females compensate for
lack of male function by having higher seed set, one
could predict that sex allocation by hermaphrodites
may respond, not only to the availability of ovules of
hermaphrodite flowers in their female phase (as predicted by Brunet & Charlesworth 1995) but also to the
availability of ovules on female plants.
Thymus vulgaris is a gynodioecious species with a
nuclear-cytoplasmic male sterility system (Belhassen
et al. 1991). Females carry a cytoplasmic male sterility
gene that prevents the production of functional
anthers. Male function is restored by the presence
of the appropriate nuclear restorer alleles. Variation in
both male sterility genes and restorer alleles exists.
Mismatches between male sterility genes and nuclear
restorer alleles are believed to be important for the high
variation in female frequency (6 – 93%) observed among
populations (Belhassen et al. 1989; Manicacci et al.
1996; Manicacci et al. 1997). Newly established populations often have high female frequencies, probably
because of the absence of nuclear alleles that restore
male fertility of the local cytoplasmic types. When
appropriate restorer alleles arrive at the population
and increase in frequency, female frequency declines
(Couvet et al. 1986; Belhassen et al. 1989; Manicacci
et al. 1992; Manicacci et al. 1996).
Although hermaphrodites show genetic variation
for both pollen (Atlan et al. 1992) and viable seed
(Thompson & Tarayre 2000) production, previous
studies of different populations that vary widely in sex
ratio have shown no evidence that hermaphrodites
adjust their male function in relation to female frequency (Manicacci et al. 1998; Thompson & Tarayre
2000). This lack of correlation between hermaphrodite
male function and female frequency among populations may be explained by rapid population turnover
and the longevity of individual plants preventing an
evolutionary response in hermaphrodite male function
at the population level (Manicacci et al. 1998). However, if female and hermaphrodite plants show genderspecific flowering phenology that is consistent across
populations, this may create a more predictable temporal variation in mating environment that could select
for a response in hermaphrodite sex allocation within
populations across a flowering season.
Hermaphrodite flowers are protandrous, with male
and female phases of roughly the same duration (2–
3 days). Individual plants flower for up to 6 weeks. As
females have a two to five times higher seed set than
hermaphrodites in natural populations (Assouad et al.
1978) and after controlled pollination (Thompson &
Tarayre 2000), pollen that fertilizes the ovules of a
female plant will have a greater chance of successfully
siring a seed than pollen fertilizing a hermaphrodite
plant. We would thus predict that, across a flowering
season, sex allocation in hermaphrodite flowers of T.
vulgaris may respond to both variation in the number
of hermaphrodite flowers in their female phase and to
the flowering phenology of female plants.
We quantified the flowering phenology of females
and hermaphrodites of T. vulgaris in order to examine
whether temporal variation in the mating environment
due to gender-specific flowering phenology occurs.
We examined how hermaphrodites of similar age and
size originating from 29 different families sampled
from four populations vary in sex allocation across one
flowering season. We followed individual hermaphrodite
plants across their entire flowering period and estimated their allocation to male (pollen production) and
female (seed production and germination rate) functions at different dates. Based on the flowering phenology of females and hermaphrodites we estimated
the relative number of open female flowers and hermaphodite flowers in the male and female phases on
each of the dates on which pollen and seed production
were quantified. We were thus able to examine whether
temporal variation in sex allocation in hermaphrodites
is correlated with temporal availability of female flowers through the flowering season.
Materials and methods

Plants used in this study all originated from populations in and around the St Martin-de-Londres valley
25 km north of Montpellier. The sampling scheme and
pollination experiment used to produce these plants is
described in Thompson & Tarayre (2000). Briefly, four
natural populations with varying female frequency
(population 1, 26%; population 2, 62%; population 3,
12%; and population 4, 80%) served as seed sources.
Fruits were collected from 10 female plants in each
population (F0-parents, each giving rise to a maternal
lineage) and their seeds germinated and brought to
flowering at the CEFE-CNRS experimental garden in
Montpellier. One or two F1 females and hermaphrodites were selected from the offspring of each F0 female
parent and F2 offspring were produced from selfing,
within-family and between-family (within population)
17
Sex allocation in
thyme
Table 1 Sample sizes used to quantify flowering phenology of
hermaphrodites (H) and females (F) in the of F2 offspring of
five to eight families from each of four populations of Thymus
vulgaris grown in an experimental field
Population
1
Family
1
2
3
4
5
6
7
8
Total
2
H
F
36
12
28
30
55
33
9
37
14
11
10
7
15
16
10
4
240
87
H
26
22
31
16
22
3
F
H
4
F
H
F
–
–
–
18
13
18
20
17
–
–
–
19
21
29
9
28
7
11
v8
7
23
15
20
19
10
12
4
21
23
28
8
12
29
21
15
16
19
27
34
27
18
21
25
117
86
132
110
157
187
crosses. Due to variation in growth and flowering of
these F1 plants, the number of maternal lineages, hereafter families, derived from a population varied from
five to eight (Table 1). For each cross, seeds were germinated and up to 10 seedlings were planted in a single
randomized block in the experimental field at the
CEFE-CNRS laboratory in Montpellier, to give F2
plants. The sex ratio values for the F2 offspring were 26,
43, 46 and 53% (for populations 1–4, respectively).
All plants used were 3 years old and grew in the same
experimental field. Hence we limited the expression
of any variation in phenology and resource allocation that could be caused by ontogenetic or large-scale
environmental differences. Any variation among sexes,
populations and families thus, at least partly, reflects
genetic variation.
  
Twice weekly, from the end of March until the beginning of June 2001, all plants (c. 1200, see Table 1 for
details) were observed and classified into five phenological classes: pre-flowering, beginning of flowering,
maximum flowering, end of flowering, and post-flowering.
A plant was considered to be at maximum flowering
when more than 50% of its stems bore open flowers,
and to have ended its flowering when the last open
flower had withered. Thus, for each individual plant
data were obtained from its day of onset of flowering,
onset of maximum flowering, and end of flowering.
    
© 2004 British
Ecological Society,
Journal of Ecology,
92, 15–23
In each F2 family from each population, three hermaphrodites were chosen for the study of sex allocation
(a total of 87 hermaphrodite plants). Care was taken to
choose individuals of similar size in order to reduce any
size-dependent effects there might be on sex allocation
pattern. To prevent variation in the degree of inbreeding
within and between families confounding our results
we used only plants produced by a between-family cross.
Furthermore, as resource allocation to male and female
functions among progeny may depend on the sex of the
maternal plant (Atlan et al. 1992; Gigord et al. 1999),
all the hermaphrodites we studied had female plants as
both their maternal parent (F1) and grandparent (F0).
On each of five dates across the flowering period (i.e.
days 17, 29, 36, 42 and 48 after the day when the first
open flower was observed), four randomly chosen flower
buds from each hermaphrodite were collected just prior
to anthesis and placed in an eppendorf tube and kept at
5 °C until the end of the flowering season. Flower buds
were collected from the same individuals at each date
and any variation across the season thus represents variation in sex allocation within individuals, not between
early and late flowering individuals.
Pollen was extracted by adding 300 µL of pure sulphuric acid to each sample for 24 h, to destroy all floral
parts except the pollen grains. After adding 1.5 mL of a
2% Triton solution samples were centrifuged for 5 min
at 2000 g. The pellet was washed twice with 1.0 mL ethanol, each time centrifuged for 5 min at 2000 g, before
drying; 100 µL of counting solution (20% glycerin and
30% saccharose) was then added and samples were
kept at 5 °C until counting. Immediately before counting, samples were placed in a ultrasonic bath for 2 min
to prevent pollen grains from aggregating. Pollen grains
in a 1-µL alignment were counted under a microscope
using a stage micrometer and the mean of two counts
used as an estimate of pollen production. This procedure is that used in previous studies of male gender
in Thymus (Atlan et al. 1992; Manicacci et al. 1998).
Fruit set of flowers was estimated for three different
periods within the flowering season, i.e. beginning,
middle and end of the flowering period. On days 17, 36
and 48, four to six flower buds about to undergo anthesis were selected on the same plants from which pollen
production was estimated and their bracts marked with
paint. Mature, marked fruits were later collected and
seed set counted. As for estimates of pollen production,
any variation in fruit set across season reflects variation
in sex allocation within individuals. Some fruits had
fallen to the ground or had lost their marks prior to collection and only a total of 148, 65, 161 and 168 fruits
from populations 1–4, respectively, were analysed. All
seeds produced from these fruits (125, 86, 226 and 205,
respectively) were scarified by automatic shaking for
60 min in eppendorf tubes containing Fointainebleau
sand. Families were pooled within each population and
sampling date and seeds were sown in plastic trays containing a 1 : 1: 1 sand : humus : garden soil mixture
and left to germinate for a period of 3 months.
We used pollen production per hermaphrodite flower
as an estimate of male function and used seed set per fruit
and germination rate as estimates of female function.
We also marked flowers produced across the season
on one female plant from each family. However, because many fruits were lost or had their paint rubbed
18
B. K. Ehlers &
J. D. Thompson
off, it was not possible to present data on female seed
set. Manicacci (1993) showed that females do not vary
their seed set across flowering season, provided they are
not pollen limited, and Thompson & Tarayre (2000)
showed that seed set of females shows little variation
relative to that by hermaphrodites.
     
     

In order to determine whether pollen production of
hermaphrodites was correlated with the number of
available flowers in the female phase we estimated (a)
the number of open female flowers on female plants
and (b) the number of hermaphrodite flowers in the
male and female phase, on each of the five dates on
which pollen production was estimated. We assumed
that all plants produce the same maximum number of
flowers at maximum flowering and that the number
of flowers produced by a plant can be described as a
linearly increasing function from its first day of flowering to its date of maximum flowering and as a linearly
decreasing function from then to the end of flowering.
To estimate the total number of open flowers on
female plants from each population on a given day we
summed the number of flowers produced by all the
individual female plants within that population.
As individual plants flower continually across a season and male and female phases within an hermaphrodite flower last the same length of time, we assumed
that at any given time the number of flowers in the
female phase was half the total number of open hermaphrodite flowers (calculated as described above).
Furthermore, as hermaphrodite individuals produce
two to five times fewer seeds than females we reasoned
that one female-phase hermaphrodite flower is of
less value than a flower on a female plant, and therefore
divided the number of the former by three to give
approximately equivalent values. These estimates do
not describe the absolute number of flowers open on a
given date, but provide an estimate of their relative number
for comparison among dates within a population.
The number of male-phase flowers (the other half
of the open hermaphrodite flowers on each of the
sampling dates) was multiplied by the pollen grains
produced per flower to give a relative estimate of the
total pollen production in a population.
 
The effects of sex, population and family, and the
interactive effects of sex and population or family, on
flowering phenology were analysed by a mixed model
 with families nested within populations, using
PROC GLM (SAS 1999). To further examine the effect
of family and sex, a mixed-model  was performed for each population separately. In both analyses the effect of family and the interaction between
sex and family were specified as random effects with
denominator degrees of freedom calculated using
Satterthwaite’s approximation (SAS 1999). F-ratios
were calculated using type III sums of squares. Plots of
residual error values were inspected visually in order to
verify homogeneity of variances.
The effect of population on pollen and seed production (all dates combined) were analysed by one-way
 using PROC GLM (SAS 1999). Variation in
pollen production and seed production among populations and dates were analysed by repeated measures
 with the PROC GLM (SAS 1999) using the
REPEATED statement, which takes into account
measures taken on the same plants but at different
dates. Due to missing data within families at some dates
these analyses were performed using the family means
for each date. Hence, the family × time interaction
could not be calculated. Type III sums of squares were
calculated in all s. Differences in seed germination among populations and dates were analysed with
a contingency tables test using the computer program
JMP (SAS 1995). As the low number of families did not
meet the criteria for normal distribution of data, we
used a non-parametric Spearman correlation to examine the association between pollen production and seed
set across families.
Results
  
Analysis of variance for all populations combined (Table 2)
revealed significant population × sex interactions on
Table 2 Mixed-model  of timing (first, maximum and end) and length of flowering in hermaphrodites and females from four populations of Thymus vulgaris
First flowering
Source
d.f.
MS
Sex
1
286.1
Population
3 1290.5
Family (population)
26
164.5
Sex × population
3
106.4
©
British
Sex2004
× family
(population)
25
54.7
Ecological
Society,
Error
1049
34.3
Journal of Ecology,
*Error
degrees of freedom were 1047.
92,
15–23
Maximum flowering
End of flowering
Length of flowering
F
P
MS
F
P
MS
F
P
MS
8.3
37.6
4.8
3.1
1.6
0.004
< 0.0001
< 0.0001
0.03
0.03
8121.7
454.9
103.9
225.8
70.3
32.6
248.9
13.9
3.2
6.9
2.2
< 0.0001
< 0.0001
< 0.0001
0.0001
0.001
13876.8
956.1
130.0
77.8
70.4
37.2*
372.7
25.7
3.5
2.1
1.9
< 0.0001 43146.0
< 0.0001
172.3
< 0.0001
127.9
0.1
567.2
0.005
121.1
60.1*
F
P
718.1
2.9
2.1
9.4
2.0
< 0.0001
0.04
0.0009
< 0.0001
0.002
19
Sex allocation in
thyme
© 2004 British
Ecological Society,
Journal of Ecology,
92, 15–23
all phenological variables except end of flowering,
indicating that the effect of sex on flowering phenology
varied among populations. Differences between the two
sexual phenotypes also depended on family, as shown
by the significant sex × family interaction for all variables.
There were significant differences between the two sex
phenotypes for all phenological variables, and population
of origin had a significant effect on the timing of flowering.
The significant population × sex effect is due to the
fact that although hermaphrodites always achieve
maximum flowering sooner and end flowering later,
and thus flower for a significantly longer period than
female plants, the degree of difference between the
two sexes vary between populations (Fig. 1). Analysis of
variance for each population separately confirmed that
the response is the same for all populations: a significant effect of sex on the timing of maximum and end of
flowering, and the length of flowering, but not on its
Fig. 1 Population mean values (+ SE) for number of days to
first, maximum and end of flowering and for the length of
flowering (in days) for hermaphrodites (closed bars) and
females (open bars) in the F2 offspring from four populations
of Thymus vulgaris grown in the same field. Times are
measured as the number of days since the first open flower was
observed in a population.
onset (Table 3). Although the peak flowering period of
hermaphrodites starts before that of females (around
day 25 vs. day 36) it lasts longer and encompasses that
of females. In all populations (and families), hermaphrodites flower for a longer period than females (6 vs. 3–
4 weeks, Fig. 1, Table 3).
    
Analysis of variance of pollen production per flower
across all dates combined showed no effect of population (F3,101 = 0.96, P > 0.1) but a significant effect of
family within populations (F23,101 = 1.83, P < 0.05).
Repeated measures analysis of variance showed no
effect of population and revealed a significant effect of
time on pollen production per flower (Table 4). Thus
pollen production showed similar variation across the
flowering season in all four populations (Fig. 2a). After
a sequential Bonferonni correction for multiple (four)
comparisons, there was no significant variation in
pollen production per flower between days 17 and 29
(F1,16 = 0.84, P > 0.1), a significant increase in pollen
production between days 29 and 36 (F1,16 = 7.71,
P < 0.05), a small but significant decrease between
days 36 and 42 (F1,16 = 6.87, P < 0.05), and a highly significant decrease between days 42 and 48 (F1,16 = 9.18,
P < 0.01). On day 48 pollen production per flower was
reduced by 83%, 74%, 69%, and 71% of that on day 36
in populations 1–4, respectively (Fig. 2a).
Total pollen production (number of hermaphrodite
flowers in male phase multiplied by pollen per flower at
each sampling date; Fig. 3, bars) showed more marked
temporal variation, with a 100% increase in all populations around day 36 relative to days 17 and 48. This
temporal variation parallels the number of open female
flowers (Fig. 3, line). A positive correlation between
total pollen production and number of open female
flowers was found across all populations (pairwise
correlation: r = 0.58, n = 20, P < 0.01), and within
populations 2, 3 and 4 (Spearman rank correlation
similar for each of the three populations: rs = 0.9,
P < 0.05, n = 5) but not in population 1 (rs = 0.77,
P = 0.1, n = 5).
Overall  showed no significant effect of population (F3,108 = 2.07, P = 0.09) or family within populations (F23,108 = 1.48, P = 0.08) on seed production.
Repeated measures analysis of variance showed no
effect of population but a significant effect of time
on seed set (Table 4). In contrast to pollen production per flower, seed set per fruit on hermaphrodite
plants was maximal at the beginning of the flowering
season, showed a significant decrease at peak flowering
(F1,20 = 28.44, P < 0.0001) and a significant increase
(F1,20 = 7.15, P < 0.05) between days 36 and 48, with
similar patterns in all populations (Fig. 2b). Seed
germination patterns were similar to those for seed
production, although the late season increase was
more marked (Fig. 2c). Except for population 1 ( χ22,123
= 2.71, P > 0.1), all populations showed a significant
Table 3 Mixed model  of timing (first, maximum and end) of flowering and length of flowering in hermaphrodites and females in each of four
20
populations
B.
K. Ehlersof&Thymus vulgaris
J. D. Thompson
Population 1
Source
d.f. MS
(a) First flowering
Sex
1
Family
7
Sex × family
7
Error
312
Population 2
F
2.32
188.1
20.6
25.7
P
d.f. MS
Population 3
F
13.09
3.1
4.50
d.f. MS
P
0.09
0.76
7.32 < 0.0001
0.8
0.59
1
4
4
193
524.4
122.0
180.2
40.1
0.0004
0.02
0.002
(b) Maximum flowering
Sex
1 3262.6
Family
7
99.8
Sex × family
7
3.07
Error
310
30.3
107.6 < 0.0001
3.29
0.002
0.1
0.99
1
4
4
190
2788.6
57.35
110.3
31.8
(c) End of flowering
Sex
1 4017.7
Family
7
111.2
Sex × family
7
102.5
Error
309
35.8
112.4 < 0.0001
3.11
0.004
2.87
0.007
1
4
4
190
3854.7 104.68 < 0.0001
1 2945.5
132.6
3.60
0.007
7 148.3
71.34
1.94
0.11
7
40.86
36.8
226
39.8
(d) Length of flowering
Sex
1 14531.2
Family
7
148.8
Sex × family
7
102.5
Error
309
57.3
253.5 < 0.0001
2.60
0.01
1.79
0.09
1 13200.5
4
130.5
4
113.8
190
57
87.7 < 0.0001
1.80
0.13
3.47
0.09
231.5 < 0.0001
2.29
0.06
2.0
0.1
1
7
7
226
41.3
24.0
37.5
23.0
1 1333.7
7
62.1
7 140.4
226
31.5
1 8243.2
7 199.0
7 138.0
226
68.0
Population 4
F
d.f. MS
P
1.80
1.04
1.63
0.18
0.40
0.13
1
8
7
319
F
P
7.6
287.9
34.5
47.2
0.16
0.69
6.10 < 0.0001
0.73
0.65
42.37 < 0.0001
1.97
0.06
4.46
0.0001
1 1108.7
8 167.4
7
44.6
319
36.2
30.65 < 0.0001
4.63 < 0.0001
1.23
0.28
74.02 < 0.0001
3.73
0.0008
1.03
0.41
1 3226.5
8 129.0
7
67.23
319
37.1
87.02 < 0.0001
3.48
0.0007
1.81
0.08
121.3 < 0.0001
2.93
0.006
2.03
0.052
1 8018.7
8
46.1
7 128.9
319
59.0
135.9 < 0.0001
0.78
0.62
2.18
0.04
Fig. 2 Allocation to male (pollen production) and female (seed set and germination rate) function of hermaphrodites across the
flowering season in F2 offspring from four populations of Thymus vulgaris grown in a uniform field. (a) Mean (+ SE) pollen production
per flower. (b) Mean seed set (+ SE) per flower. (c) Germination rate. Dates are number of days after the first flower opened.
© 2004 British
Ecological Society,
Journal of Ecology,
92, 15–23
effect of date on seed germination ( χ22,84 = 7.05, P < 0.05
for population 2, χ22,130 = 9.98, P < 0.01 for population
3, and χ22,203 = 10.43, P < 0.01 for population 4).
We found a significant negative correlation between
seed set and pollen production across families pooled
over populations (Spearman rho = −0.48, P < 0.01,
n = 26, Fig. 4), as previously observed in T. vulgaris
(Atlan et al. 1992), suggesting that there may be a
genetically based trade-off between male and female
function. For the three days on which we estimated
21
Sex allocation in
thyme
Table 4 Repeated measures  for (a) pollen production and (b) seed set in families from four populations of Thymus vulgaris
grown in a uniform garden
Pollen production
Seed set
Source
d.f.
MS
F
P
d.f.
MS
Population
Error
3
16
2439.3
1250.5
1.95
0.16
3
20
0.7
0.9
0.82
0.50
Time
Time × population
Error (Time)
4
12
64
3230.4
784.9
653.3
4.94
1.2
0.002
0.30
2
6
40
6.6
0.6
0.4
14.73
1.32
< 0.0001
0.27
F
P
Discussion
Fig. 3 Relative total pollen production by hermaphrodites in
a population (histograms), relative number of open flowers
on female plants (solid line) and relative number of open
hermaphrodite flowers in female phase (dotted line) scaled to
the value of a female flower on a female plant (see text) across
one flowering season in the F2 offspring from four populations of Thymus vulgaris grown in a uniform field.
© 2004 British
Ecological Society,
Journal of Ecology,
92, 15–23
seed set, a significant negative correlation was observed
on day 17 (Spearman rho = −0.58, P < 0.05, n = 25) and
day 36 (Spearman rho = −0.45, P < 0.05, n = 26) but not
on day 48 (Spearman rho = −0.18, P > 0.1, n = 21).
The principal result of this study is that hermaphrodites show temporal variation in sex allocation across
a flowering season in plants originating from each of four
different populations. This variation is due to variation
in sex allocation among flowers produced at different
times on the same plant and indicates that the relative
gender of hermaphrodites varies in time across a single
season. In addition, gender-specific flowering phenology
results in large temporal variation in the availability of
ovules, with the number of open female flowers positively
correlated with pollen production by hermaphrodites.
Several different factors may contribute to this
temporal variation in sex allocation. On a per flower
basis, our results agree with the prediction of Brunet
& Charlesworth (1995) that relative female function
is higher in early-produced flowers (day 17) and that
relative male function is higher in later produced flowers
(day 36). However, female function predominates again
at the end of the flowering season (day 48), due to a
decrease in pollen production per flower and an
increase in seed germination rates (Fig. 2).
Although care should be taken in interpretation,
the correlation between pollen production and open
female flowers (Fig. 3) fits the prediction that malebiased sex allocation is favoured when the probability
of pollen transfer to ovules is high. Such temporal variation of allocation in hermaphrodites may thus have an
adaptive basis by maximizing male function and minimizing female function when female plants are at peak
flowering. The same temporal allocation pattern was
found in hermaphrodites in all four populations, supporting the possibility that it may have been selected for.
The temporal variation in hermaphrodite sex allocation might be expected to be greatest in the populations
with the highest female frequency as these would also
hold the highest number of available female ovules. Significant correlations between pollen production and
open female flowers were indeed found for the three
populations with the highest female frequency in the F2
generation (i.e. populations 2, 3 and 4, with female frequencies of 43%, 46% and 53%, respectively), although
in the field population 3 had the lowest estimated
female frequency (12%) of the four. Sex ratios may,
however, vary over years within populations, and this
22
B. K. Ehlers &
J. D. Thompson
Fig. 4 Plot of family means of seed and pollen production of hermaphrodites from five to eight families from four populations.
Closed diamonds, population 1; closed squares, population 2; closed triangles, population 3; open circles, population 4.
© 2004 British
Ecological Society,
Journal of Ecology,
92, 15–23
may constrain variation in sex allocation in response to
variation in sex ratio among populations.
A second factor that may influence temporal variation in sex allocation is variation in the selfing rate.
Selfing rate in hermaphrodites increases in populations with increased female frequency in several gynodioecious species (Sun & Ganders 1986; McCauley &
Taylor 1997), including T. vulgaris (Valdeyron et al. 1977;
Thompson & Tarayre 2000), and it is possible that
there may also be temporal variation in the selfing rate
of hermaphrodites over the season as female flowering
increases. In T. vulgaris, pollination is mainly by honeybees that visit many open flowers on the same plant.
Thus, levels of geitonogamous pollination may be very
high at peak flowering when the frequency of female
flowers is high and when hundreds of flowers may be
open on a single hermaphrodite plant. As selfing causes
a decline in the germination rate of seeds produced by
hermaphrodites of T. vulgaris (Assouad et al. 1978;
Thompson & Tarayre 2000) the seriously reduced germination of seeds in the middle of the flowering season
could be a consequence of inbreeding depression. The
germination rate in some populations is reduced by
almost 50% at peak flowering relative to the beginning
and end of the flowering period. If increased selfing
were to explain this result it would require an inbreeding depression on germination rate of up to 50%
assuming complete selfing at peak flowering and complete outcrossing at the beginning and end of flowering.
Even higher levels of inbreeding depression would be
needed if intermediate selfing rates are assumed. Such
high levels of inbreeding depression would be surprising
(Husband & Schemske 1996) and have not been reported
for this species (Thompson & Tarayre 2000). Thus,
variation in the selfing rate alone is unlikely to explain
the decrease in seed germination at peak flowering.
In the case of an increased selfing rate, sex allocation theory predicts a female-biased sex allocation (e.g.
Charlesworth & Charlesworth 1981; De Jong et al.
1999), whereas we observed an increase in male func-
tion when selfing was potentially highest. Although
selfing rate may vary across the season, the observed
allocation towards increased male function is probably
not a response to a potentially increased level of selfing
at peak flowering.
Finally, the reduced female function in hermaphrodites at peak flowering could be a consequence of
resource limitation. It is possible that high flower production causes a trade-off between number of flowers
and reproductive investment in individual flowers.
Mazer & Schick (1991) found that investment in female
gametes in Raphanus is indeed more sensitive to
resource limitation than male gamete production.
Although not a new result (see Atlan et al. 1992), we
found a significant negative correlation between seed
set and pollen production among families, indicating
that trade-offs between male and female function may
influence the evolution of resource allocation patterns
in hermaphrodites of T. vulgaris.
To our knowledge, this is one of the first studies to
demonstrate significant within-individual variation in
allocation to male and female function across a single
flowering season. Our data fit the predictions made by
Brunet & Charlesworth (1995) that hermaphrodites
should increase their allocation to male function when
availability of ovules increases. Deciding whether the
pattern is shaped by variation in the mating environment, selfing rate or resource depletion at the time of
peak flowering, however, requires comparative studies
of hermaphrodite and gynodioecious species.
Acknowledgements
We are grateful to M. Maistre, who counted the seeds,
C. Collin for help with plant cultivation, to B. Husband,
S. Maurice and T. Bataillon for discussion, and to T.
Bataillon, Pierre-Olivier Cheptou and anonymous
reviewers for useful comments on earlier versions of the
manuscript. The Danish National Science Foundation
(SNF) and the CNRS provided financial support.
23
Sex allocation in
thyme
© 2004 British
Ecological Society,
Journal of Ecology,
92, 15–23
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Received 20 June 2002
revision accepted 14 October 2003