Directory UMM :Data Elmu:jurnal:A:American Journal of Botany:Vol88.Issue1.Jan2001:
American Journal of Botany 88(1): 68–75. 2001.
STAMEN DIMORPHISM IN RHODODENDRON FERRUGINEUM
(ERICACEAE): DEVELOPMENT AND FUNCTION1
NATHALIE ESCARAVAGE,2 ELISABETH FLUBACKER,2 ANDRÉ PORNON,3
BERNARD DOCHE,2 AND IRÈNE TILL-BOTTRAUD2
Laboratoire de Biologie des Populations d’Altitude CNRS-UMR 5553, Université Joseph Fourier, B.P. 53,
F-38041 Grenoble Cedex 09, France; and 3Laboratoire d’Ecologie Terrestre CNRS-UMR 5552,
Université Paul Sabatier, Zoologie Ecologie,
Bât. 4R3, 118, route de Narbonne, F-31062 Toulouse Cedex 04, France
2
The function of stamen dimorphism in the breeding system of the alpine shrub Rhododendron ferrugineum was studied in two
populations in the French Alps. This species has pentameric flowers with two whorls of stamens: an inner whorl of five long stamens
and an outer whorl of short stamens. We studied the development of stamens from buds to mature flowers (measurement of the
filament, anther, and style lengths at five successive phenological stages) and compared the size and position of reproductive organs
at maturity in control and partially emasculated flowers (removal of long-level stamens) to determine whether the presence of longlevel stamens constitutes a constraint for the development of the short-level ones. Stamen dimorphism can be observed early in stamen
development, from the bud stage of the year prior to flowering. At this early stage, meiosis had already occurred. Emasculation of the
long-level stamens induced the short-level ones to grow longer than in normal conditions. We also performed seven pollination
treatments on ten randomly chosen individuals in each population, and the number of seeds following each treatment was recorded.
Results from these treatments showed that R. ferrugineum produced spontaneous selfed seeds in the absence of pollinators. However,
no seed was produced when short-level stamens were emasculated and pollinators excluded, suggesting that long-level stamens are
not responsible for selfing in the absence of pollinators and that reproductive assurance is promoted by short-level stamens.
Key words:
Ericaceae; pollination system; reproductive assurance; Rhododendron ferrugineum; stamen development; stamen size.
In alpine and arctic environments, low pollinator activity
and diversity should promote characters that result in the assurance of seed production through self-pollination. Most temperate alpine plants need pollinators for pollen deposition on
the stigma, and therefore, are predominantly entomophilous
(Kevan, 1973; Arroyo, Primack, and Armesto, 1982; Kudo,
1993; Totland, 1994; Jacquemart and Thompson, 1995). However, some animal-pollinated plants growing in severe environmental conditions have a mixed-mating system and can
produce spontaneous selfed seeds. Outcrossing will usually occur when reproductive fitness is not limited by a paucity of
animal visits. Where such limitations exist, features that maximize within-flower selfing (autogamy) may be favored (Richards, 1997). Although autogamy is frequent, it is rarely predominant, probably because most plants growing in harsh conditions are long-lived perennials for which constraints on reproductive fitness by seed are low (Richards, 1997). In
Rhododendron parviflorum and R. redowskianum (in Siberia)
spontaneous autogamy occurs in the absence of pollinators
(Tikhmenev, 1985), however in some Vaccinium in the Belgian Ardennes (V. myrtillus, V. uliginosum, V. vitis-idaea; Jacquemart and Thompson, 1995), insect-mediated self-pollination accounts for the majority of seeds produced by selfing. In
other species, like Rhododendron aureum (in Japanese mountains), spontaneous selfing does not occur and insect pollina-
tion is necessary for seed set (Kudo, 1993). One character that
plays a key role in promoting reproductive assurance in selfcompatible species is the reduction of stigma-anther separation
(Piper, Charlesworth, and Charlesworth, 1986).
The species studied here, Rhododendron ferrugineum L.
(Ericaceae), is an evergreen shrub with a mean height of 70
cm that grows at the subalpine level from ;1600 to 2200 m.
It reproduces both vegetatively (by layering) and sexually.
Asexual propagation occurs in closed and mature populations
(Pornon et al., 1997) and preferentially down hill (Escaravage
et al., 1998). Rhododendron ferrugineum produces an inflorescence of 5–22 bright-red nectariferous flowers. The flowers
are protandrous (anthers mature before the stigma) and are
initiated the year before they mature (Escaravage et al., 1997).
Rhododendron ferrugineum is a self-compatible species, mainly pollinated by bees, bumble bees, and diptera. Spontaneous
selfing occurs in the absence of pollinators (Escaravage et al.,
1997). A study of the floral morphology revealed herkogamy
(spatial separation between anther and stigma) with different
stamen lengths: short-level stamens below or at the same
height as the style and long-level ones above the style (Escaravage et al., 1997). The term stamen dimorphism will be
used subsequently. Stamens are mediafixed (filament links to
the back of the anther) and are located on two whorls (five
stamens per whorl) with long- and short-level stamens on the
inner and outer whorls, respectively (E. Flubacker, personal
observation). This has not been described in other Rhododendron species and the question that arises is: what are the mechanisms that promote this spatial separation? Are developmental processes involved? Floral developmental studies have
been performed on heterostylous taxa (Cheung and Sattler,
1967; Ganders, 1979) and more recently Richards and Barrett
(1984, 1987) examined floral developmental differentiation
among morphs in tristylous species. In these species, the var-
Manuscript received 3 August 1999; revision accepted 11 April 2000.
The authors thank Pascal Thomas for help with the field experiments and
John D. Thompson and two anonymous referees for their critical comments
and helpful suggestions that greatly improved the first draft of the manuscript,
and Paul G. Wolf for improving the English text. This work was financially
supported by University Joseph Fourier (Grenoble), the Centre National de la
Recherche Scientifique (C.N.R.S.), and the French Ministry of the Environment (E.G.P.N. no. 95.085).
3
Author for reprint requests (e-mail: pornon@cict.fr).
1
68
January 2001]
ESCARAVAGE
ET AL.—STAMEN DIMORPHISM IN
iability caused by timing and position of stamen origin probably accounts for variation in stamen length within flowers of
heterostylous species that have two whorls of stamens (Richards and Barrett, 1992). In some arctic Primula species, homostyly has replaced heterostyly (Kelso, 1992). It is suggested
that both restrictions of pollination efficiency and developmental constraints (imposed by the need for reciprocity in organ position and to limit stigma-anther separation) are involved in this evolutionary process (Richards and Barrett,
1992).
In this study, we focused on stamen dimorphism and its
possible consequences on the breeding system of Rhododendron ferrugineum. The aims of this paper are (1) to describe
the development of the stamens in Rhododendron ferrugineum
(from the bud stage to full bloom) to determine at what phenological stage stamen dimorphism can first be detected, and
(2) to determine the role of long- and short-level stamens in
the pollination of the species.
MATERIALS AND METHODS
The study site—This study was carried out during the summer of 1997 at
two mountain sites in the northwestern French Alps (Belledonne range). The
first was ‘‘Collet d’Allevard’’ (458259 N, 68109 E) located at 1680 m on a
north-facing slope (50%), and the second was ‘‘Chamrousse’’ (458259 N,
508559 E) located at 1770 m on a west-facing slope (62%). In both sites, the
populations are composed of Rhododendron ferrugineum with a cover of 40%,
mixed with other Ericaceous shrubs, such as Vaccinium myrtillus, V. uliginosum, and V. vitis-idaea.
Experimental design—Stamen development—To study patterns of stamen
development, we randomly selected ten individuals in each population. On
each individual we conducted the following experiments: we collected three
inflorescences per individual at five successive phenological stages [inflorescence buds (t0), young inflorescences (t0 1 2 d), medium-aged inflorescences
(t0 1 4 d), advance-aged inflorescences (t0 1 6 d), and inflorescences with
flowers in full bloom (t0 1 8 d)]. One flower per inflorescence was dissected
and measured under a dissecting microscope, together with the filaments, anthers, and style.
Effects of population, individual, flower, whorl, and position of stamens on
anther and filament length in mature flowers were statistically tested by performing five-way mixed analysis of variance using PROC GLM (SAS, 1990).
Population, whorl, and position of stamens were treated as fixed effects; individual and flower were random effects. Population, individual, and flower,
and position and whorl were nested (see Table 2).
Associations between anther height of the short- and long-level stamens
and length of short- and long-level filament were tested by a correlation analysis (SAS, 1990). Data were ln transformed for normality. All these tests were
performed for each developmental stage. The mean daily growth rate, computed as the length of each stamen at the end of the last phenological stage
(full bloom) divided by the number of days elapsed between bud breaking
and full bloom (8 d), was compared by performing a Tukey HSD multiplerange test using SPSS for Windows, release 9.0.0.
Effects of partial emasculation—To determine whether the presence of
long-level stamens (inner whorl) limits the development of short-level stamens
(outer whorl), we selected three other inflorescences on the same ten individuals in which we partially emasculated three flowers at the bud stage. Anthers
of long-level stamens (inner whorl) were removed (but their filaments remained) and short-level stamens (outer whorl) were left intact. It would have
been interesting to remove the stamens of the outer whorl to compare the
results, but unfortunately this experiment was difficult to perform without
damaging the flower. The other flowers of the inflorescence were unmanipulated. When flowers reached their mature size, we collected all the treated
inflorescences. We dissected the partially emasculated flowers under a dis-
RHODODENDRON FERRUGINEUM
69
secting scope and measured the style, anther, and filament lengths of the intact
stamens of outer whorl, and the filament length of emasculated stamens (inner
whorl). We dissected and performed the same measurements on one unemasculated flower of each inflorescence as a control. In both experiments, stamens
were numbered according to their position in the flower. Stigma-anther separation was calculated as filament length minus style length.
In this experiment, the effects of population, individual, flower, treatment
(flower partially emasculated or not), and position of stamens on filament
length of each stamen (emasculated or not) were tested by five-way mixed
analysis of variance (SAS, 1990) with population, emasculation, and whorl
of stamens as fixed effects, individual and flower as random effects, and
population, individual, and flower nested. The treatment effect tested for an
effect of partial emasculation on the whole, whereas the effect of emasculation
on the longer filaments was tested by the treatment 3 whorl interaction. For
each site and treatment (partial emasculation or not) the whorl effect on filament length and stigma-anther separation was tested by one-way analysis of
variance (SAS, 1990).
At the end of the flowering period (mid-July), floral primordia for the next
flowering season are initiated. To determine when meiosis of pollen grains
occurred, we collected two flowers (from different inflorescences) per individual, on ten individuals in each population every week from mid-July to
mid-August. We dissected the flowers and crushed the anthers on a slide in
a drop of Alexander’s stain (Alexander, 1969), and observed the slide under
a microscope. As pollen is released in tetrads in the Ericaceae, observation
of single cells indicates that meiosis had not yet occurred.
Pollination trials—To study the respective roles of short- and long-level
stamens in sexual reproduction, we (1) quantified the number of pollen grains
per anther in the two whorls and (2) performed a series of pollination treatments. To quantify pollen production we collected one inflorescence bud on
each of ten individuals and preserved it in 70% ethanol in May 1997. One
flower from each bud was dissected under a dissecting scope. Each stamen
was measured according to its position (Fig. 1) and pollen grains were quantified after acid extraction from the anther using a hemacytometer according
to Escaravage et al. (1997). Pollen production was analyzed by performing
five-way mixed analysis of variance as described in the stamen development
analysis (see above). The association between the number of pollen grains
and stamen length was tested by a correlation analysis (SAS, 1990). We performed seven pollination treatments (summarized in Table 1) on ten other
randomly selected individuals in each population. Each individual received
all seven treatments, and each treatment was performed on two inflorescences,
on at least five flowers per inflorescence. We thus studied a total of 140
inflorescences and 700 flowers at each site.
In treatments T5 and T7, we emasculated the five long-level stamens (inner
whorl), while in treatments T4 and T6 we emasculated the five short-level
stamens (outer whorl). In treatments T6 and T7, nylon mesh bags were used
to exclude pollinators, whereas treatments T4 and T5 were open-pollinated.
We performed three control treatments: in treatment T3 all stamens were
emasculated (cross-pollination control), in T2 flowers were unmanipulated and
inflorescences were bagged (self-pollination control), and T1 consisted of unmanipulated (intact) inflorescences. To avoid self-pollen contamination, emasculation was performed ; 2 d after the inflorescence bud opened. At this
stage, inflorescences were very small and the flowers were still closed. Five
flowers per inflorescence (except for T1 and T2) received one of the seven
treatments. All other flowers (between one to five flowers) were removed.
This manipulation did not disturb the development of the remaining flowers
(Escaravage et al., 1997). Fruits were collected shortly before dehiscence (early September) and the seeds counted.
To detect the effects of individual, inflorescence, and treatment on the number of seeds per fruit, we performed three-way mixed analysis of variance
using PROC GLM (SAS, 1990) for each population. Treatment was a fixed
effect while inflorescence and individual were treated as random effects. Inflorescence was nested within individual. Mean comparisons between specific
treatments were also carried out by using a Scheffé multiple-range test.
70
AMERICAN JOURNAL
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[Vol. 88
TABLE 1. Pollination tests used in the study of the pollination system
of R. ferrugineum.
Level of stamens
remaining
Bagging
No. of flowers/inflo.
Long 1 short
Long 1 short
None
Long
Short
Long
Short
No
Yes
No
No
No
Yes
Yes
all
all
5
5
5
5
5
Code
T1
T2
T3
T4
T5
T6
T7
Fig. 1. Mean sizes (in cm) of the different stamens according to their
positions for different phenological stages of the flower. Odd-numbered stamens (long-level) are on the inner whorl, even-numbered stamens (short-level)
on the outer whorl. A: inflorescence buds, B: young inflorescences, C: medium-aged inflorescences, D: advance-aged inflorescences, E: inflorescences
with flowers in full bloom.
RESULTS
Stamen development—All floral organs are preformed in
July of the year preceding flowering (N. Escaravage, personal
observation). Anthers are relatively voluminous and seem
more advanced in their development than filament, style, and
petals. At this stage anthers are positioned in staggered rows
(one anther high, one low, etc.), in such a way that stamen
dimorphism can already be observed early in development.
Moreover, pollen tetrads are observed in anthers from the second week of August on. Therefore, meiosis occurred in buds
preformed in the summer the year prior to flowering.
In unmanipulated flowers, short- and long-level stamens are
located on the outer and inner whorls, respectively. Filaments
and anthers from the inner whorl are significantly longer than
those of the outer whorl (P , 0.001; Table 2). The position
within the whorl significantly affects filament length but not
anther height. Stamen number 6 was always the smallest at
each site and each phenological stage; the longest stamens
were number 1, 3, and 9. This conferred slight zygomorphy
following a plane of symmetry passing through stamens 1 and
6 (Fig. 1). Although filament length is significantly affected
by population (P , 0.05; Table 2), stamen dimorphism is preserved among populations as indicated by the non significant
interactions of population by whorl and population by position
nested in whorl (Table 2). The mean stigma-anther separation
at Chamrousse was: 0.83 mm (60.11 SD) for the outer, shortlevel whorl and 1.63 mm (60.14 SD) for the inner, long-level
whorl. At Collet d’Allevard the mean stigma–anther separation
was 0.42 mm (60.12 SD) and 1.30 mm (60.14 SD) for the
inner, short-level and outer, long-level whorls, respectively.
The slopes in Fig. 2 represent the growth rate of anthers (a)
or filaments (b) of the long-level stamens relative to those of
the short-level stamens for each developmental stage. A positive correlation exists between anther height of long- and
short-level stamens from the bud (A) to medium (C) stages (P
, 0.001; Fig. 2a) suggesting that during these stages anther
growth still occurs. However, in later phenological stages, the
growth rates, are similar until anthers have reached their final
size. In contrast, stamen elongation occurs throughout the de-
TABLE 2. Results of analysis of variance to detect the effect of population, individual, flower, whorl, and stamen position on anther and filament
lengths in mature flowers.
Filament length
Anther height
Source of variation
df
MS
F
MS
F
Population
Individual (population)
Flower [individual (population)]
Whorl
Position (whorl)
Population 3 whorl
Population 3 position (whorl)
Error
1
18
36
1
2
1
2
494
26.94
19.71
5.12
61.86
7.92
3.03
0.36
1.06
5.47*
3.84***
4.81***
58.17***
7.44***
2.85 ns
0.33 ns
0.00
0.19
0.04
1.11
0.01
0.01
0.002
0.008
0.00 ns
4.52***
5.52***
140.73***
1.99 ns
1.27 ns
0.30 ns
Note: ***P , 0.001, **P , 0.01, *P , 0.05, ns 5 non-significant.
January 2001]
ESCARAVAGE
ET AL.—STAMEN DIMORPHISM IN
RHODODENDRON FERRUGINEUM
71
Fig. 3. Mean daily stamen growth rates of each stamen. Odd-numbered
stamens are on the long-level inner whorl and even-numbered stamens are on
the short-level outer whorl. Vertical bars indicate standard deviation. Values
that share the same letter are not significantly different at 5% threshold. Means
were compared by a Tukey HSD multiple-range test.
were significantly longer than those of unemasculated flowers
(P , 0.001; Fig. 4). In unemasculated flowers, stamens of the
inner, long-level whorl showed a greater stigma-anther separation than stamens of the outer, short-level whorl (P , 0.001).
In partially emasculated flowers the increased growth of the
stamens of the outer whorl significantly increased the stigmaanther separation (Fig. 4); as a result this distance was similar
to that measured between anthers of the longest stamens (inner
whorl) and stigma in unemasculated flowers.
Fig. 2. (a) Height of anthers of the long-level stamens as a function of
height of anthers of the short-level stamens for each phenological stage (A:
r2 5 0.6319, N 5 200, y 5 0.9082x 1 0.0883, P , 0.001; B: r2 5 0.1012,
N 5 187, y 5 0.3773x 1 0.5638, P , 0.05; C: r2 5 0.1332, N 5 199, y 5
0.4371x 1 0.5353, P , 0.001; D: r2 5 0.0171, N 5 196, y 5 0.1649x 1
0.7666, non significant; E: r2 5 0.0214, N 5 194, y 5 0.1756x 1 0.214, non
significant) and (b) length of long-level filament as a function of length of
short-level filament for each phenological stage (A: r2 5 0.6122, N 5 200, y
5 0.7982x 1 0.0711, P , 0.001; B: r2 5 0.2779, N 5 189, y 5 0.5079x 1
0.650, P , 0.001; C: r2 5 0.1854, N 5 199, y 5 0.536x 1 0.8092, P ,
0.001; D: r2 5 0.2080, N 5 194, y 5 0.4802x 1 1.2314, P , 0.001; E: r2
5 0.3867, N 5 191, y 5 0.644x 1 0.9567, P , 0.001).
velopment of the flower (Fig. 2b). For each stamen, the daily
mean growth rate is shown in Fig. 3. Stamen 6 (the shortest
stamen) has the slowest growth rate, whereas stamens 1, 3 and
9 (the longest stamens) have the fastest.
In partially emasculated flowers, treatment (effect on the
whole flower) and treatment 3 whorl interaction (specific to
the emasculated whorl) have a significant effect on filament
length (Table 3; P , 0.001). At the end of the growth of
partially emasculated flowers, mean length of the filaments of
stamens on the outer whorl (unemasculated) had increased
compared to unemasculated flowers. In both populations, the
filament length difference between whorls disappeared. For
partially emasculated flowers, stamen filaments of the outer
whorl were not different from those of the inner whorl but
Number of pollen grains per stamen—Long-level stamens
contain significantly more pollen grains than short-level stamens (Table 4), and all effects are significant. A positive correlation exists between stamen length and the number of pollen
grains in both populations (P , 0.001 with r2 5 0.211 and
0.273 for Collet d’Allevard and Chamrousse, respectively). At
Collet d’Allevard the mean number of pollen grains was
16 000 (64498 SD) and 12 257 (63761 SD) for long- and
short-level stamens, respectively. At Chamrousse, mean pollen
grain production was 21 152 (64563 SD) and 15 974 (63861
SD) for long- and short-level stamens, respectively.
Effect of partial emasculation on seed production—Significantly more seeds were produced in Collet d’Allevard than
in Chamrousse (Student’s t test, P , 0.05, data not shown).
Thus populations were studied separately. For both populations, mean seed number is influenced by the treatment (P ,
TABLE 3. Results of analysis of variance to detect effects of population, individual, flower, treatment (partial emasculation of long-level stamens), and whorl on filament length in mature flowers.
Source of variation
df
MS
F
Population
Individual (population)
Flower [individual (population)]
Treatment
Treatment 3 whorl
Treatment 3 whorl 3 population
Error
1
18
36
1
2
3
883
157.81
25.68
8.73
799.67
87.26
3.26
1.22
6.13*
2.99*
7.16***
259.02***
26.09***
2.67*
Note: ***P , 0.001, *P , 0.05.
72
AMERICAN JOURNAL
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[Vol. 88
TABLE 4. Results of analysis of variance to detect the effect of whorl,
position, population, individual, and flower on the number of pollen
grains.
Source of variation
df
Whorl
1
Position (whorl)
8
Population
1
Individual (population)
18
Flower (individual 3 population)
18
Whorl 3 population
1
Whorl 3 individual (population)
18
Whorl 3 flower (individual 3 population)
18
Error
654
MS
F
31147.6
254.8
35143.6
2657.1
304.2
1301.7
236.7
297.3
98.5
316.1***
2.6**
13.3**
8.7***
3.1***
13.2***
2.4***
3.0***
Note: ***P , 0.001, **P , 0.01.
stamens were removed (T6; Fig. 5). This effect was likely due
to the extra growth of short-level stamens following removal
of long-level ones (Fig. 4).
DISCUSSION
Fig. 4. Mean filament length and anther-stigma separation for the inner
and outer stamen whorls. One-way analysis of variance indicated significant
differences among whorls in unemasculated flowers but not in flowers where
the stamens of the inner whorl were removed. Vertical bars indicate standard
deviation.
0.001, Table 5). The greatest number of seeds was produced
following open pollination and when pollinators were excluded and flowers left intact (T1 and T2; Fig. 5). At Chamrousse,
the number of seeds decreased when at least one stamen whorl
was emasculated (T4 and T5; Fig. 5), whereas at Collet
d’Allevard the mean number of seeds produced was not affected significantly. In contrast when short stamens were emasculated and inflorescences bagged, very few seeds were produced (T7; Fig. 5). The same result was obtained when long
In R. ferrugineum two stamen levels are observed: the longlevel stamens of the inner whorl and the short-level stamens
of the outer whorls. Long-level stamens produce more pollen
grains than short-level stamens. This stamen dimorphism can
be detected as early as June of the year preceding flower maturation, and pollen grains are formed in August of the same
year. When long-level stamens are removed early the flowering year, the short-level stamens grow longer. Stamen dimorphism has not been described in other Rhododendron species
but is widespread among heterostylous taxa. As in tristylous
species of Pontederia (Richards and Barrett, 1987) and Eichhornia (Richards and Barrett, 1984) and in distylous species
of Primula (Stirling, 1932), stamen dimorphism in R. ferrugineum appears in premeiotic stamens and is maintained
throughout phenological stages.
Early in flower development, anthers occupy most of the
volume in the flower while the corolla, style, and filaments
are poorly developed. Thus, arrangement in staggered rows
seems to be the most appropriate means to limit the size of
young flowers in the buds. Overwintering of floral primordia
has been described in many alpine and tundra plants species
(Bliss, 1971; Bell and Bliss, 1980; Diggle, 1997; Aydelotte
and Diggle, 1997) and allows the flower to bloom early in the
spring (Bliss, 1971; Diggle, 1997). However, it does not seem
to be a specificity of plants growing in harsh environments
(Diggle, 1997), since it occurs in plants that flower very early
in the year (Alnus glutinosa, Coryllus avellana; N. Escaravage,
TABLE 5. Results of analysis of variance for each population, to detect individual, inflorescence, and treatment effects on the seed number per
fruit.
Population
Chamrousse
Collet d’Allevard
Source of variation
df
MS
F
MS
F
Seeds/fruit
Individual
Inflorescence (individual)
Treatment
Treatment 3 inflorescence (individual)
Error
9
10
6
60
49
536.8
676.6
14053.7
280.5
261.6
0.8 ns
2.5*
53.7***
1.1 ns
1297.2
491.9
20110.3
376.9
927.6
2.6 ns
0.5 ns
21.6***
0.4 ns
Note: ***P , 0.001, *P , 0.05, ns 5 non-significant.
January 2001]
ESCARAVAGE
ET AL.—STAMEN DIMORPHISM IN
Fig. 5. Mean number of seeds produced per fruit. Vertical bars indicate
standard deviation. For each site the treatment effect was tested by a Scheffé
multiple-range test. For each site, values that share the same letter are not
significantly different at 1% threshold.
personal observation). In alpine environments, early anther
formation and spermatogenesis could be a strategy to allow
the flower to become functional rapidly and could be an adaptation to the short optimal reproductive period, allowing the
plant to flower as soon as the temperature increases. The formation of pollen grains in the season preceding flowering is
risky since a severe winter could occur and freeze the buds.
However, this risk is reduced because flowers are protected in
bud scales and plants of R. ferrugineum usually spend the
winter under thick snow cover that buffers temperature effects
(Larcher and Siegwolf, 1985).
Two main categories of causes can explain stamen dimorphism: (1) ‘‘proximate’’ and (2) ‘‘ultimate’’ causes that refer
to developmental and selective aspects, respectively. Under the
first category filament expansion can be regulated, through anthers, by growth hormones such as cytokinin (Hess and Morré,
1978), gibberellins (Greyson and Tepfer, 1967), or auxin (Koning, 1983). Greyson and Tepfer (1967) argued that inhibition
of filament growth following emasculation in Nigella hispanica could be accompanied by inhibition of both cell elongation
and cell division in the epidermis. In R. ferrugineum, emasculation of the inner whorl results in extra growth of the outer
whorl, yet emasculation does not affect the growth of the inner
whorl. This suggests that, if hormonal control is involved it is
only in the one direction. Testing for hormonal control can be
challenging because it is often difficult to distinguish between
hormonal control and homeostatic responses.
Monopolization of space by anthers of long stamens could
represent a sufficient barrier to the development of the short
stamens to create a mechanical resistance to filament cell division. Thus, the different sizes should disappear when the
spatial constraint imposed by the long stamen is removed.
However the size difference between long- and short-level stamens lies primarily in filament length and filament growth
occurs when flowers open and space is not as limiting. Daily
stamen growth rate is similar across all stamens except for the
RHODODENDRON FERRUGINEUM
73
shortest, stamen 6, and the longest ones, stamens 1, 3, and 9
(Fig. 3). We detected no difference in stamen growth rate between inner and outer whorls (data not shown). This is different from several heterostylous species (Richards and Barrett,
1984, 1987) in which differences in stamen length among
morphs arise through differences in the relative growth rate
and the duration of growth. However, some species of Pontederiaceae, Lythraceae, and Oxalidaceae initiate two temporally separated stamen sizes. The difference in time of origin
establishes a size differential between stamen series that is
maintained throughout development. This mechanism could
account for the two stamen levels in R. ferrugineum.
Short stamens might benefit from resources normally allocated to the development of long stamens (inner whorl). However, this suggestion seems unlikely since differences in nutrient availability between populations, individuals, inflorescences, and flowers would induce variation in stamen size
within the two whorls. On the contrary, stamen hierarchy in
relation to their position in whorls appears to be similar in
both populations.
In the second category, ultimate causes, the two stamen levels may have different functions in pollination in R. ferrugineum. Our results indicate that long-level stamens do not participate autonomously in self-pollination in the absence of pollinators since no seeds were produced when short stamens are
removed (T6; Fig. 5). Because unemasculated flowers could
self in the absence of pollinators, spontaneous self-pollination
is likely due to short-level stamens. However, our experiments
provided no formal proof. The treatment in which long-level
stamens were removed also produced no seeds in the absence
of pollinators (T7; Fig. 5), but, when short-level stamens were
in the corolla alone, they grew to the height of long-level
stamens. Thus they function similarly to long-level stamens
and are inefficient in autonomous selfing. Spatial separation
between the stigma and the anther of the longest stamen is on
average 1.50 mm. This distance seems to be sufficient to avoid
self-pollination (Webb and Lloyd, 1986), especially in R. ferrugineum where pollen is sticky and dehiscence poricidal.
The distance between stigmas and anthers of short-level stamens is smaller at Collet d’Allevard than at Chamrousse. This
may explain why plants at Collet d’Allevard produced more
seeds in treatment T5. The pollination mechanism in openpollinated treatment T1 is less obvious, and we do not know
the proportion of seeds sired by self- and cross-pollination, or
which stamen level is involved. However in this species the
autofertility index is high (0.92; Escaravage et al., 1997), suggesting that autogamy could account for the majority of seeds
produced.
In a previous study on the genotypic structure of a R. ferrugineum population, Escaravage et al. (1998) showed that
genotypes were closely related. These authors hypothesized
that autogamy and geitonogamy (fertilization between different flowers on the same plant) may be responsible for such a
relatively homogeneous genetic structure. However, we have
yet to estimate the proportion of outcrossed or selfed seeds.
Both fertilization modes can occur synchronously at each generation and at all reproductive stages. In alpine environments,
snow and frost often occur during periods of growth and reproduction and result in a scarcity of pollinators (Arroyo, Primack, and Armesto, 1982; Escaravage, 1997). In such a context, self-pollination can compensate for the insufficient pollinator activity and act as reproductive assurance (Arrroyo,
1974; Wyatt, 1983; Holsinger, 1992; Harder and Barrett,
74
AMERICAN JOURNAL
1995). Therefore, in R. ferrugineum, pollination by short-level
stamens could act as reproductive assurance in the absence of
pollinators that would prevent outcrossing. Reproductive assurance is promoted in numerous plants growing in severe
environments. In arctic Primula species, it is common to see
a shift in breeding system from distyly to homostyly and at
least facultative autogamy (Kelso, 1992; Mazer and Hultgard,
1993; Miller et al., 1994). Armeria maritima has also lost distyly in the tundra portion of its range (Vekemans et al., 1990).
Anther position can affect pollen removal through its influence on the timing of anther dehiscence (Harder and Barrett,
1993). However, all anthers of R. ferrugineum flowers dehisce
before the flower opens (as in R. ponticum; Percival, 1955),
whereas stigmas become receptive when the flower opens (Escaravage et al., 1997). No difference in the timing of anther
dehiscence among whorls has been observed (N. Escaravage,
personal observation). Anther position can also determine the
placement of pollen on a pollinator’s body, which influences
the likelihood of pollen loss through grooming (Faegri and van
der Pijl, 1979). It seems that the advantages of a particular
anther position depend on the morphological and behavioral
characteristics of a plant’s dominant pollinators (Harder and
Barrett, 1993). For bee-pollinated species with tubular flowers,
anther placement in the opening of the perianth mouth seems
to be the best position for pollen removal. King and Buchmann
(1995) suggested that Rhododendron spp. are seldom buzzpollinated because the flowers are too large. However, R. ferrugineum has relatively small flowers and stamens are short
and located in the perianth mouth, where bees and bumble
bees can be in contact with all of the anthers. So far, this
pollination aspect has not been addressed in R. ferrugineum
and should be studied in future investigations.
Rhododendron ferrugineum seems to fit well with the usual
theories concerning plants that grow in drastic environments:
(1) this species spreads vegetatively and (2) has a high sexual
reproduction potential (0.4–2.5 million seeds/m2 of heathland;
Pornon et al., 1997). Thus, even if seedling establishment occurs rarely, episodic recruitment is sufficient to maintain a high
level of genotypic diversity in the populations (Escaravage et
al., 1998); Also, (3) all floral organs are formed in the year
previous to flowering allowing the species to flower as soon
as conditions become favorable and (4) R. ferrugineum reproduces both through self- and cross-pollination (Escaravage et
al., 1997) with one level of stamens (the short ones) devoted
to self-pollination when pollinators are scarce.
The stamen dimorphism in flowers of R. ferrugineum has
been studied under both developmental and selective aspects.
According to Gould and Lewontin (1979), evolutionary biologists focus exclusively on immediate adaptation to local conditions and tend to ignore developmental constraints. In this
study we showed that stamen dimorphism in R. ferrugineum
flowers may originate from developmental constraints. However, it also becomes adaptive by favoring reproductive assurance.
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STAMEN DIMORPHISM IN RHODODENDRON FERRUGINEUM
(ERICACEAE): DEVELOPMENT AND FUNCTION1
NATHALIE ESCARAVAGE,2 ELISABETH FLUBACKER,2 ANDRÉ PORNON,3
BERNARD DOCHE,2 AND IRÈNE TILL-BOTTRAUD2
Laboratoire de Biologie des Populations d’Altitude CNRS-UMR 5553, Université Joseph Fourier, B.P. 53,
F-38041 Grenoble Cedex 09, France; and 3Laboratoire d’Ecologie Terrestre CNRS-UMR 5552,
Université Paul Sabatier, Zoologie Ecologie,
Bât. 4R3, 118, route de Narbonne, F-31062 Toulouse Cedex 04, France
2
The function of stamen dimorphism in the breeding system of the alpine shrub Rhododendron ferrugineum was studied in two
populations in the French Alps. This species has pentameric flowers with two whorls of stamens: an inner whorl of five long stamens
and an outer whorl of short stamens. We studied the development of stamens from buds to mature flowers (measurement of the
filament, anther, and style lengths at five successive phenological stages) and compared the size and position of reproductive organs
at maturity in control and partially emasculated flowers (removal of long-level stamens) to determine whether the presence of longlevel stamens constitutes a constraint for the development of the short-level ones. Stamen dimorphism can be observed early in stamen
development, from the bud stage of the year prior to flowering. At this early stage, meiosis had already occurred. Emasculation of the
long-level stamens induced the short-level ones to grow longer than in normal conditions. We also performed seven pollination
treatments on ten randomly chosen individuals in each population, and the number of seeds following each treatment was recorded.
Results from these treatments showed that R. ferrugineum produced spontaneous selfed seeds in the absence of pollinators. However,
no seed was produced when short-level stamens were emasculated and pollinators excluded, suggesting that long-level stamens are
not responsible for selfing in the absence of pollinators and that reproductive assurance is promoted by short-level stamens.
Key words:
Ericaceae; pollination system; reproductive assurance; Rhododendron ferrugineum; stamen development; stamen size.
In alpine and arctic environments, low pollinator activity
and diversity should promote characters that result in the assurance of seed production through self-pollination. Most temperate alpine plants need pollinators for pollen deposition on
the stigma, and therefore, are predominantly entomophilous
(Kevan, 1973; Arroyo, Primack, and Armesto, 1982; Kudo,
1993; Totland, 1994; Jacquemart and Thompson, 1995). However, some animal-pollinated plants growing in severe environmental conditions have a mixed-mating system and can
produce spontaneous selfed seeds. Outcrossing will usually occur when reproductive fitness is not limited by a paucity of
animal visits. Where such limitations exist, features that maximize within-flower selfing (autogamy) may be favored (Richards, 1997). Although autogamy is frequent, it is rarely predominant, probably because most plants growing in harsh conditions are long-lived perennials for which constraints on reproductive fitness by seed are low (Richards, 1997). In
Rhododendron parviflorum and R. redowskianum (in Siberia)
spontaneous autogamy occurs in the absence of pollinators
(Tikhmenev, 1985), however in some Vaccinium in the Belgian Ardennes (V. myrtillus, V. uliginosum, V. vitis-idaea; Jacquemart and Thompson, 1995), insect-mediated self-pollination accounts for the majority of seeds produced by selfing. In
other species, like Rhododendron aureum (in Japanese mountains), spontaneous selfing does not occur and insect pollina-
tion is necessary for seed set (Kudo, 1993). One character that
plays a key role in promoting reproductive assurance in selfcompatible species is the reduction of stigma-anther separation
(Piper, Charlesworth, and Charlesworth, 1986).
The species studied here, Rhododendron ferrugineum L.
(Ericaceae), is an evergreen shrub with a mean height of 70
cm that grows at the subalpine level from ;1600 to 2200 m.
It reproduces both vegetatively (by layering) and sexually.
Asexual propagation occurs in closed and mature populations
(Pornon et al., 1997) and preferentially down hill (Escaravage
et al., 1998). Rhododendron ferrugineum produces an inflorescence of 5–22 bright-red nectariferous flowers. The flowers
are protandrous (anthers mature before the stigma) and are
initiated the year before they mature (Escaravage et al., 1997).
Rhododendron ferrugineum is a self-compatible species, mainly pollinated by bees, bumble bees, and diptera. Spontaneous
selfing occurs in the absence of pollinators (Escaravage et al.,
1997). A study of the floral morphology revealed herkogamy
(spatial separation between anther and stigma) with different
stamen lengths: short-level stamens below or at the same
height as the style and long-level ones above the style (Escaravage et al., 1997). The term stamen dimorphism will be
used subsequently. Stamens are mediafixed (filament links to
the back of the anther) and are located on two whorls (five
stamens per whorl) with long- and short-level stamens on the
inner and outer whorls, respectively (E. Flubacker, personal
observation). This has not been described in other Rhododendron species and the question that arises is: what are the mechanisms that promote this spatial separation? Are developmental processes involved? Floral developmental studies have
been performed on heterostylous taxa (Cheung and Sattler,
1967; Ganders, 1979) and more recently Richards and Barrett
(1984, 1987) examined floral developmental differentiation
among morphs in tristylous species. In these species, the var-
Manuscript received 3 August 1999; revision accepted 11 April 2000.
The authors thank Pascal Thomas for help with the field experiments and
John D. Thompson and two anonymous referees for their critical comments
and helpful suggestions that greatly improved the first draft of the manuscript,
and Paul G. Wolf for improving the English text. This work was financially
supported by University Joseph Fourier (Grenoble), the Centre National de la
Recherche Scientifique (C.N.R.S.), and the French Ministry of the Environment (E.G.P.N. no. 95.085).
3
Author for reprint requests (e-mail: pornon@cict.fr).
1
68
January 2001]
ESCARAVAGE
ET AL.—STAMEN DIMORPHISM IN
iability caused by timing and position of stamen origin probably accounts for variation in stamen length within flowers of
heterostylous species that have two whorls of stamens (Richards and Barrett, 1992). In some arctic Primula species, homostyly has replaced heterostyly (Kelso, 1992). It is suggested
that both restrictions of pollination efficiency and developmental constraints (imposed by the need for reciprocity in organ position and to limit stigma-anther separation) are involved in this evolutionary process (Richards and Barrett,
1992).
In this study, we focused on stamen dimorphism and its
possible consequences on the breeding system of Rhododendron ferrugineum. The aims of this paper are (1) to describe
the development of the stamens in Rhododendron ferrugineum
(from the bud stage to full bloom) to determine at what phenological stage stamen dimorphism can first be detected, and
(2) to determine the role of long- and short-level stamens in
the pollination of the species.
MATERIALS AND METHODS
The study site—This study was carried out during the summer of 1997 at
two mountain sites in the northwestern French Alps (Belledonne range). The
first was ‘‘Collet d’Allevard’’ (458259 N, 68109 E) located at 1680 m on a
north-facing slope (50%), and the second was ‘‘Chamrousse’’ (458259 N,
508559 E) located at 1770 m on a west-facing slope (62%). In both sites, the
populations are composed of Rhododendron ferrugineum with a cover of 40%,
mixed with other Ericaceous shrubs, such as Vaccinium myrtillus, V. uliginosum, and V. vitis-idaea.
Experimental design—Stamen development—To study patterns of stamen
development, we randomly selected ten individuals in each population. On
each individual we conducted the following experiments: we collected three
inflorescences per individual at five successive phenological stages [inflorescence buds (t0), young inflorescences (t0 1 2 d), medium-aged inflorescences
(t0 1 4 d), advance-aged inflorescences (t0 1 6 d), and inflorescences with
flowers in full bloom (t0 1 8 d)]. One flower per inflorescence was dissected
and measured under a dissecting microscope, together with the filaments, anthers, and style.
Effects of population, individual, flower, whorl, and position of stamens on
anther and filament length in mature flowers were statistically tested by performing five-way mixed analysis of variance using PROC GLM (SAS, 1990).
Population, whorl, and position of stamens were treated as fixed effects; individual and flower were random effects. Population, individual, and flower,
and position and whorl were nested (see Table 2).
Associations between anther height of the short- and long-level stamens
and length of short- and long-level filament were tested by a correlation analysis (SAS, 1990). Data were ln transformed for normality. All these tests were
performed for each developmental stage. The mean daily growth rate, computed as the length of each stamen at the end of the last phenological stage
(full bloom) divided by the number of days elapsed between bud breaking
and full bloom (8 d), was compared by performing a Tukey HSD multiplerange test using SPSS for Windows, release 9.0.0.
Effects of partial emasculation—To determine whether the presence of
long-level stamens (inner whorl) limits the development of short-level stamens
(outer whorl), we selected three other inflorescences on the same ten individuals in which we partially emasculated three flowers at the bud stage. Anthers
of long-level stamens (inner whorl) were removed (but their filaments remained) and short-level stamens (outer whorl) were left intact. It would have
been interesting to remove the stamens of the outer whorl to compare the
results, but unfortunately this experiment was difficult to perform without
damaging the flower. The other flowers of the inflorescence were unmanipulated. When flowers reached their mature size, we collected all the treated
inflorescences. We dissected the partially emasculated flowers under a dis-
RHODODENDRON FERRUGINEUM
69
secting scope and measured the style, anther, and filament lengths of the intact
stamens of outer whorl, and the filament length of emasculated stamens (inner
whorl). We dissected and performed the same measurements on one unemasculated flower of each inflorescence as a control. In both experiments, stamens
were numbered according to their position in the flower. Stigma-anther separation was calculated as filament length minus style length.
In this experiment, the effects of population, individual, flower, treatment
(flower partially emasculated or not), and position of stamens on filament
length of each stamen (emasculated or not) were tested by five-way mixed
analysis of variance (SAS, 1990) with population, emasculation, and whorl
of stamens as fixed effects, individual and flower as random effects, and
population, individual, and flower nested. The treatment effect tested for an
effect of partial emasculation on the whole, whereas the effect of emasculation
on the longer filaments was tested by the treatment 3 whorl interaction. For
each site and treatment (partial emasculation or not) the whorl effect on filament length and stigma-anther separation was tested by one-way analysis of
variance (SAS, 1990).
At the end of the flowering period (mid-July), floral primordia for the next
flowering season are initiated. To determine when meiosis of pollen grains
occurred, we collected two flowers (from different inflorescences) per individual, on ten individuals in each population every week from mid-July to
mid-August. We dissected the flowers and crushed the anthers on a slide in
a drop of Alexander’s stain (Alexander, 1969), and observed the slide under
a microscope. As pollen is released in tetrads in the Ericaceae, observation
of single cells indicates that meiosis had not yet occurred.
Pollination trials—To study the respective roles of short- and long-level
stamens in sexual reproduction, we (1) quantified the number of pollen grains
per anther in the two whorls and (2) performed a series of pollination treatments. To quantify pollen production we collected one inflorescence bud on
each of ten individuals and preserved it in 70% ethanol in May 1997. One
flower from each bud was dissected under a dissecting scope. Each stamen
was measured according to its position (Fig. 1) and pollen grains were quantified after acid extraction from the anther using a hemacytometer according
to Escaravage et al. (1997). Pollen production was analyzed by performing
five-way mixed analysis of variance as described in the stamen development
analysis (see above). The association between the number of pollen grains
and stamen length was tested by a correlation analysis (SAS, 1990). We performed seven pollination treatments (summarized in Table 1) on ten other
randomly selected individuals in each population. Each individual received
all seven treatments, and each treatment was performed on two inflorescences,
on at least five flowers per inflorescence. We thus studied a total of 140
inflorescences and 700 flowers at each site.
In treatments T5 and T7, we emasculated the five long-level stamens (inner
whorl), while in treatments T4 and T6 we emasculated the five short-level
stamens (outer whorl). In treatments T6 and T7, nylon mesh bags were used
to exclude pollinators, whereas treatments T4 and T5 were open-pollinated.
We performed three control treatments: in treatment T3 all stamens were
emasculated (cross-pollination control), in T2 flowers were unmanipulated and
inflorescences were bagged (self-pollination control), and T1 consisted of unmanipulated (intact) inflorescences. To avoid self-pollen contamination, emasculation was performed ; 2 d after the inflorescence bud opened. At this
stage, inflorescences were very small and the flowers were still closed. Five
flowers per inflorescence (except for T1 and T2) received one of the seven
treatments. All other flowers (between one to five flowers) were removed.
This manipulation did not disturb the development of the remaining flowers
(Escaravage et al., 1997). Fruits were collected shortly before dehiscence (early September) and the seeds counted.
To detect the effects of individual, inflorescence, and treatment on the number of seeds per fruit, we performed three-way mixed analysis of variance
using PROC GLM (SAS, 1990) for each population. Treatment was a fixed
effect while inflorescence and individual were treated as random effects. Inflorescence was nested within individual. Mean comparisons between specific
treatments were also carried out by using a Scheffé multiple-range test.
70
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TABLE 1. Pollination tests used in the study of the pollination system
of R. ferrugineum.
Level of stamens
remaining
Bagging
No. of flowers/inflo.
Long 1 short
Long 1 short
None
Long
Short
Long
Short
No
Yes
No
No
No
Yes
Yes
all
all
5
5
5
5
5
Code
T1
T2
T3
T4
T5
T6
T7
Fig. 1. Mean sizes (in cm) of the different stamens according to their
positions for different phenological stages of the flower. Odd-numbered stamens (long-level) are on the inner whorl, even-numbered stamens (short-level)
on the outer whorl. A: inflorescence buds, B: young inflorescences, C: medium-aged inflorescences, D: advance-aged inflorescences, E: inflorescences
with flowers in full bloom.
RESULTS
Stamen development—All floral organs are preformed in
July of the year preceding flowering (N. Escaravage, personal
observation). Anthers are relatively voluminous and seem
more advanced in their development than filament, style, and
petals. At this stage anthers are positioned in staggered rows
(one anther high, one low, etc.), in such a way that stamen
dimorphism can already be observed early in development.
Moreover, pollen tetrads are observed in anthers from the second week of August on. Therefore, meiosis occurred in buds
preformed in the summer the year prior to flowering.
In unmanipulated flowers, short- and long-level stamens are
located on the outer and inner whorls, respectively. Filaments
and anthers from the inner whorl are significantly longer than
those of the outer whorl (P , 0.001; Table 2). The position
within the whorl significantly affects filament length but not
anther height. Stamen number 6 was always the smallest at
each site and each phenological stage; the longest stamens
were number 1, 3, and 9. This conferred slight zygomorphy
following a plane of symmetry passing through stamens 1 and
6 (Fig. 1). Although filament length is significantly affected
by population (P , 0.05; Table 2), stamen dimorphism is preserved among populations as indicated by the non significant
interactions of population by whorl and population by position
nested in whorl (Table 2). The mean stigma-anther separation
at Chamrousse was: 0.83 mm (60.11 SD) for the outer, shortlevel whorl and 1.63 mm (60.14 SD) for the inner, long-level
whorl. At Collet d’Allevard the mean stigma–anther separation
was 0.42 mm (60.12 SD) and 1.30 mm (60.14 SD) for the
inner, short-level and outer, long-level whorls, respectively.
The slopes in Fig. 2 represent the growth rate of anthers (a)
or filaments (b) of the long-level stamens relative to those of
the short-level stamens for each developmental stage. A positive correlation exists between anther height of long- and
short-level stamens from the bud (A) to medium (C) stages (P
, 0.001; Fig. 2a) suggesting that during these stages anther
growth still occurs. However, in later phenological stages, the
growth rates, are similar until anthers have reached their final
size. In contrast, stamen elongation occurs throughout the de-
TABLE 2. Results of analysis of variance to detect the effect of population, individual, flower, whorl, and stamen position on anther and filament
lengths in mature flowers.
Filament length
Anther height
Source of variation
df
MS
F
MS
F
Population
Individual (population)
Flower [individual (population)]
Whorl
Position (whorl)
Population 3 whorl
Population 3 position (whorl)
Error
1
18
36
1
2
1
2
494
26.94
19.71
5.12
61.86
7.92
3.03
0.36
1.06
5.47*
3.84***
4.81***
58.17***
7.44***
2.85 ns
0.33 ns
0.00
0.19
0.04
1.11
0.01
0.01
0.002
0.008
0.00 ns
4.52***
5.52***
140.73***
1.99 ns
1.27 ns
0.30 ns
Note: ***P , 0.001, **P , 0.01, *P , 0.05, ns 5 non-significant.
January 2001]
ESCARAVAGE
ET AL.—STAMEN DIMORPHISM IN
RHODODENDRON FERRUGINEUM
71
Fig. 3. Mean daily stamen growth rates of each stamen. Odd-numbered
stamens are on the long-level inner whorl and even-numbered stamens are on
the short-level outer whorl. Vertical bars indicate standard deviation. Values
that share the same letter are not significantly different at 5% threshold. Means
were compared by a Tukey HSD multiple-range test.
were significantly longer than those of unemasculated flowers
(P , 0.001; Fig. 4). In unemasculated flowers, stamens of the
inner, long-level whorl showed a greater stigma-anther separation than stamens of the outer, short-level whorl (P , 0.001).
In partially emasculated flowers the increased growth of the
stamens of the outer whorl significantly increased the stigmaanther separation (Fig. 4); as a result this distance was similar
to that measured between anthers of the longest stamens (inner
whorl) and stigma in unemasculated flowers.
Fig. 2. (a) Height of anthers of the long-level stamens as a function of
height of anthers of the short-level stamens for each phenological stage (A:
r2 5 0.6319, N 5 200, y 5 0.9082x 1 0.0883, P , 0.001; B: r2 5 0.1012,
N 5 187, y 5 0.3773x 1 0.5638, P , 0.05; C: r2 5 0.1332, N 5 199, y 5
0.4371x 1 0.5353, P , 0.001; D: r2 5 0.0171, N 5 196, y 5 0.1649x 1
0.7666, non significant; E: r2 5 0.0214, N 5 194, y 5 0.1756x 1 0.214, non
significant) and (b) length of long-level filament as a function of length of
short-level filament for each phenological stage (A: r2 5 0.6122, N 5 200, y
5 0.7982x 1 0.0711, P , 0.001; B: r2 5 0.2779, N 5 189, y 5 0.5079x 1
0.650, P , 0.001; C: r2 5 0.1854, N 5 199, y 5 0.536x 1 0.8092, P ,
0.001; D: r2 5 0.2080, N 5 194, y 5 0.4802x 1 1.2314, P , 0.001; E: r2
5 0.3867, N 5 191, y 5 0.644x 1 0.9567, P , 0.001).
velopment of the flower (Fig. 2b). For each stamen, the daily
mean growth rate is shown in Fig. 3. Stamen 6 (the shortest
stamen) has the slowest growth rate, whereas stamens 1, 3 and
9 (the longest stamens) have the fastest.
In partially emasculated flowers, treatment (effect on the
whole flower) and treatment 3 whorl interaction (specific to
the emasculated whorl) have a significant effect on filament
length (Table 3; P , 0.001). At the end of the growth of
partially emasculated flowers, mean length of the filaments of
stamens on the outer whorl (unemasculated) had increased
compared to unemasculated flowers. In both populations, the
filament length difference between whorls disappeared. For
partially emasculated flowers, stamen filaments of the outer
whorl were not different from those of the inner whorl but
Number of pollen grains per stamen—Long-level stamens
contain significantly more pollen grains than short-level stamens (Table 4), and all effects are significant. A positive correlation exists between stamen length and the number of pollen
grains in both populations (P , 0.001 with r2 5 0.211 and
0.273 for Collet d’Allevard and Chamrousse, respectively). At
Collet d’Allevard the mean number of pollen grains was
16 000 (64498 SD) and 12 257 (63761 SD) for long- and
short-level stamens, respectively. At Chamrousse, mean pollen
grain production was 21 152 (64563 SD) and 15 974 (63861
SD) for long- and short-level stamens, respectively.
Effect of partial emasculation on seed production—Significantly more seeds were produced in Collet d’Allevard than
in Chamrousse (Student’s t test, P , 0.05, data not shown).
Thus populations were studied separately. For both populations, mean seed number is influenced by the treatment (P ,
TABLE 3. Results of analysis of variance to detect effects of population, individual, flower, treatment (partial emasculation of long-level stamens), and whorl on filament length in mature flowers.
Source of variation
df
MS
F
Population
Individual (population)
Flower [individual (population)]
Treatment
Treatment 3 whorl
Treatment 3 whorl 3 population
Error
1
18
36
1
2
3
883
157.81
25.68
8.73
799.67
87.26
3.26
1.22
6.13*
2.99*
7.16***
259.02***
26.09***
2.67*
Note: ***P , 0.001, *P , 0.05.
72
AMERICAN JOURNAL
OF
BOTANY
[Vol. 88
TABLE 4. Results of analysis of variance to detect the effect of whorl,
position, population, individual, and flower on the number of pollen
grains.
Source of variation
df
Whorl
1
Position (whorl)
8
Population
1
Individual (population)
18
Flower (individual 3 population)
18
Whorl 3 population
1
Whorl 3 individual (population)
18
Whorl 3 flower (individual 3 population)
18
Error
654
MS
F
31147.6
254.8
35143.6
2657.1
304.2
1301.7
236.7
297.3
98.5
316.1***
2.6**
13.3**
8.7***
3.1***
13.2***
2.4***
3.0***
Note: ***P , 0.001, **P , 0.01.
stamens were removed (T6; Fig. 5). This effect was likely due
to the extra growth of short-level stamens following removal
of long-level ones (Fig. 4).
DISCUSSION
Fig. 4. Mean filament length and anther-stigma separation for the inner
and outer stamen whorls. One-way analysis of variance indicated significant
differences among whorls in unemasculated flowers but not in flowers where
the stamens of the inner whorl were removed. Vertical bars indicate standard
deviation.
0.001, Table 5). The greatest number of seeds was produced
following open pollination and when pollinators were excluded and flowers left intact (T1 and T2; Fig. 5). At Chamrousse,
the number of seeds decreased when at least one stamen whorl
was emasculated (T4 and T5; Fig. 5), whereas at Collet
d’Allevard the mean number of seeds produced was not affected significantly. In contrast when short stamens were emasculated and inflorescences bagged, very few seeds were produced (T7; Fig. 5). The same result was obtained when long
In R. ferrugineum two stamen levels are observed: the longlevel stamens of the inner whorl and the short-level stamens
of the outer whorls. Long-level stamens produce more pollen
grains than short-level stamens. This stamen dimorphism can
be detected as early as June of the year preceding flower maturation, and pollen grains are formed in August of the same
year. When long-level stamens are removed early the flowering year, the short-level stamens grow longer. Stamen dimorphism has not been described in other Rhododendron species
but is widespread among heterostylous taxa. As in tristylous
species of Pontederia (Richards and Barrett, 1987) and Eichhornia (Richards and Barrett, 1984) and in distylous species
of Primula (Stirling, 1932), stamen dimorphism in R. ferrugineum appears in premeiotic stamens and is maintained
throughout phenological stages.
Early in flower development, anthers occupy most of the
volume in the flower while the corolla, style, and filaments
are poorly developed. Thus, arrangement in staggered rows
seems to be the most appropriate means to limit the size of
young flowers in the buds. Overwintering of floral primordia
has been described in many alpine and tundra plants species
(Bliss, 1971; Bell and Bliss, 1980; Diggle, 1997; Aydelotte
and Diggle, 1997) and allows the flower to bloom early in the
spring (Bliss, 1971; Diggle, 1997). However, it does not seem
to be a specificity of plants growing in harsh environments
(Diggle, 1997), since it occurs in plants that flower very early
in the year (Alnus glutinosa, Coryllus avellana; N. Escaravage,
TABLE 5. Results of analysis of variance for each population, to detect individual, inflorescence, and treatment effects on the seed number per
fruit.
Population
Chamrousse
Collet d’Allevard
Source of variation
df
MS
F
MS
F
Seeds/fruit
Individual
Inflorescence (individual)
Treatment
Treatment 3 inflorescence (individual)
Error
9
10
6
60
49
536.8
676.6
14053.7
280.5
261.6
0.8 ns
2.5*
53.7***
1.1 ns
1297.2
491.9
20110.3
376.9
927.6
2.6 ns
0.5 ns
21.6***
0.4 ns
Note: ***P , 0.001, *P , 0.05, ns 5 non-significant.
January 2001]
ESCARAVAGE
ET AL.—STAMEN DIMORPHISM IN
Fig. 5. Mean number of seeds produced per fruit. Vertical bars indicate
standard deviation. For each site the treatment effect was tested by a Scheffé
multiple-range test. For each site, values that share the same letter are not
significantly different at 1% threshold.
personal observation). In alpine environments, early anther
formation and spermatogenesis could be a strategy to allow
the flower to become functional rapidly and could be an adaptation to the short optimal reproductive period, allowing the
plant to flower as soon as the temperature increases. The formation of pollen grains in the season preceding flowering is
risky since a severe winter could occur and freeze the buds.
However, this risk is reduced because flowers are protected in
bud scales and plants of R. ferrugineum usually spend the
winter under thick snow cover that buffers temperature effects
(Larcher and Siegwolf, 1985).
Two main categories of causes can explain stamen dimorphism: (1) ‘‘proximate’’ and (2) ‘‘ultimate’’ causes that refer
to developmental and selective aspects, respectively. Under the
first category filament expansion can be regulated, through anthers, by growth hormones such as cytokinin (Hess and Morré,
1978), gibberellins (Greyson and Tepfer, 1967), or auxin (Koning, 1983). Greyson and Tepfer (1967) argued that inhibition
of filament growth following emasculation in Nigella hispanica could be accompanied by inhibition of both cell elongation
and cell division in the epidermis. In R. ferrugineum, emasculation of the inner whorl results in extra growth of the outer
whorl, yet emasculation does not affect the growth of the inner
whorl. This suggests that, if hormonal control is involved it is
only in the one direction. Testing for hormonal control can be
challenging because it is often difficult to distinguish between
hormonal control and homeostatic responses.
Monopolization of space by anthers of long stamens could
represent a sufficient barrier to the development of the short
stamens to create a mechanical resistance to filament cell division. Thus, the different sizes should disappear when the
spatial constraint imposed by the long stamen is removed.
However the size difference between long- and short-level stamens lies primarily in filament length and filament growth
occurs when flowers open and space is not as limiting. Daily
stamen growth rate is similar across all stamens except for the
RHODODENDRON FERRUGINEUM
73
shortest, stamen 6, and the longest ones, stamens 1, 3, and 9
(Fig. 3). We detected no difference in stamen growth rate between inner and outer whorls (data not shown). This is different from several heterostylous species (Richards and Barrett,
1984, 1987) in which differences in stamen length among
morphs arise through differences in the relative growth rate
and the duration of growth. However, some species of Pontederiaceae, Lythraceae, and Oxalidaceae initiate two temporally separated stamen sizes. The difference in time of origin
establishes a size differential between stamen series that is
maintained throughout development. This mechanism could
account for the two stamen levels in R. ferrugineum.
Short stamens might benefit from resources normally allocated to the development of long stamens (inner whorl). However, this suggestion seems unlikely since differences in nutrient availability between populations, individuals, inflorescences, and flowers would induce variation in stamen size
within the two whorls. On the contrary, stamen hierarchy in
relation to their position in whorls appears to be similar in
both populations.
In the second category, ultimate causes, the two stamen levels may have different functions in pollination in R. ferrugineum. Our results indicate that long-level stamens do not participate autonomously in self-pollination in the absence of pollinators since no seeds were produced when short stamens are
removed (T6; Fig. 5). Because unemasculated flowers could
self in the absence of pollinators, spontaneous self-pollination
is likely due to short-level stamens. However, our experiments
provided no formal proof. The treatment in which long-level
stamens were removed also produced no seeds in the absence
of pollinators (T7; Fig. 5), but, when short-level stamens were
in the corolla alone, they grew to the height of long-level
stamens. Thus they function similarly to long-level stamens
and are inefficient in autonomous selfing. Spatial separation
between the stigma and the anther of the longest stamen is on
average 1.50 mm. This distance seems to be sufficient to avoid
self-pollination (Webb and Lloyd, 1986), especially in R. ferrugineum where pollen is sticky and dehiscence poricidal.
The distance between stigmas and anthers of short-level stamens is smaller at Collet d’Allevard than at Chamrousse. This
may explain why plants at Collet d’Allevard produced more
seeds in treatment T5. The pollination mechanism in openpollinated treatment T1 is less obvious, and we do not know
the proportion of seeds sired by self- and cross-pollination, or
which stamen level is involved. However in this species the
autofertility index is high (0.92; Escaravage et al., 1997), suggesting that autogamy could account for the majority of seeds
produced.
In a previous study on the genotypic structure of a R. ferrugineum population, Escaravage et al. (1998) showed that
genotypes were closely related. These authors hypothesized
that autogamy and geitonogamy (fertilization between different flowers on the same plant) may be responsible for such a
relatively homogeneous genetic structure. However, we have
yet to estimate the proportion of outcrossed or selfed seeds.
Both fertilization modes can occur synchronously at each generation and at all reproductive stages. In alpine environments,
snow and frost often occur during periods of growth and reproduction and result in a scarcity of pollinators (Arroyo, Primack, and Armesto, 1982; Escaravage, 1997). In such a context, self-pollination can compensate for the insufficient pollinator activity and act as reproductive assurance (Arrroyo,
1974; Wyatt, 1983; Holsinger, 1992; Harder and Barrett,
74
AMERICAN JOURNAL
1995). Therefore, in R. ferrugineum, pollination by short-level
stamens could act as reproductive assurance in the absence of
pollinators that would prevent outcrossing. Reproductive assurance is promoted in numerous plants growing in severe
environments. In arctic Primula species, it is common to see
a shift in breeding system from distyly to homostyly and at
least facultative autogamy (Kelso, 1992; Mazer and Hultgard,
1993; Miller et al., 1994). Armeria maritima has also lost distyly in the tundra portion of its range (Vekemans et al., 1990).
Anther position can affect pollen removal through its influence on the timing of anther dehiscence (Harder and Barrett,
1993). However, all anthers of R. ferrugineum flowers dehisce
before the flower opens (as in R. ponticum; Percival, 1955),
whereas stigmas become receptive when the flower opens (Escaravage et al., 1997). No difference in the timing of anther
dehiscence among whorls has been observed (N. Escaravage,
personal observation). Anther position can also determine the
placement of pollen on a pollinator’s body, which influences
the likelihood of pollen loss through grooming (Faegri and van
der Pijl, 1979). It seems that the advantages of a particular
anther position depend on the morphological and behavioral
characteristics of a plant’s dominant pollinators (Harder and
Barrett, 1993). For bee-pollinated species with tubular flowers,
anther placement in the opening of the perianth mouth seems
to be the best position for pollen removal. King and Buchmann
(1995) suggested that Rhododendron spp. are seldom buzzpollinated because the flowers are too large. However, R. ferrugineum has relatively small flowers and stamens are short
and located in the perianth mouth, where bees and bumble
bees can be in contact with all of the anthers. So far, this
pollination aspect has not been addressed in R. ferrugineum
and should be studied in future investigations.
Rhododendron ferrugineum seems to fit well with the usual
theories concerning plants that grow in drastic environments:
(1) this species spreads vegetatively and (2) has a high sexual
reproduction potential (0.4–2.5 million seeds/m2 of heathland;
Pornon et al., 1997). Thus, even if seedling establishment occurs rarely, episodic recruitment is sufficient to maintain a high
level of genotypic diversity in the populations (Escaravage et
al., 1998); Also, (3) all floral organs are formed in the year
previous to flowering allowing the species to flower as soon
as conditions become favorable and (4) R. ferrugineum reproduces both through self- and cross-pollination (Escaravage et
al., 1997) with one level of stamens (the short ones) devoted
to self-pollination when pollinators are scarce.
The stamen dimorphism in flowers of R. ferrugineum has
been studied under both developmental and selective aspects.
According to Gould and Lewontin (1979), evolutionary biologists focus exclusively on immediate adaptation to local conditions and tend to ignore developmental constraints. In this
study we showed that stamen dimorphism in R. ferrugineum
flowers may originate from developmental constraints. However, it also becomes adaptive by favoring reproductive assurance.
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