Biological Journal o f f h e Linnean Sociep (1986) 29: 123-137. With 2 figures
The evolution of distyly in
Primula vulgaris
JOHN PIPER
Biology Building, Uniuersily of Sussex, Falmer, Brighton BNl 9QG
AND
BRIAN CHARLESWORTH
Department of Biology, Universily of Chicago, E 57th Street, Chicago, Illinois, U.S.A.
Received 18 April 1986, accepted for publication 13 June 1986
~
~~~
Experiments on pollen flow and seed production were performed in populations of P. vulgaris in
order to examine the roles of selection for reduced self-pollination in a partially self-fertile morph,
selection for reduced stigma clogging, selection for a pollen saving effect, and selection for
disassortative pollination in the evolution of morphological distyly (reciprocal herkogamy).
Selection for reduced self-pollination and disassortative pollination were shown to have a plausible
role in the evolution of this dimorphism. Selection for reduced stigma clogging and pollen saving
appeared to have no obvious role in the evolution of morphological distyly.
KEY WORDS: -Distyly
-
stigma clogging
-
pollen saving - disassortative pollination.
CONTENTS
Introduction . . . . . . . . . . . . . . .
Testing the hypotheses
. . . . . . . . . . .
Materials and methods.
. . . . . . . . . . . .
Experimental protocols
. . . . . . . . . . .
Statistical analysis.
. . . . . . . . . . . .
Results
. . . . . . . . . . . . . . . .
Pollen grain size . . . . . . . . . . . . .
Pollen grain production and removal from anthers . . . . .
Stigmatic pollen load composition for intact and emasculated flowers
Disassortative pollination . . . . .
. . . . . .
Seed production . . . . . . . . . . . . .
Discussion . . . . . . . . . . . . . . . .
Selection for reduced self-pollination . . . . . . . .
Selection for reduced stigma clogging . . . . , . . .
Selection for pollen saving . . . . . . . . . . .
Selection for disassortative pollination . . . . . . . .
Acknowledgements
. . . . . . . . . . . . .
References.
. . . . . . . . . . . . . . .
Appendix . . . . . . . . . . . . . . . .
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01986 The Linnean
Society of London
124
J. PIPER AND B. CHARLESWORTH
INTRODUCTION
Typical distylous species segregate for two self-incompatible but inter-fertile
pin and thrum morphs, which are usually present in a one to one ratio (Darwin,
1877). Both morphs have herkogamous flowers: pins have high stigmas and low
anthers, and thrums have the reciprocal arrangement. Hence flowers of
distylous species are reciprocally herkogamous (Lloyd & Webb, unpubl.) .
Furthermore, pin anthers produce more numerous but smaller pollen than
thrum anthers. Populations of some distylous species contain the so-called long
homostyle. This is a self-fertilizing morph which has thrum male characters and
pin female characters, thus the sex organs are juxtaposed at the mouth of the
corolla tube.
Since Darwin’s work on the “forms of flowers” (Darwin, 1877) the evolution
of reciprocal herkogamy has stimulated theoretical and experimental research
(reviewed in Ganders, 1979; for distylous species see also Ornduff, 1979, 1980;
Schou, 1983; Nicholls, 1985; and for tristylous species see Barrett & Glover,
1985). Hitherto, with the exception of Charlesworth & Charlesworth (1979),
workers have failed to consider the evolution of distyly systematically, and as a
consequence they have not always identified the selective agents that might
promote the transition between the various stages that presumably occurred
during the evolution of this breeding system.
Charlesworth & Charlesworth (1979) published the only theoretical model to
date that deals with factors that could promote the evolution of distyly. After
performing numerous computer simulations they favoured the sequence of
events depicted in Fig. 1. The arguments that support their scenario run as
follows (refer to Fig. 1, and for more precise details see Charlesworth &
Charlesworth, 1979). It was assumed that the progenitors of modern distylous
species were self-fertilizing, with stigmas and anthers juxtaposed (Fig. 1, stage
1). Then, in response to selection for cross-fertilization, diallelic selfincompatibility evolved, with one of the forms experiencing partial selffertilization (Fig. 1, stage 2). Following the establishment of self-incompatibility
a change in stigma position occurred in one of the forms as a consequence of one
or a combination of three causes: ( 1 ) to reduce self-fertilization in the partially
self-fertilizing form; (2) to reduce stigma clogging (Yeo, 1975) with incompatible
pollen, thereby raising female fertility; or (3) to reduce the amount of
incompatible pollen on stigmas, thereby promoting a pollen saving effect that
results in increased male fertility (Fig. 1, stage 3). A stigma shift was favoured
by Charlesworth & Charlesworth (1979) at this stage over an anther shift
because it was assumed that altering stigma position would not affect seed set,
whereas a change in anther position would reduce male fertility. The final stage
in the model was a change in anther position in the self-incompatible
homostylous form to the height of the stigma in the thrum form, in response to
selection for improved pollen transfer, i.e. to promote disassortative pollination
(Fig. 1, stage 4). Stigma clogging and pollen saving could be involved in this
transition. However, if they acted in isolation then the evolution of floral
monomorphism would be just as effective as floral dimorphism. If the starting
point for the model was a short homostyle (sex organs juxtaposed, but concealed
in the corolla tube) then the pin form would have evolved prior to the thrum
form.
DISTYLY I N PRIMULA VULGARIS
125
I
.1
,Anther
E'igurc I ..Schrniatir representation of the Charlesworths' model for the evolution of distyly. Stage I ,
sclf-rompatihility and long homostyle floral structure. Stage 2, Di-allelic self-incompatibility, long
homostylr floral structure. 3, Di-allelir self-incompatibility, one morph with long homostyle floral
structure, and one with thrum floral structure. 4, Di-allelic self-compatibility, one morph with pin
lloral strurture, and onc with thrum floral structure. For a discussion of the mechanisms that could
havr promoted the transitions between these stages, see text. Legitimate pollination occurs between
iiiitlicrs and stigmas of the same shade.
1
I
Here we report our results from experiments conducted in order to study the
evolution of the morphological features of distyly. They were obtained from a
natural population of the distylous species Primula vulgaris that contained selffertile long homostyles. Our experiments were based on the theoretical
framework provided by the Charlesworth's model. We set out to test four
hypotheses: ( 1 ) that herkogamy reduces the incidence of self-pollination (and by
inference self-fertilization); (2) that herkogamy reduces stigmatic clogging and
improves seed set; (3) that herkogamy promotes a pollen saving effect; and (4)
that pollen transfer from anthers to stigmas at the same height is more efficient
than from anthers to stigmas at different heights, i.e. that distyly promotes
disassortative pollination.
Testing the hypotheses
Distylous species usually display a pollen dimorphism that is associated with
anther position, hence stigmatic pollen loads (s.p.1~)can be split into pin and
I26
J. PIPER AND B. CHARLESWORTH
thrum components. This facet of distyly facilitates the testing of hypotheses 1, 3
and 4 when study populations also contain a homostylous morph. The inclusion
of a third morph does not present a problem as far as s.p.1. composition is
concerned, because homostyles produce thrum-type pollen. Hereafter pollen
produced by homostyles will be referred to as thrum pollen.
For reasons of clarity it is necessary to describe the reasoning behind the
protocols adopted in order to test these hypotheses. When pins, thrums and
homostyles co-occur the self-pollination and pollen saving hypotheses can be
tested, as pin and thrum flowers are herkogamous, but homostyle flowers are
not. In order to test the self-pollination hypothesis it is necessary to compare the
movement of pollen from anthers to stigmas in the same flower (self-pollination)
for the morphs. Because thrums and homostyles share the same male
characteristics the most relevant comparison is between these two morphs. The
sizes of the self pollen loads for thrums and homostyles are calculated by
subtracting the thrum component of the s.p.1. of flowers emasculated prior to
another dehiscence from the thrum component of the s.p.1. of intact flowers.
When pins are also present homostyle flowers need not be emasculated, as
thrum s.p.ls of intact pin flowers may be used: pin flowers have the same female
characters as homostyles, but produce no thrum pollen. Thus as far as the
thrum component of pin s.p.ls is concerned, pins are equivalent to emasculated
homostyles. The differences between thrum pollen loads on stigmas of intact and
emasculated thrum flowers, and between thrum pollen loads on stigmas of intact
pin and homostyle flowers, can then be used to test the self-pollination
hypothesis.
The pollen saving hypothesis can be tested when two factors are known for
each morph; the number of pollen grains removed from anthers, and the size of
the self pollen load. Once again the thrum-homostyle comparison is the most
appropriate. The size of the component of the s.p.1. due to self-pollination can
be determined by the methods outlined above. The numbers of pollen grains
removed from anthers can be determined by subtracting estimates of pollen
grains in anthers after anthesis from numbers of pollen grains in undehisced
anthers. The proportion of pollen ‘wasted’ due to self-pollination and made
unavailable for export can then be calculated, and the degree to which
herkogamy promotes a pollen saving effect assessed.
Hitherto, most studies performed in order to investigate the status of the
disassortative pollination hypothesis (with the exception of Ganders, 1974;
Barrett & Glover, 1985) examined the pollen loads on stigmas of intact flowers.
This approach overlooks one important factor: in all co-sexual flowering plants,
with or without herkogamy, there is likely to be pollen transfer from anthers to
stigmas in the same flower, or on the same plant (geitonogamy). It is unlikely
that distyly eliminates these modes of self-pollination, and if self pollen
constitutes a significant fraction of s.p.ls then it could obscure any disassortative
pollination. Consequently, the analysis of pollen loads on stigmas of intact
flowers tells us very little about pollen flow between plants. T o overcome this
problem experiments should be performed on plants where self-pollination is
prevented by emasculating all flowers on experimental plants prior to anther
dehiscence. Emasculation of all flowers is often impractical, therefore the
procedure adopted here involved emasculation of only one flower per plant.
Because only one flower per plant was emasculated, geitonogam was not
DISTYLY IN PRIMULA VULGARIS
I27
prevented, and this means that the sizes of self pollen loads will, at worst, be
underestimates. Hence the results of the experiments outlined above will be
conservative.
Finally, pin pollen on pin and homostyle stigmas is incompatible, as is thrum
pollen on thrum stigmas. Conversely, thrum pollen on pin and homostyle
stigmas, and pin pollen on thrum stigmas, is compatible. Henceforth, in order to
Lbllow the terminology of other workers (see Ganders, 1979; Richards, 1986),
the incompatible component of s.p.ls will be described as illegitimate, and the
compatible component legitimate.
MATERIALS AND METHODS
Experimental protocols
A population of Primula vulgaris at Wyke in Somerset (GR 645339),
composed of 50% pins, 25% thrums and 25% homostyles, was chosen for study
in order to test the self-pollination, pollen saving and disassortative pollination
hypotheses. Experimental flowers were labelled with colour-coded wire on 23
April 1985. Flowers on pins and thrums were subjected to one of four
treatments, thus there was a maximum of four experimental flowers on any one
plant. Flowers in treatment 1 were allowed to be naturally pollinated. Flowers
in treatment 2 were artificially pollinated with legitimate pollen. Capsules
produced by flowers in these two treatments were collected on 17 May 1985,
and the number of seeds in each capsule was determined. Flowers in treatment 3
were labelled just prior to anther dehiscence and were allowed to be naturally
pollinated. Flowers in treatment 4 were emasculated just prior to anther
dehiscence and naturally pollinated. For pins this was done by making a slit in
the corolla tube. If performed skilfully this treatment does not affect subsequent
floral development. Flowers of treatments 3 and 4 were collected on 9 May
1985, i.e. at the end of anthesis. Each flower was stored in a 1.5 cm3 Eppendorf
vial that contained a pad of cotton wool soaked in a 1 : 1 mixture of absolute
ethanol and glacial acetic acid, which acted as a fixative. Homostyle flowers
were subjected to treatments 1 and 3 only. Buds were collected from all the
morphs on the first visit, and they were stored in the same way as flowers in
treatments 3 and 4.
In order to extract pollen grains from stigmas of intact and emasculated
flowers, from dehisced anthers of intact flowers and from undehisced anthers of
buds, the appropriate plant parts were dissected out and acetolysed (Erdtman,
1969). This process dissolves all plant material except the exines of pollen grains.
Stigmas or anthers were incubated in 0.2 cm3 of a solution comprising one part
concentrated sulphuric acid to nine parts acetic anhydride for 40 min at 90°C.
During incubation the material was broken up with a glass rod. After acetolysis
the ‘acetolysate’ was made up to 0.5 cm3 with 50% glycerol, and agitated on a
Fisons ‘whirlymixer’ in order to suspend the pollen evenly. Aliquots were
removed immediately and the pollen was counted using a compound
microscope. To estimate the numbers of pollen grains in dehisced and
undehisced anthers 20 pl aliquots were taken, and counting was performed at
x 100 magnification. To estimate the numbers of pin and thrum type pollen
grains on stigmas of intact and emasculated flowers 0.1 cm3 aliquots were taken,
128
J. PIPER AND B. CHARLESWORTH
and counted at x 100 magnification. At this magnification pin and thrum
pollen can be distinguished. To estimate the sizes of pollen produced by pins,
thrums, and homostyles, 20 pl was taken from each acetolysate of undehisced
anthers to make a bulk mixture for each morph. Aliquots of these mixtures were
analysed until 200 pollen grains had been measured. Measuring was performed
at x 1000 magnification with a microscope fitted with a graticule.
In 1984, stigma clogging experiments were performed in two Somerset
populations: Bruton (GR 692343) (pin : thrum : homostyle ratio was
0.43 : 0 : 0.57), and Cogley (GR 702347) (pin : thrum : homostyle ratio was
0.47 : 0.35 : 0.18). Flowers of pin plants were labelled with colour-coded wire
and subjected to one of three treatments: (1) natural pollination; (2) artificial
pollination with legitimate pollen followed by natural pollination; or (3)
pollination with illegitimate pollen from freshly dehisced pin anthers prior to
anther dehiscence, followed by natural pollination. At the end of the season the
seed capsules were collected, and the number of seeds each contained was
determined. At the same time, flowers of pin plants raised from seed were self- or
cross-pollinated in an insect-free greenhouse. When seed capsules produced by
these treatments were ripe they were collected, and the number of seeds in each
capsule was determined. Thrum plants were not included in this experiment as
it is not possible to pollinate them illegitimately without inflicting damage on
the flowers.
Stalislical analysis
Comparisons among means were performed by one-way analysis of variance.
Raw pollen load data was transformed using the Box-Cox procedure (Sokal &
Rohlf, 1981), thus
Y = x03- 1/0.3.
where X is the untransformed datum, and Y the transformed datum. Values
expressed as proportions were subjected to the arcsine square-root
transformation.
Estimates of disassortative pollination for pin stigmas (a,) and thrum stigmas
(a,) were derived from the model of Charlesworth & Charlesworth (1979). For
details of computation and tests of significance see the Appendix.
RESULTS
Pollen grain size
Before the compositions of s.p.ls of distylous species can be analysed, pin and
thrum pollen must be distinguishable. Figure 2 shows the mean pollen grain
size, and pollen grain size distributions of acetolysed pollen for pin and thrum
pollen. The means of the two distributions differed significantly ( P < 0.01), but
there was some overlap: 7 1yo of pin pollen was the same size as 15% of thrum
pollen. This did not present a problem for s.p.1. analysis because thrum pollen in
the smaller size classes was unfilled, and so was morphologically distinct from
pin pollen.
DISTYLY I N PRIMULA VULGARIS
I29
0
g
10T=32*85&0-40
1520-1
'
IIb'
I
I
'
15
I
I I
'
20
I
I
I
I
'
25
Graticule units
I
I
I
30
I
I
I
35
Figure 2. Acetolysed pollen grain size distributions for pins and thrums and homostyles combined.
'l'wo hundred pollen grains for each morph were measured. Size classes are in graticule units, and
thr means of these distributions are presented in pm. Although there was some overlap in the
distributions, thrum and homostyle pollen grains in the smaller size classes were unfilled, and so
morphologically distinct from pin pollen grains.
Pollen grain production and pollen removal f r o m anthers
Table 1 presents estimates of the mean numbers of pollen grains per anther
for dehisced and undehisced anthers of all three morphs. Undehisced pin
anthers contained significantly more pollen than thrum and homostyle anthers,
which produced similar numbers of pollen grains. This is usual for distylous
species. Dehisced pin anthers retained significantly more pollen grains at the
end of anthesis than those of thrums and homostyles, which retained similar
numbers of pollen grains. Subtraction of mean pollen grains per dehisced anther
from mean pollen grains per undehisced anther provides estimates of the mean
numbers of pollen grains removed from anthers, and these values are also given
in Table 1. These were compared by a series of t tests. I n each case the numbers
of pollen grains removed from anthers did not differ significantly ( P > 0.05).
Stigmatic pollen load composition for intact and
emasculatedjlowers
Table 2 presents data on s.p.1. compositions for stigmas of intact and
emasculated pin and thrum flowers, and for intact homostyle flowers. A
Table 1. Mean numbers of pollen grains in undehisced and dehisced anthers of
pins, thrums and homostyles ( &standard errors), and mean numbers of pollen
grains exported per anther. Numbers in parentheses refer to sample size
P o l l r ~ igrains per undrhiscrd anther
P(~llriigrains per dehisced anther
I'ollcn grains rrrnoved per anther
Pins
'I'hrums
Homostyles
36620.0f2147.0*
(20)
25391.3f5358.1*
(20)
I 1 228.7k5772.3
17 165.0k 1885.9
120)
7007.9f 1604.4
(19)
10 158.0f2476.0
16837.5f 1189.3
(20)
3325.0f654.4
(20)
13512.5f 1357.4
*Indicates that values for pins were significantly greater than those for thrums and homostyles (P<O.OI).
Valurs for thrums and homostyles did not differ significantly.
Thrum
258.8f63.4
(20)
267.1 k50.5:
(26)
0.61 *O.oSY
Pin
2376.6k 350.9.t
(20)
183.4k41.1
(26)
0.39k0.06
Thrum
583.0+ 143.3*?
(20)
145.0k51.3
(31)
0.33k0.05
Pin
256.1 k 75.8
(20)
304.6 k85.8:
(3')
0.67 f0.05:
Thrum
162.5f66.6
(20)
Pin
Thrum
2703.8k431.0
(20)
Homostyle
*Indicates that illegitimate pollen loads were significantly greater than legitimate pollen loads on intact flowers (P<0.05).
tlndicates that illegitimate pollen loads on stigmas of intact flowers were significantly greater than those on emasculated flowers ( P < 0.01).
:Indicates that legitimate pollen loads on emasculated flowers (in terms of the numbers of pollen grains, or proportions) were significantly greater than illegitimate
pollen loads ( P c 0.05).
Emasculated flowers
Intact flowers
Stigma type
Pollen type
Pin
Table 2. Mean stigmatic pollen load compositions for stigmas of intact and emasculated pin and thrum flowers, and intact
homostyle flowers, f their standard errors. Numbers in parentheses refer to sample size. Mean proportions of legitimate
and illegitimate pollen on stigmas are also given & standard errors
L
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i
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9
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DISTYLY IN PRIMULA VULGARIS
131
preliminary inspection of the s.p.1. composition data for stigmas of intact and
emasculated pin and thrum flowers highlights three important points. First,
stigmas from intact flowers captured significantly more illegitimate than
legitmate pollen; secondly, emasculation caused a significant reduction in the
size of the illegitimate component of s.p.ls, and thirdly, stigmas from
emasculated flowers captured more legitimate than illegitimate pollen. This last
comparison was significant for thrum stigmas only. However, when the
proportion of illegitimate and legitimate pollen on stigmas of emasculated
flowers were compared (Table 2) the difference was significant for both morphs.
Thus it is clear that self pollen accounts for a substantial proportion of
illegitimate s.p.ls, and for this reason data collected from stigmas of intact
flowers should not be used to investigate pollen flow between plants.
Further inspection of legitimate s.p.1~for intact and emasculated pin and
thrum flowers reveals that emasculation did not make flowers unattractive to
pollinators: for both morphs legitimate s.p.ls were not significantly different for
intact and emasculated flowers. These observations confirm that data on the
composition of s.p.1~from emasculated flowers, collected in this study, can be
used in order to calculate estimates of disassortative pollination, and to
investigate the self-pollination and pollen saving hypotheses.
The thrum component of s.p.1~of intact homostyle flowers was significantly
larger than that for intact thrum flowers. These morphs produced similar
numbers of pollen grains, and had similar numbers of pollen grains removed
from their anthers, and so it seems likely that thrums experienced much less selfpollination than homostyles. This view is confirmed when the components of
s.p.ls due to self-pollination are compared. The mean s.p.1. due to selfpollination for intact thrum flowers was 438+ 152.2 grains, whereas for
homostyles it was 2445f435.6 grains. These values were compared by a t test,
which showed that they differed significantly ( t = 4.35, df = 80, P < 0.01).
Clearly thrum flowers experienced much less self-pollination than homostyle
flowers, an observation which demonstrates unequivocally that herkogamy
reduces the incidence of self-pollination.
Flowers of the morphs of P. vulgaris have five anthers per flower. Therefore
pollen removal from anthers on a per flower basis can be estimated by
multiplying the values for pollen removal in Table 1 by five. Thus pins, thrums
and homostyles, on average, had 56 143.5, 50 790.0 and 67 562.5 pollen grains
respectively removed from their anthers. Using the approach outlined in the
introduction and implemented above, the self pollen component of the s.p.1~
can be estimated for each morph. This is 2193.2 for pins, 438.0 for thrums and
2436.7 for homostyles. Hence the percentage of pollen made unavailable for
pollen export due to self-pollination is 3.9% for pins, 0.9% for thrums and 3.6%
for homostyles. Thus thrums ‘wasted’ four times less pollen than pins and
homostyles.
Disassortatiue pollination
Table 3 presents values of the efficiency of distyly for pin (a,) and thrum (a,)
stigmas. Estimates of the frequencies of pin and thrum pollen in the pollen pool
are also presented, as are the probabilities of transfer of legitimate pollen to pin
(Qp) and thrum (Qt) stigmas. Before values of ap and a, can be derived
J. PIPER A N D B. CHARLESWORIH
I32
Table 3. Estimates of disassortative pollination
for pins (a,) and thrums (at),the probabilities
of transfer of legitimate pollen to pin (Q,) and
thrum (QJ stigmas, and the frequencies of pin
@I) and thrum ( 1 ) pollen in the pollen pool.
The first two statistics are presented with their
standard errors.
Pins
~~
~
a,, = 0.31f0.17*
QP=0.6I +0.06$
p =0.48
Pins
Thrums
~~
~~
a,=0.55f0.10*
Q,=0.67*0.05$
1-0.52
+Indicates that values of a were significantly greater than
zero (P< 0.05).
:Indicates that the mean proportion of legitimate pollen on a
given stigma type was significantly greater than the illegitimate
proportion (P< 0.05).
estimates of the frequencies of pin and thrum pollen in the pollen pool must be
obtained, as must estimates of Qp and Q,. Values of Qp and Q, were taken as the
proportions of legitimate pollen on the appropriate emasculated stigmas. In
order to calculate frequencies of pin and thrum pollen in the pollen pool it is
necessary to know for each morph (1) how much pollen is removed from
anthers, (2) how much removed pollen is wasted due to self-pollination, and (3)
the frequencies of the morphs in the population. Taking into account the
amount of pollen wasted through self-pollination, pins, thrums and homostyles
exported 53 950.5, 50 352.0 and 62 126.0 pollen grains per flower, respectively.
The frequencies of the morphs were 0.5 pins, 0.25 thrums and 0.25 homostyles.
Thus, flowers producing thrum pollen, on average, exported 57 739.0 pollen
grains per flower. Therefore the frequency of pin pollen was 0.48 and of thrum
pollen 0.52. In making these calculations it is assumed that a t any one time
pin, thrum and homostyle plants have the same number of flowers in anthesis.
This is usually the case for P. vulgaris (Piper, 1984).
Estimates of the frequencies of pin and thrum pollen, and Qp and Q, were
used to calculate estimates of ap and a,, and their standard errors. ap and a, were
both significantly greater than zero, demonstrating that pins and thrums
experienced significant disassortative pollination. This view is supported by the
fact that the mean proportion of thrum pollen on pin stigmas was significantly
greater than the proportion of thrum pollen on thrum stigmas, and by the fact
that the mean proportion of pin pollen on thrum stigmas was significantly
greater than the mean proportion of pin pollen on pin stigmas. Finally, the
estimates of ap and ct, did not differ significantly.
Seed production
1984
Table 4 presents acomparison of mean seed capsule fertilities of pin flowers
that were naturally pollinated, artificially pollinated with legitimate pollen, or
DISTYLY IN PRIMlJLL4 VULGARIS
133
Table 4. Mean numbers of seeds per capsule (pins) for the treatments
implemented in order to test the stigma clogging hypothesis in 1984, and to
check the abundance of pollinators in 1985
Natural
pollination
Clogging
Artificial
pollination
32.89 f 3.32
(36)
39.40 f3.35
(35)
26.62*4.53*
(21)
34.42 3.62
(31)
39.94 f 2.89
(34)
40.86 It:3.74
(35)
1984
(hgley
BI ito on
Grrcnhousr data
(:ross-pollinatcd pins
Srlf-pollinated pins
I985
Pins
'l'hrums
Honiost ylrs
+
37.13 f 2.44t
(70)
17.22k2.09
(20)
35.59+ 3.25
(27)
5 1.68 f 4.66
(25)
52.63 2.89
(41)
32.59f4.54
(29)
45.37 f5.05
(30)
* Indicates that flowers suhjerted to the clogging treatment produced significantly fewer seeds per capsule
than those subjected to artificial cross-pollination (P< 0.05).
tIndicares that pin flowers cross-pollinated in the greenhouse produced significantly more seeds per capsule
111;in sell-pollinatrd flowers (P< 0.01).
pollinated with illegitimate pollen, at Cogley and Bruton. In both populations
seed capsules produced by artificial legitimate pollination produced more seeds
than those produced by natural pollination, which in turn produced more seeds
than those produced as a result of pollination with illegitimate pollen. Only one
significant difference was obtained. At Cogley flowers pollinated artificially with
legitimate pollen produced capsules with more seeds than those that were
illegitimately pollinated. In the greenhouse self-pollinated pin flowers did
produce some seeds, but significantly less than cross-pollinated pin flowers. Seed
set by self-pollinated pin plants cannot be attributed to accidental crosspollination by insects, as untreated flowers produced no seeds.
1985
Table 4 also presents mean numbers of seeds per capsule for pins, thrums and
homostyles at Wyke in 1985. Although the number of seeds per capsule clearly
differs among morphs, artificial legitimate pollination of the cross-fertilizing
morphs had no effect on seed capsule fertilities. These observations imply that in
this season pollinators were abundant.
DISCUSSION
The theoretical framework on which this study was based considered four
factors that could be involved in the evolution of' the morphological aspects of
distyly. These were selection for ( 1 ) reduction in the incidence of self-pollination
134
J. PIPER AND B. CHARLESWORTH
in a partially self-fertile form, (2) reduction in stigma clogging, (3) the
promotion of a pollen saving effect, and (4) disassortative pollination.
Selection for reduced self-pollination
Analysis of the self-pollen component of s.p.ls of thrums and homostyles
demonstrated that herkogamy causes a substantial reduction in the incidence of
self-pollination, which presumably could result in a reduction in self-fertilization
in a partially self-fertilizing morph. This is almost certainly true for P. vulgaris.
Cahalan & Gliddon (1985) have shown that the pin form of P. vulgaris
experiences no self-fertilization, despite the fact that, as this study has shown, it
is partially self-fertile. Therefore, it is possible that selection for reduced selfpollination played a major role in the first morphological transition (stages 2-3
in Fig. 1) in the evolution of distyly in this species.
Selection for reduced stigma clogging
Computer simulations performed by Charlesworth & Charlesworth ( 1979)
indicated that stigma clogging did not promote either of the morphological
transitions that occurred during the evolution of distyly (stages 2-3 and 3-4 in
Fig. 1 ) . Their results implied that if stigma clogging caused a significant
reduction in seed set, then neither the self-incompatibility mechanism, nor the
later morphological changes would have evolved. Unfortunately the results
presented here do not rigorously test the stigma clogging hypothesis, for two
reasons: first, pin plants are partially self-fertile, and secondly, when pollinators
are scarce pin plants produce small seed crops. Consequently seed set by
‘clogged’ flowers can be equivalent to that by naturally pollinated flowers, even
if there is a significant clogging effect. This could have been the case here, as in
this region of Somerset, pollinator availability limited seed set in the crossfertilizing morphs of P. vulgaris (Piper, Charlesworth & Charlesworth, 1986).
Hence there may have been a significant stigma clogging effect, but due to
partial self-fertility in pins and low levels of pollinator service we were unable to
detect it. Shore & Barrett (1984), working in the laboratory on the distylous
species Turnera ulmifolia, only obtained a significant reduction in seed set when
illegitimate pollen from numerous anthers was applied to stigmas. Barrett &
Glover (1985) were unable to detect a significant clogging effect in natural
populations of the tristylous species Pontederia cordata. Thus other workers have
provided evidence that suggests that stigma clogging is unimportant in the
evolution of morphological floral polymorphisms.
Selection for pollen saving
I t has been postulated that selection for pollen saving could have been a
factor involved in both morphological transitions in the evolution of distyly
(stages 2-3 and 3-4 in Fig. 1). The data presented here show that thrums
experienced a significant pollen saving effect when compared to homostyles, due
to herkogamy. Although the saving is significant, it is slight: homostyles wasted
D I STYLY IN PRIM ULA VULGARIS
135
3.6% of their pollen, and thrums 0.9%. The difference, or the ‘pollen saving
effect’ was 2.5%. This represents a tiny proportion of the pollen exported, and it
is unlikely that it is biologically significant, as in absolute terms the mean
numbers of pollen grains exported by thrums and homostyles did not differ.
Selection for disassortative pollination
Pins and thrums experienced significant disassortative pollination, thus the
transfer of pollen from anthers to stigmas at the same height is more efficient
than between anthers and stigmas at different heights. This observation
supports the view that the final stage in the evolution of distyly depicted in
Fig. 1 was a shift in anther position which increased the male fertility of the selfincompatible homostylous form. Thrum stigmas tend to experience better
disassortative pollination than pins (Ganders, 1974; Nicholls, 1985; Piper
unpubl. obs.) and although the difference was not significant, this is also true for
this population of P. vulgris. Accounting for this asymmetry presents no
problem. Most workers agree that pin stigmas, due to their position, are not
able to discriminate between pin and thrum pollen to the same extent as thrum
stigmas. Our results do not contradict this hypothesis.
The data collected in order to calculate estimates of disassortative pollination
provide some insight into whether the first morphological change was a shift in
stigma or anther position. The Charlesworths argued in favour of a stigma shift
first, and our results are in accordance with this view. Assuming that there was
minimal geitonogamous pollination, our results showed that a large component
of s.p.1. was illegitimate, and in the order of 100-200 pollen grains. From the
foregoing paragraphs it is clear that a shift in stigma position does reduce the
component of the pollen load captured from anthers at a different height, but if
it were legitimate it would probably still be sufficient to ensure high seed set.
This suggests that a stigma position shift as the first morphological transition in
the evolution of distyly would not reduce seed set. In contrast, the results of the
disassortative pollination analysis show that a change in anther position could
cause a 20% reduction in male fertility, which would be highly maladaptive,
and eliminated by selection. This reasoning also explains why it is unlikely that
disassortative pollination is not totally efficient. If it was the initial stigma shift
would be highly deleterious, and the distyly dimorphism would never evolve.
Finally, we did not set out to investigate the selective agents for the evolution
of pollen sizelnumber dimorphisms common to many distylous species.
However, our results on pollen export provide an intriguing insight into this
problem. Pin anthers are concealed within the corolla tube, and as a result are
relatively inaccessible to pollinators. Thrum anthers, in contrast, are not. Our
results show that pins and thrums experienced differential pollen export.
Ornduff (1979, 1980) made similar observations for P . vulgaris and P. veris, but
based on a much smaller sample size, as has Piper (unpubl. obs.) for the
distylous species Primula furinosu and P . veris. It is tempting to speculate that
selection has increased pollen grain number in pins (or decreased it in thrums)
and as a consequence decreased (or increased) pollen grain size, in order to
overcome the problem of the poor accessibility of pin anthers.
I36
J. PIPER AND B. CHARLESWORTH
ACKNOWLEDGEMENTS
We thank Chris and Jill Curtis for providing accommodation for J. P.
during part of the field work phase of this study. We also thank Melanie Piper
and Kath Evans for assistance in the field. This work was sponsored by a
S.E.R.C. grant awarded to Brian Charlesworth and John Maynard Smith.
REFERENCES
BARRETT, S. C. H. & GLOVER, D. E., 1985. O n the Darwinian hypothesis of the adaptive significance of
distyly. Evolution, 39: 766-774.
CAHALAN, C. M. & GLIDDON, C., 1985. Genetic neighbourhood sizes in Primula vulgaris. Heredib, 54:
65-70.
CHARLESWORTH, B. & CHARLESWORTH, D., 1979. A model for the evolution of distyly. American
Naturalist, 114: 467-498.
DARWIN, C., 1877. The diyerent forms ofjlowers on plants of the same species. London: John Murray.
ERDTMAN, G., 1969. Handbook of Palynology. Copenhagen: Munksgaard.
GANDERS, F. R., 1974. Disassortative pollination in the distylyous plant Jepsonia heterandra. Canadian Journal
of Botany, 52: 2401 -2406.
GANDERS, F. R., 1979. The biology of heterostyly. N e w zealand Journal of Botany, 17: 607-635.
NICHOLLS, M. S., 1985. Pollen flow, population composition, and the adaptive significance of distyly in
Linum tenuifolium L. (Linaceae). Biological Journal of the Linnean Sociey, 25: 235-242.
ORNDUFF, R., 1979. Pollen flow in a population of Primula vulgaris Huds. Botanical Journal of the Linirean
Socieb, 78: I - 10.
ORNDUFF, R., 1980. Pollen flow in Primula ueris Primulaceae. Plant Sysfematics and Evolution, 135: 89-93.
PIPER, J. G., 1984. Breeding system evolution in Primula vulgaris. D. Phil. thesis, University of Sussex.
PIPER, J. G., CHARLESWORTH, B. & CHARLESWORTH, D., 1986. Breeding system evolution of
Primula vulgaris, and thc role of reproductive assurance. Heredib 56: 207-217.
KICHARDS, A. J., 1986. Plant Breeding Systems. London: Allen & Unwin.
SCHOU, O., 1983. The distyly of Primula elatior (L.) Hill (Primulaceae), with a study of flowering phenology
and pollen flow. Botanical Journal of the Linnean Sociely, 86: 261-274.
SHORE, J . S. & BARRETT, S. C. H., 1984 The effect of pollination intensity and incompatible pollen on
seed set in Turnera ulmifolia (Turneraceae). Canadian Journal of Botany, 62: 1298-1303.
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APPENDIX
Estimation of disassortativepollination
Charlesworth & Charlesworth (1979) derived an expression that describes the
probability of transfer of legitimate pollen to stigmas:
Q = l/l+i(l-a),
where Q is the probability of transfer of legitimate pollen to a given stigma type,
1 and i are the proportions of legitimate and illegitimate pollen in the pollen
pool respectively, and a is the value of disassortative pollination promoted by
distyly (efficiency of distyly) . When mating is disassortative, the lower boundary
of a is zero, and the upper boundary is one. This formula can be rearranged to
obtain expressions for a:
a=1
+ (l/i)2(1 - l/Q).
Estimates of variance for a were obtained thus:
V(a) = (l/i) x V(Q)x l/Q4,
DISTYLY IN PRIMULA VULGARIS
137
where V ( a )is the variance of a, V(Q) is the variance of Q,and I, i and Q are
the same as above. Estimates of V(Q) were obtained thus:
V(Q) = szQU;
where s*Q is the variance of the probability of legitimate pollen being
transferred to a given stigma (sample variance), andfis the number of stigmas
in the sample.
In order to test whether or not distyly promoted significant disassortative
pollination, values of a for pins and thrums were compared to zero by t tests.
The square root of V(Q) was taken as the appropriate standard error for these
tests.