Arrash M Senior Honors Thesis

1

Sources of variation in pollen deposition and pollen tube attrition in two Clarkia species:
the effects of style length, seasonal timing, and method of estimating attrition
By
Arrash Moghaddasi
Submitted to the University of California, Santa Barbara
Department of Ecology, Evolution and Marine Biology
June 4, 2013

2

Introduction
Due to their sessile nature, plants differ from animals in that plants cannot
undergo active mate choice, in which female plants select which pollen-producing plants
may pollinate them. Following pollination, both the male and the female genotypes
influence which matings will be successful. Male gametophytes may compete with each
other to reach the base of the style, and male-female interactions may influence which
pollen genotypes perform best within the styles of particular maternal flowers. (Hormaza
and Herrero 1994; Aronen et al. 2002).
Pollen tube attrition – or the failure of germinated pollen tubes to reach the base
of the stigma or the base of the style – represents one measure of pollen and pollen tube
performance. The rate of pollen tube attrition (the proportion of pollen grains or pollen
tubes that fail to fertilize an ovule) may be determined by multiple factors. For example,
male gametophytes with high-quality genotypes are more likely to fertilize an ovule than
low quality genotypes when pollen deposition exceeds the number of ovules. This does
not mean, however, that pollen tube attrition occurs only when this condition applies.
Pollen tube attrition may also occur whenever fatal alleles prevent successful pollen tube
growth.
The number of pollen tubes observed in a style often decreases from the stigma to
the base of the style, particularly when pollen loads are greater than the number ovules
available in the ovary (Erbar 2003; Ockendon and Gates 1975; Herrero and Dickinson
1980; Pimienta et al. 1983; Herscovitch and Martin 1990). Given that pollen tube
attrition occurs within the style, Plitmann (1993) proposes that the degree of pollen tube
attrition in outcrossing individuals should be higher than in self-fertilizing individuals

3

because pollen grains that land on stigmas of outcrossing individuals are more genetically
diverse. In self-fertilizing flowers, pollen tube attrition is most likely to be determined by
physiological or environmental factors (Plitmann 1993). In other words, the rate of
pollen tube attrition is likely to be influenced by the type of mating system (Plitmann
1993). In this study, Plitmann studied 17 outcrossing species, 11 inbreeding species, and
4 species with a mixed mating system in the Mustard family, genus Brassica. The author
found that the outcrossing species exhibited higher rates of pollen tube attrition than the
selfing species, resulting in stronger sexual selection on pollen donors in outcrossing
species.
In addition to mating system, post-pollination competition can be influenced by
pollen load composition and deposition patterns on the stigma (Bowman 1987; Jones
1994; Niesenbaum and Schueller 1997). Pollen load sizes, timing of delivery, and
deposition patterns may affect the intensity of pollen competition for access to ovules
(Németh and Huerta 2003; Niesenbaum and Schueller 1997). Effects of pollen
composition on pollen tube growth and attrition are evident in mixtures of self and
outcross pollen and mixtures of outcross pollen from different sources (Marshall et al.
1996; Snow and Spira 1991). For example, in radishes, Raphanus sativus, pollen from
one donor may interfere with pollen tube growth of another donor, possibly due to a
chemical factor (Marshall et al. 1996). Evidence supports the idea that pollen deposition
patterns can affect germination and lead to pollen tube attrition of competing pollen
genotypes (Marshall et al. 1996).
There have been relatively few studies designed to detect and compare sources of
variation in pollen tube attrition in wild angiosperm species. Several studies have

4

detected effects of male-female gametophytic interactions on attrition (Cheung 1995;
Cruzan 1986; Erbar 2003) and have measured the dynamics of pollen tube growth under
different mating systems (Plitmann 1993; Hormaza and Herrero 1996), different
pollination regimes (Huerta 1997; Snow and Spira 1991), and different deposition
patterns (Németh and Huerta 2002; Niesenbaum and Schueller 1997). However, no
studies to date have examined temporal variation in pollen tube attrition within or across
flowering seasons, although one study concluded that the intensity of competition in
Clarkia unguiculata may vary within a season (Németh and Huerta 2003).
The goal of my senior thesis has been to examine the effects of several factors
that may influence pollen attrition rates in natural populations of two species of annual
wildflowers: Clarkia unguiculata and C. xantiana subspecies xantiana. In particular, I’m
interested in five potential causes of variation: style length, pollen load, the time during
the flowering season when flowers are sampled, the method used to estimate pollen
attrition rate, and species identity.
Accordingly, I plan to explore these sources of variation by asking the following
questions:
1. When examining samples taken in 2009 and 2010, does style length have a
significant effect on the amount of pollen received? We predicted that flowers
with longer styles will receive higher pollen loads because a stigma on a longer
style might have a higher probability of being contacted by pollinators or because
longer styles may be produced by relatively large flowers, which may be more
attractive to potential pollinators.

5

2. Is there evidence of interference (negative interactions) among pollen tubes? For
example, as pollen load increases, does the rate of pollen tube failure following
germination increase as well? Furthermore, does pollen tube failure increase with
high pollen tube density? We predicted that if many pollen tubes enter the style,
they might interfere with each other by physically taking up space in the stylar
tissue, or by producing an allelopathic chemical that negatively affects their
competitors.
3. Do estimates of pollen tube attrition depend on the way in which it is measured?
Here I compare three different measures of pollen tube attrition: (a) the proportion
of pollen grains deposited on the stigma that fail to reach the stigma-style
junction; (b) the proportion of pollen grains deposited on the stigma that fail to
reach the base of the style; and (c) the proportion of pollen tubes that reach the
stigma-style junction that fail to reach the base of the style.
4. Do pollen attrition rates change during the season, and is this related to pollen
load? If flowers that receive relatively high quantities of pollen on their stigmas
experience greater rates of pollen tube attrition than flowers with low pollen
loads, then if the mean level of pollen deposition differs between sampling dates,
then pollen attrition rates should also change over time. Alternatively, higher
pollen tube failure may be due to the conditions in which the pollen recipient
grows. For example, if plants become more nutrient stressed or water-limited later
in the season, they may be less able to support pollen tube growth. We might
therefore expect lower numbers of pollen tubes to enter the stigma-style junction
and style base in flowers sampled relatively late in the season. Here, we examine

6

relationships between pollen load and pollen attrition between early and late
sampling dates in each of our focal taxa.
Methods and Materials
Study species
Clarkia (Onagraceae) is a genus comprised of ~41 winter annual and herbaceous taxa
(Mazer et al. 2010; Delesalle et al. 2008; Dudley et al. 2007). Clarkia unguiculata
occupies different habitats throughout California ranging from woodland slopes to
grasslands including the western slopes of the Sierra Nevada, the eastern and western
slopes of the Coastal ranges, and the Tehachapi, Western Transverse, and Peninsular
ranges. Relative to C. unguiculata, Clarkia xantiana ssp. xantiana is more
geographically restricted, occupying rocky hillsides in the southern Sierra Nevada, the
Tehachapi Mountains, and the Western Transverse Ranges (Mazer et al. 2010). Both of
these outcrossers are protandrous (the anthers develop faster and dehisce first relative to
the stigma), highly herkogamous (high spatial separation between the sexual organs) and
produce large, well-developed flowers with individual life spans ranging from 2.5-3
months (Delesalle et al. 2008).
Sample collection in the field
During the flowering season of 2009 and 2010, individual flowers of C. unguiculata and
C. xantiana ssp. xantiana were collected from the southern Sierra Nevada region,
California, USA (Hickman 1993). Four populations of C. xantiana ssp. xantiana and
four populations of C. unguiculata were sampled (refer to Table 1 for populations). From
each population, one flower was collected from fifty individual plants, at the time of each
flowers’ senescence. The samples were removed from the stem by cutting just below the

7

ovary. Each flower was immediately put into an eppendorf tube containing formalin-
acetic acid (FAA), which causes all physiological activity to arrest, including pollen tube
growth. As a result, we can preserve a snapshot of physiological activity within the style.
A rack of 50 eppendorf tubes, with each tube containing the individual flower sample,
was sealed in a plastic bag and on each bag the following information was recorded:
timing of style harvest, taxon and population. Samples were stored for 2-3 years before
subsequent processing.

8

Note. Summary of focal taxa with according sample dates, sample sizes and coordinates of C. xantiana
ssp. xantiana and C. unguiculata. Sample sizes of each were initially 50, but after data analysis
samples sizes were trimmed. In 2009, two populations were sampled twice, but were not classified as
“early” or “late” (Borel Road and Sawmill Road).

9

Style processing
After carefully removing the style from the other floral parts, while maintaining the base
of the style, the style was rinsed twice with de-ionized water in order to wash off any
FAA. In the same eppendorf tube, the sample was immersed in 8M NaOH for 30-40
hours in order to soften the style. These samples were stored in a dark drawer to prevent
degradation by exposure to light. After soaking, styles were rinsed twice with de-ionized
water and stained for 2 hours in a 0.1 N K2HPO4 solution with 0.1% aniline blue dye
(Martin 1959). Following staining, each style was placed on a microscope slide,
straightened, and the length of its style recorded to the nearest 0.5 mm. On each
microscope slide, a label was placed and marked with the plant identification number.
The taxon and population corresponding to each sample ID was written in our data sheet
and referred to when needed. To facilitate viewing of the style we used a scalpel to
separate the stigma from the style. The stigma and style were gently squashed using
cover slips. As soon as style squashing was complete, the samples were taken to a
fluorescence microscope for viewing. We used the DAPI (4',6-diamidino-2-
phenylindole) excitation filter on the fluorescence microscope in order to detect callose
plugs, which appear noticeable as bright blue dots. We used the Picture Frame software
application in conjunction with the fluorescence microscope with a 4x objective lens. In
order to quantify the pollen tube attrition rate, at 40x, we used a manual thumb counter to
record the number of callose plugs in the basal and distal 1mm-intervals of the style.
In Clarkia, as pollen tubes progress through the style, they produce callose plugs
at ~1-mm intervals, which ensure that the cytoplasm and all of its contents are enclosed
within the growing tip of the pollen tube (Franklin-Tong 1999). We estimated the

10

number of pollen tubes by counting the number of callose plugs in the 1mm-interval at
the tip of style to estimate the number of tubes that entered the stigma-style junction, and
the number of callose plugs in the 1mm-interval at the base of the style to estimate the
number of tubes that had reached the ovary. A transparency with a printed grid was
placed over the computer screen in order to keep track of previously counted callose
plugs within subsections of these 1mm-intervals.
After counting callose plugs at the tip and base of the style, microscope slides
were placed under refrigerations; within two weeks of slide preparation, slides were
viewed under a dissecting microscope to record the number of pollen grains visible on the
stigma.
Statistical Analyses
The effects of style length on pollen load
In order to determine the relationship between style length and the amount of
pollen that was deposited on the stigma, while considering the year and timing of style
harvest, we conducted regression analyses on each species categorized by year and by
timing of style harvest (early vs. late). We performed regression plots of pollen load on
style length (mm) for C. xantiana ssp. xantiana and C. unguiculata separately. For styles
collected in 2009, all styles were pooled; for styles collected in 2010, separate analyses
were conducted for styles collected early vs. late in the flowering season.

11

The effects of timing of style harvest on pollen load
Analysis of variances (ANOVA) were conducted to detect significant effects of
the timing of style harvest on the mean number of pollen grains deposited on the stigma.
In order to do this, we excluded samples from 2009 and analyzed the styles collected in
2010 by taxon, pooling all populations sampled within each taxon.
Temporal differences in the number of pollen tubes at the stigma-style junction and at the
style base
Since samples in 2009 were not classified as “early” vs. “late”, we used only
samples collected in 2010 to detect significant effects of timing of style harvest on the
mean number of callose plugs located at the stigma-style junction and at the style base.
Analyses of variance were conducted within each species. Similar analyses were done on
the number of callose plugs at the style base.
Temporal differences in pollen tube attrition
Within each taxon, we conducted ANOVAs to detect significant effects of the
timing of style harvest on the mean rate of pollen attrition from the stigma to the stigma-
style junction. We excluded samples from 2009 and pooled all styles collected from each
species early and late in the season in 2010. We used the equation (1) to determine the
attrition rate of pollen tubes that fail to enter the stigma-style junction (ssj):
(1) Attrition rate (stigma to ssj) =
(# pollen grains deposited on the stigma - # callose plugs at ssj)
# pollen grains deposited on the stigma

12

Similarly, we conducted the same ANOVA as above to detect the effects of the timing of
style harvest on the attrition rate from the stigma-style junction to the style base using the
equation (2):
(2) Attrition rate (ssj to style base) =
(# callose plugs at ssj - # callose plugs at base)
# pollen grains deposited at ssj
Lastly, in order to test the attrition rate from the stigma to the style base in an ANOVA,
we used the equation (3):
(3) Attrition rate (stigma to style base) =
(# pollen grains deposited on the stigma - # callose plugs at base)
# pollen grains deposited on the stigma
The effects of pollen load and number of callose plugs at the stigma-style junction on the
three measures of attrition rate
Here we analyzed two independent variables (the number of pollen grains
deposited on the stigma, and the number of callose plugs at the stigma-style junction) to
estimate three measures of attrition: 1) stigma to ssj, 2) ssj to style base, and 3) stigma to
style base. We examined each taxon independently, and within each taxon, we pooled all
styles representing all populations and combined both years. We conducted regression
analyses in each taxon to detect whether pollen load or the number of pollen tubes at the
ssj had a significant effect on any of the three attrition rates. We calculated lines of best
fit to determine the slope of each regression, which indicated one of three processes: 1) if
the slope was significantly different from zero and positive, then we interpreted this as

13

evidence of interference among pollen tubes; 2) if the slope was not significantly
different from zero, then the x variable did not have a significant effect on the failure rate
in the given interval within the style; and 3) if the slope was significantly different from
zero and negative, then we interpreted this pattern as evidence of facilitation,
characterized by a lower failure rate with increasing pollen load or number of pollen
tubes. Here, we used the same equations (1-3) in our calculations of the attrition rate in
each regression analyses.
Results
The relationship between pollen deposition and style length differed between taxa
and sampling dates. Where style length predicted the number of pollen grains deposited
on stigmas, the relationship was always positive; flowers with relatively long styles

14

received more pollen than those with shorter styles. In Clarkia unguiculata, style length
did not have a statistically significant effect on pollen load among styles sampled in 2009
(Fig. 1A) (n = 70, r 2
= 0.0103, P = 0.403) or in early 2010 (Fig. 1B) (n = 213, r 2
=
0.0119, P = 0.112). Style length did have a significant and positive effect, however, in
late 2010 (Fig. 1C) (n = 213, r 2
= 0.344, P < 0.001*). In Clarkia xantiana ssp. xantiana,
style length had a significant and positive effect on pollen load in 2009 (Fig. 1D) (n =
239, r 2
= 0.0515, P < 0.004*). In 2010, style length did not have a significant effect on
pollen load early in the season (Fig. 1E) (n = 90, r 2
= 0.0211, P = 0.172), but did have a
significant positive effect late in the season (Fig. 1F) (n = 172, r 2
= 0.0715, P < 0.004*).
The effects of timing of style harvest on pollen load
The effect of the timing of style harvest on pollen deposition in 2010 differed
between taxa (Fig. 2). The timing of style harvest of C. unguiculata had no significant
effect on the mean pollen load (Early season mean = 271.9 (SD = 11.5) pollen grains vs.
Late season mean = 293.0 (SD = 17.9); n = 300, r 2
= 0.0033, P = 0.321) (Fig. 2A). By
contrast, in C. xantiana ssp. xantiana, styles sampled early in the season received
significantly more pollen than styles sampled late in the season (Early season mean =
428.3 (SD = 17.7) vs. Late season mean = 246.4 (SD = 12.8); n = 262, r 2
= 0.210, P <
0.001*) (Fig. 2B).

15

Temporal differences in the number of pollen tubes at the stigma-style junction and at the
style base
In both taxa, the mean number of pollen tubes at the stigma-style junction (ssj)
and the style base decreased from early to late in the season in 2010 (Fig. 3). In C.
unguiculata, even though the number of pollen grains on the stigma did not change over
time w, the mean number of pollen tubes at the ssj (Early mean = 96.8 (SD = 4.01), Late
mean = 71.2 (SD = 6.27)) and style base (Early mean = 53.1 (SD = 1.82), Late mean =
42.8 (SD = 2.85)) were significantly higher Early than Late in the season (ssj: n = 300, r 2
= 0.0381, P = 0.0007*; base: n = 300, r 2
= 0.0305, P = 0.0024*) (Fig. 3A, B). Similarly,
in C. xantiana ssp. xantiana, there were more pollen tubes at the ssj (Early mean = 154
(SD = 6.93), Late mean = 92.6 (SD = 5.01) and style base (Early mean = 99.1 (SD =

16

4.35), Late mean = 61.9 (SD = 3.15)) earlier in the season (ssj: n = 262, r 2
= 0.165, P <
0.0001*; base: n = 262, r 2
= 0.155, P < 0.0001*) (Fig 3C, D).
Pollen tube attrition from the stigma to the SSJ: The rate of pollen tube attrition
during germination and early pollen tube growth differed between styles sampled Early
vs. Late in C. unguiculata but not in C. xantiana. In C. unguiculata, the pollen attrition
rate increased significantly between samples collected Early and Late in the season (Early
mean = 0.579 (SD = 0.0166), Late mean = 0.663 (SD = 0.0261); n = 299, r 2
= 0.0243, P
= 0.0069*) (Fig. 4A). There was no significant effect of timing of style harvest on the
pollen tube attrition rate in C. xantiana ssp. xantiana (Early mean = 0.584 (SD = 0.0270),
Late mean = 0.554 (SD = 0.0196); n = 261, r 2
= 0.00309, P = 0.371) (Fig. 4B).
Pollen tube attrition from the SSJ to the style base: The timing of style harvest
had no significant effect on the mean attrition rate from the SSJ to the style base in either
taxon (Fig. 5). There was no significant effect seen in C. unguiculata (Early mean =
0.407 (SD = 0.0177), Late mean = 0.372 (SD = 0.0278); n = 300, r 2
= 0.00374, P =

17

0.291) (Fig. 5A) or C. xantiana ssp. xantiana (Early mean = 0.335 (SD = 0.0267), Late
mean = 0.318 (SD = 0.0192); n = 260, r 2
= 0.00108, P = 0.598) (Fig. 5B).
Pollen tube attrition from the stigma to the style base: The timing of style harvest had a
significant effect on pollen tube attrition throughout the entire length of the style in C.
unguiculata (Early mean = 0.771 (SD = 0.00863), Late mean = 0.811 (SD = 0.0136); n =
299, r 2
= 0.0198, P = 0.0149*) (Fig. 6A) but not in C. xantiana ssp. xantiana (Early
mean = 0.721 (0.0369), Late mean = 0.673 (SD = 0.0268); n = 261, r 2
= 0.00422, P =
0.2956) (Fig. 6B).

18

The effects of pollen load and number of pollen tubes at the stigma-style junction on the
In both species, pollen tube attrition rates tended to increase significantly with the
number of pollen grains deposited and the number of growing pollen tubes. In C.
unguiculata, the number of pollen tubes at the stigma-style junction had a positive,
significant effect on the attrition rate from the SSJ to the base of the style (n = 370, r 2
=
0.161, P < 0.0001*) (Fig. 7A). Similarly, the number of pollen grains deposited on the
stigma had a positive, significant effect on the attrition rate from the stigma to the SSJ
and from the stigma to the style base (stigma-ssj: n = 369, r 2
= 0.199, P < 0.0001*;
stigma-style base: n = 369, r 2
= 0.190, P < 0.0001*) (Fig. 7C, E). By contrast, the
number pollen grains deposited on the stigma had a significant negative effect on the
attrition rate from the SSJ to the style base (n = 370, r 2
= 0.0139, P = 0.0235*) (Fig. 7G).
In C. xantiana ssp. xantiana, the number of pollen tubes at the SSJ had a
significant positive effect on the attrition rate from the SSJ junction to the style base (n =
500, r 2
= 0.231, P < 0.0001*) (Fig. 7B). Similarly, the number of pollen grains deposited
on the stigma had a significant positive effect on the attrition rate from the stigma to the
SSJ and from the stigma to the style base (stigma-ssj: n = 501, r 2
= 0.206, P < 0.0001*;
stigma-style base: n = 498, r 2
= 0.241, P < 0.0001*) (Fig. 7D, F). By contrast, we did
not see any significant effect of pollen load on the attrition rate from the SSJ to the style
base (n = 497, r 2
= 0.000241, P = 0.730) (Fig. H).

19

!
-0.1
0.1
0.3
0.5
0.7
0.9
1.1
AttritionRate-
ssjtostylebase
0 100 200 300
# callose plugs at stigma-
style junction (ssj)
C. unguiculata
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
AttritionRate-
ssjtostylebase
0 100 200 300 400
# callose plugs at stigma-
style junction (ssj)
C. xantiana ssp. xantiana
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
AttritionRate-(pollen
depositedtossj)
0 100 300 500 700 900 1100
# pollen grains on stigma
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
AttritionRate-(pollen
depositedtossj)
0 100 300 500 700 900
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
AttritionRate-pollen
depositedtostylebase
0 100 300 500 700 900 1100
-0.2
0
0.2
0.4
0.6
0.8
1
AttritionRate-pollen
depositedtostylebase
0 100 300 500 700 900
-0.1
0.1
0.3
0.5
0.7
0.9
1.1
AttritionRate-
ssjtostylebase
-100 100 300 500 700 900
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
AttritionRate-
ssjtostylebase
0 100 300 500 700 900
* *
* *
* *
*
Fig. 7 Regression analyses showing the effect of the number of callose plugs at the ssj (A, B) or
pollen load (C-H) on particular attrition rate. Red lines indicate the line of best fit with (*) denoting a
significant effect between the two variables. !
A B
!
A
C D
E F
G H

20

Discussion
In our experiment, we detected several sources of variation in pollen deposition
and pollen tube attrition that have not been previously measured. These sources of
variation include both intrinsic factors, such as style length, and extrinsic factors, such as
timing of style harvest.
Firstly, we found that style length had a significant and positive effect on pollen
deposition. One explanation for this pattern is that flowers with longer styles have a
higher probability of being touched by pollinators. Also, longer styles may be produced
by relatively large flowers, which may be more attractive to potential pollinators.
Secondly, we observed a significant decrease in the number of pollen grains deposited on
the stigma from early to late in the season in one of the two taxa. This pattern may be
due to pollinator scarcity or plant population fragmentation (and poorer pollinator
service) later in the season. Both of these possibilities would account for lower pollen
loads observed later in the flowering season. In both of our focal taxa, the mean number
of pollen tubes from the stigma-style junction to the base of the style showed a significant
decrease from early to late in the season. Here we explained that pollen tube growth
through the style depends on both pollen and the host sporophyte. In addition, pollen
tube growth can be affected by environmental factors acting on the sporophyte.
Furthermore, our study discovered the importance of interference among pollen
grains or pollen tubes in the three measures of attrition. We interpreted this interference
to be caused by either a physical or chemical factor.

21

Style length had a significant and positive effect on pollen deposition. Several
studies have supported this finding, including Aguilar et al. (2008) who reported that as
style length increased in Solanum carolinense (Solanacease), pollen load and the number
of contacts made by the bumblebee, Bombus impatiens, increased. The authors argued
that long-styled flowers have higher pollen loads because they may be touched by the
bee’s body more frequently than short-styled flowers. This finding supports our
predictions that longer styles may be more accessible to pollinators or have a higher
probability of being touched. Aguilar et al. (2008) found that among 141 flowers, those
with longer styles (> 8mm) had a 35% greater chance of receiving pollen than short-
styled flowers. The authors also state that long-styled flowers serve primarily as pollen
recipients, while short-styled flowers serve as pollen donors. In our study, we did not
measure the functional relationship between style length and siring success, so further
investigation is required to detect this pattern in Clarkia.
Another study, which examined style length in the fig, Ficus maxima, also
observed a positive relationship between style length, pollen deposition, and stigma
length (Jousselin et al. 2004). In addition, they report that longer-styled flowers have a
much larger receptive surface than short-styled flowers, which increases the probability
of pollination.
Alternatively, we hypothesized that longer styles may receive higher pollen loads
because they may be produced by relatively larger flowers, which may be more attractive
to potential pollinators. According to Bai et al. (2011), larger corollas in the
hermaphroditic flowers of the gynodioecious species Glechoma longituba (Lamiaceae)

22

were more attractive to pollinators than hermaphroditic flowers with smaller corollas. In
fact, their study suggests that corolla size is the most important factor in attracting
pollinators because larger corollas provide larger landing platforms. Partial excisions of
the corolla, and consequent reduction of the landing platform, changed the relative
positions of anther, stigma and flower tube opening, resulting in reduced pollination (Bai
et al. 2011). Thus, flower size may play an important role in Clarkia pollinations.
Temporal variation in pollen load and in the number of pollen tubes at the stigma-style
junction and at the style base
Effects of the time of style harvest on pollen load: In Clarkia xantiana ssp. xantiana,
styles sampled early in the season received significantly more pollen than styles sampled
later in the season. This could be due to pollinator scarcity as the flowering season
progresses, which could have caused a decrease in pollen deposition (Mazer et al. 2010).
Internicola and Harder (2012) found a similar pattern in the orchid, Calypso bulbosa,
reporting that flowers produced early are longer-lived and received significantly more
pollinator visits than flowers produced later in the season. The authors suggest that
selection may favor early anthesis and long-lived lived flowers because it maximizes
opportunities for pollination and mating (Internicola and Harder 2011).
Since we sampled the flowers of both Clarkia taxa from multiple locations, it is
possible that as the flowering season transitioned from early to late within each taxon,
isolated patches that were fragmented from larger habitats received lower amounts of
pollen deposited on their stigmas (Cunningham 2000; Steffan-Dewenter and Tscharntke
1999; Colling et al. 2004). Cunningham observed that Acacia brachybotrya and

23

Eremophila glabra had lower levels of pollination in fragmented strips compared to their
nearby remnants when studied over two seasons. Similarly, Steffan-Dewenter and
Tscharntke (1999) found that the abundance and species richness of flowering-visiting
wild bees declined significantly with increasing distance from the nearest grassland. The
authors measured the abundance and richness of wild bees at distances 0-1600 meters
away from the main grassland.
Timing of style harvest on the number of pollen tubes at the stigma-style junction
(ssj) and base: For both taxa, the mean number of pollen tubes at the ssj and style base
decreased from early to late in the season. Erbar (2003) observed similar results in a
variety of angiosperm species, stating that pollen tube attrition was strongest within a
very short zone beneath the stigma. The number of pollen tubes was further reduced in
the middle portion of the style and only a fraction (on average 1.6 pollen tubes) of the
original (on average 18.6) pollen tubes that entered the style successfully made it to the
base.
There have been numerous studies confirming the occurrence of interference,
where an allelopathic chemical negatively affects other pollen donors (Jimenez et al.
1983; Marshall et al. 1996; Kanchan and Chandra 1980; Murphy and Aarssen 1995).
However, other studies support the idea that, following germination, further stages of
pollen tube growth depend on both pollen and the stylar tissue (Hulskamp et al 1995;
Erbar 2003). As a result, pollen tube growth throughout the style is not a passive process;
rather it is influenced by the genotype of the sporophyte. Further studies have concluded
that there is a progressive reduction in the width of the transmitting tissue from the
stigma to the ovary (Herrero and Hormaza 1996; Hormaza and Herrero 1996; Smith-

24

Huerta 1997; Modlibowska 1942). In this case, the number of pollen tubes that can enter
the stigma-style junction and style base depends on the physical space within the style.
Pollen tube germination and growth can also be affected by environmental
factors experienced by the sporophyte. In Clarkia, the nutrient status of pollen recipients
has the potential to influence the success of pollen grains as pollen tubes continue to
grow down the style (Smith Huerta et al. 2007). Specifically, further pollen tube growth
depends on the resources available in the style tissue. Logically, one would expect added
nutrients to enhance pollen tube growth because the excess amount could be used to the
pollen tube’s advantage. In this study, however, the authors did not observe this to be the
case. They found significantly increased germination and pollen tube growth in Clarkia
unguiculata Lindley (Onagraceae) in pollen recipients that received no added nutrients
relative to the treatment provided with extra nutrients. Water is an essential nutrient to
plants and does seem to have an impact on pollen tube growth. Many Californian
populations of Clarkia occupy habitats that experience seasonal drought (Mazer et al.
2010). Pollen tube growth in water-stressed plants may be slower as a result of this
(Marshall and Diggle 2001). As presented here, there are likely a variety of factors that
contribute to the decrease in pollen tubes from the stigma-style junction to the style base.
To the best of my knowledge, this study is the first to quantify and to compare the
attrition rate of pollen tubes at different stages of their growth through the stigma and
style, and at different times during the flowering season. In C. xantiana ssp. xantiana,
the timing of style harvest had no effect on any measure of attrition rate, even though the

25

number of pollen grains and tubes did differ between early and late sampling times (early
samples had higher numbers of pollen grains and pollen tubes than late samples). In
xantiana, it is possible that the mean number of pollen grains or tubes did not influence
the mean attrition rates on a given date. However, where timing of style harvest did have
a significant effect on the mean attrition rate in C. unguiculata, we predicted competitive
interference among pollen grains or tubes. Other studies have observed similar patterns
(Jimenez et al. 1983; Marshall et al. 1996; Kanchan and Chandra 1980; Murphy and
Aarssen 1995; Thomson 1989). Thomson (1989) argues that application of high pollen
loads can result in reduced pollen germination suggesting interference by physical means.
Germination occurred last in pollen grains located at the tops of clumps deposited
(Thomson 1989). In this study on Erythronium grandiflorum (Liliaceae), Thomson
(1989) also found that pollen grains located in clumps along the outer fringes of papillae
had germinated last. This supported our hypothesis that at high pollen loads, a proportion
of the pollen grains may be located in microenvironments where germination is unlikely,
resulting in higher attrition rates.
Other studies have examined the chemical nature of interference (Jimenez et al.
1983; Kanchan and Chandra 1980; Murphy and Aarssen 1995). Flavonols and
phytosulphokine-α have been identified as possible factors involved in density-dependent
pollen germination (Taylor and Hepler 1997; Chen et al. 2000). Murphy and Aarssen
(1995) isolated acidic, basic and neutral fractions from pollen as well as extract from
intact pollen from Phleum pratense (Aveneae: Poaceae) to test the allelopathic effect of
pollen on germination in vitro on a variety of other sympatric species of Poaceae. The
authors found that increasing extract concentrations from acidic fractions decreased

26

germination in the other sympatric species. Therefore, they concluded that
allelochemicals produced by pollen might be acidic.
The effects of pollen load and number of pollen tubes at the stigma-style junction on the
In addition to examining the effects of timing of style harvest on pollen attrition
rates, we independently compared the effects of pollen load and number of pollen tubes
at the stigma-style junction on the three different estimates of attrition rates. We
interpreted any significant positive effects of pollen load or pollen tube number (at the
ssj) on attrition rates as evidence of interference among pollen grains or tubes. As
previously stated, higher pollen loads and pollen tubes can result in reduced germination
of pollen grains either by physical or chemical means (Thomson 1989; Jimenez et al.
1983; Kanchan and Chandra 1980; Murphy and Aarssen 1995).
In spite of our prediction that high pollen deposition would result in greater
interference among male gametophytes, we observed a significant negative effect of
pollen load on the attrition rate from the stigma-style junction to the style base in C.
unguiculata, suggesting lower pollen tube failure rate at higher pollen loads. Some
studies have supported this finding, where rates of pollen germination can increase as
pollen density increases from very low to moderately high (Brink 1924; Schemske and
Fenster 1983; Cruzan 1986; Bjorkman 1995; Zhang et al. 2010). Therefore, where high
pollen loads result in higher germination rates, a higher proportion of pollen grains will
generate more pollen tubes at the ssj and style base. For example, Zhang et al. (2010),
found that treatments with the highest pollen density in the Japanese pear, Pyrus

27

pyrifolia, showed significant increases in germination rate and pollen tube growth, both
in vivo and in vitro. Alternatively, higher pollen loads could have increased selectivity
among gametes before and during fertilization by increased pollen competition or female
choice (Colling et al. 2004; Winsor et al. 2000; Kalla and Ashman 2002). If this is the
case, then lower attrition rates from the ssj to the base of the style could be due to the
higher quality of pollen tubes reaching the ssj.
The results presented in this study are only the first step toward determining
whether intrinsic and extrinsic factors may influence pollen loads and attrition rates in
natural populations of angiosperms. Further studies are required in other flowering
species to determine if similar results are observed and whether attrition rates are affected
by the same variables that we found in our study. Our study could have been more
effective had we categorized samples taken in 2009 as “early” or “late”. This would help
compare sources of variation for pollen load and attrition rates concerning timing of style
harvest between flowering seasons. Furthermore, we only collected one sample per plant
and did not know the maternal or paternal genotypes. Future studies should collect
multiple samples from each plant and determine the genotypes of the maternal
sporophyte and paternal gametophyte.
Acknowledgments
I would like to express my deepest gratitude to Dr. Susan J. Mazer for critical
reading of my thesis and for her mentorship. I am indebted to my fellow undergraduates
Brandon Wallace and Alexandra Bello, who have assisted me throughout the years and
peer-reviewed my drafts. Lastly, I would like to thank all of my colleagues who have
supported me throughout my two and a half years in the Mazer lab.

28

Literature Cited
Aguilar AQ, Kalisz S, Ashman TL 2008. Flower morphology and pollinator dynamics in
Solanum carolinense (Solanaceae): implications for the evolution of
andromonoecy. American Journal of Botany 95:974-984.
Aronen T, Nikkanen T, Harju A, Tiimonen H 2002 Pollen competition and seed-siring
success in Picea abies. Theor Appl Genet 104:638-642.
Bai YP, Zhang YW, Gituru RW, Zhoa JM, Li JD 2011. Sexual differences in
reproductive characters and pollinator attractiveness in gynodioecious Glechoma
longituba (Lamiaceae). Plant Species Biology 26:33-42.
Bjorkman T 1995. The effect of pollen load and pollen grain size competition on
fertilization success and progeny performance in Fagopyrum esculentum.
Euphytica 83:47-52.
Bowman RN 1987 Cryptic self-incompatibility and the breeding system of Clarkia
unguiculata (Onagraceae). Am J Bot 74:471-476.
Brink RA 1924. The physiology of pollen. IV. Chemotropism effects on growth of
grouping grains: formation and function of callose plugs; summary and
conclusions. American Journal of Botany 11:417-436.
Chen Y, Matsubayashi Y, Sakagami Y 2000. Peptide growth factor phytosulfokine-α
contributes to the pollen population effect. Planta 211:752-755.
Cheung AY 1995 Pollen-pistil interactions in compatible pollination. Proc. Natl. Acad.
Sci. 92:3077-3080.

29

Colling G, Reckinger C, Matthies D (2004). Effects of pollen quantity and quality on
reproduction and offspring vigor in the rare plant Scorzonera humilis
(Asteraceae). American Journal of Botany 91:1174-1782.
Cruzan MB 1986. Pollen-pollen and pollen-style interactions during pollen tube growth
in Erythronium grandiflorum (Liliaceae). Am J Bot 77:116-122.
Cruzan MB 1986. Pollen tube distributions in Nicotiana glauca: evidence for density
dependence growth. American Journal of Botany 73:902-907.
Cunningham SA 2000. Depressed pollination in habitat fragments causes low fruit set.
Proceeding of the Royal Society London 267:1149-1152.
Delesalle VA, Mazer SJ, Paz H 2008. Temporal variation in the pollen:ovule ratios of
Clarkia (Onagraceae) taxa with contrasting mating systems: field populations. J
Evol Biol 21:310-323.
Dudley LS, Mazer SJ, Galusky P 2007. The joint evolution of mating system, floral traits
and life history in Clarkia (Onagraceae): genetic constraints vs. independent
evolution. J Evol Biol 20:2200-2218.
Erbar C 2003 Pollen tube transmitting tissue: place of competition of male gametophytes.
Int J Plant Sci 164:265-277.
Franklin-Tong, VE 1999. Signaling and the Modulation of Pollen Tube Growth. The
Plant Cell 11:727-738.
Herrero M, Dickinson HG 1980 Pollen tube growth following compatible and
incompatible intraspecific pollinations in Petunia hybrida. Planta 148:217-221.
Herrero M, Hormaza JI 1996. Pistil strategies controlling pollen tube growth. Sexual
plant Reproduction 9:343-347.

30

Herscovitch JC, Martin ARH 1990 Pollen-pistil interactions in Grevillea-banksii. Grana
29:5-17.
Hickman, JC 1993. The Jepson manual: higher plants of California. University of
California Press, Berkeley and Los Angeles, CA.
Hormaza JI, Herrero M 1996. Dynamics of pollen tube growth under different
competition regimes. Sexual Plant Reproduction 9:153-160.
Hormaza JI, Herrero M 1994 Gametophytic competition and selection. In: Williams EG,
Clarke AE, Knox RB (eds) Genetic control of self-incompatibility and
reproductive development in flowering plants. Kluwer, Dordrecht, pp 372-400.
Huerta NLS 1997 Pollen tube attrition in Clarkia Tembloriensis (Onagraceae). Int. J.
Plant Sci. 158(5):519-524.
Hulskamp M, Schneitz K, Pruitt RE 1995. Genetic evidence for a long-range activity that
directs pollen tube guidance in Arabidopsis. Plant Cell 7:57-64.
Internicola AI, Harder LD 2012. Bumble-bee learning selects for both early and long
flowering in food-deceptive plants. Proceedings of The Royal Society 279:1538-
1543.
Jimenez JJ, Schultz K, Anaya AL, Hernandez J, Espejo O 1983. Allelopathic potential of
corn pollen. Journal of Chemical Ecology 9:1011-1025.
Jones KN 1994 Nonrandom mating in Clarkia gracilis (Onagraceae): a case of cryptic
self-incompatibility. Am J Bot 81:195-198.
Jousselin E, Kjellberg F, Herre EA 2004. Flower specialization in a passively pollinated
monoecious fig: a question of style and stigma? International Journal of Plant
Sciences 165:587-593.

31

Kalla SE, Ashman TL 2002. The effects of pollen competition on progeny vigor in
Fragaria virginiana (Rosaceae) depend on progeny growth environment.
International Journal of Plant Sciences 163:335-340.
Kanchan S, Chandra J 1980. Pollen allelopathy-a new phenomenon. New Phytologist
84:739-746.
Marshall DL, Diggle PK 2001. Mechanisms of differential pollen donor performance in
wild radish, Raphanus sativus (Brassicaceae). American Journal of Botany
88:242-257.
Marshall DL, Folsom MW, Hatfield C, Bennett T 1996 Does interference competition
among pollen grains occur in wild radish? Evolution 50:1842-1848.
Martin, FW 1959. Staining and observing pollen tubes in the style by means of
fluorescence. Stain Technology 34:125-128.
Mazer SJ, Dudley LS, Hove AA, Emms SK, Verhoeven AS 2010. Physiological
performance in Clarkia sister taxa with contrasting mating systems: do early-
flowering autogamous taxa avoid water stress relative to their pollinator-
dependent counterparts? Int. J. Plant Sci. 171:1029-1047.
Murphy SD, Aarssen LW 1995. Allelopathic pollen extract from Phleum pratense L.
(Poaceae) reduces germination, in vitro, of pollen of sympatric species.
Modlibowksa I 1942. Bimodality of crowded pollen tubes in Primula obconica. The
Journal of Heredity 33:187-190.

32

Németh BM, Huerta NLS 2002 Effects of pollen load composition and deposition pattern
on pollen performance in Clarkia unguiculata (Onagraceae). Int J Plant Sci
163(5):795-802.
Németh BM, Huerta NLS 2003 Pollen deposition, pollen tube growth, seed production,
and seedling performance in natural populations of Clarkia unguiculata
(Onagraceae). Int J Plant Sci 164(1):153-164.
Niesenbaum RA, Schueller SK 1997 Effects of pollen competitive environment on pollen
performance in Mirabilis jalapa (Nyctaginaceae). Sex Plant Reprod 10:101-106.
Ockendon DJ, Gates PJ 1975 Growth of cross- and self-pollen tubes in the styles of
Brassica oleracea. New Phytol 75:155-160.
Pimienta E, Polito VS, Kester DE 1983 Pollen-tube growth in cross-pollinated and self-
pollinated nonpareil almond. Journal of the American Society for Horticultural
Science 108:643-647.
Plitmann U 1993 Pollen tube attrition as related to breeding systems in Brassicaceae.
Plant Syst Evol 188:65-72.
Schemske DW, Fenster C 1983. Pollen grain interactions in a neotropical Costus: effects
of clump size and competitors. Pollen: Biology and implications for plant
breeding pg. 405-410.
Scribailo RW, Barrett CH 1991. Pollen-pistil interactions in tristylous Pontederia
sagittata (Pontederiaceae) II. Patterns of pollen tube growth. American Journal of
Botany 78:1662-1682.

33

Smith-Huerta NL, Carrino-Kyker SR, Huerta AJ 2007. The effects of maternal and
paternal nutrient status on pollen performance in the wildflower Clarkia
unguiculata (Onagraceae). Journal of the Torrey Botanical Society 134:451-457.
Smith-Huerta NL 1997. Pollen tube attrition in Clarkia tembloriensis (Onagraceae).
Snow AA, Spira TP 1991 Differential pollen tube growth rates and nonrandom
fertilization in Hibiscus moscheutos (Malvaceae). Am J Bot 78:1419-1426.
Steffan-Dewenter I, Tscharntke T 1999. Effects of habitat isolation on pollinator
communities and seed set. Oecologia 121:432-440.
Taylor LP, Hepler PK 1997. Pollen germination and tube growth. Annual Review of
Plant Physiology and Plant Molecular Biology 48:461-491.
Thomson JD 1989. Germination schedules of pollen grains: implications for pollen
selection. Evolution 43:220-223.
Winsor JA, Peretz S, Stephenson AG 2000. Pollen competition in a natural population of
Cucurbita foetidissima (Cucurbitaceae). American Journal of Botany 87:527-532.
Zhang C, Tateishi N, Tanabe K 2010. Pollen density on the stigma affects endogenous
gibberellin metabolism, seed and fruit set, and fruit quality in Pyrus pyrifolia.
Journal of Experimental Botany 61:4291-4302.

Arrash M Senior Honors Thesis

Recommandé

Recommandé

Contenu connexe

Tendances

Tendances (20)

En vedette

En vedette (12)

Similaire à Arrash M Senior Honors Thesis

Similaire à Arrash M Senior Honors Thesis (20)

Arrash M Senior Honors Thesis