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 effects of style length on pollen load

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).

Temporal differences in pollen tube attrition

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

three measures of attrition rate

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

Att

riti

on R

ate

-ss

j to

styl

e bas

e

0 100 200 300# callose plugs at stigma-

style junction (ssj)

C. unguiculata

00.10.20.30.40.50.60.70.80.9

1

Att

riti

on R

ate

-ss

j to

styl

e ba

se

0 100 200 300 400# callose plugs at stigma-

style junction (ssj)

C. xantiana ssp. xantiana

00.10.20.30.40.50.60.70.80.9

1

Att

riti

on R

ate

- (p

olle

ndep

osi

ted t

o s

sj)

0 100 300 500 700 900 1100# pollen grains on stigma

00.10.20.30.40.50.60.70.80.9

1

Att

riti

on R

ate

- (p

olle

ndep

osi

ted t

o s

sj)

0 100 300 500 700 900# pollen grains on stigma

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Att

riti

on R

ate

- polle

ndep

osi

ted t

o s

tyle

bas

e

0 100 300 500 700 900 1100# pollen grains on stigma

-0.2

0

0.2

0.4

0.6

0.8

1

Att

riti

on R

ate

- polle

ndep

osi

ted t

o s

tyle

bas

e

0 100 300 500 700 900# pollen grains on stigma

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

Att

riti

on R

ate

-ss

j to

sty

le b

ase

-100 100 300 500 700 900# pollen grains on stigma

00.10.20.30.40.50.60.70.80.9

1

Att

riti

on R

ate

-ss

j to

sty

le b

ase

0 100 300 500 700 900# pollen grains on stigma

*

*

*

*

*

*

*

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  

The effects of style length on pollen load

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.

Temporal differences in pollen tube attrition

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

three measures of attrition rate

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.

International Journal of Plant Sciences 156:425-434.

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).

International Journal of Plant Sciences 158:519-524.

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.