Investigation of Wind Pollination in the Humboldt Bay

Wallflower (Erysimum menziesii ssp. eurekense) at the

Lanphere-Christensen Dunes

Joshua Der, Sarah Jordan, Veronica Vega

Senior Thesis in Biology
Humboldt State University, Arcata, CA 95521
May 2003

Final report submitted to Humboldt Bay National Wildlife Refuge.

Abstract
We examined wind pollination as a potential contributor to the
pollination success of the Humboldt Bay wallflower. We selected 15
plants each on dune ridges and in dune hollows to test the effects of
different wind regimes. Plants were emasculated and caged to exclude
insect pollinators and ensure that wind was the only mechanism available
for pollination. Stigmas were collected five days after emasculation and
pollen loads were counted. We found no significant differences in the
number of Erysimum or heterospecific pollen grains, or the ratio of each
on the stigmas between ridges and hollows. There may not have been
enough of a difference between treatments to affect pollen deposition. We
found more pollen in both treatments than can be attributed to
contamination from emasculation. This suggests that wind may play a
role in the pollination system of Erysimum.
 Wind Pollination in the Humboldt Bay Wallflower

Introduction
Plants use a variety of mechanisms to achieve pollination. These pollination
systems are variable, cryptic, and often poorly understood in most plants. Plants often
utilize more than one pollination system in order to ensure pollination success and
increase fecundity (Whitehead, 1968; Goodwillie 1999). Ambophily is a pollination
system that uses both insects and wind as vectors of pollen transfer (Culley et al., 2002).
Ambophily has evolved in several unrelated families of plants including the Asteraceae,
Brassicaceae, Ericaceae, Salixaceae and others (Culley et al., 2002 and references
therein). Recent research suggests that ambophily is a transitional phase between
exclusively animal and wind pollinated systems (Culley et al., 2002). Ambophily may be
selected for by low insect visitation, high winds, and semelparity (Goodwillie, 1999).
Wind pollination is generally considered to be a primitive pollination system, and
the diversification of angiosperms has been associated with the evolution of insects as
pollinators. However, morphological adaptations of flowers in some animal pollinated
plants may promote wind pollination. Flattened or reflexed corolla lobes and exerted or
elongating stigmas are adaptations which increase the likelihood of wind pollination
(Goodwillie, 1999). The contribution of wind pollination to reproductive fitness varies
widely between different plant groups. The proportion of the total pollen transferred by
wind varies from 8% to as much as 93% (Culley et al., 2002).
The Humboldt Bay wallflower (Erysimum menziesii ssp. eurekense, Brassicaceae)
is a small Endangered herb endemic to the windy coastal foredunes near Humboldt Bay,
California. This habitat faces increasing pressure from off road vehicle use and invasive
plants. Erysimum produces a small inflorescence as early as mid-February, when
pollinator abundance is low. The flowers are small and have many characteristics
commonly associated with bee-pollinated plants: showy yellow flowers, abundant pollen,
nectar rewards, and petals that provide a landing platform for bees. Erysimum also has
flattened petals and an elongated style exposing the stigma, increasing the opportunity for
wind pollination. Although insect visitation is limited, fruit and seed set are nearly
complete (M. Mesler and J. Sawyer, unpublished). These factors suggest that Erysimum
may utilize wind as a vector of pollination in addition to insect pollination.

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 Wind Pollination in the Humboldt Bay Wallflower

Understanding the reproductive biology of this Endangered plant, allows us to

develop effective management strategies to conserve the species and preserve the
integrity of its rare community. Since there has been little research on ambophily to date,
it is important to examine this as a potential mechanism of pollination in plants
traditionally thought to be exclusively insect pollinated.
The purpose of this study was to determine if Erysimum utilizes wind pollination
as part of its pollination system. We expected to find a higher rate of pollination in
exposed areas than in sheltered areas when we emasculated plants and excluded insect
pollinators.

Methods
Study Species and Site
Erysimum menziesii ssp. eurekense is a Federally and State Endangered species,
endemic to the Humboldt Bay area. It is found only on the Lanphere and the Samoa
dunes. It typically flowers between mid February and mid June, with peak flowering
between March and April. Insect and self-pollination have both been observed in
Erysimum. An average of 29 flowers are produced by an inflorescence over its lifetime
and these flowers open acropetally at a rate of 2.2 flowers per day (M. Mesler and J.
Sawyer, unpublished). Plants usually display inflorescences 10-25 cm tall, and larger
plants sometimes branch. Plants are monocarpic and inflorescences bolt from a basal
whorl of leaves after 1-2 years of vegetative growth.
Erysimum’s most abundant insect pollinator is the solitary bee, Habropoda
misirabilis (M. Mesler and J. Sawyer, unpublished). Other important pollinators include
Bombus melanopygus, B. mixtus, B. vosnesenskii and Lasioglossum pavanotum.
Occasionally ants and Elaterid beetles will visit the flowers.
The Lanphere Dunes Preserve is a unit of the Humboldt Bay National Wildlife
Refuge located west of Arcata in Northern California and is characterized by two general
habitat types: foredunes and dune forest. The foredunes are characterized by a series of
dune ridges and hollows. Erysimum occurs predominantly in exposed sandy areas on the
foredunes in the northern section of the preserve and appears to have a clumped
distribution (personal observation). The preserve is an important site that protects these

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 Wind Pollination in the Humboldt Bay Wallflower

habitats and their rare vegetation from encroachment by invasive plants. Invasive plants
have been actively and thoroughly eradicated from the preserve, providing a sharp
contrast to the neighboring dune habitat. The surrounding areas invaded by yellow bush
lupine and European beach grass no longer support Erysimum, but population
demographic studies within the Lanphere Dunes Preserve have shown the population to
be increasing (lambda=1.10) (E. Jules, personal communication).

Pollen Trapping
We collected airborne pollen using pollen traps. These were constructed from
glass microscope slides, coated with a thin layer of Vaseline®, and mounted at flower
level on wire supports (Kearns and Inouye, 1993). Four traps were placed in two
locations: at 1 m and 5 m downwind from a flowering Erysimum in hollows and on ridge
tops. We collected pollen twice on sunny days in late February, each for a 48-hour
period. Slides were visually scanned at 40X with a compound scope to detect the
presence of pollen.

Sample Size and Plant Selection

We selected 15 plants each from dune ridges and hollows. We chose these two
locations because they seemed to experience relatively different wind regimes: windy and
calm, respectively. Plants were selected based on four fundamental criteria: 1) plants
with 7-15 unopened flowers remaining, 2) unbranched inflorescences, 3) were 0.25-10 m
away from the nearest flowering Erysimum, and 4) were approximately the same size and
age.

Emasculation and Insect Exclosure

We emasculated the unopened flowers from each plant in order to eliminate
autonomous self-pollination. We also removed remaining pollen-bearing anthers to
avoid geitonogamy. Immediately after emasculation we caged each plant. We
constructed cages from 1/4 inch steel mesh hardware cloth and three stakes made from
1/4 inch welding rod. Cages were cylinders 0.3 m in diameter and 0.6 m tall. They were
large enough that plants could sway freely without contacting the sides. Stakes extended

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 Wind Pollination in the Humboldt Bay Wallflower

approximately 20-25 cm into the sand ensuring that cages would not be blown over when
exposed to high winds. The mesh was fine enough to exclude most common insects, but
would still allow wind to pass through the cage.

Study Period and Pollen Analysis

Plants were caged for five days beginning March 25-26, 2003, with ridge plants
emasculated on March 25th and hollow plants on March 26th. March 25th was rainy; each
subsequent day in the study period was clear with long periods of strong winds. At the
conclusion of the five-day exposure period, we removed stigmas from all flowers that had
been unopened at the start of the study period. We mounted stigmas on microscope
slides in fuchsin gel, and set them with a coverslip using a lighter to heat and melt the gel.
We visually compared pollen found on the collected stigmas with voucher Erysimum
pollen collected from anthers and mounted in fuchsin gel. We also collected voucher
pollen from three common plants flowering during this study (Fragaria, Salix, Solidago)
to attempt identification of heterospecific pollen found on stigmas. We counted the
number of Erysimum and heterospecific pollen grains on each stigma at 100X
magnification.

Emasculation Technique Calibration

In order to evaluate the level of potential contamination due to our emasculation
techniques we performed a series of control emasculations. Each member of the field
team emasculated a series of three flowers. Each person emasculated a young flower
bud, a ready to open flower, and a newly opened flower. After emasculation, we
immediately removed the stigmas and mounted them on a microscope slide in fuchsin
gel. We counted the number of pollen grains on these stigmas to evaluate the level of
contamination each person was responsible for during emasculation.

Data Analysis
We calculated mean numbers of pollen grains for each plant to make plants the
unit of observation. We compared the mean number of Erysimum and heterospecific
pollen grains per flower for each plant between ridges and hollows using a t-test. We

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 Wind Pollination in the Humboldt Bay Wallflower

also compared the mean proportions of Erysimum pollen found on stigmas from ridges
and hollows using a t-test. The level of contamination was compared to the number of
Erysimum pollen on stigmas in ridges and hollows with a t-test.

Results
Pollen traps collected very little pollen from any species. Pollen grains that were
caught on the microscope slides were not Erysimum pollen. In our emasculation
calibration procedure we found an average of 4.0 Erysimum pollen grains on each stigma.
This was significantly lower than the number of Erysimum pollen grains found on
treatment stigmas (p=0.01, df=35). The mean total number of pollen grains received on
stigmas per plant ranged from 3.57 to 196.67 among plants in both ridges and hollows.
Due to this wide range in pollen loads, the variance of our means for the total number of
pollen grains was high – 2388 and 747 for ridges and hollows, respectively. The mean
number of Erysimum, heterospecific, and total pollen grains as well as the proportion of
pollen grains was not different between ridges and hollows (Table 1, Figure 1).

Table 1: Mean number of pollen grains per stigma ± standard error. P-value from a two-
tailed t-test.
Erysimum Heterospecific Total Ratio Erysimum:Heterospecific
Ridge 13.9±4.3 38.8±8.6 52.7±12.6 0.33±0.04
Hollow 11.0±4.2 29.5±5.1 40.6±7.1 0.56±0.2
P-value 0.64 0.36 0.41 0.31

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 Wind Pollination in the Humboldt Bay Wallflower

Mean Number of Pollen Grains

70

60 Ridge
Hollow
Number of Grains

50

40

30

20

10

0
Erysimum Pollen Heterospecific Pollen Total Pollen
Pollen Type

Figure 1: Mean number of pollen grains found on stigmas on ridges and in hollows. Error
bars indicate standard error. Differences were not significant.

Discussion
The lack of pollen found on the pollen traps compared to the levels of pollen
found on stigmas suggests that the pollen traps were not efficient at catching airborne
pollen. Another possible explanation for the low pollen counts could be attributed to the
method used to examine pollen grains. This method was different than that used to count
pollen grains on stigmas. Because we didn’t stain the pollen grains on the traps, they
were difficult to see, and since we were only able to look at the slides under 40X
magnification, these pollen counts may be conservative.
We chose to examine the pollen loads on the ridges and in the hollows because
we expected to find a difference in the wind regimes and subsequently a difference in the
contribution of wind to pollination among these two sites. As we spent time on the
dunes, however, we observed that during strong winds, the ridges appeared to funnel the
wind into the hollows. We did not directly measure wind speed and are unable to
quantify any difference in wind regimes between our two treatments. Because we found
no difference in the pollen loads between ridges and hollows, this may suggest that there
was no difference in our treatments.

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 Wind Pollination in the Humboldt Bay Wallflower

We emasculated all of the plants on the ridge on March 25th when it was raining
and windy. These plants were more prone to damage during emasculation due to these
harsh conditions. This damage may have impacted the level of receptivity of the stigmas
and resulted in an underestimation of the number of pollen grains normally recieved by
wind. Plants in our hollow treatment group were emasculated on March 26 when
weather was clear and of moderate temperatures, thus they were not exposed to the same
conditions as the ridge plants had been immediately after emasculation. The weather was
constant for the remainder of our study period.
Lasioglossum is the only known visitor of Erysimum small enough to pass
through the mesh of our cages, but Lasioglossum is skittish and cages could potentially
discourage visitation by acting as a physical barrier around the plant. Lasioglossum was
commonly observed at the field site on Erysimum, but was never seen inside or near the
cages. We did see ants and Elaterid beetles on some of the plants. While these insects
could potentially move pollen from anthers to stigmas within a flower, or between
flowers on a single plant, it is unlikely that they could move pollen between plants. The
integument of these insects is smooth and pollen does not seem to stick to it well.
We collected voucher specimens of pollen to aid in identifying the pollen found
on stigmas. Voucher pollen was made from fresh anthers and mounted in fuchsin gel
immediately after collection. Stigmas remained exposed for up to five days and pollen
on the stigmas may have become dehydrated and changed shape while they were
exposed. As a result, we may have counted some of the Erysimum pollen erroneously as
heterospecific pollen because it didn’t match our Erysimum voucher pollen. While some
of the heterospecific pollen could be positively identified, the majority of the
heterospecific pollen was not similar to any pollen we looked at, and may have been from
Erysimum. In order to positively identify it, we would need to examine the texture and
arrangement of pores and furrows on the microspore wall at high magnification and
compare this with a comprehensive collection from all plants flowering at the Lanphere
preserve during our study.
Improvements to this study may be able to demonstrate more definitively the role
of wind in the pollination of the Humboldt Bay wallflower. The design of the pollen
traps should be improved to effectively document the presence of airborne pollen. Wind

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 Wind Pollination in the Humboldt Bay Wallflower

speed should be measured to ascertain that there is a real difference between ridges and
hollows. If there is no difference in the wind regimes we need to devise a treatment that
controls for differing levels of wind. Pollen identification techniques need to be
improved to increase the accuracy of pollen counts. Though ants and beetles were not
expected to act as pollinators in this experiment, a better cage could exclude them
completely. In order to determine the effectiveness of our cages in excluding
Lasioglossum, we could conduct more extensive observations of these bees interacting
with the cages.
The level of contamination in plants due to error in the emasculation technique
was not great enough to explain the high pollen loads observed on the stigmas. The
plants were caged immediately after emasculation to prevent autogamy, ensuring wind as
the only pollen vector (Gomez and Zamora, 1996). Despite the shortcomings of this
study, the fact that we found substantial amounts of Erysimum pollen on our stigmas is a
significant finding which cannot be explained by contamination. In addition, our
Erysimum pollen counts may be conservative. These points demonstrate that Erysimum
can collect airborne pollen and suggests that wind may indeed play a substantial role in
the pollination of Erysimum.

Acknowledgements
We would like to acknowledge Dr. Michael Mesler for his guidance, funding, and
support throughout this project. Andrea Pickart provided site maps and use permits to the
Lanphere Christensen Unit of the Humboldt Bay National Wildlife Refuge. Anthony
Baker supplied microscope coverslips, engineered a specialized microscope slide storage
box, and provided other equipment services. Kristal Watrous, Molly Alles, Justin
Garwood, James Sclafani, Kim McFarland, and Andrew Jordan helped carry cages and
provided moral support.

Literature Cited
Culley, T. M; S. G. Weller; A. K. Sakai. 2002. The Evolution of Wind Pollination in
Angiosperms. TRENDS in Ecology & Evolution. 17(8): 361-369.

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 Wind Pollination in the Humboldt Bay Wallflower

Gomez, J. M. and R. Zamora. 1996. Wind Pollination in High-Mountain Populations of

Horathophylla spinosa (Cruciferae). American Journal of Botany. 83(5):580-585.
Goodwillie, C. 1999. Wind Pollination and Reproductive Assurance in Linanthus
parviflorus (Polemoniaceae), A Self-Incompatible Annual. American Journal of
Botany. 86(7): 948-954.
Kearns, C. A., and D. W. Inouye. 1993. Techniques for Pollination Biologists. University
Press of Colorado, Niwot, CO.
Whitehead, D. R. 1968. Wind Pollination in the Angiosperms: Evolutionary and
Environmental Considerations. Evolution 23: 28-35.

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