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thesis entitled

RECONSTRUCTING THE OVULIFEROUS SCALE IN PICEA PUNGENS TO

EXAMINE THE MECHANISMS SURROUNDING POLLINATION
DROP SECRETION.

presented by

GEOFFREY LLOYD WILLIAMS

has been accepted towards fulfillment
of the requirements for

MASTER QE SCIENCE degree in BOTANY AND PLANT PATHOLOGY

 

Major professor

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6/01 cJCIRCIDateDuepGS-p. 15

Reconstructing the ovuliferous scale in Picea pungens to
examine the mechanisms surrounding pollination drop
secretion.

BY

Geoffrey Lloyd Williams

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Botany and Plant Pathology

2000

ABSTRACT

Reconstructing the ovuliferous scale in Picea pungens to
examine the mechanisms surrounding pollination drop
secretion.

BY

Geoffrey Lloyd Williams

The pollination drop is the primary mechanism in Picea
pungens and other Picea species by which the pollen is
brought into the micropyle. Three-dimensional
reconstruction using Laser Scanning Confocal Microscope was
used to elucidate the proximity and involvement of
ovuliferous scale vasculature in the pollination drop
emergence and disappearance in Picea pungens. The
development of the xylem trace from the main ovuliferous
scale vasculature to the proximity of the base of the ovule
is consistent with pollination drop emergence, in that the
pollination drop does not emerge until the trace is
complete. The drop disappears as the trace breaks from
ovuliferous scale cellular expansion. These data support
the supposition that the pollination drop emerges once the
xylem vascular trace completes its differentiation from the
cone axis to the base of the ovule and integument, and
ceases as the scale cells expand, severing the mature xylem

elements supporting the pollination drop.

Copyright by
Geoffrey Lloyd Williams
2000

This is dedicated to all those who provided support and
patience through the process, with a special dedication to
my wife, Erin.

ACKNOWLEDGMENTS

I would like to begin by acknowledging the person who
provided the support to pursue this project, my advisor,
Karen Klomparens. I owe her more than can be expressed
here, mostly for giving me a chance and an opportunity. The
Forestry Department at MSU deserves special recognition,
specifically Paul Bloese, Greg Kowalewski, and Karen
Bushouse for providing support with information and
materials without which the collection of samples would not
have been possible. John W. Heckman Jr., thank you for
providing the initial guidance along this project and for
all the help sectioning and in setting up collection.

Joanne Whallon and the LSM lab here at MSU deserves thanks
for providing support and instrumentation that made the bulk
of this unique. I would like to acknowledge my Masters
committee; Karen Klomparens, Frank Telewski, and Ray
Hammerschmidt. For the rest of you. . . well you all know
who you are, thank you. There are so many that need to be
acknowledged, my students, fellow TAs, co-workers, that I am
afraid that if I begin a list someone would be left off so a

big general thank you.

TABLE OF CONTENTS

LIST OF FIGURES ....................................... VII
LIST OF TABLES ........................................ VIII
INTRODUCTION ............................................. 1
MATERIALS AND METHODS .................................. 12
RESULTS .................................................. 23
DISCUSSION AND CONCLUSIONS ............................... 35
BIBLIOGRAPHY ............................................. 43
APPENDIX A ............................................... 47
APPENDIX B ............................................... 57

vi

LIST OF FIGURES

FIGURE 1. NATURAL RANGE OF PICEA PUNGENS IN THE UNITED STATES. ....... 13
FIGURE 2. COMPARTMENT MAP OF W. K. KELLOGG FOREST. . . . . . . . . . . . . . . . 15
FIGURE 3. SCHEMATIC DIAGRAM OF A PICEA OVULIFEROUS SCALE. ........... 21

FIGURE 4 . OVULIFEROUS SCALES AT IDENTICAL STAGES OF DEVELOPMENT SECTIONED
AT 10 pm AND EXAMINED USING SEM AND LSCM. 24

FIGURE 5. SINGLE LSCM CONFOCAL, FLUORESCENT, DIGITAL IMAGES REPRESENTING
THE FIRST AND LAST IMAGES IN EACH OF THE THREE SERIAL SECTIONED
OVULIFEROUS SCALES ........................................ 26

FIGURE 6. VOLUME REPRESENTED AS A STEREO PAIR RENDERED FROM THE DIGITALLY
RECONSTRUCTED SERIAL SECTIONS IN FIGURE 5A AND B. .............. 30

FIGURE 7. VOLUME REPRESENTED AS A STEREO PAIR RENDERED FROM THE DIGITALLY
RECONSTRUCTED SERIAL SECTIONS IN FIGURE 5C AND D. . . . . . . . . . . . . . . 31

FIGURE 8 . VOLUME REPRESENTED As A STEREO PAIR RENDERED PROM THE DIGITALLY
RECONSTRUCTED SERIAL SECTIONS IN FIGURE 5E AND F. . . . . . . . . . . . . . . 32

FIGURE 9. STEREO PAIR REPRESENTATION OF A LSCM COLLECTED SERIES OF DIGITAL
IMAGES OFAGROUP OF XYLEM CELLS 34

vii

LIST OF TABLES

TABLE 1. OVULIFEROUS CONE DEVELOPMENTAL STAGE SUMMARY OF KEY FEATURES. . . 6

TABLE 2 . COLLECTION TIMES FOR THE 1997 FIELD SEASON . . . . . . . . . . . . . . . 17

viii

INTRODUCTION

One of the least understood aspects Of the pollination
mechanism in the Coniferae is the development and subsequent
disappearance of the pollination drop. The pollination
mechanism, as defined by Owens and Blake (1984) refers to
the structure of the ovule tip and the process by which
pollen is taken into the micropyle. The pollination drop is
the primary mechanism in Picea pungens and other Picea
Species by which the pollen is brought into the micropyle.
The drop consists of a small volume of fluid that exudes
from inside the micropyle and fills the pollen capture
chamber. The origin Of the fluid in the pollination drop,
and how it fills the micropylar canal, have not been fully
explained for the genus Picea, but may be related to the
vasculature in the ovuliferous scale in the female, or seed—

bearing, cone.

The last time coniferous species vasculature was
investigated in depth was at the turn of the last century.
Now 100 years later, new investigative tools allow a more
detailed examination of the development in the female
reproductive system of the conifers. This study focuses
Specifically on Picea pungens, as it has been least studied

of the Picea genus over the last 25 years.

AS new imaging techniques that increasingly involve
computers and modeling take the fore front, they allow us to
expand knowledge in the area of structure and function.
These same technologies can also be used to re-examine data
in previously published studies, to aid in instruction, and
to provide more developmental structure information Of the
general anatomy of well—known and economically important

families in the Plant Kingdom.

Research into the vasculature in coniferous cones can
be traced back to the last century. Van Tieghem's 1869
publication was the first to report on the development and
evolution of the female cone with particular emphasis on the
vasculature. In his paper, Van Tieghem did a comparative
anatomical study Of the female “flower” and the fruit of the
"Cycadées, des Coniferes et des Gnétacées," or as they are
referred to now as the Cycads, the Conifera, and the
Gnetophyta. Of particular interest in this study were his
figures and description of the Coniferes, and Picea nigra in
particular. The focus on the fibers and the tracheids in
relation to the female cone were very useful in organizing

this study.

Van Tieghem was focused on trying to comprehend whether
the scales on the cone were the result Of modified branches

or modified leaves. He described vascular bundles as

independent units. The upper bundles in the scale form an
arc with inverted orientation, (xylem directed outwards and
downwards) and the lower bundles with normal orientation
belong to the bract. He concluded that the arrangement of
the bundles in an arc showed that the ovuliferous scale is a
leaf and not a branch and the orientation of the arc showed
that the leaf is diametrically opposite the bract. So this
modified leaf belongs to an axilary branch of which it is
the first and only appendage, and it is this 'leaf' that
bears the ovules on its dorsal surface. The pollination
mechanisms might not have been completely understood for the
species examined. Van Tieghem’s work was the foundation for

continued studies by both Wordsell and Aase.

W. C. Wordsell (1900) wrote that understanding the
structure Of the female 'flower' in Coniferae "will occupy a
paramount place in the minds of the foremost botanists of
the day." This did not seem to be the case. Very little
published work examined the full range of the Coniferae.
Many papers examined various genera but, as mentioned
before, Picea was the least studied then as it is now, with

the main focus still on trees in the Abies genus.

Wordsell (1899, 1900) provides an interpretation of the
work of Van Tieghem after 30 years of research in the field

had elapsed. Wordsell compiled an extensive review in this

paper covering the various theories Of development of the
“female ‘flower’ in Coniferae” from 1682 to 1897. Much Of
the information presented and analyzed by Wordsell has been
forgotten or become accepted as dogma in the modern study of
botany yet pollination mechanisms were not the focus Of
study during that time. His 1900 publication is a
historical collection Of data and analyses that are of great
value to the modern study of conifers, providing perspective

as to how far the science has progressed.

Even Wordsell (1899) himself, in his publication
describing Observations on the vascular system of the
‘female flowers’ of Coniferae, did not describe any Picea
cones. He did, however, provide diagramatic evidence of
vascular support for the ovule in Sciadoptis verticillata.
His study was primarily focused on describing the anatomical
characters in general and using these characters to throw
“some light upon the phylogenetic relationship[s] of the

order as a whole” (Wordsell 1899).

Similarly, Hanna C. Aase (1915) published a review and
investigation into the vascular anatomy Of the coniferous
megasporophyll. As with Wordsell, Aase's paper did not
include an examination Of the vasculature of any Picea
species. Although Aase did not solidify the understanding

of Picea, her publication began to cement the wild variation

in interpretation of cone morphology into a more modern
understanding, that emphasized the importance Of the

vasculature.

Over a century after Van Tieghem, we still do not have
a complete examination of the vasculature Of Picea. There
are, however, publications that indirectly describe the
nature of the vasculature in the ovuliferous cone or
'flower,‘ most of which are from the last 25 years. Most of
these examine the reproductive cycle of various members of

the genus Picea.

The events surrounding reproduction and pollination
have been studied in Picea glauca (Owens and Molder, 1977;
Owens and Molder, 1979), P. stikensis (Owens and Blake,
1984; Owens and Molder, 1980), P. orientalis (Runions et al.
1999), P. engelmannii (Runions et al. 1995; Runions and
Owens, 1996; Owens and Simpson, 1987; Owens et al., 1987;
Harrison and Owens, 1983; Singh and Owens 1981) and in a

limited scope, P. pungens (Cram, 1984).

Picea glauca, P. stikensis, and P. engelmannii,
represent the more important economic trees in the southwest
of British Columbia, Canada, where the preeminent
laboratories for the study of conifer sexual reproduction,
that of Dr. John Owens, is located. The contribution to the

understanding Of spruce reproduction and cone development is

nearly complete from the papers listed above. These papers
are primarily a collection of descriptive articles detailing
almost all aspects of observable development. Owens and
Molder (1977), in their paper with an examination Of cone
differentiation and early development of the bud in Picea
glauca, concluded that one can accurately estimate or track
seed cone differentiation based on lateral Shoot elongation,
which proved to be an invaluable aid to track bud
development in Picea pungens for the research presented in

this thesis.

Owens et al. (1987) classified six stages to describe
the development of Picea engelmannii ovulate cones. These
stages were slightly modified after further investigation by

Runions et al. 1995 (Table 1). Stage one begins from the

 

Stage 4:

0Continued axis elongation
00v. scales reflexed

°even more receptive

Stage 1:
°After dormancy till
°Ovuliferous scales grow

 

 

Stage 2: Stage 5:

~Micropylar arms develop °Basal OV. scales begin to close
~Cones elongate -Pollination drops emerge

Stage 3:

Stage 6:
0Cones become pendant
0Bract basal tissue expands

00v. scales enlarged
0Micropylar arms elongate
-Receptive to pollen

 

 

 

 

Table 1. Ovuliferous cone developmental stage summary of
key features. Table adapted from Owens et al. 1987, and
Runions et al. 1995.

developmental point at which the bud breaks dormancy to the
point at which the bud stalk and axis curve upwards and
begin elongation. From the time the cone just partially
emerges from the bud scales to when the distal ovuliferous
scales expand is deemed stage two. Stage two cones are not
yet receptive to pollen, but the micropylar arms are almost
fully developed. The cone axis in stage three continues to
elongate, enlarging the spaces between the ovuliferous scale
wide enough for pollen to begin to enter and settle in the
hydrophilic region in the axis below the micropylar arms.
Pollen also beings to collect on the secretory droplets on
the micropylar arms in this stage, but the pollination drop
has not yet emerged. Stage 4 cones undergo further axis
elongation, widely separating and exposing more of the
ovuliferous scales. The ovuliferous scales are reflexed
with distinctly curled margins. The wind born pollen easily
collects on and around the micropylar arms. At the end of
this stage, pollination drops begin to emerge in the basal
scales of the cone and continue acropetally, immediately
prior to the ovuliferous scales bending upwards and closing.
The closing of the ovuliferous scale creates a closed
environment for the pollination drop to fill the space out
Side the micropylar arms to efficiently collect any pollen
that is in the space. Stage 5 is the time at which the

scale closure continues and the cone starts to bend down.

After the scales close they elongate and thicken, and become
tightly appressed. At this stage of development, the cone
is no longer receptive. In stage six all of the ovuliferous
scales are tightly appressed and begin to fill the air
space previously occupied by the pollination drop. This
last stage is when the cones begin to enlarge and become

completely pendant.

Picea pungens is most closely related to Picea
engelmannii (Wright, 1955), the species examined by Owens et
al., (1987) which prompted the most interesting questions
about the pollination drop. Specifically this statement
piqued my interest: “No vascular tissue extended into the
tip Of the nucellus. Only a weak vascular strand terminated
in nucellar tissue at the base [chalazal end] Of the ovule”
(Owens et al. 1987). The hypothesis introduced to explain
the pollination drop withdrawal was that an increased
surface area caused by the pollen in the micropyle
accelerated the evaporation of the drop (attributed
originally to Doyle, 1945). Later publications (Runions et
al., 1995; Runions and Owens 1996) explained this process
based on further Observations of interior spruce, the term
used for Picea engelmannii, Picea glauca and hybrids of the
two growing in mixed stands. These papers identified the
importance Of ambient and secreted moisture in the

pollination mechanism Of these species, but without

explaining how the pollination drop fluid appeared. Runions
and Owens (1996) concluded that the pollination mechanism in
certain environments may have provided the selection

pressure for the evolution of the pollination drop.

While the most significant accumulation of information
about pollination mechanisms exists for Picea engelmannii,
P. pungens has been largely unexplored. Jonathan Wright
(1955) was focused on the relationship and distribution Of
spruce in relation to species crossability. Wright found
that Picea pungens did not successfully cross with P.
gluaca, P. sitchensis, or P. orientalis, yet he published
morphological data that suggested that P. pungens was most
similar to P. engelmannii. Daubenmire (1972), examined the
relation of Picea pungens and P. engelmannii in the Rocky
Mountains, and theorized that a recent single mutation in P.
engelmannii produced a derivative (P. pungens) that was at
once incompatible and had differing environmental

requirements.

Mitton and Andalora (1981), used genetic and
morphological data to explore the relationship between these
two species in the Colorado Front Range, (the eastern edge
of the Central Rocky Mountains where the mountains rise out
of the Great Plains, also the eastern most distribution of

Picea pungens). They found that genetically there were no

hybrids between Picea pungens and P. engelmannii in an area
of suspected introgression, yet discriminant analysis of
morphological data did not resolve two groups from the data.
While these publications did not help with our understanding
of the specifics of the pollination mechanism in Picea
pungens, they provided a reasonable foundation to use P.
engelmannii as a model system to begin an investigation into

the pollination mechanism of P. pungens.

Research examining the pollination drop in other
coniferous species (Tomlinson et al., 1991, Tomlinson, 1992,
Takaso, 1990) has not fully elucidated the cellular
processes associated with the functions and activities Of
the pollination drop. Takaso (1990) gives a brief summary
Of the pollen capture mechanisms, but concludes that there
is no consensus on pollination drop exudation and
withdrawal. Tomlinson et al., (1991) introduced the concept
of “pollen scavenging”, describing the process of the
pollination drop extending over adjacent bract surface or
cone axis, passively collecting pollen that landed prior to
drop secretion. The drop, therefore, functionally provides
a pathway for the pollen to float into the micropyle,
extending the process of pollination in time and space.
Tomlinson’s studies provided a new aspect to examine in all
species with a pollination drop, but there was no mention of

mechanism for the timing Of the event.

10

In summary, previous work on pollination in Picea and
other gymnosperms has resulted in no clear understanding of
the mechanisms providing the relatively large amount Of
fluid needed for the pollination drop. Picea pungens is
ideal for examining this mechanism as cones are generally
larger than Picea engelmannii, the most studied Species, but
the ovuliferous scales are small enough to allow easy
cellular and tissue investigation using a variety of

microscopy techniques.

New microscope techniques such as Laser Scanning
Confocal Microscopy (LSCM) can, of course, give us more
information than was possible to Obtain in 1869, and more
than was obtainable even into the late 1980's. An
associated technique, three-dimensional (3-D)
reconstruction, is a powerful tool for examination of
morphology and developmental events. This thesis takes
advantage of 3—D reconstruction using LSCM to elucidate the
proximity and involvement of ovuliferous scale vasculature
in the pollination drop in Picea pungens. The objective was
to begin to clarify the pollination mechanisms in Picea
pungens specifically the events surrounding the development
and disappearance of the pollination drop. This was tested
by examining and reconstructing, digitally, in three
dimensions, a time series of ovuliferous scales from

developing cones.

11

Materials and Methods

Collection

Sample collection was done at W. K. Kellogg Forest,
Augusta, Michigan located in Ross TWP T18-R9W. It is an
ideal site for collecting Picea pungens samples, as the
trees are just becoming reproductive (30 y Old), which
simplified collection. The other advantage is the wide
genetic diversity and known geographic origin of the trees
in the plantation. The plantation is a collection Of half—
sib progeny tests from seed collected all through the

natural range (Fig. 1) Of P. pungens.

Eight seed sources were selected for consistent
reproductive set and uniform morphology. Trees were selected
for high cone yields in the late spring of 1997. One tree
from each MICHCOTIP (Michigan Cooperative Tree Improvement
Program) stock numbers 67318097 and 67318201, specifically
locations RR—20 in MSFG-P-1—7l (Michigan State Forest
Genetics Plantation Number One planted in 1971) and 8-94 in
MSFG-P-l-73, were selected for the primary study (Fig. 2).
The source of tree RR-20 was Open pollinated seed collected
by Ross E. Mosier (on Sept. 6, 1969) from a green Picea
pungens tree (height = 60' and D.B.H. = 14") located near

thinning cabins on Mushroom Gulch Road in Chaffee Country,

12

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1. Natural range of Picea pungens (gray shaded area)

in the United States. Map adapted from Daubenmire, 1972.

13

Figure 2. Compartment map of W. K. Kellogg Forest in
Augusta, Michigan identifying the specific location of the

trees used for cone collection in 1997, 8-94 and RR-20.

Courtesy Of the W. K. Kellogg Forest.

14

W. K. KELLOGG FOREST
u AUGUSTA. MICHIGAN

KALAMAIOO COUNTY
R058 TWP. - TIS ROW

 

RR _'20 LEGEND
-— COHPARTHENT LINES
I BUILD‘NGS "N FOOT PATHS 9-- TRAILS
SCALE — =——__—_—

4“ I!” me about

3/78

15

CO, Sec. NE 1/4 1, T14S, R77W, elevation 9400'. The source
of tree B-94 was open pollinated seed collected by L. R.
Rich, G. J. Gottfried and J. B. Ryan (on Sept. 24, 1969)
from a green Picea pungens tree (height = 55') located near
Buffalo crossing and Big Lake Road in Apache County, AZ,
Sec. 14, T5N, R28E, elevation 8520'. In both cases Pinus
ponderosa and Pseudotsuga menzesii occurred in the area but

Picea engelmannii did not.

Collection times were Optimized to examine the key
developmental stages surrounding the pollination drop
release. The first year of collection, 1996, was used as a
guide to understand P. pungens reproduction at the Kellogg
Forest site in comparison to the published literature on P.
engelmannii and other Picea species. Unfortunately the
weather Of 1996 resulted in a very irregular pattern of cone
development, and it wasn’t possible to collect a complete
sequence of development from a Single tree source (Appendix
A). However, the process resulted in material that was used
to optimize fixation, embedding and examining techniques.
The data collected in the first year were not included in

the results.

The second year of collection, 1997, was timed to
collect and sample ovuliferous cones in the necessary stages

for completion of this study. Ovuliferous cone samples were

16

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Collection
Date Sample # Tree # Field evaluation
5/20/97 A3 B94 early stage 1
5/20/97 A2* B94 stage 1
5/20/97 A1 B94 stage 1-2
5/22/97 Bl&2 B94 one cone, stage 2-3
5/25/97 C1&2* B94 one cone two vials, stage 4-5
5/28/97 D* B94 late stage 5, early stage 6
6/1/97 E B94 stage 6 and on
6/3/97 F B94
6/5/97 G B94
6/5/97 Z RR20
6/8/97 Y RR20 (could not access B94)
6/10/97 X RR20
6/10/97 H B94
6/12/97 I B94
6/12/97 W RR20 becoming pendant
6/17/97 V RR20 nearly II to ground
6/17/97 J B94 almost pointed down
6/26/97 K B94 Pendant and enlarging
6/26/97 U RR20 Pendant and enlarging
* = cones used for full reconstruction
Table 2. Collection times, sample allocations, location

source and field notes for the 1997 field season at Kellogg
Forest.

collected from the selected trees from the time they broke
dormancy until seed maturation began. Dates and times of
collection are noted here (Table 2), but the second field
year was another unusual year for weather patterns at
Kellogg Forest (Appendix A). The weather, while unusual was
not disruptive once the cones broke dormancy. The most
Significant factor affecting the onset of cone development
was the delay in degree day accumulation in 1997 (Appendix

A).

17

One to two ovuliferous cones were collected per tree
every two or three days. All the samples were examined,
however, one tree (8—94), with the most consistent cone
development and highest cone set, was used for full three-

dimensional reconstruction and analysis.
Fixation

The fixation protocol was the same for every sample
regardless of the microscope used for examination. Once the
cones were collected, they were immediately dissected in the
field into the primary fixation solution, which was based on
research on developing spruce meristems (Heckman, 1985).

The distal and basal ends of the cones were discarded to
keep collection times as developmentally Uniform as
possible. The primary fixative was 1% paraformaldehyde, 3%
glutaraldehyde in a 0.05 M cacodylate buffer. The fixative
temperature was held between 18 and 25°C. After immersion
in the solution a low vacuum (0.05 MPa) was applied for 20
min then vented and repeated five times in the field using a
hand-Operated piston vacuum pump, for a total fixation time
of 2 h. The samples were washed twice for 2 h each, in
0.05M cacodylate buffer, at room temperature and stored in
buffer overnight at 4°C. The samples were transported at
ambient temperature to the Center for Electron Optics, on

the campus of Michigan State University, East Lansing,

18

Michigan. Samples were then divided into subsets for
Scanning Electron Microscopy, and Laser Scanning Confocal

Microscopy.

Scanning Electron Microscopy

After fixation, whole or partially dissected
ovuliferous scales were dehydrated to 100% ethanol in a
graded series over the period of 5 days. They were then
critical point dried, and sputter coated with gold.
Alternatively images from 10 pm thick sections were Obtained
from material processed for the Laser Scanning Confocal
Microscope (see below). Series of Slides were selected and
placed in a xylene bath to remove the paraffin. The slides
were then air-dried and sputter coated with gold. Imaging
of all samples was done at 15 kV on a JEOL 64OOV. Images
were collected and stored digitally and processed with Adobe

Photoshop version 4.0 software.

Laser Scanning Confocal Microscopy

In preparation for LSCM, the ovuliferous scales were
dehydrated to 50% in a graded ethanol series and then into a
100% t-butyl alcohol at 35°C. They were then infiltrated
with paraplast over 7d before embedding. The samples from
each set were serially sectioned at 10 pm on a rotary

microtome. The scales were sectioned tangentially in

19

relation to the cone axis from the micropylar arms to end Of
the seed wing primordia. Sections were then mounted on
Fisher Superfrost Plus slides for staining and Viewing with
a Zeiss LSM 210. Sections were stained with 1% aqueous
Safranin O for 15 min and mounted under N0. 1 B coverslips
with Fluka DPX. Confocal, fluorescent images Of the serial
sections (from A to B in Fig. 3), were collected and hand
registered using a 5x Plan—NeOfluar lens (NA 0.15) with an
Optical section thickness about 2 microns thicker than the
serial sections. Images used in the detailed reconstruction
Of small groups Of individual xylem cells were collected
using a 40x Plan-Neofluar Oil (NA 1.30) lens. Once the
fluorescent images of the serial sections were collected,

they were treated as a series of optically sectioned images.
Computer Reconstruction and Image Processing

Images for 3-D reconstruction were all processed using
the same procedure on the Silicon Graphics Inc. (SGI)
workstation. Raw images were transferred from the Zeiss LSM
210 to an intermediate computer between the microscope and
the SGI for conversion to TIFF images. From there the
sequence was imported to the SGI and a “volume” was created

from this sequence in Vital Images Voxe1_View_E software.

20

 

 

 

 

 

 

 

Figure 3. Schematic diagram of a Picea ovuliferous scale

illustrating the starting (A), the axis end, and ending (B)

points, chalazal end, for the serial sections examined. (0)

ovule, (n) nucellus, (i) integument, (ma) micropylar arms,

(a) cone axis, (b) subtending bract, (os) ovuliferous scale.

21

The volume is a serial collection of two—dimensional
digital images that have been given a third dimension and
stacked on top of each other in order, so that pixels become
voxels (a voxel is a pixel that has volume and is equi-
dimensional). The volume aspect ratio was corrected and
fractional interpolations were added to correct for the
voxel size in the 'Z' dimension. Turning off voxels, in the
whole volume, with a value dissimilar to those of the xylem,
created the xylem trace movies and stereo pairs. Voxels
that had similar values but that obscured the rotational
View of the xylem trace and the fibers were individually
removed to enhance the images. No digital filters were used

to create the images.

The images appear as they were collected, except for
their combination into a 3-dimensional volume. Once the
volume was correctly sized, movies and images were generated
and were transferred to a Windows NT workstation where they
could be converted from SGI formats to Microsoft Video Clip
format (AVI) and Tagged Image File Format (TIFF) using Adobe

Photoshop 5.0 and After Effects 4.0 software.

22

Results

The SEM examination of the sections provided more
information than available with traditional or advanced
light microscopy techniques. Specially prepared slides for

SEM were used to examine the fibers and xylem in individual
10 um sections (Fig. 4). Sections from similar stages of

development showed fibers with identical patterns (Figs 4A
and 48). There was much more information in the confocal,
fluorescent image (Fig. 4B) than in the standard SEM image
(Fig. 4A) at this magnification. Additional SEM images
(Fig. 4C and 4D) were collected from the area shown at the
arrows in Figure 4A. At these higher magnifications, the
cell type was apparent, confirming that the lower
fluorescent bundles were xylem (X) and the upper fluorescent
bundles were fibers (F). Phlouroglucinol—HCL tests verified
the presence of lignin in the vessels and fiber tissues

(data not Shown).

The first and last serial section of each selected area
of the ovuliferous scale at three key developmental times,
represented the basis for the reconstruction (Fig. 5). The
ovuliferous scales from cones that were chosen for specific

stages of development from tree 8-94 were used (Fig. 5).

23

(1.5mm

 

Figure 4. Ovuliferous scales of Picea pungens at identical
stages of development sectioned at 10 um and examined using
SEM and LSCM. A. SEM digital image of a scale section. B.
LSCM confocal, fluorescent digital image of a scale. C. SEM
detail of the xylem at the }{ arrow in Fig. 4A. D. SEM
detail of the fibers at the F arrow in Fig. 4A. For all

images in this figure: (F) fiber, (X) xylem.

24

Figure 5. Single LSCM images representing the first and
last images 1h] each <xf the three serial-sectioned
ovuliferous scales. The double arrow identifies fiber
fluorescence and the single arrow identifies the xylem
fluorescence. A and B are from an ovuliferous scale in
developmental stage 3-4, C and D are from a late stage 5

scale, and E and F are from a stage 6 scale.

25

(l 5) mm

 

26

The samples were from three sequential collection dates and
were selected from the middle section of each cone. These
single, digitally collected, confocal, fluorescent images of
the ovuliferous scale, were combined to create the three
dimensional structure used to examine the xylem pathways in

the ovuliferous scale at the chalazal end of the ovule.

The vasculature on the right side of these images was
selected for examination for simplicity and clarity. The
movies (on the accompanying digital media Appendix B Fig.
5AB, Fig. 5CD, Fig. 5EF) are “slice parades”, showing the
series of serial sections that start at the axis end (A,
Fig. 3) and progress through to the chalazal end (B, Fig.
3). This series of images (soon to be a “volume”)
represents the entire area between A and B in Fig. 3, which
was about 250 microns thick, and consisted of 25 serial
mechanically-sectioned images (these vary depending on the

volume).

The highest fluorescent signal comes from the vasculature
(xylem) and the fibers. The fibers on top (double arrows in
Fig. 5) are elastic and provide the primary mechanical
support for the mature ovuliferous scale and the mechanism
for ovule opening and on the bottom is the xylem (single
arrows in Fig. 5). The ovuliferous scales showed a variety

of ovule shapes and orientation, even though they were

27

selected from a single tree. This variety, however, was not
detected in the vasculature inside the scale. The patterns
illustrated by these three series of images, or volumes, are

representative.

When compared to the stages of development presented by
Runions et al. (1995), the Picea pungens cones studied here
represented a similar progression. The first series of
images is from a late stage 3, early stage 4, cone (Fig. 5A
and SB). In stage 3-4 cones, the xylem was still developing
and had not yet completed differentiation to the base of the
ovule. The second set of images (Fig. 5C and 5D) is from a
larger cone in late stage 5, at the approximate time of
pollination drop secretion. Here the xylem had completed
the pathway for the fluid in the xylem to make the
pollination drop. Stage 6 is represented by the last series
of images (Fig. 5E and SF). This scale had started to

elongate and the seed was beginning to develop.

The next set of figures is constructed from the data
collected from the serial sections represented in Fig. 5E
and 5F. There are no data missing from the reconstruction.
All depictions are in the same orientation, left to right in
the image (Fig. 6) is the same orientation as A to B in Fig
3. Figures 6, 7, and 8 are stereo pair representations of

the vasculature at the chalazal end of the ovule on the same

28

side in each volume. The volumes were rendered from the
same perspective and the stereo nature was produced by using
projections of the volume with 15 degrees of separation.
These volumes were constructed by either digitally removing
the surrounding cells leaving the fibers (darker gray) and
the xylem (lighter gray and white) (Figs. 6 and 7) or by
digitally removing most of the fibers, allowing clearer

presentation of the xylem (Fig. 8).

In the first stereo pair image (Fig. 6), the xylem
shown had barely begun to differentiate 'up' to the base of
the integument and ovule through the middle of a distinct
group of intensely stained cells (for Fig. 6, 7, 8, and 9
full digital movies can be viewed on the accompanying
digital media Attached as Appendix B). The fibers had
branched just below the base of the ovule and the xylem
showed a Slight bend up through the fibers. The fibers
would eventually form a sheath around the single xylem
strand, which is shown in the next volume (Fig. 7, Appendix

B Fig. 7).

In the next stereo pair image (Fig. 7, Appendix B Fig.
7), the xylem shown had developed to the base of the ovule,
thereby completing the supply of water to the ovule and
providing vascular support for the production of the
pollination drop. These vascular traces appeared thin (i.e.

Fig. 9, Appendix B Fig. 9), because they were made up of a

29

 

Figure 6. A volume represented as a stereo pair rendered
from the digitally reconstructed serial sections from the
LSCM shown in Figure 5A and B, illustrating the path of the
xylem and fibers in an early pre—pollination drop
ovuliferous scale. (f) fiber, (x) xylem. The length of
this sample is approximately 200 um edge to edge of one of

the stereo pairs (A to B in Fig. 3)

3O

 

Figure 7. A volume represented as a stereo pair rendered
from the digitally reconstructed serial sections from the
LSCM in Figure 5C and D, illustrating the path of the xylem
and fibers during pollination drop secretion. The unlabeled
arrows identify the xylem trace as it passes through the
fiber sheath. (f) fiber, (x) xylenL The length. of this
sample is approximately 200 um edge to edge of one of the

stereo pairs.

31

 

Figure 8. A volume represented as a stereo pair rendered
from the digitally reconstructed serial sections from the
LSCM in Figure 5E and F, illustrating the path of the xylem
and fibers after pollination drop secretion. (f) fiber, (x)
xylem. The length of this sample is approximately 200 pm

edge to edge of one of the stereo pairs.

32

continuous one to two cell thick, connected line of xylem
elements. In all scales that were examined there was always
a xylem element paired with a fiber bundle that was
associated with the base of the ovule at the point at which

the fiber bundle branches.

The last stereo pair image (Fig. 8, Appendix B Fig. 8)
demonstrates an ovuliferous scale at developmental stage 6.
At this stage the scale had grown and expanded, disrupting
the single xylem cell trace connecting the cone vasculature
to the point of pollination drop exudation. The
developmental break in the xylem cells resulted in the

retraction of the pollination drop.

The vascular traces in each of the three volumes (Fig. 6,
7, and 8, Appendix B Fig. 6, 7, and 8) were structurally
identical with small, short xylem cells. A stereo pair
detail from a single section of the mature xylem cells from
stage 5 is representative of the xylem in the vascular
traces supporting the pollination drop. The vasculature
that provides primary support for the rest of the
ovuliferous scale was comprised of tracheids with more
regular and even, helical thickenings and two to three times

longer than those shown in Figure 9 (Appendix B Fig. 9).

33

 

Figure 9. Stereo pair representation of an LSCM—collected

series of digital images of a group of tracheids from a
single 10 um section of an ovuliferous scale in

developmental s tage 5 .

34

Discussion and Conclusions

There are six stages of cone development related to
receptivity of the ovules as proposed by Owens et al (1987).
Runions et al. (1995) suggested minor changes in this model
that affect stages 4, 5 and 6, specifically that the
pollination drop was released later in cone development and
that it persisted after the scales became appressed. These
six stages generally occur in Michigan's Lower Peninsula any
where between late April to the beginning of June, depending
on the weather. The research presented here focuses on the
development represented by stage 5 (Runions et al., 1995).
In stage 5, the basal ovuliferous scale begins to close. It

is at this point that the pollination drop begins to emerge.

Runions and Owens (1996) have described in detail the
pollination drop and pollen scavenging for interior Spruce
(Picea engelmannii, Picea glauca, or their hybrid). They
reported that the pollination drop did not emerge until just
before the ovuliferous scale closes. The closed scale
creates a chamber reducing evaporation allowing the drop to
get larger, collecting more pollen. When the ovuliferous
scale closes, the ends of the scale have drops of resin,
which help to seal the chamber. My field observations

provide additional support for Runions and Owens results as

35

Picea pungens pollen scavenging followed similar patterns as

interior spruce.

Data (the reconstructed images) presented here, clarify
the events relating to pollination drop release and
cessation in P. pungens. From this research, the serially
reconstructed images, in particular, provide support for the
concept that the cause of the pollination drop formation and
subsequent disappearance results from the various stages in
development of a particular xylem trace as related to the

growth and expansion of each ovuliferous scale.

In all cases, the development of the xylem trace is
consistent with pollination drop emergence, in that the
pollination drop does not emerge until the trace is complete
and it begins to disappear as soon as the trace breaks. The
actual observance of the pollination drop, or the presence
of pollen in the micropylar canal combined with the
degradation of the nucellar tip, was used to determine

pollination drop occurrence.

Once the pollination drop liquid moves to the end of
the vascular pathway, how does it move micropyle? One
pathway might be via the intercellular space separating the
nucellar tissue from the integument on all sides except for
the chalazal end of the nucellus. This space is

sufficiently large for the fluid to move through and

36

completely continuous and may provide the pathway for the
fluid. AS the pollination drop fluid fills the space,
nucellar tip degradation occurs which could account for the
elevated glucose and fructose content in the drop compared

to the xylem sap as observed by Owens et al., (1987).

The vasculature in the ovuliferous scale is Similar to
most modified leaf structures in that there are vascular
bundles with both phloem and xylem (tracheids). These
bundles also contain a group of fibers providing support as
well as the mechanism for opening the ovuliferous scales to
disperse the seeds at maturity. At seed maturity the cone
desiccates completely, resulting in the fibers dehydrating
and mechanically opening the scales. The tracheids are dead
at cellular maturity (tracheids have to be dead to be
functional), and once mature there is little flexibility
with the short cell length found in the ovuliferous scale.
The overall cell length is much shorter than in the axis
xylem or the fibers, and most importantly, the xylem matures

before the ovuliferous scale completes its development.

There are three types of xylem cells in the ovuliferous
cone and scale. The first type is in the cone axis. This
.xylem branches off to form the primary connection to the
(Jvuliferous scale. These xylem cells are extremely long and

fiave regular thickenings with few cell junctions. The

37

second type branches out of the cone axis and provides the
vascular support for the ovuliferous scale. These cells
retain many of the characteristics of the axis, i.e. longer
cells, and somewhat orderly thickenings of the cell wall.
The third type is xylem that develops and connects from the
main vasculature to the base of the ovule. These xylem
cells have slightly different characteristics, as these
xylem traces develop not from meristematic initial cells,
but rather are differentiated from parenchymous cells. These
xylem trace cells resemble, in all forms, cells grown in
tracheid inducing cultures in Zinnia (Williams 1994, Liskova

1985 and Fukuda 1997).

The sequence of the xylem trace development is a rapid
event in the overall timing of the development of an
ovuliferous scale, 2-4 days from the start of its
development to drop emergence. The timing of this
development correlates closely to the differentiation of
isolated parenchymous cells into tracheids in an in vitro
system (such as Zinnia elegans). The in Vitro
differentiation generally occurs in 24-36 hours, start to
finish, with the first complete cells visible as soon as 8
hours (Williams, 1994). An assumption could be made that in
Vivo systems are much more efficient and possibly faster at

differentiating than in vitro systems and thus it could be

38

surmised that the single cell trace can develop quite

rapidly.

The completion of the xylem pathway corresponds to the
most rapid phase of cell expansion in the ovuliferous scale
and this thin 1 to 2 cell branch of xylem supporting the
pollination drop is fragmented and disrupted by ovuliferous
scale cell expansion. It is important to remember that the
xylem cells are dead and rigid at maturity, and are unable
to keep pace with the cell expansion occurring in the rest
of the ovuliferous scale. It is at this developmental stage
that the pollination drop begins to disappear as the xylem
path is no longer intact. This ovuliferous scale cell
expansion does not break the other vascular pathways because
they are protected by the fibers. The primary direction of
expansion is in the Z direction, parallel to the cone axis,
hence the fibers reduce most of the expansion in the X—Y
direction, perpendicular to the cone axis. These fibers
provide more than just the mechanism for opening the scale
upon maturity, they protect the primary xylem and phloem

vasculature.

An comprehesion of where particular xylem originates is
important to understand the different morphological types of
xylem present in the cone. The vasculature in the cone axis

results from a uniform meristem, giving rise to an ordered

39

and specific orientation. As the cone develops the axial
vasculature branches out to support the primordial
ovuliferous scales. This vasculature is slightly more
irregular in shape than the xylem in the cone axis, but it
still resembles xylem developing from a meristematic
initial. The tiny vascular traces are distinct from the
first two types of xylem as discussed above. From the
results presented here and observations made on the tissue
during examinations this difference appears to be due to the

manner in which the xylem develops.

There may be many explanations for the origin of the
vascular trace. Following Runions and Owens (1996)
statement that "the conifer pollination drop may not be
ancestral but derived from a pollination mechanism in which
rain water was required for pollen delivery," it is possible
to theorize that the xylem trace developed to support a
pollination drop as a mechanism to supplement rainwater and

then worked so well that it became a distinct advantage.

Another possible explanation is that the xylem trace
differentiates to the base of the ovule along a signal
gradient. In cell culture an elevated auxin level in a cell
suspension triggers formation of tracheids (Williams 1994,
Liskova 1985 and Fukuda 1997). It is possible that the

ovule is producing elevated levels of a specific hormone or

40

combinations that may be carried through the distinct cell
types at the chalazal end of the ovule, signaling the
nearest vascular bundle to grow towards the ovule. As the
xylem pathway, or trace, forms, it creates the path for the
pollination drop. This signal may be involved in the
selection of the pollination drop mechanism in coniferous

species.

The final potential explanation is that the pollination
drop existed prior to the fusing of the ovules into a cone
(ancestral), and that it has been there all along in the
evolution of Picea from an ancestral form. Those coniferous
genera without a pollination drop may have lost it due to an
environmental factor such as a migration or ecological
change to a wet climate. The pollination drop may prove to
be an additional tool in the understanding of the evolution

of the Conifers.

Van Tieghem's figures (1869) of selected schematic line
drawings, in a general way support the evidence presented in
this thesis. His drawings of sections through the
ovuliferous scales of Picea nigra are of particular
importance despite the fact that the are primarily fully
mature cones (very late stage 6). His observations of the

orientation of the arc bundles, the vascular support for the

41

ovule was documented but not directly correlated with the

pollination drop.

The research presented here supports the supposition
that the pollination drop emerges once the xylem vascular
trace completes its differentiation from the cone axis to
the base of the ovule and integument. The pollination drop
ceases as the scale continues its development and growth,
thereby severing the mature xylem elements supporting the

supply of fluid into the pollination drop.

Further experiments need to be conducted to further
elucidate the pollination drop mechanism and to provide
correlative data for the anatomical and structural data
presented here. These might include using tracers that can
be fixed in place inside the xylem tracheids and pollination
drop to conclusively link the differentiation of the xylem
with the emergence of the pollination drop. The difficulty
is that unlike the wide open stem/cone xylem the tracheid
trace is individual cells linked together, any tracer
particle size must be small. Ideally an ultra-high
resolution MRI and a detectable tracer would be perfect
allow the imaging of the xylem and contents on a whole cone
basis as there are no other traditional techniques available

to follow the dynamics of the whole system in vivo.

42

BIBLIOGRAPHY

Bibliography

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DAUBENMIRE, R. 1972. On the relation between Picea pungens
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46

 

Appendix.A

Weather Data from Kellogg Forest

The data presented in this section was collect as
standard procedure by the staff at The W. K. Kellogg Forest
in Augusta, Michigan over the years the forestry station has
been in operation. Some of the incomplete years have been
removed for simplicity, specifically 1941 through 1946 and
the early data from 2000. The information was compiled and
provided by Karen Bushouse. The years material was
collected for this research have been shaded in following
tables listed below. The degree data was only collected
starting in 1992 but it provides an explanation for the

difference in bud break dates between 1996 and 1997.

LIST OF TABLES

TABLE 1. SUMMARY OF MONTHLY MEAN MAX. , MEAN MIN. , HIGH, AND Low,

TEMPERATURES FOR THE YEARS 1947-1999 (IN 4 PARTS). ........... 57
TABLE 2. DEGREE DAY DATA FOR THE YEARS 1992-1999. ............... 61
TABLE 3. RAIN PRECIPITATION BY MONTH FOR THE YEARS 1947-1999. ...... 62

TABLE 4 . SNOWFALL AMOUNTS FOR THE WINTER MONTHS OCTOBER THROUGH MAY 194 6—
47 TO 1998—99. ........................................ 63

TABLE 5. YEARLY MEANS AND TOTALS FOR THE YEARS 1946-1999. ......... 64

48

Table 1. Summary of Monthly Temperatures (part 1 of 4).
JAN. JAN JAN JAN FEB FEB FEB FEB MAR MAR MAR MAR

Max. Min. Max. Min. Max. Min.
7

 

49

Table 1. Summary of Monthly Temperatures (part 2 of 4).

APRIL APRIL APRIL APRIL MAY MAY MAY MAY JUNE JUNE JUNE JUNE

Aux Mm. hum Mm. NMx Mm.

56.6 36.4 76.9 21.5 69.5 45.8 85.6 31.5 79.3 56.1 93.4
.7 17 4 7

 

50

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

51

 

 

 

 

 

 

Table 1. Summary of Monthly Temperatures (part 3 of 4).
JULY JULY JULY JULY AUG AUG AUG AUG SEPT SEPT SEPT SEPT
Mean Mean High Low Mean Mean High Low Mean Mean High Low
Max. Min. Max. Min. Max. Min.
1947 79.8 52.2 93.0 35.0 89.2 58.8 >99 40.0 74.3 49.0 88.0 25.0
1948 82.9 55.0 89.0 41.0 82.9 49.5 96.0 37.0 76.4 45.8 90.0 31.0
1949 85.7 59.5 96.0 43.0 82.8 53.4 94.0 37.0 67.5 41.2 82.0 27.0
1950 79.8 49.1 88.0 35.0 78.8 49.9 87.0 31.0 71.0 46.9 83.0 25.0
1951 80.8 52.8 88.0 38.0 77.9 51.4 90.0 35.0 70.5 44.3 85.0 24.0
1952 84.1 55.6 94.0 40.0 81.2 51.5 92.0 33.0 75.0 43.9 93.0 31.0
1953 86.4 54.3 95.0 37.0 84.9 52.2 97.0 38.0 77.4 42.5 99.0 28.0
1954 82.3 52.5 93.0 39.0 79.0 51.5 93.0 38.0 74.2 48.2 95.0 29.0
1955 88.5 58.7 97.0 46.0 86.3 57.1 97.0 44.0 77.1 44.1 91.0 29.0
1956 80.1 54.8 91.0 41.0 81.4 53.2 94.0 35.0 75.0 42.6 90.0 25.0
1957 84.1 54.9 92.0 44.0 82.3 54.8 90.0 44.0 72.7 45.9 89.0 28.0
1958 80.6 56.5 87.0 42.0 82.2 53.9 90.0 40.0 73.2 47.1 84.0 31.0
1959 84.2 55.0 91.0 42.0 86.2 61.8 93.0 44.0 78.6 51.3 93.0 28.0
1960 82.0 53.7 89.0 43.0 81.7 55.5 89.0 46.0 78.2 52.5 95.0 31.0
1961 83.6 55.5 90.0 43.0 80.8 56.2 89.0 41.0 77.9 54.6 89.0 29.0
1962 80.0 54.8 91.0 43.0 83.2 52.9 91.0 40.0 75.5 45.2 86.0 28.0
1963 84.8 53.2 95.0 37.0 79.0 52.0 87.0 38.0 75.0 44.9 85.0 32.0
1964 84.8 58.2 93.0 40.0 79.3 51.9 99.0 35.0 73.5 48.1 89.0 29.0
1965 81.6 52.5 93.0 40.0 78.5 54.7 95.0 36.0 73.5 51.9 86.0 28.0
1966 85.8 56.4 94.0 42.0 79.5 52.9 87.0 41.0 71.5 45.9 86.0 29.0
1967 79.4 54.5 87.0 43.0 78.3 51.5 87.0 39.0 73.3 44.4 82.0 33.0
1968 82.0 55.7 91.0 42.0 81.6 57.6 93.0 40.0 73.6 50.0 83.0 34.0
1969 81.8 58.4 90.0 46.0 82.5 54.8 88.0 42.0 73.0 49.9 85.0 33.0
1970 81.9 58.5 92.0 42.0 81.2 55.2 89.0 43.0 72.8 50.8 86.0 29.0
1971 78.3 56.3 85.0 45.0 78.8 53.5 88.0 41.0 73.1 55.3 87.0 36.0
1972 79.6 57.7 90.0 41.0 77.4 58.1 86.0 42.0 70.2 52.9 81.0 36.0
1973 81.3 59.4 91.0 49.0 80.2 59.4 91.0 45.0 73.2 52.2 88.0 36.0
1974 83.0 58.1 93.0 46.0 79.5 56.6 89.0 45.0 70.2 46.3 83.0 33.0
1975 80.3 57.3 90.0 45.0 79.1 59.5 90.0 46.0 65.7 47.2 77.0 33.0
1976 81.5 58.2 92.0 44.0 79.2 52.8 87.0 38.0 71.5 46.6 86.0 29.0
1977 83.8 59.9 92.0 45.0 76.9 57.1 87.0 40.0 70.0 54.6 84.0 41.0
1978 79.2 57.5 87.0 43.0 78.6 57.2 86.0 43.0 75.3 52.9 89.0 34.0
1979 73.8 55.3 85.0 44.0 75.1 56.9 88.0 40.0 73.6 49.0 81.0 34.0
1980 80.6 60.1 91.0 43.0 78.7 61.2 85.0 51.0 72.3 52.3 80.0 35.0
1981 80.3 59.5 88.0 47.0 79.4 57.0 84.0 41.0 68.8 49.9 82.0 31.0
1982 81.9 59.9 89.0 44.0 78.2 55.3 86.0 37.0 68.0 51.1 85.0 36.0
1983 86.1 61.6 96.0 40.0 83.9 60.1 92.0 49.0 73.1 50.0 89.0 31.0
1984 82.9 56.5 93.0 43.0 81.5 55.8 90.0 43.0 70.5 50.0 85.0 29.0
1985 80.3 58.0 92.0 43.0 75.0 54.2 84.0 46.0 70.0 52.9 88.0 37.0
1986 81.1 59.6 92.0 46.0 73.9 52.9 82.0 38.0 67.0 53.0 78.0 36.0
1987 83.1 60.7 93.0 47.0 76.5 58.0 89.0 59.0 67.9 49.8 79.0 36.0
1988 85.8 58.4 98.0 42.0 80.4 57.7 96.0 42.0 67.9 45.8 78.0 38.0
1989 80.5 59.0 91.0 48.0 76.9 54.4 87.0 42.0 65.6 42.5 79.0 29.0
1990 80.8 60.7 97.0 50.0 78.7 60.2 91.0 48.0 72.4 53.1 88.0 38.0
1991 82.8 64.1 93.0 51.0 78.4 60.0 89.0 53.0 68.2 50.9 84.0 32.0
1992 72.7 56.6 86.0 48.0 71.7 53.7 87.0 44.0 66.6 49.5 78.0 35.0
1993 78.6 62.1 90.0 51.0 75.8 60.2 86.0 45.0 56.5 43.9 74.0 30.0
1994 75.1 57.7 84.0 48.0 78.1 58.9 92.0 46.0 72.3 55.1 87.0 39.0
1995 87.3 62.9 100.0 45.0 87.3 66.7 95.0 54.0 693 47.0 85.0 35.0
t ‘ 1566 813' 55.1 * SETS {2'58 “8'33 RE 95.8" “50:53 - 70:9 " “5119“ 84.1 '3 .1
1......1997 83.1 58.7 95.7. 45.3 75.9 56.2 86.1 43.1 . . 7.0.4 .49-7 81-0 37.4
1998 84.9 59.5 93.0 51.1 84.6 60.6 94.9 47.0 80.6 52.0 92.9 35.7
1999 92.6 64.1 103.8 49.8 86.7 57.5 99.2 47.1 88.1 49.3 105.3 35.6
:33? 81.8 60.0 93.3 48.2 79.8 58.8 91.2 47.2 71.0 49.5 85.3 34.8
avg 82.1 57.3 91.7 43.6 80.2 55.9 90.2 42.3 72.3 48.8 85.9 32.1

 

Table 1. Summary of Monthly Temperatures (part 4 of 4).

OCT OCT OCT OCT NOM NOM NON’ NOV DEC
wa1 hmmw
flux NML hmx Mm.

DEC DEC DEC

NMx Mm.

 

52

Table 2.

Degree Day Data for the years 1992-1999.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Jan Feb March April May June July August Sept Oct Nov Dec :52:
1992 0.00 0.00 4.00 50.10 214.50 370.00 450.50 395.00 266.00 50.00 4.00 0.00 1804.10
1993 0.00 0.00 0.00 37.50 244.00 366.00 632.00 559.50 106.00 20.50 3.00 0.00 1968.50
1994 0.00 0.00 0.00 55.00 121.00 407.00 510.50 575.00 413.00 204.50 64.50 7.00 2357.50
1995 0.00 0.00 40.00 78.50 298.00 641.50 778.50 837.50 268.50 137.50 2.00 0.00 3082.00
L1997 0.00 , 0.00 _ 0.65 30.10? 84.70 551.65 647.95 496.401.301.40 175.30 8.50 0.00 2296.65;
1998 0.45 0.00 74.95 51.45 462.50 597.50 689.05 699.55 488.90 111.25 10.05 25.70 3211.35
1999 0.00 2.55 10.75 97.10 456.75 682.90 879.05 685.08 561.10 100.25 23.05 1.80 3500.38

 

 

53

Table 3. Rain precipitation by month for the years 1947-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1999.

JAN. FEB. MAR. ”I!" MAY JUNE JULY AUG. SEPT. OCT. NOV. DEC. TOE“ $3;
1947 2.68 0.84 2.04 7.44 5.18 5.62 1.81 3.27 4.73 1.13 2.52 1.68 38.94 3.25
1948 1.20 2.40 4.66 3.51 5.59 3.37 1.94 1.69 2.99 1.05 2.83 3.20 34.43 2.87
1949 3.51 2.73 3.41 2.24 2.43 3.48 2.72 3.59 3.70 3.21 1.68 4.57 37.27 3.11
1950 4.08 3.49 2.47 7.41 1.32 5.00 4.80 2.71 7.92 0.56 3.01 2.25 45.02 3.75
1951 2.67 1.76 2.34 3.38 3.28 4.17 3.71 3.61 3.72 4.63 2.71 3.50 39.48 3.29
1952 2.18 1.10 2.40 3.09 4.99 2.58 4.21 3.54 2.06 0.78 3.18 2.11 32.22 2.69
1953 1.93 1.53 2.49 3.23 3.58 2.56 2.78 2.42 1.36 1.56 1.49 1.42 26.35 2.20
1954 1.95 4.10 3.51 2.81 1.37 7.27 2.52 3.72 2.63 8.06 2.70 2.18 42.82 3.57
1955 1.54 2.02 2.17 2.59 1.75 4.49 4.57 3.15 1.45 5.52 3.65 0.50 33.40 2.78
1956 0.59 2.68 2.45 5.84 3.62 2.35 2.79 1.81 0.56 0.35 1.52 1.14 25.70 2.14
1957 2.25 1.42 2.09 5.52 3.79 2.49 3.82 3.44 1.39 4.47 3.64 2.65 36.97 3.08
1958 1.37 0.94 0.84 1.83 1.55 5.71 3.92 3.52 2.42 1.80 2.37 0.74 27.01 2.25
1959 2.75 2.13 2.22 3.78 2.67 1.53 5.92 3.13 3.63 5.36 3.20 2.40 38.72 3.23
1960 4.26 3.35 1.27 2.68 5.14 4.54 3.46 1.73 2.56 1.65 2.03 0.60 33.27 2.77
1961 0.52 1.50 2.97 4.41 1.62 2.60 1.96 4.32 6.68 2.54 1.95 1.81 32.88 2.74
1962 2.94 1.73 1.58 1.75 3.47 4.22 3.51 1.48 3.98 2.53 0.88 2.36 30.43 2.54
1963 1.03 0.51 2.96 2.32 3.97 1.47 3.90 1.59 1.07 0.94 1.52 1.19 22.47 1.87
1964 1.05 0.79 2.87 4.34 2.34 2.49 4.13 5.39 4.36 1.02 2.75 1.94 33.47 2.79
1965 3.58 2.56 3.47 2.11 2.73 3.23 1.45 4.61 4.83 2.12 2.71 4.58 37.98 3.17
1966 0.87 1.28 3.32 4.07 3.52 2.42 2.08 4.50 1.52 1.01 5.61 3.70 33.90 2.83
1967 3.02 1.83 1.35 5.12 1.98 5.89 2.33 1.78 2.49 4.42 2.91 4.39 37.51 3.13
1968 1.58 2.12 1.31 4.31 2.82 6.66 3.89 2.76 3.08 3.25 3.91 3.68 39.37 3.28
1969 2.70 0.26 1.86 4.29 3.66 5.87 4.82 1.04 1.97 4.63 3.23 0.71 35.04 2.92
1970 1.03 0.77 3.06 4.14 3.96 2.42 8.91 1.88 3.47 4.88 3.63 2.09 40.24 3.35
1971 1.11 2.89 2.26 0.91 1.96 2.12 6.46 1.33 5.86 3.51 2.00 4.03 34.44 2.87
1972 1.73 0.90 3.50 3.07 2.95 2.90 3.77 4.21 4.92 3.01 3.15 4.59 38.70 3.23
1973 1.52 1.43 2.93 3.79 6.68 3.43 4.06 1.28 4.94 2.41 3.29 3.58 39.34 3.28
1974 3.27 2.49 4.07 4.12 5.11 3.90 1.19 1.75 3.59 1.52 3.08 2.29 36.38 3.03
1975 3.49 2.27 2.17 6.63 5.59 4.08 1.54 11.33 1.74 1.05 3.11 4.16 47.16 3.93
1976 1.54 2.33 3.55 3.88 3.45 3.22 3.62 0.55 2.19 2.63 1.85 1.26 30.07 2.51
1977 1.25 0.99 3.14 3.28 1.41 4.08 2.26 5.61 4.95 1.72 2.01 2.78 33.48 2.79
1978 2.88 0.39 1.43 2.31 2.36 11.03 2.35 2.15 5.85 2.99 2.07 3.34 39.15 3.26
1979 2.87 0.78 2.75 5.11 3.25 7.19 2.52 4.98 0.00 2.84 4.62 2.76 39.67 3.31
1980 1.04 1.34 2.49 3.70 2.56 5.25 4.96 5.54 4.92 2.11 1.32 4.09 39.32 3.28
1981 0.53 2.14 0.85 5.45 3.62 3.65 1.72 3.85 5.29 2.80 1.54 1.62 33.06 2.76
1982 3.08 1.04 3.45 1.66 5.05 3.39 5.47 2.12 1.49 1.08 4.09 4.56 36.48 3.04
1983 0.90 1.04 3.40 4.59 6.19 1.90 3.41 5.63 5.31 2.22 3.80 3.42 41.81 3.48
1984 0.94 1.19 2.91 2.44 4.16 0.78 4.08 1.75 7.06 3.66 3.39 4.15 36.51 3.04
1985 3.09 4.06 3.13 3.07 3.18 1.57 6.40 4.49 2.54 5.14 6.13 1.89 44.69 3.72
1986 2.04 3.25 1.65 5.20 3.96 6.43 7.40 3.82 9.92 3.67 1.59 1.40 50.33 4.19
1987 1.62 0.07 1.32 2.13 2.00 2.75 4.02 7.66 4.21 2.41 2.30 4.44 34.93 2.91
1988 2.00 2.28 1.51 3.82 0.98 1.60 2.89 4.61 7.63 4.23 4.18 2.32 38.05 3.17
1989 1.41 3.29 2.16 2.00 5.91 4.76 2.05 3.31 3.51 0.86 3.21 1.13 33.60 2.80
1990 2.35 3.56 1.96 2.36 6.21 5.24 2.63 2.60 3.70 6.53 6.56 2.88 46.58 3.88
1991 1.71 3.28 4.99 5.95 3.41 1.93 3.94 3.31 2.30 8.92 4.32 3.24 47.30 3.94
1992 1.67 1.24 2.05 3.09 1.42 1.26 5.37 3.47 5.00 2.88 4.82 2.21 34.48 2.87
1993 3.40 1.81 2.64 4.70 1.82 5.91 3.17 3.08 6.01 3.55 1.53 1.94 39.56 3.30
1994 2.10 1.75 1.78 3.95 1.25 5.88 4.36 6.19 1.20 1.82 4.52 1.40 36.20 3.02
1995 2.22 1.00 . . . 3.31 2.85 5.03 2.13 3.75 4.73 1.11 35.30 2.94
- ~ 151531058" ' " ;' ~.- «1.49- 1:33""32.77:“:3W5-#25136 -"3:31-' 458235 2%“
3119977,.332; ~2.68 __2.52_. 1.34 43.88-— 3.50__ ,_.0.85 3.41, 4.82;...123, 0.98 1.58 730.11 2.514.;
1998 3.71 1.39 2.98 4.29 1.83 3.12 3.95 1.56 1.36 2.34 1.37 1.16 29.06 2.42
1999 4.62 0.54 1.60 6.06 1.38 4.43 2.89 1.67 1.91 0.96 1.30 3.22 30.58 2.55
130V: 2.51 1.96 2.24 3.62 3.11 4.09 3.05 3.18 3.16 3.31 3.22 2.11 35.56
avg 2.07 1.75 2.37 3.59 3.12 3.63 3.29 3.13 3.42 2.73 2.81 2.46 33.68

 

 

 

 

 

 

 

54

 

 

 

 

 

 

 

Table 4. Snowfall Amounts for the winter months October
through May 1946-47 to 1998-99.

Oct. Nov. Dec. Jan. Feb. Mar. April May Total/Snow
1946-47 0.00 0.00 9.75 11.50 20.00 12.25 1.50 0.00 55.00
1947-48 0.00 9.50 8.25 13.75 5.25 7.75 0.00 0.00 44.50
1948-49 0.00 0.00 5.00 3.25 5.25 4.00 0.50 0.00 18.00
1949-50 0.00 12.50 5.00 4.00 17.00 10.00 5.00 0.00 53.50
1950-51 0.00 17.00 12.00 14.00 7.00 7.00 2.00 0.00 59.00
1951-52 0.00 17.50 38.00 8.00 4.00 5.00 5.00 0.00 77.50
1952-53 0.00 2.00 5.00 7.00 5.50 1.25 0.00 0.00 20.75
1953-54 0.00 8.75 7.50 17.25 15.50 20.00 0.50 1.00 70.50
1954-55 3.00 11.00 10.00 10.50 11.50 19.00 0.00 0.00 65.00
1955-56 0.00 20.25 7.75 7.00 16.00 21.50 5.00 0.00 77.50
1956-57 0.00 12.00 5.00 30.75 7.00 6.00 5.00 0.00 65.75
1957-58 0.50 12.25 13.25 23.50 18.00 12.50 0.00 0.00 80.00
1958-59 0.00 11.00 13.25 25.00 7.00 13.25 0.00 0.00 69.50
1959-60 0.00 13.50 10.63 17.25 28.25 17.00 1.75 0.25 88.63
1960-61 0.00 8.00 11.00 15.50 14.25 2.25 13.75 0.00 64.75
1961-62 0.00 2.50 18.25 18.75 19.75 7.50 5.00 0.00 71.75
1962-63 3.00 0.00 41.50 23.00 16.50 15.00 1.00 0.00 100.00
1963-64 0.00 0.00 16.00 7.50 11.50 12.00 1.00 0.00 48.00
1964-65 0.00 10.50 26.00 14.75 33.25 27.25 7.00 0.00 118.75
1965-66 0.00 7.50 8.75 14.75 7.25 11.50 0.00 0.00 49.75
1966-67 0.00 17.25 21.50 35.25 29.50 9.50 5.00 0.00 118.00
1967—68 4.00 10.00 10.00 15.00 7.75 15.50 0.00 0.00 62.25
1968-69 0.00 9.50 32.50 27.00 4.75 7.75 0.50 0.00 82.00
1969-70 0.00 14.00 9.75 15.50 11.50 20.50 9.25 0.00 80.50
1970-71 0.00 14.50 19.50 16.25 8.25 19.50 1.50 0.00 79.50
1971-72 0.00 14.00 3.50 15.00 12.50 9.25 0.00 0.00 54.25
1972-73 0.25 13.75 20.50 6.75 14.75 10.50 4.25 0.00 70.75
1973-74 0.00 2.00 19.75 14.50 12.25 6.25 0.00 0.25 55.00
1974-75 0.00 6.50 17.50 9.75 18.50 7.75 9.00 0.00 69.00
1975-76 0.00 7.50 15.25 26.75 4.00 2.75 2.50 0.00 58.75
1976-77 1.75 8.00 23.50 23.00 4.00 7.50 3.25 0.00 71.00
1977-78 0.00 10.00 22.50 47.50 5.75 2.50 0.00 0.00 88.25
1978-79 0.00 8.00 20.25 34.50 4.75 3.25 6.50 0.00 77.25
1979-80 0.00 8.25 5.50 12.75 9.50 10.00 4.50 0.00 50.50
1980-81 0.50 8.00 10.50 12.50 19.75 0.75 0.00 0.00 52.00
1981-82 0.75 2.00 8.75 29.00 15.50 11.00 0.00 0.00 67.00
1982-83 0.00 1.50 3.25 2.25 5.25 8.75 2.00 0.00 23.00
1983-84 0.00 4.00 32.25 12.75 0.75 9.25 0.00 0.00 59.00
1984-85 0.00 0.50 12.00 30.50 24.25 0.50 2.00 0.00 69.75
1985-86 0.00 2.00 27.25 10.50 16.75 0.00 0.00 0.00 56.50
1986-87 0.00 5.00 5.25 14.00 0.00 4.00 0.00 0.00 28.25
1987-88 0.00 0.00 10.20 6.50 16.50 2.00 0.00 0.00 35.20
1988-89 0.00 2.00 6.25 7.25 20.26 1.00 1.00 0.00 37.76
1989-90 3.25 6.25 10.50 2.00 21.00 1.00 0.00 0.00 44.00
1990-91 0.00 0.00 10.25 16.00 6.00 0.00 0.00 0.00 32.25
1991-92 0.00 6.00 14.75 18.00 1.00 14.25 0.00 0.00 54.00
1992-93 0.00 12.00 11.00 18.00 13.00 8.00 0.00 0.00 62.00
1993-94 0.00 1.00 8.50 12.25 19.00 0.50 0.00 0.00 41.25
1994-95 0.00 0.00 12.50 15.75 3.50 0.00 0.00 0.00 31.75
:1 1995556 0.061 - 11.50 » - ‘ 6325 ‘6.00'-'- 35? 10.00 000 ‘~ 0.0? - 37:2?” 1;
31996-97, ,, ._ 0.00 4.007 , 13.25”. $38.75 . $5.75 1.00 0.00 , _ 0.00 . 62.75 . _‘;
1997-98 2.00 0.00 8.75 11.25 0.00 10.00 0.00 0.00 32.00
' 1998-99 0.00 0.00 1.50 31.85 3.00 10.50 0.00 0.00 46.85
10 yr avg 0.48 3.89 9.41 16.10 8.73 5.11 0.09 0.00 43.81
Average 0.36 7.26 13.70 16.63 11.55 8.62 1.99 0.03 59.03

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

55

Table 5. Yearly Means and Totals for the years 1946—1999.

Mean Mean Mean Total Total
Max.T Mln.T T Snow
1 . .1 1

55.99 . 45.36 55.00
57.68 45.35 34. 44.50

7 1
55.74 44.54 53.50
56.16 44.93 59.00
58.45 46.41 77.50
61 44 .75
09 46.79 70.50

            

16 46.79 . 77.50
58. 46.99 75

47.74 69.50
46.57

47. 1

46.44

45.

47.98

47.18

46.22

47

Average 57.68 36.67 47.17

56

Appendix B

Attached CD-R Digital Media

There is an attached CD-R containing the digital movie
complements to Figures 5, 6, 7, 8, and 9 in some copies of
this thesis. Those who do not have a copy of the digital
media and wish to View the digital movies may contact the
author to obtain versions of the data.

Specifications:

Recorded with a Smart and FriendlyTM CD-R 4012 using the
program "Easy CD Creator" on a Windows NT 4.0 workstation
administered by Dr. Stanley L. Flegler at the Center for
Advanced Microscopy, Michigan State University.

Movies recorded on the CD as .AVI using the current Video
for Windows format in Adobe After Effects version 4.0.

Additional copies of the digital movies are included in
various formats.

58