son. The group presumed to be of ‘intermediate’ age, species, expressed an intermediate level of
emergence. The demonstration in that paper of a possible
relationship between phylogenetic time, which is only inferred, and the degree of emergence raises
this question: is there a similar relationship be- tween the degree of emergence and time when
time is assessed by the real time of development? More specifically, will older needle primordia
show a greater degree of emergence than younger? That is the purpose of this paper, to report on an
analysis of emergence in an ontogenetic study of differently aged needle primordia of Douglas fir
Pseudotsuga menziesii Mirb. Franco.
A broader and more abstract way to envision the real time of ontogeny consists of starting with
the histogenetic events that occur during needle development; the division, differentiation and
elongation of cells in the needle primordia. As these cells mature, they act as ‘clocks,’ ‘clocks’
referring to a means to measure or perceive time, creating their own local time as they interact with
other cells and respond to previous stages as ontogeny progresses. Histogenetic events occur-
ring later in the process reference earlier steps in development, or, the cellular communication
through which development is coordinated gener- ates local, asymmetric unidirectional time Mat-
suno, 1988. The many cells, behaving as ‘clocks’ when they interact with other cells, form the
integrated unit we recognize as a conifer needle primordium. A needle primordium, when used as
a point of reference, can also be viewed as a higher level ‘clock’ comprised of many component
‘clocks’ formed from individual cells and tissues. This communication, through which form un-
folds, also references ‘global,’ or ‘in the record’ phylogenetic information that exists because of
the unique history of Douglas fir as a species, as a conifer, and as a vascular plant Matsuno, 1988;
Matsuno and Salthe, 1995. In the context of this study, the initiation of a needle primordium starts
a ‘clock’ which forms the reference for subsequent ‘clocks’ that appear with more ontogenetic events.
As more needle primordia are initiated, they refer- ence the ‘clocks’ generated by events that pre-
ceded them. Upon reaching some threshold, the primordia move beyond a generalized mass of
cells and form a structure that is distinctively a Douglas fir needle. Matsuno’s approach to view-
ing ontogeny local scale and phylogeny globally encompassing as different tiers or sets of time
emphasizes the hierarchical nature of biological organization Matsuno, 1988. This view includes,
but is not limited or reduced to, the expression of genetic information. Parts of a bud rely on the
‘clocks’ within a bud, the buds on a tree rely on ‘clocks’ established within a tree, a tree relies on
the ‘clocks’ that existed in its parents and so on back through its phylogenetic history. The reverse
process does not occur, that is, phylogenetic time does not reference local, developmental events
because it is part of the historical record. Thus, what we are studying here, while it is focused on
needle primordia in buds, is part of an integrated series with a long history. Using these concepts of
time serves to emphasize the historical interrelat- edness of all botanical structures. As well, these
self-same concepts identify the kind of compari- sons Goethe identified as the holistic approach
necessary to understand botanical events Arber, 1946.
2. Materials and methods
2
.
1
. Plants The material for this study came from four
full-sib families that were part of a partial diallel of Douglas fir Maze and Banerjee, 1989. For the
plants analyzed terminal buds were taken from the uppermost whorl of branches, with one to
four branches being sampled per plant. There were 115 plants, 28 from one family and 29 from
the other three, for a total of 230 buds. The buds were sampled in March 1988, fixed in formalin-
acetic acid-alcohol 90 cm
3
ethanol, 6 cm
3
forma- lin, 4 cm
3
glacial acetic acid, embedded in paraplast, sectioned at 15 mm, and stained with
safranin and fast green Johansen, 1940. Mea- surements were made from the projection head of
a Zeiss Ultraphot II microscope. The measure- ments made on the needle primordia were the
thickness at the point of attachment b, thickness
at the midpoint perpendicular to the main axis of the primordium m and length of the needle
primordium along a line from the midpoint of the attachment to the tip l Fig. 1II. There were
two criteria used in picking needle primordia to measure. First, they had to be from sagittal sec-
tions of buds as determined by being able to see the cytohistological zonation in the apical meris-
tem and the clarity of image of cell walls. The second criterion was applied to the specific needle
primordia. For the youngest, which usually lack procambium, we relied on clarity of cell wall
image. For the older we chose those primordia in which the procambium could be seen extending
into the stem and also had clear cell wall images. Young, middle age and older needle primordia
were chosen by taking two needle primordia on opposite sides of the incipient stem from the
uppermost needle primordium in sagittal section, the young, the bottom-most the old, and at the
midpoint of the bud the middle age. Fig. 1 also includes a diagram of a bud showing the relative
positions of the primordia chosen I. Labeling the needle primordia closest to the apical meris-
tem as young and those farthest away as old is appropriate even though they all coexist in one
bud. The needle primordia at the base of the bud were formed first while the bud was developing in
the year before it was sampled, the needle primor- dia nearest the apical meristem were those formed
last in the same year. There were 424, 427 and 440 needle primordia for the young, middle age and
old, respectively. The difference in numbers is due to the inability to measure certain needle primor-
dia because of artifacts of sectioning.
2
.
2
. Analyses The needle primordia for all four families were
combined for the analyses, which gave us a much larger sample size, an advantage when performing
the indirect and complicated analyses we used. As well, the families were not strongly different.
Based on average r
2
values for the three variables measured, the families accounted for only 2.3, 6.3
and 10.4 of the variation in the data for the young, middle age and old, respectively.
Each set of differently-aged needle primordia was used to create two hierarchical levels using
the same approach as in Maze and Bohm 1997, Maze 1998, 1999. The differently-aged needle
primordia sets, the young, middle age and old, were bootstrapped 50 times to create data sets
with a complete set of variables for 400 needle primordia; these 50 bootstrapped samples repre-
sented the higher hierarchical level for subsequent analyses. When the 50 samples were drawn, the
individual plants from each of the families were scattered, without apparent order, within the orig-
inal data file. This was done to eliminate any effect the four different families may have had on
the outcome of the analyses even though the family effect was small. In order to create the
lower hierarchical level each bootstrapped sample was divided in half. Each of these halves repre-
sented a subgroup, the lower hierarchical level, and the entire sample, the whole, the higher hi-
erarchical level. The statistic calculated for the
Fig. 1. I, Diagram of bud. am, apical meristem; y, young needle primordia; m, middle age needle primordia; o, old
needle primordia. II, Diagram of single needle primordium showing measurements made. b, width at point of attachment;
m, width at middle; l, length.
different hierarchical levels was the angle between first eigenvectors, derived from a principal com-
ponents analysis PCA of a correlation matrix, and a vector of isometry. Only the first eigenvec-
tor was used since it alone was consistently derived from a data matrix that was not spherical
as determined by either Bartlett’s or Anderson’s test Pimentel, 1993. Once this statistic was calcu-
lated, the degree of emergence was determined as the difference in that angle between the subgroups
and the whole and is expressed as the average degree of emergence AVGD, i.e. the average
difference in angles with a vector of isometry between each subgroup, the lower hierarchical
levels, and the entire sample, the higher hierarchi- cal level.
In order to explore the relationship between the average degree of emergence and properties of the
needle primordia, a multiple regression analysis was done with AVGD as the dependent variable
and two estimates of variation as the independent. One of these estimates was variation in size, or the
spread of individual structures around a mean value. This is the usual way variation is assessed
in a biological context. This was estimated as the within-age group variation in PCA axis scores
from the original data set which combined all needle primordia, the young, middle age and old.
Again, only the first axis was used. The other estimate was variation in integration and refers to
the variation in organization among the variables measured. This is variation in growth rate among
the variables relative to each other and could also be called variation in allometry. In spite of the
significance of allometry in biological studies, its variation is often not directly addressed. The vari-
ation in integration was approximated by the variance in eigenvector loadings on the first PCA
axis from the original data sets from which the bootstrapped samples were gathered, i.e. from an
analysis of all the young, middle age and old needle primordia. Before the multiple regression
analysis was done the estimates of variation in size and integration were standardized so that
their coefficients could be directly compared through converting the means of both to 0.0. This
is the same analysis as was done in Maze 1999. The bootstrapping was done using the random
sample generator in
SYSTAT
4.1 Wilkinson, 1988 and the angles with a vector of isometry were
calculated by Pimentel’s
MPCA
program. Calcula- tion of angles with a vector of isometry in Pi-
mentel’s program
relies on
the standard
calculation of the angle between vectors, i.e. the ratio of the dot product to the product of the
norms of the vectors. Pimentel’s program also calculates the statistics for the entire data set, the
within-groups analysis which represents the higher hierarchical level, from the within-groups disper-
sion matrix, the weighted average of the group dispersions Pimentel, 1993. This approach is less
sensitive to assumptions about the within-group dispersions, e.g. equality, that describe the lower
hierarchical levels. Details are presented in Pi- mentel 1979. Comparisons of AVGD among the
differently-aged needle primordia was done using Tukey’s multiple comparison in
SYSTAT
4.1. The probability level for rejection of similar AVGD
values was set at a conservative 0.0001. The analyses done appear to be very compli-
cated, but the basic idea behind them is simple. The organizational properties, as expressed in a
correlation matrix of an entire data set, are de- scribed, as are the organizational properties, also
expressed as correlation matrices, of two subsets of that entire data set. The description of those
organizational properties for the entire data set and the two subsets are then compared. To de-
scribe the organizational properties, we used an angle with a vector of isometry, which is related
to the properties of a correlation matrix.
We also generated notched box plots for each measured variable for the three differently-aged
needles. This offers the most direct graphical rep- resentation of the data and allows simple observa-
tions to be made of how patterns of variation change through time for each variable.
Another analysis, a PCA of each of the differ- ently-aged needle primordia, was done. The pur-
pose here
was to
describe the
changing relationships among the variables as expressed in
eigenvector elements. This is an assay of the spe- cific ontogenetic changes that are summarized,
first in angles with a vector of isometry and second with AVGD. Again, only the first axis was
used since it alone was consistently derived from a non-spherical data set.
3. Results
